# Patent

## Title
Screening methods

## Abstract

The present invention provides polypeptide binding agents, e.g. antibodies, that exhibit the ability to kinetically modulate the binding and signaling of biological signaling complexes, e.g., receptor-ligand complexes; methods of identifying such polypeptide binding agents, methods of making such polypeptide binding agents, compositions comprising such polypeptide binding agents, and methods of using such polypeptide binding agents.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims the priority benefit of U.S. Provisional Patent Application No. 61/246,079, filed Sep. 25, 2009 and U.S. Provisional Patent Application No. 61/306,324, filed Feb. 19, 2010, each of which is incorporated by reference in its entirety.


FIELD
The present invention relates to methods of screening for polypeptide binding agents, e.g. antibodies, that exhibit the ability to kinetically modulate the binding and signaling of biological signaling complexes, e.g., receptor-ligand complexes. The invention also relates to specific polypeptide binding agents characterized by desired kinetic modulating properties.
INCORPORATION OF APPENDIX
This application includes a table, Appendix A, “41726_SecretedProteins.txt”, 255 KB in size, created 25 Sep. 2009, submitted with this application. The material included in this ASCII text file is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. §1.52(e)(5). 







TABLES




The patent contains table(s) that have been included at the end of the specification.






BACKGROUND
Most antibody drugs are conventionally identified by screening for antibodies that bind to either a cell-surface receptor or its cognate ligand, and identifying antibodies that specifically block or stimulate the receptor signaling activity. Many antibody drugs block signaling pathways by binding to either the ligand or receptor, thereby eliminating the ability of the ligand to bind to and activate the receptor. Such blocking antibodies mediate their effect stoichiometrically by preventing the formation of receptor-ligand complex. Conversely, some antibody drugs bind to and activate signaling of a receptor. Such agonist antibodies may mediate their effects by mimicking the natural activity of the ligand and thus do not require the presence of ligand to activate signaling.
SUMMARY
The invention provides novel categories of polypeptide binding agents, designated “kinetic modulating drugs” or “kinetic modulators,” that have desirable properties for modulating, either positively or negatively, cellular pathway activity involving a target and its signaling partner. The target and/or its signaling partner may be an endogenous or exogenous compound, either proteinaceous or non-proteinaceous in nature, but which optionally may exclude ions and salts. The invention also provides novel methods of identifying such kinetic modulators, based on their effect on binding kinetics between the target and its signaling partner, or based on differential binding of the kinetic modulator for the target (and/or its signaling partner) in complexed form versus uncomplexed form. The polypeptide binding agent may bind the target, its signaling partner and/or a complex comprising the target and its signaling partner. This discovery allows biophysical screening assays to be designed which can identify modulators of cellular pathway activity suitable for therapeutic use.
In some aspects, assays are provided to identify polypeptide binding agents which modulate the binding kinetics between a target and its signaling partner. Nonlimiting examples of targets include, e.g. a secreted protein of any of the Accession nos. as set forth in Appendix A (or SEQ ID NOS: 1-88). These secreted proteins include a number of secreted membrane-bound receptors. Appendix A herewith lists human secreted proteins as compiled by the Swissprot/EMBL database (see e.g., Boeckmann et al. “The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003”, Nucleic Acids Res. 31:365-370 (2003). Appendix A sets out the Swissprot accession number for the amino acid sequence of the secreted protein, the name of the protein (and all acronyms or related names) and the length of the amino acid sequence in the database. As used herein, a “signaling partner” is a binding partner of a target that, when bound to the target, forms a signaling complex or is part of a signaling complex that activates or inhibits a cellular pathway. The presence of such kinetic modulator polypeptide binding agents alters (strengthens or weakens) the apparent binding affinity of the target for its signaling partner, thus altering the dose-response of the target for activating the cellular pathway. Alternatively, a kinetic modulator polypeptide binding agent that alters (increases or decreases) the on-rate or alters (decreases or increases) the off-rate of the target for its signaling partner can also change (increase or decrease) the residency time of the target complexed with the signaling partner, change the rate of receptor internalization and/or change the degree of phosphorylation of signaling proteins that are activated or deactivated by the signaling partner complex. Such changes could significantly alter the relative activation of different signaling pathways by the complexation of target and signaling partner and thus alter the dose-response of the target for activating the cellular pathway. Such kinetic modulators are expected to have advantages over conventional therapeutic drugs, including improved safety profiles, altered clearance rates, broader therapeutic windows and less frequent dosing. Where the target is an exogenous compound that is being administered to the patient, administration of the kinetic modulator as an adjunct therapy with the target can alter (e.g., decrease) the total amount (daily, weekly or monthly) and/or the frequency of dosing of the target.
The invention provides methods of identifying candidate kinetic modulating drugs that are polypeptide binding agents, excluding traditional small molecule drugs such as non-polymeric organic chemical compounds having a molecular weight of about 1000 daltons or less. Examples of specifically contemplated polypeptide binding agents include antibodies, including antigen-binding fragments thereof, peptibodies, polypeptides and peptides, optionally conjugated to other peptide moieties or non-peptidic moieties. Examples of antibodies include monoclonal antibodies, tetrameric immunoglobulins comprising two heavy chains and two light chains, single chain antibodies, single domain antibodies, antibody fragments, scFv, Fab, CDRs, rodent antibodies, mammalian antibodies, human antibodies, chimeric antibodies and humanized antibodies.
The invention provides methods of identifying a candidate polypeptide binding agent, e.g. an antibody, that modulates binding between first and second components of a signaling complex (the target and signaling partner, or vice versa). Examples of such first and/or second components include any of the secreted proteins of Appendix A (or SEQ ID NOS: 1-88) and endogenous or exogenous signaling partners of such secreted proteins, or any of the ligands or receptors or transmembrane proteins described herein. In some embodiments, the first and second components are polypeptides. In exemplary specific embodiments, the first and second components are endogenous.
In one aspect, the methods of identifying a candidate kinetic modulating drug include (a) measuring a binding affinity or binding rate parameter of said first component for said second component, in the presence of a test polypeptide binding agent, e.g. antibody, (b) measuring a binding affinity or binding rate parameter of said first component for said second component in the absence of said test polypeptide binding agent; and (c) identifying said test polypeptide binding agent as a candidate kinetic modulating drug when said test polypeptide binding agent exhibits at least a 1.5-fold difference in a binding affinity or binding rate parameter measured in steps (a) and (b). FIG. 1 shows a schematic diagram illustrating some exemplary embodiments. In some embodiments, the difference in binding affinity or binding rate parameter ranges from about 1.5-fold (i.e., 50%) to, optionally, about 1000-fold, or about 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about 2-fold to 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, or up to 30-fold. In some embodiments, the test polypeptide binding agent is identified as a candidate positive modulator if the test polypeptide agent strengthens a binding affinity or binding rate parameter between said first component and said second component (e.g., reduced KD, or increased KA, or reduced ratio of off-rate/on-rate, or increased ratio of on-rate/off-rate, or increased on-rate, or decreased off-rate). In other embodiments, the test polypeptide agent is identified as a candidate negative modulator if the test polypeptide agent weakens a binding affinity or binding rate parameter between said first component and said second component (e.g., increased KD, or decreased KA, or increased ratio of off-rate/on-rate, or decreased ratio of on-rate/off-rate, or decreased on-rate, or increased off-rate).
In some alternative embodiments, where a stronger binding rate parameter (e.g., increased association or residency time, via increased on-rate or decreased off-rate) results in increased relative activation of the desired signaling pathway, even when binding affinity is not detectably changed, the test polypeptide binding agent is identified as a candidate positive modulator by identifying the desired-fold strengthening in binding rate parameter. Where a weaker binding rate parameter (e.g., decreased association or residency time, via decreased on-rate or increased off-rate) results in increased relative activation of the desired signaling pathway, even when binding affinity is not detectably changed, the test polypeptide binding agent is identified as a candidate positive modulator by identifying the desired-fold weakening in binding rate parameter. Similarly, where a stronger binding rate parameter (e.g., increased association or residency time, via increased on-rate or decreased off-rate) results in decreased relative activation of the desired signaling pathway, even when binding affinity is not detectably changed, the test polypeptide binding agent is identified as a candidate negative modulator by identifying the desired-fold strengthening in binding rate parameter. Where a weaker binding rate parameter (e.g., decreased association or residency time, via decreased on-rate or increased off-rate) results in decreased relative activation of the desired signaling pathway, even when binding affinity is not detectably changed, the test polypeptide binding agent is identified as a candidate negative modulator by identifying the desired-fold weakening in binding rate parameter.
In another aspect, the methods of identifying a candidate kinetic modulating drug include (a) (i) measuring a binding affinity or binding rate parameter of a test polypeptide binding agent, e.g. antibody, for said first component in the presence of said second component, or (ii) measuring a binding affinity or binding rate parameter of a test polypeptide binding agent for said second component in the presence of said first component; and (b) (i) measuring a binding affinity or binding rate parameter of said test polypeptide binding agent for said first component in the absence of said second component, or (ii) measuring a binding affinity or binding rate parameter of said test polypeptide binding agent for said second component in the absence of said first component; and (c) identifying said test polypeptide binding agent as a candidate kinetic modulating drug when said test polypeptide binding agent exhibits a 1.5-fold to 100-fold difference in the binding affinity or binding rate parameter measured in steps (a) and (b). FIG. 2 shows a schematic diagram illustrating some exemplary embodiments, in which interaction is measured in the presence and absence of the second component.
In some embodiments, the difference in binding affinity or binding rate parameter measured in steps (a) and (b) ranges from about 1.5-fold (i.e., 50%) to, optionally, about 1000-fold, or about 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, or up to 30-fold. In some embodiments, the test polypeptide binding agent is identified as a candidate positive modulator if the binding affinity or binding rate parameter measured in step (a) is stronger than the binding affinity or binding rate parameter measured in step (b). In other embodiments, the test polypeptide binding agent is identified as a candidate negative modulator if the binding affinity or binding rate parameter measured in step (b) is stronger than the binding affinity or binding rate parameter measured in step (a).
Any of the foregoing methods can be carried out as high throughput assays, in which multiple polypeptide binding agents (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, 150, 200, 500, 1,000, 10,000, or 25,000) are screened simultaneously or sequentially. In some embodiments, the methods further involve assaying a plurality of test polypeptide binding agents, e.g. antibodies, for binding affinity to any one of (a) the first component, (b) the second component, or (c) a complex comprising the first component and second component, optionally prior to measuring differences in binding affinity or binding rate parameter. Such prescreening of libraries can also be carried out as high throughput assays, in which multiple polypeptide binding agents (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, 150, 200, 500 or 1000) are screened simultaneously or sequentially. In some embodiments, the plurality of test polypeptide binding agents screened are variants of a parent polypeptide binding agent made by introducing one or more different mutations into a parent polypeptide binding agent.
In further embodiments, the polypeptide binding agents may be screened for selectivity of effect for the first or second component, compared to a different binding partner such as a decoy receptor, clearance receptor, or alternate signal pathway component. Such methods may involve identifying a polypeptide binding agent that does not significantly change the binding affinity or binding rate parameter of the first or second component for a different binding partner, such binding partner being neither the first nor second component.
Any of the preceding measurements of binding affinity or binding rate parameter may be carried out in assays where one or more of the first component, second component and polypeptide binding agent are in solution, or in assays where one or more of the first component, second component and polypeptide binding agent are linked to a solid phase (covalently or noncovalently), or in assays where one or more of the first component, second component and polypeptide binding agent are expressed on a cell surface. The first and/or second components may each themselves be complexes of multiple compounds. The first and/or second components (e.g., target or signaling partner or vice versa) may be soluble or membrane-bound ligands or receptors, including but not limited to 7-transmembrane receptors, G-protein coupled receptors (GPCRs), adrenergic receptors, neurotransmitter receptors, olfactory receptors, opioid receptors, chemokine receptors, rhodopsin, receptor tyrosine kinases, growth factor receptors, integrins, and toll-like receptors, enzymes, or substrates.
Any of the preceding methods may further include recloning and expressing, or synthesizing and expressing, or synthesizing, the candidate kinetic modulating polypeptide binding agent; purifying and/or sequencing the kinetic modulator; adding or replacing an Fc region or fragment thereof; formulating the kinetic modulator or a variant, e.g. an antibody comprising at least three or six of the same CDRs of the parent antibody, in a sterile composition with a sterile pharmaceutically acceptable diluent; and/or administering the kinetic modulator or a variant to an animal.
Any of the preceding methods may further include measuring the level of signaling mediated by the signaling complex in the presence and absence of the test polypeptide binding agent, and determining whether the test polypeptide binding agent is additionally an agonist, partial agonist, antagonist or partial antagonist. In certain embodiments, the agonist or partial agonist is an allosteric agonist.
In related aspects, the invention provides a polypeptide binding agent, e.g. an antibody, identified by any of the preceding methods or any of the methods described elsewhere herein.
In a separate aspect, the invention also provides polypeptide binding agents with desired characteristics. In some embodiments, the invention provides a positive modulator that (a) binds to the target, e.g., a secreted protein of any of Appendix A (or SEQ ID NOS: 1-88) with an equilibrium dissociation constant KD of about 10−5M or less, e.g., 10−6M or less, or 10−7M or less, or 10−8M or less (wherein a lower number indicates higher binding affinity), and (b) is capable of improving the binding affinity KD between said target and its signaling partner by about 1.5-fold (i.e., 50%) to, optionally, about 1000-fold, or about 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, or up to 30-fold. In other embodiments, the invention provides a negative modulator that (a) binds to the target, e.g., secreted protein of any of Appendix A (or SEQ ID NOS: 1-88) with an equilibrium dissociation constant KD of about 10−5M or less, e.g., 10−6M or less, or 10−7M or less, or 10−8M or less, and (b) is capable of reducing the binding affinity KD between said target and its signaling partner by about 1.5-fold (i.e., 50%) to, optionally, about 1000-fold, or about 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, or up to 30-fold.
Any of such polypeptide binding agents may be further subject to purification, to obtain a substantially homogeneous composition, e.g. at least about 90%, 95%, 97%, 98%, 99% or 99.5% pure.
The invention further provides methods of preparing a sterile pharmaceutical composition comprising adding a sterile pharmaceutically acceptable diluent to such polypeptide binding agents, sterile compositions of such polypeptide binding agents, e.g., in a therapeutically effective amount, and methods of administering such sterile compositions, e.g. to modulate (increase or decrease) signaling of a complex comprising the secreted protein.
It is understood that each feature or embodiment, or combination, described herein is a non-limiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein. For example, where features are described with language such as “one embodiment”, “some embodiments”, “further embodiment”, “specific exemplary embodiments”, and/or “another embodiment”, each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination. Such features or combinations of features apply to any of the aspects of the invention. Similarly, where a method describes identifying polypeptide binding agents, such as antibodies, characterized by certain features, polypeptide binding agents characterized by those features are also contemplated by the invention. Where examples of values falling within ranges are disclosed, any of these examples are contemplated as possible endpoints of a range, any and all numeric values between such endpoints are contemplated, and any and all combinations of upper and lower endpoints are envisioned.
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently preferred embodiments thereof. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this application, are incorporated herein by reference, in their entireties.



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-1F are schematic diagrams to illustrate binding assay configurations for measurement of binding performed in the presence or absence of test polypeptide binding agent.
FIGS. 2A-2F are schematic diagrams to illustrate binding assay configurations for measurement of binding performed in the presence or absence of a second complex component.
FIGS. 3A-3C show simulated data to show predicted effects of kinetic modulators on signaling activity at (FIG. 3A) varying ligand concentrations; (FIG. 3B) varying modulator concentrations (non-agonist antibody); and (FIG. 3C) varying modulator concentrations (agonist antibody).
FIG. 4 shows simulated data from an equilibrium solution affinity measurement method to detect modulation of a protein-protein interaction.
FIGS. 5A-5B show the effects of XOMA 052 on the affinity of IL-1β binding to IL-1 sRI (FIG. 5A), and to IL-1 sRII (FIG. 5B). XOMA 052 reduces the affinity of IL-1β binding to IL-1 sRI (A), but has no effect on the affinity of IL-1β binding to IL-1 sRII (B).
FIGS. 6A-6C show (FIG. 6A) neutralization of IL-1β activity by XOMA 052 at the EC50 of IL-1β for the cell assay, and (FIGS. 6B, 6C) that negative affinity modulation of the IL-1β to IL-1 sRI interaction results in an altered cellular dose-response to IL-1β resulting in an increase in the IC50. Negative affinity modulation of the IL-1 to IL-1 sRI interaction results in an altered cellular dose-response to IL-1.
FIG. 7 shows the amount of total IL-1β remaining in circulation 48 hours following injection of antibody/IL-1β complexes.
FIG. 8 is an illustration of the regulation of IL-1β activity by different drug types in T2D.
FIGS. 9A-9C show results from solid phase affinity measurement assays to identify antibodies which modulate the GCSF-GCSFR binding interaction as described in Example 2.
FIGS. 10A-10B show GCSF-dependent binding of A10(B6) antibody to GCSFR-transfected BAF3 cells.
FIGS. 11A-E show sample results from a cell-based affinity measurement assay to identify antibodies which modulate the hINS-INSR binding interaction. FIGS. 11A-E represent example results from receptor occupancy screen showing test antibody binding to IM-9 cells in the presence and absence of insulin.
FIGS. 12A-D show sample results from a cell-based affinity measurement assay to measure modulation of the hINS-INSR binding interaction by test antibodies. FIGS. 12A-D represent example results from biotinylated ligand screen showing the effects of test antibodies on insulin binding to insulin receptor.
FIG. 13 shows example results from an assay measuring the ability of test anti-INSR antibodies to stimulate pIRS-1 phosphorylation.
FIG. 14 is a table showing insulin EC50 values from the pIRS-1 assay in the presence or absence of fixed concentrations of various test antibodies.
FIGS. 15A-B show blood glucose levels in 20 week old DIO mice fed a high fat diet and treated with partial agonist anti-INSR antibodies: FIG. 15A. Line graph of glucose levels. FIG. 15B. Bar chart of glucose levels showing statistically significant reduction in blood glucose after injection of partial agonist anti-INSR antibody.
FIGS. 16A-C illustrate that administration of a partial agonist anti-INSR antibody improves glycemic control in DIO mice: FIG. 16A. Glucose tolerance test timecourse; FIG. 16B. Fasting blood glucose levels; FIG. 16C. Glucose tolerance test; area under curve (AUC).
FIGS. 17A-C show that a positive modulator anti-INSR antibody improves insulin sensitivity in DIO mice: FIG. 17A. Insulin tolerance test timecourse; FIG. 17B. Fasting blood glucose levels; FIG. 17C. Insulin tolerance test; area under curve (AUC).
FIGS. 18A-C show that a positive modulator anti-INSR antibody improves glycemic control in DIO mice: FIG. 18A. Glucose tolerance test timecourse; FIG. 18B. Fasting blood glucose levels; FIG. 18C. Glucose tolerance test; area under curve (AUC).
FIGS. 19A-B illustrate the dose response from a partial allosteric agonist in comparison to the dose response to the endogenous ligand (FIG. 19A) or activation by ligand in the presence or absence of the allosteric agonist (FIG. 19B).
FIGS. 20A-B show the dose response from a positive allosteric modulator antibody in comparison to the dose response to the endogenous ligand (FIG. 20A) or the dose response of an endogenous ligand in the presence and absence of a positive allosteric modulator antibody (FIG. 20B).
FIG. 21 illustrates the activation parameters for a set of partial allosteric agonists alone relative to the endogenous ligand insulin. Data obtained from measurements of percent Akt phosphorylation at Ser473.
FIG. 22 illustrates the activation properties of insulin in the presence of 10 ug/ml partial allosteric agonist antibodies relative to the maximal response to the endogenous ligand in the presence of a negative control antibody. Data obtained from measurements of percent Akt phosphorylation at Ser473.



DETAILED DESCRIPTION
The invention provides kinetic modulating drugs that are polypeptide binding agents, uses thereof, and various methods of identifying kinetic modulating drugs. These kinetic modulators can induce either a positive or negative effect on the cellular response by altering the kinetic rate constants for assembly and dissociation of signaling complex components or by other mechanisms including altering the structural state of the signaling complex, e.g., by binding to a transition state and accelerating the activation of signaling.
Modulation of a signaling complex can result in an increase or decrease in sensitivity to signal input and concomitant increases or decreases in signal transduction. Administration of these kinetic modulators increases or decreases the sensitivity of the cellular pathway and/or absolute levels of the cellular response. The kinetic modulators of the invention, depending on their properties, can function as a modulator, potentiator, regulator, effector or sensitizer.
Many antibody drugs act to block signaling pathways by binding to either a cell-surface receptor or its cognate ligand and eliminating the ability of the ligand to bind to and activate the receptor. Such blocking drugs mediate their effect stoichiometrically by preventing the formation of receptor-ligand complex. However, most pathways that have been linked to disease when abnormally activated also have normal developmental or homeostatic roles in normal biology. This observation is particularly true for the immune system, where highly potent cytokines such as TNF-α and IL-6 drive inflammation in pathological contexts but also have important beneficial roles in the control of infections. Successful treatment of some diseases may therefore require attenuation rather than complete inhibition of signaling pathways to restore a normal physiological state with acceptable side-effect profiles. The kinetic modulators provided by the invention are expected to provide such advantages.
Other therapeutic drugs affect cellular signaling pathways by binding to a cell-surface receptor and altering the activity of the receptor. Such direct agonist drugs may mediate their effects by mimicking the natural activity of the ligand and thus have inherent activity i.e. they do not require the presence of ligand to mediate their effects. Further therapeutic drugs affect cellular signaling pathways by binding to a ligand. Such indirect agonist drugs may mediate their effects by altering ligand stability or valency.
Biological processes are generally regulated in a continuous rather than binary manner, and thus in many cases modulation of pathway activity may be a more appropriate therapeutic strategy than complete pathway blockade or stimulation. Performing functional, cell-based screens for modulation of pathway activity, rather than for complete pathway blockade or stimulation, is laborious and may not readily be readily performed in a high throughput manner, since such screens generally require a known concentration of test compound and may be sensitive to any impurities in the test compound preparation. In particular, the ability to perform high throughput functional, cell-based screens for modulation of pathway activity is restricted for cell-impermeable molecules which are unable to enter the intracellular environment, and especially for recombinant biological molecules which may have different expression levels, degrees of purity and stability in the production system used. In addition, some binding interactions may have no signaling output to measure in a functional screen (e.g. in the case of decoy receptors, decoy substrates, or inactive forms of a target) making it difficult to identify agents that perturb these interactions.
The present invention overcomes these disadvantages and provides a means for identifying positive and negative kinetic modulators of the desired activity and desired potency in a high throughput manner.
Definitions
The term “compound” refers to any chemical compound, organic or inorganic, endogenous or exogenous, including, without limitation, polypeptides, proteins, peptides, small molecules, nucleic acids (e.g. DNA and RNA), carbohydrates, lipids, fatty acids, steroids, purines, pyrimidines, peptidomimetics, polyketides and derivatives, structural analogs or combinations thereof. “Endogenous” means naturally occurring in a mammal, while “exogenous” means not naturally occurring in the mammal, e.g. an administered foreign compound.
The term “polypeptide binding agent” refers to a polypeptide that is capable of specifically binding an antigen, e.g. a target or its signaling partner, or that is capable of binding an antigen with a measurable binding affinity. Examples of polypeptide binding agents include antibodies, peptibodies, polypeptides and peptides, optionally conjugated to other peptide moieties or non-peptidic moieties. Antigens to which a polypeptide binding agent may bind include any proteinaceous or non-proteinaceous molecule that is capable of eliciting an antibody response, or that is capable of binding to a polypeptide binding agent with detectable binding affinity greater than non-specific binding. The antigen to which a kinetic modulating polypeptide binding agent binds may include a target, a signaling partner of a target, and/or a complex comprising the target and its signaling partner.
The term “antibody” is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind an antigen (e.g., Fab′, F′(ab)2, Fv, single chain antibodies, diabodies), and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity. An “immunoglobulin” or “tetrameric antibody” is a tetrameric glycoprotein that consists of two heavy chains and two light chains, each comprising a variable region and a constant region. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antibody fragments or antigen-binding portions include, inter alia, Fab, Fab′, F(ab′)2, Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibody, linear antibody; chelating recombinant antibody, a tribody or bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), a antigen-binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a variant or a derivative thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide, such as one, two, three, four, five, or six CDR sequences, as long as the antibody retains the desired biological activity.
“Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
“Antibody variant” as used herein refers to an antibody polypeptide sequence that contains at least one amino acid substitution, deletion, or insertion in the variable region of the natural antibody variable region domains. Variants may be substantially homologous or substantially identical to the unmodified antibody.
A “chimeric antibody,” as used herein, refers to an antibody containing sequence derived from two different antibodies (see, e.g., U.S. Pat. No. 4,816,567) which typically originate from different species. Most typically, chimeric antibodies comprise human and rodent antibody fragments, generally human constant and mouse variable regions.
A “neutralizing antibody” is an antibody molecule which is able to eliminate or significantly reduce a biological function of an antigen to which it binds. Accordingly, a “neutralizing” antibody is capable of eliminating or significantly reducing a biological function, such as enzyme activity, ligand binding, or intracellular signaling.
An “isolated” antibody is one that has been identified and separated and recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
As used herein, an antibody that “specifically binds” is “antigen specific”, is “specific for” antigen or is “immunoreactive” with an antigen refers to an antibody or polypeptide binding agent of the invention that binds an antigen with greater affinity than other antigens of similar sequence. In one aspect, the polypeptide binding agents of the invention, or fragments, variants, or derivatives thereof, will bind with a greater affinity to human antigen as compared to its binding affinity to similar antigens of other, i.e., non-human, species, but polypeptide binding agents that recognize and bind orthologs of the target are within the scope of the invention.
For example, a polypeptide binding agent that is an antibody or fragment thereof “specific for” its cognate antigen indicates that the variable regions of the antibodies recognize and bind the desired antigen with a detectable preference (e.g., where the desired antigen is a polypeptide, the variable regions of the antibodies are able to distinguish the antigen polypeptide from other known polypeptides of the same family, by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between family members). It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of a polypeptide binding agent, e.g. antibody, for use in the methods of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies for use in the invention can be produced using any method known in the art.
The term “epitope” refers to that portion of any molecule capable of being recognized by and bound by a selective binding agent at one or more of the antigen binding regions. Epitopes usually consist of chemically active surface groupings of molecules, such as, amino acids or carbohydrate side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes as used herein may be contiguous or non-contiguous.
The term “derivative” when used in connection with polypeptide binding agents and polypeptides of the invention refers to polypeptides chemically modified by such techniques as ubiquitination, conjugation to therapeutic or diagnostic agents, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins. Derivatives retain the binding properties of underivatized molecules of the invention.
“Detectable moiety” or a “label” refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to another labeled nucleic acid molecule. The detectable moiety often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantitate the amount of bound detectable moiety in a sample.
“Peptides” or “oligopeptides” are short amino acid sequences, typically between 3 and 100 amino acid residues in length and encompass naturally occurring amino acid residues and non-naturally occurring analogs of residues which may be used singly or in combination with naturally occurring amino acid residues in order to give the peptide a particular conformational specificity or a particular biological activity, such as resistance to proteolysis. Peptides include repeats of peptide sequences and may include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of an amino acid sequence arranged head-to-tail or head-to-head. Peptides may be conjugated to non-peptidic moieties, e.g. [expand]. Peptides include dimers, trimers or higher order multimers, e.g. formed through conjugation to other polymeric or non-polymeric moieties, such as PEG.
“Polypeptides” are longer amino acid sequences, typically 100 or more amino acid residues in length, and encompass naturally occurring amino acid residues and non-naturally occurring analogs of residues which may be used singly or in combination with naturally occurring amino acid residues in order to give the polypeptide a particular conformational specificity or a particular biological activity, such as resistance to proteolysis.
As used herein, a “peptibody” is a fusion polypeptide comprising one or more peptides fused to all or a portion of an immunoglobulin (Ig) constant region. See, e.g., U.S. Pat. No. 6,660,843. The peptide may be any naturally occurring or recombinantly prepared or chemically synthesized peptide that binds to the antigen. The peptide may be repeated and may include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of an amino acid sequence arranged head-to-tail or head-to-head. The portion of the Ig constant region may include at least one constant region domain (e.g., CH1, CH2, CH3, and/or CH4), multiple domains (e.g., CH2 with CH3), multiple copies of domains (e.g., CH2-CH2), any fragment of a constant domain that retains the desired activity, e.g. the salvage receptor epitope responsible for the prolonged half-life of immunoglobulins in circulation, or any combinations thereof.
A “small” molecule or “small” organic molecule is defined herein as a non-polymeric organic chemical compound having a molecular weight of about 1000 Daltons or less.
As used herein, a “signaling complex” is an assembly of proteins and/or endogenous or exogenous compounds that mediate the transduction of a cellular signal. Examples of a signaling complex include, but are not limited to, a ligand bound to a membrane bound receptor, an enzyme bound to a substrate or any cellular molecules that associate to propagate biochemical reactions that are involved in a signal cascade. Signaling complexes can also include coreceptors, cofactors, scaffold proteins, allosteric modulators and numerous other types of proteins and molecules that are involved in cellular signal transduction. Signaling complexes can be formed transiently or can be long lived. The molecular constituents or components of a signaling complex can vary over time and can be dependent on activation state of each component and the cellular environment. Signaling complexes can undergo chemical modification and regulation that can induce a spectrum of effects on the complex including subtle changes in transduction activity, complete inactivation and constitutive activation or both positive and negative modulation. A component of a signaling complex may be a protein, e.g. a secreted protein of any of Appendix A (or SEQ ID NOS: 1-88), that can exist in association with other proteins and/or compounds in a complex (“complexed”) or separately therefrom (“uncomplexed”).
The term “therapeutically effective amount” is used herein to indicate the amount of kinetic modulator composition of the invention that is effective to ameliorate or lessen symptoms or signs of disease associated with abnormal (e.g. abnormally high or abnormally low) signaling of the signaling complex.
As used herein “binding” is the physical association between two or more distinct molecular entities that results from a specific network of non-covalent interactions consisting of one or more of the weak forces including hydrogen bonds, Van der Waals, ion-dipole and hydrophobic interactions and the strong force ionic bonds. The level or degree of binding may be measured in terms of affinity. Affinity, or “binding affinity”, is a measure of the strength of the binding interaction between two or more distinct molecular entities that can be defined by equilibrium binding constants or kinetic binding rate parameters. Examples of suitable constants or parameters and their measurement units are well known in the art and include but are not limited to equilibrium association constant (KA), e.g. about 105M−1 or higher, about 106M−1 or higher, about 107M−1 or higher, about 108M−1 or higher, about 109M−1 or higher, about 1010M−1 or higher, about 1011M−1 or higher or about 1012M−1 or higher; equilibrium dissociation constant (KD), e.g., about 10−5M or less, or about 10−6M or less, or about 10−7M or less, or about 10−8M or less, or about 10−9M or less, or about 10−10M or less, or about 10−11M or less, or about 10−12M or less; on-rate (e.g., sec−1, mol−1) and off-rate (e.g., sec-1)). In the case of KA, higher values mean “stronger” or “strengthened” binding affinity while in the case of KD, lower values mean “stronger” or “strengthened” binding affinity. As used herein, a “strengthened” binding rate parameter means increased residency time, faster association or slower dissociation. As used herein, a “weakened” binding rate parameter means decreased residency time, slower association or faster dissociation. In the case of on-rate, higher values mean faster or more frequent association and thus generally result in strengthened binding affinity. In the case of off-rate, lower values generally mean slower dissociation and thus generally result in stronger binding affinity. However, it is the ratio of the on-rate and off-rate that indicates binding affinity, as explained in further detail later.
Affinity between two compounds, e.g. between an antibody and an antigen, or between first and second components of a signaling complex, may be measured directly or indirectly. Indirect measurement of affinity may be performed using surrogate properties that are indicative of, and/or proportional to, affinity. Such surrogate properties include: the quantity or level of binding of a first component to a second component of a signaling complex, or a biophysical characteristic of the first component or the second component that is predictive of or correlated to the apparent binding affinity of the first component for the second component. Specific examples include measuring the quantity or level of binding of first component to a second component at a subsaturating, concentration of either the first or the second component. Other biophysical characteristics that can be measured include, but are not limited to, the net molecular charge, rotational activity, diffusion rate, melting temperature, electrostatic steering, or conformation of one or both of the first and second components. Yet other biophysical characteristics that can be measured include determining stability of a binding interaction to the impact of varying temperature, pH, or ionic strength.
Measured affinity is dependent on the exact conditions used to make the measurement including, among many other factors, concentration of binding components, assay setup, valence of binding components, buffer composition, pH, ionic strength and temperature as well as additional components added to the binding reaction such as allosteric modulators and regulators. Quantitative and qualitative methods may be used to measure both the absolute and relative strength of binding interactions.
Apparent affinity is a measure of the strength of the binding interaction between two or more distinct molecular entities under conditions where the affinity is altered by conditions or components in the binding reaction such as allosteric modulators, inhibitors, binding component valence etc.
As used herein a “subsaturating concentration” is a concentration of one or more components in a binding reaction that is significantly below the binding affinity KD and/or a concentration of one component in a binding reaction that is less than is required to occupy all of the binding sites of the other component(s). Under subsaturating conditions a significant percentage of one of the binding components in the binding reaction has available binding sites.
As used herein a “biophysical assay” is any method that measures, in an absolute or relative fashion, the binding, association, dissociation, binding affinity, binding level, or binding rate parameters between at least two compounds. Biophysical assays are generally performed in vitro and may be conducted with purified binding components, unpurified components, cell associated components as well as a combination of purified and cell associated components.
An agonist is a term used to describe a type of ligand or drug that binds and activates signaling of a signaling complex component. The ability to alter the activity of a signaling complex component (e.g. a receptor), also known as the agonist's efficacy, is a property that distinguishes it from antagonists, a type of receptor ligand which also binds a signaling complex component but which does not activate signaling of the signaling complex component. The efficacy of an agonist may be positive, causing an increase in the signaling complex component's activity, or negative causing a decrease in the signaling complex component's activity. Full agonists bind and activate a signaling complex component, displaying full efficacy at that signaling complex component. Partial agonists also bind and activate a given signaling complex component, but have only partial efficacy at the signaling complex component relative to a full agonist. An inverse agonist is an agent which binds to the same signaling complex component binding-site as an agonist for that signaling complex component and reverses constitutive activity of the signaling complex component. Inverse agonists exert the opposite pharmacological effect of an agonist. A co-agonist works with other co-agonists to produce the desired effect together.
In a different aspect, the agonists disclosed herein act as allosteric agonists. They bind to a portion of a receptor that is distinct from the active ligand-binding site, and do not appreciably change the binding affinity of ligand and receptor, e.g. they alter binding affinity by less than 2-fold or 3-fold. They also do not appreciably affect the EC50 of ligand activation of its receptor, e.g. they alter EC50 by less than 2-fold or 3-fold. Such allosteric agonists constitutively activate the receptor with a maximal agonist response that is 80% or less of the maximal agonist response of ligand, for example 15%-80%, 20-80%, 20-60%, 20%-40% or 15%-30%. In certain embodiments, the allosteric agonists constitutively activate the receptor with a maximal agonist response that at least about 15%, 20%, 25%, 30%, 35%, 40%; and up to 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% of the maximal agonist response of ligand. It is understood that any combination of any of these range endpoints is contemplated without having to recite each possible combination. In further embodiments, the invention provides an allosteric agonist that binds to a receptor with a KD affinity of 10−5, 10−6, 10−7, 10−8, 10−9 M or less (wherein a lower number indicates higher binding affinity). Without being bound by a theory of the invention, the weak agonist activity of allosteric agonists serves to mimic the effect of natural basal ligand activation levels, while permitting exogenously administered ligand to have its normal effect. In certain embodiments, an allosteric agonist is a partial allosteric agonist. An antagonist blocks a receptor from activation by agonists. A selective agonist is selective for one certain type of signaling complex component. It can additionally be of any of the aforementioned types. In exemplary embodiments, the invention provides an allosteric agonist polypeptide binding agent, e.g. antibody, that binds to any of the secreted proteins in Appendix A (or SEQ ID NOS: 1-88) with an affinity of at least 10−5, 10−6, 10−7, 10−8, 10−9 M or less (wherein a lower number indicates greater binding affinity), and (a) exhibits maximal agonist activity that is 20%-80% that of the native ligand's maximal agonist activity when measured in an in vitro assay, (b) when present does not alter the EC50 of ligand for receptor by more than 2-fold, and (c) when present does not alter the KD of ligand for receptor by more than 2-fold.
The potency of an agonist is usually defined by the inverse of its EC50 value. This can be calculated for a given agonist by determining the concentration of agonist needed to elicit half of the maximum biological response of the agonist. The lower the EC50, the greater the potency of the agonist.
An antagonist is a type of ligand or drug that does not provoke a biological response itself upon binding to a signaling complex component (e.g. a receptor), but blocks or dampens agonist-mediated responses. Antagonists may have affinity but no efficacy for their cognate signaling complex component, and binding will disrupt the interaction and inhibit the function of an agonist or inverse agonist at receptors. Antagonists mediate their effects by binding to the active site or to allosteric sites on signaling complex components, or they may interact at unique binding sites not normally involved in the biological regulation of the signaling complex component's activity. Antagonist activity may be reversible or irreversible depending on the longevity of the antagonist-receptor complex, which, in turn, depends on the nature of antagonist binding to signaling complex component. The majority of antagonists achieve their potency by competing with endogenous ligands or substrates at structurally-defined binding sites on receptors.
Antagonists display no efficacy to activate the signaling complex components they bind. Once bound, however, antagonists may inhibit the function of agonists, inverse agonists and partial agonists. In functional antagonist assays, a dose-response curve measures the effect of the ability of a range of concentrations of antagonists to reverse the activity of an agonist. The potency of an antagonist is usually defined by its IC50 value. This can be calculated for a given antagonist by determining the concentration of antagonist needed to elicit half inhibition of the maximum biological response of an agonist. The lower the IC50, the greater the potency of the antagonist.
Competitive antagonists reversibly bind to signaling complex components at the same binding site (active site) as the ligand or agonist, but without activating the signaling complex component, thereby competing with agonist for the same binding site on the signaling complex component. Non-competitive, or allosteric, antagonists bind to a separate binding site from the agonist, exerting their action to that signaling complex component via that separate binding site. Thus, they do not compete with agonists for binding. Uncompetitive antagonists differ from non-competitive antagonists in that they require signaling complex component activation by an agonist before they can bind to a separate allosteric binding site.
Methods of Identifying Kinetic Modulators
Without being bound by a theory of the invention, the present disclosure provides that kinetic perturbation of an interaction between two components (first component, C1 and second component, C2) of a signaling complex with a kinetic modulator (M) can be described mathematically as:




  
    K
    
      C
      ⁢
      
        
      
      ⁢
      1
      ⁢
      C
      ⁢
      
        
      
      ⁢
      2
    
    ′
  
  =
  
    
      K
      
        C
        ⁢
        
          
        
        ⁢
        1
        ⁢
        C
        ⁢
        
          
        
        ⁢
        2
      
    
    ⁢
    
      
        
          (
          
            1
            +
            
              M
              /
              
                K
                
                  MC
                  ⁢
                  
                    
                  
                  ⁢
                  1
                
              
            
          
          )
        
        ⁢
        
          (
          
            1
            +
            
              M
              /
              
                K
                
                  MC
                  ⁢
                  
                    
                  
                  ⁢
                  2
                
              
            
          
          )
        
      
      
        (
        
          1
          +
          
            M
            /
            
              K
              
                
                  [
                  
                    C
                    ⁢
                    
                      
                    
                    ⁢
                    1
                    ⁢
                    C
                    ⁢
                    
                      
                    
                    ⁢
                    2
                  
                  ]
                
                ⁢
                M
              
            
          
        
        )
      
    
  




where the change in binding equilibrium constant between the components (K′C1C2) is a function of equilibrium constant between the components (KC1C2), kinetic modulator concentration (M), kinetic modulator affinity for the complex (K[C1C2]M) and kinetic modulator affinity for either the first component (KMC1) or the second component (KMC2).

In cases where the signaling complex is a receptor-ligand complex, and the modulator is an antibody, the kinetic perturbation of the receptor-ligand interaction with an antibody can be described mathematically as:




  
    K
    RL
    ′
  
  =
  
    
      K
      RL
    
    ⁢
    
      
        
          (
          
            1
            +
            
              A
              
                K
                AR
              
            
          
          )
        
        ⁢
        
          (
          
            1
            +
            
              A
              
                K
                AL
              
            
          
          )
        
      
      
        (
        
          1
          +
          
            A
            
              
                K
                ⁢
                
                  [
                  RL
                
                ]
              
              A
            
          
        
        )
      
    
  




where the change in receptor-ligand binding equilibrium constant (K′RL) is a function of receptor-ligand equilibrium constant (KRL), antibody concentration (A), antibody affinity for the complex (K[RL]A) and antibody affinity for either the receptor (KAR) or ligand (KAL).

A kinetic modulator binds the target, or its signaling partner, or a complex of the target and signaling partner, in such a manner that the binding affinity or binding rate parameter of the target for its signaling partner is weakened or strengthened. For example, where the target is either a receptor or ligand, the binding affinity or binding rate parameter of the ligand for its receptor is weakened or strengthened in the presence of the kinetic modulator. A kinetic modulator with complete blocking activity represents a boundary condition in this analysis, since when K[C1C2]M is sufficiently high, K′C1C2 approaches infinity. One implication of this model is that the degree of signaling modulation is independent of kinetic modulator concentration when the concentration of kinetic modulator ([M]) is sufficiently above the equilibrium dissociation constant (KD) for the kinetic modulator/antigen interaction to be saturating for binding ligand. Hence, modulation of the interaction is related to the ratio of affinities for the complex versus the components where [M]>KD for the modulator and its antigen.
The present disclosure provides that the biophysical properties of a kinetic modulator's interactions with a target and/or its signaling partner can be used to predict the functional effect of the kinetic modulator on the target signaling pathway. Kinetic modulators which alter the signaling pathway can therefore be identified based on their relative affinity for target (and/or its signaling partner) in complexed versus uncomplexed form. The invention contemplates that kinetic perturbation of an interaction between two components (first component, C1 and second component, C2) of a signaling complex with a kinetic modulator (M) can be predicted in the following manner:

    
    
        K[C1C2]M or K[MC2]C1 or K[MC1]C2<KMC2 or KMC1 leads to positive kinetic modulation
        K[C1C2]M or K[MC2]C1 or K[MC1]C2=KMC2 or KMC1 leads to no kinetic modulation
        K[C1C2]M or K[MC2]C1 or K[MC1]C2>KMC2 or KMC1 leads to negative kinetic modulation
    
    


In cases where the signaling complex is a receptor (R)-ligand (L) complex, and the kinetic modulator is an antibody (A), the kinetic perturbation can be predicted in the Following manner:

    
    
        K[RL]A or K[AL]R or K[AR]L<KAL or KAR leads to positive kinetic modulation
        K[RL]A or K[AL]R or K[AR]L=KAL or KAR leads to no kinetic modulation
        K[RL]A or K[AL]R or K[AR]L>KAL or KAR leads to negative kinetic modulation
    
    


In some embodiments, a kinetic modulator, such as an antibody (A) can be identified by its ability to alter a binding interaction, such as a receptor (R)-ligand (L) interaction at any given sub-saturating concentration of the first or second component (e.g. ligand (L) concentration), as depicted in FIG. 3A. The data in FIG. 3A was generated from a reversible interaction model, assuming an affinity of the receptor ligand interaction of either 10 pM, 500 pM or 10 nM. A kinetic modulator could effectively shift the affinity and the corresponding dose response of the receptor ligand interaction from the 500 pM interaction to either the 10 pM (positive modulator) or 10 nM (negative modulator) as depicted. In some embodiments the kinetic modulator will produce a higher level of R-L binding at a given ligand concentration, shifting the assay curve to the left (positive modulation). In other embodiments the kinetic modulator will produce a lower level of R-L binding at a given ligand concentration, shifting the assay curve to the right (negative modulation). In some embodiments the shift is uniform, as shown in FIG. 3A. In other embodiments the shift is non-uniform, reflecting the involvement of other factors e.g. accessory proteins in the complex, receptor internalization, etc. The data from FIG. 3A at a 500 pM affinity was used to generate FIGS. 3B and 3C in which the effects of various concentrations of non-agonist (FIG. 3B) or agonist (FIG. 3C) antibodies on signaling were depicted, assuming a fixed concentration of antigen.
The correlation of binding characteristics to functional effect is depicted in Table 1 below for an illustrative target, insulin receptor.








TABLE 1







Target Binding



Characteristics











R
L
R − L
KD ratios
Functional effect





−
−
+
K[RL]A < KR, KL
Positive modulation


−
+
+
K[AL]R < KL
Positive modulation


+
−
+
K[AR]L < KR
Positive modulation


−
+
+
K[AL]R > KL
Negative modulation


+
−
+
K[AR]L > KR
Negative modulation









Illustrative examples of data showing the predicted effects match the binding characteristics are shown in Table 2 below.











TABLE 2











Functional





effect (pAKT





assay, fold-





decrease in





insulin EC50



Target Binding

relative to



Characteristics

isotype control












Ab
R
L
R-L
KD ratios
Ab)#





Predicted
−
−
+
K[RL]A <
Positive






KR, KL
modulation


Ab078
Out of

 3.4e−10

3.3



Range*


Ab085
No Binding

  2e−10

8.9


Predicted
+
−
+
K[AR]L < KR
Positive







modulation


Ab001
1.2e−8

1.16e−10
103.4
9.7


Ab079
9.6e−9

4.96e−10
19.4
6.7


Ab080
1.2e−8

 6.8e−10
17.6
8.4


Ab083
7.6e−9

3.76e−10
20.2
8.5


Predicted
+
−
+
K[AR]L = KR
Non-







Modulators


Ab037
1.08e−10

  8e−11
1.4
No change


Ab053
1.48e−10

 9.6e−11
1.5
No change


Ab062
1.24e−10

1.08e−10
1.1
No change





*Binding of this clone in the absence of insulin is evident, but insufficiently potent to be accurately measured in this assay.


#Assay run at saturating concentrations of test antibody (2-20 ug/ml). Insulin


EC50 in the presence of 10 ug/ml isotype control Ab = 0.44 nM.






Thus, the binding properties of the interaction(s) between the modulator and the target, its signaling partner and/or a complex comprising the target and its signaling partner, are generally predictive of the functional effect of the kinetic modulator on the target signaling pathway. Depending on the target being studied, certain other factors may need to be considered. These include: (1) the concentration of the kinetic modulator, the concentration of the target, and/or the concentration of its signaling partner (e.g., the prediction is optimized if the kinetic modulator concentration ([M]) is significantly greater than the KD of the binding between kinetic modulator and its antigen), (2) the structural form of the kinetic modulator used e.g. monovalent vs divalent or bivalent, (3) inter/intra target crosslinking, which may restrict the conformation of target and/or cause target activation, (4) the kinetic modulator's ability to alter assembly or docking, or to alter additional components of the signaling complex by steric or allosteric mechanisms, (5) signaling pathway specific properties such as alterations in the signal pathway due to disease that introduce a “bottleneck,” (6) negative/positive feedback regulation of the signaling pathway, (7) alteration of clearance/internalization rates of the components of the signaling complex, (8) alterations in the target that uncouple or differentially alter ligand binding and activation e.g. a modulator enhances ligand binding but traps its receptor in a desensitized state, or a modulator attenuates ligand binding but induces a conformational change in its receptor that is activating.
In some aspects the invention provides methods for measuring the differential binding of a first component of a signaling complex for a second component of the signaling complex in the presence or absence of a test polypeptide agent. In these aspects, differential binding is preferably observed when there are sub-saturating concentrations of the first or second component. In some preferred embodiments the concentration of the first or second component may be reduced to provide sub-saturating conditions.
In some aspects the invention provides methods for measuring the differential binding of a test polypeptide binding agent, e.g. antibody, to target and/or its signaling partner, in complexed and uncomplexed form. In these aspects, differential binding is preferably observed when there are sub-saturating concentrations of test polypeptide binding agent. In some preferred embodiments the concentration of test polypeptide binding agent may be reduced to provide sub-saturating conditions.
In some embodiments, testing in the absence of a test polypeptide agent is performed using a control compound which is preferably a compound belonging to a similar structural class as the test polypeptide agent, but which binds to a different antigen that has no effect on the signaling complex being tested. For example, a control for a test antibody may be an isotype-matched antibody binding to an unrelated antigen, e.g. keyhole limpet hemocyanin (KLH).
For positive modulators, at a given sub-saturating concentration of C1, higher C1 affinity will be reflected in a higher signal for C1 binding to C2 in the presence of the positive modulator. Preferential binding of the kinetic modulator will be reflected in a higher signal for the complex comprising C1 and C2, compared to the signal for either C1 alone or C2 alone. In some aspects, there may be binding of the kinetic modulator to the complex of C1 and C2, but no measurable binding to either C1 alone or C2 alone.
For negative modulators, at a given sub-saturating concentration of C1, lower C1 affinity will be reflected in a lower signal for C1 binding to C2 in the presence of the modulator. Preferential binding of the kinetic modulator will be reflected in a higher signal for binding of the kinetic modulator to C1 alone, or to C2 alone, compared to the signal for binding of the kinetic modulator to the complex of C1 and C2.
The invention provides methods of identifying a candidate polypeptide binding agent, e.g. an antibody, that modulates binding between first and second components of a signaling complex. Examples of such first and/or second components include any of the secreted proteins of Appendix A (or SEQ ID NOS: 1-88) and endogenous or exogenous signaling partners of such secreted proteins, which may be proteinaceous or non-proteinaceous but which optionally may exclude ions and salts. In some embodiments, the first and second components are polypeptides. In exemplary specific embodiments, the first and second components are endogenous.
Other examples include any one of TNFα, CD3, CD4, CD20, VEGF-A, CD25, HER-2, EGFR, CD33, CD52, EPO, insulin, INSR, human growth hormone, GM-CSF, G-CSF, IL-2, TPO, neurotrophic factors (NGF, NT-3, NT-4, GDNF), IFNβ, TGFβ, TNFα, FGFR4, CETP, Leptin Receptor, IL-10, IL-10 receptor alpha, IL-10 receptor beta, Growth hormone receptor, IL-13 receptor, IL-18 receptor, IL-2 receptor alpha subunit, complement factor C5a, IL-17 receptor, IL-20 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-9 receptor, Interferon type I receptor 1 (IFNAR1), Interferon type I receptor 2 (IFNAR2), Lymphocyte function antigen-3 receptor, Monocyte chemotactic protein 1 ligand, NGF receptor, IL-6, IL-6 receptor. Their sequences are well known in the art and representative Accession Numbers and amino acid sequences from NCBI's Genbank database are set forth below. NCBI handbook [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2002 October Chapter 18, The Reference Sequence (RefSeq) Project. Reference to any of the proteins set forth in Appendix A or SEQ ID NOS: 1-88 herein includes reference to any naturally occurring human allelic variant thereof, such as those comprising amino acid sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the representative sequence of any of SEQ ID NOs: 1-88, or comprising amino acid sequences encoded by nucleic acid molecules that can be obtained from human genomic DNA or cDNA libraries using nucleic acid molecules that encode any of SEQ ID NOs: 1-88 or fragments thereof that are at least about 20, 30, 40, 50 or more bases in length, e.g., under stringent hybridization conditions such as 42° C. in 50% formamide, 5×SSC, 20 mM Na.PO4, pH 6.8; and washing in 1×SSC at 55° C. for 30 minutes.












Target
Accession Number
SEQ ID NO:

















TNFα
NP_000585
1


T cell receptor beta chain CD3 region; TCR CD3
AAB27501
2


CD3 antigen, delta subunit isoform B precursor
NP_001035741
3


CD3 antigen, delta subunit isoform A precursor
NP_000723
4


T-cell surface glycoprotein CD3 gamma chain
P09693
5


T-cell surface glycoprotein CD3 gamma chain
ACA05963
6


precursor


T-cell surface glycoprotein CD3 epsilon chain
P07766
7


T-cell surface glycoprotein CD3 delta chain
P04234
8


T-cell surface glycoprotein CD3 delta chain
ACA05962
9


precursor


T-cell surface glycoprotein CD3 zeta chain
P20963
10


CD4
P01730
11


CD4 antigen (p55), isoform CRA_a
EAW88739
12


CD20
P11836
13



NP_068769.2 or



NP_690605.1


membrane-spanning 4-domains, subfamily A,
NP_690605
14


member 1


membrane-spanning 4-domains, subfamily A,
NP_006129
15


member 3 isoform a


membrane-spanning 4-domains, subfamily A,
NP_001026979
16


member 3 isoform b


membrane-spanning 4-domains, subfamily A,
NP_001026836
17


member 3 isoform c


VEGF-A
P15692
18


vascular endothelial growth factor A isoform a
NP_001020537
19


precursor


vascular endothelial growth factor A isoform b
NP_003367
20


precursor


vascular endothelial growth factor A isoform c
NP_001020538
21


precursor


vascular endothelial growth factor A isoform d
NP_001020539
22


precursor


vascular endothelial growth factor A isoform e
NP_001020540
23


precursor


vascular endothelial growth factor A isoform f
NP_001020541
24


precursor


vascular endothelial growth factor A isoform g
NP_001028928
25


precursor


CD25 (interleukin 2 receptor, alpha chain precursor)
NP_000408
26


HER-2
AAA75493
27


EGFR
AAH94761;
28


epidermal growth factor receptor isoform a precursor
NP_005219 or
29



P00533


epidermal growth factor receptor isoform b
NP_958439
30


precursor


epidermal growth factor receptor isoform c precursor
NP_958440
31


epidermal growth factor receptor isoform d
NP_958441
32


precursor


CD33 antigen isoform 1 precursor
NP_001763
33


CD33 antigen isoform 2 precursor
NP_001076087
34


CD33 antigen (gp67), isoform CRA_a
EAW71994
35


CD33 antigen (gp67), isoform CRA_b
EAW71995
36


CD33 antigen (gp67), isoform CRA_c
EAW71996
37


CD52 antigen precursor
NP_001794
38


EPO
CAA26095
39


insulin
AAA59172
40


INSR
P06213
41


insulin receptor isoform Short precursor
NP_001073285
42


insulin receptor isoform Long precursor
NP_000199
43


human growth hormone
AAA72260
44


GM-CSF
AAA52578
45


G-CSF
P09919
46


IL-2
AAB46883
47


TPO
AAB33390
48


NGF
AAH32517
49


nerve growth factor, beta polypeptide precursor
NP_002497
50


NT-3
P20783
51


neurotrophin 3 isoform 1 preproprotein
NP_001096124
52


neurotrophin 3 isoform 2 preproprotein
NP_002518
53


NT-4
AAA60154
54


Chain A, Brain Derived Neurotrophic Factor,
1B8M_A
55


Neurotrophin-4


Chain B, Brain Derived Neurotrophic Factor,
1B8M_B
56


Neurotrophin-4


GDNF
P39905
57


glial cell derived neurotrophic factor isoform 1
NP_000505
58


preproprotein


glial cell derived neurotrophic factor isoform 2
NP_954701
59


precursor


glial cell derived neurotrophic factor isoform 3
NP_954704
60


IFNβ
P01574
61


TGFβ
AAA36738
62


FGFR4
AAB25788
63


fibroblast growth factor receptor 4 isoform 1
NP_998812
64


precursor


fibroblast growth factor receptor 4 isoform 2
NP_075252
65


precursor


CETP
P11597
66


Leptin Receptor
P48357
67


IL-10
P22301
68


IL-10 receptor alpha
EAW67343
69


IL-10 receptor beta
AAH01903
70


Growth hormone receptor
P10912
71


IL-13 receptor
CAA70021
72


IL-18 receptor
AAH93977
73


IL-2 receptor alpha subunit
P01589
74


complement factor C5a
NP_001726
75


IL-17 receptor
AAB99730
76


IL-20 receptor
Q9UHF4
77


IL-3 receptor
AAA59148
78


IL-4 receptor
CAA36672
79


IL-5 receptor
CAA01794
80


IL-9 receptor
AAB30844
81


Interferon type I receptor 1 (IFNAR1)
P17181
82


Interferon type I receptor 2 (IFNAR2)
P48551
83


Lymphocyte function antigen-3 receptor
P19256
84


Monocyte chemotactic protein 1 ligand
P13500 or
85



NP_002973


NGF receptor
AAB59544
86


IL-6
NP_000591
87


IL-6 receptor
NP_000556
88









In one aspect, the methods of identifying a candidate kinetic modulating drug include (a) measuring a binding affinity or binding rate parameter of said first component for said second component, in the presence of a test polypeptide binding agent, e.g. antibody, (b) measuring a binding affinity or binding rate parameter of said first component for said second component in the absence of said test polypeptide binding agent; and (c) identifying said test polypeptide binding agent as a candidate kinetic modulating drug when said test polypeptide binding agent exhibits at least a 1.5-fold difference in the binding affinity or binding rate parameter measured in steps (a) and (b). In some embodiments, the difference in binding affinity or binding rate parameter ranges from about 1.5-fold (i.e., 50%) to, optionally, about 1000-fold, or about 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold.
In some embodiments, the test polypeptide binding agent is identified as a candidate positive modulator if the test polypeptide agent strengthens the binding affinity or binding rate parameter between said first component and said second component. In other embodiments, the test polypeptide agent is identified as a candidate negative modulator if the test polypeptide agent weakens the binding affinity or binding rate parameter between said first component and said second component.
Whether a change (increase or decrease) in a particular binding affinity value or binding rate parameter value represents “strengthened” (or stronger) or “weakened” (or weaker) binding affinity or binding rate parameter depends on the value of the parameter and its units, and is well known in the art. For example, in the case of the parameter KA, higher values mean “strengthened” binding affinity, such that a KA of about 106M−1 is stronger than a KA of about 105M−1. As another example, in the case of the parameter KD, lower values mean “strengthened” binding affinity, such that a KD of about 10−6M is stronger than a KD of about 10−5M. Conversely, in the case of KA, lower values mean “weakened” binding affinity, such that a KA of about 105M−1 is a weakened binding affinity compared to a KA of about 106M−1. As another example, in the case of KD, higher values mean “weakened” binding affinity, such that a KD of about 10−5M is weakened binding affinity compared to a KD of about 10−6M.
As used herein, a “strengthened” binding rate parameter means increased residency time, faster association or slower dissociation. As used herein, a “weakened” binding rate parameter means decreased residency time, slower association or faster dissociation.
Binding affinity can also be determined through the ratio of the on-rate and off-rate binding rate parameters. Generally, in the case of on-rate, higher values mean faster or stronger association or increased residence time, and typically result in stronger binding affinity. Conversely, lower values for on-rate mean slower or weaker association or decreased residence time, and typically result in weaker binding affinity. Generally, in the case of off-rate, higher values mean faster dissociation or decreased residence time, and typically result in weaker binding affinity. Conversely, lower values for off-rate mean slower dissociation or increased residence time, and typically result in stronger binding affinity. This is because the ratio of off-rate to on-rate, or on-rate to off-rate, indicates binding affinity as displayed in the equations below.




  Affinity
  ⁢
  
    {
    
      
        
          
            
              
                
                  
                    K
                    D
                  
                  =
                  
                    
                      
                        
                          [
                          A
                          ]
                        
                        ⁡
                        
                          [
                          L
                          ]
                        
                      
                      
                        [
                        AL
                        ]
                      
                    
                    =
                    
                      
                        off
                        -
                        rate
                      
                      
                        on
                        -
                        rate
                      
                    
                  
                
              
            
            
              
                
                  
                    K
                    A
                  
                  =
                  
                    
                      
                        [
                        AL
                        ]
                      
                      
                        
                          [
                          A
                          ]
                        
                        ⁡
                        
                          [
                          L
                          ]
                        
                      
                    
                    =
                    
                      
                        on
                        -
                        rate
                      
                      
                        off
                        -
                        rate
                      
                    
                  
                
              
            
          
          ⁢
          
            

          
          ⁢
          where
          ⁢
          
            

          
          ⁢
          A
        
        +
        L
      
      ⁢
      
        
          ⇄
          
            K
            on
          
        
        
          K
          off
        
      
      ⁢
      AL
    
  




Even when binding affinity is not detectably or significantly altered, however, the change in residence time, i.e. an increased residence time via increased on-rate or decreased off-rate, or a decreased residence time via a decreased on-rate or increased off-rate, may still result in differential activation of signaling pathways. For example, in some instances where a receptor may activate two different pathways, the pathways differ in the degree of receptor activation required for a full effect. One signaling pathway can be fully activated at low levels of receptor activation or residence time, while full activation of the second pathway requires higher levels of receptor activation or residence time.
In another aspect, the methods of identifying a candidate kinetic modulating drug include (a) (i) measuring a binding affinity or binding rate parameter of a test polypeptide binding agent, e.g. antibody, for said first component in the presence of said second component, or (ii) measuring a binding affinity or binding rate parameter of a test polypeptide binding agent for said second component in the presence of said first component; and (b) (i) measuring a binding affinity or binding rate parameter of said test polypeptide binding agent for said first component in the absence of said second component, or (ii) measuring a binding affinity or binding rate parameter of said test polypeptide binding agent for said second component in the absence of said first component; and (c) identifying said test polypeptide binding agent as a candidate kinetic modulating drug when said test polypeptide binding agent exhibits at least a 1.5-fold (i.e., 50%) difference in the binding affinity or binding rate parameters measured in steps (a) and (b).
In some embodiments, the test polypeptide binding agent is identified as a candidate positive modulator if the binding affinity or binding rate parameter measured in step (a) is at least 1.5-fold (i.e., 50%) stronger than the binding affinity or binding rate parameter measured in step (b). In specific embodiments, the binding affinity or binding rate parameter measured in step (a) compared to that measured in step (b) is about 1.5-fold (i.e., 50%) to, optionally, about 1000-fold stronger for step (a) vs. step (b), or about 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, or up to 30-fold.
In other embodiments, the test polypeptide binding agent is identified as a candidate negative modulator if the binding affinity or binding rate parameter measured in step (b) is at least 1.5-fold (i.e., 50%) stronger than the binding affinity or binding rate parameter measured in step (a). In specific embodiments, the binding affinity or binding rate parameter measured in step (b) compared to that measured in step (a) is about 1.5-fold (i.e., 50%) to, optionally, about 1000-fold stronger for step (b) vs. step (a), or about 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, or up to 30-fold.
In some embodiments, the binding affinity or binding rate parameter of the test polypeptide binding agent for the first component alone is measured. In some embodiments, the binding affinity or binding rate parameter of the test polypeptide binding agent for the second component alone is measured.
In some embodiments, the test polypeptide binding agent is identified as a candidate positive modulator if one or more binding affinity or binding rate parameters selected from the group consisting of (A) the binding affinity or binding rate parameter of the test polypeptide binding agent for a complex comprising the first and second components, optionally K[C1C2]M, (B) the binding affinity or binding rate parameter of the first component for a complex comprising the polypeptide binding agent and the second component, optionally K[MC2]C1, or (C) the binding affinity or binding rate parameter of the second component for a complex comprising the polypeptide binding agent and the first component, optionally K[MC1]C2, is at least about 1.5-fold stronger than one or more binding affinity or binding rate parameter selected from the group consisting of (1) the binding affinity or binding rate parameter of the test polypeptide binding agent for the second component alone, optionally KMC2 or (2) the binding affinity or binding rate parameter of the test polypeptide binding agent for the first component alone, optionally KMC1. In some embodiments, the specific binding affinity or binding rate parameter of any one or more of (A), (B) or (C) is about 1.5-fold (i.e., 50%) to, optionally, about 1000-fold stronger than the binding affinity or binding rate parameter of any one or more of (1) or (2); or alternatively, about 1.5-fold to about 100-fold stronger, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, or up to 30-fold. For example, in some embodiments, the binding affinity or binding rate parameter of any one or more of (A), (B) or (C) is stronger than the binding affinity or binding rate parameter of both (1) and (2). In some embodiments, the binding affinity or binding rate parameter of (1) is stronger than the binding affinity or binding rate parameter of (2). In other embodiments, the binding affinity or binding rate parameter of (2) is stronger than the binding affinity or binding rate parameter of (1). In some embodiments, two or more binding affinity or binding rate parameters are measured and compared, e.g. off-rate and on-rate, or KA and KD, or any combination thereof.
In specific embodiments, wherein the binding affinity measured is the equilibrium dissociation constant KD, any of K[C1C2]M, K[MC2]C1, or K[MC1]C2 is lower, e.g., about 1.5-fold to, optionally, 1000-fold lower, than any of KMC2 or KMC1. Similarly, wherein the binding affinity measured is the off-rate, any of the off-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C) [MC1] and C2 are lower, e.g. about 1.5-fold to, optionally, 1000-fold lower, than any of the off-rates between (1) M and C2 or (2) M and C1. In one exemplary embodiment, K[C1C2]M is about 1.5-fold to, optionally, 1000-fold lower than KMC2. In another exemplary embodiment, K[MC2]C1 is about 1.5-fold to, optionally, 1000-fold lower than KMC2. In another exemplary embodiment, K[MC1]C2 is about 1.5-fold to, optionally, 1000-fold lower than KMC2. In another exemplary embodiment, K[C1C2]M is about 1.5-fold to, optionally, 1000-fold lower than KMC1. In another exemplary embodiment, K[MC2]C1 is about 1.5-fold to, optionally, 1000-fold lower than KMC1. In yet another exemplary embodiment, K[MC1]C2 is about 1.5-fold to, optionally, 1000-fold lower than KMC1. Similar examples can be envisioned for each of the off-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C) [MC1] and C2, compared to each of the off-rates between (1) M and C2 or (2) M and C1.
Conversely, where the binding affinity measured is the equilibrium association constant KA, any of K[C1C2]M, K[MC2]C1, or K[MC1]C2 is higher, e.g., about 1.5-fold to, optionally, 1000-fold higher, than any of KMC2 or KMC1. Similarly, wherein the binding affinity measured is the on-rate, any of the on-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C) [MC1] and C2 are higher, e.g. about 1.5-fold to, optionally, 1000-fold higher, than any of the on-rates between (1) M and C2 or (2) M and C1. In one exemplary embodiment, K[C1C2]M is about 1.5-fold to, optionally, 1000-fold higher than KMC2. In another exemplary embodiment, K[MC2]C1 is about 1.5-fold to, optionally, 1000-fold higher than KMC2. In another exemplary embodiment, K[MC1]C2 is about 1.5-fold to, optionally, 1000-fold higher than KMC2. In another exemplary embodiment, K[C1C2]M is about 1.5-fold to, optionally, 1000-fold higher than KMC1. In another exemplary embodiment, K[MC2]C1 is about 1.5-fold to, optionally, 1000-fold higher than KMC1. In yet another exemplary embodiment, K[MC1]C2 is about 1.5-fold to, optionally, 1000-fold higher than KMC1. Similar examples can be envisioned for each of the on-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C) [MC1] and C2, compared to each of the on-rates between (1) M and C2 or (2) M and C1.
In some embodiments, the test polypeptide binding agent is identified as a candidate negative modulator if one or more binding affinity or binding rate parameters selected from the group consisting of (1) the binding affinity or binding rate parameter of the test polypeptide binding agent for the second component alone, optionally KMC2, or (2) the binding affinity or binding rate parameter of the test polypeptide binding agent for the first component alone, optionally KMC1, is at least about 1.5-fold stronger than one or more binding affinity or binding rate parameter selected from the group consisting of (A) the binding affinity or binding rate parameter of the test polypeptide binding agent for a complex comprising the first and second components, optionally K[C1C2]M, (B) the binding affinity or binding rate parameter of the first component for a complex comprising the polypeptide binding agent and the second component, optionally K[MC2]C1, or (C) the binding affinity or binding rate parameter of the second component for a complex comprising the polypeptide binding agent and the first component, optionally K[MC1]C2. In some embodiments, the specific binding affinity or binding rate parameter of any one or more of (1) or (2) is about 1.5-fold (i.e., 50%) to, optionally, about 1000-fold stronger than the binding affinity or binding rate parameter of any one or more of (A), (B) or (C); or alternatively, about 1.5-fold to about 100-fold stronger, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, or up to 30-fold. In some embodiments, the binding affinity or binding rate parameter of any of (1) or (2) is stronger than the binding affinity or binding rate parameter of all of (A), (B) and (C). In some embodiments, the binding affinity or binding rate parameter of (1) is stronger than the binding affinity or binding rate parameter of (2). In other embodiments, the binding affinity or binding rate parameter of (2) is stronger than the binding affinity or binding rate parameter of (1). In some embodiments, two or more binding affinity or binding rate parameters are measured and compared, e.g. off-rate and on-rate, or KA and KD, or any combination thereof.
In specific embodiments, where the binding affinity measured is the equilibrium dissociation constant KD, any of KMC2 or KMC1 is lower, e.g., about 1.5-fold to, optionally, 1000-fold lower, than any of K[C1C2]M, K[MC2]C1, or K[MC1]C2. Similarly, wherein the binding affinity measured is the off-rate, any of the off-rates between (1) M and C2 or (2) M and C1 are lower, e.g. about 1.5-fold to, optionally, 1000-fold lower, than any of the off-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C) [MC1] and C2. In one exemplary embodiment KMC2 is about 1.5-fold to, optionally, 1000-fold lower than K[C1C2]M. In another exemplary embodiment, KMC2 is about 1.5-fold to, optionally, 1000-fold lower than K[MC2]C1. In another exemplary embodiment, KMC2 is about 1.5-fold to, optionally, 1000-fold lower than K[MC1]C2. In another exemplary embodiment, KMC1 is about 1.5-fold to, optionally, 1000-fold lower than K[C1C2]M. In another exemplary embodiment, KMC1 is about 1.5-fold to, optionally, 1000-fold lower than K[MC2]C1. In yet another exemplary embodiment, KMC1 is about 1.5-fold to, optionally, 1000-fold lower than K[MC1]C2. Similar examples can be envisioned for each of the off-rates between (1) M and C2 or (2) M and C1, compared to each of the off-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C) [MC1] and C2.
Conversely, wherein the binding affinity is the equilibrium association constant KA, any of KMC2 or KMC1 is higher, e.g., about 1.5-fold to, optionally, 1000-fold higher, than any of K[C1C2]M, K[MC2]C1, or K[MC1]C2. Similarly, wherein the binding affinity measured is the on-rate, any of the on-rates between (1) M and C2 or (2) M and C1 are higher, e.g. about 1.5-fold to, optionally, 1000-fold higher, than any of the on-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C) [MC1] and C2. In one exemplary embodiment KMC2 is about 1.5-fold to, optionally, 1000-fold higher than K[C1C2]M. In another exemplary embodiment, KMC2 is about 1.5-fold to, optionally, 1000-fold higher than K[MC2]C1. In another exemplary embodiment, KMC2 is about 1.5-fold to, optionally, 1000-fold higher than K[MC1]C2. In another exemplary embodiment, KMC1 is about 1.5-fold to, optionally, 1000-fold higher than K[C1C2]M. In another exemplary embodiment, KMC1 is about 1.5-fold to, optionally, 1000-fold higher than K[MC2]C1. In yet another exemplary embodiment, KMC1 is about 1.5-fold to, optionally, 1000-fold higher than K[MC1]C2. Similar examples can be envisioned for each of the on-rates between (1) M and C2 or (2) M and C1, compared to each of the on-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C) [MC1] and C2.
In any of these embodiments, the test polypeptide binding agent and second component can be contacted with multiple different concentrations of said first component. In any of these embodiments, the test polypeptide binding agent and first component can be contacted with multiple different concentrations of said second component. In any of these embodiments, multiple different concentrations of the test polypeptide binding agent can be contacted with said first component and said second component.
When the effect of test polypeptide binding agent on the binding interaction between the first component and second component is determined, in some specific embodiments, when the antigen for the test polypeptide binding agent is the first component, e.g., ligand, the test polypeptide binding agent is at a saturating concentration compared to the concentration of the first component. Alternatively, when the antigen for the test polypeptide binding agent is the second component, e.g., receptor, the test polypeptide binding agent is at a saturating concentration compared to the concentration of the second component. In some embodiments, the concentration of the test polypeptide binding agent is greater than or equal to the KD of the test polypeptide binding agent for a complex comprising the first component and the second component. In further embodiments, the concentration of the second component is less than the KD of the test polypeptide binding agent for the first component, e.g., ligand. In yet further embodiments, the concentration of the first component, e.g., ligand, is at a subsaturating concentration for the binding of first component to second component, e.g., receptor. In some embodiments, the concentration of the first component, e.g., ligand is within the range of about EC20 to EC80 for the interaction of the first component with the second component. In some embodiments, one or more concentrations of the test polypeptide binding agent is contacted with multiple different concentrations of the first component, e.g., ligand, in the presence of one or more concentrations of the second component, e.g., receptor. In some embodiments, one or more concentrations of the test polypeptide binding agent is contacted with multiple different concentrations of the second component, e.g., receptor, in the presence of one or more concentrations of the first component, e.g., ligand.
When differential binding of test polypeptide binding agent to complexed vs uncomplexed target and/or signaling partner is determined in order to identify a positive modulator, in some embodiments, the test polypeptide binding agent is at a saturating concentration for a complex comprising the first component and the second component. In some embodiments, the concentration of test polypeptide binding agent is greater than or equal to the KD of the test polypeptide binding agent for a complex comprising the first component, e.g., ligand, and the second component, e.g., receptor. In further embodiments, the concentration of the second component, e.g., receptor is greater than the KD of the second component, e.g., receptor, for the first component, e.g., ligand. In further embodiments, the concentration of the first component, e.g., ligand, is a saturating concentration for the second component, e.g., receptor. In yet further embodiments, the test polypeptide binding agent is at a subsaturating concentration for a complex comprising the first component and the second component. In some embodiments, the concentration of the polypeptide binding agent is within the range of about EC20 to EC80 for the interaction of the first component with the second component. In some embodiments, the concentration of the second component, e.g., receptor, is greater than the KD of the second component, e.g., receptor, for the first component, e.g., ligand. In some embodiments, the concentration of the first component, e.g., ligand, is a saturating concentration for the second component, e.g., receptor.
When differential binding of test polypeptide binding agent to complexed vs uncomplexed target and/or signaling partner is determined in order to identify a negative modulator, in some embodiments, when the antigen to which the test polypeptide binding agent binds is the first component, e.g., ligand, the test polypeptide binding agent is at a subsaturating concentration for the first component. When the antigen to which the test polypeptide binding agent binds is the second component, e.g., receptor, the test polypeptide binding agent is at a subsaturating concentration for the second component. In further embodiments, the concentration of the polypeptide binding agent is within the range of about EC20 to EC80 for the interaction of the first component with the second component. In further embodiments, the concentration of the second component, e.g., receptor, is greater than the KD of the second component, e.g., receptor, for the first component, e.g., ligand. In further embodiments, the concentration of the first component, e.g., ligand, is a saturating concentration for the second component, e.g., receptor.
In some embodiments, the methods further involve assaying a plurality of test polypeptide binding agents, e.g. antibodies, for binding affinity to any one of (a) the first component, (b) the second component, or (c) a complex comprising the first component and second component. In some specific embodiments, the polypeptide binding agents have a binding affinity characterized, e.g., by an equilibrium dissociation constant KD of about 10−5M or less, or about 10−6M or less, or about 10−7M or less, or about 10−8M or less, where a lower KD means stronger binding affinity. In some embodiments, the plurality of test polypeptide binding agents screened are variants of a parent polypeptide binding agent made by introducing one or more different mutations into a parent polypeptide binding agent.
In further embodiments, the polypeptide binding agents may be screened for selectivity of effect for the first or second component, compared to a different binding partner such as a decoy receptor, clearance receptor, or alternate signal pathway component. Such methods may involve identifying a polypeptide binding agent that does not significantly change the binding affinity or binding rate parameter of the first or second component for a different binding partner, such binding partner being neither the first nor second component. In some embodiments, the presence of the polypeptide binding agent changes the binding affinity or binding rate parameter of the first or second component for a different binding partner no more than 5-fold, or no more than 10-fold, or no more than 20-fold, or no more than 30-fold, or no more than 40-fold, or no more than 50-fold.
Any of the preceding methods may further include measuring the level of signaling mediated by the signaling complex in the presence and absence of the test polypeptide binding agent, and determining whether the test polypeptide binding agent is additionally an agonist, partial agonist, antagonist or partial antagonist. Antagonism or agonism can be measured in any in vitro or in vivo assay known in the art, including but not limited to signaling in a phosphorylation assay, ion flux assay, molecular transport assay, or gene expression assay.
In some embodiments, the test polypeptide binding agent shifts (positively or negatively) the dose-response curve of the interaction of the first component, e.g. ligand, with the second component, e.g. receptor. The shift may manifest as an increased or decreased EC50 by at least about 1.5-fold, e.g. about 1.5-fold to about 1000-fold. In some embodiments, the test polypeptide binding agent does not significantly change the maximal agonist response of the signal produced by interaction of the first and second components of the signaling complex. In other embodiments, the test polypeptide binding agent itself acts as an antagonist (e.g., reduces the maximal agonist response of the signaling produced by said signaling complex) or agonist (e.g. increases the maximal agonist response of the signaling produced by said signaling complex).
Where the test polypeptide binding agent acts as an antagonist or partial antagonist, the maximal agonist response may be decreased, e.g., by about 1.5-fold to about 100-fold, or about 2-fold to about 25-fold, or about 1.5-fold to about 50-fold; or, decreased by about 10%, 25%, 50% (1.5-fold), 75%, 2-fold, 3-fold, or 4-, 5-, 6-, 7-, 8-, 9- or 10-fold. Alternatively, where the test polypeptide binding agent acts as an agonist or partial agonist, the maximal agonist response may be increased, e.g. by at least about 10%, 25%, 50% (1.5-fold), 75%, 2-fold, 3-fold, or 4-, 5-, 6-, 7-, 8-, 9- or 10-fold. Moreover, when the test polypeptide binding agent acts as an antagonist or partial antagonist, the IC50 may be 1×10−5 or less. The test polypeptide binding agent may exhibit further desirable characteristics, e.g. the test polypeptide binding agent does not significantly decrease clearance of said first component, or said second component, or said signaling complex comprising said first and second components.
In a related aspect, the invention provides an antibody identified by any of the methods described above or anywhere in the present application.
Polypeptide Binding Agents with Desired Characteristics
The invention also provides polypeptide binding agents, e.g., antibodies, that possess certain desirable characteristics. In some embodiments, the invention provides a positive modulator that (a) binds to the target, e.g., the secreted protein of any of Appendix A (or SEQ ID NOS: 1-88) or any of the ligands, receptors or components described herein, with an equilibrium dissociation constant KD of about 10−5M or less, e.g., 10−6M or less, or 10−7M or less, or 10−8M or less, and (b) is capable of improving the binding affinity KD between said target and its signaling partner by at least about 1.5-fold (i.e., 50%); or by about 1.5-fold to, optionally, about 1000-fold, or 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to about 500-fold, or up to about 200-fold, or up to about 150-fold, or up to about 100-fold, or up to about 90-fold, or up to about 80-fold, or up to about 70-fold, or up to about 60-fold, or up to about 50-fold, or up to about 40-fold, or up to about 30-fold.
In other embodiments, the invention provides a negative modulator that (a) binds to the target, e.g. the secreted protein of any of Appendix A (or SEQ ID NOS: 1-88) or any of the ligands, receptors or components described herein, with an equilibrium dissociation constant KD of about 10−5M or less, e.g., 10−6M or less, or 10−7M or less, or 10−8M or less, and (b) is capable of reducing the binding affinity KD between said secreted protein and its signaling partner by at least about 1.5-fold (i.e., 50%); or by about 1.5-fold to, optionally, about 1000-fold, or 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to about 500-fold, or up to about 200-fold, or up to about 150-fold, or up to about 100-fold, or up to about 90-fold, or up to about 80-fold, or up to about 70-fold, or up to about 60-fold, or up to about 50-fold, or up to about 40-fold, or up to about 30-fold.
In some embodiments, the invention provides a positive modulating antibody that strengthens the binding of a first component (C1) to a second component (C2) of a signaling complex, said antibody characterized by the following equilibrium dissociation constant KD binding properties: (i) said antibody binds with an equilibrium dissociation constant KD of about 10−5M or less, e.g., 10−6M or less, or 10−7M or less, or 10−8M or less, to any one of C1, C2, or a complex comprising C1 and C2 (C1C2); and (ii) any of K[C1C2]A, K[AC2]C1, or K[AC1]C2 is at least about 50% (1.5-fold) lower than any of KAC2 or KAC1, wherein C1 or C2 is a target and its signaling partner, optionally the secreted protein of any of Appendix A (or SEQ ID NOS: 1-88). In some embodiments any of K[C1C2]A, K[AC2]C1, or K[AC1]C2 is about 1.5-fold to, optionally, about 1000-fold lower than any of KAC2 or KAC1; or 1.5-fold to about 100-fold lower, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to about 500-fold, or up to about 200-fold, or up to about 150-fold, or up to about 100-fold, or up to about 90-fold, or up to about 80-fold, or up to about 70-fold, or up to about 60-fold, or up to about 50-fold, or up to about 40-fold, or up to about 30-fold lower. In some embodiments, any of K[C1C2]A, K[AC2]C1, or K[AC1]C2 is at least about 1.5-fold lower than both of KAC2 or KAC1; or 1.5-fold to, optionally, about 1000-fold lower, or 1.5-fold to about 100-fold lower, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to about 500-fold, or up to about 200-fold, or up to about 150-fold, or up to about 100-fold, or up to about 90-fold, or up to about 80-fold, or up to about 70-fold, or up to about 60-fold, or up to about 50-fold, or up to about 40-fold, or up to about 30-fold lower.
In some embodiments, the invention provides a negative modulating antibody that weakens the binding of a first component (C1) to a second component (C2) of a signaling complex, said antibody characterized by the following equilibrium dissociation constant KD binding properties: (i) said antibody binds with an equilibrium dissociation constant KD of about 10−5M or less, e.g., 10−6M or less, or 10−7M or less, or 10−8M or less, to any one of C1, C2, or a complex comprising C1 and C2 (C1C2), and (ii) any of KAC2 or KAC1 is at least about 50% (1.5-fold) lower than any of K[C1C2]A, K[AC2]C1, or K[AC1]C2, wherein C1 or C2 is a target and its signaling partner, optionally the secreted protein of any of Appendix A (or SEQ ID NOS: 1-88). In some embodiments, any of KAC2 or KAC1 is at least about 1.5-fold to, optionally, 1000-fold lower than any of K[C1C2]A, K[AC2]C1, or K[AC1]C2, or 1.5-fold to about 100-fold lower, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to about 500-fold, or up to about 200-fold, or up to about 150-fold, or up to about 100-fold, or up to about 90-fold, or up to about 80-fold, or up to about 70-fold, or up to about 60-fold, or up to about 50-fold, or up to about 40-fold, or up to about 30-fold lower. In some embodiments, any of KAC2 or KAC1 is at least about 1.5-fold lower than all of K[C1C2]A, K[AC2]C1, or K[AC1]C2; or 1.5-fold to, optionally, about 1000-fold lower, or 1.5-fold to about 100-fold lower, or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to about 500-fold, or up to about 200-fold, or up to about 150-fold, or up to about 100-fold, or up to about 90-fold, or up to about 80-fold, or up to about 70-fold, or up to about 60-fold, or up to about 50-fold, or up to about 40-fold, or up to about 30-fold lower.
Any of such polypeptide binding agents are preferably purified and substantially homogeneous, e.g. at least about 90%, 95%, 97%, 98%, 99% or 99.5% pure. In some examples, the polypeptide binding agents are monoclonal antibodies.
The invention further provides methods of preparing a sterile pharmaceutical composition comprising adding a sterile pharmaceutically acceptable diluent to such polypeptide binding agents, sterile compositions of such polypeptide binding agents, e.g., in a therapeutically effective amount, and methods of administering such sterile compositions, e.g. to modulate (increase or decrease) signaling of a complex comprising the secreted protein.
Signaling Complexes
Activation of genes, alterations in metabolism, the continued proliferation and death of the cell, and the stimulation or suppression of locomotion, are some of the cellular responses to extracellular stimulation that may be mediated by signaling complexes. Gene activation leads to further cellular effects, since the protein products of many of the responding genes include enzymes and transcription factors themselves. Transcription factors produced as a result of a signal transduction cascade can, in turn, activate yet more genes. Therefore an initial stimulus can trigger the expression of an entire cohort of genes, and this, in turn, can lead to the activation of any number of complex physiological events. These events include the increased uptake of glucose from the blood stream stimulated by insulin and the migration of neutrophils to sites of infection stimulated by bacterial products.
Neurotransmitters are ligands that are capable of binding to ion channel proteins to form a complex, resulting in their opening to allow the rapid flow of a particular ion across the plasma membrane. This results in an altering of the cell's membrane potential and is important for processes such as the neural conduction of electrochemical impulses. For example, binding of the neurotransmitter acetylcholine at certain synapses opens channels that admit Na+ and initiate a nerve impulse or muscle contraction. Ligands can be freely soluble, or can be found on the surface of other cells or within the extracellular matrix. Such cell surface or extracellular matrix ligands signal between cells when they come in contact with each other, such as when a phagocytic cell presents antigens to lymphocytes, or upon adhesion to the extracellular matrix, as when integrins at the cell surface of fibroblasts engage fibronectin.
Most mammalian cells require stimulation to control not only cell division but also survival. In the absence of growth factor stimulation, programmed cell death ensues in most cells. Such requirements for extra-cellular stimulation are necessary for controlling cell behavior in the context of both unicellular and multicellular organisms. Signal transduction pathways are perceived to be central to biological processes and a large number of diseases have been attributed to their dysregulation.
Signal transduction may be mediated via receptors, which may be located intracellularly e.g. those for steroid hormones, thyroid hormone, retinoic acid, and derivatives of vitamin D3, or on the cell-surface, or may occur both at the cell-surface or intracellularly e.g. ligand-gated ion channel receptors. Signal transduction may also be mediated by transmembrane transporters that transport small molecules, e.g. glucose transporters, or ion channels such as sodium channels, potassium channels, calcium channels, or other positive ion channels, or chloride channels or bicarbonate channels or other anion channels. Many ion channels open or close in response to binding a small signaling molecule or ligand. Some ion channels are gated by extracellular ligands; some by intracellular ligands. Generally, the ligand is not the substance that is transported when the channel opens. ABC (“ATP-Binding Cassette”) transporters are transmembrane proteins that expose a ligand-binding domain at one surface and a ATP-binding domain at the other surface. Some examples of these ABC transporters include cystic fibrosis transmembrane conductance regulator (CFTR); sulfonylurea receptor (SUR) TAP, the transporter associated with antigen processing; SPGP, the transporter that liver cells use to pump the salts of bile acids out into the bile; and the multidrug resistance (MDR) transporter that pumps chemotherapeutic drugs out of cancer cells thus reducing their effectiveness. Mutations of genes in this family have been linked to various diseases including: ALD gene-adrenoleukodystrophy, SUR gene-diabetes, CFTR gene-cystic fibrosis, MDR gene-multidrug resistance in cancer. A list of ABC transporters, their aliases (if any), chromosomal location, and putative function appears below (see Luckie et al., Current Genomics, 2003, 4, 109-121):
ABCA1 ABC1 9q31.1 Ubiquitous Cholesterol efflux onto HDL
ABCA2 ABC2 9q34 2 Brain Drug resistance
ABCA3 ABC3, ABCC 16p13.3 Lung
ABCA4 ABCR 1p22.1-p21 Rod photoreceptors N-retinylidiene-PE efflux
ABCA5 17q24 Muscle, heart, testes
ABCA6 17q24 Liver
ABCA7 19p13.3 Spleen, thymus
ABCA8 17q24 Ovary
ABCA9 17q24 Heart
ABCA10 17q24 Muscle, heart
ABCA12 2q34 Stomach
ABCA13 7p11-q11 Low in all tissues
ABCB1 PGY1, MDR 7p21 Adrenal, kidney, brain Multidrug resistance
ABCB2 TAP1 6p21 A11 cells Peptide transport
ABCB3 TAP2 6p21 A11 cells Peptide transport
ABCB4 PGY3 7q21.1 Liver PC transport
ABCB5 7p14 Ubiquitous
ABCB6 MTABC3 2q36 Mitochondria Iron transport
ABCB7 ABC7 Xq12-q13 Mitochondria Fe/S cluster transport
ABCB8 MABC1 7q36 Mitochondria
ABCB9 12q24 Heart, brain
ABCB10 MTABC2 1q42 Mitochondria
ABCB11 SPGP 2q24 Liver Bile salt transport
ABCC1 MRP1 16p13.1 Lung, testes, PBMC Drug resistance
ABCC2 MRP2 10q24 Liver Organic anion efflux
ABCC3 MRP3 17q21.3 Lung, intestine, liver Drug resistance
ABCC4 MRP4 13q32 Prostate Nucleoside transport
ABCC5 MRP5 3q27 Ubiquitous Nucleoside transport
ABCC6 MRP6 16p13.1 Kidney, liver
CFTR ABCC7 7q31.2 Exocrine tissues Chloride ion channel
ABCC8 SUR1 11p15.1 Pancreas Sulfonylurea receptor
ABCC9 SUR2 12p12.1 Heart, muscle
ABCC10 MRP7 6p21 Low in all tissues
ABCC11 16q11-q12 Low in all tissues
ABCC12 16q11-q12 Low in all tissues
ABCD1 ALD Xq28 Peroxisomes VLCFA transport regulation
4 Current Genomics, 2003, Vol. 4, No. 3 Luckie et al.
(Table 1) contd . . . .
Symb Alias Location Expression Function
ABCD2 ALDL1, ALDR 12q11-q12 Peroxisomes
ABCD3 PXMP1, PMP70 1p22 p21 Peroxisomes
ABCD4 PMP69, P70R 14q24.3 Peroxisomes
ABCE1 OABP, RNS4I 4q31 Ovary, testes, spleen Oligoadenylate binding protein
ABCF1 ABC50 6p21.33 Ubiquitous
ABCF2 7q36 Ubiquitous
ABCF3 3q25 Ubiquitous
ABCG1 ABC8, White 21q22.3 Ubiquitous Cholesterol transport
ABCG2 ABCP, MXR, BCRP 4q22 Placenta, intestine Toxin efflux, drug resistance
ABCG4 White2 11q23 5 59 Liver
ABCG5 White3 2p21 17 Liver, intestine Sterol transport
ABCG8 2p21 17 Liver, intestine Sterol transport. Cell-surface receptors recognize the vast majority of extracellular signaling molecules. Transmembrane receptors span the plasma membrane of the cell, with one part of the receptor on the outside of the cell (the extracellular domain), and the other on the inside of the cell (the intracellular domain). Signal transduction generally occurs as a result of the binding of a ligand to its extracellular domain.
Binding of a ligand to a cell-surface receptor generally stimulates a series of events inside the cell, with different types of receptor stimulation of different intracellular responses. Receptors typically respond to only the binding of a specific ligand. Upon binding, the ligand generally initiates the transmission of a signal across the plasma membrane by inducing a change in the shape or conformation of the intracellular part of the receptor. Often, such changes in conformation either result in the activation of an enzymatic activity contained within the receptor or expose a binding site for other signaling proteins within the cell. Once these proteins bind to the receptor, they themselves may become active and propagate the signal into the cytoplasm.
In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction generally possess an enzymatic activity. These enzymes include tyrosine kinases, heterotrimeric G proteins, small GTPases, various serine/threonine protein kinases, phosphatases, lipid kinases, and hydrolases. Some receptor-stimulated enzymes create specific second messengers including cyclic nucleotides, such as cyclic AMP (cAMP) and cyclic GMP (cGMP), phosphatidylinositol derivatives, such as phosphatidylinositol-triphosphate (PIP3), diacylglycerol (DAG) and inositol-triphosphate (IP3). Other activated proteins interact with adapter proteins. Adapter proteins facilitate interactions between other signaling proteins, and coordinate the formation of further signaling complexes necessary to produce an appropriate cellular response to a particular stimulus. Enzymes and adapter proteins are both responsive to various second messenger molecules.
There are many different classes of transmembrane receptor that recognize different extracellular signaling molecules. Examples include: G-protein coupled receptors, (GPCRs) e.g. adrenergic receptors, neurotransmitter receptors, olfactory receptors, opioid receptors, chemokine receptors, and rhodopsin; receptor tyrosine kinases, e.g., growth factor receptors; integrins; and toll-like receptors.
In some cases, a signaling complex component may be a member of more than one signaling complex, each comprising different complex components and performing different signaling functions (e.g. a ligand may bind more than one cognate receptor). In some cases a ligand may bind one or more decoy receptors. A decoy receptor is a receptor that binds a ligand, inhibiting it from binding to its normal receptor. For instance, the receptor VEGF-1 can prevent vascular endothelial growth factor (VEGF) from binding to the VEGFR-2. Differential modulation of the binding of a signaling complex component with one binding partner versus another should allow highly targeted regulation of biological signaling. In some instances a signaling complex component may be a mutant or variant form that is trapped in a particular conformational form, for example rendering the complex constitutively active or inactive. In some instances a receptor may be trapped in a specific conformation such as the ligand bound conformation. In some instances a signaling complex component may be a mutant or variant form, or a mimetic or analog, of a ligand.
Types and Sources of Test Polypeptide Binding Agents: Peptides and Polypeptides
Numerous libraries of natural or random peptides or polypeptides are available commercially or are readily synthesized. Alternatively, libraries of natural peptides or polypeptides in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Additional derivatization or modifications may be carried out, such as acylation, alkylation, esterification, amidification.
Libraries of protein scaffolds capable of specifically binding to an antigen are also available. These include: Adnectins, Affibodies, Anticalins, DARPins, engineered Kunitz-type inhibitors, tetranectins, A-domain proteins, lipocalins, repeat proteins such as ankyrin repeat proteins, immunity proteins, α2p8 peptide, insect defensin A, PDZ domains, charybdotoxins, PHD fingers, TEM-I β-lactamase, fibronectin type III domains, CTLA-4, T-cell receptors, knottins, neocarzinostatin, carbohydrate binding module 4-2, green fluorescent protein, thioredoxin (Gebauer & Skerra, Curr. Opin. Chem. Biol. 13:245-55 (2009); Gill & Damle, Curr. Opin. Biotech 17: 653-58 (2006); Hosse et al, Protein Sci. 15:14-27 (2006); Skerra, Curr. Opin. Biotech 18: 295-3-4 (2007)).
A number of different approaches for screening in combinatorial libraries are known in the art, including: biological libraries, spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
Peptides that bind to a signaling complex or to a component thereof may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening peptide libraries for peptides that are capable of specifically binding to an antigen are well known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Moth., 102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).
Peptides may be chemically synthesized using known peptide synthesis methodology or may be prepared and purified using recombinant technology. Peptides are usually at least about 3 amino acids in length, alternatively at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such peptides that are capable of binding, preferably specifically, to signaling complex or to a component thereof.
In this regard, bacteriophage (phage) display is one well known technique which allows one to screen large peptide libraries to identify member(s) of those libraries which are capable of specifically binding to an antigen. Phage display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to an antigen with high affinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been used for screening millions of polypeptides or peptides for ones with specific binding, properties (Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).
Although most phage display methods have used filamentous phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024), T4 phage display systems (Ren, Z J. et al. (1998) Gene 215:439; Zhu, Z. (1997) CAN 33:534; Jiang, J. et al. (1997) can 128:44380; Ren, Z J. et al. (1997) CAN 127:215644; Ren, Z-J. (1996) Protein Sci. 5:1833; Efimov, V. P. et al. (1995) Virus Genes 10:173) and T7 phage display systems (Smith, G. P. and Scott, J. K. (1993) Methods in Enzymology, 217, 228-257; U.S. Pat. No. 5,766,905) are also known.
Methods of generating peptide libraries and screening these libraries are also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and 5,723,323.
Types and Sources of Test Polypeptide Binding Agents: Antibodies
The term “antibody” is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind an antigen (e.g., Fab′, F′(ab)2, Fv, single chain antibodies, diabodies), and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity. An “immunoglobulin” or “tetrameric antibody” is a tetrameric glycoprotein that consists of two heavy chains and two light chains, each comprising a variable region and a constant region. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antibody fragments or antigen-binding portions include, inter alia, Fab, Fab′, F(ab′)2, Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibody, linear antibody; chelating recombinant antibody, a tribody or bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), a antigen-binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a variant or a derivative thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide, such as 1, 2, 3, 4, 5 or all 6 CDR sequences, as long as the antibody retains the desired biological activity.
In a naturally-occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa (κ) and lambda (λ) light chains. Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.
Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Chothia et al., J. Mol. Biol. 196:901-917, 1987).
Immunoglobulin variable domains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, (J. Mol. Biol. 196:901-917, 1987); Chothia et al., (Nature 342:878-883, 1989).
The hypervariable region of an antibody refers to the CDR amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a CDR (residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a hypervariable loop (residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain as described by Chothia et al., J. Mol. Biol. 196: 901-917 (1987).
Framework or FR residues are those variable domain residues other than the hypervariable region residues.
“Heavy chain variable region” as used herein refers to the region of the antibody molecule comprising at least one complementarity determining region (CDR) of said antibody heavy chain variable domain. The heavy chain variable region may contain one, two, or three CDRs of said antibody heavy chain.
“Light chain variable region” as used herein refers to the region of an antibody molecule, comprising at least one complementarity determining region (CDR) of said antibody light chain variable domain. The light chain variable region may contain one, two, or three CDRs of said antibody light chain, which may be either a kappa or lambda light chain depending on the antibody.
Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes, IgA, IgD, IgE, IgG and IgM, which may be further divided into subclasses or isotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions; for example, IgG1 and IgG3 isotypes have ADCC activity. An antibody of the invention, if it comprises a constant domain, may be of any of these subclasses or isotypes, or a variant or consensus sequence thereof, or a hybrid of different isotypes (e.g., IgG1/IgG2 hybrid).
In exemplary embodiments, an antibody of the invention can comprise a human kappa (κ) or a human lambda (λ) light chain or an amino acid sequence derived therefrom, or a hybrid thereof, optionally together with a human heavy chain or a sequence derived therefrom, or both heavy and light chains together in a single chain, dimeric, tetrameric (e.g., two heavy chains and two light chains) or other form.
Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are, generally highly specific, and may be directed against a single antigenic site, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the homogeneous culture, uncontaminated by other immunoglobulins with different specificities and characteristics.
Monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., (Nature, 256:495-7, 1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., (Nature 352:624-628, 1991) and Marks et al., (J. Mol. Biol. 222:581-597, 1991).
In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization (Harlow & Lane; Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1988).
Recombinant Production of Antibodies
The present invention also encompasses nucleic acid molecules encoding antibodies of the invention. In some embodiments, different nucleic acid molecules encode a heavy chain variable region and a light chain variable region of an antigen-specific antibody. In other embodiments, the same nucleic acid molecule encodes a heavy chain and a light chain variable regions of an antigen-specific antibody.
DNA encoding a monoclonal antibody of the invention may be isolated and sequenced from a hybridoma cell secreting the antibody using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). Sequence determination will generally require isolation of at least a portion of the gene or cDNA of interest. Usually this requires cloning the DNA or, preferably, mRNA (i.e., cDNA) encoding the monoclonal antibodies. Cloning is carried out using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, which is incorporated herein by reference). For example, a cDNA library may be constructed by reverse transcription of polyA+ mRNA, preferably membrane-associated mRNA, and the library screened using probes specific for human immunoglobulin polypeptide gene sequences. Nucleotide probe reactions and other nucleotide hybridization reactions are carried out at conditions enabling the identification of polynucleotides which hybridize to each other under specified conditions.
One exemplary set of conditions is as follows: stringent hybridization at 42° C. in 50% formamide, 5×SSC, 20 mM Na.PO4, pH 6.8; and washing in 1×SSC at 55° C. for 30 minutes. Formula for calculating equivalent hybridization conditions and/or selecting other conditions to achieve a desired level of stringency are well known. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51
In one embodiment, the polymerase chain reaction (PCR) is used to amplify cDNAs (or portions of full-length cDNAs) encoding an immunoglobulin gene segment of interest (e.g., a light chain variable segment). The amplified sequences can be readily cloned into any suitable vector, e.g., expression vectors, minigene vectors, or phage display vectors. It will be appreciated that the particular method of cloning used is not critical, so long as it is possible to determine the sequence of some portion of the immunoglobulin polypeptide of interest. As used herein, an “isolated” nucleic acid molecule or “isolated” nucleic acid sequence is a nucleic acid molecule that is either (1) identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid or (2) cloned, amplified, tagged, or otherwise distinguished from background nucleic acids such that the sequence of the nucleic acid of interest can be determined, is considered isolated. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
One source for RNA used for cloning and sequencing is a hybridoma produced by obtaining a B cell from the transgenic mouse and fusing the B cell to an immortal cell. Alternatively, RNA can be isolated from B cells (or whole spleen) of the immunized animal. When sources other than hybridomas are used, it may be desirable to screen for sequences encoding immunoglobulins or immunoglobulin polypeptides with specific binding characteristics. One method for such screening is the use of phage display technology. Phage display is described further herein and is also well-known in the art. See e.g., Dower et al., WO 91/17271, McCafferty et al., WO 92/01047, and Caton and Koprowski, (Proc. Natl. Acad. Sci. USA, 87:6450-54 (1990)), each of which is incorporated herein by reference. In one embodiment, cDNA from an immunized transgenic mouse (e.g., total spleen cDNA) is isolated, the polymerase chain reaction is used to amplify a cDNA sequences that encode a portion of an immunoglobulin polypeptide, e.g., CDR regions, and the amplified sequences are inserted into a phage vector. cDNAs encoding peptides of interest, e.g., variable region peptides with desired binding characteristics, are identified by standard phage display techniques such as panning.
The sequence of the amplified or cloned nucleic acid is then determined. Typically the sequence encoding an entire variable region of the immunoglobulin polypeptide is determined, however, it will sometimes by adequate to sequence only a portion of a variable region, for example, the CDR-encoding portion. Typically the portion sequenced will be at least 30 bases in length, more often based coding for at least about one-third or at least about one-half of the length of the variable region will be sequenced.
Sequencing can be carried out on clones isolated from a cDNA library, or, when PCR is used, after subcloning the amplified sequence or by direct PCR sequencing of the amplified segment. Sequencing is carried out using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467, which is incorporated herein by reference). By comparing the sequence of the cloned nucleic acid with published sequences of human immunoglobulin genes and cDNAs, one of skill will readily be able to determine, depending on the region sequenced, (i) the germline segment usage of the hybridoma immunoglobulin polypeptide (including the isotype of the heavy chain) and (ii) the sequence of the heavy and light chain variable regions, including sequences resulting from N-region addition and the process of somatic mutation. One source of immunoglobulin gene sequence information is the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.
Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, human embryonic kidney 293 cells (e.g., 293E cells), Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is well known in the art.
Expression control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
In an alternative embodiment, the amino acid sequence of an immunoglobulin of interest may be determined by direct protein sequencing. Suitable encoding nucleotide sequences can be designed according to a universal codon table.
Amino acid sequence variants of the desired antibody may be prepared by introducing appropriate nucleotide changes into the encoding DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibodies. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.
Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.
The invention also provides isolated nucleic acid encoding antibodies of the invention, optionally operably linked to control sequences recognized by a host cell, vectors and host cells comprising the nucleic acids, and recombinant techniques for the production of the antibodies, which may comprise culturing the host cell so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture or culture medium. Various systems and methods for antibody production are reviewed by Birch & Racher (Adv. Drug Deliv. Rev. 671-685 (2006)).
For recombinant production of the antibody, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selective marker genes, an enhancer element, a promoter, and a transcription termination sequence.
Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger. 
Suitable host cells for the expression of glycosylated antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, tobacco, lemna, and other plant cells can also be utilized as hosts.
Examples of useful mammalian host cell lines are Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen Virol. 36: 59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, (Biol. Reprod. 23: 243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
Host cells are transformed or transfected with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In addition, novel vectors and transfected cell lines with multiple copies of transcription units separated by a selective marker are particularly useful and preferred for the expression of antibodies that bind the desired antigen.
Host cells containing desired antibody nucleic acid sequences may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al (Meth. Enz. 58: 44, 1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S. Pat. Re. No. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium, including from microbial cultures. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Better et al. (Science 240:1041-43, 1988; ICSU Short Reports 10:105 (1990); and Proc. Natl. Acad. Sci. USA 90:457-461 (1993) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. [See also, (Carter et al., Bio/Technology 10:163-167 (1992)].
The antibody composition prepared from microbial or mammalian cells can be purified using, for example, hydroxylapatite chromatography cation or avian exchange chromatography, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13, 1983). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH 3 domain, the Bakerbond ABXTMresin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
Antibody Fragments
Antibody fragments comprise a portion of an intact full length antibody, preferably an antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecific, trispecific, etc. antibodies (e.g., diabodies, triabodies, tetrabodies); minibody; chelating recombinant antibody; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins; camelized antibodies; VHH containing antibodies; and other polypeptides formed from antibody fragments. See for example Holliger & Hudson (Nat. Biotech. 23 (9) 1126-36 (2005))
Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, monovalent fragments consisting of the VL, VH, CL and CH domains each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, that has two “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding, resulting in a single-chain antibody (scFv), in which a VL and VH region are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain (Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). For a review of sFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). An Fd fragment consists of the VH and CH1 domains.
Additional antibody fragments include a domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989) which consists of a VH domain. Diabodies are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., EP 404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994). Diabodies can be bispecific or monospecific.
Functional heavy-chain antibodies devoid of light chains are naturally occurring in nurse sharks (Greenberg et al., Nature 374:168-73, 1995), wobbegong sharks (Nuttall et al., Mol Immunol. 38:313-26, 2001) and Camelalae (Hamers-Casterman et al., Nature 363: 446-8, 1993; Nguyen et al., J. Mol. Biol. 275: 413, 1998), such as camels, dromedaries, alpacas and llamas. The antigen-binding site is reduced to a single domain, the VHH domain, in these animals. These antibodies form antigen-binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only having the structure H2L2 (referred to as “heavy-chain antibodies” or “HCAbs”). Camelid VHH reportedly recombines with IgG2 and IgG3 constant regions that contain hinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman et al., supra). For example, llama IgG1 is a conventional (H2L2) antibody isotype in which VH recombines with a constant region that contains hinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 are heavy chain-only isotypes that lack CH1 domains and that contain no light chains. Camelid VHH domains have been found to bind to antigen with high affinity (Desmyter et al., J. Biol. Chem. 276:26285-90, 2001) and possess high stability in solution (Ewert et al., Biochemistry 41:3628-36, 2002). Classical VH-only fragments are difficult to produce in soluble form, but improvements in solubility and specific binding can be obtained when framework residues are altered to be more VHH-like. (See, e.g., Reichman, et al., J Immunol Methods 1999, 231:25-38.) Methods for generating antibodies having camelid heavy chains are described in, for example, in U.S. Patent Publication Nos. 20050136049 and 20050037421.
The variable domain of an antibody heavy-chain is has a molecular mass of 15 kDa, and is referred to as a nanobody (Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004). A nanobody library may be generated from an immunized dromedary as described in Conrath et al., (Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinant methods as described in Revets et al, Expert Opin. Biol. Ther. 5 (1): 111-24 (2005).
Production of bispecific Fab-scFv (“bibody”) and trispecific Fab-(scFv)(2) (“tribody”) are described in Schoonjans et al., (J Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFv molecule is fused to one or both of the VL-CL (L) and VH-CH1 (Fd) chains, e.g., to produce a tribody two scFvs are fused to C-term of Fab while in a bibody one scFv is fused to C-term of Fab.
A “minibody” consisting of scFv fused to CH3 via a peptide linker (hingeless) or via an IgG hinge has been described in Olafsen, et al., Protein Eng Des Sel. 2004 April; 17 (4):315-23.
Intrabodies are single chain antibodies which demonstrate intracellular expression and can manipulate intracellular protein function (Biocca, et al., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA. 101:17616-21, 2004). Intrabodies, which comprise cell signal sequences which retain the antibody construct in intracellular regions, may be produced as described in Mhashilkar et al (EMBO J. 14:1542-51, 1995) and Wheeler et al., (FASEB J. 17:1733-5. 2003). Transbodies are cell-permeable antibodies in which a protein transduction domains (PTD) is fused with single chain variable fragment (scFv) antibodies Heng et al., (Med Hypotheses. 64:1105-8, 2005).
Further contemplated are antibodies that are SMIPs or binding domain immunoglobulin fusion proteins specific for an antigen. These constructs are single-chain polypeptides comprising antigen binding domains fused to immunoglobulin domains necessary to carry out antibody effector functions. See e.g., WO03/041600, U.S. Patent publication 20030133939 and US Patent Publication 20030118592.
One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest.
Thus, a variety of compositions comprising one, two, and/or three CDRs of a heavy chain variable region or a light chain variable region of an antibody may be generated by techniques known in the art.
Multispecific Antibodies
In some embodiments, it may be desirable to generate multispecific (e.g. bispecific) antibodies of the invention having binding specificities for at least two different epitopes of the same or different molecules. Exemplary bispecific antibodies may bind to two different epitopes of the antigen. Alternatively, an antigen-specific antibody arm may be combined with an arm which binds to a cell surface molecule, such as a T-cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the desired antigen. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express or take up the desired antigen. These antibodies possess an antigen-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-60, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab′)2 bispecific antibodies).
According to another approach for making bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. See WO96/27011 published Sep. 6, 1996.
Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., (Science 229:81-83, 1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. In yet a further embodiment, Fab′-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies. (Shalaby et al., J. Exp. Med. 175:217-225 (1992))
Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor antigens.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. (Kostelny et al., J. Immunol. 148:1547-1553, 1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al. (Proc. Natl. Acad. Sci. USA 90:6444-48, 1993) has provided an alternative mechanism for making bispecific antibody fragments.
The fragments comprise a heavy chain variable region (VH) connected to a light-chain variable region (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).
Alternatively, the bispecific antibody may be a “linear antibody” produced as described in Zapata et al. Protein Eng. 8:1057-62 (1995). Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
In a further embodiment, the bispecific antibody may be a chelating recombinant antibody (CRAb). A chelating recombinant antibody recognizes adjacent and non-overlapping epitopes of the antigen, and is flexible enough to bind to both epitopes simultaneously (Neri et al., J Mol. Biol. 246:367-73, 1995).
Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. (Tutt et al., J. Immunol. 147:60, 1991).
Chimeric and Humanized Antibodies
Because chimeric or humanized antibodies are less immunogenic in humans than the parental mouse monoclonal antibodies, they can be used for the treatment of humans with far less risk of anaphylaxis.
Chimeric monoclonal antibodies, in which the variable Ig domains of a mouse monoclonal antibody are fused to human constant Ig domains, can be generated using standard procedures known in the art (See Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6841-6855 (1984); and, Boulianne et al, Nature 312, 643-646, (1984)). Although some chimeric monoclonal antibodies have proved less immunogenic in humans, the mouse variable Ig domains can still lead to a significant human anti-mouse response.
Humanized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-human complementarity determining regions (CDRs) onto a human framework and constant region (a process referred to in the art as humanizing through “CDR grafting”) (2) transplanting the entire non-human variable domains, but “cloaking” them with a human-like surface by replacement of surface residues (a process referred to in the art as “veneering”), or, alternatively, (3) substituting human amino acids at positions determined to be unlikely to adversely effect either antigen binding or protein folding, but likely to reduce immunogenicity in a human environment (a process referred to in the art as HUMAN ENGINEERINGTM). In the present invention, humanized antibodies will include both “humanized”, “veneered” and “HUMAN ENGINEEREDTM”, antibodies. These methods are disclosed in, e.g., Jones et al., Nature 321:522 525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immunol. 31:169-217 (1994); Kettleborough et al., Protein Eng. 4:773-783 (1991); Studnicka et al. U.S. Pat. No. 5,766,886; Studnicka et al., (Protein Eng 7: 805-814, 1994) each of which is incorporated herein by reference.
Human Antibodies from Transgenic Animals
Human antibodies to antigen can also be produced using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci. For example, WO 98/24893 discloses transgenic animals having a human Ig locus wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci. WO 91/00906 also discloses transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin encoding loci are substituted or inactivated. WO 96/30498 and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system to modify the immunoglobulin locus in a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecule. WO 94/02602 discloses non-human mammalian hosts having inactivated endogenous Ig loci and functional human Ig loci. U.S. Pat. No. 5,939,598 discloses methods of making transgenic mice in which the mice lack endogenous heavy chains, and express an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions. See also, U.S. Pat. Nos. 6,114,598 6,657,103 and 6,833,268.
Using a transgenic animal described above, an immune response can be produced to a selected antigen, and antibody producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies. Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse as described in WO 96/33735. This publication discloses monoclonal antibodies against a variety of antigens including IL-6, IL-8, TNFa, human CD4, L selectin, gp39, and tetanus toxin. The monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein. WO 96/33735 discloses that monoclonal antibodies against IL-8, derived from immune cells of transgenic mice immunized with IL-8, blocked IL-8 induced functions of neutrophils. Human monoclonal antibodies with specificity for the antigen used to immunize transgenic animals are also disclosed in WO 96/34096 and U.S. patent application no. 20030194404; and U.S. patent application no. 20030031667.
Additional transgenic animals useful to make monoclonal antibodies include the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429 and Fishwild, et al. (Nat. Biotechnol. 14:845-851 (1996)), which contains gene sequences from unrearranged human antibody genes that code for the heavy and light chains of human antibodies. Immunization of a HuMAb-MOUSE® enables the production of fully human monoclonal antibodies to the antigen.
Also, Ishida et al. (Cloning Stem Cells. 4:91-102 (2002)) describes the TransChromo Mouse (TCMOUSETM) which comprises megabase-sized segments of human DNA and which incorporates the entire human immunoglobulin (hIg) loci. The TCMOUSETM has a fully diverse repertoire of hIgs, including all the subclasses of IgGs (IgG1-G4). Immunization of the TCMOUSETM with various human antigens produces antibody responses comprising human antibodies.
See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993); and U.S. Pat. No. 5,591,669, U.S. Pat. No. 5,589,369, U.S. Pat. No. 5,545,807; and U.S Patent Publication No. 20020199213. U.S. Patent Publication No. 20030092125 describes methods for biasing the immune response of an animal to the desired epitope. Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
Human Antibodies from Display Technology
The development of technologies for making repertoires of recombinant human antibody genes, and the display of the encoded antibody fragments on the surface of filamentous bacteriophage, has provided a means for making human antibodies directly. The antibodies produced by phage technology are produced as antigen binding fragments—usually Fv or Fab fragments—in bacteria and thus lack effector functions. Effector functions can be introduced by one of two strategies: The fragments can be engineered either into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site capable of triggering an effector function.
The invention contemplates a method for producing antigen-specific antibody or antigen-binding portion thereof comprising the steps of synthesizing a library of human antibodies on phage, screening the library with antigen or a portion thereof, isolating phage that bind antigen, and obtaining the antibody from the phage. By way of example, one method for preparing the library of antibodies for use in phage display techniques comprises the steps of immunizing a non-human animal comprising human immunoglobulin loci with antigen or an antigenic portion thereof to create an immune response, extracting antibody producing cells from the immunized animal; isolating RNA from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using a primer, and inserting the cDNA into a phage display vector such that antibodies are expressed on the phage. Recombinant antigen-specific antibodies of the invention may be obtained in this way. In another example, antibody producing cells can be extracted from non-immunized animals, RNA isolated from the extracted cells and reverse transcribed to produce cDNA, which is amplified using a primer, and inserted into a phage display vector such that antibodies are expressed on the phage. Phage-display processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in WO 99/10494, which describes the isolation of high affinity and functional agonistic antibodies for MPL and msk receptors using such an approach. Antibodies of the invention can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. See e.g., U.S. Pat. No. 5,969,108. There are commercially available kits for generating phage display libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAPTM phage display kit, catalog no. 240612). There are also other methods and reagents that can be used in generating and screening antibody display libraries (see, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982.
In one embodiment, to isolate human antibodies specific for an antigen, with the desired binding characteristics, a human VH and VL library are screened to select for antibody fragments having the desired specificity. The antibody libraries used in this method are preferably scFv libraries prepared and screened as described herein and in the art (McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al., (Nature 348:552-554 (1990)); and Griffiths et al., (EMBO J 12:725-734 (1993)). The scFv antibody libraries preferably are screened using the antigen.
Alternatively, the Fd fragment (VH-CH1) and light chain (VL-CL) of antibodies are separately cloned by PCR and recombined randomly in combinatorial phage display libraries, which can then be selected for binding to a particular antigen. The Fab fragments are expressed on the phage surface, i.e., physically linked to the genes that encode them. Thus, selection of Fab by antigen binding co-selects for the Fab encoding sequences, which can be amplified subsequently. Through several rounds of antigen binding and re-amplification, a procedure termed panning, Fab specific for the antigen are enriched and finally isolated.
In 1994, an approach for the humanization of antibodies, called “guided selection”, was described. Guided selection utilizes the power of the phage display technique for the humanization of mouse monoclonal antibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903 (1994)). For this, the Fd fragment of the mouse monoclonal antibody can be displayed in combination with a human light chain library, and the resulting hybrid Fab library may then be selected with antigen. The mouse Fd fragment thereby provides a template to guide the selection. Subsequently, the selected human light chains are combined with a human Fd fragment library. Selection of the resulting library yields entirely human Fab.
A variety of procedures have been described for deriving human antibodies from phage-display libraries (See, for example, Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol, 222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson, T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, in vitro selection and evolution of antibodies derived from phage display libraries has become a powerful tool (See Burton, D. R., and Barbas III, C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., et al., Annu. Rev. Immunol. 12, 433-455 (1994); U.S. patent publication no. 20020004215 and WO 92/01047; U.S. patent publication no. 20030190317; and U.S. Pat. Nos. 6,054,287 and 5,877,293.
Watkins, “Screening of Phage-Expressed Antibody Libraries by Capture Lift,” Methods in Molecular Biology, Antibody Phage Display: Methods and Protocols 178:187-193 (2002), and U.S. patent publication no. 20030044772, published Mar. 6, 2003, describe methods for screening phage-expressed antibody libraries or other binding molecules by capture lift, a method involving immobilization of the candidate binding molecules on a solid support.
Fv fragments are displayed on the surface of phage, by the association of one chain expressed as a phage protein fusion (e.g., with M13 gene III) with the complementary chain expressed as a soluble fragment. It is contemplated that the phage may be a filamentous phage such as one of the class I phages: fd, M13, f1, lf1, lke, ZJ/Z, Ff and one of the class II phages Xf, Pf1 and Pf3. The phage may be M13, or fd or a derivative thereof.
Once initial human VL and VH segments are selected, “mix and match” experiments, in which different pairs of the initially selected VL and VH segments are screened for antigen binding, may be performed to select preferred VL/VH pair combinations. Additionally, to further improve the quality of the antibody, the VL and VH segments of the preferred VL/VH pair(s) can be randomly mutated, preferably within the any of the CDR1, CDR2 or CDR3 region of VH and/or VL, a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be accomplished by amplifying VL, and VH regions using PCR primers complimentary to the VH CDR1, CDR2, and CDR3, or VL CDR1, CDR2, and CDR3, respectively, which primers have been “spiked” with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VL and VH segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VL and VH segments can be rescreened for binding to antigen.
Following screening and isolation of an antigen-specific antibody from a recombinant immunoglobulin display library, nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other antibody forms of the invention, as described below. To express a recombinant human antibody isolated by screening of a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell, as described herein.
It is contemplated that the phage display method may be carried out in a mutator strain of bacteria or host cell. A mutator strain is a host cell which has a genetic defect which causes DNA replicated within it to be mutated with respect to its parent DNA. Example mutator strains are NR9046mutD5 and NR9046 mut T1.
It is also contemplated that the phage display method may be carried out using a helper phage. This is a phage which is used to infect cells containing a defective phage genome and which functions to complement the defect. The defective phage genome can be a phagemid or a phage with some function encoding gene sequences removed. Examples of helper phages are M13K07, M13K07 gene III no. 3, hyperphage; and phage displaying or encoding a binding molecule fused to a capsid protein.
Antibodies may also be generated via phage display screening methods using the hierarchical dual combinatorial approach as disclosed in WO 92/01047 in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding member is selected in accordance with phage display techniques such as those described therein. This technique is also disclosed in Marks et al, (Bio/Technology, 10:779-783 (1992)).
Methods for display of polypeptides on the surface of viruses, yeast, microbial and mammalian cells have also been used to identify antigen specific antibodies. See, for example, U.S. Pat. Nos. 5,348,867; 5,723,287; 6,699,658; Wittrup, Curr Op. Biotech. 12:395-99 (2001); Lee et al, Trends in Biotech. 21 (1) 45-52 (2003); Surgeeva et al, Adv. Drug Deliv. Rev. 58: 1622-54 (2006). Antibody libraries may be attached to yeast proteins, such as agglutinin, effectively mimicking the cell surface display of antibodies by B cells in the immune system.
In addition to phage display methods, antibodies may be isolated using in vitro display methods including ribosome display and mRNA display (Amstutz et al, Curr. Op. Biotech. 12: 400-05 (2001)). Selection of polypeptide using ribosome display is described in Hanes et al., (Proc. Natl. Acad Sci USA, 94:4937-4942 (1997)) and U.S. Pat. Nos. 5,643,768 and 5,658,754 issued to Kawasaki. Ribosome display is also useful for rapid large scale mutational analysis of antibodies. The selective mutagenesis approach also provides a method of producing antibodies with improved activities that can be selected using ribosomal display techniques.
Altered Glycosilation
Antibody variants can also be produced that have a modified glycosylation pattern relative to the parent antibody, for example, deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. The presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Thus, N-linked glycosylation sites may be added to an antibody by altering the amino acid sequence such that it contains one or more of these tripeptide sequences. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. O-linked glycosylation sites may be added to an antibody by inserting or substituting one or more serine or threonine residues to the sequence of the original antibody.
Fc glycans influence the binding of IgG to Fc receptors and C1q, and are therefore important for IgG effector functions. Antibody variants with modified Fc glycans and altered effector function may be produced. For example, antibodies with modified terminal sugars such as sialic acids, core fucose, bisecting N-acetylglucosamine, and mannose residues may have altered binding to the FcγRIIIa receptor and altered ADCC activity. In a further example, antibodies with modified terminal galactose residues may have altered binding to C1q and altered CDC activity (Raju, Curr. Opin. Immunol. 20: 471-78 (2008).
Also contemplated are antibody molecules with absent or reduced fucosylation that exhibit improved ADCC activity. A variety of ways are known in the art to accomplish this. For example, ADCC effector activity is mediated by binding of the antibody molecule to the FcγRIII receptor, which has been shown to be dependent on the carbohydrate structure of the N-linked glycosylation at the Asn-297 of the CH2 domain. Non-fucosylated antibodies bind this receptor with increased affinity and trigger FcγKIII-mediated effector functions more efficiently than native, fucosylated antibodies. For example, recombinant production of non-fucosylated antibody in CHO cells in which the alpha-1,6-fucosyl transferase enzyme has been knocked out results in antibody with 100-fold increased ADCC activity (Yamane-Ohnuki et al., Biotechnol Bioeng. 87:614-22 (2004)). Similar effects can be accomplished through decreasing the activity of this or other enzymes in the fucosylation pathway, e.g., through siRNA or antisense RNA treatment, engineering cell lines to knockout the enzyme(s), or culturing with selective glycosylation inhibitors (Rothman et al., Mol Immunol. 26:1113-23 (1989)). Some host cell strains, e.g. Lec13 or rat hybridoma YB2/0 cell line naturally produce antibodies with lower fucosylation levels. (Shields et al., J Biol. Chem. 277:26733-40 (2002); Shinkawa et al., J Biol. Chem. 278:3466-73 (2003)). An increase in the level of bisected carbohydrate, e.g. through recombinantly producing antibody in cells that overexpress GnTIII enzyme, has also been determined to increase ADCC activity (Umana et al., Nat Biotechnol. 17:176-80 (1999)). It has been predicted that the absence of only one of the two fucose residues may be sufficient to increase ADCC activity (Ferrara et al., Biotechnol Bioeng. 93:851-61 (2006)).
Variants with Altered Effector Function
Other modifications of the antibody are contemplated. In one aspect, it may be desirable to modify the antibody of the invention with respect to effector function, for example, to enhance the effectiveness of the antibody in treating cancer (Natsume et al, Drug Design Dev't & Ther. 3: 7-16 (2009). Exemplary effector functions include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. One method for modifying effector function teaches that cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., (J. Exp Med. 176: 1191-1195 (1992)) and Shopes, B. (J. Immunol. 148: 2918-2922 (1992)). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., (Cancer Research 53: 2560-2565 (1993)). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., (Anti-Cancer Drug Design 3: 219-230 (1989)). In addition, it has been shown that sequences within the CDR can cause an antibody to bind to MHC Class II and trigger an unwanted helper T-cell response. A conservative substitution can allow the antibody to retain binding activity yet lose its ability to trigger an unwanted T-cell response. Also see Steplewski et al., (Proc Natl Acad Sci USA. 85:4852-56 (1998)), which described chimeric antibodies wherein a murine variable region was joined with human gamma 1, gamma 2, gamma 3, and gamma 4 constant regions.
In certain embodiments of the invention, it may be desirable to use an antibody fragment, rather than an intact antibody, to increase tumor penetration, for example. In this case, it may be desirable to modify the antibody fragment in order to increase its serum half-life, for example, adding molecules such as PEG or other water soluble polymers, including polysaccharide polymers, to antibody fragments to increase the half-life. This may also be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis) (see, e.g., WO96/32478).
The salvage receptor binding epitope preferably constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferred, the epitope is taken from the CH2 domain of the Fc region (e.g., of an IgG) and transferred to the CH1, CH3, or VH region, or more than one such region, of the antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL region or VL region, or both, of the antibody fragment. See also International applications WO 97/34631 and WO 96/32478 which describe Fc variants and their interaction with the salvage receptor.
Thus, antibodies of the invention may comprise a human Fc portion, a human consensus Fc portion, or a variant thereof that retains the ability to interact with the Fc salvage receptor, including variants in which cysteines involved in disulfide bonding are modified or removed, and/or in which the a met is added at the N-terminus and/or one or more of the N-terminal 20 amino acids are removed, and/or regions that interact with complement, such as the C1q binding site, are removed, and/or the ADCC site is removed [see, e.g., Sarmay et al., Molec. Immunol. 29:633-9 (1992)].
Previous studies mapped the binding site on human and murine IgG for FcR primarily to the lower hinge region composed of IgG residues 233-239. Other studies proposed additional broad segments, e.g. Gly316-Lys338 for human Fc receptor I, Lys274-Arg301 and Tyr407-Arg416 for human Fc receptor III, or found a few specific residues outside the lower hinge, e.g., Asn297 and Glu318 for murine IgG2b interacting with murine Fc receptor II. The report of the 3.2-Å crystal structure of the human IgG1 Fc fragment with human Fc receptor IIIA delineated IgG1 residues Leu234-Ser239, Asp265-Glu269, Asn297-Thr299, and Ala327-Ile332 as involved in binding to Fc receptor IIIA. It has been suggested based on crystal structure that in addition to the lower hinge (Leu234-Gly237), residues in IgG CH2 domain loops FG (residues 326-330) and BC (residues 265-271) might play a role in binding to Fc receptor IIA. See Shields et al., (J. Biol. Chem., 276:6591-604 (2001)), incorporated by reference herein in its entirety. Mutation of residues within Fc receptor binding sites can result in altered effector function, such as altered ADCC or CDC activity, or altered half-life. As described above, potential mutations include insertion, deletion or substitution of one or more residues, including substitution with alanine, a conservative substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same position from a different IgG subclass (e.g. replacing an IgG1 residue with a corresponding IgG2 residue at that position).
Shields et al. reported that IgG1 residues involved in binding to all human Fc receptors are located in the CH2 domain proximal to the hinge and fall into two categories as follows: 1) positions that may interact directly with all FcR include Leu234-Pro238, Ala327, and Pro329 (and possibly Asp265); 2) positions that influence carbohydrate nature or position include Asp265 and Asn297. The additional IgG1 residues that affected binding to Fc receptor II are as follows: (largest effect) Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298, and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295, Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339, Ala378, and Lys414. A327Q, A3275, P329A, D265A and D270A reduced binding. In addition to the residues identified above for all FcR, additional IgG1 residues that reduced binding to Fc receptor IIIA by 40% or more are as follows: Ser239, Ser267 (Gly only), His268, Glu293, Gln295, Tyr296, Arg301, Val303, Lys338, and Asp376. Variants that improved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A, and A339T. Lys414 showed a 40% reduction in binding for FcRIIA and FcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIIIA, Gln419 a 30% reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23% improvement to FcRIIIA. See also Presta et al., (Biochem. Soc. Trans. 30:487-490, 2001), incorporated herein by reference in its entirety, which described several positions in the Fc region of IgG1 were found which improved binding only to specific Fc gamma receptors (R) or simultaneously improved binding to one type of Fc gamma R and reduced binding to another type. Selected IgG1 variants with improved binding to Fc gamma RIIIa were then tested in an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay and showed an enhancement in ADCC when either peripheral blood mononuclear cells or natural killer cells were used.
For example, U.S. Pat. No. 6,194,551, incorporated herein by reference in its entirety, describes variants with altered effector function containing mutations in the human IgG Fc region, at amino acid position 329, 331 or 322 (using Kabat numbering), some of which display reduced C1q binding or CDC activity. As another example, U.S. Pat. No. 6,737,056, incorporated herein by reference in its entirety, describes variants with altered effector or Fc-gamma-receptor binding containing mutations in the human IgG Fc region, at amino acid position 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 (using Kabat numbering), some of which display receptor binding profiles associated with reduced ADCC or CDC activity. Of these, a mutation at amino acid position 238, 265, 269, 270, 327 or 329 are stated to reduce binding to FcRI, a mutation at amino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 are stated to reduce binding to FcRII, and a mutation at amino acid position 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437 is stated to reduce binding to FcRIII.
U.S. Pat. No. 5,624,821, incorporated by reference herein in its entirety, reports that C1q binding activity of an murine antibody can be altered by mutating amino acid residue 318, 320 or 322 of the heavy chain and that replacing residue 297 (Asn) results in removal of lytic activity.
U.S. Patent Publication No. 20040132101, incorporated by reference herein in its entirety, describes variants with mutations at amino acid positions 240, 244, 245, 247, 262, 263, 266, 299, 313, 325, 328, or 332 (using Kabat numbering) or positions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330, or 332 (using Kabat numbering), of which mutations at positions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330, or 332 may reduce ADCC activity or reduce binding to an Fc gamma receptor.
Chappel et al. (Proc Natl Acad Sci USA. 88:9036-40 (1991)), incorporated herein by reference in its entirety, report that cytophilic activity of IgG1 is an intrinsic property of its heavy chain CH2 domain. Single point mutations at any of amino acid residues 234-237 of IgG1 significantly lowered or abolished its activity. Substitution of all of IgG1 residues 234-237 (LLGG) into IgG2 and IgG4 were required to restore full binding activity. An IgG2 antibody containing the entire ELLGGP sequence (residues 233-238) was observed to be more active than wild-type IgG1.
Isaacs et al. (J Immunol. 161:3862-9 (1998)), incorporated herein by reference in its entirety, report that mutations within a motif critical for Fc gammaR binding (glutamate 233 to proline, leucine/phenylalanine 234 to valine, and leucine 235 to alanine) completely prevented depletion of target cells. The mutation glutamate 318 to alanine eliminated effector function of mouse IgG2b and also reduced the potency of human IgG4.
Armour et al. (Mol Immunol. 40:585-93 (2003)), incorporated by reference herein in its entirety, identified IgG1 variants which react with the activating receptor, FcgammaRIIa, at least 10-fold less efficiently than wildtype IgG1 but whose binding to the inhibitory receptor, FcgammaRIIb, is only four-fold reduced. Mutations were made in the region of amino acids 233-236 and/or at amino acid positions 327, 330 and 331. See also WO 99/58572, incorporated by reference herein in its entirety.
Xu et al. (J Biol Chem. 269:3469-74 (1994)), incorporated by reference herein in its entirety, report that mutating IgG1 Pro331 to Ser markedly decreased C1q binding and virtually eliminated lytic activity. In contrast, the substitution of Pro for Ser331 in IgG4 bestowed partial lytic activity (40%) to the IgG4 Pro331 variant.
Schuurman et al. (Mol Immunol. 38:1-8 (2001)), incorporated by reference herein in its entirety, report that mutating one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, to serine resulted in a more stable inter-heavy chain linkage. Mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent interaction between the heavy chains.
Angal et al. (Mol Immunol. 30:105-8 (1993)), incorporated by reference herein in its entirety, report that mutating the serine at amino acid position 241 in IgG4 to proline (found at that position in IgG1 and IgG2) led to the production of a homogeneous antibody, as well as extending serum half-life and improving tissue distribution compared to the original chimeric IgG4.
Covalent Modifications
Covalent modifications of the polypeptide binding agents of the invention, e.g., antibodies, are also included within the scope of this invention. They may be made by chemical synthesis or by enzymatic or chemical cleavage of the polypeptide binding agent, if applicable. Other types of covalent modifications of the polypeptide binding agent are introduced into the molecule by reacting targeted amino acid residues of the polypeptide binding agent with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.
Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, .alpha.-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing .alpha.-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125I or 131I to prepare labeled proteins for use in radioimmunoassay.
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R-N.dbd.C.dbd.N-R′), where R and R′ are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated form of these residues falls within the scope of this invention.
Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification involves chemically or enzymatically coupling glycosides to the polypeptide binding agent. These procedures are advantageous in that they do not require production of the polypeptide binding agent in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO87/05330 and in Aplin and Wriston, (CRC Crit. Rev. Biochem., pp. 259-306 (1981)).
Removal of any carbohydrate moieties present on the polypeptide binding agent may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the polypeptide binding agent to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide binding agent intact. Chemical deglycosylation is described by Hakimuddin, et al., (Arch. Biochem. Biophys. 259: 52 (1987)) and by Edge et al., (Anal. Biochem. 118: 131 (1981)). Enzymatic cleavage of carbohydrate moieties on polypeptide binding agents can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., (Meth. Enzymol. 138: 350 (1987)).
Another type of covalent modification of the polypeptide binding agent comprises linking the polypeptide binding agent to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran. Such methods are known in the art, see, e.g. U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337, 4,766,106, 4,179,337, 4,495,285, 4,609,546 or EP 315 456.
Derivatives
Derivative refers to polypeptide binding agents, including antibodies, chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine. Derivatives of the polypeptide binding agents of the invention, such as an antibody, are also useful as therapeutic agents and may be produced by the method of the invention
The conjugated moiety can be incorporated in or attached to a polypeptide binding agent either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin.
Polyethylene glycol (PEG) may be attached to the polypeptide binding agents to provide a longer half-life in vivo. The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG will preferably range from about 2 kiloDalton (“kD”) to about 100 kDa, more preferably from about 5 kDa to about 50 kDa, most preferably from about 5 kDa to about 10 kDa. The PEG groups will generally be attached to the polypeptide binding agents of the invention via acylation or reductive alkylation through a natural or engineered reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the polypeptide binding agent (e.g., an aldehyde, amino, or ester group). Addition of PEG moieties to polypeptide binding agents can be carried out using techniques well-known in the art. See, e.g., International Publication No. WO 96/11953 and U.S. Pat. No. 4,179,337.
Ligation of the polypeptide binding agent with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated substances are purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.
Antibody Conjugates
A polypeptide binding agent may be administered in its “naked” or unconjugated form, or may be conjugated directly to other therapeutic or diagnostic agents, or may be conjugated indirectly to carrier polymers comprising such other therapeutic or diagnostic agents. In some embodiments the polypeptide binding agent is conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate). Suitable chemotherapeutic agents include: daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra). Suitable toxins include: bacterial toxins such as diphtheria toxin; plant toxins such as ricin; small molecule toxins such as geldanamycin (Mandler et al J. Natl. Cancer Inst. 92 (19):1573-81 (2000); Mandler et al Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et al Bioconjugate Chem. 13.786-91 (2002)), maytansinoids (EP 1391213; Liu et al, Proc. Natl. Acad. Sci. USA 93:8618-23 (1996)), auristatins (Doronina et al, Nat. Biotech. 21: 778-84 (2003) and calicheamicin (Lode et al Cancer Res. 58:2928 (1998); Hinman et al Cancer Res. 53:3336-3342 (1993)).
Polypeptide binding agents can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent or luminescent or bioluminescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are well known in the art; for example, see (Sternberger, L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).
Conjugation of polypeptide binding agent moieties is described in U.S. Pat. No. 6,306,393. General techniques are also described in Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106 (1990); and Shih et al., U.S. Pat. No. 5,057,313. This general method involves reacting a polypeptide binding agent component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drug, toxin, chelator, boron addends, or other therapeutic agent. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
The carrier polymer may be, for example, an aminodextran or polypeptide of at least 50 amino acid residues. Various techniques for conjugating a drug or other agent to the carrier polymer are known in the art. A polypeptide carrier can be used instead of aminodextran, but the polypeptide carrier should have at least 50 amino acid residues in the chain, preferably 100-5000 amino acid residues. At least some of the amino acids should be lysine residues or glutamate or aspartate residues. The pendant amines of lysine residues and pendant carboxylates of glutamine and aspartate are convenient for attaching a drug, toxin, immunomodulator, chelator, boron addend or other therapeutic agent. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier and conjugate.
Alternatively, conjugated polypeptide binding agents can be prepared by directly conjugating a polypeptide binding agent component with a therapeutic agent. The general procedure is analogous to the indirect method of conjugation except that a therapeutic agent is directly attached to an oxidized polypeptide binding agent component. For example, a carbohydrate moiety of a polypeptide binding agent can be attached to polyethyleneglycol to extend half-life.
Alternatively, a therapeutic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation, or using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56:244 (1994). General techniques for such conjugation are well-known in the art. See, for example, Wong, Chemistry Of Protein Conjugation and Cross-Linking (CRC Press 1991); Upeslacis et al., “Modification of Antibodies by Chemical Methods,” in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterization of Synthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 60-84 (Cambridge University Press 1995). A variety of bifunctional protein coupling agents are known in the art, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Antibody Fusion Proteins
Methods of making antibody fusion proteins are well known in the art. See, e.g., U.S. Pat. No. 6,306,393. Antibody fusion proteins comprising an interleukin-2 moiety are described by Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al., Cancer Gene Ther. 2:161 (1995), Becker et al. Proc. Nat'l Acad. Sci. USA 93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and Hu et al., Cancer Res. 56:4998 (1996). In addition, Yang et al., (Hum. Antibodies Hybridomas 6:129 (1995)), describe a fusion protein that includes an F(ab′)2 fragment and a tumor necrosis factor alpha moiety. Further examples of antibody fusion proteins are described by Pastan et al, Nat. Reviews Cancer 6: 559-65 (2006).
Methods of making antibody-toxin fusion proteins in which a recombinant molecule comprises one or more antibody components and a toxin or chemotherapeutic agent also are known to those of skill in the art. For example, antibody-Pseudomonas exotoxin A fusion proteins have been described by Chaudhary et al., Nature 339:394 (1989), Brinkmann et al., Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et al., Proc. Nat'l Acad. Sci. USA 89:5867 (1992), Friedman et al., J. Immunol. 150:3054 (1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J. Biol. Chem. 271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996), and Schmidt et al., Int. J. Can. 65:538 (1996). Antibody-toxin fusion proteins containing a diphtheria toxin moiety have been described by Kreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem. 268:5302 (1993), Thompson et al, J. Biol. Chem. 270:28037 (1995), and Vallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting 1:177 (1995), have described an antibody-toxin fusion protein having an RNase moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994), produced an antibody-toxin fusion protein comprising a DNase I component. Gelonin was used as the toxin moiety in the antibody-toxin fusion protein of Wang et al., Abstracts of the 209th ACS National Meeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a further example, Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994), reported an antibody-toxin fusion protein comprising Staphylococcal enterotoxin-A.
Illustrative of toxins which are suitably employed in the preparation of such fusion proteins are ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, C A—A Cancer Journal for Clinicians 44:43 (1994). Other suitable toxins are known to those of skill in the art.
Antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, See WO81/01145) to an active anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No. 4,975,278.
The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
Enzymes that are useful in the this invention include, but are not limited to: alkaline phosphatase; arylsulfatase; cytosine deaminase, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L); D-alanylcarboxypeptidases; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase; β-lactamase; and penicillin amidases, such as penicillin V amidase or penicillin G amidase. Alternatively, antibodies with enzymatic activity, also known in the art as abzymes, can be used to convert the prodrugs of the invention into free active drugs (See, e.g., Massey, Nature 328: 457-458 (1987). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
The enzymes above can be covalently bound to the antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (See, e.g., Neuberger et al., Nature 312: 604-608 (1984))
Formulation of Pharmaceutical Compositions
To administer polypeptide binding agents of the invention to human or test mammals, it is preferable to formulate the polypeptide binding agent in a sterile composition comprising one or more sterile pharmaceutically acceptable carriers. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce allergic, or other adverse reactions when administered using routes well-known in the art, as described below. “Pharmaceutically acceptable carriers” include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
The polypeptide binding agent is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or subcutaneous administration. Preferably the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Other administration methods are contemplated, including topical, particularly transdermal, transmucosal, rectal, oral or local administration e.g. through a catheter placed close to the desired site.
Pharmaceutical compositions of the present invention containing a polypeptide binding agent of the invention as an active ingredient may contain sterile pharmaceutically acceptable carriers or additives depending on the route of administration. Examples of such carriers or additives include water, a pharmaceutical acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like. Additives used are chosen from, but not limited to, the above or combinations thereof, as appropriate, depending on the dosage form of the present invention. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers. A variety of aqueous carriers are suitable, e.g., sterile phosphate buffered saline solutions, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include other proteins for enhanced stability, such as albumin, lipoprotein, globulin, etc., subjected to mild chemical modifications or the like.
Therapeutic formulations of the polypeptide binding agent are prepared for storage by mixing the polypeptide binding agent having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
The concentration of polypeptide binding agent in these formulations can vary widely, for example from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Thus, a typical pharmaceutical composition for parenteral injection could be made up to contain 1 ml sterile buffered water, and 50 mg of polypeptide binding agent. A typical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 150 mg of polypeptide binding agent. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980). An effective dosage of polypeptide binding agent is within the range of 0.01 mg to 1000 mg per kg of body weight per administration.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous, oleaginous suspension, dispersions or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, vegetable oils, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Compositions useful for administration may be formulated with uptake or absorption enhancers to increase their efficacy. Such enhancers include for example, salicylate, glycocholate/linoleate, glycholate, aprotinin, bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci., 85:1282-1285 (1996)) and Oliyai and Stella (Ann. Rev. Pharmacol. Toxicol., 32:521-544 (1993)).
Biophysical Assays
Complex biological events can be studied via molecular biophysical approaches which consider them as systems of interacting units which can be understood in terms of statistical mechanics, thermodynamics and chemical kinetics.
Nuclear magnetic resonance (NMR), Isothermal Titration calorimetry, dynamic light scattering, surface plasmon resonance, dual polarisation interferometry are commonly used to assess whether the compound binds effectively to the test antigen, the stoichiometry of binding, any associated conformational change and to identify promiscuous inhibitors. (See, for example, Correia J. J & Detrich H. W. (eds) “Biophysical Tools for Biologists vol. 2.” Methods in Cell Biol 89 (2) (2008))
Fluorescent imaging techniques, as well as electron microscopy, x-ray crystallography, NMR spectroscopy and atomic force microscopy (AFM) are often used to visualize structures of biological significance. Conformational changes in structure can be measured using techniques such as dual polarisation interferometry and circular dichroism. Direct manipulation of molecules using optical tweezers or AFM can also be used to monitor biological events where forces and distances are at the nanoscale.
A number of techniques are available for use in biophysics, including: atomic force microscopy, biophotonics, biosensor and bioelectronics, calcium imaging, calorimetry, circular dichroism, cryobiology, dual polarisation interferometry, electrophysiology, fluorescence, microscopy, neuroimaging, neutron spin echo spectroscopy, patch clamping, nuclear magnetic resonance spectroscopy, x-ray crystallography. In certain embodiments, the assays of the present invention may employ a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a measurable signal, such as a radioactive, chromogenic, luminescence, or fluorescent signal, which can be used to quantitate the amount of bound detectable moiety or label in a sample. Detectable labels known in the art include radioisotopes, such as 3H, 14C, 32P, 35S, or 125I, electrochemiluminescent labels (such as Ruthenium (Ru)-based catalyst in conjunction with substrates, etc.), luminescent or bioluminescent labels (e.g., Europium, Vanadium), fluorescent or chemiluminescent compounds, such as fluorescein isothiocyanate, rhodamine, or luciferin, enzymes (e.g., enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase), colorimetric labels such as colloidal gold, colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.), paramagnetic atoms or magnetic agents, electron-dense reagents, a nano- or micro-bead containing a fluorescent dye, nanocrystals, a quantum dot, a quantum bead, a nanotag, dendrimers with a fluorescent label, a micro-transponder, an electron donor molecule or molecular structure, or a light reflecting particle. the microparticles may be nanocrystals or quantum dots. Nanocrystals are substances that absorb photons of light, then re-emit photons at a different wavelength (fluorophores). In addition, additional florescent labels, or secondary antibodies may be conjugated to the nanocrystals. Nanocrystals are commercially available from sources such as Invitrogen and Evident Technologies (Troy, N.Y.). Other labels include E)-5-[2-(methoxycarbonyl)ethenyl]cytidine, which is a nonfluorescent molecule that when subjected to ultraviolet (UV) irradiation yields a product, 3-.beta.-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine, which displays a strong fluorescence signal. Bar code labels are described in U.S. Patent Publication No. US 20070037195.
A variety of assay methods known in the art may be employed in the present invention, such as competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays, fluorescent resonance energy transfer (FRET), electroimmunoassays surface plasmon resonance (SPR), and nanoparticle-derived techniques.
Competitive binding assays rely on the ability of a labeled standard (e.g., an antigen or a fragment thereof to which a polypeptide binding agent binds) to compete with antigen in the test sample for binding to the polypeptide binding agent. The amount of antigen in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies typically are insolubilized before or after the competition, so that the bound antigen may conveniently be separated from the unbound antigen. In alternative embodiments, competitive binding assays measure the ability of a labeled polypeptide binding agent to compete with unlabeled polypeptide binding agent for binding to antigen or a fragment thereof.
Sandwich assays typically involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected and/or quantitated. In a sandwich assay, the analyte in the test sample is typically bound by a first polypeptide binding agent which is immobilized on a solid phase, and thereafter a second polypeptide binding agent binds to the analyte, thus forming an insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. The second polypeptide binding agent may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assays). For example, one type of sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in which case the detectable moiety is an enzyme. See, for example, chapter 18, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, New York, N.Y. (1995).
Yet another example of an assay method involves fluorescent resonance energy transfer (FRET) emissions. For example, one compound is labeled with a FRET donor molecule and its binding partner is labeled with a FRET acceptor molecule, or vice versa. When binding occurs between the binding partners, the FRET donor and FRET acceptor molecules are brought into proximity and emit fluorescence at a certain wavelength. A narrow band pass filter can be used to block all wavelengths except that of the label. FRET molecule pairs are commercially available in the art (e.g., from Invitrogen), and may be used according to the manufacturer's protocol. FRET emissions are detected using optical imaging techniques, such as a CCD camera.
Yet another example of an assay method is bioluminescence resonance energy transfer (BRET), for example using biosensors as described in WO/06086883.
Another type of assay involves labeling with an electron donor. One molecule is labeled with an electron donor and the interacting molecule is bound to an electrical contact, or vice versa. When binding occurs between the binding partners, the label donates electrons to the electrical contact. See, for example, Ghindilis, Biochem Soc Trans. 28:84-9, (2000) and Dai et al., Cancer Detect Prev. 29:233-40 (2005), which describe methods for electro immunoassays. The electron contact would then be read by an A to D (analog to digital) converter and quantified. The higher the electron count the more interactions took place.
One embodiment of a label capable of single molecule detection is the use of plasmon-resonant particles (PRPs) as optical reporters, as described in Schultz et al., Proc. Natl. Acad. Sci. USA 97:996-1001 (2000), incorporated herein by reference. PRPs are metallic nanoparticles, e.g. 40-100 nm in diameter, which scatter light because of a collective resonance of the conduction electrons in the metal (the surface plasmon resonance). The magnitude, peak wavelength, and spectral bandwidth of the plasmon resonance associated with a nanoparticle are dependent on the particle's size, shape, and material composition, as well as the local environment. By influencing these parameters during preparation, PRPs can be formed that have scattering peak anywhere in the visible range of the spectrum. For spherical PRPs, both the peak scattering wavelength and scattering efficiency increase with larger radius, providing a means for producing differently colored labels. Populations of silver spheres, for example, can be reproducibly prepared for which the peak scattering wavelength is within a few nanometers of the targeted wavelength, by adjusting the final radius of the spheres during preparation. Because PRPs are bright, yet nanosized, they are used as indicators for single-molecule detection; that is, the presence of a bound PRP in a field of view can indicate a single binding event. An example of a surface plasmon resonance detector system is the BIAcore assay system. See, e.g., Malmquist, J Molec Recognition, 7:1-7 (1994).
Molecular interactions may also be detected using nanoparticle-derived techniques. See, for example, Ao et al., Anal Chem. 78:1104-6 (2006), which describes gold nanoparticle quenching, Tang et al., Biosens Bioelectron. 2005 Nov. 30, which describes SiO(2)/Au nanoparticle surfaces in antibody detection, and Lieu et al., J Immunol Methods. 307:34-40 (2005), which describes silicon dioxide nanoparticles containing dibromofluorescein for use in solid substrate-room temperature phosphorescence immunoassay (SS-RTP-IA).
Any of the preceding measurements of binding affinity or binding rate parameter may be carried out in assays where one or more of the first component, second component and polypeptide binding agent are in solution, or in assays where one or more of the first component, second component and polypeptide binding agent are linked to a solid phase (covalently or noncovalently), or in assays where one or more of the first component, second component and polypeptide binding agent are expressed on a cell surface. The first and/or second components may each themselves be complexes of multiple compounds.
Solution Phase Biophysical Assays
In some embodiments, kinetic modulators can be identified using solution phase biophysical assays. By “solution phase” assay is meant an assay wherein the interaction to be measured takes place in a liquid. Solution affinity assays are useful tools to measure the equilibrium dissociation constant (also referred to as “affinity” or “KD”) of interactions at equilibrium. If a modulator can bind to proteins that form a complex, (e.g. a receptor/ligand interaction) and the modulator alters the affinity of that interaction, then a solution affinity assay can be used to determine the affinity of the interaction in the presence and absence of the kinetic modulator. It can also be used to measure a dose response to the modulator over a fixed concentration range of the complex components.
The present disclosure provides novel applications for equilibrium solution affinity measurements. Such assays enable the characterization and stratification of lead candidates on the basis of their ability to differentially modulate the affinity of the target-signaling partner interaction. Previous work on monitoring the reduction in affinity caused by inhibitory or steric-hindering drugs has utilized an excess of drug pre-complexed to one of the binding partners and has typically been done with surface plasmon resonance (SPR) technology or radio-ligand assay technology. In some aspects, the present disclosure allows for the use of drugs that bind epitopes that only exist when the ligand and receptor or other protein binding partners have formed their complex.
Example 1 describes methods for equilibrium solution affinity measurement using a model protein-protein complex. All reversible binding interactions could theoretically be affinity modulated by a drug and monitored in an assay very similar to the format described. The roles of the ligand and receptor in the assay format could be switched and doing so would serve as verification that the system was functioning properly. Any pair of interacting binding partners could be used, not just receptor-ligand pairs.
Such techniques serve as a highly sensitive method for the quantitation of the free (unbound) binding partner being interrogated. When this analysis is performed with a fixed concentration of one binding partner (B) and the other (A) is varied over a wide concentration range at least one log above and below the KD value of the interaction, then the amount of unbound B can be measured and the data fitted to a model that gives the KD value for the interaction. This experiment could be performed in the presence and absence of varying concentrations of the kinetic modulating drug, allowing for detailed characterization of the affinity enhancement. By measuring the free binding partner B and not the drug of interest, the monitoring of the A-B interaction affinity can be determined independently from the drug-complex interaction.
Such assays can be used to screen for drugs which modulate the affinity of binding interactions. This can have applications in the development of drug therapies and more sensitive diagnostic and analytical assays. Furthermore the assays allow the extent of the affinity modulation to be characterized in detail, even to drugs that recognize epitopes which are created only after a complex has formed. The present disclosure allows for the stratification and ranking of multiple drug candidates for their potency in affinity modulation.
Solid-Phase Biophysical Assays
In some embodiments, kinetic modulators can be identified using solid phase biophysical assays. By “solid phase” is meant a non-aqueous, inert matrix to which a test compound or complex component of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol, silicones metal, metal-alloy, anopol, polymers, nylon, or microarrays such as protein chips. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate, a filter, a membrane, a chromatographic resin, or a bead. This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
In some embodiments, a test compound or complex component is attached or linked to a solid phase using any means known in the art, including but not limited to covalent bonding or indirect attachment through selective binding pairs (e.g., biotin with avidin, glutathione with GST, histidine tag with Ni).
A number of solid phase assay formats are known in the art, including microplates, beads, resins, chips etc. For example, the FMATTM (fluorimetric microvolume assay technology; Applied Biosystems, Foster City, Calif.) system could use cells or receptor bound to beads with either a fluorescently labeled or biotinylated ligand. This has the potential to be run as a completely homogeneous assay format. A virtually identical format could be employed using the LuminexTM system (Luminex Corp., Austin, Tex.) where a similar bead based system is used. The main difference between the FMATTM and LuminexTM assay formats is in the method of detection. The LuminexTM platform flows the beads through an optical flow cell to measure fluorescent emission from the beads and the FMATTM images the fluorescence in a narrow plane very near the base of the well of the microplate. Alternatively electrochemiluminescence systems would be readily applicable to screening for affinity modulators due to their high sensitivity and homogenous capacity. These systems, such as the TricorderTM (BioVeris Corp., Washington D.C.) use ruthenium derived electrochemiluminescent detection technologies. The assay formats that could be optimized for this system would be very similar to the other systems with the primary difference being the use of electrochemiluminescent labeling technology and detection instrumentation.
Suitable assays include microplate-based assays designed to detect modulation of the affinity of a labeled (e.g., biotinylated) target for its binding partner by an interacting modulator. The assay may be homogeneous or semi-homogeneous. A homogeneous assay is an assay where all the components are mixed together, incubated, and then analyzed. A semi-homogeneous assay is one where the majority of the reaction takes place as a complex mixture, but a washing step is required prior to the addition of a final reagent and analysis, in contrast to a typical stepwise assembly sandwich assay where each component is added then washed off before the next component is added. In some embodiments the assay is an immunoassay. In certain embodiments the assay is a semi-homogeneous Enzyme Immuno-Assay (EIA), allowing modulations of the equilibrium binding constants of binding partners to be monitored by using the amount of labeled (e.g., biotinylated) analyte captured on the EIA plate as a readout. At labeled analyte concentrations well below saturation in a equilibrium, or almost equilibrium environment, changes in equilibrium affinity constants result in more or less of the analyte being retained on the microplate.
In some embodiments, assays for kinetic modulators may be structured as follows. The binding partner for the analyte is immobilized on a microplate such as an EIA plate. The plate is then blocked with an irrelevant protein or blocking agent (e.g., bovine serum albumin, casein, ChemiBlockTM (Millipore, Billerica, Mass.), irrelevant IgG, etc or any combination of blocking agents). Following blocking, the plate is washed and added to the wells is a mixture of modulator, labeled analyte (e.g. biotinylated analyte) and secondary detection reagent (e.g. streptavidin) conjugated to a detectable moiety such as an enzyme or fluorophore. Various detectable moieties are known in the art including enzymes such as alkaline phosphatase or horseradish peroxidase, acting on a variety of colorimetric, fluorescent or luminescent substrates. Europium may be used for time resolved fluorescence detection and fluorophores may be used for direct fluorescence. Anti-ligand detection antibodies with various labels may also be employed, avoiding the need for biotinylated ligand as long as the antibodies used could detect receptor-bound ligand. In other embodiments, ligands which are directly conjugated to fluorophores or to colorimetric enzymes may be used.
The plates are generally incubated for several hours to allow the various interactions to approach equilibrium. After the incubation the plate is washed and developed immediately with a reporter substrate (e.g. colorimetric, fluorescent or luminescent substrates). The more labeled analyte captured on the plate, the higher the signal from the secondary detection reagent (e.g. streptavidin-alkaline phosphatase). If the modulator increases the affinity of the interaction, the amount of labeled (e.g. biotinylated) analyte bound to the plate will be greater than it would be in the absence of the kinetic modulator. If the modulator decreases the affinity of the interaction then the amount of labeled (e.g. biotinylated) analyte bound to the plate will be lower than it would be in the absence of the kinetic modulator.
Microplate assays may be used to monitor affinity modulation in several ways. In some embodiments a fixed polypeptide binding agent concentration is used and the labeled analyte titrated across various wells. This shows the affinity modulation effect as a shift in the titration curve versus the same analyte titration curve without the kinetic modulator present.
In some embodiments, once a titration of labeled analyte has been performed without modulator, a concentration can be selected for use in single labeled analyte concentration assay. This concentration should fall just above the bottom of the analyte's titration curve (EC10-20). This allows for any enhancing modulations in the affinity of the ligand-receptor interaction to create an easily observable shift in the signal of the assay. Once a sensitive concentration of labeled analyte is determined, the assay can be performed in a screening mode where crude or purified modulator samples can be tested in single point analysis for affinity modulating effect. Also the assay can be performed with a titration of modulator at a fixed concentration of labeled analyte to demonstrate dose response of the modulator effect.
In some embodiments the labeling (e.g. biotinylation) of an analyte molecule may impact the binding ability or stability of that molecule. If this is the case an alternative assay format could be employed wherein unlabelled analyte is used in the experimental/equilibrium phase. When the samples are washed off before detection, a step could be added where an optimized amount of labeled analyte is added for a shorter amount of time, washed off then detected. This would allow the analyte that was bound in the equilibrium phase to act as competitor of the labeled analyte. Affinity enhancing antibodies would then yield a lower assay signal as the amount of unlabelled analyte prebound in the equilibrium step was higher, reducing the ability of the labeled analyte to bind to the plate. Other forms of semi-homogenous assays could also be developed using secondary reporter molecules or addition of tiered detection reagents to enhance the signal. These types of modifications are known to those skilled in the art. An example solid phase affinity assay is described in Example 2.
Cell-Based Biophysical Assays
In some embodiments kinetic modulators can be identified using cell-based biophysical assays. By “cell-based” is meant an assay in which at least one of the components of the signaling complex being tested is present on the surface of a cell. Such assays may be particularly advantageous for detecting modulators of binding interactions involving conformationally-sensitive proteins such as transmembrane receptors, including: G protein-coupled receptors (GPCRs; e.g. muscarinic acetylcholine receptor, adenosine receptors, adrenoreceptors, GABA receptors, angiotensin receptors, cannaboid receptors, cholecystokinin receptors, dopamine receptors, glucagons receptors, metabotropic glutamate receptors, histamine receptors, olfactory receptors, opiod receptors, rhodosin receptors, secretin receptors, serotonin receptors, somatostatin receptors, calcium-sensing receptor, chemokine receptors, sphingosine-1-phosphate (S1P) receptors); receptor tyrosine kinases (e.g. erythropoietin receptor, insulin receptor, insulin-like growth factor 1 receptor, Eph receptors); guanylyl cyclase receptors (e.g. receptors for natriuretic peptides, guanylin receptor); and ionotropic receptors (e.g. nicotinic acetylcholine receptor, glycine receptor, 5-HT3 receptor, P2X receptors). A number of human plasma membrane receptors is listed at the Human Plasma Membrane Receptome database maintained by a group at Stanford University (Ben-Shlomo et al., “Signaling Receptome: A Genomic and Evolutionary Perspective of Plasma Membrane Receptors Involved in Signal Transduction” (Science Signaling STKE, Vol. 2003, Issue 187, pp. re9, 17 Jun. 2003); see also Ben-Shlomo et al., Molecular Endocrinology 21 (8): 2009-2014); each entry of the publication and database is incorporated herein by reference in its entirety. Additional databases for databases for receptor tyrosine kinase receptors (Grassot et al., 2003, “RTKdb: database of receptor tyrosine kinase” Nucleic Acids Res 31:353-358), G protein-coupled receptors (Horn et al., 2003, “GPCRDB information system for G protein-coupled receptors” Nucleic Acids Res 31:294-297), olfactory receptors (Skoufos et al., 2000, “Olfactory receptor database: a sensory chemoreceptor resource” Nucleic Acids Res 28:341-343), thyrotropin receptor mutations (Fuhrer et al., 2003 “The thyrotropin receptor mutation database: update 2003” Thyroid 13:1123-1126), nuclear receptors (Patterson et al., 1994 “The androgen receptor gene mutations database” Nucleic Acids Res 22:3560-3562; Gottlieb et al., 1998, “The androgen receptor gene mutations database” Nucleic Acids Res 26:234-238), and endocrine disruptor receptors (Nakata et al., 1999, “Development of the receptor database (RDB): application to the endocrine disruptor problem” Bioinformatics 15:544-552) are known; each entry of each of these publications and databases is incorporated herein by reference in its entirety. The Database of Ligand-Receptor Partners maintained by a group at University of California-Los Angeles (http://dip.doe-mbi.ucla.edu/dip/DLRP.cgi) contains subgroups of receptors for chemokines, TNF, fibroblast growth factor (FGF), and TGFβ ligands; each entry of the publication and database is incorporated herein by reference in its entirety. The Alliance for Cellular Signaling database contains extensive information on many signaling genes; each entry of the publication and database is incorporated herein by reference in its entirety. Likewise, the reactome database (Joshi-Tope et al., 2005 “Reactome: a knowledgebase of biological pathways” Nucleic Acids Res 33:D428-D432) and the Human Protein Reference Database (Peri et al., 2003, “Development of human protein reference database as an initial platform for approaching systems biology in humans,” Genome Res 13:2363-2371) represent curated resources of protein-protein interactions for core pathways and reactions in human biology; each entry of each of these publications and databases is incorporated herein by reference in its entirety.
A variety of cell types may be used, so long as one component of the signaling complex to be modulated is present on the surface or in the membrane. The cells may express the signaling complex component from an endogenous, or an exogenous gene. Methods for introducing exogenous genes into host cells such that the host cells express the gene products are well known in the art. [See e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor, N.Y. (2001)]. Adherent or suspension cells may be used. Primary cell cultures, cell lines or engineered cell lines may be used. Suitable cell types may include: CHO, IM-9, HEK 293, and 3T3.
Several assay formats may be employed. In some embodiments, a receptor occupancy assay may be used. In certain embodiments cells are serum-starved to remove any bound ligand and modulator binding to the cells pre-incubated in either the presence or absence of ligand is then measured. Modulator binding may be detected using any suitable detection system. For example, the modulator may be tagged with an affinity or epitope tag and detected with a cognate binding species such as a metal ion, glutathione, anti-tag antibody. Suitable tags are well known in the art and include: c-myc, FLAG, poly-His, V5, HA, glutathione-S-transferase (GST), calmodulin-binding peptide (CBP), covalent yet dissociable NorpD peptide (CYD), strep-tag, heavy chain of protein C (HPC), and maltose-binding protein (MBP). Alternatively, the modulator may be detected with a specific antibody, e.g. a species-specific antibody or an anti-Fc antibody, conjugated to a detection agent such as a colorimetric enzyme (e.g. alkaline phosphatase, horseradish peroxidase), a fluorescent protein (e.g. phycoerythrin) a fluorescent dye (e.g. Alexa Fluor®, Invitrogen Corp., Carlsbad, Calif.) or other suitable agent. Positive kinetic modulators are expected to show higher binding affinity for the cells which were exposed to ligand than for the cells which were not. Negative kinetic modulators are expected to show lower binding affinity for the cells which were exposed to ligand than for the cells which were not. An example receptor occupancy assay is shown in Example 4.
In other embodiments, a labeled ligand assay may be used to measure differential ligand binding to cells in the presence or absence of test polypeptide binding agents. An example of a labeled ligand assay is described in Example 5.
Signaling Assays
The positive or negative modulatory activities of agents identified in the biophysical screens of the present disclosure may be confirmed by measuring the level of signaling in the presence and absence of the test polypeptide binding agent.
Signal transduction refers to any process by which a cell converts one kind of signal or stimulus into another. Intracellular signal transduction is largely carried out by second messenger molecules such as: calcium; lipophilic molecules e.g. diacylglycerol, ceramide, eicosanoids, and lysophosphatidic acid; nitric oxide. Thus a change in the level or location of second messenger may be used to measure signaling.
Examples of common signaling pathways include: the cAMP dependent pathway (in humans, cAMP works by activating protein kinase A), the MAPK/ERK pathway (a pathway that couples intracellular responses to the binding of growth factors to cell surface receptors); and the IP3/DAG pathway (phospholipase C cleaves the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) yielding diacyl glycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG remains bound to the membrane, and IP3 is released as a soluble structure into the cytosol. IP3 then diffuses through the cytosol to bind to IP3 receptors, particular calcium channels in the endoplasmic reticulum. These channels are specific to calcium and only allow the passage of calcium to move through. This causes the cytosolic concentration of calcium to increase, causing a cascade of intracellular changes and activity. In addition, calcium and DAG together work to activate protein kinase C, which goes on to phosphorylate other molecules, leading to altered cellular activity).
Signaling assays may, for example, detect the level, location, interactions or post-translational modification of cellular proteins. Gene expression may also be used to measure signaling.
A number of assays for measuring the level of signaling mediated by a signaling complex are available in the art. See, for example: Dove, Nat. Methods 3: 223-229 (2006);
The choice of assay will depend on the nature of the signaling pathway to be modulated. Assay kits available include: kits for assaying c-Fos, c-Jun, G Proteins, G Protein chimera clones, GPCRs, NF-kB p50, NF-kB p50/p65, NF-kB p65, and p38 MAPK; phosphoprotein assay kits such as for phosphothreonine and phosphotyrosine; kits for assaying second messengers, such as calcium, cAMP, cGMP and PIP3; kits for assaying s mall GTPase's such as Cdc42, Rac, Rap, Ras and Rho; and STAT assay kits (see e.g. www.biocompare.com). Available kits include ELISAs and phosphospecific ELISAs to detect non-phosphorylated or phosphorylated proteins of interest, isolation kits to extract subcellular components, enzymatic assays utilizing numerous detection methods, and targeted assays. Kits are available to measure signaling in pathways such as those relating to apoptosis, cytoskeleton/extracellular matrix, neuroscience, nitric oxide/cell stress, protein phosphorylation (see e.g. sigmaaldrich.com).
Effects of test compounds on signaling activity may be measured at a single ligand, or test compound concentration point. Alternatively or additionally, signaling assays may be performed using multiple ligand or test compound concentration points.
Exemplary methods for measuring signaling include, but are not limited to calcium flux assays, phosphorylation assays, gene expression assays, molecular transport assays, and other methods known to one of skill in the art.
Methods for determining changes in intracellular calcium due to cell signaling are well-known in the art. See e.g., Walsh et al. J. Biol. Chem., 283:16971-16984, 2008 and Janas et al., Clin Exp Immunol. 139:439-446, 2005. Briefly, appropriate cells are first cultured with an agent that inhibits calcium accumulation in the cell, such as indomethacin, fluorescent dyes, such as URA-2, FLUO-3, FLUO-4, Calcium-3, Calcium 4, Calcium 5 and Calcium Green-1/AM (Molecular Devices, Sunnyvale, Calif.), Rhod-4 NW (ABD Bioquest, Sunnyvale, Calif.) that report increases in intracellular calcium due to changes in fluorescence signal upon calcium binding, or biosensor photoproteins, e.g., aequorin and PHOTINA® (Perkin Elmer, Waltham, Mass.), which provide a luminescent signal in response to elevation in intracellular calcium. Cells are then contacted with a polypeptide of the invention, and the changes in intracellular calcium levels determined using such techniques as flow cytometry, Fluorometric Imaging Plate Reader (FLIPR), confocal fluorescent microscopy, calcium chip methods (e.g., Cell Kinetics, Lod, Israel) or other calcium detection methods known in the art.
Tyrosine or serine phosphorylation assays are often used to determine activation of cellular pathways involving receptor activation, and methods for detection of phosphotyrosine are readily available in the art. Sample protocols are disclosed in, e.g., Walsh et al., J. Biol. Chem., 283:16971-16984, 2008 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., Ch. 18.4.1-18.4.7, 1997. Appropriate cells are contacted with a polypeptide of the invention for a sufficient period of time to induce cell activation, for example 15 to 30 minutes, the cells are lysed and proteins resolved on an SDS-PAGE gel. The gel is then probed with an anti-phosphotyrosine or anti-phosphoserine antibody, and the level, type and protein-specific phosphorylation assessed using techniques in the art, such as chemiluminescence. Induction of phosphorylation is also measurable using antibodies specific for phosphphotyrosine or phosphoserine, as well as antibodies specific for receptors and/or other proteins in the phosphorylated state in assays such as enzyme linked immunosorbant assay (ELISA) and other microplate-based assays such as kits available from Meso Scale Discovery, Gaithersburg, Md.), or flow cytometry based assays from BD Biosciences, (San Jose, Calif.), or Millipore (Billerica, Mass.).
Methods for detecting gene expression are well-known in the art. Gene expression induced by contacting cells with a polypeptide of the invention is determined using techniques known in the art, including, but not limited to, Northern blot detection of mRNA for downstream signaling events or a transcript of interest, gene reporter assays, differential display, subtractive DNA assays and serial analysis of gene expression (SAGE) (See e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., Ch. 4 and 25, 1996, 2001 and 2007). Additionally, gene arrays are available, e.g., GeneChip Human Genome U133 Plus 2.0 Arrays, to determine levels of gene expression in a cell population or subject after contacting with a polypeptide of the invention.
Molecular transport induced by contact of cells with a polypeptide of the invention are assayed using techniques known in the art, including, but not limited to, lipid raft assays and pinocytosis assays. In one embodiment, cells are contacted with a polypeptide of the invention for a sufficient period of time to allow for signal induction, the cells are lysed and lipid and non-lipid material is separated by sucrose density gradient. Signaling proteins are isolatable from the different lipid raft fractions resolved in the sucrose gradient, and the ability of a polypeptide of the invention to affect signaling is determined by a change in the membrane bound protein composition of the lipid rafts isolated before and after contacting with a polypeptide of the invention. See e.g., Petrie et al., J. Immunol. 165:1220-7, 2000; Chamberlain et al., Proc Natl Acad Sci USA. 98:5619-24, 2001; Janas et al., Clin Exp Immunol. 139:439-446, 2005. Pinocytosis and transport of molecules is detectable using electron microscopy, by measuring the uptake of [14C]sucrose (Chow et al., The FASEB Journal. 12:823-830, 1998), using flow cytometry, and other techniques known in the art.
An example of a phosphorylation assay is shown in Example 6.
In some aspects, biophysical screens are combined with functional screens to identify polypeptide binding agents with only kinetic modulating properties, i.e. without further antagonist or agonist properties, or to identify kinetic modulators that additionally have agonistic or antagonistic properties. Drugs with both kinetic modulating and agonistic properties can affect both affinity and efficacy of the endogenous target, thereby significantly expanding the repertoire of therapeutic intervention possibilities for a given target.
Methods of Producing Kinetic Modulating Drugs from Existing Compounds
Any polypeptide binding agents may be tested in the assays described herein to determine their kinetic modulating properties. In one embodiment, if a polypeptide binding agent is determined to have no kinetic modulating properties, it may be used in complex with its antigen, for immunization, panning etc, in order to obtain other polypeptide binding agents that bind to different epitopes and that may be more likely to produce kinetic modulating effects.
Variants of parental polypeptide binding agents may be produced by introducing mutations or chemical derivatization, including conjugation, using any methods known to those skilled in the art. The variants may then be screened in the assays disclosed herein, in order to identify those with desired kinetic modulating properties. The parental polypeptide binding agents may have no kinetic modulating activities, or may preferably have existing kinetic modulating properties that are desired to be increased, decreased, or altered in some other manner.
A number of methods for producing variants of parental drugs or drug candidates are available in the art.
Preparing Amino Acid Sequence Variants
It is contemplated that modified polypeptide compositions comprising one, two, three, four, five, and/or six CDRs of an antibody or polypeptide binding agent are generated, wherein a CDR or non-CDR region is altered to provide increased specificity or affinity to the antigen, or to provide increased modulation of binding affinity between the target and its signaling partner. For example, sites within antibody CDRs are typically modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid substituted for a non-identical hydrophobic amino acid) and then with more dissimilar choices (e.g., hydrophobic amino acid substituted for a charged amino acid), and then deletions or insertions may be made at the targeted site. For example, using the conserved framework sequences surrounding the CDRs, PCR primers complementary to these consensus sequences are generated to amplify the antigen-specific CDR sequence located between the primer regions. Techniques for cloning and expressing nucleotide and polypeptide sequences are well-established in the art [see e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989)]. The amplified CDR sequences are ligated into an appropriate plasmid. The plasmid comprising one, two, three, four, five and/or six cloned CDRs optionally contains additional polypeptide encoding regions linked to the CDR.
Polypeptide binding agents comprising the modified CDRs are screened for binding affinity for the original antigen. Additionally, the antibody or polypeptide is further tested for its ability to neutralize the activity of its antigen. For example, antibodies of the invention may be analyzed as set out in the Examples to determine their ability to interfere with the biological activity of the target.
Modifications may be made by conservative or non-conservative amino acid substitutions described in greater detail below. “Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids. The variation may be introduced by systematically making substitutions of amino acids in an antibody polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity. Nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Methods for altering antibody sequences and expressing antibody polypeptide compositions useful in the invention are described in greater detail below.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody (including antibody fragment) fused to an epitope tag or a salvage receptor epitope. Other insertional variants of the antibody molecule include the fusion to a polypeptide which increases the serum half-life of the antibody, e.g. at the N-terminus or C-terminus.
The term “epitope tagged” refers to the antibody fused to an epitope tag. The epitope tag polypeptide has enough residues to provide an epitope against which an antibody there against can be made, yet is short enough such that it does not interfere with activity of the antibody. The epitope tag preferably is sufficiently unique so that the antibody there against does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8-50 amino acid residues (preferably between about 9-30 residues). Examples include the flu hemagglutinin (HA) tag polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol. 8: 2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Mol. Cell. Biol. 5:3610-16 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering 3:547-53 (1990)). Other exemplary tags are a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation. Other labels and tags, such as the FLAG® tag (Eastman Kodak, Rochester, N.Y.), well known and routinely used in the art, are embraced by the invention.
As used herein, the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Substitutional mutagenesis within any of the hypervariable or CDR regions or framework regions is contemplated. Conservative substitutions involve replacing an amino acid with another member of its class. Non-conservative substitutions involve replacing a member of one of these classes with a member of another class.
Conservative amino acid substitutions are made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine (Ala, A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine (Met, M); polar neutral amino acids include glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N), and glutamine (Gln, Q); positively charged (basic) amino acids include arginine (Arg, R), lysine (Lys, K), and histidine (His, H); and negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E).
Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
Affinity Maturation
Affinity maturation generally involves preparing and screening antibody variants that have substitutions within the CDRs of a parent antibody and selecting variants that have improved biological properties such as stronger binding affinity relative to the parent antibody. A convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity). See e.g., WO 92/01047, WO 93/112366, WO 95/15388 and WO 93/19172.
Current antibody affinity maturation methods belong to two mutagenesis categories: stochastic and nonstochastic. Error prone PCR, mutator bacterial strains (Low et al., J. Mol. Biol. 260, 359-68 (1996)), and saturation mutagenesis (Nishimiya et al., J. Biol. Chem. 275:12813-20 (2000); Chowdhury, P. S. Methods Mol. Biol. 178, 269-85 (2002)) are typical examples of stochastic mutagenesis methods (Rajpal et al., Proc Natl Acad Sci USA. 102:8466-71 (2005)). Nonstochastic techniques often use alanine-scanning or site-directed mutagenesis to generate limited collections of specific variants. Some methods are described in further detail below.
Affinity Maturation Via Panning Methods—
Affinity maturation of recombinant antibodies is commonly performed through several rounds of panning of candidate antibodies in the presence of decreasing amounts of antigen. Decreasing the amount of antigen per round selects the antibodies with the highest affinity to the antigen thereby yielding antibodies of high affinity from a large pool of starting material. Affinity maturation via panning is well known in the art and is described, for example, in Huls et al. (Cancer Immunol Immunother. 50:163-71 (2001)). Methods of affinity maturation using phage display technologies are described elsewhere herein and known in the art (see e.g., Daugherty et al., Proc Natl Acad Sci USA. 97:2029-34 (2000)).
Look-Through Mutagenesis—
Look through mutagenesis (LTM) (Rajpal et al., Proc Natl Acad Sci USA. 102:8466-71 (2005)) provides a method for rapidly mapping the antibody-binding site. For LTM, nine amino acids, representative of the major side-chain chemistries provided by the 20 natural amino acids, are selected to dissect the functional side-chain contributions to binding at every position in all six CDRs of an antibody. LTM generates a positional series of single mutations within a CDR where each “wild type” residue is systematically substituted by one of nine selected amino acids. Mutated CDRs are combined to generate combinatorial single-chain variable fragment (scFv) libraries of increasing complexity and size without becoming prohibitive to the quantitative display of all variants. After positive selection, clones with stronger binding affinity are sequenced, and beneficial mutations are mapped.
Error-Prone PCR—
Error-prone PCR involves the randomization of nucleic acids between different selection rounds. The randomization occurs at a low rate by the intrinsic error rate of the polymerase used but can be enhanced by error-prone PCR (Zaccolo et al., J. Mol. Biol. 285:775-783 (1999)) using a polymerase having a high intrinsic error rate during transcription (Hawkins et al., J Mol Biol. 226:889-96 (1992)). After the mutation cycles, clones with stronger binding affinity for the antigen are selected using routine methods in the art.
DNA Shuffling—
Nucleic acid shuffling is a method for in vitro or in vivo homologous recombination of pools of shorter or smaller polynucleotides to produce variant polynucleotides. DNA shuffling has been described in U.S. Pat. No. 6,605,449, U.S. Pat. No. 6,489,145, WO 02/092780 and Stemmer, Proc. Natl. Acad. Sci. USA, 91:10747-51 (1994). Generally, DNA shuffling is comprised of 3 steps: fragmentation of the genes to be shuffled with DNase I, random hybridization of fragments and reassembly or filling in of the fragmented gene by PCR in the presence of DNA polymerase (sexual PCR), and amplification of reassembled product by conventional PCR.
DNA shuffling differs from error-prone PCR in that it is an inverse chain reaction. In error-prone PCR, the number of polymerase start sites and the number of molecules grows exponentially. In contrast, in nucleic acid reassembly or shuffling of random polynucleotides the number of start sites and the number (but not size) of the random polynucleotides decreases over time.
In the case of an antibody, DNA shuffling allows the free combinatorial association of all of the CDR1s with all of the CDR2s with all of the CDR3s, for example. It is contemplated that multiple families of sequences can be shuffled in the same reaction. Further, shuffling generally conserves the relative order, such that, for example, CDR1 will not be found in the position of CDR2. Rare shufflants will contain a large number of the best (e.g. highest affinity) CDRs and these rare shufflants may be selected based on their superior affinity.
The template polynucleotide which may be used in DNA shuffling may be DNA or RNA. It may be of various lengths depending on the size of the gene or shorter or smaller polynucleotide to be recombined or reassembled. Preferably, the template polynucleotide is from 50 bp to 50 kb. The template polynucleotide often should be double-stranded.
It is contemplated that single-stranded or double-stranded nucleic acid polynucleotides having regions of identity to the template polynucleotide and regions of heterology to the template polynucleotide may be added to the template polynucleotide, during the initial step of gene selection. It is also contemplated that two different but related polynucleotide templates can be mixed during the initial step.
Alanine Scanning—
Alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Cunningham and Wells, (Science 244:1081-1085 (1989)). A residue or group of targeted residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
Computer-Aided Design—
Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen, or to use computer software to model such contact points. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
Advantages of Kinetic Modulating Drugs
Kinetic modulating drugs are expected to provide several advantages as therapeutic agents in comparison to standard therapies such as competitive antagonists, agonists and ligand replacement. Such advantages may include those outlined below.
(1) Self-limiting activity. At saturating levels, kinetic modulating drugs have decreased potential for toxic effects due to concentration-independent limits on the activity of the drug. Modulators with limited cooperativity will have a ceiling level to their effect, irrespective of the administered dose. Unlike conventional drugs, kinetic modulators achieve saturation beyond which increases in the concentration of the modulator have no additional effect on function. The effect is limited by the extent of cooperativity (either positive or negative) between the modulator and the endogenous signaling complex component (e.g. ligand). The cooperativity-defined boundary enhances the safety profile of kinetic modulating drugs with respect to dosing regimens. Hence, kinetic modulating drugs could be given in larger more frequent doses than conventional drugs, while maintaining a favorable overall safety profile, providing increased efficacy due to the ability to maintain continuously saturating levels of drug.
(2) Complex component-dependent activation/inhibition limits side-effects. Unlike agonists or antagonists, a kinetic modulator can be inactive in the absence of the endogenous complex component (e.g. ligand) and active only when appropriate, such as when the complex component (e.g. ligand) is present. If a kinetic modulator does not possess appreciable agonism or antagonism, it can provide another powerful therapeutic advantage over conventional ligands, namely the ability to selectively tune up or down tissue responses only when the endogenous agonist is present. These properties enable kinetic modulating drugs to augment the endogenous spatial and temporal regulation of the target.
(3) Reduced off-target effects in vivo from greater target selectivity, tissue-, or subtype-, specificity. Kinetic modulating drugs may bind to sites other than the sites of interaction between the complex components. Such allosteric binding sites have not faced the same evolutionary pressure as orthosteric sites so are more diverse. Therefore, greater selectivity may be obtained by targeting allosteric sites. This is a particular advantage when there is a high degree of sequence conservation of the orthosteric site across target subtypes.
(4) Broader therapeutic window. Positive kinetic modulators can discriminate between activated and non-activated receptor states, while agonists indiscriminately activate all receptor states. Kinetic modulators that do not have appreciable agonism therefore have a broader therapeutic window than agonists. In addition, positive kinetic modulators may carry a reduced liability for receptor desensitization and/or tolerance, which can significantly expand the range of possible therapeutic applications. Negative kinetic modulators enable the reduction of signaling without completely blocking it. This may be useful, for example, where a receptor mediates pathological functions while at the same time mediating physiologically useful functions.
EXAMPLES
Example 1
Use of Equilibrium Solution Affinity Measurement Methods to Determine Receptor-Ligand Affinity in the Presence or Absence of Test Compounds
A. Model System
This example describes methods for equilibrium solution affinity measurement using a model signaling complex, such as a receptor-ligand complex. The model system described here will use beads with an immobilized ligand, so the free receptor will be assayed and detected with an anti-receptor Cy5 labeled polyclonal antibody.
One of the binding partners, in this case the ligand, is immobilized to a solid phase that can be suspended by an automatic stirrer and allowed to form a small column bed. Typically the support is a bead of polymethylmethacrylate (PMMA), agarose, or other compatible material. The other binding partner, in this case the receptor, is detected in its bound state by a fluorescently-labeled, biotinylated, or otherwise-tagged molecule.
The KinExaTM instrument from Sapidyne Instruments can be used to perform the affinity analysis by kinetic exclusion assay (KinExA). In short, the interacting complex components, in this case the receptor and ligand, are mixed together in varying known concentrations in the presence or absence of test polypeptide binding agent and allowed to come to an equilibrium. The sample containing ligand, free receptor, and ligand-receptor complex is pulled rapidly through a small bead column that has been coupled or coated with one of the complex components, in this case the ligand, or an equivalent competitive binder. Free receptor is bound to ligand on the beads. Secondary label such as Cy5 fluorescently labeled secondary antibody to the receptor is then passed through the column. Labeled secondary antibody binds to the bound receptor. A buffer wash removes excess label, leaving fluorescence signal on the bead column directly proportional to the amount of free receptor in the original sample. The bead bed is positioned near a fluorescent detector to allow readout of the level of fluorescence signal.
A batch of beads with the immobilized ligand is prepared. A variety of bead types can be used, including PMMA, agarose, polystyrene etc. The coupling of the ligand to the beads can be performed using a variety of methods known to those skilled in the art. A stock of receptor is prepared at a concentration below the predicted KD of the receptor-ligand interaction. An experiment should be run with a variety of concentrations of receptor without ligand to determine the lowest concentration of receptor that can be used. By lowering the receptor concentration in the assay the affinity determinations become increasingly accurate. For a 500 pM interaction a receptor concentration of 50 pM should allow for accurate KD measurements. Enough receptor must be used to achieve an adequate signal and provide the dynamic range required.
Once an optimized receptor concentration is determined, a 2× stock of receptor is prepared (if final desired concentration is 50 pM then a 100 pM solution is made up), with and without test compound.
A serial dilution of ligand at 2× concentration is created. This dilution series should ideally contain points at least 10-fold above and below the KD values of the interaction. A 12-point titration plus a zero-ligand sample is usually sufficient to cover this type of range at a 1:2 dilution series.
Equal volumes of the ligand titration series are mixed with the receptor and receptor-plus-test compound samples and these are allowed to come to equilibrium. This can take from hours to several weeks depending on the kinetics of the interaction. If any of the kinetics of the interaction are known, they can be used to estimate time to equilibrium. The reaction approaches true equilibrium at a slow, but exponential rate, so it is likely not necessary to wait for a high affinity interaction to reach true equilibrium, since in a few days it is often >95 percent complete. It is, however, important to understand this relationship and evaluate it critically.
Once the required incubation time has been reached a dilution of the Cy5 labeled anti-receptor antibody is prepared. It is important that the labeled molecule is able to bind its antigen, in this case the receptor, when it is bound to its binding partner, in this case the ligand, or else the assay will have little to no signal. For the assay format described here, a 1 ug/mL solution of an anti-receptor Cy5 labeled polyclonal antibody is suitable.
The KinExa sample inlet tubes are placed into the sample vials. The KinExa instrument then analyzes the concentration of free receptor in every sample and plots it as percent free receptor over concentration of ligand. The curves are fitted to a model and the KD Value is determined. Example results are shown in FIG. 4 which are generated using the below equation where R is the receptor concentration and L is the ligand concentration.




  
    R
    free
  
  =
  
    
      
        (
        
          
            R
            tot
          
          -
          
            L
            tot
          
          -
          
            K
            D
          
        
        )
      
      2
    
    ±
    
      
        
          
            
              (
              
                
                  L
                  tot
                
                +
                
                  R
                  tot
                
                +
                
                  K
                  D
                
              
              )
            
            2
          
          2
        
        -
        
          
            L
            tot
          
          ·
          
            R
            tot
          
        
      
    
  




B. IL-1β Signaling Complex

IL-1β is a highly potent cytokine that drives the acute phase inflammatory response and has an essential role in the innate immune response. While high levels of IL-1β have been implicated in inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, acute respiratory distress syndrome, and Type 2 diabetes, low levels have beneficial effects on pancreatic beta cell function, proliferation, and survival, intestinal epithelial cell survival, and neuronal response to injury. As in many receptor-ligand systems, IL-1β signaling is complex, with multiple ligands interacting with membrane-bound and soluble forms of several receptors (Dinarello, Arthritis Rheum. 52 (7): 1960-1967, 2005). IL-1β signaling activity is mediated by a single receptor, IL-1 Receptor type I (IL-1RI) and its co-receptor IL-1 Receptor Accessory Protein (IL-1RAcP). A second IL-1 family member, IL-1α, signals through the same receptor complex but has not been implicated in inflammatory diseases. IL-1β activity is under tight physiological control, with multiple levels of negative regulation including: neutralization and endocytosis of excess IL-1β mediated by the decoy receptor IL-1 Receptor type II (IL-1RII); inhibition of circulating IL-1β mediated by multiple soluble forms of its receptors (sRI, RII, and sRAcP); and competitive inhibition by an inhibitory IL-1 homologue, IL-1 Receptor Antagonist (IL-1Ra). The complexity of this receptor-ligand system presents a challenge for the production of anti-IL-1β antibodies. It is proposed that the optimal therapeutic agent modulating this pathway would selectively reduce high-level IL-1β signaling to lower, beneficial levels while allowing neutralization of IL-1β activity by soluble receptors and clearance of IL-1β by receptor-mediated pathways without interfering with IL-1α signaling or IL-1Ra activity.
Biophysical Assay
A KinExA assay was configured to measure the concentration of free ligand (IL-1β) in samples of ligand-receptor (IL-1sRI or IL-1sRII) mixtures in the presence or absence of a test compound (anti-IL-1β antibody XOMA052, see U.S. Pat. No. 7,531,166)
The equilibrium dissociation constants (KD) of IL-1β±XOMA 052 binding to soluble IL-1 receptors (IL-1sRI: R&D Systems, cat#260-100/CF, and IL-1sRII: R&D Systems, cat#263-2R-050/CF) were determined using KinExA technology (Sapidyne, Inc). Equilibrium experiments were conducted by serially diluting soluble receptors from 150 nM to 4 pM in PBS (0.01M phosphate, pH 7.4, 0.15M NaCl, 0.02% azide) with 1% BSA sample buffer, into a constant binding site concentration (1 to 5 nM) of IL-1β alone or mixed with XOMA 052. To obtain KD-controlled data, the binding site concentration was no more than two-fold above the KD (IL-1β=1 nM and IL-1β±XOMA 052=5 nM). For all experiments where XOMA 052 was present, the antibody concentration was maintained at a 100-fold molar excess over IL-1β to ensure that all of the cytokine was bound by XOMA 052. The IL-1β (±XOMA 052) plus receptor mixtures were incubated at room temperature ( ̃22° C.) for 12-24 hours prior to assay initiation to allow complex formation to reach equilibrium. Following the incubation period, the mixtures containing receptor, IL-1β±XOMA 052, and IL-1sR/IL-1β complexes±XOMA 052, were drawn through a solid phase, consisting of receptor-blocking anti-IL-1β antibody-coupled beads, to capture IL-1β (±XOMA 052) not bound to receptor. The capture antibody was verified to completely compete with the receptor and not with XOMA 052. The captured IL-1β is directly proportional to the concentration of free IL-1β not bound by receptor remaining in the equilibrium reaction and was detected using a polyclonal anti-IL-1β antibody (R&D Systems, AB-201-NA), followed by a phycoerythrin-conjugated anti-goat IgG secondary antibody (Jackson ImmunoResearch laboratory cat #705-116-147) in the sample buffer. The bound signals were converted into relative values as a proportion of control in the absence of receptors. Two replicates of each sample were measured for all equilibrium experiments. The equilibrium titration data were fit to a 1:1 binding model using KinExA software (Version 2.4; Sapidyne Instruments). These measurements were repeated a total of five times for sRI and three times for sRII. The results show that XOMA 052 weakens the binding affinity of IL-1β binding to IL-1 sRI from 2 to 10 nM, but has no effect on the binding affinity of IL-1β binding to IL-1 sRII, which remains at 2 nM (FIG. 5). Measurements using surface plasmon resonance (SPR) were consistent with these results.
Another variation of this assay could be performed to rank potential drug candidates amongst each other. Once the full KD solution equilibrium experiment has been run, the conditions needed to generate a signal on the log linear portion of the curve are known. A ligand concentration is selected a little below the EC50 value of the interaction without a kinetic modulating drug. Each drug candidate can then be tested at several concentrations. This allows for analysis of dose response to the drug and relative potency comparisons between potential drug candidates.
Functional Assays
Functional assays were performed to confirm the prediction that the reduction in the affinities of signal complex components will cause a shift in the cellular dose-response to IL-1β.
MRC-5 IL-6 Release Assay
MRC-5 human lung fibroblast cells (ATCC, Manassas, Va.) were seeded into a sterile 96-well tissue culture plate at 5000 cells per well in MEM complete growth medium (Invitrogen) with 10% fetal bovine serum (FBS; Hyclone). After an overnight incubation at 37° C. with 5% CO2, supernatants were removed and replaced with growth medium containing recombinant human IL-1β (Peprotech, cat. 200-001B) plus either control IL-1β-blocking antibody (WO 2006/081139), anti-KLH (Keyhole Limpet Hemocyanin) isotype control antibody (IgG2; clone KLH8.G2 (XOMA)), or XOMA 052 at the concentrations indicated.
An antibody potency assay was performed (FIG. 6A) whereby recombinant human IL-1β was pre-incubated with the indicated antibody for 1 hour at 37° C. prior to addition to the MRC-5 cells, and added at a final concentration of 100 pg/ml IL-1β.
An IL-1β dose-response assay was performed (FIG. 6B) using increasing amounts of IL-β pre-incubated overnight at room temperature with a 100-fold molar excess of antibody prior to addition to the MRC-5 cells.
Following a 20 hour incubation at 37° C. with 5% CO2, cell supernatants were removed and diluted according to estimated IL-6 concentration and assayed for human IL-6 by ELISA (QuantikineTM human IL-6 ELISA, R&D Systems, cat# D6050) according to the manufacturer's instructions. All samples were set up and assayed in duplicate or triplicate.
Whole Blood IL-8 Induction Assay
Normal human blood was collected by venipuncture into collection tubes containing heparin sulfate. An IL-1β dose-response assay was performed using increasing amounts of IL-β preincubated with a 10-fold molar excess of XOMA 052 for 1 hour at 37° C. in RPMI (Invitrogen) with 10% FBS prior to addition to whole blood. Samples were incubated for 6 hours at 37° C. in 96-well round bottom plates (Corning Costar, cat#3799) then lysed with Triton X-100 at a final concentration of 0.5% for 10 minutes. Lysates were centrifuged for 5 minutes at 2000 rpm to remove debris and transferred to a clean plate. After repeating the centrifugation step, lysates were transferred to a −80° C. freezer for overnight storage. The following morning lysates were thawed and tested for human IL-8 by ELISA (Quantikine human IL-8 ELISA, R&D Systems, cat# D8000C) according to manufacturer's instructions. All samples were set up and assayed in duplicate or triplicate. Results are shown in FIG. 6C.
Results
While XOMA 052 binding to IL-1β does not abrogate signal complex formation at high concentrations of IL-1β, it nonetheless is a potent inhibitor of IL-1β activity at physiological and pathologically relevant concentrations. XOMA 052 completely neutralizes 100 pg/mL of IL-1β in an MRC-5 cytokine release assay with an observed IC50 in the low pM range. This is comparable to neutralization observed with a control blocking antibody (FIG. 6A), and around 10-fold more potent than recombinant IL-1ra.
The kinetic perturbation model predicts that the reduction in the affinities of signal complex components will cause a shift in the cellular dose-response to IL-1β. When XOMA 052 is in molar excess of the concentration of IL-1β, the antibody increases the EC50 of the IL-1β dose response curve up to 60-fold in the MRC-5 cell IL-6 release assay relative to that of IL-1β in the presence of an isotype control antibody (EC50 values of 815 pM versus 12 pM). Under the same conditions an IL-1β blocking antibody almost completely ablates cellular response across a broad range of IL-1β concentrations (FIG. 6B).
Similar dose-response shifts with XOMA 052 are seen in stimulation of IL-8 expression in whole blood (FIG. 6C), cinq1 in rat NRKE cells, and IL-8 in PBMCs, demonstrating that this effect is not unique to a particular assay system. Thus, under physiological conditions where high levels of IL-1β cause pathology (in pancreatic beta-cells, for instance), XOMA 052 will neutralize excess IL-1β while potentially allowing continued low-level beneficial signaling. Furthermore, XOMA 052 may allow for better responsiveness of the innate immune system to infection as compared to a complete blockade of IL-1β activity. The degree of signaling attenuation mediated by XOMA 052 is independent of the concentration of antibody when its concentration is sufficiently high to bind all available IL-1β. In those conditions the concomitant signaling output depends only on the concentration of ligand.
While reducing IL-1β affinity for IL-1RI causes attenuation of signaling, maintenance of efficient binding to IL-1RII is important because IL-1RII functions as a decoy receptor on responding cells to attenuate sensitivity to IL-1β. In addition, IL-1RII mediated internalization of IL-1β is an important pathway for clearance of IL-1β. When binding of a therapeutic antibody to its antigen of interest interrupts physiological clearance pathways, the prolonged half-life and high affinity of the antibody can cause the accumulation of antibody/antigen complexes. While such complexes are typically inactive, it may be necessary to maintain excess levels of antibody in order to ensure that any antigen that dissociates from the antibody is rapidly rebound by free antibody.
We have demonstrated that while XOMA 052 weakens the affinity of IL-1β binding to IL-1RI, it does not similarly weaken its binding to IL-1RII. Furthermore, XOMA 052 does not block binding of sRAcP to IL-1β/sRII complex. The ability of the IL-1β/XOMA 052 complex to bind IL-1RII and RAcP may allow clearance and neutralization of the cytokine by normal physiological mechanisms and thus reduce accumulation of long-lived complexes. In addition, retention of binding of the neutralizing soluble receptors to the XOMA 052/IL-1β complex should allow these molecules to regulate activity and thus avoid potential systemic activity by complexes before their clearance.
To test whether the ability of IL-1β/XOMA 052 complexes to bind IL-1 receptors affects clearance rates, we injected mice with recombinant human IL-1β, either free (incubated with a non-binding isotype control antibody) or pre-complexed with either XOMA 052 or a control blocking anti-IL-1β antibody (BM5), and measured the amount of total measurable IL-1β remaining in circulation after 48 hours by ELISA (BD OptEIATM human IL-1β ELISA set, BD Biosciences, San Jose, Calif., cat#557953) as per kit instructions. In two separate experiments, C57B/6 mice were injected (n=4 per group) intravenously with 100 μl each of antibody pre-complexed with human IL-1β (1 mg/kg antibody with 20 ng IL-1β per mouse). Data for the circulating levels of BM5-IL-1β and XOMA 052-IL-1β-complexed antibodies after 48 hrs are shown in FIG. 7. In study 1, by 48 hours post injection, 7.7% of the IL-1β-complexed to BM5 remained in circulation; however, over the same time period, only 1.7% of the XOMA 052-IL-1β complex remained (p=0.006). In study 2, by 48 hours post injection, 8.1% of the IL-1β-complexed to BM5 remained in circulation while only 1.8% of the XOMA 052-IL-1β complex remained (p=0.02). While IL-1β complexed with either antibody cleared more slowly from circulation than free IL-1β, IL-1β bound to XOMA 052 cleared more rapidly than IL-1β bound to a blocking antibody. After 48 hours, an average of 4.6-fold less IL-1β bound to XOMA 052 was detected in the serum as compared to IL-1β bound to a blocking antibody. At the same time, concentrations of the total antibodies did not differ from each other, indicating that the difference did not result from differential clearance of the different antibodies.
The approach we outline here to attenuate rather than block IL-1β may yield important insights into the optimal reduction of IL-1β signaling required for disease modification in Type 2 diabetes, which is increasingly regarded as an inflammatory disease. There is evidence that high levels of IL-1β underlie the pathology of this disease by causing beta-cell dysfunction and apoptosis. However, low levels of IL-1β activity are required for maximum levels of stimulated insulin release and beta-cell proliferation and survival. Treatment of patients with ANAKINRA® (rhu IL-1ra) improves insulin secretion and glucose control but requires frequent dosing due to the rapid clearance of drug. A blocking antibody that completely neutralizes IL-1β activity will not allow low level signaling of IL-1β. A kinetic modulating (or regulatory) antibody such as XOMA 052 should reduce IL-1β activity, allowing a lower level of signaling at higher concentrations of IL-1β, thus regulating IL-1β activity within a beneficial range in T2D patients (see FIG. 8).
We have described here a recombinant antibody that differentially tunes the affinity of a ligand for binding to multiple receptors, allowing for context-dependent attenuation of ligand activity. There is increasing appreciation that many receptor-ligand systems are comprised of multiple ligands and receptors that generate complicated and context-dependent cellular effects. For some receptor systems these effects are beneficial at low levels and pathological at high levels, and have been difficult to approach mechanistically with monoclonal antibody therapeutics. The ability to use antibodies therapeutically as “rheostats” rather than “binary switches” introduces an additional level of subtlety and sophistication in therapeutic antibody design for regulating the activity of disease-relevant targets.
Example 2
Identification of Kinetic Modulators Using a Solid Phase Affinity Measurement Method
The assay described here could be utilized for any two interacting binders of which one can be labeled (e.g. biotinylated) and the other immobilized on an EIA plate. This example uses granulocyte colony stimulating factor (GCSF) binding to its receptor (GCSFR) as a model system. The procedure used for this system is described below. Various conditions that may need to be optimized if different systems were to be utilized would include; plate coating conditions (both time, temperature, concentration, and buffer), analyte labeling conditions, and concentration of labeled (e.g. biotinylated) analyte.
Antibody modulators of the GCSF-GCSFR binding interaction were identified using the assay described below.
Purified GCSF (R&D Systems Minneapolis, Minn.) was biotinylated through activated NHS chemistry using PEG4 Biotin (Pierce Protein Research Products cat#21329, Rockford, Ill.). GCSFR (R&D Systems Minneapolis, Minn.) was coated on an EIA plate (Nune, Rochester, N.Y.) in PBS at 2 ug/mL, 100 uL/well at 37° C. for 1 hour on a shaker. The plate was then blocked with a bovine serum albumin (BSA) and ChemiBlockTM (Millipore Billerica, Mass.) blocking solution for at least 1⁄2 hour at room temperature on a shaker. Any blocking solution that does not interfere with the reactants and sufficiently blocks nonspecific binding on the EIA plate could be used for the assay.
Samples were prepared on a dilution plate. Fab antibody fragments from an OmniclonalTM phage display library generated from mice immunized with GCSF/GCSFR complex (Biosite Inc., San Diego, Calif.) were screened as periplasmic extracts (PPE). For the single point assay, PPE samples were mixed 1:1 with biotinylated GCSF prepared at 0.15 ug/mL in blocking buffer and 50 uL of this solution was loaded onto the EIA plate. Potential hits from the single point assay (FIG. 9A) were purified and further tested at 1.8 ug/mL Fab concentration using titrations of ligand (FIG. 9B), or tested at various titrations at a fixed ligand concentration of 0.075 ug/mL (FIG. 9C). 50 uL/well of this antibody-GCSF-biotin solution was added to the blocked EIA plate and incubated at room temperature for >1 hour on a shaker. Negative controls of PBS and blocking buffer alone were included in the single point assay to establish a background signal level of roughly 0.83 OD405 nm. The purified Fab F5 was also included as 5 ug/mL as positive control and was selected from earlier rounds of screening. 50 uL of streptavidin labeled alkaline phosphatase (Zymed South San Francisco, Calif.) at 5 ug/mL in blocking buffer was then added to all the wells. The biotinylated analyte and antibody mixture remained in the well and was not removed or washed out. The plate was then incubated at room temperature on a shaker for an additional >1 hour (total reaction time of 3-4 hours was used in most assays; however longer incubations allow slower interactions to achieve equilibrium). The plate was then thoroughly washed and the developed with 100 uL/well p-NitroPhenyl Phosphate (PNPP; Pierce Protein Research Products Rockford, Ill.). After allowing the plate to develop for 5 to 15 minutes the reaction was stopped, using 100 uL/well of 1M NaOH. The absorbance was read on a microplate reader at 405 nm.
FIG. 9A shows the results of the single point in triplicate screening assay of the anti-GCSF/GCSFR Fab PPEs. Enhancement or inhibition of biotinylated GSCF binding to GCSFR compared to control was observed in the presence of several of the Fab clones tested at a fixed concentration of biotinylated GCSF:Fab mixture indicating the identification of both positive and negative modulating antibodies by the screen. FIG. 9B shows the results from a titration of GCSF-biotin in the presence and absence of one of the positive modulating Fabs, B2(E4). The biotinylated GCSF binding curve was left shifted in the presence of antibody B2(E4). This suggests enhanced affinity of the ligand receptor interaction in the presence of the antibody. FIG. 9C shows the results from titration of a further positive modulating Fab, B4(F5) as well as a weakly or non-modulating Fab B6(A10), against a fixed concentration of biotinylated GCSF. The B4(F5) Fab enhances binding of biotinylated GCSF to the plate-bound receptor in an antibody dose dependent manner.
Example 3
Identification of Kinetic Modulators of GCSF-GCSFR Binding Using a Cell Binding Measurement Method
This example describes the use of FACS based assays to measure differential test compound (e.g. antibody) binding to GCSFR-transfected cells in the presence or absence of ligand (recombinant human GCSF (rhGCSF), R&D Systems, Minneapolis, Minn.). Anti-GCSF/GCSFR antibodies from phage display libraries (see example 2) were screened in ELISA assays to identify antibodies specific for binding to GCSF-GCSFR complexes. Because these antibodies are complex-specific, the mathematical model predicts that they will, modulate the kinetics of GCSF binding to GCSFR.
BaF3 is a murine lymphocytic cell line that does not respond to human GCSF but does respond to other cytokines in the family. This cell line is maintained in RPMI (Gibco/Invitrogen)/10% FBS (Hyclone/Thermo Scientific, Waltham, Mass.)/+2 ng/mL murine IL-3 (R&D Systems). The human GCSFR gene (Origene, Rockville, Md.) was stably transfected into the BaF3 cell line (Alexion AAC 621) using electroporation followed by G418 (Invitrogen, Carlsbad, Calif.) selection. Expression was confirmed by FACS analysis using a phycoerythrin-conjugated anti-GCSFRα (CD114) antibody [554538 (LMM741) BD Biosciences, San Jose, Calif.]. Stimulation of the transfected cells with human GCSF resulted in a proliferative response indicating a functional ligand/receptor interaction.
BaF3/GCSFR cells (1×106 cells/sample) were washed 2× with PBS/2% FBS/0.1% azide, then incubated for 15 min on ice with rhGCSF diluted into the same buffer. GCSF/GCSFR test antibodies at 5 ug/mL in the presence or absence of rhGCSF (0, 1, 10 or 100 ng/mL) were added and incubated for 1 hour on ice, followed by addition of 10 ug/mL of phycoerythrin-labeled goat anti-human IgG (H+L) (Jackson Labs, Bar Harbor, Me.) and another 1 hour incubation on ice. If the test antibodies bound to the GCSF/GCSFR complex, the secondary antibody then bound to the test antibody and stained the cells. Several test antibodies were observed to bind to BaF3/GCSFR cells in the presence, but not in the absence of GCSF. These antibodies were shown not to bind to GCSFR or to GCSF, and therefore bound only to the GCSF/GCSFR signaling complex, suggesting that they may positively modulate signaling of the GCSF/GCSFR complex. Example data for antibody A10(B6) (see example 2) is shown in FIG. 10. Treatment of cells with increasing concentrations of rhGCSF and a fixed concentration of A10(B6) led to a dose dependent increase in mean fluorescence index (MFI) readout in the assay as compared to cells stained with rhGCSF and an irrelevant antibody (KLH8-G2).
Example 4
Identification of Kinetic Modulators of INS-INSR Binding Using a Cell-Based Antibody Affinity Measurement Method
This example describes the use of flow cytometric (FACS) based assays to measure differential antibody binding to cells in the presence or absence of human insulin (hINS). Anti-insulin receptor (INSR) antibodies from phage display libraries were screened in the assays to identify modulators of INS-INSR binding.
IM-9 cells were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640+10% FBS. Prior to use in assays cells were washed in serum-free RPMI 1640, counted and the concentration adjusted to 2×106 cells/ml in RPMI 1640+0.5% BSA (Sigma-Aldrich). The cells were cultured overnight in this media and as such were designated as “serum-starved.” These cells were washed once and resuspended at 2×106 cells/ml in PBS containing 0.5% BSA and 0.01% sodium azide (FACS buffer).
Cells exposed to insulin were resuspended in FACS buffer supplemented with 70 nM human insulin (Sigma-Aldrich, St. Louis, Mo.). Both cell populations (+hINS) or (−hINS) were incubated at 4° C. for 30 minutes, washed once with FACS buffer and resuspended at 2×106 cells/ml in FACS buffer. Twenty five microliter aliquots of cells were plated into 96 well plates, mixed with 25 ul of antibody or PPE and incubated on ice for 1 h.
The cells were then washed once with FACS buffer and the binding of the antibody was detected by the addition of 25 ul of an appropriate fluorochrome-conjugated secondary antibody. If the initial incubation had been with PPE containing a myc-tagged antibody, 25 ul of a 1/1000 dilution of an anti-c-myc antibody (Roche) was added to the wells and the cells incubated on ice for 30 mins. The cells were then washed once with FACS buffer and the binding of the anti-c-myc revealed by the addition of a phycoerythrin-conjugated anti-mouse IgG. After a final 15 min incubation on ice the cells were washed and the pellets resuspended in FACS buffer. The cells were analyzed on a FACScanTM (Becton-Dickinson, Milipitas, Calif.) and the data analysed in both FlowJoTM (Treestar, Ashland, Oreg.) and Microsoft ExcelTM.
This assay allowed the detection of four types of antibody, examples of which are shown in FIG. 11:

    1. Antibodies that only bind to IM-9 cells if they have been exposed to human insulin (bind exclusively to INS/INSR complex)
    2. Antibodies that bind more strongly to IM-9 cells if they have been exposed to human insulin (bind preferentially to INS/INSR complex)
    3. Antibodies that bind less strongly to IM-9 cells if they have been exposed to human insulin (bind preferentially to uncomplexed INSR)
    4. Antibodies that bind to IM-9 cells independent of the exposure of the IM9 cells to human insulin (bind equally to uncomplexed INSR and INS/INSR complex)


Antibodies were scored as predicted positive modulators if the ratio of antibody binding to INS/INSR complex: antibody binding to uncomplexed INSR was greater than 1.3. Antibodies were scored as predicted negative modulators if the ratio of antibody binding to INS/INSR complex: antibody binding to uncomplexed INSR was less than 0.6. Antibodies were scored as predicted non-modulators if the ratio of antibody binding to INS/INSR complex: antibody binding to uncomplexed INSR was greater than 0.9 but less than 1.1.
Experiments were performed as described above except that suspension adapted CHO-K1 transfected with either hINSR or muINSR were used instead of IM-9 cells, and cells exposed to insulin were resuspended in FACS buffer supplemented with 150 nM rather than 70 nM human insulin.
FACS binding data for a number of anti-INSR antibodies having positive or negative modulating activity in functional assays was reviewed. Negative modulators were found to have a binding ratio +insulin/−insulin of approximately 0.7 or less, with the majority having a ratio of 0.5 or less. Positive modulators were found to have a binding ratio +insulin/−insulin of approximately 1.0 or more, with the majority having a ratio of 1.3 or more. The binding ratios are shown in Table 3 below. Thus the ratio of differential receptor binding activity by a modulating antibody in the presence of absence of ligand is generally predictive of its modulating function (positive or negative).









TABLE 3







FACS area under log




transformed data MFI ratio of



Functional
clones binding to human INSR



Activity (by pAKT
CHO-K1 with insulin and


Antibody
and/or pIRS-1 assay)
without insulin (+insulin/−insulin)

















Ab050
Negative modulator
0.32


Ab052
Negative modulator
0.30


Ab055
Negative modulator
0.64


Ab057
Negative modulator
0.56


Ab061
Negative modulator
0.29


Ab063
Weak Negative
0.71



modulator


Ab065
Negative modulator
0.28


Ab070
Negative modulator
0.53


Ab072
Negative modulator
0.37


Ab074
Negative modulator
0.44


Ab081
Negative modulator
0.42


Ab059
Positive modulator
34.25


Ab076
Positive modulator
0.98


Ab077
Positive modulator
2.12


Ab078
Positive modulator
66


Ab079
Positive modulator
1.30


Ab080
Positive modulator
1.37


Ab083
Positive modulator
3.03









Example 5
Identification of Kinetic Modulators Using a Cell-Based Ligand Affinity Measurement Method
This example describes the use of FACS based assays to measure differential ligand (human insulin) binding to cells in the presence or absence of test compounds (antibodies against the INSR). INSR antibodies from phage display libraries were screened in the assays to identify modulators of the INS-INSR complex.
IM 9 cells were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640+10% FBS. Prior to use in assays cells were washed in serum-free RPMI 1640, counted and the concentration adjusted to 2×106 cells/ml in RPMI 1640+0.5% BSA (Sigma-Aldrich). The cells were cultured overnight in this media and as such were designated as “serum-starved.” These cells were washed once and resuspended at 2×106 cells/ml in PBS containing 0.5% BSA (binding buffer).
Serum-starved cells were pre-exposed to INSR antibodies at room temperature for 15 minutes and then incubated with various concentrations of biotinylated human insulin purchased from R&D Systems for a further 30 minutes at room temperature. The binding of the biotinylated insulin was revealed by the addition of a 1/100 dilution of streptavidin-phycoerythrin to this mixture for a further 15 minutes at room temperature. The cells were then washed once with binding buffer and resuspended in equal volumes of PBS containing 0.5% BSA, 0.1% sodium azide and 2% paraformaldehyde. The cells were analyzed on a FACScanTM (Becton-Dickinson, Milipitas, Calif.) and the data analyzed in both FlowJoTM (Treestar, Ashland, Oreg.) and Microsoft ExcelTM.
FIG. 12 shows the binding of biotinylated insulin to IM9 cells in the presence or absence of anti-INSR antibodies at different insulin concentrations. Antibody 83-7 enhanced binding of biotinylated insulin; antibody MA-20 diminished binding of biotinylated insulin; control mouse IgG had no effect on binding of biotinylated insulin.
Example 6
Confirmation of Kinetic Modulation Using a Phosphorylation Assay
The substrate proteins which are phosphorylated by the INSR include a protein called insulin receptor substrate 1 (IRS-1). IRS-1 phosphorylation to form pIRS-1 eventually leads to an increase in the high affinity glucose transporter (Glut4) molecules on the outer membrane of insulin-responsive tissues, and therefore to an increase in the uptake of glucose from blood into these tissues. A pIRS-1 assay was developed using the Luminex® technology platform (Luminex Corp., Austin, Tex.). Two modes of assay were developed: (a) titration of test antibody at a fixed concentration of insulin, and (b) titration of insulin at a fixed concentration of antibody. Anti-insulin receptor (INSR) antibodies selected on the basis of their differential binding to complexed and uncomplexed INSR (see examples 4 and 5) were tested in the assays to identify modulators of the INS-INSR complex.
Cell Treatment and Lysis
IM-9 cells were serum starved for 16-20 hours by counting, centrifuging, washing once with PBS and re-suspending at about 2×106 cells/ml in RPMI+0.5% Sigma Cohn V BSA (10% stock in RPMI, filter sterilized, stored 4° C.).
2× concentrated solutions of insulin (Sigma I-9278 (10 mg/ml) 1.77 mM liquid stock stored at 4′ C) dilutions were prepared in RPMI+0.5% BSA. A standard insulin titration may include 4-fold serial dilutions of for example: 6.25 nM, 1.56 nM, 0.39 nM, 0.097 nM, 0.024 nM, 0.006 nM, 0.0015 nM, 0 nM.
Milliplex MAP Cell Signalling Buffer and Detection Kit (Millipore catalog #48-602) and Phospho-IRS-1 MAP Mates (Millipore catalog #46-627) were employed for the detection of pIRS-1 levels, according to the manufacturer's instructions. Briefly, V-bottomed plates containing 50 ul/well of 2× treatment media (RPMI containing 0.5% BSA+/−test antibody) were prepared and 1×106 cells serum-starved IM-9 cells resuspended in 50 ul RPMI+0.5% BSA were added per well. Antibody pretreatment was performed for 15 minutes prior to insulin treatment, either (a) as a bulk antibody/cell mixture at a single antibody concentration that was then applied to wells containing serial dilutions of insulin, or (b) by adding cells directly to wells containing serial dilutions of antibody and spiking in insulin at 0.1 nM. Plates were placed in a 37° C. incubator and centrifuged at 1500 rpm at RT for the last 3 minutes of treatment time (total of 15 minutes). Supernatant was removed by inversion and gentle blotting and treated cell pellets were lysed by triturating 3 times using a multi channel pipette with 100 ul Lysis Buffer prepared according to Table 1 below (labile components, i.e. protease inhibitors and benzonase, were added just prior to use). Plates were placed on a shaker at RT for 30 minutes and centrifuged at 3000 rpm for 10 minutes to clarify the lysate and remove any air bubbles that may have occurred during trituration. 50 ul of cleared lysate was removed and diluted 1:1 in 50 uL Assay Buffer-1 (AB-1) from the Detection Kit, triturated 2-3 times to mix and 50 ul was loaded onto a filter plate membrane on top of the 25 ul/well of diluted beads (see below).







TABLE 1







Lysis buffer components























100



10 wells
20 wells
25 wells 
30 wells 
40 wells
50 wells
60 wells
wells


Lysis Buffer
1 ml
2 mls
2.5 mls
3 mls
4 mls
5 mls
6 mls
10 mls


















Lysis Buffer (Millipore cat.
1
2
2.5
3
4
5
6
10


#43-040)










SDS 20% stock
0.045
0.09
0.1125
0.135
0.18
0.225
0.27
0.45


MgCl 50 mM (Invitrogen
0.02
0.04
0.05
0.06
0.08
0.1
0.12
0.2


cat. #Y02016)










Protease inhibitors (50X)
0.02
0.04
0.05
0.06
0.08
0.1
0.12
0.2


(Millipore cat. #20-201)










Benzonase EMD
0.004
0.008
0.01
0.012
0.016
0.02
0.024
0.04


1.01697.0002 @ 250 ug/m1









Filter plate membranes (Millipore Catalog# MABVN1250) were pre-wet with 25 ul AB-1/well. Pre-wetting buffer was aspirated from the filter plate using a Millipore vacuum manifold, being careful not to dry the membranes, and any remaining liquid was blotted from the bottom of the filter plate. 25 ul of 1× bead suspension was added per well (pIRS-1 beads (Millipore catalog #46-627) were pre-prepared by diluting from 20× concentrate into AB-1 buffer and alternately vortexing and sonicating for 5 seconds 3 times each).
Filter plate wells were covered with a plate sealer, covered in aluminum foil to prevent light exposure, and incubated on a plate shaker (setting 7-8 on a Labline, Bella) plate shaker or similar model) at either RT for 2 hours or alternatively at 4° C. overnight.
Luminex Detection
The filter plates were aspirated and their bottoms blotted. The beads remained in the well and were washed with 100 μl of AB-1 and placed on shaker for 1-2 minutes. Plates were aspirated, and the wash step was repeated.
25 ul per well 1× biotinylated detection antibody, diluted from a 20× stock into AB-1 buffer, was added and plates were incubated on a shaker at RT for 1 hour. Plates were aspirated and their bottoms blotted. 25 ul per well 1× streptavidin phycoerythrin diluted from a 25× stock into AB-1 buffer, was added and plates were incubated on a shaker at RT for 15 minutes. 25 ul of Amplification Buffer (Millipore catalog #48-602) was added to each well, and plates were incubated on a shaker at RT for further 15 minutes. The plates were aspirated and the beads were resuspended in 150 uL AB-1 and read on the Luminex® instrument.
Results
FIG. 13 shows pIRS-1 assay results from titrations of insulin in the presence of fixed concentrations of representative test antibodies. MFIs were normalized such that the curve fit maximum was adjusted to 100%. Some antibodies (positive modulators) shifted the insulin titration curve to the left. Other antibodies (negative modulators) shifted the insulin titration curve to the right. Varying magnitudes of modulation were observed. The data in FIG. 13 shows antibodies producing up to a 9-fold increase, or up to a 24-fold decrease, in insulin sensitivity.
FIG. 14 is a table showing insulin EC50 values from the pIRS-1 assay in the presence or absence of fixed concentrations of various test antibodies. The results are ranked according to EC50 ratio +Ab/−Ab.
Example 7
Identification of Kinetic Modulators of TNF-α/TNFR Binding
A desired property of TNFα modulators would be to attenuate signal transduction of pathologic levels of TNFα while allowing sufficient signaling to support the innate immune response. Identification of such TNFα modulators is accomplished through the selection of polypeptide binding agents (e.g. antibodies) that reduce the affinity of TNFα for one or both of its receptor(s). The reduced affinity of TNFα for its receptor(s) is reflected in a number of standard analytical measurements such as faster off-rate, slower on-rate, lower association constant and higher dissociation constant. Alternatively, this can be detected by preferential binding (e.g. higher binding signal) of the polypeptide binding agents to TNFα alone compared to the TNFα-TNFR complex. Absence of binding signal for the TNFα-TNFR complex is indicative of a complete blocker and is not selected.
This example describes the use of assays to measure differential ligand (human TNFα) binding to TNFR1 or TNFR2 in the presence or absence of test compounds (TNFα-binding polypeptides). Because TNFR1 and TNFR2 are believed to play different roles in biological pathways it may be desirable to selectively modulate the activity of these two receptors (e.g. TNFα induces inflammatory responses primarily through interaction with TNFR1, while TNFR2 expression provides protection in an inflammatory colitis model; upregulation of TNFR1 provides neuroprotection in an ischemic preconditioning model). Selective modulation of the binding of TNFα to the two receptors could provide benefits in different diseases (Schneider et al Eur. J. Immunol. 39 (7):1743-53 (2009); Mukai et al, J. Biochem. 146 (2):167-72 (2009); Pradillo et al J. Cereb. Flow Metab. 25 (2):193-203 (2005)). TNFα-binding polypeptides are obtained de novo from sources such as naive phage display libraries and hybridomas, or are generated as variants of known of TNFα-binding polypeptides, such as infliximab, adalimumab, certolizumab pegol, HUMICADETM, golimumab, atacicept, and etanercept.
Antibody modulators of the TNFα-TNFR binding interaction can be identified using the assay described below.
Purified TNF (cat #210-TA-010, R&D Systems Minneapolis, Minn.) is biotinylated through activated NHS chemistry using PEG4 Biotin (Pierce Protein Research Products cat#21329, Rockford, Ill.). TNFR1 or TNFR2 (cat #636-R1-025 or 1089-R2-025, R&D Systems Minneapolis, Minn.) is coated on an EIA plate (Nunc, Rochester, N.Y.) in PBS at 2 ug/mL, 100 uL/well at 37° C. for 1 hour on a shaker. The plate is then blocked with a bovine serum albumin (BSA) and ChemiBlockTM (Millipore Billerica, Mass.) blocking solution for at least 1⁄2 hour at room temperature on a shaker. Any blocking solution that does not interfere with the reactants and sufficiently blocks nonspecific binding on the EIA plate can be used for the assay.
Samples are prepared on a dilution plate. TNFα-binding scFv or Fab can be screened as periplasmic extracts (PPE) or hybridoma supernatants. For a single point assay, PPE samples are mixed 1:1 with biotinylated TNF prepared at 0.15 ug/mL in blocking buffer and 50 uL of this solution is loaded onto the EIA plate. Potential hits from the single point assay are purified and further tested using titrations of ligand, or tested at various titrations at a fixed ligand concentration. 50 uL/well of this antibody-TNF-biotin solution is added to the blocked EIA plate and incubated at room temperature for >1 hour on a shaker. Negative controls of PBS and blocking buffer alone are included in the single point assay to establish a background signal level. The biotinylated analyte and antibody mixture remains in the well and is not removed or washed out. The plate is then incubated at room temperature on a shaker for an additional >1 hour (total reaction time of 3-4 hours is used in most assays; however longer incubations allow slower interactions to achieve equilibrium). The plate is then thoroughly washed and the developed with 100 uL/well p-NitroPhenyl Phosphate (PNPP; Pierce Protein Research Products Rockford, Ill.). After allowing the plate to develop for 5 to 15 minutes the reaction is stopped, using 100 μL/well of 1M NaOH. The absorbance is read on a microplate reader at 405 nm.
Functional assays are performed to confirm that the reduction in the affinities of signal complex components will cause as shift in the cellular dose-response to TNFα. A variety of cell-based assays may be used for this purpose, including for example, assays described in U.S. Pat. Nos. 7,524,502 and 7,179,893 and U.S. application 2009/0155205.
Neutralization of TNFα-Induced Cytotoxicity in L929 Cells
TNFα-sensitive L929 mouse fibroblasts cells are seeded into 96-well tissue culture plates at a density of 5×104 cells in RPMI medium containing 10% fetal bovine serum (FBS). An antibody potency assay is performed by adding to the L929 cells RPMI+FBS media containing recombinant TNFα (500 pg/mL), pre-incubated for 1 hour at 37° C. with either anti-TNFα antibody or anti-KLH (Keyhole limpet hemocyanin) isotype control antibody at desired test concentrations. An TNFα dose-response assay is performed using increasing amounts of TNFα pre-incubated overnight at room temperature with 100-fold molar excess of antibody prior to addition to the L929 cells. The plates are then incubated overnight (18-24 hours) at 37° C. in 5% CO2.
To determine the effect on TNFα-induced cell cytotoxicity, 100 uL of medium is removed from each well and 50 uL of 5 mg/mL 3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, Mo.) in PBS is added. The plates are then incubated for 4 hours at 37° C. Fifty uL of 20% sodium dodecyl sulfate (SDS) is then added to each well and the plates are incubated overnight at 37° C. The optical density at 570/630 nm is measured, curves are plotted for each sample and IC50 are determined by standard methods. All samples are set up and assayed in duplicate or triplicate.
Inhibition of ELAM-1 and/or ICAM-1 Expression on HUVEC
TNFα induces surface expression of endothelial cell adhesion molecules such as ELAM-1 and ICAM-1. The ability of anti-TNFα antibodies to neutralize TNFα stimulated production in human umbilical vein endothelial cells (HUVEC) of membrane bound ICAM-1 and or ELAM-1, is tested in an in vitro assay
Briefly, HUVEC (ATCC No. CRL 1730) are grown in a 96-well plate in the presence of TNFα and varying concentrations of test or control antibody. The quantitative relative expression of membrane bound ICAM-1 and/or ELAM-1 is subsequently assessed by cell lysis and an enzyme linked immunoabsorbance assay (ELISA) using a commercially available detection reagents. HUVEC are seeded in 96-well plates at a density of 5×104 cells per well and allowed to adhere to the plate by incubation at 37° C., 5% CO2 for at least 2 hours.
An antibody potency assay is performed by adding to the HUVEC media containing recombinant TNFα, pre-incubated for 1 hour at 37° C. with either anti-TNFα antibody or anti-KLH (Keyhole limpet hemocyanin) isotype control antibody at desired test concentrations. A TNFα dose-response assay is performed using increasing amounts of TNFα pre-incubated overnight at room temperature with 100-fold molar excess of antibody prior to addition to the HUVEC. The plates are then incubated for 24 hours at 37° C. in 5% CO2. Following incubation, the medium is removed and the cells washed with PBS. The cells are lysed and the lysate assayed for the presence of ICAM-1 and/or ELAM-1. For assay, cleared lysate is analyzed for the presence of solubilized ICAM-1 or ELAM-1 by standard ELISA using commercially available reagents (e.g., sICAM-1 Module set; Bender Medsystems, Towcester, UK). All samples are set up and assayed in duplicate or triplicate.
Inhibition TNFα Induced Up-Regulation of IL-6 in Hs 27 Cells
Human foreskin fibroblast cells can be induced to produce IL-6 by exposure to TNFα. The ability of anti-TNFα antibodies to inhibit this up-regulation of expression is assessed by co-incubation of the cells with recombinant TNFα and the test antibodies, followed by a determination of the subsequent IL-6 levels secreted into the medium using a commercially available IL-6 detection system.
TNFα-sensitive human foreskin fibroblast cells Hs 27 (e.g., from the European Collection of Animal Cell Cultures (ECACC no. 94041901)), are seeded into 96-well tissue culture plates at a density of 2×I04 cells in DMEM+Glutamax containing 10% fetal calf serum medium and allowed to adhere to the plate by overnight incubation at 37° C. An antibody potency assay is performed by adding to the Hs 27 cells media containing recombinant TNFα, pre-incubated for 1 hour at 37° C. with either anti-TNFα antibody or anti-KLH (Keyhole limpet hemocyanin) isotype control antibody at desired test concentrations. A TNFα dose-response assay is performed using increasing amounts of TNFα pre-incubated overnight at room temperature with 100-fold molar excess of antibody prior to addition to the Hs 27 cells. The plates are then incubated overnight (18-24 hours) at 37° C. in 5% CO2. Following incubation, the medium is removed from and transferred to a U-bottomed 96 well plate for assay. The medium is analyzed for the presence of IL-6 using a commercial ELISA system (e.g., R&D Systems, as described above). All samples are set up and assayed in duplicate or triplicate.
Example 8
Effects of Partial Agonist Anti-INSR Antibodies on Glycemic Control in DIO Mice
In the diet-induced obesity (DIO) model, C57BL/6 mice can become insulin resistant after approximately 12-14 weeks on a high-fat diet (HFD). Anti-INSR antibodies demonstrated to behave as partial agonists or positive modulators in vitro were evaluated in this model to determine if these antibodies improved insulin sensitivity and/or glycemic control in vivo.
To determine whether partial agonist anti-INSR antibodies reduce fasted blood glucose, 20 week-old DIO mice (14 weeks on HFD; n=8/group) were fasted for 5 hours and challenged intravenously with partial agonist antibodies Ab030 and Ab037, or an isotype control (5 mg/kg). In additional control studies, DIO mice were treated with insulin (0.5 U/kg), or age-matched mice fed a normal diet (ND) were dosed with isotype control (5 mg/kg). Blood glucose was sampled prior to injection (time=0) and 1, 2 and 4 hours post-administration. Compared to age-matched controls, increased blood glucose was observed in DIO mice (HFD-fed/isotype control) at the 1-hour time point, consistent with insulin resistance in animals fed HFD (FIG. 15A). Administration of insulin or either of the partial agonist antibodies resulted in a statistically significant reduction (p<0.05; one-tailed t-test) in blood glucose (FIG. 15B). Neither antibody induced hypoglycemia at any time point (defined as blood glucose <36 mg/dL). These results suggest that anti-INSR partial agonist antibodies safely and effectively reduce fasting blood glucose.
To further evaluate the effect of a partial agonist anti-INSR antibody on glycemic control, 18-week old DIO mice (12 weeks on HFD; n=8/group) were injected intraperitoneally (IP) with Ab037 (0.1, 1.0 or 9 mg/kg) or isotype control (1.0 mg/kg). As additional controls, age-matched control mice were dosed with isotype control (1.0 mg/kg) or DIO animals were given insulin (0.75 U/kg; IP). A glucose tolerance test (GTT) was performed 24 hours after antibody administration (30 min after insulin) by fasting the animals for 16 hours (beginning approximately 8 hours after antibody administration), injecting glucose (1.0 U/kg) and following blood glucose over 2 hours. In this experiment, HFD did not have a significant impact on fasting glucose (FIG. 16B) or post-bolus peak glucose (FIG. 16A). Nevertheless, in DIO mice, partial agonist antibody significantly reduced fasting blood glucose relative to isotype control when dosed at or above 1.0 mg/kg (FIG. 16B) and reduced GTT area under the curve (AUC) at 9.0 mg/kg (FIG. 16C).
This outcome demonstrates that an anti-INSR partial agonist antibody can reduce fasting glucose and improve glycemic control in vivo.
Example 9
Effects of Positive Modulator Anti-INSR Antibodies on Glycemic Control in DIO Mice
To determine if a positive modulator anti-INSR antibody improves insulin sensitivity in vivo, 18-week old DIO mice (n=8/group) were given IP injections of Ab001 (positive modulator) (0.1, 1.0 or 10 mg/kg), partial agonist antibody (Ab037) (10 mg/kg) or isotype control (1.0 mg/kg). Age-matched mice fed ND dosed with isotype control (1.0 mg/kg) served as an additional control (FIG. 17A). Twenty-four hours later, an insulin tolerance test (ITT) was carried out by administering insulin (0.5 U/kg) after a 5 hour fast and monitoring blood glucose levels over 2 hours. A HFD did not have a significant impact on fasting glucose (FIG. 17B) or ITT AUC (FIG. 17C) relative to regular diet, and neither partial agonist antibody (Ab037) nor positive modulator antibody (Ab001) administration resulted in a statistically significant lower AUC ITT, relative to isotype control treated DIO animals (FIG. 17C). Partial agonist antibody Ab037 significantly reduced fasting glucose, while positive modulator antibody Ab001 induced a non-statistically significant, dose-dependent trend towards reduced fasting glucose.
The following week, a GTT was carried out on the same animals after an additional dose of antibody (FIG. 18A). In this study, HFD resulted in a non-statistical increase in fasting glucose (FIG. 18B) and GTT AUC (FIG. 18C) compared to control fed animals. Compared to isotype control-treated DIO mice, partial agonist antibody and positive modulator antibody significantly reduced fasting glucose at all doses tested. In addition, both partial agonist antibody and positive modulator antibody significantly reduced GTT AUC at 10 mg/kg relative to isotype control.
These results suggest that partial agonist and positive modulator antibodies specific for the INSR improve glycemic control in diabetic subjects.
Example 10
Panning for Allosteric Agonist Antibodies Against a Receptor
Selection of agonist antibodies that exhibit greater binding to the complex of receptor/ligand than to the free receptor enhances the probability of identifying antibodies that are noncompetitive with the ligand and do not block or diminish binding of the ligand to the orthosteric site of the receptor. An antibody of this type, that binds to a site on the target receptor distinct from the endogenous binding site, is known as an allosteric agonist (Kenakin et al., J Receptors and Signal Transduction, 27:247-259, 2007; Jahns et al., J Am Coll Cardiol. 36:1280-87, 2000; May et al., Ann Rev Toxicol. 47: 1-51, 2007).
Methods described above to screen for agonist antibodies are also useful to screen for allosteric agonists. Preferential binding of the test antibody to the receptor ligand complex is consistent with allosteric activity whereas preferential binding of the test antibody to the free receptor is consistent with an antibody that competes with insulin for the orthosteric site. The screen is useful to enrich the pool of candidate clones for allosteric agonists by eliminating the some if not all competitive agonists.
Allosteric antibodies are less likely to interfere with the binding affinity and efficacy of the ligand and therefore, are less likely to interfere with the maximum ligand signaling or maximum sensitivity to ligand. Allosteric antibodies can exhibit a range of agonism from weak partial agonists to agonism levels similar to the endogenous ligand. A partial allosteric agonist will elicit a maximum signaling response that is of significantly lower in magnitude than the maximum response of the endogenous ligand. In some applications, where sustained sub maximal signal activation is preferred over maximum signal activation, a partial agonist antibody is preferable to a full agonist antibody. The distinguishing characteristics between a partial allosteric agonist and a positive allosteric modulator are evident from a comparison of the dose response curves shown in FIGS. 19 and 20, which show the different binding curves for a partial allosteric agonist (FIG. 19) and a positive allosteric modulator (sensitizer) antibody (FIG. 20), as exemplified using an antibody specific for INSR.
FIG. 19A illustrates an example of the dose response from a partial allosteric agonist in comparison to the dose response to the endogenous ligand (insulin) and FIG. 19B demonstrates activation by ligand in the presence or absence of the allosteric agonist. FIG. 20A shows the dose response from a positive allosteric modulator antibody in comparison to the dose response to the endogenous ligand while FIG. 20B shows a dose response curve of an endogenous ligand (insulin) in the presence and absence of a positive allosteric modulator antibody that binds INSR. FIG. 21 provides the activation parameters for a set of partial allosteric agonists relative to the endogenous ligand. The nature of signal activation by the partial allosteric agonists is distinct from that of an allosteric modulator obtained from the same primary screening approach.
A non-competitive partial allosteric agonist antibody may offer a therapeutic advantage over a competitive agonist where it is beneficial to have independent signal activation by both the partial agonist and an endogenous ligand simultaneously. For example, and not to be bound by theory, a partial allosteric agonist can be used to elevate the basal activation of a signaling pathway while still allowing response from transient fluctuations in endogenous ligand levels. In certain instances, under conditions where a partial allosteric agonist of this sort is present, the endogenous ligand dose response will exhibit an increase in the baseline (constitutive or basal) signaling level and will achieve the same or greater maximal response to the endogenous ligand with little or no significant change in the ligand EC50. For example, FIG. 19B shows the dose response of an endogenous ligand in the presence and absence of a partial allosteric agonist and FIG. 22 shows the maximal activation of insulin in the presence partial allosteric agonist antibodies relative to the maximal response to the endogenous ligand in the presence of a negative control antibody. FIG. 22 demonstrates that the partial allosteric agonist antibodies Ab037 and Ab040 have little or no significant impact on the EC50 of the dose response and maximum phosphorylation of Akt at Ser473 by insulin when compared to a negative control antibody within the same assay.
Example 21
Assay to Measure Modulation of Insulin Binding Affinity for INSR by Anti-INSR Antibodies
To determine the ability of the modulating antibodies to affect the binding of insulin to the insulin receptor, the affinity of unmodified insulin binding to human INSR expressed on the surface of serum starved CHOK1 cells (hINSR8-CHOK1) was measured in the presence and absence of monoclonal antibodies to INSR. A KinExA assay was developed to measure very low levels of insulin in cell culture media. This assay allowed the binding of insulin to cells expressing INSR to be measured by determining the level of insulin depletion from the cell culture media. As insulin became bound to the cells, the concentration of insulin in the cell culture media dropped. By using a titration of cells expressing INSR and measuring the percent free insulin, the affinity of the INS-INSR interaction could be estimated using KinExA software. This assay was used to measure the degree of modulation of insulin binding activity shown by various anti-INSR antibodies.
hINSR8-CHOK1 cells were serum starved overnight and then prepared for assay by pelleting cells and resuspending at a concentration of 2× the final assay concentration for the highest dilutions (between 3.5×107 and 2.0×107 cells/mL in assay dilution buffer of PBS (Teknova, Hollister Calif.) with 500 μg/mL BSA and 0.1% sodium azide (Sigma Aldrich, St. Louis, Mo.)). A two-fold serial dilution of cells was prepared creating a ten-point dilutions series and a no-cells control was also used. Cell suspensions were aliquoted into polypropylene assay tubes in 2 mL volume each. To these cell suspensions 1 mL of 40 ug/mL test antibody (or 100 ug/mL for Ab078) was added to each tube, gently mixed and incubated for 30-45 minutes on ice. The antibodies used were tested in comparison to the negative control human IgG2 anti-KLH antibody. 1 mL of 200 pM insulin was added to each tube to establish a final insulin concentration of 50 pM (300 pg/mL) (Sigma-Aldrich, St. Louis, Mo.). Samples were incubated overnight at 4° C. for 18 hours then centrifuged to pellet cells and supernatants were removed for testing.
KinExA 3000 analysis was performed using beads coated with an anti-insulin monoclonal antibody. 2 grams of poly(methyl methacrylate) (PMMA) beads (Sapidyne, Boise, Id.) was suspended in 9 mL of assay buffer PBS containing 65 ug/mL of clone D6C4 mouse anti-insulin monoclonal antibody (Fitzgerald Industries, Acton Mass.). Beads were rotated at room temperature for 6 hours then allowed to settle. Supernatant was replaced with PBS with 50 mg/mL BSA Fraction V (Sigma-Aldrich, St. Louis, Mo.) and rotated overnight at 4° C. Detection solution used was biotinylated mouse anti-insulin clone D3E7 (Fitzgerald Industries, Acton Mass.) at 0.15 μg/mL in assay dilution buffer with Streptavidin-PE at 1 ug/mL (Invitrogen, Carlsbad, Calif.). On the KinExA 3000 the sample was injected at 0.25 mL/minute for 240 seconds, then rinsed for 60 seconds in running buffer (PBS with 0.05% sodium azide), then 240 seconds of the detection solution was injected, followed by a final 90 second wash at 1 mL/minute. The difference in voltage from an early initial time-point and a time point near the end of the run was measured and used to calculate affinities. The INSR concentration on the cells was estimated at 2.5×105 receptors/cell. Affinity was determined using the KinExA software (Sāpidyne, Boise Id.) and EC50's were calculated by non-linear fit in Prism (GraphPad Software, La Jolla Calif.).
A number of anti-INSR antibodies enhanced the affinity of insulin for the cells, as shown in Table 4 below. One of the tested antibodies decreased the affinity of insulin for the cells by approximately three-fold.







TABLE 4







Insulin Affinity and IC50 Table















Fold Shift in



Antibody
KD (pM)
EC50 (pM)
Affinity
















IgG2-KLH
272
365
1.0



Ab037
271
471
1.0



Ab001
49
104
+5.6



Ab053
228
33
+1.2



Ab062
762
760
−2.8



Ab078
41
80
+6.6



Ab079
12.1
40
+22.5



Ab080
11.2
34
+24.3



Ab083
13.7
39
+19.9



Ab085
34
70
+8.0










These data illustrate that the screening methods described herein produce positive modulator antibodies that strengthen the binding affinity of insulin for insulin receptor.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications, variations and combinations of embodiments may be made without deviating from the spirit and scope of the invention. Such modifications, variations and combinations are intended to be encompassed by the following claims.












Q8NFU4


Follicular dendritic cell secreted peptide (FDC secreted protein) (FDC-SP)


85


Q6E0U4


Dermokine (Epidermis-specific secreted protein SK30/SK89)


476


Q8N474


Secreted frizzled-related protein 1 (sFRP-1) (Frizzled-related protein 1) (FRP-1) (Secreted apoptosis-related protein 2) (SARP-2)


314


Q92838


Ectodysplasin-A (Ectodermal dysplasia protein) (EDA protein) [Cleaved into: Ectodysplasin-A, membrane form; Ectodysplasin-A, secreted form]


391


Q96HF1


Secreted frizzled-related protein 2 (sFRP-2) (Secreted apoptosis-related protein 1) (SARP-1) (Frizzled-related protein 2) (FRP-2)


295


Q9HD89


Resistin (Cysteine-rich secreted protein FIZZ3) (Adipose tissue-specific secretory factor) (ADSF) (C/EBP-epsilon-regulated myeloid-specific


secreted cysteine-rich protein) (Cysteine-rich secreted protein A12-alpha-like 2)


108


P10451


Osteopontin (Bone sialoprotein 1) (Secreted phosphoprotein 1) (SPP-1) (Urinary stone protein) (Nephropontin) (Uropontin)


314


Q5T4F7


Secreted frizzled-related protein 5 (sFRP-5) (Secreted apoptosis-related protein 3) (SARP-3) (Frizzled-related protein 1b) (FRP-1b)


317


Q6FHJ7


Secreted frizzled-related protein 4 (sFRP-4) (Frizzled protein, human endometrium) (FrpHE)


346


Q13103


Secreted phosphoprotein 24 (Spp-24) (Secreted phosphoprotein 2)


211


Q9Y625


Glypican-6 [Cleaved into: Secreted glypican-6]


555


Q92765


Secreted frizzled-related protein 3 (sFRP-3) (Frizzled-related protein 1) (FrzB-1) (Frezzled) (Fritz)


325


P13236


C-C motif chemokine 4 (Small-inducible cytokine A4) (Macrophage inflammatory protein 1-beta) (MIP-1-beta) (MIP-1-beta(1-69))


(T-cell activation protein 2) (ACT-2) (PAT 744) (Protein H400) (SIS-gamma) (Lymphocyte activation gene 1 protein) (LAG-1) (HC21)


(G-26 T-lymphocyte-secreted protein) [Cleaved into: MIP-1-beta(3-69)]


92


P40967


Melanocyte protein Pmel 17 (Silver locus protein homolog) (Melanocyte lineage-specific antigen GP100) (Melanoma-associated ME20 antigen)


(ME20-M) (ME20M) [Cleaved into: ME20-S (Secreted melanoma-associated ME20 antigen) (ME20S) (95 kDa melanocyte-specific secreted


glycoprotein)]


661


P55000


Secreted Ly-6/uPAR-related protein 1 (SLURP-1) (ARS component B) (ARS(component B)-81/S) (Anti-neoplastic urinary protein) (ANUP)


103


P08118


Beta-microseminoprotein (Prostate secreted seminal plasma protein) (Prostate secretory protein PSP94) (PSP-94) (Seminal plasma beta-inhibin)


(Immunoglobulin-binding factor) (IGBF) (PN44)


114


O75487


Glypican-4 (K-glypican) [Cleaved into: Secreted glypican-4]


556


O95150


Tumor necrosis factor ligand superfamily member 15 (Vascular endothelial cell growth inhibitor) (TNF ligand-related molecule 1)


[Cleaved into: Tumor necrosis factor ligand superfamily member 15, membrane form; Tumor necrosis factor ligand superfamily member 15,


secreted form]


251


Q9BQ08


Resistin-like beta (RELMbeta) (Cysteine-rich secreted protein FIZZ2) (Colon and small intestine-specific cysteine-rich protein)


(Cysteine-rich secreted protein A12-alpha-like 1) (Colon carcinoma-related gene protein)


111


O43508


Tumor necrosis factor ligand superfamily member 12 (TNF-related weak inducer of apoptosis) (TWEAK) (APO3 ligand)


[Cleaved into: Tumor necrosis factor ligand superfamily member 12, membrane form; Tumor necrosis factor ligand superfamily member 12,


secreted form]


249


P09486


SPARC (Secreted protein acidic and rich in cysteine) (Osteonectin) (ON) (Basement-membrane protein 40) (BM-40)


303


P78333


Glypican-5 [Cleaved into: Secreted glypican-5]


572


O95994


Anterior gradient protein 2 homolog (hAG-2) (AG-2) (Secreted cement gland protein XAG-2 homolog) (HPC8)


175


Q9Y2B0


Protein canopy homolog 2 (MIR-interacting saposin-like protein) (Transmembrane protein 4) (Putative secreted protein ZSIG9)


182


P35052


Glypican-1 [Cleaved into: Secreted glypican-1]


558


Q5GFL6


von Willebrand factor A domain-containing protein 2 (A domain-containing protein similar to matrilin and collagen) (AMACO)


(Colon cancer secreted protein 2) (CCSP-2)


755


O95388


WNT1-inducible-signaling pathway protein 1 (WISP-1) (Wnt-1-induced secreted protein)


367


P22362


C-C motif chemokine 1 (Small-inducible cytokine A1) (T lymphocyte-secreted protein I-309)


96


Q9H3U7


SPARC-related modular calcium-binding protein 2 (Secreted modular calcium-binding protein 2) (SMOC-2)


(Smooth muscle-associated protein 2) (SMAP-2)


446


Q9Y240


C-type lectin domain family 11 member A (Stem cell growth factor) (Lymphocyte secreted C-type lectin) (p47)


(C-type lectin superfamily member 3)


323


Q86SR0


Secreted Ly-6/uPAR-related protein 2 (SLURP-2)


97


P51654


Glypican-3 (Intestinal protein OCI-5) (GTR2-2) (MXR7) [Cleaved into: Secreted glypican-3]


580


Q8WVN6


Secreted and transmembrane protein 1 (Protein K12)


248


Q9Y6I9


Testis-expressed sequence 264 protein (Putative secreted protein ZSIG11)


313


Q8N158


Glypican-2 [Cleaved into: Secreted glypican-2]


579


O95084


Serine protease 23 (EC 3.4.21.-) (Putative secreted protein ZSIG13)


383


Q7Z7F7


39S ribosomal protein L55, mitochondrial (L55mt) (MRP-L55)


128


Q9UFN0


Protein NipSnap homolog 3A (NipSnap3A) (NipSnap4) (Target for Salmonella secreted protein C) (TassC)


247


P02751


Fibronectin (FN) (Cold-insoluble globulin) (CIG) [Cleaved into: Ugl-Y1; Ugl-Y2; Ugl-Y3]


2,386


Q9UMX5


Neudesin (Neuron-derived neurotrophic factor) (Secreted protein of unknown function) (SPUF protein) (Cell immortalization-related protein 2)


172


P21802


Fibroblast growth factor receptor 2 (FGFR-2) (EC 2.7.10.1) (Keratinocyte growth factor receptor 2) (CD antigen CD332)


821


P23280


Carbonic anhydrase 6 (EC 4.2.1.1) (Carbonic anhydrase VI) (CA-VI) (Carbonate dehydratase VI) (Secreted carbonic anhydrase)


(Salivary carbonic anhydrase)


308


Q9UGM3


Deleted in malignant brain tumors 1 protein (Glycoprotein 340) (Gp-340) (Surfactant pulmonary-associated D-binding protein) (Hensin)


(Salivary agglutinin) (SAG)


2,413


Q9H4F8


SPARC-related modular calcium-binding protein 1 (Secreted modular calcium-binding protein 1) (SMOC-1)


434


P05067


Amyloid beta A4 protein (Alzheimer disease amyloid protein) (ABPP) (APPI) (APP) (PreA4) (Cerebral vascular amyloid peptide) (CVAP)


(Protease nexin-II) (PN-II) [Cleaved into: N-APP; Soluble APP-alpha (S-APP-alpha); Soluble APP-beta (S-APP-beta); C99; Beta-amyloid protein 42


(Beta-APP42); Beta-amyloid protein 40 (Beta-APP40); C83; P3(42); P3(40); Gamma-secretase C-terminal fragment 59 (Gamma-CTF(59))


(Amyloid intracellular domain 59) (AICD-59) (AID(59)); Gamma-secretase C-terminal fragment 57 (Gamma-CTF(57)) (Amyloid intracellular 


domain 57) (AICD-57) (AID(57)); Gamma-secretase C-terminal fragment 50 (Gamma-CTF(50)) (Amyloid intracellular domain 50) (AICD-50)


(AID(50)); C31]


770


P25445


Tumor necrosis factor receptor superfamily member 6 (FASLG receptor) (Apoptosis-mediating surface antigen FAS) (Apo-1 antigen) (CD


antigen CD95)


335


Q9UBS5


Gamma-aminobutyric acid type B receptor subunit 1 (GABA-B receptor 1) (GABA-B-R1) (Gb1)


961


Q02297


Pro-neuregulin-1, membrane-bound isoform (Pro-NRG1) [Cleaved into: Neuregulin-1 (Neu differentiation factor) (Heregulin) (HRG)


(Breast cancer cell differentiation factor p45) (Acetylcholine receptor-inducing activity) (ARIA) (Sensory and motor neuron-derived factor)


(Glial growth factor)]


640


Q96T91


Glycoprotein hormone alpha-2 (Thyrostimulin subunit alpha) (Putative secreted protein ZSIG51)


129


Q495T6


Membrane metallo-endopeptidase-like 1 (EC 3.4.24.11) (Membrane metallo-endopeptidase-like 2) (Neprilysin-2) (Neprilysin II) (NL2) (NEPII) 


(NEP2(m)) [Cleaved into: Membrane metallo-endopeptidase-like 1, soluble form (Neprilysin-2 secreted) (NEP2(s))]


779


Q7L513


Fc receptor-like A (Fc receptor-like and mucin-like protein 1) (Fc receptor homolog expressed in B-cells) (Fc receptor-related protein X) (FcRX)


(Fc receptor-like protein)


359


Q8NHW4


C-C motif chemokine 4-like (Small-inducible cytokine A4-like) (Lymphocyte activation gene 1 protein) (LAG-1) (Macrophage inflammatory protein 


1-beta) (MIP-1-beta) (Monocyte adherence-induced protein 5-alpha)


92


Q08345


Epithelial discoidin domain-containing receptor 1 (Epithelial discoidin domain receptor 1) (EC 2.7.10.1) (Tyrosine kinase DDR) (Discoidin receptor


tyrosine kinase )(Tyrosine-protein kinase CAK) (Cell adhesion kinase) (TRK E) (Protein-tyrosine kinase RTK 6) (HGK2) (CD167 antigen-like 


family member A) (CD antigen CD167a)


913


P15941


Mucin-1 (MUC-1) (Polymorphic epithelial mucin) (PEM) (PEMT) (Episialin) (Tumor-associated mucin) (Carcinoma-associated mucin)


(Tumor-associated epithelial membrane antigen) (EMA) (H23AG) (Peanut-reactive urinary mucin) (PUM) (Breast carcinoma-associated antigen DF3)


(CD antigen CD227) [Cleaved into: Mucin-1 subunit alpha (MUC1-alpha) (MUC1-NT); Mucin-1 subunit beta (MUC1-beta) (MUC1-CT)]


1,255


P15692


Vascular endothelial growth factor A (VEGF-A) (Vascular permeability factor) (VPF)


232


P23142


Fibulin-1 (FIBL-1)


703


P16471


Prolactin receptor (PRL-R)


622


P29122


Proprotein convertase subtilisin/kexin type 6 (EC 3.4.21.-) (Paired basic amino acid cleaving enzyme 4) (Subtilisin/kexin-like protease PACE4)


(Subtilisin-like proprotein convertase 4) (SPC4)


969


Q9NNX6


CD209 antigen (Dendritic cell-specific ICAM-3-grabbing non-integrin 1) (DC-SIGN1) (DC-SIGN) (C-type lectin domain family 4 member L) 


(CD antigen CD209)


404


P13942


Collagen alpha-2(XI) chain


1,736


P15509


Granulocyte-macrophage colony-stimulating factor receptor subunit alpha (GM-CSF-R-alpha) (GMR) (CDw116) (CD antigen CD116)


400


P15502


Elastin (Tropoelastin)


786


Q13261


Interleukin-15 receptor subunit alpha (IL-15R-alpha) (IL-15RA) [Cleaved into: Soluble interleukin-15 receptor subunit alpha (sIL-15R-alpha)


(sIL-15RA)]


267


P21741


Midkine (MK) (Neurite outgrowth-promoting protein) (Midgestation and kidney protein) (Amphiregulin-associated protein) (ARAP) (Neurite


outgrowth-promoting factor 2)


143


Q13683


Integrin alpha-7 [Cleaved into: Integrin alpha-7 heavy chain; Integrin alpha-7 light chain]


1,181


Q99102


Mucin-4 (MUC-4) (Pancreatic adenocarcinoma mucin) (Testis mucin) (Ascites sialoglycoprotein) (ASGP) (Tracheobronchial mucin) [Cleaved into: 


Mucin-4 alpha chain (Ascites sialoglycoprotein 1) (ASGP-1); Mucin-4 beta chain (Ascites sialoglycoprotein 2) (ASGP-2)]


2,169


P19021


Peptidyl-glycine alpha-amidating monooxygenase (PAM) [Includes: Peptidylglycine alpha-hydroxylating monooxygenase (PHM) (EC 1.14.17.3);


Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (EC 4.3.2.5) (Peptidylamidoglycolate lyase) (PAL)]


973


P02647


Apolipoprotein A-I (Apo-AI) (ApoA-I) [Cleaved into: Apolipoprotein A-I(1-242)]


267


Q92954


Proteoglycan 4 (Lubricin) (Megakaryocyte-stimulating factor) (Superficial zone proteoglycan) [Cleaved into: Proteoglycan 4 C-terminal part]


1,404


Q93038


Tumor necrosis factor receptor superfamily member 25 (WSL-1 protein) (Apoptosis-mediating receptor DR3) (Apoptosis-mediating receptor TRAMP)


(Death domain receptor 3) (WSL protein) (Apoptosis-inducing receptor AIR) (Apo-3) (Lymphocyte-associated receptor of death) (LARD)


417


O75882


Attractin (Mahogany homolog) (DPPT-L)


1,429


Q1L6U9


Prostate-associated microseminoprotein (PC3-secreted microprotein)


139


P04745


Alpha-amylase 1 (EC 3.2.1.1) (1,4-alpha-D-glucan glucanohydrolase 1) (Salivary alpha-amylase)


511


P23560


Brain-derived neurotrophic factor (BDNF) (Abrineurin)


247


Q9H2X3


C-type lectin domain family 4 member M (CD209 antigen-like protein 1) (Dendritic cell-specific ICAM-3-grabbing non-integrin 2) (DC-SIGN2)


(DC-SIGN-related protein) (DC-SIGNR) (Liver/lymph node-specific ICAM-3-grabbing non-integrin) (L-SIGN) (CD antigen CD299)


399


Q99062


Granulocyte colony-stimulating factor receptor (G-CSF-R) (CD antigen CD114)


836


P01344


Insulin-like growth factor II (IGF-II) (Somatomedin-A) [Cleaved into: Insulin-like growth factor II Ala-25 Del; Preptin]


180


P13688


Carcinoembryonic antigen-related cell adhesion molecule 1 (Biliary glycoprotein 1) (BGP-1) (CD antigen CD66a)


526


Q9BXN2


C-type lectin domain family 7 member A (Dendritic cell-associated C-type lectin 1) (DC-associated C-type lectin 1) (Dectin-1) (Beta-glucan receptor)


(C-type lectin superfamily member 12)


247


Q8TDQ1


CMRF35-like molecule 1 (CLM-1) (CD300 antigen-like family member F) (Immune receptor expressed on myeloid cells 1) (IREM-1)


(Immunoglobulin superfamily member 13) (IgSF13) (NK inhibitory receptor) (CD antigen CD300f)


290


Q53GD3


Choline transporter-like protein 4 (Solute carrier family 44 member 4)


710


Q8IWL2


Pulmonary surfactant-associated protein A1 (SP-A1) (SP-A) (PSP-A) (PSPA) (Alveolar proteinosis protein) (35 kDa pulmonary surfactant-associated


protein)


248


Q9NWM0


Spermine oxidase (EC 1.5.3.n1) (EC 1.5.3.n2) (Polyamine oxidase 1) (PAO-1) (PAOh1)


555


Q11203


CMP-N-acetylneuraminate-beta-1,4-galactoside alpha-2,3-sialyltransferase (EC 2.4.99.6) (N-acetyllactosaminide alpha-2,3-sialyltransferase)


(Gal beta-1,3(4) GlcNAc alpha-2,3 sialyltransferase) (ST3GalIII) (ST3N) (Sialyltransferase 6)


375


O60259


Kallikrein-8 (hK8) (EC 3.4.21.118) (Neuropsin) (NP) (Ovasin) (Serine protease TADG-14) (Tumor-associated differentially expressed gene 14 protein)


(Serine protease 19)


260


P00750


Tissue-type plasminogen activator (t-plasminogen activator) (t-PA) (tPA) (EC 3.4.21.68) (Alteplase) (Reteplase) [Cleaved into: Tissue-type plasminogen


activator chain A; Tissue-type plasminogen activator chain B]


562


Q6QHF9


Peroxisomal N(1)-acetyl-spermine/spermidine oxidase (EC 1.5.3.n3) (EC 1.5.3.n4) (EC 1.5.3.n10) (Polyamine oxidase)


649


Q9NPG8


Probable palmitoyltransferase ZDHHC4 (EC 2.3.1.-) (Zinc finger DHHC domain-containing protein 4) (DHHC-4) (Zinc finger protein 374)


344


P11597


Cholesteryl ester transfer protein (Lipid transfer protein I)


493


P01892


HLA class I histocompatibility antigen, A-2 alpha chain (MHC class I antigen A*2)


365


P02458


Collagen alpha-1(II) chain (Alpha-1 type II collagen) [Cleaved into: Chondrocalcin]


1,487


Q14005


Pro-interleukin-16 [Cleaved into: Interleukin-16 (IL-16) (Lymphocyte chemoattractant factor) (LCF)]


1,332


P48357


Leptin receptor (LEP-R) (OB receptor) (OB-R) (HuB219) (CD antigen CD295)


1,165


P07585


Decorin (Bone proteoglycan II) (PG-S2) (PG40)


359


P48061


Stromal cell-derived factor 1 (SDF-1) (hSDF-1) (C-X-C motif chemokine 12) (Pre-B cell growth-stimulating factor) (PBSF) (hIRH) [Cleaved into:


SDF-1-beta(3-72); SDF-1-alpha(3-67)]


93


P24821


Tenascin (TN) (Tenascin-C) (TN-C) (Hexabrachion) (Cytotactin) (Neuronectin) (GMEM) (JI) (Myotendinous antigen) (Glioma-associated-extracellular


matrix antigen) (GP 150-225)


2,201


O43184


Disintegrin and metalloproteinase domain-containing protein 12 (ADAM 12) (EC 3.4.24.-) (Meltrin-alpha)


909


Q76LX8


A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS-13) (ADAM-TS 13) (ADAM-TS13) (EC 3.4.24.-)


(von Willebrand factor-cleaving protease) (vWF-cleaving protease) (vWF-CP)


1,427


P01732


T-cell surface glycoprotein CD8 alpha chain (T-lymphocyte differentiation antigen T8/Leu-2) (CD antigen CD8a)


235


Q96AW1


EGFR-coamplified and overexpressed protein (ECop) (Glioblastoma-amplified secreted protein) (Putative NF-kappa-B-activating protein 055N)


172


Q676U5


Autophagy-related protein 16-1 (APG16-like 1)


607


O75815


Breast cancer anti-estrogen resistance protein 3 (SH2 domain-containing protein 3B) (Novel SH2-containing protein 2)


825


Q9H251


Cadherin-23 (Otocadherin)


3,354


P09603


Macrophage colony-stimulating factor 1 (CSF-1) (MCSF) (M-CSF) (Lanimostim) [Cleaved into: Processed macrophage colony-stimulating factor 1]


554


P15328


Folate receptor alpha (FR-alpha) (Folate receptor 1) (Folate receptor, adult) (Adult folate-binding protein) (FBP) (Ovarian tumor-associated antigen 


MOv18) (KB cells FBP)


257


Q14114


Low-density lipoprotein receptor-related protein 8 (Apolipoprotein E receptor 2)


963


Q6UWE0


E3 ubiquitin-protein ligase LRSAM1 (EC 6.3.2.-) (Leucine-rich repeat and sterile alpha motif-containing protein 1) (Tsg101-associated ligase) (hTAL)


723


P13591


Neural cell adhesion molecule 1 (NCAM-1) (N-CAM-1) (CD antigen CD56)


858


Q8N0W4


Neuroligin-4, X-linked (Neuroligin X) (HNLX)


816


Q9Y2I2


Netrin-G1 (Laminet-1)


539


Q969N2


GPI transamidase component PIG-T (Phosphatidylinositol-glycan biosynthesis class T protein)


578


P21583


Kit ligand (C-kit ligand) (Stem cell factor) (SCF) (Mast cell growth factor) (MGF)


273


Q9BYH1


Seizure 6-like protein


1,024


Q9NQ25


SLAM family member 7 (CD2-like receptor-activating cytotoxic cells) (CRACC) (Protein 19A) (CD2 subset 1) (Novel Ly9) (Membrane protein 


FOAP-12) (CD antigen CD319)


335


P01375


Tumor necrosis factor (TNF-alpha) (Tumor necrosis factor ligand superfamily member 2) (TNF-a) (Cachectin) [Cleaved into: Tumor necrosis factor,


membrane form; Tumor necrosis factor, soluble form]


233


P07911


Uromodulin (Tamm-Horsfall urinary glycoprotein) (THP)


640


O75888


Tumor necrosis factor ligand superfamily member 13 (A proliferation-inducing ligand) (APRIL) (TNF- and APOL-related leukocyte expressed ligand 2)


(TALL-2) (TNF-related death ligand 1) (TRDL-1) (CD antigen CD256)


250


O75629


Protein CREG1 (Cellular repressor of E1A-stimulated genes 1)


220


Q99944


EGF-like domain-containing protein 8 (Epidermal growth factor-like protein 8) (Multiple EGF-like domain protein 8) (Vascular endothelial-statin 2)


(VE-statin-2)


293


Q12904


Aminoacyl tRNA synthetase complex-interacting multifunctional protein 1 (Multisynthetase complex auxiliary component p43) [Cleaved into


Endothelial monocyte-activating polypeptide 2 (EMAP-II) (Small inducible cytokine subfamily E member 1)]


312


Q6UY14


ADAMTS-like protein 4 (ADAMTSL-4) (Thrombospondin repeat-containing protein 1)


1,074


P03951


Coagulation factor XI (FXI) (EC 3.4.21.27) (Plasma thromboplastin antecedent) (PTA) [Cleaved into: Coagulation factor XIa heavy chain;


Coagulation factor XIa light chain]


625


Q969J5


Interleukin-22 receptor subunit alpha-2 (IL-22R-alpha-2) (Interleukin-22-binding protein) (IL22BP) (Cytokine receptor family class II member 10)


(CRF2-10) (Cytokine receptor family type 2, soluble 1) (CRF2-S1)


263


O75398


Deformed epidermal autoregulatory factor 1 homolog (Nuclear DEAF-1-related transcriptional regulator) (NUDR) (Suppressin) (Zinc finger MYND


domain-containing protein 5)


565


P41222


Prostaglandin-H2 D-isomerase (EC 5.3.99.2) (Lipocalin-type prostaglandin-D synthase) (Glutathione-independent PGD synthetase) (Prostaglandin-D2


synthase) (PGD2 synthase) (PGDS2) (PGDS) (Beta-trace protein) (Cerebrin-28)


190


P01009


Alpha-1-antitrypsin (Alpha-1 protease inhibitor) (Alpha-1-antiproteinase) [Cleaved into: Short peptide from AAT (SPAAT)]


418


P01011


Alpha-1-antichymotrypsin (ACT) (Cell growth-inhibiting gene 24/25 protein) [Cleaved into: Alpha-1-antichymotrypsin His-Pro-less]


423


P07108


Acyl-CoA-binding protein (ACBP) (Diazepam-binding inhibitor) (DBI) (Endozepine) (EP)


87


P00751


Complement factor B (EC 3.4.21.47) (C3/C5 convertase) (Properdin factor B) (Glycine-rich beta glycoprotein) (GBG) (PBF2) [Cleaved into:


Complement factor B Ba fragment; Complement factor B Bb fragment]


764


P01233


Choriogonadotropin subunit beta (CG-beta) (Chorionic gonadotrophin chain beta)


165


P13611


Versican core protein (Large fibroblast proteoglycan) (Chondroitin sulfate proteoglycan core protein 2) (PG-M) (Glial hyaluronate-binding protein)


(GHAP)


3,396


P00533


Epidermal growth factor receptor (EC 2.7.10.1) (Receptor tyrosine-protein kinase ErbB-1)


1,210


Q16206


Ecto-NOX disulfide-thiol exchanger 2 (Tumor-associated hydroquinone oxidase) (tNOX) (Cytosolic ovarian carcinoma antigen 1) (APK1 antigen)


[Includes: Hydroquinone [NADH]oxidase (EC 1.-.-.-); Protein disulfide-thiol oxidoreductase (EC 1.-.-.-)]


610


Q12794


Hyaluronidase-1 (Hyal-1) (EC 3.2.1.35) (Hyaluronoglucosaminidase-1) (LUCA-1)


435


P18510


Interleukin-1 receptor antagonist protein (IL-1ra) (IL-1RN) (IRAP) (IL1 inhibitor) (ICIL-1RA) (Anakinra)


177


P40189


Interleukin-6 receptor subunit beta (IL-6R-beta) (Interleukin-6 signal transducer) (Membrane glycoprotein 130) (gp130) (CDw130) (Oncostatin-M 


receptor subunit alpha) (CD antigen CD130)


918


Q01638


Interleukin-1 receptor-like 1 (Protein ST2)


556


P01308


Insulin [Cleaved into: Insulin B chain; Insulin A chain]


110


P09466


Glycodelin (GD) (Pregnancy-associated endometrial alpha-2 globulin) (PAEG) (PEG) (Placental protein 14) (PP14) (Progesterone-associated


endometrial protein) (Progestagen-associated endometrial protein)


180


P12272


Parathyroid hormone-related protein (PTH-rP) (PTHrP) [Cleaved into: PTHrP[1-36]; PTHrP[38-94]; Osteostatin (PTHrP[107-139])]


177


Q8IXM6


Nurim (Nuclear rim protein) (Nuclear envelope membrane protein)


262


O15460


Prolyl 4-hydroxylase subunit alpha-2 (EC 1.14.11.2) (4-PH alpha-2) (Procollagen-proline,2-oxoglutarate-4-dioxygenase subunit alpha-2)


535


Q5ZPR3


CD276 antigen (Costimulatory molecule) (B7 homolog 3) (B7-H3) (4Ig-B7-H3) (CD antigen CD276)


534


Q96HD1


Cysteine-rich with EGF-like domain protein 1


420


Q96PZ7


CUB and sushi domain-containing protein 1 (CUB and sushi multiple domains protein 1)


3,565


Q15485


Ficolin-2 (Ficolin-B) (Ficolin-beta) (L-ficolin) (Collagen/fibrinogen domain-containing protein 2) (Serum lectin p35) (37 kDa elastin-binding protein)


(EBP-37) (Hucolin)


313


Q96RD9


Fc receptor-like protein 5 (FcR-like protein 5) (FcRL5) (Fc receptor homolog 5) (FcRH5) (Immunoglobulin receptor translocation-associated protein 2)


(BXMAS1) (CD antigen CD307)


977


Q9Y302


Protein GPR89 (Putative MAPK-activating protein PM01) (Putative NF-kappa-B-activating protein 90)


455


Q9Y624


Junctional adhesion molecule A (JAM-A) (Junctional adhesion molecule 1) (JAM-1) (Platelet adhesion molecule 1) (PAM-1) (Platelet F11 receptor)


(CD antigen CD321)


299


Q8IWT6


Leucine-rich repeat-containing protein 8A


810


Q9UNW1


Multiple inositol polyphosphate phosphatase 1 (EC 3.1.3.62) (Inositol (1,3,4,5)-tetrakisphosphate 3-phosphatase) (Ins(1,3,4,5)P(4) 3-phosphatase)


487


O14786


Neuropilin-1 (Vascular endothelial cell growth factor 165 receptor) (CD antigen CD304)


923


Q9HCM2


Plexin-A4


1,894


O43157


Plexin-B1 (Semaphorin receptor SEP)


2,135


Q15063


Periostin (PN) (Osteoblast-specific factor 2) (OSF-2)


836


Q7Z5B4


Protein RIC-3


369


Q9H156


SLIT and NTRK-like protein 2


845


Q8NBK3


Sulfatase-modifying factor 1 (EC 1.8.99.-) (C-alpha-formylglycine-generating enzyme 1)


374


Q8NBJ7


Sulfatase-modifying factor 2 (C-alpha-formylglycine-generating enzyme 2)


301


Q8IU80


Transmembrane protease, serine 6 (EC 3.4.21.-) (Matriptase-2)


811


P01374


Lymphotoxin-alpha (LT-alpha) (TNF-beta) (Tumor necrosis factor ligand superfamily member 1)


205


O14763


Tumor necrosis factor receptor superfamily member 10B (Death receptor 5) (TNF-related apoptosis-inducing ligand receptor 2)


(TRAIL receptor 2) (TRAIL-R2) (CD antigen CD262)


440


Q03405


Urokinase plasminogen activator surface receptor (uPAR) (U-PAR) (Monocyte activation antigen Mo3) (CD antigen CD87)


335


Q7Z7D3


V-set domain-containing T-cell activation inhibitor 1 (Immune costimulatory protein B7-H4) (B7h.5) (T-cell costimulatory molecule B7x)


(Protein B7S1)


282


P01275


Glucagon [Cleaved into: Glicentin; Glicentin-related polypeptide (GRPP); Oxyntomodulin (OXY) (OXM); Glucagon; Glucagon-like peptide 1


(GLP-1); Glucagon-like peptide 1(7-37) (GLP-1(7-37)); Glucagon-like peptide 1(7-36) (GLP-1(7-36)); Glucagon-like peptide 2 (GLP-2)]


180


Q96PD5


N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) (Peptidoglycan recognition protein long) (PGRP-L) (Peptidoglycan recognition protein 2)


576


O00391


Sulfhydryl oxidase 1 (hQSOX) (EC 1.8.3.2) (Quiescin Q6)


747


Q9BTZ2


Dehydrogenase/reductase SDR family member 4 (EC 1.1.1.184) (Short-chain dehydrogenase/reductase family member 4) (NADPH-dependent carbonyl


reductase/NADP-retinol dehydrogenase) (PHCR) (CR) (Peroxisomal short-chain alcohol dehydrogenase) (PSCD) (NADPH-dependent retinol


dehydrogenase/reductase) (NRDR) (humNRDR) (SCAD-SRL)


260


O95998


Interleukin-18-binding protein (IL-18BP) (Tadekinig-alfa)


194


Q6UWL6


Kin of IRRE-like protein 2 (Kin of irregular chiasm-like protein 2) (Nephrin-like protein 3)


708


Q96S97


Myeloid-associated differentiation marker (SB135)


322


Q9NPP4


NLR family CARD domain-containing protein 4 (Caspase recruitment domain-containing protein 12) (Ice protease-activating factor) (Ipaf)


(CARD, LRR, and NACHT-containing protein) (Clan protein)


1,024


Q9NRZ7


1-acyl-sn-glycerol-3-phosphate acyltransferase gamma (EC 2.3.1.51) (1-acylglycerol-3-phosphate O-acyltransferase 3) (1-AGP acyltransferase 3)


(1-AGPAT 3) (Lysophosphatidic acid acyltransferase gamma) (LPAAT-gamma)


376


Q15109


Advanced glycosylation end product-specific receptor (Receptor for advanced glycosylation end products)


404


Q9H2X0


Chordin


955


O14511


Pro-neuregulin-2, membrane-bound isoform (Pro-NRG2) [Cleaved into: Neuregulin-2 (NRG-2) (Neural- and thymus-derived activator for ERBB


kinases) (NTAK) (Divergent of neuregulin-1) (DON-1)]


850


Q6PDA7


Sperm-associated antigen 11A (Protein EP2) (Sperm antigen HE2)


123


A8MZH6


Oocyte-secreted protein 1 homolog


123


P02768


Serum albumin


609


O43405


Cochlin (COCH-5B2)


550


P05155


Plasma protease C1 inhibitor (C1 Inh) (C1Inh) (C1 esterase inhibitor) (C1-inhibiting factor)


500


P16871


Interleukin-7 receptor subunit alpha (IL-7R-alpha) (CDw127) (CD antigen CD127)


459


Q86UX2


Inter-alpha-trypsin inhibitor heavy chain H5 (Inter-alpha-inhibitor heavy chain 5) (ITI heavy chain H5)


942


Q9GZP0


Platelet-derived growth factor D (PDGF-D) (Iris-expressed growth factor) (Spinal cord-derived growth factor B) (SCDGF-B) [Cleaved into:


Platelet-derived growth factor D, latent form (PDGFD latent form); Platelet-derived growth factor D, receptor-binding form (PDGFD 


receptor-binding form)]


370


P31151


Protein S100-A7 (S100 calcium-binding protein A7) (Psoriasin)


101


Q96LC7


Sialic acid-binding Ig-like lectin 10 (Siglec-10) (Siglec-like protein 2)


697


Q9GZM7


Tubulointerstitial nephritis antigen-like (Tubulointerstitial nephritis antigen-related protein) (TIN Ag-related protein) (TIN-Ag-RP)


(Glucocorticoid-inducible protein 5) (Oxidized LDL-responsive gene 2 protein) (OLRG-2)


467


Q9Y275


Tumor necrosis factor ligand superfamily member 13B (TNF- and APOL-related leukocyte expressed ligand 1) (TALL-1) (B lymphocyte stimulator)


(BLyS) (B cell-activating factor) (BAFF) (Dendritic cell-derived TNF-like molecule) (CD antigen CD257) [Cleaved into: Tumor necrosis factor


ligand superfamily member 13b, membrane form; Tumor necrosis factor ligand superfamily member 13b, soluble form]


285


Q9ULC5


Long-chain-fatty-acid--CoA ligase 5 (EC 6.2.1.3) (Long-chain acyl-CoA synthetase 5) (LACS 5)


683


O95500


Claudin-14


239


Q8TCA0


Leucine-rich repeat-containing protein 20


184


Q9NZH6


Interleukin-1 family member 7 (IL-1F7) (Interleukin-1 zeta) (IL-1 zeta) (FIL1 zeta) (Interleukin-1 homolog 4) (IL-1H4) (Interleukin-1-related protein)


(IL-1RP1) (IL-1X protein)


218


Q8NDX9


Lymphocyte antigen 6 complex locus protein G5b


201


P22303


Acetylcholinesterase (AChE) (EC 3.1.1.7)


614


Q99217


Amelogenin, X isoform


191


P03950


Angiogenin (EC 3.1.27.-) (Ribonuclease 5) (RNase 5)


147


P58335


Anthrax toxin receptor 2 (Capillary morphogenesis gene 2 protein) (CMG-2)


489


P07355


Annexin A2 (Annexin-2) (Annexin II) (Lipocortin II) (Calpactin I heavy chain) (Chromobindin-8) (p36) (Protein I) (Placental anticoagulant protein IV)


(PAP-IV)


339


P02656


Apolipoprotein C-III (Apo-CIII) (ApoC-III)


99


O14791


Apolipoprotein L1 (Apolipoprotein L-I) (ApoL-I) (Apolipoprotein L) (Apo-L) (ApoL)


398


P02745


Complement C1q subcomponent subunit A


245


P02747


Complement C1q subcomponent subunit C


245


P01258


Calcitonin [Cleaved into: Calcitonin; Katacalcin (Calcitonin carboxyl-terminal peptide) (CCP) (PDN-21)]


141


Q8WVQ1


Soluble calcium-activated nucleotidase 1 (SCAN-1) (EC 3.6.1.6) (Apyrase homolog) (Putative MAPK-activating protein PM09) (Putative


NF-kappa-B-activating protein 107)


401


Q16663


C-C motif chemokine 15 (Small-inducible cytokine A15) (Macrophage inflammatory protein 5) (MIP-5) (Chemokine CC-2) (HCC-2) (NCC-3)


(MIP-1 delta) (Leukotactin-1) (LKN-1) (Mrp-2b) [Cleaved into: CCL15(22-92); CCL15(25-92); CCL15(29-92)]


113


P48960


CD97 antigen (Leukocyte antigen CD97) (CD antigen CD97) [Cleaved into: CD97 antigen subunit alpha; CD97 antigen subunit beta]


835


Q8IWV2


Contactin-4 (Brain-derived immunoglobulin superfamily protein 2) (BIG-2)


1,026


P06681


Complement C2 (EC 3.4.21.43) (C3/C5 convertase) [Cleaved into: Complement C2b fragment; Complement C2a fragment]


752


Q14031


Collagen alpha-6(IV) chain


1,691


P12110


Collagen alpha-2(VI) chain


1,019


P12107


Collagen alpha-1(XI) chain


1,806


P82279


Crumbs homolog 1


1,406


P01034


Cystatin-C (Cystatin-3) (Neuroendocrine basic polypeptide) (Gamma-trace) (Post-gamma-globulin)


146


P59665


Neutrophil defensin 1 (HNP-1) (HP-1) (HP1) (Defensin, alpha 1) [Cleaved into: HP 1-56; Neutrophil defensin 2 (HNP-2) (HP-2) (HP2)]


94


P14138


Endothelin-3 (ET-3) (Preproendothelin-3) (PPET3)


238


P06734


Low affinity immunoglobulin epsilon Fc receptor (Lymphocyte IgE receptor) (Fc-epsilon-RII) (BLAST-2) (Immunoglobulin E-binding factor)


(CD antigen CD23) [Cleaved into: Low affinity immunoglobulin epsilon Fc receptor membrane-bound form; Low affinity immunoglobulin


epsilon Fc receptor soluble form]


321


P22607


Fibroblast growth factor receptor 3 (FGFR-3) (EC 2.7.10.1) (CD antigen CD333)


806


P22455


Fibroblast growth factor receptor 4 (FGFR-4) (EC 2.7.10.1) (CD antigen CD334)


802


Q14393


Growth arrest-specific protein 6 (GAS-6) (AXL receptor tyrosine kinase ligand)


721


P10912


Growth hormone receptor (GH receptor) (Somatotropin receptor) [Cleaved into: Growth hormone-binding protein (GH-binding protein) (GHBP)


(Serum-binding protein)]


638


Q9Y223


Bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc-2-epimerase/ManAc kinase) [Includes:


UDP-N-acetylglucosamine 2-epimerase (EC 5.1.3.14) (Uridine diphosphate-N-acetylglucosamine-2-epimerase) (UDP-GlcNAc-2-epimerase);


N-acetylmannosamine kinase (EC 2.7.1.60) (ManAc kinase)]


7


Q969P0


Immunoglobulin superfamily member 8 (CD81 partner 3) (Glu-Trp-Ile EWI motif-containing protein 2) (EWI-2) (Keratinocytes-associated


transmembrane protein 4) (KCT-4) (LIR-D1) (CD antigen CD316)


613


P24394


Interleukin-4 receptor alpha chain (IL-4R-alpha) (CD antigen CD124) [Cleaved into: Soluble interleukin-4 receptor alpha chain (sIL4Ralpha/prot)


(IL-4-binding protein) (IL4-BP)]


825


Q9P0G3


Kallikrein-14 (hK14) (EC 3.4.21.-) (Kallikrein-like protein 6) (KLK-L6)


267


Q92876


Kallikrein-6 (EC 3.4.21.-) (Protease M) (Neurosin) (Zyme) (SP59) (Serine protease 9) (Serine protease 18)


244


Q13751


Laminin subunit beta-3 (Laminin 5 beta 3) (Laminin B1k chain) (Kalinin B1 chain)


1,172


Q9UIQ6


Leucyl-cystinyl aminopeptidase (Cystinyl aminopeptidase) (EC 3.4.11.3) (Oxytocinase) (OTase) (Insulin-regulated membrane aminopeptidase)


(Insulin-responsive aminopeptidase) (IRAP) (Placental leucine aminopeptidase) (P-LAP) [Cleaved into: Leucyl-cystinyl aminopeptidase, pregnancy


serum form]


1,025


P48740


Mannan-binding lectin serine protease 1 (EC 3.4.21.-) (Mannose-binding lectin-associated serine protease-1) (MASP-1) (Mannose-binding protein-


associated serine protease) (Complement factor MASP-3) (Serine protease 5) (Complement-activating component of Ra-reactive factor) (Ra-reactive 


factor serine protease p100) (RaRF) [Cleaved into: Mannan-binding lectin serine protease 1 heavy chain; Mannan-binding lectin serine protease


1 light chain]


699


O00187


Mannan-binding lectin serine protease 2 (EC 3.4.21.104) (Mannose-binding protein-associated serine protease 2) (MASP-2) (MBL-associated serine


protease 2) [Cleaved into: Mannan-binding lectin serine protease 2 A chain; Mannan-binding lectin serine protease 2 B chain]


686


P80188


Neutrophil gelatinase-associated lipocalin (NGAL) (p25) (25 kDa alpha-2-microglobulin-related subunit of MMP-9) (Lipocalin-2) (Oncogene 24p3)


198


P01303


Neuropeptide Y [Cleaved into: Neuropeptide Y (Neuropeptide tyrosine) (NPY); C-flanking peptide of NPY (CPON)]


97


P16112


Aggrecan core protein (Cartilage-specific proteoglycan core protein) (CSPCP) (Chondroitin sulfate proteoglycan core protein 1) [Cleaved into:


Aggrecan core protein 2]


2,415


P07988


Pulmonary surfactant-associated protein B (SP-B) (Pulmonary surfactant-associated proteolipid SPL(Phe)) (18 kDa pulmonary-surfactant protein)


(6 kDa protein)


381


Q15223


Poliovirus receptor-related protein 1 (Herpes virus entry mediator C) (HveC) (Nectin-1) (Herpesvirus Ig-like receptor) (HIgR) (CD antigen CD111)


517


P00797


Renin (EC 3.4.23.15) (Angiotensinogenase)


406


P07998


Ribonuclease pancreatic (EC 3.1.27.5) (RNase 1) (RNase A) (RNase UpI-1) (RIB-1) (HP-RNase)


156


P04279


Semenogelin-1 (Semenogelin I) (SGI) [Cleaved into: Alpha-inhibin-92; Alpha-inhibin-31; Seminal basic protein]


462


Q13291


Signaling lymphocytic activation molecule (IPO-3) (CDw150) (CD antigen CD150)


335


P10646


Tissue factor pathway inhibitor (TFPI) (Lipoprotein-associated coagulation inhibitor) (LACI) (Extrinsic pathway inhibitor) (EPI)


304


O75445


Usherin (Usher syndrome type-2A protein) (Usher syndrome type IIa protein)


5,202


Q9GZV9


Fibroblast growth factor 23 (FGF-23) (Tumor-derived hypophosphatemia-inducing factor) (Phosphatonin) [Cleaved into: Fibroblast growth factor 23


N-terminal peptide; Fibroblast growth factor 23 C-terminal peptide]


251


Q9NPF7


Interleukin-23 subunit alpha (IL-23 subunit alpha) (Interleukin-23 subunit p19) (IL-23p19)


189


P61916


Epididymal secretory protein E1 (Niemann-Pick disease type C2 protein) (hE1)


151


Q8N6G6


ADAMTS-like protein 1 (ADAMTSL-1) (Punctin-1)


525


Q9UBR5


Chemokine-like factor (C32)


152


Q9UHF1


EGF-like domain-containing protein 7 (Multiple EGF-like domain protein 7) (Multiple epidermal growth factor-like domain protein 7) (Vascular


endothelial statin) (VE-statin) (NOTCH4-like protein) (ZNEU1)


273


Q2MV58


Tectonic-1


587


Q86YD3


Transmembrane protein 25


366


O75752


UDP-GalNAc:beta-1,3-N-acetylgalactosaminyltransferase 1 (EC 2.4.1.79) (Beta-3-GalNAc-T1) (Beta-1,3-galactosyltransferase 3) (Beta-1,3-GalTase 3)


(Beta3Gal-T3) (b3Gal-T3) (Beta-3-Gx-T3) (Galactosylgalactosylglucosylceramide beta-D-acetyl-galactosaminyltransferase)


(UDP-N-acetylgalactosamine:globotriaosylceramide beta-1,3-N-acetylgalactosaminyltransferase) (Globoside synthase)


331


P40933


Interleukin-15 (IL-15)


162


A6BM72


Multiple epidermal growth factor-like domains 11 (Multiple EGF-like-domains 11)


1,044


Q9HBB8


Mucin and cadherin-like protein (Mu-protocadherin)


845


P34096


Ribonuclease 4 (RNase 4) (EC 3.1.27.-)


147


Q8N5H7


SH2 domain-containing protein 3C (Novel SH2-containing protein 3)


860


Q11206


CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase (Beta-galactoside alpha-2,3-sialyltransferase) (EC 2.4.99.-)


(Alpha 2,3-sialyltransferase IV) (Alpha 2,3-ST) (Gal-NAc6S) (STZ) (SIAT4-C) (ST3Gal III) (SAT-3) (ST-4)


333


Q9P0T7


Transmembrane protein 9 (Dermal papilla-derived protein 4)


183


Q14508


WAP four-disulfide core domain protein 2 (Major epididymis-specific protein E4) (Epididymal secretory protein E4) (Putative protease inhibitor WAP5)


124


P33765


Adenosine A3 receptor


318


Q96BI3


Gamma-secretase subunit APH-1A (APH-1a) (Aph-1alpha) (Presenilin-stabilization factor)


265


P35613


Basigin (Leukocyte activation antigen M6) (Collagenase stimulatory factor) (Extracellular matrix metalloproteinase inducer) (EMMPRIN) (5F7)


(Tumor cell-derived collagenase stimulatory factor) (TCSF) (OK blood group antigen) (CD antigen CD147)


385


Q96CA5


Baculoviral IAP repeat-containing protein 7 (Kidney inhibitor of apoptosis protein) (KIAP) (Melanoma inhibitor of apoptosis protein) (ML-IAP) 


(Livin) (RING finger protein 50)


298


O43852


Calumenin (Crocalbin) (IEF SSP 9302)


315


P47710


Alpha-S1-casein [Cleaved into: Casoxin-D]


185


Q9UK58


Cyclin-L1 (Cyclin-L)


526


P08603


Complement factor H (H factor 1)


1,231


P01243


Chorionic somatomammotropin hormone (Choriomammotropin) (Lactogen)


217


P78310


Coxsackievirus and adenovirus receptor (Coxsackievirus B-adenovirus receptor) (HCVADR) (hCAR) (CVB3-binding protein)


365


Q13822


Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 (E-NPP 2) (EC 3.1.4.39) (Extracellular lysophospholipase D) (LysoPLD)


(Autotaxin)


863


Q96HE7


ERO1-like protein alpha (ERO1-L-alpha) (ERO1-L) (EC 1.8.4.-) (Oxidoreductin-1-L-alpha) (Endoplasmic oxidoreductin-1-like protein)


468


P00740


Coagulation factor IX (EC 3.4.21.22) (Christmas factor) (Plasma thromboplastin component) (PTC) [Cleaved into: Coagulation factor IXa light chain;


Coagulation factor IXa heavy chain]


461


O75015


Low affinity immunoglobulin gamma Fc region receptor III-B (IgG Fc receptor III-1) (Fc-gamma RIII-beta) (Fc-gamma RIIIb) (FcRIIIb)


(Fc-gamma RIII) (FcRIII) (FcR-10) (CD antigen CD16b)


233


O75636


Ficolin-3 (Collagen/fibrinogen domain-containing protein 3) (Collagen/fibrinogen domain-containing lectin 3 p35) (Hakata antigen)


299


Q96LA5


Fc receptor-like protein 2 (FcR-like protein 2) (FcRL2) (Fc receptor homolog 2) (FcRH2) (Immunoglobulin receptor translocation-associated protein 4)


(IFGP family protein 4) (SH2 domain-containing phosphatase anchor protein 1)


508


Q9NZU0


Leucine-rich repeat transmembrane protein FLRT3 (Fibronectin-like domain-containing leucine-rich transmembrane protein 3)


649


P06396


Gelsolin (Actin-depolymerizing factor) (ADF) (Brevin) (AGEL)


782


P22749


Granulysin (Protein NKG5) (Lymphokine LAG-2) (T-cell activation protein 519)


145


Q14956


Transmembrane glycoprotein NMB (Transmembrane glycoprotein HGFIN)


572


P28799


Granulins (Proepithelin) (PEPI) [Cleaved into: Acrogranin; Paragranulin; Granulin-1 (Granulin G); Granulin-2 (Granulin F); Granulin-3 (Granulin B);


Granulin-4 (Granulin A); Granulin-5 (Granulin C); Granulin-6 (Granulin D); Granulin-7 (Granulin E)]


593


Q9Y251


Heparanase (EC 3.2.-.-) (Heparanase-1) (Hpa1) (Endo-glucoronidase) [Cleaved into: Heparanase 8 kDa subunit; Heparanase 50 kDa subunit]


543


P01871


Ig mu chain C region


452


P01583


Interleukin-1 alpha (IL-1 alpha) (Hematopoietin-1)


271


Q8NI17


Interleukin-31 receptor A (IL-31RA) (Cytokine receptor-like 3) (Gp130-like monocyte receptor) (HGLM-R) (GLM-R) (Gp130-like receptor)


732


P57087


Junctional adhesion molecule B (JAM-B) (Junctional adhesion molecule 2) (Vascular endothelial junction-associated molecule) (VE-JAM)


(CD antigen CD322)


298


P31025


Lipocalin-1 (Von Ebner gland protein) (VEG protein) (Tear prealbumin) (TP) (Tear lipocalin) (Tlc)


176


Q9HBX8


Leucine-rich repeat-containing G-protein coupled receptor 6


967


Q14766


Latent-transforming growth factor beta-binding protein 1 (LTBP-1) (Transforming growth factor beta-1-binding protein 1) (TGF-beta1-BP-1)


1,721


O15232


Matrilin-3


486


O95297


Myelin protein zero-like protein 1 (Protein zero-related)


269


Q6ZNJ1


Neurobeachin-like protein 2


2,754


Q92542


Nicastrin


709


Q9BW91


ADP-ribose pyrophosphatase, mitochondrial (EC 3.6.1.13) (ADP-ribose diphosphatase) (Adenosine diphosphoribose pyrophosphatase) (ADPR-PPase)


(ADP-ribose phosphohydrolase) (Nucleoside diphosphate-linked moiety X motif 9) (Nudix motif 9)


350


Q96QU1


Protocadherin-15


1,955


P49763


Placenta growth factor (PlGF)


221


Q8WZA1


Protein O-linked-mannose beta-1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) (EC 2.4.1.-) (UDP-GlcNAc:alpha-D-mannoside


beta-1,2-N-acetylglucosaminyltransferase I.2) (GnT I.2)


660


Q8N490


Probable hydrolase PNKD (EC 3.-.-.-) (Paroxysmal nonkinesiogenic dyskinesia protein) (Myofibrillogenesis regulator 1) (MR-1) (Trans-activated by


hepatitis C virus core protein 2)


385


P21246


Pleiotrophin (PTN) (Heparin-binding growth-associated molecule) (HB-GAM) (Heparin-binding growth factor 8) (HBGF-8) (Osteoblast-specific factor 1)


(OSF-10) (Heparin-binding neurite outgrowth-promoting factor 1) (HBNF-1) (Heparin-binding brain mitogen) (HBBM)


168


P15151


Poliovirus receptor (Nectin-like protein 5) (Necl-5) (CD antigen CD155)


417


Q9BZR6


Reticulon-4 receptor (Nogo receptor) (NgR) (Nogo-66 receptor)


473


P02735


Serum amyloid A protein (SAA) [Cleaved into: Amyloid protein A (Amyloid fibril protein AA); Serum amyloid protein A(2-104); Serum amyloid


protein A(3-104); Serum amyloid protein A(2-103); Serum amyloid protein A(2-102); Serum amyloid protein A(4-101)]


122


P04278


Sex hormone-binding globulin (SHBG) (Sex steroid-binding protein) (SBP) (Testis-specific androgen-binding protein) (ABP)


(Testosterone-estrogen-binding globulin) (Testosterone-estradiol-binding globulin) (TeBG)


402


Q96DU3


SLAM family member 6 (NK-T-B-antigen) (NTB-A) (Activating NK receptor)


332


Q14BN4


Sarcolemmal membrane-associated protein (Sarcolemmal-associated protein)


828


Q4LDE5


Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 (Polydom) (Selectin-like osteoblast-derived protein)


(SEL-OB) (CCP module-containing protein 22) (Serologically defined breast cancer antigen NY-BR-38)


3,574


P05543


Thyroxine-binding globulin (T4-binding globulin) (Serpin A7)


415


Q9UM00


Transmembrane and coiled-coil domain-containing protein 1 (Xenogeneic cross-immune protein PCIA3) (Transmembrane and coiled-coil


domains protein 4)


188


P57727


Transmembrane protease, serine 3 (EC 3.4.21.-) (Tumor-associated differentially-expressed gene 12 protein) (Serine protease TADG-12)


454


O14798


Tumor necrosis factor receptor superfamily member 10C (Decoy receptor 1) (DcR1) (Decoy TRAIL receptor without death domain)


(TNF-related apoptosis-inducing ligand receptor 3) (TRAIL receptor 3) (TRAIL-R3) (Trail receptor without an intracellular domain)


(Lymphocyte inhibitor of TRAIL) (Antagonist decoy receptor for TRAIL/Apo-2L) (CD antigen CD263)


259


O00300


Tumor necrosis factor receptor superfamily member 11B (Osteoprotegerin) (Osteoclastogenesis inhibitory factor)


401


P02787


Serotransferrin (Transferrin) (Siderophilin) (Beta-1 metal-binding globulin)


698


P20231


Tryptase beta-2 (Tryptase-2) (EC 3.4.21.59) (Tryptase II)


275


O95292


Vesicle-associated membrane protein-associated protein B/C (VAMP-associated protein B/C) (VAMP-B/VAMP-C) (VAP-B/VAP-C)


243


Q969M3


Protein YIPF5 (YIP1 family member 5) (YPT-interacting protein 1 A) (Five-pass transmembrane protein localizing in the Golgi apparatus and


the endoplasmic reticulum 5) (Smooth muscle cell-associated protein 5) (SMAP-5)


257


B2RDL6


cDNA, FLJ96669, highly similar to Homo sapiens secreted protein, acidic, cysteine-rich (osteonectin)(SPARC), mRNA


303


A8KAM5


cDNA FLJ77519, highly similar to Homo sapiens secreted frizzled related protein mRNA


313


P30203


T-cell differentiation antigen CD6 (T12) (TP120) (CD antigen CD6)


668


Q63HQ2


Pikachurin (EGF-like, fibronectin type-III and laminin G-like domain-containing protein)


1,017


Q08830


Fibrinogen-like protein 1 (Hepatocyte-derived fibrinogen-related protein 1) (HFREP-1) (Hepassocin) (Liver fibrinogen-related protein 1)


(LFIRE-1) (HP-041)


312


P24001


Interleukin-32 (IL-32) (Natural killer cells protein 4) (Tumor necrosis factor alpha-inducing factor)


234


O95460


Matrilin-4


622


Q08648


Sperm-associated antigen 11B (Protein EP2) (Sperm antigen HE2)


103


P00748


Coagulation factor XII (EC 3.4.21.38) (Hageman factor) (HAF) [Cleaved into: Coagulation factor XIIa heavy chain; Beta-factor XIIa part 1;


Beta-factor XIIa part 2; Coagulation factor XIIa light chain]


615


P81172


Hepcidin (Liver-expressed antimicrobial peptide) (LEAP-1) (Putative liver tumor regressor) (PLTR) [Cleaved into: Hepcidin-25 (Hepc25);


Hepcidin-20 (Hepc20)]


84


Q9UEF7


Klotho (EC 3.2.1.31) [Cleaved into: Klotho peptide]


1,012


P10124


Serglycin (Secretory granule proteoglycan core protein) (Platelet proteoglycan core protein) (P.PG) (Hematopoetic proteoglycan core protein)


158


Q9UIK5


Tomoregulin-2 (Transmembrane protein with EGF-like and two follistatin-like domains) (TR-2) (Hyperplastic polyposis protein 1)


374


Q6WN34


Chordin-like protein 2 (Chordin-related protein 2) (Breast tumor novel factor 1) (BNF-1)


429


Q8WWZ1


Interleukin-1 family member 10 (IL-1F10) (Interleukin-1 receptor antagonist-like FIL1 theta) (Interleukin-1 theta) (IL-1 theta) (FIL1 theta)


(Interleukin-1 HY2) (IL-1HY2)


152


Q9UBX7


Kallikrein-11 (hK11) (EC 3.4.21.-) (Hippostasin) (Trypsin-like protease) (Serine protease 20) [Cleaved into: Kallikrein-11 inactive chain 1;


Kallikrein-11 inactive chain 2]


282


Q9NP55


Protein Plunc (Palate lung and nasal epithelium clone protein) (Lung-specific protein X) (Nasopharyngeal carcinoma-related protein)


(Tracheal epithelium-enriched protein) (Secretory protein in upper respiratory tracts) (Von Ebner protein Hl)


256


Q7Z5L7


Podocan


613


O95407


Tumor necrosis factor receptor superfamily member 6B (Decoy receptor for Fas ligand) (Decoy receptor 3) (DcR3) (M68)


300


Q96A57


UPF0414 transmembrane protein C20orf30


120


Q8WTT0


C-type lectin domain family 4 member C (C-type lectin superfamily member 7) (Blood dendritic cell antigen 2 protein) (BDCA-2) (Dendritic lectin)


(CD antigen CD303)


213


Q7Z3D6


UPF0317 protein C14orf159, mitochondrial


616


Q96CW9


Netrin-G2 (Laminet-2)


530


Q8NFT2


Metalloreductase STEAP2 (EC 1.16.1.-) (Six-transmembrane epithelial antigen of prostate 2) (SixTransMembrane protein of prostate 1)


(Prostate cancer-associated protein 1) (Protein upregulated in metastatic prostate cancer) (PUMPCn)


490


Q5VX71


Sushi domain-containing protein 4


490


Q5BJH7


Protein YIF1B (YIP1-interacting factor homolog B)


314


Q9NRN5


Olfactomedin-like protein 3 (HNOEL-iso) (hOLF44)


406


O95445


Apolipoprotein M (Apo-M) (ApoM) (Protein G3a)


188


P20851


C4b-binding protein beta chain


252


P10966


T-cell surface glycoprotein CD8 beta chain (CD antigen CD8b)


210


P16619


C-C motif chemokine 3-like 1 (Small-inducible cytokine A3-like 1) (Tonsillar lymphocyte LD78 beta protein) (LD78-beta(1-70))


(G0/G1 switch regulatory protein 19-2) (G0S19-2 protein) (PAT 464.2) [Cleaved into: LD78-beta(3-70); LD78-beta(5-70)]


93


P55075


Fibroblast growth factor 8 (FGF-8) (Heparin-binding growth factor 8) (HBGF-8) (Androgen-induced growth factor) (AIGF)


233


Q04900


Sialomucin core protein 24 (MUC-24) (Multi-glycosylated core protein 24) (MGC-24) (Endolyn) (CD antigen CD164)


197


Q9BQ51


Programmed cell death 1 ligand 2 (Programmed death ligand 2) (PD-L2) (PD-1-ligand 2) (PDCD1 ligand 2) (Butyrophilin B7-DC) (B7-DC)


(CD antigen CD273)


273


B2R7H0


Secreted and transmembrane 1 (cDNA, FLJ93441, Homo sapiens secreted and transmembrane 1 (SECTM1), mRNA)


248


Q9BXI9


Complement C1q tumor necrosis factor-related protein 6


259


Q9BY15


EGF-like module-containing mucin-like hormone receptor-like 3 (EGF-like module-containing mucin-like receptor EMR3) [Cleaved into:


EGF-like module-containing mucin-like hormone receptor-like 3 subunit alpha; EGF-like module-containingmucin-like hormone receptor-like 3


subunit beta]


652


Q96PB7


Noelin-3 (Olfactomedin-3) (Optimedin)


478


Q9NPH6


Odorant-binding protein 2b (OBPIIb)


170


O95399


Urotensin-2 (Urotensin-II) (U-II) (UII)


124


Q6UXI7


Vitrin


678


O95389


WNT1-inducible-signaling pathway protein 3 (WISP-3)


354


A8K7E3


cDNA FLJ75759, highly similar to Homo sapiens follistatin-like 3 (secreted glycoprotein) (FSTL3), mRNA (Follistatin-like 3 (Secreted glycoprotein),


isoform CRA_a)


263


Q9BYF1


Angiotensin-converting enzyme 2 (EC 3.4.17.-) (ACE-related carboxypeptidase) (Angiotensin-converting enzyme homolog) (ACEH)


(Metalloprotease MPROT15) [Cleaved into: Processed angiotensin-converting enzyme 2]


805


Q15848


Adiponectin (Adipocyte, C1q and collagen domain-containing protein) (30 kDa adipocyte complement-related protein) (Adipocyte complement-related


30 kDa protein) (ACRP30) (Adipose most abundant gene transcript 1 protein) (apM-1) (Gelatin-binding protein)


244


Q9BY76


Angiopoietin-related protein 4 (Angiopoietin-like 4) (Hepatic fibrinogen/angiopoietin-related protein) (HFARP)


406


Q6Q788


Apolipoprotein A-V (Apo-AV) (ApoA-V) (Apolipoprotein A5) (Regeneration-associated protein 3)


366


Q9BXN1


Asporin (Periodontal ligament-associated protein 1) (PLAP-1)


380


P17213


Bactericidal permeability-increasing protein (BPI) (CAP 57)


487


Q9H5V8


CUB domain-containing protein 1 (Transmembrane and associated with src kinases) (Membrane glycoprotein gp140) (Subtractive immunization


M plus HEp3-associated 135 kDa protein) (SIMA135) (CD antigen CD318)


836


O75339


Cartilage intermediate layer protein 1 (CILP-1) (Cartilage intermediate-layer protein) [Cleaved into: Cartilage intermediate layer protein 1 C1;


Cartilage intermediate layer protein 1 C2]


1,184


Q96KN2


Beta-Ala-His dipeptidase (EC 3.4.13.20) (Carnosine dipeptidase 1) (CNDP dipeptidase 1) (Serum carnosinase) (Glutamate carboxypeptidase-like 


protein 2)


507


P20908


Collagen alpha-1(V) chain


1,838


Q9BXS0


Collagen alpha-1(XXV) chain (CLAC-P) (Alzheimer disease amyloid-associated protein) (AMY) [Cleaved into: Collagen-like Alzheimer amyloid


plaque component (CLAC)]


654


Q8NBQ5


Estradiol 17-beta-dehydrogenase 11 (EC 1.1.1.62) (17-beta-hydroxysteroid dehydrogenase 11) (17-beta-HSD 11) (17betaHSD11) (17bHSD11)


(17-beta-HSD XI) (17betaHSDXI) (Dehydrogenase/reductase SDR family member 8) (Retinal short-chain dehydrogenase/reductase 2) (retSDR2)


(Cutaneous T-cell lymphoma-associated antigen HD-CL-03) (CTCL tumor antigen HD-CL-03)


300


Q8IXB1


DnaJ homolog subfamily C member 10 (ER-resident protein ERdj5) (Macrothioredoxin) (MTHr)


793


Q8IUX8


EGF-like domain-containing protein 6 (Multiple EGF-like domain protein 6) (Multiple epidermal growth factor-like domain protein 6)


(MAM and EGF domains-containing gene protein)


553


P00488


Coagulation factor XIII A chain (Coagulation factor XIIIa) (EC 2.3.2.13) (Protein-glutamine gamma-glutamyltransferase A chain)


(Transglutaminase A chain)


732


P06744


Glucose-6-phosphate isomerase (GPI) (EC 5.3.1.9) (Phosphoglucose isomerase) (PGI) (Phosphohexose isomerase) (PHI) (Autocrine motility factor)


(AMF) (Neuroleukin) (NLK) (Sperm antigen 36) (SA-36)


558


Q9UBU3


Appetite-regulating hormone (Growth hormone secretagogue) (Growth hormone-releasing peptide) (Motilin-related peptide) (M46 protein)


[Cleaved into: Ghrelin-27; Ghrelin-28 (Ghrelin); Obestatin]


117


P29460


Interleukin-12 subunit beta (IL-12B) (IL-12 subunit p40) (Cytotoxic lymphocyte maturation factor 40 kDa subunit) (CLMF p40)


(NK cell stimulatory factor chain 2) (NKSF2)


328


Q9GZX6


Interleukin-22 (IL-22) (IL-10-related T-cell-derived-inducible factor) (IL-TIF)


179


Q8WWA0


Intelectin-1 (ITLN-1) (Intestinal lactoferrin receptor) (Galactofuranose-binding lectin) (Endothelial lectin HL-1) (Omentin)


313


O95970


Leucine-rich glioma-inactivated protein 1 (Epitempin-1)


557


Q9Y6Y9


Lymphocyte antigen 96 (Protein MD-2) (ESOP-1)


160


P09237


Matrilysin (EC 3.4.24.23) (Pump-1 protease) (Uterine metalloproteinase) (Matrix metalloproteinase-7) (MMP-7) (Matrin)


267


Q8N307


Mucin-20 (MUC-20)


709


Q8NBP7


Proprotein convertase subtilisin/kexin type 9 (EC 3.4.21.-) (Proprotein convertase PC9) (Subtilisin/kexin-like protease PC9) (Neural apoptosis-regulated


convertase 1) (NARC-1)


692


O75594


Peptidoglycan recognition protein (PGRP-S)


196


P05161


Interferon-induced 17 kDa protein [Cleaved into: Ubiquitin cross-reactive protein (hUCRP) (Interferon-induced 15 kDa protein)]


165


P56705


Protein Wnt-4


351


Q9BQI0


Allograft inflammatory factor 1-like (Ionized calcium-binding adapter molecule 2)


150


Q9UH62


Armadillo repeat-containing X-linked protein 3 (Protein ALEX3) (ARM protein lost in epithelial cancers on chromosome X 3)


379


Q8TDX6


Chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CsGalNAcT-1) (EC 2.4.1.174) (Chondroitin beta-1,4-N-acetylgalactosaminyltransferase 1)


(Beta4GalNAcT-1)


532


Q13231


Chitotriosidase-1 (EC 3.2.1.14) (Chitinase-1)


466


Q9NY35


Claudin domain-containing protein 1 (Membrane protein GENX-3745)


253


O94905


Erlin-2 (Endoplasmic reticulum lipid raft-associated protein 2) (Stomatin-prohibitin-flotillin-HflC/K domain-containing protein 2)


(SPFH domain-containing protein 2)


339


Q68CQ7


Glycosyltransferase 8 domain-containing protein 1 (EC 2.4.1.-)


371


Q8NBJ4


Golgi membrane protein 1 (Golgi phosphoprotein 2) (Golgi membrane protein GP73)


401


Q8IWK6


Probable G-protein coupled receptor 125


1,321


Q9UHF4


Interleukin-20 receptor alpha chain (IL-20R-alpha) (IL-20R1) (Cytokine receptor class-II member 8) (Cytokine receptor family 2 member 8)


(CRF2-8) (ZcytoR7)


553


P47929


Galectin-7 (Gal-7) (HKL-14) (PI7) (p53-induced gene 1 protein)


136


Q8TD07


NKG2D ligand 4 (NKG2DL4) (N2DL-4) (Retinoic acid early transcript 1E) (Lymphocyte effector toxicity activation ligand) (RAE-1-like transcript 4)


(RL-4)


263


Q96RQ9


L-amino-acid oxidase (LAAO) (LAO) (EC 1.4.3.2) (Interleukin-4-induced protein 1) (IL4-induced protein 1) (Protein Fig-1) (hFIG1)


567


Q32P28


Prolyl 3-hydroxylase 1 (EC 1.14.11.7) (Leucine- and proline-enriched proteoglycan 1) (Leprecan-1) (Growth suppressor 1)


736


Q5H8A4


GPI ethanolamine phosphate transferase 2 (EC 2.-.-.-) (Phosphatidylinositol-glycan biosynthesis class G protein) (PIG-G) (GPI7 homolog) (hGPI7)


983


Q8TEQ8


GPI ethanolamine phosphate transferase 3 (EC 2.-.-.-) (Phosphatidylinositol-glycan biosynthesis class O protein) (PIG-O)


1,089


Q6KCM7


Calcium-binding mitochondrial carrier protein SCaMC-2 (Small calcium-binding mitochondrial carrier protein 2) (Mitochondrial ATP-Mg/Pi carrier


protein 3) (Mitochondrial Ca(2+)-dependent solute carrier protein 3) (Solute carrier family 25 member 25)


469


Q8IWL1


Pulmonary surfactant-associated protein A2 (SP-A2) (SP-A) (PSP-A) (PSPA) (Alveolar proteinosis protein) (35 kDa pulmonary surfactant-associated


protein)


248


Q8N2M8


Splicing factor, arginine/serine-rich 16 (Suppressor of white-apricot homolog 2)


674


Q969X2


Alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 6 (EC 2.4.99.-) (GalNAc alpha-2,6-sialyltransferase VI) (hST6GalNAc VI) (ST6GalNAc VI)


(Sialyltransferase 7F)


333


Q6IA17


Single Ig IL-1-related receptor (Single Ig IL-1R-related molecule) (Single immunoglobulin domain-containing IL1R-related protein)


(Toll/interleukin-1 receptor 8) (TIR8)


410


Q6NUS6


Tectonic-3


607


O14788


Tumor necrosis factor ligand superfamily member 11 (Receptor activator of nuclear factor kappa B ligand) (RANKL) (TNF-relatedactivation-induced


cytokine) (TRANCE) (Osteoprotegerin ligand) (OPGL) (Osteoclast differentiation factor) (ODF) (CD antigen CD254) [Cleaved into: Tumor necrosis


factor ligand superfamily member 11, membrane form; Tumor necrosis factor ligand superfamily member 11, soluble form]


317


Q9NS68


Tumor necrosis factor receptor superfamily member 19 (Toxicity and JNK inducer) (TRADE)


423


Q9Y397


Palmitoyltransferase ZDHHC9 (EC 2.3.1.-) (Zinc finger DHHC domain-containing protein 9) (DHHC-9) (DHHC9) (Zinc finger protein 379)


364


Q9UBX5


Fibulin-5 (FIBL-5) (Developmental arteries and neural crest EGF-like protein) (Dance) (Urine p50 protein) (UP50)


448


Q9UK55


Protein Z-dependent protease inhibitor (PZ-dependent protease inhibitor) (PZI) (Serpin A10)


444


P01023


Alpha-2-macroglobulin (Alpha-2-M) (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 5)


1,474


O00253


Agouti-related protein


132


P04746


Pancreatic alpha-amylase (PA) (EC 3.2.1.1) (1,4-alpha-D-glucan glucanohydrolase)


511


P16860


Natriuretic peptides B (Gamma-brain natriuretic peptide) [Cleaved into: Brain natriuretic peptide 32 (BNP-32) (BNP(1-32)); BNP(1-30); BNP(1-29);


BNP(1-28); BNP(2-31); BNP(3-32); BNP(3-30); BNP(3-29); BNP(4-32); BNP(4-31); BNP(4-30); BNP(4-29); BNP(4-27); BNP(5-32);


BNP(5-31); BNP(5-29)]


134


P01160


Atrial natriuretic factor (ANF) (Atrial natriuretic peptide) (ANP) (Prepronatriodilatin) (CDD-ANF) [Cleaved into: Cardiodilatin-related peptide (CDP)]


153


Q9NR71


Neutral ceramidase (N-CDase) (NCDase) (EC 3.5.1.23) (Acylsphingosine deacylase 2) (N-acylsphingosine amidohydrolase 2) (Non-lysosomal


ceramidase) (BCDase) (LCDase) (hCD) [Cleaved into: Neutral ceramidase soluble form]


780


P61769


Beta-2-microglobulin [Cleaved into: Beta-2-microglobulin form pI 5.3]


119


P12644


Bone morphogenetic protein 4 (BMP-4) (BMP-2B)


408


P43251


Biotinidase (Biotinase) (EC 3.5.1.12)


543


Q86VB7


Scavenger receptor cysteine-rich type 1 protein M130 (Hemoglobin scavenger receptor) (CD antigen CD163) [Cleaved into: Soluble CD163 (sCD163)]


1,156


Q96IY4


Carboxypeptidase B2 (EC 3.4.17.20) (Carboxypeptidase U) (CPU) (Thrombin-activable fibrinolysis inhibitor) (TAFI) (Plasma carboxypeptidase B)


(pCPB)


423


Q66K79


Carboxypeptidase Z (CPZ) (EC 3.4.17.-)


652


P13501


C-C motif chemokine 5 (Small-inducible cytokine A5) (T-cell-specific protein RANTES) (SIS-delta) (T cell-specific protein P228) (TCP228)


(Eosinophil-chemotactic cytokine) (EoCP) [Cleaved into: RANTES(3-68); RANTES(4-68)]


91


P80098


C-C motif chemokine 7 (Small-inducible cytokine A7) (Monocyte chemoattractant protein 3) (Monocyte chemotactic protein 3) (MCP-3) (NC28)


99


P80075


C-C motif chemokine 8 (Small-inducible cytokine A8) (Monocyte chemoattractant protein 2) (Monocyte chemotactic protein 2) (MCP-2) (HC14)


[Cleaved into: MCP-2(6-76)]


99


P13987


CD59 glycoprotein (Membrane attack complex inhibition factor) (MACIF) (MAC-inhibitory protein) (MAC-IP) (Protectin) (MEM43 antigen)


(Membrane inhibitor of reactive lysis) (MIRL) (20 kDa homologous restriction factor) (HRF-20) (HRF20) (1F5 antigen) (CD antigen CD59)


128


P05156


Complement factor I (EC 3.4.21.45) (C3B/C4B inactivator) [Cleaved into: Complement factor I heavy chain; Complement factor I light chain]


583


P10909


Clusterin (Complement-associated protein SP-40,40) (Complement cytolysis inhibitor) (CLI) (NA1/NA2) (Apolipoprotein J) (Apo-J)


(Testosterone-repressed prostate message 2) (TRPM-2) (Ku70-binding protein 1) (Aging-associated gene 4 protein) [Cleaved into: Clusterin beta


chain (ApoJalpha) (Complement cytolysis inhibitor a chain); Clusterin alpha chain (ApoJbeta) (Complement cytolysis inhibitor b chain)]


449


P08123


Collagen alpha-2(I) chain (Alpha-2 type I collagen)


1,366


P02461


Collagen alpha-1(III) chain


1,466


P02462


Collagen alpha-1(IV) chain (Arresten)


1,669


Q01955


Collagen alpha-3(IV) chain (Goodpasture antigen) [Cleaved into: Tumstatin]


1,670


P29400


Collagen alpha-5(IV) chain


1,685


P12111


Collagen alpha-3(VI) chain


3,177


P13671


Complement component C6


934


P20849


Collagen alpha-1(IX) chain


921


Q03692


Collagen alpha-1(X) chain


680


Q9UMD9


Collagen alpha-1(XVII) chain (Bullous pemphigoid antigen 2) (180 kDa bullous pemphigoid antigen 2) [Cleaved into: 120 kDa linear IgA disease


antigen (120 kDa linear IgA dermatosis antigen) (Linear IgA disease antigen 1) (LAD-1); 97 kDa linear IgA disease antigen (97 kDa linear IgA


bullous dermatosis antigen) (97 kDa LAD antigen) (97-LAD) (Linear IgA bullous disease antigen of 97 kDa) (LABD97)]


1,497


Q96P44


Collagen alpha-1(XXI) chain


957


P53621


Coatomer subunit alpha (Alpha-coat protein) (Alpha-COP) (HEP-COP) (HEPCOP) [Cleaved into: Xenin (Xenopsin-related peptide); Proxenin]


1,224


P17927


Complement receptor type 1 (C3b/C4b receptor) (CD antigen CD35)


2,039


P01037


Cystatin-SN (Cystatin-1) (Salivary cystatin-SA-1) (Cystain-SA-I)


141


P24855


Deoxyribonuclease-1 (EC 3.1.21.1) (Deoxyribonuclease I) (DNase I) (Dornase alfa)


282


Q16610


Extracellular matrix protein 1 (Secretory component p85)


540


P12259


Coagulation factor V (Activated protein C cofactor) (Proaccelerin, labile factor) [Cleaved into: Coagulation factor V heavy chain;


Coagulation factor V light chain]


2,224


P08709


Coagulation factor VII (EC 3.4.21.21) (Serum prothrombin conversion accelerator) (SPCA) (Proconvertin) (Eptacog alfa)


[Cleaved into: Factor VII light chain; Factor VII heavy chain]


466


P24071


Immunoglobulin alpha Fc receptor (IgA Fc receptor) (CD antigen CD89)


287


P08637


Low affinity immunoglobulin gamma Fc region receptor III-A (IgG Fc receptor III-2) (Fc-gamma RIII-alpha) (Fc-gamma RIIIa) (FcRIIIa)


(Fc-gamma RIII) (FcRIII) (FcR-10) (CD16a antigen) (CD antigen CD16a)


254


P02771


Alpha-fetoprotein (Alpha-1-fetoprotein) (Alpha-fetoglobulin)


609


P09038


Heparin-binding growth factor 2 (HBGF-2) (Basic fibroblast growth factor) (BFGF)


210


P02679


Fibrinogen gamma chain


453


P43026


Growth/differentiation factor 5 (GDF-5) (Cartilage-derived morphogenetic protein 1) (CDMP-1) (Radotermin)


501


P39905


Glial cell line-derived neurotrophic factor (Astrocyte-derived trophic factor) (ATF) (hGDNF)


211


P17936


Insulin-like growth factor-binding protein 3 (IGF-binding protein 3) (IGFBP-3) (IBP-3)


291


P01343


Insulin-like growth factor IA (IGF-IA) (Somatomedin-C) (Mechano growth factor) (MGF)


153


P01857


Ig gamma-1 chain C region


330


P01859


Ig gamma-2 chain C region


326


P01860


Ig gamma-3 chain C region (Heavy chain disease protein) (HDC)


377


Q8N6C5


Immunoglobulin superfamily member 1 (Inhibin-binding protein) (InhBP) (Immunoglobulin-like domain-containing protein 1)


(Pituitary gland specific factor 2) (p120)


1,336


P05112


Interleukin-4 (IL-4) (B-cell stimulatory factor 1) (BSF-1) (Lymphocyte stimulatory factor 1) (Binetrakin) (Pitrakinra)


153


P08887


Interleukin-6 receptor subunit alpha (IL-6R-alpha) (IL-6R 1) (Membrane glycoprotein 80) (gp80) (CD antigen CD126)


468


P05231


Interleukin-6 (IL-6) (B-cell stimulatory factor 2) (BSF-2) (Interferon beta-2) (Hybridoma growth factor) (CTL differentiation factor) (CDF)


212


P10145


Interleukin-8 (IL-8) (C-X-C motif chemokine 8) (Monocyte-derived neutrophil chemotactic factor) (MDNCF) (T-cell chemotactic factor)


(Neutrophil-activating protein 1) (NAP-1) (Protein 3-10C) (Granulocyte chemotactic protein 1) (GCP-1) (Monocyte-derived neutrophil-activating


peptide) (MONAP) (Emoctakin) [Cleaved into: MDNCF-a (IL8/NAP1 form I) (GCP/IL-8 protein IV); Interleukin-8 (IL-8(1-77)) (MDNCF-b)


(IL8/NAP1 form II) (GCP/IL-8 protein II) ((Ala-IL-8)77); IL-8(5-77); IL-8(6-77) (Lymphocyte-derived neutrophil-activating factor) (LYNAP)


(Neutrophil-activating factor) (NAF) (MDNCF-c) (IL8/NAP1 form III) (GCP/IL-8 protein I) ((Ser-IL-8)72); IL-8(7-77) (IL8/NAP1 form IV)


(GCP/IL-8 protein V); IL-8(8-77) (IL8/NAP1 form V) (GCP/IL-8 protein VI); IL-8(9-77) (IL8/NAP1 form VI) (GCP/IL-8 protein III)]


99


P51460


Insulin-like 3 (Leydig insulin-like peptide) (Ley-I-L) (Relaxin-like factor) [Cleaved into: Insulin-like 3 B chain; Insulin-like 3 A chain]


131


Q14624


Inter-alpha-trypsin inhibitor heavy chain H4 (Inter-alpha-inhibitor heavy chain 4) (ITI heavy chain H4) (Inter-alpha-trypsin inhibitor family heavy


chain-related protein) (IHRP) (Plasma kallikrein sensitive glycoprotein 120) (PK-120) (GP120) [Cleaved into: 70 kDa inter-alpha-trypsin inhibitor


heavy chain H4; 35 kDa inter-alpha-trypsin inhibitor heavy chain H4]


930


Q14667


UPF0378 protein KIAA0100 (Breast cancer overexpressed gene 1 protein) (Antigen MLAA-22)


2,235


P01042


Kininogen-1 (High molecular weight kininogen) (HMWK) (Williams-Fitzgerald-Flaujeac factor) (Fitzgerald factor) (Alpha-2-thiol proteinase inhibitor)


[Cleaved into: Kininogen-1 heavy chain; T-kinin (Ile-Ser-Bradykinin); Bradykinin (Kallidin I); Lysyl-bradykinin (Kallidin II); Kininogen-1 light chain;


Low molecular weight growth-promoting factor]


644


P24043


Laminin subunit alpha-2 (Laminin M chain) (Merosin heavy chain)


3,122


Q16363


Laminin subunit alpha-4


1,823


P07942


Laminin subunit beta-1 (Laminin B1 chain)


1,786


P28300


Protein-lysine 6-oxidase (EC 1.4.3.13) (Lysyl oxidase)


417


P08581


Hepatocyte growth factor receptor (HGF receptor) (EC 2.7.10.1) (Scatter factor receptor) (SF receptor) (HGF/SF receptor) (Met proto-oncogene tyrosine


kinase) (c-Met)


1,390


P20774


Mimecan (Osteoglycin) (Osteoinductive factor) (OIF)


298


Q99542


Matrix metalloproteinase-19 (MMP-19) (EC 3.4.24.-) (Matrix metalloproteinase RASI) (MMP-18)


508


Q13201


Multimerin-1 (Endothelial cell multimerin) (EMILIN-4) (Elastin microfibril interface located protein 4) (Elastin microfibril interfacer 4) [Cleaved into:


Platelet glycoprotein Ia*; 155 kDa platelet multimerin (p-155) (p155)]


1,228


P01185


Vasopressin-neurophysin 2-copeptin (AVP-NPII) [Cleaved into: Arg-vasopressin; Neurophysin 2 (Neurophysin-II); Copeptin]


164


P14543


Nidogen-1 (NID-1) (Entactin)


1,247


P14555


Phospholipase A2, membrane associated (EC 3.1.1.4) (Phosphatidylcholine 2-acylhydrolase) (Group IIA phospholipase A2) (GIIC sPLA2)


(Non-pancreatic secretory phospholipase A2) (NPS-PLA2)


144


P14222


Perforin-1 (P1) (Lymphocyte pore-forming protein) (PFP) (Cytolysin)


555


P80108


Phosphatidylinositol-glycan-specific phospholipase D (PI-G PLD) (EC 3.1.4.50) (Glycoprotein phospholipase D) (Glycosyl-phosphatidylinositol-specific


phospholipase D) (GPI-specific phospholipase D) (GPI-PLD)


840


Q8TCZ9


Fibrocystin (Polycystic kidney and hepatic disease 1 protein) (Polyductin) (Tigmin)


4,074


P55058


Phospholipid transfer protein (Lipid transfer protein II)


493


P15309


Prostatic acid phosphatase (EC 3.1.3.2)


386


P01236


Prolactin (PRL)


227


P27918


Properdin (Complement factor P)


469


P07225


Vitamin K-dependent protein S


676


P22891


Vitamin K-dependent protein Z


400


P02810


Salivary acidic proline-rich phosphoprotein 1/2 (PRP-1/PRP-2) (Parotid proline-rich protein 1/2) (Pr1/Pr2) (Protein C) (Parotid acidic protein) (Pa)


(Parotid isoelectric focusing variant protein) (PIF-S) (Parotid double-band protein) (Db-s) [Cleaved into: Salivary acidic proline-rich phosphoprotein 1/2;


Salivary acidic proline-rich phosphoprotein 3/4 (PRP-3/PRP-4) (Protein A) (PIF-F) (Db-F); Peptide P-C]


166


P11464


Pregnancy-specific beta-1-glycoprotein 1 (PSBG-1) (Pregnancy-specific beta-1 glycoprotein C/D) (PS-beta-C/D) (Fetal liver non-specific cross-reactive 


antigen 1/2) (FL-NCA-1/2) (PSG95) (CD66 antigen-like family member F) (CD antigen CD66f)


419


P20742


Pregnancy zone protein (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 6)


1,482


P04090


Prorelaxin H2 [Cleaved into: Relaxin B chain; Relaxin A chain]


185


P78509


Reelin (EC 3.4.21.-)


3,460


P02753


Retinol-binding protein 4 (Plasma retinol-binding protein) (PRBP) (RBP) [Cleaved into: Plasma retinol-binding protein(1-182); Plasma retinol-binding


protein(1-181); Plasma retinol-binding protein(1-179); Plasma retinol-binding protein(1-176)]


201


Q92854


Semaphorin-4D (BB18) (A8) (GR3) (CD antigen CD100)


862


O94813


Slit homolog 2 protein (Slit-2) [Cleaved into: Slit homolog 2 protein N-product; Slit homolog 2 protein C-product]


1,529


O75443


Alpha-tectorin


2,155


P22105


Tenascin-X (TN-X) (Hexabrachion-like protein)


4,289


Q07654


Trefoil factor 3 (Intestinal trefoil factor) (hITF) (Polypeptide P1.B) (hP1.B)


80


P02786


Transferrin receptor protein 1 (TfR1) (TfR) (TR) (Trfr) (T9) (p90) (CD antigen CD71) [Cleaved into: Transferrin receptor protein 1, serum form (sTfR)]


760


P01135


Protransforming growth factor alpha [Cleaved into: Transforming growth factor alpha (TGF-alpha) (EGF-like TGF) (ETGF) (TGF type 1)]


160


P61812


Transforming growth factor beta-2 (TGF-beta-2) (Glioblastoma-derived T-cell suppressor factor) (G-TSF) (BSC-1 cell growth inhibitor) (Polyergin) 


(Cetermin)


414


Q9UJW2


Tubulointerstitial nephritis antigen (TIN-Ag)


476


P48023


Tumor necrosis factor ligand superfamily member 6 (Fas antigen ligand) (Fas ligand) (CD95L protein) (Apoptosis antigen ligand) (APTL) (CD antigen 


CD178) [Cleaved into: Tumor necrosis factor ligand superfamily member 6, membrane form; Tumor necrosis factor ligand superfamily member 6, 


soluble form]


281


P20333


Tumor necrosis factor receptor superfamily member 1B (Tumor necrosis factor receptor 2) (TNF-R2) (Tumor necrosis factor receptor type II) (p75)


(p80 TNF-alpha receptor) (CD antigen CD120b) (Etanercept) [Cleaved into: Tumor necrosis factor receptor superfamily member 1b, membrane form;


Tumor necrosis factor-binding protein 2 (TBPII) (TBP-2)]


461


P25942


Tumor necrosis factor receptor superfamily member 5 (CD40L receptor) (B-cell surface antigen CD40) (Bp50) (CDw40) (CD antigen CD40)


277


P40225


Thrombopoietin (Megakaryocyte colony-stimulating factor) (Myeloproliferative leukemia virus oncogene ligand) (C-mpl ligand) (ML)


(Megakaryocyte growth and development factor) (MGDF)


353


Q9NP99


Triggering receptor expressed on myeloid cells 1 (Triggering receptor expressed on monocytes 1) (TREM-1)


234


P01282


VIP peptides [Cleaved into: Intestinal peptide PHV-42; Intestinal peptide PHM-27 (Peptide histidine methioninamide 27); Vasoactive intestinal peptide 


(VIP) (Vasoactive intestinal polypeptide)]


170


Q9BZP6


Acidic mammalian chitinase (AMCase) (EC 3.2.1.14) (TSA1902)


476


P16562


Cysteine-rich secretory protein 2 (CRISP-2) (Testis-specific protein TPX-1) (Cancer/testis antigen 36) (CT36)


243


P00739


Haptoglobin-related protein


348


Q15238


Pregnancy-specific beta-1-glycoprotein 5 (PSBG-5) (Fetal liver non-specific cross-reactive antigen 3) (FL-NCA-3)


335


Q9BXJ4


Complement C1q tumor necrosis factor-related protein 3 (Secretory protein CORS26)


246


Q9BZM5


NKG2D ligand 2 (UL16-binding protein 2) (NKG2DL2) (N2DL-2) (ALCAN-alpha) (Retinoic acid early transcript 1H)


246


Q9Y258


C-C motif chemokine 26 (Small-inducible cytokine A26) (Eotaxin-3) (Macrophage inflammatory protein 4-alpha) (MIP-4-alpha) (Thymic stroma


chemokine-1) (TSC-1) (CC chemokine IMAC)


94


Q9BWP8


Collectin-11 (Collectin kidney protein 1) (CL-K1)


271


Q6UXH1


Cysteine-rich with EGF-like domain protein 2


353


O75462


Cytokine receptor-like factor 1 (Cytokine-like factor 1) (CLF-1) (ZcytoR5)


422


Q9H2A7


C-X-C motif chemokine 16 (Small-inducible cytokine B16) (Transmembrane chemokine CXCL16) (SR-PSOX) (Scavenger receptor for


phosphatidylserine and oxidized low density lipoprotein)


254


Q14512


Fibroblast growth factor-binding protein 1 (FGF-binding protein 1) (FGF-BP1) (FGFBP-1) (FGF-BP) (17 kDa heparin-binding growth factor-binding


protein) (17 kDa HBGF-binding protein) (HBp17)


234


Q9UBH0


Interleukin-1 family member 5 (IL-1F5) (Interleukin-1 delta) (IL-1 delta) (FIL1 delta) (Interleukin-1-like protein 1) (IL-1L1) (Interleukin-1 HY1)


(IL-1HY1) (Interleukin-1 receptor antagonist homolog 1) (IL-1ra homolog 1) (IL-1-related protein 3) (IL-1RP3)


155


Q9NZH8


Interleukin-1 family member 9 (IL-1F9) (Interleukin-1 homolog 1) (IL-1H1) (Interleukin-1 epsilon) (IL-1 epsilon) (IL-1-related protein 2) (IL-1RP2)


169


Q9Y337


Kallikrein-5 (EC 3.4.21.-) (Stratum corneum tryptic enzyme) (Kallikrein-like protein 2) (KLK-L2)


293


O00339


Matrilin-2


956


Q8TD46


Cell surface glycoprotein CD200 receptor 1 (Cell surface glycoprotein OX2 receptor 1) (CD200 cell surface glycoprotein receptor)


325


Q9NPH0


Lysophosphatidic acid phosphatase type 6 (EC 3.1.3.2) (Acid phosphatase 6, lysophosphatidic) (Acid phosphatase-like protein 1) (PACPL1)


428


Q9H173


Nucleotide exchange factor SIL1 (BiP-associated protein) (BAP)


461


O75094


Slit homolog 3 protein (Slit-3) (Multiple epidermal growth factor-like domains 5)


1,523


Q6X4U4


Sclerostin domain-containing protein 1 (Ectodermal BMP inhibitor) (Ectodin) (Uterine sensitization-associated gene 1 protein) (USAG-1)


206


Q86WD7


Serpin A9 (Germinal center B-cell-expressed transcript 1 protein)


417


Q6ZMP0


Thrombospondin type-1 domain-containing protein 4


1,018


O76076


WNT1-inducible-signaling pathway protein 2 (WISP-2) (Connective tissue growth factor-like protein) (CTGF-L) (Connective tissue growth


factor-related protein 58)


250


Q9H8M2


Bromodomain-containing protein 9 (Rhabdomyosarcoma antigen MU-RMS-40.8)


597


Q8TCZ2


CD99 antigen-like protein 2 (MIC2-like protein 1) (CD antigen CD99)


262


Q96HA4


Uncharacterized protein C1orf159


380


Q9NRB3


Carbohydrate sulfotransferase 12 (EC 2.8.2.5) (Chondroitin 4-O-sulfotransferase 2) (Chondroitin 4-sulfotransferase 2) (C4ST2) (C4ST-2)


(Sulfotransferase Hlo)


414


Q9H3G5


Probable serine carboxypeptidase CPVL (EC 3.4.16.-) (Carboxypeptidase, vitellogenic-like) (Vitellogenic carboxypeptidase-like protein)


(VCP-like protein) (HVLP)


476


Q9NQ79


Cartilage acidic protein 1 (68 kDa chondrocyte-expressed protein) (CEP-68) (ASPIC)


661


Q86UP6


CUB and zona pellucida-like domain-containing protein 1 (CUB and ZP domain-containing protein 1) (Transmembrane protein UO-44)


607


Q96FL9


Polypeptide N-acetylgalactosaminyltransferase 14 (EC 2.4.1.41) (Polypeptide GalNAc transferase 14) (pp-GaNTase 14) (GalNAc-T14)


(Protein-UDP acetylgalactosaminyltransferase 14) (UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 14)


552


Q2KHT4


Germ cell-specific gene 1 protein


349


Q9HBE5


Interleukin-21 receptor (IL-21R) (Novel interleukin receptor)


538


O00182


Galectin-9 (HOM-HD-21) (Ecalectin)


355


Q8N6Y2


Leucine-rich repeat-containing protein 17 (p37NB)


441


O75325


Leucine-rich repeat neuronal protein 2 (Leucine-rich repeat neuronal protein 5) (Glioma amplified on chromosome 1 protein)


713


O95803


Bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 3 (EC 2.8.2.8) (Glucosaminyl N-deacetylase/N-sulfotransferase 3) (NDST-3) (hNDST-3)


(N-heparan sulfate sulfotransferase 3) (N-HSST 3) [Includes: Heparan sulfate N-deacetylase 3 (EC 3.-.-.-); Heparan sulfate N-sulfotransferase 3


(EC 2.8.2.-)]


873


Q9H3S1


Semaphorin-4A (Semaphorin-B) (Sema B)


761


Q9HAV5


Tumor necrosis factor receptor superfamily member 27 (X-linked ectodysplasin-A2 receptor) (EDA-A2 receptor)


297


Q9NNX1


Tuftelin


390


O95258


Brain mitochondrial carrier protein 1 (BMCP-1) (Mitochondrial uncoupling protein 5) (UCP 5) (Solute carrier family 25 member 14)


325


Q8IX30


Signal peptide, CUB and EGF-like domain-containing protein 3


993


P31947


14-3-3 protein sigma (Stratifin) (Epithelial cell marker protein 1)


248


P02763


Alpha-1-acid glycoprotein 1 (AGP 1) (Orosomucoid-1) (OMD 1)


201


P19652


Alpha-1-acid glycoprotein 2 (AGP 2) (Orosomucoid-2) (OMD 2)


201


P08697


Alpha-2-antiplasmin (Alpha-2-AP) (Alpha-2-plasmin inhibitor) (Alpha-2-PI)


491


Q13443


Disintegrin and metalloproteinase domain-containing protein 9 (ADAM 9) (EC 3.4.24.-) (Metalloprotease/disintegrin/cysteine-rich protein 9)


(Myeloma cell metalloproteinase) (Meltrin-gamma) (Cellular disintegrin-related protein)


819


P02760


Protein AMBP [Cleaved into: Alpha-1-microglobulin (Protein HC) (Complex-forming glycoprotein heterogeneous in charge) 


(Alpha-1 microglycoprotein); Inter-alpha-trypsin inhibitor light chain (ITI-LC) (Bikunin) (HI-30) (Uronic-acid-rich protein) (EDC1); Trypstatin]


352


P01019


Angiotensinogen (Serpin A8) [Cleaved into: Angiotensin-1 (Angiotensin I) (Ang I); Angiotensin-2 (Angiotensin II) (Ang II); Angiotensin-3


(Angiotensin III) (Ang III) (Des-Asp[1]-angiotensin II)]


485


Q9HCJ1


Progressive ankylosis protein homolog (ANK)


492


Q9HDC9


Adipocyte plasma membrane-associated protein (Protein BSCv)


416


P02652


Apolipoprotein A-II (Apo-AII) (ApoA-II) [Cleaved into: Apolipoprotein A-II(1-76)]


100


P06727


Apolipoprotein A-IV (Apo-AIV) (ApoA-IV)


396


P02655


Apolipoprotein C-II (Apo-CII) (ApoC-II)


101


P02749


Beta-2-glycoprotein 1 (Beta-2-glycoprotein I) (Beta(2)GPI) (B2GPI) (Apolipoprotein H) (Apo-H) (Activated protein C-binding protein) (APC inhibitor)


(Anticardiolipin cofactor)


345


Q6UW56


Apoptosis-related protein 3 (APR-3) (p18)


229


Q9Y5Z0


Beta-secretase 2 (EC 3.4.23.45) (Beta-site APP-cleaving enzyme 2) (Aspartyl protease 1) (Asp 1) (ASP1) (Membrane-associated aspartic protease 1) 


(Memapsin-1) (Aspartic-like protease 56 kDa) (Down region aspartic protease)


518


P16442


Histo-blood group ABO system transferase (NAGAT) (Glycoprotein-fucosylgalactoside alpha-N-acetylgalactosaminyltransferase) (EC 2.4.1.40) 


(Fucosylglycoprotein alpha-N-acetylgalactosaminyltransferase) (Histo-blood group A transferase) (A transferase) (Glycoprotein-fucosylgalactoside 


alpha-galactosyltransferase) (EC 2.4.1.37) (Fucosylglycoprotein 3-alpha-galactosyltransferase) (Histo-blood group B transferase) (B transferase) 


[Cleaved into: Fucosylglycoprotein alpha-N-acetylgalactosaminyltransferase soluble form]


354


O60911


Cathepsin L2 (EC 3.4.22.43) (Cathepsin V) (Cathepsin U)


334


P10147


C-C motif chemokine 3 (Small-inducible cytokine A3) (Macrophage inflammatory protein 1-alpha) (MIP-1-alpha) (Tonsillar lymphocyte LD78 alpha


protein) (G0/G1 switch regulatory protein 19-1) (G0S19-1 protein) (SIS-beta) (PAT 464.1) [Cleaved into: MIP-1-alpha(4-69) (LD78-alpha(4-69))]


92


Q8N6Q3


CD177 antigen (Polycythemia rubra vera protein 1) (PRV-1) (NB1 glycoprotein) (NB1 GP) (Human neutrophil alloantigen 2a) (HNA-2a) 


(CD antigen CD177)


437


P00450


Ceruloplasmin (EC 1.16.3.1) (Ferroxidase)


1,065


P36222


Chitinase-3-like protein 1 (Cartilage glycoprotein 39) (CGP-39) (GP-39) (hCGP-39) (39 kDa synovial protein) (YKL-40)


383


Q9P126


C-type lectin domain family 1 member B (C-type lectin-like receptor 2) (CLEC-2)


229


A8K7I4


Calcium-activated chloride channel regulator 1 (Calcium-activated chloride channel family member 1) (hCLCA1) (Calcium-activated chloride channel


protein 1) (CaCC-1) (hCaCC-1)


914


P23946


Chymase (EC 3.4.21.39) (Alpha-chymase) (Mast cell protease I)


247


P12109


Collagen alpha-1(VI) chain


1,028


P07357


Complement component C8 alpha chain (Complement component 8 subunit alpha)


584


P02748


Complement component C9 [Cleaved into: Complement component C9a; Complement component C9b]


559


P02775


Platelet basic protein (PBP) (C-X-C motif chemokine 7) (Small-inducible cytokine B7) (Leukocyte-derived growth factor) (LDGF) (Macrophage-


derived growth factor) (MDGF) [Cleaved into: Connective tissue-activating peptide III (CTAP-III) (Low-affinity platelet factor IV) (LA-PF4); TC-2;


Connective tissue-activating peptide III(1-81) (CTAP-III(1-81)); Beta-thromboglobulin (Beta-TG); Neutrophil-activating peptide 2(74) (NAP-2(74)); 


Neutrophil-activating peptide 2(73) (NAP-2(73)); Neutrophil-activating peptide 2 (NAP-2); TC-1; Neutrophil-activating peptide 2(1-66)


(NAP-2(1-66)); Neutrophil-activating peptide 2(1-63) (NAP-2(1-63))]


128


O14625


C-X-C motif chemokine 11 (Small-inducible cytokine B11) (Interferon-inducible T-cell alpha chemoattractant) (I-TAC) (Interferon-gamma-inducible 


protein 9) (IP-9) (H174) (Beta-R1)


94


Q9UBS4


DnaJ homolog subfamily B member 11 (ER-associated dnaJ protein 3) (ERj3p) (ERdj3) (ER-associated Hsp40 co-chaperone) (ER-associated DNAJ)


(HEDJ) (hDj9) (PWP1-interacting protein 4) (APOBEC1-binding protein 2) (ABBP-2)


358


Q6UWV6


Ectonucleotide pyrophosphatase/phosphodiesterase family member 7 (E-NPP7) (NPP-7) (EC 3.1.4.12) (Alkaline sphingomyelin phosphodiesterase)


(Intestinal alkaline sphingomyelinase) (Alk-SMase)


458


P19235


Erythropoietin receptor (EPO-R)


508


Q9NZ08


Endoplasmic reticulum aminopeptidase 1 (EC 3.4.11.-) (Adipocyte-derived leucine aminopeptidase) (A-LAP) (ARTS-1) (Aminopeptidase PILS) 


(Puromycin-insensitive leucyl-specific aminopeptidase) (PILS-AP) (Type 1 tumor necrosis factor receptor shedding aminopeptidase regulator)


941


P21860


Receptor tyrosine-protein kinase erbB-3 (c-erbB3) (EC 2.7.10.1) (Tyrosine kinase-type cell surface receptor HER3)


1,342


Q9BS26


Endoplasmic reticulum resident protein ERp44 (Thioredoxin domain-containing protein 4)


406


Q9Y6R7


IgGFc-binding protein (Fcgamma-binding protein antigen) (FcgammaBP)


5,405


Q03591


Complement factor H-related protein 1 (FHR-1) (H factor-like protein 1) (H-factor-like 1) (H36)


330


Q10471


Polypeptide N-acetylgalactosaminyltransferase 2 (EC 2.4.1.41) (Polypeptide GalNAc transferase 2) (pp-GaNTase 2) (GalNAc-T2) (Protein-UDP 


acetylgalactosaminyltransferase 2) (UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2) [Cleaved into: Polypeptide


N-acetylgalactosaminyltransferase 2 soluble form]


571


P22352


Glutathione peroxidase 3 (EC 1.11.1.9) (GSHPx-3) (GPx-3) (Extracellular glutathione peroxidase) (Plasma glutathione peroxidase) (GSHPx-P) (GPx-P)


226


P02790


Hemopexin (Beta-1B-glycoprotein)


462


Q04756


Hepatocyte growth factor activator (HGF activator) (HGFA) (EC 3.4.21.-) [Cleaved into: Hepatocyte growth factor activator short chain; Hepatocyte 


growth factor activator long chain]


655


P15516


Histatin-3 (Histidine-rich protein 3) (PB) (Basic histidine-rich protein) (Hst) [Cleaved into: Histatin-3; Histatin-3 1/25 (Histatin-6); Histatin-3 1/24


(Histatin-5); Histatin-3 1/13; Histatin-3 1/12; Histatin-3 1/11; Histatin-3 5/13; Histatin-3 5/12 (Histatin-11); Histatin-3 5/11 (Histatin-12);


Histatin-3 6/13; Histatin-3 6/11; Histatin-3 7/13; Histatin-3 7/12; Histatin-3 7/11; Histatin-3 12/32 (Histatin-4); Histatin-3 12/25 (Histatin-9);


Histatin-3 12/24 (Histatin-7); Histatin-3 13/25 (Histatin-10); Histatin-3 13/24 (Histatin-8); Histatin-3 14/25; Histatin-3 14/24; Histatin-3 15/25;


Histatin-3 15/24; Histatin-3 26/32; Histatin-3 28/32; Histatin-3 29/32]


51


Q95460


Major histocompatibility complex class I-related gene protein (MHC class-I related-gene protein) (Class I histocompatibility antigen-like protein)


341


P00738


Haptoglobin [Cleaved into: Haptoglobin alpha chain; Haptoglobin beta chain]


406


P24592


Insulin-like growth factor-binding protein 6 (IGF-binding protein 6) (IGFBP-6) (IBP-6)


240


P01880


Ig delta chain C region


384


P01584


Interleukin-1 beta (IL-1 beta) (Catabolin)


269


P05154


Plasma serine protease inhibitor (Serpin A5) (Protein C inhibitor) (PCI) (Plasminogen activator inhibitor 3) (PAI-3) (PAI3) (Acrosomal serine protease


inhibitor)


406


Q9BX67


Junctional adhesion molecule C (JAM-C) (Junctional adhesion molecule 3) (JAM-3) (JAM-2)


310


Q5VV43


Uncharacterized protein KIAA0319


1,072


P00709


Alpha-lactalbumin (Lactose synthase B protein) (Lysozyme-like protein 7)


142


O15230


Laminin subunit alpha-5


3,695


P11047


Laminin subunit gamma-1 (Laminin B2 chain)


1,609


Q08380


Galectin-3-binding protein (Lectin galactoside-binding soluble 3-binding protein) (Mac-2-binding protein) (Mac-2 BP) (MAC2BP) (Tumor-associated 


antigen 90K) (Basement membrane autoantigen p105)


585


P06858


Lipoprotein lipase (LPL) (EC 3.1.1.34)


475


Q7Z4F1


Low-density lipoprotein receptor-related protein 10


713


P03956


Interstitial collagenase (EC 3.4.24.7) (Matrix metalloproteinase-1) (MMP-1) (Fibroblast collagenase) [Cleaved into: 22 kDa interstitial collagenase;


27 kDa interstitial collagenase]


469


P14780


Matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) (92 kDa type IV collagenase) (92 kDa gelatinase) (Gelatinase B) (GELB) [Cleaved into:


67 kDa matrix metalloproteinase-9; 82 kDa matrix metalloproteinase-9]


707


Q8WXI7


Mucin-16 (MUC-16) (Ovarian carcinoma antigen CA125) (Ovarian cancer-related tumor marker CA125) (CA-125)


22,152


Q02817


Mucin-2 (MUC-2) (Intestinal mucin-2)


5,179


Q9HC84


Mucin-5B (MUC-5B) (Mucin-5 subtype B, tracheobronchial) (High molecular weight salivary mucin MG1) (Sublingual gland mucin) (Cervical mucin)


5,703


Q99972


Myocilin (Trabecular meshwork-induced glucocorticoid response protein)


504


P78380


Oxidized low-density lipoprotein receptor 1 (Ox-LDL receptor 1) (Lectin-type oxidized LDL receptor 1) (Lectin-like oxidized LDL receptor 1) 


(Lectin-like oxLDL receptor 1) (hLOX-1) (LOX-1) [Cleaved into: Oxidized low-density lipoprotein receptor 1, soluble form]


273


Q13093


Platelet-activating factor acetylhydrolase (PAF acetylhydrolase) (EC 3.1.1.47) (PAF 2-acylhydrolase) (LDL-associated phospholipase A2) (LDL-PLA(2))


(2-acetyl-1-alkylglycerophosphocholine esterase) (1-alkyl-2-acetylglycerophosphocholine esterase)


441


Q13219


Pappalysin-1 (EC 3.4.24.79) (Pregnancy-associated plasma protein A) (PAPP-A) (Insulin-like growth factor-dependent IGF-binding protein 4 protease)


(IGF-dependent IGFBP-4 protease) (IGFBP-4ase)


1,627


P01833


Polymeric immunoglobulin receptor (Poly-Ig receptor) (PIGR) (Hepatocellular carcinoma-associated protein TB6) [Cleaved into: Secretory component]


764


Q9UKY0


Prion-like protein doppel (PrPLP) (Prion protein 2)


176


Q86YD1


Prostate tumor overexpressed gene 1 protein (PTOV-1) (Activator interaction domain-containing protein 2)


416


O43353


Receptor-interacting serine/threonine-protein kinase 2 (EC 2.7.11.1) (RIP-like-interacting CLARP kinase) (Receptor-interacting protein 2) (RIP-2)


(CARD-containing interleukin-1 beta-converting enzyme-associated kinase) (CARD-containing IL-1 beta ICE-kinase)


540


Q96TC7


Regulator of microtubule dynamics protein 3 (RMD-3) (hRMD-3) (Protein FAM82A2) (Protein FAM82C) (Protein tyrosine phosphatase-interacting 


protein 51) (TCPTP-interacting protein 51) (Cerebral protein 10)


470


Q96S37


Solute carrier family 22 member 12 (Urate anion exchanger 1) (Renal-specific transporter) (RST) (Organic anion transporter 4-like protein)


553


Q9BZD2


Equilibrative nucleoside transporter 3 (hENT3) (Solute carrier family 29 member 3)


475


P49908


Selenoprotein P (SeP)


381


P35247


Pulmonary surfactant-associated protein D (SP-D) (PSP-D) (Lung surfactant protein D)


375


Q9BX79


Stimulated by retinoic acid gene 6 protein homolog


667


Q9UMX1


Suppressor of fused homolog (SUFUH)


484


Q8IWU5


Extracellular sulfatase Sulf-2 (EC 3.1.6.-) (HSulf-2)


870


P20062


Transcobalamin-2 (TC-2) (Transcobalamin II) (TC II) (TCII)


427


P04155


Trefoil factor 1 (Protein pS2) (Polypeptide P1.A) (hP1.A) (Breast cancer estrogen-inducible protein) (PNR-2)


84


P48307


Tissue factor pathway inhibitor 2 (TFPI-2) (Placental protein 5) (PP5)


235


P00734


Prothrombin (EC 3.4.21.5) (Coagulation factor II) [Cleaved into: Activation peptide fragment 1; Activation peptide fragment 2; Thrombin light chain;


Thrombin heavy chain]


622


Q9NR96


Toll-like receptor 9 (CD antigen CD289)


1,032


Q92956


Tumor necrosis factor receptor superfamily member 14 (Herpesvirus entry mediator A) (Tumor necrosis factor receptor-like 2) (TR2)


283


O14773


Tripeptidyl-peptidase 1 (TPP-1) (EC 3.4.14.9) (Tripeptidyl-peptidase I) (TPP-I) (Tripeptidyl aminopeptidase) (Lysosomal pepstatin-insensitive protease) 


(LPIC) (Cell growth-inhibiting gene 1 protein)


563


Q86YW5


Trem-like transcript 1 protein (TLT-1) (Triggering receptor expressed on myeloid cells-like protein 1)


311


Q5T4W7


Artemin (Enovin) (Neublastin)


220


Q99715


Collagen alpha-1(XII) chain


3,063


Q05707


Collagen alpha-1(XIV) chain (Undulin)


1,796


O15263


Beta-defensin 2 (BD-2) (hBD-2) (Defensin, beta 2) (Skin-antimicrobial peptide 1) (SAP1)


64


Q12805


EGF-containing fibulin-like extracellular matrix protein 1 (Fibulin-3) (FIBL-3) (Fibrillin-like protein) (Extracellular protein S1-5)


493


Q14773


Intercellular adhesion molecule 4 (ICAM-4) (Landsteiner-Wiener blood group glycoprotein) (LW blood group protein) (CD antigen CD242)


271


Q9UHD0


Interleukin-19 (IL-19) (Melanoma differentiation-associated protein-like protein) (NG.1)


177


Q6H9L7


Isthmin-2 (Thrombospondin and AMOP domain-containing isthmin-like protein 1) (Thrombospondin type-1 domain-containing protein 3)


571


Q96J84


Kin of IRRE-like protein 1 (Kin of irregular chiasm-like protein 1) (Nephrin-like protein 1)


757


O43240


Kallikrein-10 (EC 3.4.21.-) (Protease serine-like 1) (Normal epithelial cell-specific 1)


276


Q8N2S1


Latent-transforming growth factor beta-binding protein 4 (LTBP-4)


1,624


O60462


Neuropilin-2 (Vascular endothelial cell growth factor 165 receptor 2)


931


Q7Z3S9


Notch homolog 2 N-terminal-like protein


236


Q9UKJ1


Paired immunoglobulin-like type 2 receptor alpha (Inhibitory receptor PILR-alpha) (Cell surface receptor FDF03)


303


Q15262


Receptor-type tyrosine-protein phosphatase kappa (Protein-tyrosine phosphatase kappa) (R-PTP-kappa) (EC 3.1.3.48)


1,439


Q06141


Regenerating islet-derived protein 3 alpha (Reg III-alpha) (Pancreatitis-associated protein 1)


175


Q9BYZ8


Regenerating islet-derived protein 4 (Reg IV) (REG-like protein) (Gastrointestinal secretory protein)


158


Q99942


E3 ubiquitin-protein ligase RNF5 (EC 6.3.2.-) (RING finger protein 5) (HsRma1) (Protein G16)


180


P20366


Protachykinin-1 (PPT) [Cleaved into: Substance P; Neurokinin A (NKA) (Substance K) (Neuromedin L); Neuropeptide K (NPK); Neuropeptide gamma;


C-terminal-flanking peptide]


129


Q86UU9


Tachykinin-4 (Preprotachykinin-C) (PPT-C) [Cleaved into: Endokinin-A (EKA); Endokinin-A/B (EKA/B); Endokinin-C (EKC)]


113


Q6ZSL4


Secreted frizzled-related protein 1, isoform CRA_a (cDNA FLJ45402 fis, clone BRHIP3029409, moderately similar to Homo sapiens secreted


frizzled-related protein 1 (SFRP1))


178


B2RDA1


Secreted phosphoprotein 1 (Osteopontin, bone sialoprotein I, early T-lymphocyte activation 1), isoform CRA_c (cDNA, FLJ96520, Homo sapiens


secreted phosphoprotein 1 (osteopontin, bonesialoprotein I, early T-lymphocyte activation 1) (SPP1), mRNA)


300


Q02325


Plasminogen-related protein B


96


P17931


Galectin-3 (Galactose-specific lectin 3) (Mac-2 antigen) (IgE-binding protein) (35 kDa lectin) (Carbohydrate-binding protein 35) (CBP 35) 


(Laminin-binding protein) (Lectin L-29) (L-31) (Galactoside-binding protein) (GALBP)


250


Q969W1


Probable palmitoyltransferase ZDHHC16 (EC 2.3.1.-) (Zinc finger DHHC domain-containing protein 16) (DHHC-16)


377


P60171


Pre-small/secreted glycoprotein (pre-sGP) [Cleaved into: Small/secreted glycoprotein (sGP); Delta-peptide]


364


O95841


Angiopoietin-related protein 1 (Angiopoietin-like 1) (Angiopoietin-3) (ANG-3)


491


Q9NR16


Scavenger receptor cysteine-rich type 1 protein M160 (CD163 antigen-like 1) (CD antigen CD163b)


1,453


Q9BXJ0


Complement C1q tumor necrosis factor-related protein 5


243


Q5UCC4


UPF0510 protein C19orf63 (Hematopoietic signal peptide-containing membrane domain-containing protein 1)


262


Q6UWP2


Dehydrogenase/reductase SDR family member 11 (EC 1.-.-.-)


260


Q9UBP4


Dickkopf-related protein 3 (Dickkopf-3) (Dkk-3) (hDkk-3)


350


Q9BV94


ER degradation-enhancing alpha-mannosidase-like 2


578


Q5XG92


Carboxylesterase 8 (EC 3.1.1.-)


561


Q8IW92


Beta-galactosidase-1-like protein 2 (EC 3.2.1.-)


636


Q8NFR9


Interleukin-17 receptor E (IL-17 receptor E) (IL-17RE)


667


Q9NYY1


Interleukin-20 (IL-20) (Four alpha helix cytokine Zcyto10)


176


Q9H293


Interleukin-25 (IL-25) (Interleukin-17E) (IL-17E)


177


Q9UKR0


Kallikrein-12 (EC 3.4.21.-) (Kallikrein-like protein 5) (KLK-L5)


248


Q8N387


Mucin-15 (MUC-15)


334


Q5EBL8


PDZ domain-containing protein 11


140


Q96GW7


Brevican core protein (Brain-enriched hyaluronan-binding protein) (Protein BEHAB)


911


Q8N131


Porimin (Transmembrane protein 123) (Pro-oncosis receptor inducing membrane injury) (Keratinocytes-associated transmembrane protein 3) (KCT-3)


208


Q6UW15


Regenerating islet-derived protein 3 gamma (Reg III-gamma) (Pancreatitis-associated protein 1B) (PAP IB)


175


Q8WXF3


Relaxin-3 (Prorelaxin H3) (Insulin-like peptide INSL7) (Insulin-like peptide 7) [Cleaved into: Relaxin-3 B chain; Relaxin-3 A chain]


142


Q9HB40


Retinoid-inducible serine carboxypeptidase (EC 3.4.16.-) (Serine carboxypeptidase 1)


452


Q5VYX0


Renalase (EC 1.4.-.-)


342


Q6UXX9


R-spondin-2 (Roof plate-specific spondin-2) (hRspo2)


243


Q96DR5


Short palate, lung and nasal epithelium carcinoma-associated protein 2 (Parotid secretory protein) (PSP)


249


Q9BQ16


Testican-3 (SPARC/osteonectin, CWCV, and Kazal-like domains proteoglycan 3)


436


Q9Y5U5


Tumor necrosis factor receptor superfamily member 18 (Glucocorticoid-induced TNFR-related protein) (Activation-inducible TNFR family receptor)


241


Q8TCV5


WAP four-disulfide core domain protein 5 (Putative protease inhibitor WAP1) (p53-responsive gene 5 protein)


224


Q9NRA1


Platelet-derived growth factor C (PDGF-C) (Spinal cord-derived growth factor) (SCDGF) (Fallotein) (VEGF-E) [Cleaved into: Platelet-derived growth 


factor C, latent form (PDGFC latent form); Platelet-derived growth factor C, receptor-binding form (PDGFC receptor-binding form)]


345


Q9BZ11


Disintegrin and metalloproteinase domain-containing protein 33 (ADAM 33) (EC 3.4.24.-)


813


O60513


Beta-1,4-galactosyltransferase 4 (Beta-1,4-GalTase 4) (Beta4Gal-T4) (b4Gal-T4) (EC 2.4.1.-) (UDP-galactose:beta-N-acetylglucosamine


beta-1,4-galactosyltransferase 4) (UDP-Gal:beta-GlcNAc beta-1,4-galactosyltransferase 4) [Includes: N-acetyllactosamine synthase (EC 2.4.1.90)


(Nal synthetase); Beta-N-acetylglucosaminyl-glycolipid beta-1,4-galactosyltransferase (EC 2.4.1.-)]


344


Q9BWV1


Brother of CDO (Protein BOC)


1,114


Q9NW68


BSD domain-containing protein 1


430


Q8N126


Cell adhesion molecule 3 (Immunoglobulin superfamily member 4B) (Nectin-like protein 1) (TSLC1-like protein 1) (Synaptic cell adhesion 


(molecule 3) > Brain immunoglobulin receptor)


398


O75419


CDC45-related protein (Cdc45) (PORC-PI-1)


566


Q9H9P2


Chondrolectin (Transmembrane protein MT75)


273


Q96PD7


Diacylglycerol O-acyltransferase 2 (EC 2.3.1.20) (Diglyceride acyltransferase 2)


388


P56937


3-keto-steroid reductase (EC 1.1.1.270) (Estradiol 17-beta-dehydrogenase 7) (EC 1.1.1.62) (17-beta-hydroxysteroid dehydrogenase 7) (17-beta-HSD 7)


341


Q9BPW9


Dehydrogenase/reductase SDR family member 9 (EC 1.1.-.-) (3-alpha hydroxysteroid dehydrogenase) (3alpha-HSD) (Short-chain dehydrogenase/


reductase retSDR8) (NADP-dependent retinol dehydrogenase/reductase) (RDH-E2) (RDHL)


319


O00548


Delta-like protein 1 (Drosophila Delta homolog 1) (Delta1) (H-Delta-1)


723


P20827


Ephrin-A1 (EPH-related receptor tyrosine kinase ligand 1) (LERK-1) (Immediate early response protein B61) (Tumor necrosis factor, alpha-induced 


protein 4)


205


Q8N441


Fibroblast growth factor receptor-like 1 (FGF receptor-like protein 1) (Fibroblast growth factor receptor 5) (FGFR-like protein) (FGF homologous


factor receptor)


504


O60609


GDNF family receptor alpha-3 (GFR-alpha-3)


400


Q9H7M9


Platelet receptor Gi24


311


Q9BQS7


Hephaestin (EC 1.-.-.-)


1,158


Q8NAC3


Interleukin-17 receptor C (IL-17 receptor C) (IL-17RC) (Interleukin-17 receptor-like protein) (IL-17RL) (Interleukin-17 receptor homolog) (IL17Rhom)


791


Q8NFM7


Interleukin-17 receptor D (IL-17 receptor D) (IL-17RD) (IL17Rhom) (Interleukin-17 receptor-like protein) (Sef homolog) (hSef)


739


Q8N201


Integrator complex subunit 1 (Int1)


2,190


Q86YT9


Junctional adhesion molecule-like (Dendritic cell-specific protein CREA7-1) (Adhesion molecule interacting with CXADR antigen 1)


394


P49862


Kallikrein-7 (hK7) (EC 3.4.21.117) (Stratum corneum chymotryptic enzyme) (hSCCE) (Serine protease 6)


253


Q13753


Laminin subunit gamma-2 (Laminin 5 gamma 2 subunit) (Kalinin/nicein/epiligrin 100 kDa subunit) (Laminin B2t chain) (Cell-scattering factor 140 kDa 


subunit) (CSF 140 kDa subunit) (Large adhesive scatter factor 140 kDa subunit) (Ladsin 140 kDa subunit)


1,193


Q9H0V9


VIP36-like protein (Lectin mannose-binding 2-like) (LMAN2-like protein)


348


Q6UX01


Protein LMBR1L (Lipocalin-1-interacting membrane receptor) (Lipocalin-interacting membrane receptor) (Limb region 1 protein homolog-like)


489


Q8TBB1


E3 ubiquitin-protein ligase LNX (EC 6.3.2.-) (Numb-binding protein 1) (Ligand of Numb-protein X 1)


728


Q86VH5


Leucine-rich repeat transmembrane neuronal protein 3


581


Q9UKM7


Endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase (EC 3.2.1.113) (ER alpha-1,2-mannosidase) (Mannosidase alpha class 1B


member 1) (Man9GlcNAc2-specific-processing alpha-mannosidase)


699


Q9H0U3


Magnesium transporter protein 1 (MagT1) (Implantation-associated protein) (IAP)


335


Q9NZL9


Methionine adenosyltransferase 2 subunit beta (Methionine adenosyltransferase II beta) (MAT II beta) (Methionine adenosyltransferase 2 beta subunit)


(DTDP-4-keto-6-deoxy-D-glucose 4-reductase)


334


Q5SSG8


Mucin-21 (MUC-21) (Epiglycanin)


566


Q9NZ53


Podocalyxin-like protein 2 (Endoglycan)


605


Q8N271


Prominin-2 (PROM-2) (Prominin-like protein 2) (hPROML2)


834


Q6UX71


Plexin domain-containing protein 2 (Tumor endothelial marker 7-related protein)


529


Q8WZ75


Roundabout homolog 4 (Magic roundabout)


1,007


Q9NPR2


Semaphorin-4B


832


Q9P283


Semaphorin-5B


1,151


Q9NRX5


Serine incorporator 1 (Tumor differentially expressed protein 2) (Tumor differentially expressed 1 protein-like)


453


O43556


Epsilon-sarcoglycan (Epsilon-SG)


437


Q9UNP4


Lactosylceramide alpha-2,3-sialyltransferase (EC 2.4.99.9) (CMP-NeuAc:lactosylceramide alpha-2,3-sialyltransferase) (Ganglioside GM3 synthase) 


(ST3Gal V) (Sialyltransferase 9)


418


Q8NBJ9


SID1 transmembrane family member 2


832


P29508


Serpin B3 (Squamous cell carcinoma antigen 1) (SCCA-1) (Protein T4-A)


390


Q6UWL2


Sushi domain-containing protein 1


747


Q9UL54


Serine/threonine-protein kinase TAO2 (EC 2.7.11.1) (Thousand and one amino acid protein 2) (Prostate-derived STE20-like kinase 1) (PSK-1)


(Kinase from chicken homolog C) (hKFC-C)


1,235


Q9BXR5


Toll-like receptor 10 (CD antigen CD290)


811


Q9NR97


Toll-like receptor 8 (CD antigen CD288)


1,041


O15393


Transmembrane protease, serine 2 (EC 3.4.21.-) (Serine protease 10) [Cleaved into: Transmembrane protease, serine 2 non-catalytic chain; 


Transmembrane protease, serine 2 catalytic chain]


492


Q9BVT8


Transmembrane and ubiquitin-like domain-containing protein 1 (Hepatocyte odd protein shuttling protein) (Ubiquitin-like protein SB144) 


(Ubiquitin-like protein DULP)


246


Q9Y320


Thioredoxin-related transmembrane protein 2 (Thioredoxin domain-containing protein 14) (Proliferation-inducing gene 26 protein)


296


Q15661


Tryptase beta-1 (Tryptase-1) (EC 3.4.21.59) (Tryptase I)


275


Q9BZJ3


Tryptase delta (EC 3.4.21.59) (Delta-tryptase) (Mast cell mMCP-7-like) (Tryptase-3) (HmMCP-3-like tryptase III)


242


Q8IZJ1


Netrin receptor UNC5B (Protein unc-5 homolog B) (Unc-5 homolog 2) (p53-regulated receptor for death and life protein 1)


945


Q8NBZ7


UDP-glucuronic acid decarboxylase 1 (EC 4.1.1.35) (UDP-glucuronate decarboxylase 1) (UXS-1) (UGD)


420


Q9BQB6


Vitamin K epoxide reductase complex subunit 1 (EC 1.1.4.1) (Vitamin K1 2,3-epoxide reductase subunit 1)


163


Q9BXJ1


Complement C1q tumor necrosis factor-related protein 1 (G protein-coupled receptor-interacting protein) (GIP)


281


Q9BRX8


Uncharacterized protein C10orf58


229


Q9UHF0


Tachykinin-3 (ZNEUROK1) [Cleaved into: Neurokinin-B (NKB) (Neuromedin-K)]


121


Q4W597


Secreted phosphoprotein 1 (cDNA FLJ78337, highly similar to Homo sapiens secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early 


T-lymphocyte activation 1), transcript variant 1, mRNA) (Putative uncharacterized protein SPP1) (Secreted phosphoprotein 1 (Osteopontin, bone


sialoprotein I, early T-lymphocyte activation 1), isoform CRA_b)


314


Q9BQB4


Sclerostin


213


Q86TH1


ADAMTS-like protein 2 (ADAMTSL-2)


951


P59510


A disintegrin and metalloproteinase with thrombospondin motifs 20 (ADAMTS-20) (ADAM-TS 20) (ADAM-TS20) (EC 3.4.24.-)


1,910


Q9NZK5


Cat eye syndrome critical region protein 1


511


A5D8T8


C-type lectin domain family 18 member A (Mannose receptor-like protein 2)


446


Q9P218


Collagen alpha-1(XX) chain


1,329


Q9BU40


Chordin-like protein 1 (Neuralin-1) (Ventroptin) (Neurogenesin-1)


450


P54108


Cysteine-rich secretory protein 3 (CRISP-3) (SGP28 protein)


245


Q9HC73


Cytokine receptor-like factor 2 (Thymic stromal lymphopoietin protein receptor) (TSLP receptor) (Cytokine receptor-like 2) (CRL2) (IL-XR)


371


P81534


Beta-defensin 103 (Defensin, beta 103) (Beta-defensin 3) (BD-3) (hBD-3) (HBD3) (DEFB-3) (Defensin-like protein)


67


Q8N104


Beta-defensin 106 (Defensin, beta 106) (Beta-defensin 6) (BD-6) (DEFB-6)


65


O43820


Hyaluronidase-3 (Hyal-3) (EC 3.2.1.35) (Hyaluronoglucosaminidase-3) (LUCA-3)


417


Q8IU57


Interleukin-28 receptor alpha chain (IL-28R-alpha) (IL-28RA) (Cytokine receptor class-II member 12) (Cytokine receptor family 2 member 12)


(CRF2-12) (Interferon lambda receptor 1) (IFN-lambda R1) (Likely interleukin or cytokine receptor 2)


520


Q9NRE1


Matrix metalloproteinase-26 (MMP-26) (EC 3.4.24.-) (Matrilysin-2) (Endometase)


261


P41271


Neuroblastoma suppressor of tumorigenicity 1 (Zinc finger protein DAN) (N03) (DAN domain family member 1)


180


Q9NY56


Odorant-binding protein 2a (OBPIIa)


170


Q8IUK5


Plexin domain-containing protein 1 (Tumor endothelial marker 7) (Tumor endothelial marker 3)


500


Q2MKA7


R-spondin-1 (Roof plate-specific spondin-1) (hRspo1)


263


Q92824


Proprotein convertase subtilisin/kexin type 5 (EC 3.4.21.-) (Proprotein convertase PC5) (Subtilisin/kexin-like protease PC5) (hPC6) (PC6)


913


P12821


Angiotensin-converting enzyme (ACE) (EC 3.4.15.1) (EC 3.2.1.-) (Dipeptidyl carboxypeptidase I) (Kininase II) (CD antigen CD143) [Cleaved into:


Angiotensin-converting enzyme, soluble form]


1,306


P01008


Antithrombin-III (ATIII)


464


P04114


Apolipoprotein B-100 (Apo B-100) [Cleaved into: Apolipoprotein B-48 (Apo B-48)]


4,563


P05090


Apolipoprotein D (Apo-D) (ApoD)


189


P02649


Apolipoprotein E (Apo-E)


317


P15291


Beta-1,4-galactosyltransferase 1 (Beta-1,4-GalTase 1) (Beta4Gal-T1) (b4Gal-T1) (EC 2.4.1.-) (UDP-galactose:beta-N-acetylglucosamine


beta-1,4-galactosyltransferase 1) (UDP-Gal:beta-GlcNAc beta-1,4-galactosyltransferase 1) [Cleaved into: Processed beta-1,4-galactosyltransferase 1]


[Includes: Lactose synthase A protein (EC 2.4.1.22); N-acetyllactosamine synthase (EC 2.4.1.90) (Nal synthetase); Beta-N-


acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase (EC 2.4.1.38); Beta-N-acetylglucosaminyl-glycolipid beta-1,4-galactosyltransferase


(EC 2.4.1.-)]


398


Q15582


Transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) (Kerato-epithelin) (RGD-containing collagen-associated protein) (RGD-CAP)


683


P18075


Bone morphogenetic protein 7 (BMP-7) (Osteogenic protein 1) (OP-1) (Eptotermin alfa)


431


P02746


Complement C1q subcomponent subunit B


251


P04003


C4b-binding protein alpha chain (C4bp) (Proline-rich protein) (PRP)


597


P27797


Calreticulin (CRP55) (Calregulin) (HACBP) (ERp60) (grp60)


417


P08185


Corticosteroid-binding globulin (CBG) (Transcortin) (Serpin A6)


405


P15085


Carboxypeptidase A1 (EC 3.4.17.1)


419


P48052


Carboxypeptidase A2 (EC 3.4.17.15)


417


P51671


Eotaxin (C-C motif chemokine 11) (Small-inducible cytokine A11) (Eosinophil chemotactic protein)


97


Q99616


C-C motif chemokine 13 (Small-inducible cytokine A13) (Monocyte chemoattractant protein 4) (Monocyte chemotactic protein 4) (MCP-4)


(CK-beta-10) (NCC-1) [Cleaved into: C-C motif chemokine 13, long chain; C-C motif chemokine 13, medium chain; C-C motif chemokine 13, 


short chain]


98


P55774


C-C motif chemokine 18 (Small-inducible cytokine A18) (Macrophage inflammatory protein 4) (MIP-4) (Pulmonary and activation-regulated chemokine)


(CC chemokine PARC) (Alternative macrophage activation-associated CC chemokine 1) (AMAC-1) (Dendritic cell chemokine 1) (DC-CK1) 


[Cleaved into: CCL18(1-68); CCL18(3-69); CCL18(4-69)]


89


P78556


C-C motif chemokine 20 (Small-inducible cytokine A20) (Macrophage inflammatory protein 3 alpha) (MIP-3-alpha) (Liver and activation-regulated


chemokine) (CC chemokine LARC) (Beta chemokine exodus-1) [Cleaved into: CCL20(1-67); CCL20(1-64); CCL20(2-70)]


96


P55773


C-C motif chemokine 23 (Small-inducible cytokine A23) (Macrophage inflammatory protein 3) (MIP-3) (Myeloid progenitor inhibitory factor 1)


(MPIF-1) (CK-beta-8) (CKB-8) [Cleaved into: CCL23(19-99); CCL23(22-99); CCL23(27-99); CCL23(30-99)]


120


P13500


C-C motif chemokine 2 (Small-inducible cytokine A2) (Monocyte chemoattractant protein 1) (Monocyte chemotactic protein 1) (MCP-1) 


(Monocyte chemotactic and activating factor) (MCAF) (Monocyte secretory protein JE) (HC11)


99


P29965


CD40 ligand (CD40-L) (Tumor necrosis factor ligand superfamily member 5) (TNF-related activation protein) (TRAP) (T-cell antigen Gp39) 


(CD antigen CD154) [Cleaved into: CD40 ligand, membrane form; CD40 ligand, soluble form]


261


Q15517


Corneodesmosin (S protein)


529


P00746


Complement factor D (EC 3.4.21.46) (C3 convertase activator) (Properdin factor D) (Adipsin)


253


P10645


Chromogranin-A (CgA) (Pituitary secretory protein I) (SP-I) [Cleaved into: Vasostatin-1 (Vasostatin I); Vasostatin-2 (Vasostatin II); EA-92; ES-43;


Pancreastatin; SS-18; WA-8; WE-14; LF-19; AL-11; GV-19; GR-44; ER-37]


457


P02452


Collagen alpha-1(I) chain (Alpha-1 type I collagen)


1,464


P01024


Complement C3 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 1) [Cleaved into: Complement C3 beta chain; Complement C3


alpha chain; C3a anaphylatoxin; Complement C3b alpha' chain; Complement C3c alpha' chain fragment 1; Complement C3dg fragment;


Complement C3g fragment; Complement C3d fragment; Complement C3f fragment; Complement C3c alpha' chain fragment 2]


1,663


P08572


Collagen alpha-2(IV) chain [Cleaved into: Canstatin]


1,712


P53420


Collagen alpha-4(IV) chain


1,690


P0C0L4


Complement C4-A (Acidic complement C4) (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 2) [Cleaved into: Complement C4


beta chain; Complement C4-A alpha chain; C4a anaphylatoxin; C4b-A; C4d-A; Complement C4 gamma chain]


1,744


P0C0L5


Complement C4-B (Basic complement C4) (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 3) [Cleaved into: Complement C4 


beta chain; Complement C4-B alpha chain; C4a anaphylatoxin; C4b-B; C4d-B; Complement C4 gamma chain]


1,744


P05997


Collagen alpha-2(V) chain


1,499


P01031


Complement C5 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 4) [Cleaved into: Complement C5 beta chain; Complement


C5 alpha chain; C5a anaphylatoxin; Complement C5 alpha' chain]


1,676


Q02388


Collagen alpha-1(VII) chain (Long-chain collagen) (LC collagen)


2,944


P10643


Complement component C7


843


P07358


Complement component C8 beta chain (Complement component 8 subunit beta)


591


P07360


Complement component C8 gamma chain


202


P39059


Collagen alpha-1(XV) chain [Cleaved into: Endostatin (Endostatin-XV) (Restin) (Related to endostatin)]


1,388


Q07092


Collagen alpha-1(XVI) chain


1,604


P39060


Collagen alpha-1(XVIII) chain [Cleaved into: Endostatin]


1,754


Q14993


Collagen alpha-1(XIX) chain (Collagen alpha-1(Y) chain)


1,142


P49747


Cartilage oligomeric matrix protein (COMP)


757


P02741


C-reactive protein [Cleaved into: C-reactive protein(1-205)]


224


P04141


Granulocyte-macrophage colony-stimulating factor (GM-CSF) (Colony-stimulating factor) (CSF) (Sargramostim) (Molgramostin)


144


P09919


Granulocyte colony-stimulating factor (G-CSF) (Pluripoietin) (Filgrastim) (Lenograstim)


207


O00622


Protein CYR61 (Cysteine-rich angiogenic inducer 61) (Insulin-like growth factor-binding protein 10) (Protein GIG1)


381


P01040


Cystatin-A (Cystatin-AS) (Stefin-A)


98


P01036


Cystatin-S (Cystatin-4) (Salivary acidic protein 1) (Cystatin-SA-III)


141


P59666


Neutrophil defensin 3 (HNP-3) (HP-3) (HP3) (Defensin, alpha 3) [Cleaved into: HP 3-56; Neutrophil defensin 2 (HNP-2) (HP-2) (HP2)]


94


P27487


Dipeptidyl peptidase 4 (EC 3.4.14.5) (Dipeptidyl peptidase IV) (DPP IV) (T-cell activation antigen CD26) (TP103) (Adenosine deaminase complexing


protein 2) (ADABP)(CD antigen CD26) [Cleaved into: Dipeptidyl peptidase 4 membrane form (Dipeptidyl peptidase IV membrane form); Dipeptidyl


peptidase 4 soluble form (Dipeptidyl peptidase IV soluble form)]


766


Q9NZW4


Dentin sialophosphoprotein [Cleaved into: Dentin phosphoprotein (Dentin phosphophoryn) (DPP); Dentin sialoprotein (DSP)]


1,301


P05305


Endothelin-1 (Preproendothelin-1) (PPET1) [Cleaved into: Endothelin-1 (ET-1); Big endothelin-1]


212


P98172


Ephrin-B1 (EPH-related receptor tyrosine kinase ligand 2) (LERK-2) (ELK ligand) (ELK-L) (EFL-3)


346


P19957


Elafin (Elastase-specific inhibitor) (ESI) (Skin-derived antileukoproteinase) (SKALP) (Peptidase inhibitor 3) (WAP four-disulfide core domain


protein 14) (Protease inhibitor WAP3)


117


Q9Y6C2


EMILIN-1 (Elastin microfibril interface-located protein 1) (Elastin microfibril interfacer 1)


1,016


P14625


Endoplasmin (Heat shock protein 90 kDa beta member 1) (94 kDa glucose-regulated protein) (GRP94) (gp96 homolog) (Tumor rejection antigen 1)


803


P29320


Ephrin type-A receptor 3 (EC 2.7.10.1) (Tyrosine-protein kinase receptor ETK1) (HEK) (HEK4) (Tyrosine-protein kinase TYRO4)


983


O15197


Ephrin type-B receptor 6 (Tyrosine-protein kinase-defective receptor EPH-6) (HEP)


1,006


P01588


Erythropoietin (Epoetin)


193


P00742


Coagulation factor X (EC 3.4.21.6) (Stuart factor) (Stuart-Prower factor) [Cleaved into: Factor X light chain; Factor X heavy chain; Activated factor 


Xa heavy chain]


488


P00451


Coagulation factor VIII (Procoagulant component) (Antihemophilic factor) (AHF) [Cleaved into: Factor VIIIa heavy chain, 200 kDa isoform; 


Factor VIIIa heavy chain, 92 kDa isoform; Factor VIII B chain; Factor VIIIa light chain]


2,351


P35555


Fibrillin-1


2,871


P35556


Fibrillin-2


2,912


P02765


Alpha-2-HS-glycoprotein (Ba-alpha-2-glycoprotein) (Alpha-2-Z-globulin) (Fetuin-A) [Cleaved into: Alpha-2-HS-glycoprotein chain A; 


Alpha-2-HS-glycoprotein chain B]


367


O15520


Fibroblast growth factor 10 (FGF-10) (Keratinocyte growth factor 2)


208


P02671


Fibrinogen alpha chain [Cleaved into: Fibrinopeptide A]


866


P02675


Fibrinogen beta chain [Cleaved into: Fibrinopeptide B]


491


P01225


Follitropin subunit beta (Follicle-stimulating hormone beta subunit) (FSH-beta) (FSH-B) (Follitropin beta chain)


129


P01350


Gastrin [Cleaved into: Gastrin-71 (Gastrin component I); Gastrin-52 (G52); Big gastrin (Gastrin-34) (G34) (Gastrin component II); Gastrin (Gastrin-17) 


(G17) (Gastrin component III); Gastrin-14 (G14); Gastrin-6 (G6)]


101


P01215


Glycoprotein hormones alpha chain (Anterior pituitary glycoprotein hormones common subunit alpha) (Follitropin alpha chain) (Follicle-stimulating 


hormone alpha chain) (FSH-alpha) (Lutropin alpha chain) (Luteinizing hormone alpha chain) (LSH-alpha) (Thyrotropin alpha chain)


(Thyroid-stimulating hormone alpha chain) (TSH-alpha) (Choriogonadotropin alpha chain) (Chorionic gonadotrophin subunit alpha) (CG-alpha)


116


Q3T906


N-acetylglucosamine-1-phosphotransferase subunits alpha/beta (EC 2.7.8.17) (GlcNAc-1-phosphotransferase subunits alpha/beta) (Stealth protein


GNPTAB) (UDP-N-acetylglucosamine-1-phosphotransferase subunits alpha/beta) [Cleaved into: N-acetylglucosamine-1-phosphotransferase subunit


alpha; N-acetylglucosamine-1-phosphotransferase subunit beta]


1,256


P12544


Granzyme A (EC 3.4.21.78) (Granzyme-1) (Cytotoxic T-lymphocyte proteinase 1) (Hanukkah factor) (H factor) (HF) (CTL tryptase) (Fragmentin-1)


262


P26927


Hepatocyte growth factor-like protein (Macrophage stimulatory protein) (Macrophage-stimulating protein) (MSP) [Cleaved into: Hepatocyte growth


factor-like protein alpha chain; Hepatocyte growth factor-like protein beta chain]


711


P10997


Islet amyloid polypeptide (Amylin) (Diabetes-associated peptide) (DAP) (Insulinoma amyloid peptide)


89


P08833


Insulin-like growth factor-binding protein 1 (IGF-binding protein 1) (IGFBP-1) (IBP-1) (Placental protein 12) (PP12)


259


P18065


Insulin-like growth factor-binding protein 2 (IGF-binding protein 2) (IGFBP-2) (IBP-2)


328


P22692


Insulin-like growth factor-binding protein 4 (IGF-binding protein 4) (IGFBP-4) (IBP-4)


258


Q9BYX4


Interferon-induced helicase C domain-containing protein 1 (EC 3.6.1.-) (Interferon-induced with helicase C domain protein 1) (Helicase with


2 CARD domains) (Helicard) (Melanoma differentiation-associated protein 5) (MDA-5) (RNA helicase-DEAD box protein 116) 


(Murabutide down-regulated protein)


1,025


P01562


Interferon alpha-1/13 (Interferon alpha-D) (LeIF D)


189


P01563


Interferon alpha-2 (Interferon alpha-A) (LeIF A)


188


P01574


Interferon beta (IFN-beta) (Fibroblast interferon)


187


P01579


Interferon gamma (IFN-gamma) (Immune interferon)


166


P05019


Insulin-like growth factor IB (IGF-IB) (Somatomedin-C) (Mechano growth factor) (MGF)


195


Q14623


Indian hedgehog protein (IHH) (HHG-2) [Cleaved into: Indian hedgehog protein N-product; Indian hedgehog protein C-product]


411


P22301


Interleukin-10 (IL-10) (Cytokine synthesis inhibitory factor) (CSIF)


178


P35225


Interleukin-13 (IL-13)


146


P60568


Interleukin-2 (IL-2) (T-cell growth factor) (TCGF) (Aldesleukin)


153


P05113


Interleukin-5 (IL-5) (T-cell replacing factor) (TRF) (Eosinophil differentiation factor) (B-cell differentiation factor I)


134


P08476


Inhibin beta A chain (Activin beta-A chain) (Erythroid differentiation protein) (EDF)


426


P00995


Pancreatic secretory trypsin inhibitor (Serine protease inhibitor Kazal-type 1) (Tumor-associated trypsin inhibitor) (TATI)


79


P19827


Inter-alpha-trypsin inhibitor heavy chain H1 (Inter-alpha-inhibitor heavy chain 1) (ITI heavy chain H1) (Inter-alpha-trypsin inhibitor complex


component III) (Serum-derived hyaluronan-associated protein) (SHAP)


911


P19823


Inter-alpha-trypsin inhibitor heavy chain H2 (Inter-alpha-inhibitor heavy chain 2) (ITI heavy chain H2) (Inter-alpha-trypsin inhibitor complex


component II) (Serum-derived hyaluronan-associated protein) (SHAP)


946


Q06033


Inter-alpha-trypsin inhibitor heavy chain H3 (Inter-alpha-inhibitor heavy chain 3) (ITI heavy chain H3) (ITI-HC3) (Serum-derived 


hyaluronan-associated protein) (SHAP)


890


Q15726


Metastasis-suppressor KiSS-1 (Kisspeptin-1) [Cleaved into: Metastin (Kisspeptin-54); Kisspeptin-14; Kisspeptin-13; Kisspeptin-10]


145


P07288


Prostate-specific antigen (PSA) (EC 3.4.21.77) (Kallikrein-3) (Semenogelase) (Gamma-seminoprotein) (Seminin) (P-30 antigen)


261


Q9Y5K2


Kallikrein-4 (EC 3.4.21.-) (Prostase) (Kallikrein-like protein 1) (KLK-L1) (Enamel matrix serine proteinase 1) (Serine protease 17)


254


P03952


Plasma kallikrein (EC 3.4.21.34) (Plasma prekallikrein) (Kininogenin) (Fletcher factor) [Cleaved into: Plasma kallikrein heavy chain; 


Plasma kallikrein light chain]


638


P18428


Lipopolysaccharide-binding protein (LBP)


481


P09382


Galectin-1 (Lectin galactoside-binding soluble 1) (Beta-galactoside-binding lectin L-14-I) (Lactose-binding lectin 1) (S-Lac lectin 1) (Galaptin)


(14 kDa lectin) (HPL) (HBL) (Putative MAPK-activating protein PM12)


135


P41159


Leptin (Obesity factor) (Obese protein)


167


P15018


Leukemia inhibitory factor (LIF) (Differentiation-stimulating factor) (D factor) (Melanoma-derived LPL inhibitor) (MLPLI) (Emfilermin)


202


P11150


Hepatic triacylglycerol lipase (Hepatic lipase) (HL) (EC 3.1.1.3) (Lipase member C)


499


P16233


Pancreatic triacylglycerol lipase (Pancreatic lipase) (PL) (EC 3.1.1.3)


465


Q05315


Eosinophil lysophospholipase (EC 3.1.1.5) (Charcot-Leyden crystal protein) (Lysolecithin acylhydrolase) (CLC) (Galectin-10)


142


P01229


Lutropin subunit beta (Lutropin beta chain) (Luteinizing hormone subunit beta) (LSH-beta) (LSH-B) (LH-B)


141


P55145


Mesencephalic astrocyte-derived neurotrophic factor (Protein ARMET) (Arginine-rich protein)


179


P55081


Microfibrillar-associated protein 1


439


P08493


Matrix Gla protein (MGP) (Cell growth-inhibiting gene 36 protein)


103


P08253


72 kDa type IV collagenase (EC 3.4.24.24) (72 kDa gelatinase) (Matrix metalloproteinase-2) (MMP-2) (Gelatinase A) (TBE-1)


660


P08254


Stromelysin-1 (SL-1) (EC 3.4.24.17) (Matrix metalloproteinase-3) (MMP-3) (Transin-1)


477


P22894


Neutrophil collagenase (EC 3.4.24.34) (Matrix metalloproteinase-8) (MMP-8) (PMNL collagenase) (PMNL-CL)


467


Q13421


Mesothelin (Pre-pro-megakaryocyte-potentiating factor) (CAK1 antigen) [Cleaved into: Megakaryocyte-potentiating factor (MPF); Mesothelin, 


cleaved form]


630


P98088


Mucin-5AC (Mucin-5 subtype AC, tracheobronchial) (Tracheobronchial mucin) (TBM) (Major airway glycoprotein) (Gastric mucin) (Lewis B


blood group antigen) (LeB) (Fragments)


5,030


Q6W4X9


Mucin-6 (MUC-6) (Gastric mucin-6)


2,392


Q00604


Norrin (Norrie disease protein) (X-linked exudative vitreoretinopathy 2 protein)


133


P01178


Oxytocin-neurophysin 1 (OT-NPI) [Cleaved into: Oxytocin (Ocytocin); Neurophysin 1]


125


P01138


Beta-nerve growth factor (Beta-NGF)


241


Q13253


Noggin


232


P20783


Neurotrophin-3 (NT-3) (Neurotrophic factor) (HDNF) (Nerve growth factor 2) (NGF-2)


257


P13725


Oncostatin-M (OSM)


252


P05121


Plasminogen activator inhibitor 1 (PAI-1) (PAI) (Endothelial plasminogen activator inhibitor)


402


P05120


Plasminogen activator inhibitor 2 (PAI-2) (Placental plasminogen activator inhibitor) (Monocyte Arg-serpin) (Urokinase inhibitor)


415


Q15113


Procollagen C-endopeptidase enhancer 1 (Procollagen COOH-terminal proteinase enhancer 1) (Procollagen C-proteinase enhancer 1) (PCPE-1)


(Type I procollagen COOH-terminal proteinase enhancer) (Type 1 procollagen C-proteinase enhancer protein)


449


P04085


Platelet-derived growth factor subunit A (PDGF subunit A) (Platelet-derived growth factor A chain) (Platelet-derived growth factor alpha polypeptide)


(PDGF-1)


211


P01127


Platelet-derived growth factor subunit B (PDGF subunit B) (Platelet-derived growth factor B chain) (Platelet-derived growth factor beta polypeptide)


(PDGF-2) (c-sis) (Becaplermin)


241


P07237


Protein disulfide-isomerase (PDI) (EC 5.3.4.1) (Prolyl 4-hydroxylase subunit beta) (Cellular thyroid hormone-binding protein) (p55)


508


P36955


Pigment epithelium-derived factor (PEDF) (Serpin-F1) (EPC-1)


418


P00790


Pepsin A (EC 3.4.23.1)


388


P20142


Gastricsin (EC 3.4.23.3) (Pepsinogen C)


388


P98160


Basement membrane-specific heparan sulfate proteoglycan core protein (HSPG) (Perlecan) (PLC)


4,391


Q96LB9


Peptidoglycan recognition protein I-alpha (PGRP-I-alpha) (Peptidoglycan recognition protein intermediate alpha) (PGLYRPIalpha) (Peptidoglycan


recognition protein 3)


341


P21810


Biglycan (Bone/cartilage proteoglycan I) (PG-S1)


368


P12273


Prolactin-inducible protein (Prolactin-induced protein) (Secretory actin-binding protein) (SABP) (Gross cystic disease fluid protein 15)


(GCDFP-15) (gp17)


146


P02776


Platelet factor 4 (PF-4) (C-X-C motif chemokine 4) (Oncostatin-A) (Iroplact) [Cleaved into: Platelet factor 4, short form]


101


P00747


Plasminogen (EC 3.4.21.7) [Cleaved into: Plasmin heavy chain A; Activation peptide; Angiostatin; Plasmin heavy chain A, short form;


Plasmin light chain B]


810


P27169


Serum paraoxonase/arylesterase 1 (PON 1) (EC 3.1.1.2) (EC 3.1.8.1) (Serum aryldialkylphosphatase 1) (A-esterase 1) (Aromatic esterase 1) (K-45)


355


P05187


Alkaline phosphatase, placental type (EC 3.1.3.1) (PLAP-1) (Alkaline phosphatase Regan isozyme)


535


P23284


Peptidyl-prolyl cis-trans isomerase B (PPIase) (Rotamase) (EC 5.2.1.8) (Cyclophilin B) (S-cyclophilin) (SCYLP) (CYP-S1)


216


P13727


Bone marrow proteoglycan (BMPG) (Proteoglycan 2) [Cleaved into: Eosinophil granule major basic protein (EMBP) (MBP) (Pregnancy-associated


major basic protein)]


222


P04280


Basic salivary proline-rich protein 1 (Salivary proline-rich protein) [Cleaved into: Proline-rich peptide II-2; Basic peptide IB-6; Peptide P-H]


392


P11686


Pulmonary surfactant-associated protein C (SP-C) (SP5) (Pulmonary surfactant-associated proteolipid SPL(Val))


197


P01270


Parathyroid hormone (PTH) (Parathyrin) (Parathormone)


115


P02743


Serum amyloid P-component (SAP) (9.5S alpha-1-glycoprotein) [Cleaved into: Serum amyloid P-component(1-203)]


223


P05060


Secretogranin-1 (Secretogranin I) (SgI) (Chromogranin-B) (CgB) [Cleaved into: GAWK peptide; CCB peptide]


677


Q15465


Sonic hedgehog protein (SHH) (HHG-1) [Cleaved into: Sonic hedgehog protein N-product; Sonic hedgehog protein C-product]


462


P03973


Antileukoproteinase (ALP) (Secretory leukocyte protease inhibitor) (HUSI-1) (Seminal proteinase inhibitor) (BLPI) (Mucus proteinase inhibitor) (MPI) 


(WAP four-disulfide core domain protein 4) (Protease inhibitor WAP4)


132


Q9UQE7


Structural maintenance of chromosomes protein 3 (Chondroitin sulfate proteoglycan 6) (Chromosome-associated polypeptide) (hCAP) (Basement


membrane-associatedchondroitin proteoglycan) (Bamacan)


1,217


P08294


Extracellular superoxide dismutase [Cu-Zn](EC-SOD) (EC 1.15.1.1)


240


P01241


Somatotropin (Growth hormone) (GH) (GH-N) (Pituitary growth hormone) (Growth hormone 1)


217


P36952


Serpin B5 (Protease inhibitor 5) (Maspin)


375


Q9HCB6


Spondin-1 (F-spondin) (Vascular smooth muscle cell growth-promoting factor)


807


Q9NY15


Stabilin-1 (Fasciclin, EGF-like, laminin-type EGF-like and link domain-containing scavenger receptor 1) (FEEL-1) (MS-1 antigen)


2,570


Q16623


Syntaxin-1A (Neuron-specific antigen HPC-1)


288


P05452


Tetranectin (TN) (C-type lectin domain family 3 member B) (Plasminogen kringle 4-binding protein)


202


P01137


Transforming growth factor beta-1 (TGF-beta-1) [Cleaved into: Latency-associated peptide (LAP)]


390


P01266


Thyroglobulin (Tg)


2,768


P01033


Metalloproteinase inhibitor 1 (Tissue inhibitor of metalloproteinases) (TIMP-1) (Erythroid-potentiating activity) (EPA) (Fibroblast collagenase


inhibitor) (Collagenase inhibitor)


207


P16035


Metalloproteinase inhibitor 2 (Tissue inhibitor of metalloproteinases 2) (TIMP-2) (CSC-21K)


220


P35625


Metalloproteinase inhibitor 3 (Tissue inhibitor of metalloproteinases 3) (TIMP-3) (Protein MIG-5)


211


P19438


Tumor necrosis factor receptor superfamily member 1A (p60) (TNF-R1) (TNF-RI) (TNFR-I) (p55) (CD antigen CD120a) [Cleaved into: Tumor necrosis


factor receptor superfamily member 1A, membrane form; Tumor necrosis factor-binding protein 1 (TBPI)]


455


P02788


Lactotransferrin (Lactoferrin) (EC 3.4.21.-) (Talalactoferrin) [Cleaved into: Kaliocin-1; Lactoferroxin-A; Lactoferroxin-B; Lactoferroxin-C]


710


P07477


Trypsin-1 (EC 3.4.21.4) (Trypsin I) (Cationic trypsinogen) (Serine protease 1) (Beta-trypsin) [Cleaved into: Alpha-trypsin chain 1; Alpha-trypsin chain 2]


247


P02766


Transthyretin (Prealbumin) (TBPA) (TTR) (ATTR)


147


P00749


Urokinase-type plasminogen activator (U-plasminogen activator) (uPA) (EC 3.4.21.73) [Cleaved into: Urokinase-type plasminogen activator long


chain A; Urokinase-type plasminogen activator short chain A; Urokinase-type plasminogen activator chain B]


431


P17948


Vascular endothelial growth factor receptor 1 (VEGFR-1) (EC 2.7.10.1) (Vascular permeability factor receptor) (Tyrosine-protein kinase receptor FLT) 


(Flt-1) (Tyrosine-protein kinase FRT) (Fms-like tyrosine kinase 1)


1,338


P02774


Vitamin D-binding protein (DBP) (VDB) (Group-specific component) (Gc-globulin)


474


P04004


Vitronectin (Serum-spreading factor) (S-protein) (V75) [Cleaved into: Vitronectin V65 subunit; Vitronectin V10 subunit; Somatomedin-B]


478


P04275


von Willebrand factor (vWF) [Cleaved into: von Willebrand antigen 2 (von Willebrand antigen II)]


2,813


O15537


Retinoschisin (X-linked juvenile retinoschisis protein)


224


P25311


Zinc-alpha-2-glycoprotein (Zn-alpha-2-glycoprotein) (Zn-alpha-2-GP)


295


Q9H972


Uncharacterized protein C14orf93


538


Q5SRR4


Lymphocyte antigen 6 complex locus protein G5c


150


P11465


Pregnancy-specific beta-1-glycoprotein 2 (PSBG-2) (Pregnancy-specific beta-1 glycoprotein E) (PS-beta-E)


335


B2RAD4


C-type lectin domain family 11, member A, isoform CRA_b (cDNA, FLJ94843, Homo sapiens stem cell growth factor; lymphocyte secreted C-type


lectin (SCGF), mRNA)


323


P14174


Macrophage migration inhibitory factor (MIF) (EC 5.3.2.1) (Phenylpyruvate tautomerase) (L-dopachrome tautomerase) (EC 5.3.3.12) (L-dopachrome


isomerase) (Glycosylation-inhibiting factor) (GIF)


115


Q9H553


Alpha-1,3-mannosyltransferase ALG2 (EC 2.4.1.-) (GDP-Man:Man(1)GlcNAc(2)-PP-dolichol mannosyltransferase) (Asparagine-linked glycosylation


protein 2)


416


Q9BWW8


Apolipoprotein L6 (Apolipoprotein L-VI) (ApoL-VI)


343


Q8WY98


UPF0546 membrane protein C1orf91


164


Q9NS85


Carbonic anhydrase-related protein 10 (Carbonic anhydrase-related protein X) (CA-RP X) (CARP X) (Cerebral protein 15)


328


P06307


Cholecystokinin (CCK) [Cleaved into: Cholecystokinin-58 (CCK58); Cholecystokinin-58 desnonopeptide ((1-49)-CCK58); Cholecystokinin-39


(CCK39); Cholecystokinin-33 (CCK33); Cholecystokinin-25 (CCK25); Cholecystokinin-18 (CCK18); Cholecystokinin-12 (CCK12);


Cholecystokinin-8 (CCK8); Cholecystokinin-7 (CCK7); Cholecystokinin-5 (CCK5)]


115


Q8IWY9


Codanin-1


1,227


Q6UWU4


Uncharacterized protein C6orf89


347


Q9P2E5


Chondroitin sulfate glucuronyltransferase (EC 2.4.1.226) (N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase) (Chondroitin 


glucuronyltransferase II) (CSGlcA-T)


772


Q9BWS9


Chitinase domain-containing protein 1 (Stabilin-1-interacting chitinase-like protein) (SI-CLP)


393


Q9UGQ2


Transmembrane protein C9orf7


172


Q6UXG3


CMRF35-like molecule 9 (CLM-9) (Triggering receptor expressed on myeloid cells 4) (TREM-4) (CD300 antigen-like family member G)


(CD antigen CD300g)


332


Q96SQ9


Cytochrome P450 2S1 (EC 1.14.14.1) (CYPIIS1)


504


Q9BUF7


Crumbs protein homolog 3


120


Q9Y394


Dehydrogenase/reductase SDR family member 7 (EC 1.1.-.-) (Retinal short-chain dehydrogenase/reductase 4) (retSDR4)


339


Q6UWH4


Protein ENED (Expressed in nerve and epithelium during development)


519


Q92496


Complement factor H-related protein 4 (FHR-4)


331


Q96DB9


FXYD domain-containing ion transport regulator 5 (Dysadherin)


178


Q8WTR4


Glycerophosphodiester phosphodiesterase domain-containing protein 5 (EC 3.1.-.-) (Glycerophosphodiester phosphodiesterase 2)


605


Q6ZMI3


Gliomedin


551


Q8IZF5


Probable G-protein coupled receptor 113 (G-protein coupled receptor PGR23)


1,079


Q8IZF4


Probable G-protein coupled receptor 114 (G-protein coupled receptor PGR27)


528


Q86UL3


Glycerol-3-phosphate acyltransferase 4 (GPAT4) (EC 2.3.1.15) (Acyl-CoA:glycerol-3-phosphate acyltransferase 4) (1-acylglycerol-3-phosphate


O-acyltransferase 6) (1-AGP acyltransferase 6) (1-AGPAT 6) (Lysophosphatidic acid acyltransferase zeta) (LPAAT-zeta)


456


O60565


Gremlin-1 (Cysteine knot superfamily 1, BMP antagonist 1) (Increased in high glucose protein 2) (IHG-2) (Down-regulated in Mos-transformed 


cells protein) (DAN domain family member 2) (Cell proliferation-inducing gene 2 protein)


184


Q96T54


Potassium channel subfamily K member 17 (TWIK-related alkaline pH-activated K(+) channel 2) (2P domain potassium channel Talk-2)


(TWIK-related acid-sensitive K(+) channel 4) (TASK-4)


332


Q7Z4H8


KDEL motif-containing protein 2


507


Q6UX15


Layilin


382


Q6P9F7


Leucine-rich repeat-containing protein 8B (T-cell activation leucine repeat-rich protein)


803


Q7L1W4


Leucine-rich repeat-containing protein 8D


858


Q6UXM1


Leucine-rich repeats and immunoglobulin-like domains protein 3 (LIG-3)


1,119


Q86UE6


Leucine-rich repeat transmembrane neuronal protein 1


522


Q8N2G4


Ly6/PLAUR domain-containing protein 1 (Putative HeLa tumor suppressor) (PHTS)


141


Q6UWN5


Ly6/PLAUR domain-containing protein 5


251


O95772


MLN64 N-terminal domain homolog (STARD3 N-terminal-like protein)


234


Q6UXD7


Major facilitator superfamily domain-containing protein 7 (Myosin light polypeptide 5 regulatory protein) (MYL5)


560


Q86VF5


2-acylglycerol O-acyltransferase 3 (EC 2.3.1.22) (EC 2.3.1.20) (Monoacylglycerol O-acyltransferase 3) (Acyl CoA:monoacylglycerol acyltransferase 3)


(MGAT3) (Diacylglycerol acyltransferase 2-like protein 7) (Diacylglycerol O-acyltransferase candidate 7) (hDC7)


341


Q9NZJ7


Mitochondrial carrier homolog 1 (Presenilin-associated protein)


389


Q9NX14


NADH dehydrogenase [ubiquinone]1 beta subcomplex subunit 11, mitochondrial (NADH-ubiquinone oxidoreductase ESSS subunit) (Complex I-ESSS)


(CI-ESSS) (Neuronal protein 17.3) (Np17.3) (p17.3)


153


Q9UN73


Protocadherin alpha-6 (PCDH-alpha-6)


950


O60330


Protocadherin gamma-A12 (PCDH-gamma-A12) (Cadherin-21) (Fibroblast cadherin-3)


932


Q9H490


Phosphatidylinositol glycan anchor biosynthesis class U protein (GPI transamidase component PIG-U) (Cell division cycle protein 91-like 1)


(Protein CDC91-like 1)


435


Q6NUM9


All-trans-retinol 13,14-reductase (EC 1.3.99.23) (All-trans-13,14-dihydroretinol saturase) (RetSat)


610


Q96LZ7


Regulator of microtubule dynamics protein 2 (RMD-2) (hRMD-2) (Protein FAM82A1)


410


Q2I0M5


R-spondin-4 (Roof plate-specific spondin-4) (hRspo4)


234


Q5K4L6


Long-chain fatty acid transport protein 3 (Fatty acid transport protein 3) (FATP-3) (EC 6.2.1.-) (Very long-chain acyl-CoA synthetase homolog 3)


(VLCS-3) (Solute carrier family 27 member 3)


730


Q9BRL7


Vesicle-trafficking protein SEC22c (SEC22 vesicle-trafficking protein homolog C) (SEC22 vesicle-trafficking protein-like 3)


303


Q11201


CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase (Beta-galactoside alpha-2,3-sialyltransferase) (Alpha 2,3-ST) (EC 2.4.99.4)


(Gal-NAc6S) (Gal-beta-1,3-GalNAc-alpha-2,3-sialyltransferase) (ST3GalA.1) (ST3GalIA) (ST3Gal I) (ST3O) (SIAT4-A) (SIATFL)


340


O43699


Sialic acid-binding Ig-like lectin 6 (Siglec-6) (Obesity-binding protein 1) (OB-BP1) (CD33 antigen-like 1) (CDw327) (CD antigen CD327)


453


Q96A28


SLAM family member 9 (CD2 family member 10) (CD2F-10) (CD84 homolog 1) (CD84-H1)


289


P02814


Submaxillary gland androgen-regulated protein 3B (Proline-rich protein 3) (Proline-rich peptide P-B) [Cleaved into: Peptide P-A; Peptide D1A]


79Q9NY57


Serine/threonine-protein kinase 32B (EC 2.7.11.1) (YANK2)


414


Q71RG4


Transmembrane and ubiquitin-like domain-containing protein 2


321


Q9UNG2


Tumor necrosis factor ligand superfamily member 18 (Glucocorticoid-induced TNF-related ligand) (hGITRL) (Activation-inducible TNF-related ligand)


(AITRL)


177


P15157


Tryptase alpha-1 (Tryptase-1) (EC 3.4.21.59)


275


Q96N46


Tetratricopeptide repeat protein 14 (TPR repeat protein 14)


770


Q96DZ1


XTP3-transactivated gene B protein (ER lectin) (Erlectin)


483


Q96MV8


Palmitoyltransferase ZDHHC15 (EC 2.3.1.-) (Zinc finger DHHC domain-containing protein 15) (DHHC-15)


337


P21754


Zona pellucida sperm-binding protein 3 (Zona pellucida glycoprotein ZP3) (Zona pellucida protein C) (Sperm receptor) (ZP3A/ZP3B) [Cleaved into:


Processed zona pellucida sperm-binding protein 3]


424


Q96DD0


Leucine-rich repeat-containing protein 39 (Densin hlg)


335


O95867


Lymphocyte antigen 6 complex locus protein G6c


125


Q8IV31


Transmembrane protein 139


216


O75173


A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) (ADAM-TS 4) (ADAM-TS4) (EC 3.4.24.82) (Aggrecanase-1)


(ADMP-1)


837


Q8N4F0


Bactericidal/permeability-increasing protein-like 1 (Long palate, lung and nasal epithelium carcinoma-associated protein 2) (RYSR)


458


O00585


C-C motif chemokine 21 (Small-inducible cytokine A21) (Beta chemokine exodus-2) (6Ckine) (Secondary lymphoid-tissue chemokine) (SLC)


134


O43866


CD5 antigen-like (SP-alpha) (CT-2) (IgM-associated peptide)


347


Q7L1S5


Carbohydrate sulfotransferase 9 (EC 2.8.2.-) (N-acetylgalactosamine-4-O-sulfotransferase 2) (GalNAc-4-O-sulfotransferase 2) (GalNAc-4-ST2)


443


Q14CN2


Calcium-activated chloride channel regulator 4 (Calcium-activated chloride channel family member 4) (hCLCA4) (Calcium-activated chloride channel


protein 2) (CaCC-2) (hCaCC-2) [Cleaved into: Calcium-activated chloride channel regulator 4, 110 kDa form; Calcium-activated chloride channel


regulator 4, 30 kDa form]


919


Q9NZV1


Cysteine-rich motor neuron 1 protein (CRIM-1) (Cysteine-rich repeat-containing protein S52) [Cleaved into: Processed cysteine-rich motor 


neuron 1 protein]


1,036


P29279


Connective tissue growth factor (Hypertrophic chondrocyte-specific protein 24)


349


Q5T1H1


Protein eyes shut homolog (Protein spacemaker homolog) (EGF-like domain-containing protein 10) (EGF-like domain-containing protein 11)


3,165


O95967


EGF-containing fibulin-like extracellular matrix protein 2 (Fibulin-4) (FIBL-4) (Protein UPH1)


443


O95750


Fibroblast growth factor 19 (FGF-19)


216


O95633


Follistatin-related protein 3 (Follistatin-like 3) (Follistatin-related gene protein)


263


Q96QV1


Hedgehog-interacting protein (HHIP) (HIP)


700


Q9NRM6


Interleukin-17 receptor B (IL-17 receptor B) (IL-17RB) (Interleukin-17B receptor) (IL-17B receptor) (IL-17 receptor homolog 1) (IL-17Rh1)


(IL17Rh1) (Cytokine receptor CRL4)


502


Q8N145


Leucine-rich repeat LGI family member 3 (Leucine-rich glioma-inactivated protein 3) (LGI1-like protein 4)


548


Q9Y5X9


Endothelial lipase (EC 3.1.1.3) (Endothelial cell-derived lipase) (EDL) (EL)


500


Q96DR8


Mucin-like protein 1 (Small breast epithelial mucin) (Protein BS106)


90


Q9NRC9


Otoraplin (Fibrocyte-derived protein) (Melanoma inhibitory activity-like protein)


128


Q9NZ20


Group 3 secretory phospholipase A2 (EC 3.1.1.4) (Group III secretory phospholipase A2) (GIII sPLA2) (Phosphatidylcholine 2-acylhydrolase GIII)


(sPLA2-III)


509


Q8NCC3


Group XV phospholipase A2 (EC 2.3.1.-) (1-O-acylceramide synthase) (ACS) (Lysosomal phospholipase A2) (Lysophospholipase 3) (LPLA2)


(LCAT-like lysophospholipase) (LLPL)


412


Q6UXH9


Inactive serine protease PAMR1 (Peptidase domain-containing protein associated with muscle regeneration 1) (Regeneration-associated muscle 


protease homolog)


720


O75051


Plexin-A2 (Semaphorin receptor OCT)


1,894


O95428


Papilin


1,278


P58294


Prokineticin-1 (Endocrine-gland-derived vascular endothelial growth factor) (EG-VEGF) (Mambakine)


105


Q9H1E1


Ribonuclease 7 (RNase 7) (EC 3.1.27.-) (Skin-derived antimicrobial protein 2) (SAP-2)


156


O43278


Kunitz-type protease inhibitor 1 (Hepatocyte growth factor activator inhibitor type 1) (HAI-1)


529


Q9BUD6


Spondin-2 (Mindin) (Differentially expressed in cancerous and non-cancerous lung cells 1) (DIL-1)


331


Q92563


Testican-2 (SPARC/osteonectin, CWCV, and Kazal-like domains proteoglycan 2)


424


O43557


Tumor necrosis factor ligand superfamily member 14 (Herpesvirus entry mediator-ligand) (HVEM-L) (CD antigen CD258) [Cleaved into: Tumor 


necrosis factor ligand superfamily member 14, membrane form; Tumor necrosis factor ligand superfamily member 14, soluble form]


240


Q5JU69


Torsin-2A (Torsin family 2 member A) (Torsin-related protein 1)


321


Q7L8A9


Vasohibin-1


365


Q6EMK4


Vasorin (Protein slit-like 2)


673


Q86Y38


Xylosyltransferase 1 (EC 2.4.2.26) (Xylosyltransferase I) (XylT-I) (XT-I) (Peptide O-xylosyltransferase 1)


959


O95832


Claudin-1 (Senescence-associated epithelial membrane protein)


211


Q96CD2


Phosphopantothenoylcysteine decarboxylase (PPC-DC) (EC 4.1.1.36) (CoaC)


204


Q9BUN8


Derlin-1 (Der1-like protein 1) (Degradation in endoplasmic reticulum protein 1) (DERtrin-1)


251


Q9Y287


Integral membrane protein 2B (Transmembrane protein BRI) [Cleaved into: ABri/ADan amyloid peptide]


266


Q16651


Prostasin (EC 3.4.21.-) (Serine protease 8) [Cleaved into: Prostasin light chain; Prostasin heavy chain]


343


Q9UL52


Transmembrane protease, serine 11E (EC 3.4.21.-) (Serine protease DESC1) [Cleaved into: Transmembrane protease, serine 11E non-catalytic chain;


Transmembrane protease, serine 11E catalytic chain]


423


Q9Y5W5


Wnt inhibitory factor 1 (WIF-1)


379


Q29960


HLA class I histocompatibility antigen, Cw-16 alpha chain (MHC class I antigen Cw*16)


366


P23582


C-type natriuretic peptide [Cleaved into: CNP-22; CNP-29; CNP-53]


126


O15123


Angiopoietin-2 (ANG-2)


496


Q9UKP5


A disintegrin and metalloproteinase with thrombospondin motifs 6 (ADAMTS-6) (ADAM-TS 6) (ADAM-TS6) (EC 3.4.24.-)


1,117


Q8WXQ8


Carboxypeptidase A5 (EC 3.4.17.1)


436


Q16627


C-C motif chemokine 14 (Small-inducible cytokine A14) (Chemokine CC-1/CC-3) (HCC-1/HCC-3) (HCC-1(1-74)) (NCC-2) [Cleaved into:


HCC-1(3-74); HCC-1(4-74); HCC-1(9-74)]


93


P08217


Chymotrypsin-like elastase family member 2A (EC 3.4.21.71) (Elastase-2A)


269


O00533


Neural cell adhesion molecule L1-like protein (Close homolog of L1) [Cleaved into: Processed neural cell adhesion molecule L1-like protein]


1,208


P19875


C-X-C motif chemokine 2 (Macrophage inflammatory protein 2-alpha) (MIP2-alpha) (Growth-regulated protein beta) (Gro-beta) [Cleaved into:


GRO-beta(5-73) (GRO-beta-T) (SB-251353) (Hematopoietic synergistic factor) (HSF)]


107


P42830


C-X-C motif chemokine 5 (Small-inducible cytokine B5) (Epithelial-derived neutrophil-activating protein 78) (Neutrophil-activating peptide ENA-78)


(ENA-78(1-78)) [Cleaved into: ENA-78(8-78); ENA-78(9-78)]


114


Q13609


Deoxyribonuclease gamma (DNase gamma) (EC 3.1.21.-) (Deoxyribonuclease I-like 3) (DNase I-like 3) (DNase I homolog protein DHP2)


(Liver and spleen DNase) (LS-DNase) (LSD)


305


Q9NRM1


Enamelin


1,142


P98095


Fibulin-2 (FIBL-2)


1,184


O00602


Ficolin-1 (Ficolin-A) (Ficolin-alpha) (M-ficolin) (Collagen/fibrinogen domain-containing protein 1)


326


P49771


SL cytokine (Fms-related tyrosine kinase 3 ligand) (Flt3 ligand) (Flt3L)


235


Q5H8C1


FRAS1-related extracellular matrix protein 1 (Protein QBRICK)


2,179


P19883


Follistatin (FS) (Activin-binding protein)


344


Q10472


Polypeptide N-acetylgalactosaminyltransferase 1 (EC 2.4.1.41) (Protein-UDP acetylgalactosaminyltransferase 1) (UDP-GalNAc:polypeptide 


N-acetylgalactosaminyltransferase 1) (Polypeptide GalNAc transferase 1) (pp-GaNTase 1) (GalNAc-T1) [Cleaved into: Polypeptide 


N-acetylgalactosaminyltransferase 1 soluble form]


559


P10915


Hyaluronan and proteoglycan link protein 1 (Proteoglycan link protein) (Cartilage link protein) (LP)


354


P01570


Interferon alpha-14 (Interferon alpha-H) (LeIF H) (Interferon lambda-2-H)


189


Q96PD4


Interleukin-17F (IL-17F) (Interleukin-24) (IL-24) (Cytokine ML-1)


163


Q9NPH3


Interleukin-1 receptor accessory protein (IL-1 receptor accessory protein) (IL-1RAcP)


570


Q9NQ38


Serine protease inhibitor Kazal-type 5 (Lympho-epithelial Kazal-type-related inhibitor) (LEKTI) [Cleaved into: Hemofiltrate peptide HF6478; 


Hemofiltrate peptide HF7665]


1,064


Q9H2R5


Kallikrein-15 (EC 3.4.21.-) (ACO protease)


256


Q8N6C8


Leukocyte immunoglobulin-like receptor subfamily A member 3 (Leukocyte immunoglobulin-like receptor 4) (LIR-4) (Immunoglobulin-like transcript 6) 


(ILT-6) (Monocyte inhibitory receptor HM43/HM31) (CD85 antigen-like family member E) (CD antigen CD85e)


439


Q9NS15


Latent-transforming growth factor beta-binding protein 3 (LTBP-3)


1,303


P45452


Collagenase 3 (EC 3.4.24.-) (Matrix metalloproteinase-13) (MMP-13)


471


P51512


Matrix metalloproteinase-16 (MMP-16) (EC 3.4.24.-) (Membrane-type matrix metalloproteinase 3) (MT-MMP 3) (MTMMP3) (Membrane-type-3 


matrix metalloproteinase) (MT3-MMP) (MT3MMP) (MMP-X2)


607


P18509


Pituitary adenylate cyclase-activating polypeptide (PACAP) [Cleaved into: PACAP-related peptide (PRP-48); Pituitary adenylate cyclase-activating


polypeptide 27 (PACAP-27) (PACAP27); Pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38) (PACAP38)]


176


Q9HC23


Prokineticin-2 (PK2) (Protein Bv8 homolog)


129


Q00888


Pregnancy-specific beta-1-glycoprotein 4 (PSBG-4) (PSBG-9)


419


P01286


Somatoliberin (Growth hormone-releasing factor) (GRF) (Growth hormone-releasing hormone) (GHRH) (Somatocrinin) (Somatorelin) (Sermorelin)


108


O75093


Slit homolog 1 protein (Slit-1) (Multiple epidermal growth factor-like domains 4)


1,534


P01242


Growth hormone variant (GH-V) (Placenta-specific growth hormone) (Growth hormone 2)


217


Q03167


TGF-beta receptor type III (TGFR-3) (Transforming growth factor beta receptor III) (Betaglycan)


850


P01222


Thyrotropin subunit beta (Thyrotropin beta chain) (Thyroid-stimulating hormone subunit beta) (TSH-beta) (TSH-B) (Thyrotropin alfa)


138


Q9BXJ5


Complement C1q tumor necrosis factor-related protein 2


285


Q1ZYL8


Uncharacterized protein C19orf36 (Sperm 22 kDa protein c113)


232


Q96L15


Ecto-ADP-ribosyltransferase 5 (EC 2.4.2.31) (NAD(P)(+)--arginine ADP-ribosyltransferase 5) (Mono(ADP-ribosyl)transferase 5)


292


Q8TAA1


Probable ribonuclease 11 (RNase 11) (EC 3.1.27.-)


199


Q9NS62


Thrombospondin type-1 domain-containing protein 1 (Transmembrane molecule with thrombospondin module)


852


Q8N302


Angiogenic factor with G patch and FHA domains 1 (Angiogenic factor VG5Q) (Vasculogenesis gene on 5q protein) (hVG5Q) (G patch 


domain-containing protein 7)


714


Q9Y5C1


Angiopoietin-related protein 3 (Angiopoietin-like 3) (Angiopoietin-5) (ANG-5)


460


O43827


Angiopoietin-related protein 7 (Angiopoietin-like 7) (Angiopoietin-like factor) (Cornea-derived transcript 6 protein)


346


P58397


A disintegrin and metalloproteinase with thrombospondin motifs 12 (ADAMTS-12) (ADAM-TS 12) (ADAM-TS12) (EC 3.4.24.-)


1,593


O75493


Carbonic anhydrase-related protein 11 (CA-RP XI) (CARP XI) (CA-XI) (Carbonic anhydrase-related protein 2) (CARP-2) (CA-RP II)


328


Q6UXH8


Collagen and calcium-binding EGF domain-containing protein 1 (Full of fluid protein homolog)


406


Q96SM3


Probable carboxypeptidase X1 (EC 3.4.17.-) (Metallocarboxypeptidase CPX-1)


734


Q9H0B8


Cysteine-rich secretory protein LCCL domain-containing 2 (LCCL domain-containing cysteine-rich secretory protein 2) (Cysteine-rich secretory


protein 11) (CRISP-11)


497


O95715


C-X-C motif chemokine 14 (Small-inducible cytokine B14) (Chemokine BRAK)


99


Q9H1M4


Beta-defensin 127 (Defensin, beta 127) (Beta-defensin 27) (DEFB-27)


99


Q9H1M3


Beta-defensin 129 (Defensin, beta 129) (Beta-defensin 29) (DEFB-29)


183


P81605


Dermcidin (Preproteolysin) [Cleaved into: Survival-promoting peptide; DCD-1]


110


Q7Z5P4


17-beta hydroxysteroid dehydrogenase 13 (EC 1.1.-.-) (Short-chain dehydrogenase/reductase 9)


300


O94907


Dickkopf-related protein 1 (Dickkopf-1) (Dkk-1) (hDkk-1) (SK)


266


P52798


Ephrin-A4 (EPH-related receptor tyrosine kinase ligand 4) (LERK-4)


201


Q96BQ1


Protein FAM3D


224


Q9NSA1


Fibroblast growth factor 21 (FGF-21)


209


Q9BTY2


Plasma alpha-L-fucosidase (EC 3.2.1.51) (Alpha-L-fucoside fucohydrolase 2) (Alpha-L-fucosidase 2)


467


Q9NS71


Gastrokine-1 (18 kDa antrum mucosa protein) (AMP-18) (Protein CA11)


199


Q86XP6


Gastrokine-2 (Blottin) (Trefoil factor interactions(z) 1) (Down-regulated in gastric cancer)


184


Q96SL4


Glutathione peroxidase 7 (EC 1.11.1.9) (CL683)


187


Q96JK4


HHIP-like protein 1


782


Q9P0W0


Interferon kappa (IFN-kappa)


207


Q9Y5Q6


Insulin-like peptide INSL5 (Insulin-like peptide 5) [Cleaved into: Insulin-like peptide INSL5 B chain; Insulin-like peptide INSL5 A chain]


135


O14498


Immunoglobulin superfamily containing leucine-rich repeat protein


428


Q96I82


Kazal-type serine protease inhibitor domain-containing protein 1


304


O75610


Left-right determination factor 1 (Protein lefty-1) (Left-right determination factor B) (Protein lefty-B)


366


Q8N135


Leucine-rich repeat LGI family member 4 (Leucine-rich glioma-inactivated protein 4) (LGI1-like protein 3)


537


Q8TDL5


Long palate, lung and nasal epithelium carcinoma-associated protein 1 (Von Ebner minor salivary gland protein) (VEMSGP)


484


Q86TE4


Leucine zipper protein 2


346


O95157


Neurexophilin-3


252


Q99983


Osteomodulin (Osteoadherin) (OSAD) (Keratan sulfate proteoglycan osteomodulin) (KSPG osteomodulin)


421


Q9UKZ9


Procollagen C-endopeptidase enhancer 2 (Procollagen COOH-terminal proteinase enhancer 2) (Procollagen C-proteinase enhancer 2) (PCPE-2)


415


Q8IXA5


Sperm acrosome membrane-associated protein 3 (Sperm lysozyme-like protein 1) (Lysozyme-like protein 3) (Lysozyme-like acrosomal sperm-specific 


secretory protein ALLP-17) (Sperm protein reactive with antisperm antibodies) (Sperm protein reactive with ASA) (Cancer/testis antigen 54) (CT54) 


[Cleaved into: Sperm acrosome membrane-associated protein 3, membrane form; Sperm acrosome membrane-associated protein 3, processed form]


215


Q96QR1


Secretoglobin family 3A member 1 (Uteroglobin-related protein 2) (Cytokine HIN-1) (High in normal 1) (Pneumo secretory protein 2) (PnSP-2)


104


Q8WUA8


Tsukushin (Tsukushi) (Leucine-rich repeat-containing protein 54) (E2-induced gene 4 protein)


353


P57739


Claudin-2 (SP82)


230


Q8IWY4


Signal peptide, CUB and EGF-like domain-containing protein 1


988


P02654


Apolipoprotein C-I (Apo-CI) (ApoC-I)


83


Q6UWZ7


BRCA1-A complex subunit Abraxas (Coiled-coil domain-containing protein 98) (Protein FAM175A)


409


Q53EP0


Fibronectin type III domain-containing protein 3B (Factor for adipocyte differentiation 104) (HCV NS5A-binding protein 37)


1,204


Q9UBK5


Hematopoietic cell signal transducer (Transmembrane adapter protein KAP10) (DNAX-activation protein 10) (Membrane protein DAP10)


93


Q96FE5


Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 1 (Leucine-rich repeat and immunoglobilin-domain 


containing protein 1) (Leucine-rich repeat neuronal protein 6A) (Leucine-rich repeat neuronal protein 1)


620


Q5JRA6


Melanoma inhibitory activity protein 3 (Transport and Golgi organization protein 1) (TANGO1) (C219-reactive peptide) (D320)


1,907


Q9H3N1


Thioredoxin-related transmembrane protein 1 (Thioredoxin domain-containing protein 1) (Transmembrane Trx-related protein)


280


Q8TAD4


Zinc transporter 5 (ZnT-5) (ZnT-like transporter 1) (hZTL1) (Solute carrier family 30 member 5)


765


Q8TC27


Disintegrin and metalloproteinase domain-containing protein 32 (ADAM 32)


787


Q6UXC1


Apical endosomal glycoprotein (MAM domain-containing protein 4)


1,216


Q9BT22


Chitobiosyldiphosphodolichol beta-mannosyltransferase (EC 2.4.1.142) (GDP-mannose-dolichol diphosphochitobiose mannosyltransferase) 


(GDP-Man:GlcNAc2-PP-dolichol mannosyltransferase) (Beta-1,4-mannosyltransferase) (Mannosyltransferase-1) (Hmat-1) (MT-1)


(Asparagine-linked glycosylation protein 1)


464


Q9Y2A9


UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3 (Beta3Gn-T3) (BGnT-3) (EC 2.4.1.-) (Core 1 extending


beta-1,3-N-acetylglucosaminyltransferase) (Core1-beta3GlcNAcT) (Beta-1,3-galactosyltransferase 8) (Beta-1,3-GalTase 8) (Beta3Gal-T8) (b3Gal-T8) 


(Beta-3-Gx-T8) (UDP-galactose:beta-N-acetylglucosamine beta-1,3-galactosyltransferase 8) (UDP-Gal:beta-GlcNAc beta-1,3-galactosyltransferase 8)


372


Q9UBV7


Beta-1,4-galactosyltransferase 7 (Beta-1,4-GalTase 7) (Beta4Gal-T7) (b4Gal-T7) (EC 2.4.1.-) (UDP-galactose:beta-N-acetylglucosamine 


beta-1,4-galactosyltransferase 7) (UDP-Gal:beta-GlcNAc beta-1,4-galactosyltransferase 7) [Includes: Xylosylprotein 4-beta-galactosyltransferase 


(EC 2.4.1.133) (UDP-galactose:beta-xylose beta-1,4-galactosyltransferase) (Xylosylprotein beta-1,4-galactosyltransferase) (XGPT) 


(Proteoglycan UDP-galactose:beta-xylose beta1,4-galactosyltransferase I) (XGalT-1)]


327


Q9BUT1


3-hydroxybutyrate dehydrogenase type 2 (EC 1.1.1.30) (R-beta-hydroxybutyrate dehydrogenase) (Dehydrogenase/reductase SDR family member 6) 


(Oxidoreductase UCPA)


245


Q96EU7


C1GALT1-specific chaperone 1 (Core 1 beta3-galactosyltransferase-specific molecular chaperone) (Beta1,3-galactosyltransferase 2) (C1Gal-T2)


(C1GalT2) (C38H2-like protein 1) (C38H2-L1)


318


P05814


Beta-casein


226


P07498


Kappa-casein


182


Q8N357


Transmembrane protein C2orf18


371


P15169


Carboxypeptidase N catalytic chain (CPN) (EC 3.4.17.3) (Carboxypeptidase N polypeptide 1) (Carboxypeptidase N small subunit) 


(Lysine carboxypeptidase) (Arginine carboxypeptidase) (Kininase-1) (Serum carboxypeptidase N) (SCPN) (Anaphylatoxin inactivator)


(Plasma carboxypeptidase B)


458


Q9NPF0


CD320 antigen (8D6 antigen) (FDC-signaling molecule 8D6) (FDC-SM-8D6) (Transcobalamin receptor) (TCblR) (CD antigen CD320)


282


Q86X52


Chondroitin sulfate synthase 1 (EC 2.4.1.175) (Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase 1) 


(N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase 1) (EC 2.4.1.226) (Chondroitin glucuronyltransferase II)


(N-acetylgalactosaminyltransferase II)


802


Q8IZ52


Chondroitin sulfate synthase 2 (EC 2.4.1.175) (Glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-beta-N-acetylgalactosaminyltransferase II)


(N-acetylgalactosaminyl-proteoglycan 3-beta-glucuronosyltransferase II) (EC 2.4.1.226) (Chondroitin glucuronyltransferase II)


(N-acetylgalactosaminyltransferase) (Chondroitin-polymerizing factor)


775


A8K4G0


CMRF35-like molecule 7 (CLM-7) (CMRF35-A2) (Immune receptor expressed on myeloid cells 3) (IREM-3) (Triggering receptor expressed on 


myeloid cells 5) (TREM-5) (Leukocyte mono-Ig-like receptor 5) (CD300 antigen-like family member B) (CD antigen CD300b)


201


Q9BT09


Protein canopy homolog 3 (Trinucleotide repeat-containing gene 5 protein) (CTG repeat protein 4a) (Expanded repeat-domain protein CAG/CTG 5)


(Protein associated with TLR4)


278


O75911


Short-chain dehydrogenase/reductase 3 (EC 1.1.-.-) (Retinal short-chain dehydrogenase/reductase 1) (retSDR1) (DD83.1)


302


Q9NR61


Delta-like protein 4 (Drosophila Delta homolog 4) (Delta4)


685


Q8NFT8


Delta and Notch-like epidermal growth factor-related receptor


737


Q9UPQ8


Dolichol kinase (EC 2.7.1.108) (Transmembrane protein 15)


538


O95672


Endothelin-converting enzyme-like 1 (EC 3.4.24.-) (Xce protein)


775


P60508


HERV-FRD_6p24.1 provirus ancestral Env polyprotein (Envelope polyprotein) (HERV-FRD) (Syncytin-2) [Cleaved into: Surface protein (SU); 


Transmembrane protein (TM)]


538


O14638


Ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (E-NPP 3) (Phosphodiesterase I/nucleotide pyrophosphatase 3) 


(Phosphodiesterase I beta) (PD-Ibeta) (CD antigen CD203c) [Includes: Alkaline phosphodiesterase I (EC 3.1.4.1); Nucleotide pyrophosphatase


(NPPase) (EC 3.6.1.9)]


875


Q96AP7


Endothelial cell-selective adhesion molecule


390


P36980


Complement factor H-related protein 2 (FHR-2) (H factor-like protein 2) (H factor-like 3) (DDESK59)


270


Q02985


Complement factor H-related protein 3 (FHR-3) (H factor-like protein 3) (DOWN16)


330


O43155


Leucine-rich repeat transmembrane protein FLRT2 (Fibronectin-like domain-containing leucine-rich transmembrane protein 2)


660


Q12841


Follistatin-related protein 1 (Follistatin-like 1)


308


Q8N5D6


Globoside alpha-1,3-N-acetylgalactosaminyltransferase 1 (EC 2.4.1.-) (Forssman glycolipid synthetase-like protein)


347


Q92820


Gamma-glutamyl hydrolase (EC 3.4.19.9) (Gamma-Glu-X carboxypeptidase) (Conjugase) (GH)


318


Q8N3T1


Polypeptide N-acetylgalactosaminyltransferase-like protein 2 (EC 2.4.1.41) (Polypeptide GalNAc transferase-like protein 2) (pp-GaNTase-like protein 2) 


(GalNAc-T-like protein 2) (Protein-UDP acetylgalactosaminyltransferase-like protein 2) (UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-


like protein 2)


639


Q53EU6


Glycerol-3-phosphate acyltransferase 3 (GPAT3) (EC 2.3.1.15) (1-acylglycerol-3-phosphate O-acyltransferase 9) (1-AGP acyltransferase 9) (1-AGPAT 9) 


(Lysophosphatidic acid acyltransferase theta) (LPAAT-theta) (Acyl-CoA:glycerol-3-phosphate acyltransferase 3) (hGPAT3) (Lung cancer metastasis-


associated protein 1) (MAG-1)


434


Q9Y653


G-protein coupled receptor 56 (Protein TM7XN1)


693


Q14520


Hyaluronan-binding protein 2 (EC 3.4.21.-) (Plasma hyaluronan-binding protein) (Hepatocyte growth factor activator-like protein) (Factor VII-activating


protease) (Factor seven-activating protease) (FSAP) [Cleaved into: Hyaluronan-binding protein 2 50 kDa heavy chain; Hyaluronan-binding protein 2 


50 kDa heavy chain alternate form; Hyaluronan-binding protein 2 27 kDa light chain; Hyaluronan-binding protein 2 27 kDa light chain alternate form]


560


Q99075


Proheparin-binding EGF-like growth factor [Cleaved into: Heparin-binding EGF-like growth factor (HB-EGF) (HBEGF) (Diphtheria toxin receptor)]


(DT-R)


208


P04196


Histidine-rich glycoprotein (Histidine-proline-rich glycoprotein) (HPRG)


525


Q9Y663


Heparan sulfate glucosamine 3-O-sulfotransferase 3A1 (EC 2.8.2.30) (Heparan sulfate D-glucosaminyl 3-O-sulfotransferase 3A1) (Heparan sulfate


3-O-sulfotransferase 3A1) (h3-OST-3A)


406


Q6UXL0


Interleukin-20 receptor beta chain (IL-20R-beta) (IL-20R2)


311


P24593


Insulin-like growth factor-binding protein 5 (IGF-binding protein 5) (IGFBP-5) (IBP-5)


272


Q16270


Insulin-like growth factor-binding protein 7 (IGF-binding protein 7) (IGFBP-7) (IBP-7) (MAC25 protein) (Prostacyclin-stimulating factor)


(PGI2-stimulating factor) (IGFBP-rP1) (Tumor-derived adhesion factor) (TAF)


282


P29622


Kallistatin (Kallikrein inhibitor) (Protease inhibitor 4) (Serpin A4)


427


Q8IZU9


Kin of IRRE-like protein 3 (Kin of irregular chiasm-like protein 3) (Nephrin-like 2)


778


P42702


Leukemia inhibitory factor receptor (LIF receptor) (LIF-R) (CD antigen CD118)


1,097


Q9HAP6


Lin-7 homolog B (Lin-7B) (hLin7B) (Mammalian lin-seven protein 2) (MALS-2) (Vertebrate lin-7 homolog 2) (Veli-2 protein) (hVeli2)


207


Q9HBW1


Leucine-rich repeat-containing protein 4 (Brain tumor-associated protein BAG) (Nasopharyngeal carcinoma-associated gene 14 protein)


653


Q6UXK5


Leucine-rich repeat neuronal protein 1 (Neuronal leucine-rich repeat protein 1) (NLRR-1)


716


O95274


Ly6/PLAUR domain-containing protein 3 (GPI-anchored metastasis-associated protein C4.4A homolog) (Matrigel-induced gene C4 protein) (MIG-C4)


346


Q9Y5Y7


Lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1) (Cell surface retention sequence-binding protein 1) (CRSBP-1) (Hyaluronic acid


receptor) (Extracellular link domain-containing protein 1)


322


Q9UQ53


Alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase B (EC 2.4.1.145) (UDP-N-acetylglucosamine: alpha-1,3-D-mannoside


beta-1,4-N-acetylglucosaminyltransferase IVb) (N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase IVb)


(N-acetylglucosaminyltransferase IVb) (GlcNAc-T IVb) (GnT-IVb)


548


Q16674


Melanoma-derived growth regulatory protein (Melanoma inhibitory activity)


131


Q8IXL7


Methionine-R-sulfoxide reductase B3, mitochondrial (MsrB3) (EC 1.8.4.-)


185


Q8NC67


Neuropilin and tolloid-like protein 2 (Brain-specific transmembrane protein containing 2 CUB and 1 LDL-receptor class A domains protein 2)


525


Q86WC4


Osteopetrosis-associated transmembrane protein 1


334


Q9UHG3


Prenylcysteine oxidase 1 (EC 1.8.3.5) (Prenylcysteine lyase)


505


Q9UBV8


Peflin (PEF protein with a long N-terminal hydrophobic domain) (Penta-EF hand domain-containing protein 1)


284


Q9Y3C6


Peptidyl-prolyl cis-trans isomerase-like 1 (PPIase) (Rotamase) (EC 5.2.1.8)


166


O43653


Prostate stem cell antigen


123


Q9Y6C5


Protein patched homolog 2 (PTC2)


1,203


P35542


Serum amyloid A-4 protein (Constitutively expressed serum amyloid A protein) (C-SAA)


130


Q9UBV2


Protein sel-1 homolog 1 (Suppressor of lin-12-like protein 1) (Sel-1L)


794


P15907


Beta-galactoside alpha-2,6-sialyltransferase 1 (EC 2.4.99.1) (CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,6-sialyltransferase 1) (Alpha 2,6-ST)


(Sialyltransferase 1) (ST6Gal I) (B-cell antigen CD75)


406


Q9Y336


Sialic acid-binding Ig-like lectin 9 (Siglec-9) (Protein FOAP-9)


463


Q96PX8


SLIT and NTRK-like protein 1 (Leucine-rich repeat-containing protein 12)


696


P02808


Statherin


62


Q9Y6M0


Testisin (EC 3.4.21.-) (Serine protease 21) (Eosinophil serine protease 1) (ESP-1)


314


Q6UXY8


Transmembrane channel-like protein 5


1,006


Q9NRS4


Transmembrane protease, serine 4 (EC 3.4.21.-) (Membrane-type serine protease 2) (MT-SP2)


437


O75509


Tumor necrosis factor receptor superfamily member 21 (TNFR-related death receptor 6) (Death receptor 6)


655


Q9UBN6


Tumor necrosis factor receptor superfamily member 10D (Decoy receptor 2) (DcR2) (TNF-related apoptosis-inducing ligand receptor 4)


(TRAIL receptor 4) (TRAIL-R4) (TRAIL receptor with a truncated death domain) (CD antigen CD264)


386


Q5T2D2


Trem-like transcript 2 protein (TLT-2) (Triggering receptor expressed on myeloid cells-like protein 2)


321


O95881


Thioredoxin domain-containing protein 12 (EC 1.8.4.2) (Thioredoxin-like protein p19) (Endoplasmic reticulum protein ERp19) (ERp18) (hTLP19)


172


P49765


Vascular endothelial growth factor B (VEGF-B) (VEGF-related factor) (VRF)


207


P49767


Vascular endothelial growth factor C (VEGF-C) (Vascular endothelial growth factor-related protein) (VRP) (Flt4 ligand) (Flt4-L)


419


Q9Y279


V-set and immunoglobulin domain-containing protein 4 (Protein Z39Ig)


399


Q9BXJ2


Complement C1q tumor necrosis factor-related protein 7


289


P98173


Protein FAM3A (2-19 protein)


230


Q9Y334


Protein G7c


891


Q8TDF5


Neuropilin and tolloid-like protein 1 (Brain-specific transmembrane protein containing 2 CUB and 1 LDL-receptor class A domains protein 1)


533


Q9UQ72


Pregnancy-specific beta-1-glycoprotein 11 (PSBG-11) (Pregnancy-specific beta-1-glycoprotein 13) (PSBG-13)


332


O75077


Disintegrin and metalloproteinase domain-containing protein 23 (ADAM 23) (Metalloproteinase-like, disintegrin-like, and cysteine-rich protein 3) 


(MDC-3)


832


O15204


ADAM DEC1 (EC 3.4.24.-) (A disintegrin and metalloproteinase domain-like protein decysin 1) (ADAM-like protein decysin 1)


470


Q9BRR6


ADP-dependent glucokinase (ADP-GK) (ADPGK) (EC 2.7.1.147) (RbBP-35)


497


P19961


Alpha-amylase 2B (EC 3.2.1.1) (1,4-alpha-D-glucan glucanohydrolase 2B) (Carcinoid alpha-amylase)


511


Q9H324


A disintegrin and metalloproteinase with thrombospondin motifs 10 (ADAMTS-10) (ADAM-TS 10) (ADAM-TS10) (EC 3.4.24.-)


1,103


Q8WXS8


A disintegrin and metalloproteinase with thrombospondin motifs 14 (ADAMTS-14) (ADAM-TS 14) (ADAM-TS14) (EC 3.4.24.-)


1,223


Q13410


Butyrophilin subfamily 1 member A1 (BT)


526


P35070


Probetacellulin [Cleaved into: Betacellulin (BTC)]


178


P10092


Calcitonin gene-related peptide 2 (Calcitonin gene-related peptide II) (CGRP-II) (Beta-type CGRP)


127


P16870


Carboxypeptidase E (CPE) (EC 3.4.17.10) (Carboxypeptidase H) (CPH) (Enkephalin convertase) (Prohormone-processing carboxypeptidase)


476


Q9UBD9


Cardiotrophin-like cytokine factor 1 (B cell-stimulating factor 3) (BSF-3) (Novel neurotrophin-1) (NNT-1)


225


P25067


Collagen alpha-2(VIII) chain (Endothelial collagen)


703


Q5IJ48


Crumbs homolog 2 (Crumbs-like protein 2)


1,285


Q13316


Dentin matrix acidic phosphoprotein 1 (Dentin matrix protein 1) (DMP-1)


513


O60469


Down syndrome cell adhesion molecule (CHD2)


2,012


Q9BXX0


EMILIN-2 (Elastin microfibril interface-located protein 2) (Elastin microfibril interfacer 2) (Protein FOAP-10)


1,053


Q5JZY3


Ephrin type-A receptor 10 (EC 2.7.10.1)


1,008


Q9UBQ6


Exostosin-like 2 (EC 2.4.1.223) (Glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase)


(Alpha-1,4-N-acetylhexosaminyltransferase EXTL2) (Alpha-GalNAcT EXTL2) (EXT-related protein 2) [Cleaved into: Processed exostosin-like 2]


330


Q6MZW2


Follistatin-related protein 4 (Follistatin-like 4)


842


Q8N475


Follistatin-related protein 5 (Follistatin-like 5)


847


Q7Z5L2


Growth inhibition and differentiation-related protein 88 (Putative mitochondrial space protein 32.1)


792


Q96RW7


Hemicentin-1 (Fibulin-6) (FIBL-6)


5,635


P01861


Ig gamma-4 chain C region


327


Q13007


Interleukin-24 (Suppression of tumorigenicity 16 protein) (Melanoma differentiation-associated gene 7 protein) (MDA-7)


206


O00515


Ladinin-1 (Lad-1) (Linear IgA disease antigen) (LADA)


517


Q6XZB0


Lipase member I (EC 3.1.1.-) (Membrane-associated phosphatidic acid-selective phospholipase A1-beta) (mPA-PLA1 beta) (LPD lipase)


(Cancer/testis antigen 17) (CT17)


460


P54315


Pancreatic lipase-related protein 1 (PL-RP1) (EC 3.1.1.3)


467


O95711


Lymphocyte antigen 86 (Protein MD-1)


162


O75095


Multiple epidermal growth factor-like domains 6 (EGF-like domain-containing protein 3) (Multiple EGF-like domain protein 3)


1,541


P55083


Microfibril-associated glycoprotein 4


255


Q9UM21


Alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A (EC 2.4.1.145) (UDP-N-acetylglucosamine: alpha-1,3-D-mannoside


beta-1,4-N-acetylglucosaminyltransferase IVa) (N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase IVa) 


(N-acetylglucosaminyltransferase IVa) (GlcNAc-T IVa) (GnT-IVa) [Cleaved into: Alpha-1,3-mannosyl-glycoprotein


4-beta-N-acetylglucosaminyltransferase A soluble form]


535


Q9NR99


Matrix-remodeling-associated protein 5 (Adhesion protein with leucine-rich repeats and immunoglobulin domains related to perlecan) (Adlican)


2,828


Q9HB63


Netrin-4 (Beta-netrin) (Hepar-derived netrin-like protein)


628


P08949


Neuromedin-B [Cleaved into: Neuromedin-B-32; Neuromedin-B]


121


P56975


Pro-neuregulin-3, membrane-bound isoform (Pro-NRG3) [Cleaved into: Neuregulin-3 (NRG-3)]


720


Q9GZU5


Nyctalopin


481


P02818


Osteocalcin (Gamma-carboxyglutamic acid-containing protein) (Bone Gla protein) (BGP)


100


Q04118


Basic salivary proline-rich protein 3 (Parotid salivary glycoprotein G1) (Proline-rich protein G1)


309


Q15235


Pregnancy-specific beta-1-glycoprotein 10 (PSBG-10) (Pregnancy-specific beta-1-glycoprotein 12) (PSBG-12)


424


P04808


Prorelaxin H1 [Cleaved into: Relaxin B chain; Relaxin A chain]


185


Q9BXY4


R-spondin-3 (Roof plate-specific spondin-3) (hRspo3) (Thrombospondin type-1 domain-containing protein 2) (Protein with TSP type-1 repeat)


(hPWTSR)


272


Q9BZZ2


Sialoadhesin (Sialic acid-binding Ig-like lectin 1) (Siglec-1) (CD antigen CD169)


1,709


P35030


Trypsin-3 (EC 3.4.21.4) (Trypsin III) (Brain trypsinogen) (Mesotrypsinogen) (Trypsin IV) (Serine protease 3) (Serine protease 4)


304


Q5TIE3


von Willebrand factor A domain-containing protein 5B1


1,220


Q9UBV4


Protein Wnt-16


365


Q93097


Protein Wnt-2b (Wnt-13)


391


Q6UX41


Butyrophilin-like protein 8


500


Q86UP0


Cadherin-24


819


Q9H4A9


Dipeptidase 2 (EC 3.4.13.19)


486


Q6UXU4


Germ cell-specific gene 1-like protein (GSG1-like protein)


331


Q6UY01


Leucine-rich repeat-containing protein 31


552


Q86VH4


Leucine-rich repeat transmembrane neuronal protein 4


590


Q9BXS4


Transmembrane protein 59 (Liver membrane-bound protein)


323


Q9BUR5


Apolipoprotein O (Protein FAM121B)


198


Q96CG8


Collagen triple helix repeat-containing protein 1 (NMTC1 protein)


243


Q9UKF2


Disintegrin and metalloproteinase domain-containing protein 30 (ADAM 30) (EC 3.4.24.-)


790


Q86WK7


Amphoterin-induced protein 3 (AMIGO-3) (Alivin-3)


504


Q8WW43


Gamma-secretase subunit APH-1B (APH-1b) (Aph-1beta) (Presenilin-stabilization factor-like)


257


P55056


Apolipoprotein C-IV (Apo-CIV) (ApoC-IV)


127


Q96EG1


Arylsulfatase G (ASG) (EC 3.1.6.-)


525


Q9H040


Zinc finger RAD18 domain-containing protein C1orf124


489


Q9ULX7


Carbonic anhydrase 14 (EC 4.2.1.1) (Carbonic anhydrase XIV) (CA-XIV) (Carbonate dehydratase XIV)


337


P57730


Caspase recruitment domain-containing protein 18 (Caspase-1 inhibitor Iceberg)


90


Q8NCH0


Carbohydrate sulfotransferase 14 (EC 2.8.2.-) (Dermatan 4-sulfotransferase 1) (D4ST-1) (hD4ST)


376


Q86T13


C-type lectin domain family 14 member A (Epidermal growth factor receptor 5) (EGFR-5)


490


Q86VU5


Catechol-O-methyltransferase domain-containing protein 1 (EC 2.1.1.-)


262


O95406


Protein cornichon homolog (T-cell growth-associated molecule 77) (TGAM77)


144


Q8N4M1


Choline transporter-like protein 3 (Solute carrier family 44 member 3)


653


Q9BPX1


17-beta-hydroxysteroid dehydrogenase 14 (EC 1.1.1.-) (Dehydrogenase/reductase SDR family member 10) (17-beta-hydroxysteroid dehydrogenase


DHRS10) (Retinal short-chain dehydrogenase/reductase retSDR3)


270


Q8N5I4


Dehydrogenase/reductase SDR family member on chromosome X (EC 1.1.-.-) (DHRSXY)


330


Q8TBM8


DnaJ homolog subfamily B member 14


379


Q9HBW9


EGF, latrophilin and seven transmembrane domain-containing protein 1 (EGF-TM7-latrophilin-related protein) (ETL protein)


690


Q8WW52


Protein FAM151A


585


Q8TC84


Fibronectin type 3 and ankyrin repeat domains protein 1


345


Q9NRD0


F-box only protein 8 (F-box/SEC7 protein FBS)


319


Q14314


Fibroleukin (Fibrinogen-like protein 2) (pT49)


439


Q9NWM8


FK506-binding protein 14 (EC 5.2.1.8) (Peptidyl-prolyl cis-trans isomerase) (PPIase) (Rotamase) (22 kDa FK506-binding protein) (FKBP-22)


211


Q9NZU1


Leucine-rich repeat transmembrane protein FLRT1 (Fibronectin-like domain-containing leucine-rich transmembrane protein 1)


646


Q9H0Q3


FXYD domain-containing ion transport regulator 6 (Phosphohippolin)


95


Q9Y3E0


Vesicle transport protein GOT1B (Golgi transport 1 homolog B) (hGOT1a) (Putative NF-kappa-B-activating protein 470)


138


Q5T9L3


Integral membrane protein GPR177 (Protein wntless homolog) (Protein evenness interrupted homolog) (EVI) (Putative NF-kappa-B-activating


protein 373)


541


Q96P69


Probable G-protein coupled receptor 78


363


A8MVW5


HEPACAM family member 2


462


Q6UWB1


Interleukin-27 receptor subunit alpha (IL-27R-alpha) (WSX-1) (Type I T-cell cytokine receptor) (TCCR) (Protein CRL1)


636


Q6UXK2


Immunoglobulin superfamily containing leucine-rich repeat protein 2


745


O75578


Integrin alpha-10


1,167


Q8NBL1


KTEL motif-containing protein 1 (CAP10-like 46 kDa protein) (Myelodysplastic syndromes relative protein)


392


P83111


Serine beta-lactamase-like protein LACTB, mitochondrial (EC 3.4.-.-)


547


Q6UWP7


Lysocardiolipin acyltransferase 1 (EC 2.3.1.51) (EC 2.3.1.-) (Acyl-CoA:lysocardiolipin acyltransferase 1) (1-acylglycerol-3-phosphate 


O-acyltransferase 8) (1-AGP acyltransferase 8) (1-AGPAT 8)


414


Q7L985


Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 2 (Leucine-rich repeat neuronal protein 6C)


(Leucine-rich repeat neuronal protein 3)


606


A6NI73


Leukocyte immunoglobulin-like receptor subfamily A member 5 (Leukocyte immunoglobulin-like receptor 9) (LIR-9) (Immunoglobulin-like


transcript 11) (ILT-11) (CD85 antigen-like family member F) (CD antigen CD85f)


299


Q9BTN0


Leucine-rich repeat and fibronectin type-III domain-containing protein 3


628


Q8NA29


Major facilitator superfamily domain-containing protein 2


543


Q9HCJ2


Netrin-G1 ligand (NGL-1) (Leucine-rich repeat-containing protein 4C)


640


Q96RD7


Pannexin-1


426


Q9NPG4


Protocadherin-12 (Vascular cadherin-2) (Vascular endothelial cadherin-2) (VE-cadherin-2) (VE-cad-2)


1,184


Q9Y5I2


Protocadherin alpha-10 (PCDH-alpha-10)


948


Q9UN67


Protocadherin beta-10 (PCDH-beta-10)


800


Q9Y5F0


Protocadherin beta-13 (PCDH-beta-13)


798


Q9UHG2


ProSAAS (pro-SAAS) (Proprotein convertase subtilisin/kexin type 1 inhibitor) (Proprotein convertase 1 inhibitor) [Cleaved into: KEP; 


Big SAAS (b-SAAS); Little SAAS (l-SAAS) (N-proSAAS); Big PEN-LEN (b-PEN-LEN) (SAAS CT(1-49)); PEN; Little LEN (l-LEN); 


Big LEN (b-LEN) (SAAS CT(25-40))]


260


Q6UW60


Proprotein convertase subtilisin/kexin type 4 (EC 3.4.21.-) (Proprotein convertase PC4)


755


Q96FM1


Post-GPI attachment to proteins factor 3 (PER1-like domain-containing protein 1) (Gene coamplified with ERBB2 protein) (hCOS16)


320


Q96S52


GPI transamidase component PIG-S (Phosphatidylinositol-glycan biosynthesis class S protein)


555


Q5FWE3


Proline-rich transmembrane protein 3


981


Q6ZRP7


Sulfhydryl oxidase 2 (EC 1.8.3.2) (Quiescin Q6-like protein 1) (Neuroblastoma-derived sulfhydryl oxidase)


698


Q96D15


Reticulocalbin-3 (EF-hand calcium-binding protein RLP49)


328


O60939


Sodium channel subunit beta-2


215


Q9HCN8


Stromal cell-derived factor 2-like protein 1 (SDF2-like protein 1) (PWP1-interacting protein 8)


221


Q9BRG2


SH2 domain-containing protein 3A (Novel SH2-containing protein 1)


576


Q6S5L8


SHC-transforming protein 4 (Src homology 2 domain-containing-transforming protein C4) (SH2 domain protein C4) (Rai-like protein) (RaLP) (hShcD)


630


P48594


Serpin B4 (Squamous cell carcinoma antigen 2) (SCCA-2) (Leupin)


390


Q9Y4P3


Transducin beta-like protein 2 (WS beta-transducin repeats protein) (WS-betaTRP) (Williams-Beuren syndrome chromosomal region 13 protein)


447


Q5VZ19


Tudor domain-containing protein 10


366


Q9NYK1


Toll-like receptor 7


1,049


Q8N3G9


Transmembrane protein 130


435


Q9BSN7


Transmembrane protein 204 (Claudin-like protein 24)


226


Q9HD45


Transmembrane 9 superfamily member 3 (SM-11044-binding protein) (EP70-P-iso)


589


Q8TDI7


Transmembrane channel-like protein 2 (Transmembrane cochlear-expressed protein 2)


906


Q6UWJ1


Transmembrane and coiled-coil domain-containing protein 3 (Putative LAG1-interacting protein)


677


Q96BY9


Transmembrane protein 66 (Protein FOAP-7) (HBV X-transactivated gene 3 protein)


339


Q9H1E5


Thioredoxin-related transmembrane protein 4 (Thioredoxin domain-containing protein 13)


349


Q9H2S6


Tenomodulin (TeM) (hTeM) (Chondromodulin-I-like protein) (ChM1L) (hChM1L) (Myodulin) (Tendin)


317


O43657


Tetraspanin-6 (Tspan-6) (Transmembrane 4 superfamily member 6) (T245 protein) (Tetraspanin TM4-D) (A15 homolog)


(Putative NF-kappa-B-activating protein 321)


245


Q8NBS9


Thioredoxin domain-containing protein 5 (Thioredoxin-like protein p46) (Endoplasmic reticulum protein ERp46)


432


O43915


Vascular endothelial growth factor D (VEGF-D) (c-fos-induced growth factor) (FIGF)


354


Q96IQ7


V-set and immunoglobulin domain-containing protein 2 (CT-like protein) (Cortical thymocyte-like protein)


327


Q13275


Semaphorin-3F (Semaphorin IV) (Sema IV) (Sema III/F)


785


Q8TE99


Acid phosphatase-like protein 2 (EC 3.1.3.2)


480


Q6UXV4


Apolipoprotein O-like (Protein FAM121A)


268


Q96EE4


Coiled-coil domain-containing protein 126


140


O00230


Cortistatin [Cleaved into: Cortistatin-29; Cortistatin-17]


105


Q9H7Y0


UPF0672 protein CXorf36


433


Q8N690


Beta-defensin 119 (Defensin, beta 119) (Beta-defensin 19) (DEFB-19) (Beta-defensin 120) (Defensin, beta 120) (Beta-defensin 20) (DEFB-20)


84


Q9UJA9


Ectonucleotide pyrophosphatase/phosphodiesterase family member 5 (E-NPP5) (NPP-5) (EC 3.1.-.-)


477


Q6UXB0


Protein FAM131A


335


P58499


Protein FAM3B (Cytokine-like protein 2-21)


235


O76093


Fibroblast growth factor 18 (FGF-18) (zFGF5)


207


Q6UWU2


Beta-galactosidase-1-like protein (EC 3.2.1.-)


654


Q8N1E2


Lysozyme g-like protein 1 (EC 3.2.1.-)


194


Q86Y78


Ly6/PLAUR domain-containing protein 6


171


Q96QH8


Sperm acrosome-associated protein 5 (EC 3.2.1.17) (Lysozyme-like protein 5) (Sperm-specific lysozyme-like protein X) (SLLP-X)


159


O75951


Lysozyme-like protein 6 (EC 3.2.1.17)


148


Q9NQ76


Matrix extracellular phosphoglycoprotein (Osteoblast/osteocyte factor 45) (OF45)


525


Q6UW10


Surfactant-associated protein 2 (Surfactant-associated protein G)


78


Q96A54


Adiponectin receptor protein 1 (Progestin and adipoQ receptor family member I)


375


P58166


Inhibin beta E chain (Activin beta-E chain)


350


P05408


Neuroendocrine protein 7B2 (Secretory granule endocrine protein I) (Secretogranin-5) (Secretogranin V) (Pituitary polypeptide)


[Cleaved into: N-terminal peptide; C-terminal peptide]


212


P04217


Alpha-1B-glycoprotein (Alpha-1-B glycoprotein)


495


P02750


Leucine-rich alpha-2-glycoprotein (LRG)


347


P19801


Amiloride-sensitive amine oxidase [copper-containing](Diamine oxidase) (DAO) (EC 1.4.3.22) (Amiloride-binding protein) (ABP) (Histaminase)


(Kidney amine oxidase) (KAO)


751


Q6PIU2


Arylacetamide deacetylase-like 1 (EC 3.1.1.-) (Neutral cholesterol ester hydrolase) (NCEH)


408


P35318


ADM [Cleaved into: Adrenomedullin (AM); Proadrenomedullin N-20 terminal peptide (ProAM N-terminal 20 peptide) (ProAM-N20) (PAMP)]


185


P43652


Afamin (Alpha-albumin) (Alpha-Alb)


599


O00468


Agrin


2,045


P35858


Insulin-like growth factor-binding protein complex acid labile chain (ALS)


605


P42127


Agouti-signaling protein (ASP) (Agouti switch protein)


132


Q9UHI8


A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS-1) (ADAM-TS 1) (ADAM-TS1) (EC 3.4.24.-) (METH-1)


967


O95972


Bone morphogenetic protein 15 (BMP-15) (Growth/differentiation factor 9B) (GDF-9B)


392


P12645


Bone morphogenetic protein 3 (BMP-3) (Osteogenin) (BMP-3A)


472


Q10589


Bone marrow stromal antigen 2 (BST-2) (Tetherin) (HM1.24 antigen) (CD antigen CD317)


180


P55285


Cadherin-6 (Kidney cadherin) (K-cadherin)


790


P49913


Cathelicidin antimicrobial peptide (18 kDa cationic antimicrobial protein) (CAP-18) (hCAP-18) [Cleaved into: Antibacterial protein FALL-39 


(FALL-39 peptide antibiotic); Antibacterial protein LL-37]


170


Q16568


Cocaine- and amphetamine-regulated transcript protein [Cleaved into: CART(1-39); CART(42-89)]


116


P15086


Carboxypeptidase B (EC 3.4.17.2) (Pancreas-specific protein) (PASP)


417


O15467


C-C motif chemokine 16 (Small-inducible cytokine A16) (IL-10-inducible chemokine) (Chemokine LEC) (Liver-expressed chemokine)


(Monotactin-1) (MTN-1) (Chemokine CC-4) (HCC-4) (NCC-4) (Lymphocyte and monocyte chemoattractant) (LMC) (LCC-1)


120


Q92583


C-C motif chemokine 17 (Small-inducible cytokine A17) (Thymus and activation-regulated chemokine) (CC chemokine TARC)


94


O00626


C-C motif chemokine 22 (Small-inducible cytokine A22) (Macrophage-derived chemokine) (MDC(1-69)) (Stimulated T-cell chemotactic protein 1)


(CC chemokine STCP-1) [Cleaved into: MDC(3-69); MDC(5-69); MDC(7-69)]


93


O00175


C-C motif chemokine 24 (Small-inducible cytokine A24) (Myeloid progenitor inhibitory factor 2) (MPIF-2) (CK-beta-6) (Eosinophil chemotactic


protein 2) (Eotaxin-2)


119


Q6PJG6


HEAT repeat-containing protein C7orf27


821


Q14055


Collagen alpha-2(IX) chain


689


Q14050


Collagen alpha-3(IX) chain


684


P04118


Colipase


112


Q8IZC6


Collagen alpha-1(XXVII) chain


1,860


P22792


Carboxypeptidase N subunit 2 (Carboxypeptidase N polypeptide 2) (Carboxypeptidase N 83 kDa chain) (Carboxypeptidase N regulatory subunit)


(Carboxypeptidase N large subunit)


545


P80162


C-X-C motif chemokine 6 (Small-inducible cytokine B6) (Granulocyte chemotactic protein 2) (GCP-2) (Chemokine alpha 3) (CKA-3)


[Cleaved into: Small-inducible cytokine B6, N-processed variant 1; Small-inducible cytokine B6, N-processed variant 2; Small-inducible


cytokine B6, N-processed variant 3]


114


P02778


C-X-C motif chemokine 10 (Small-inducible cytokine B10) (10 kDa interferon-gamma-induced protein) (Gamma-IP10) (IP-10)


[Cleaved into: CXCL10(1-73)]


98


P28325


Cystatin-D (Cystatin-5)


142


O76096


Cystatin-F (Leukocystatin) (Cystatin-7) (Cystatin-like metastasis-associated protein) (CMAP)


145


P09228


Cystatin-SA (Cystatin-2) (Cystatin-S5)


141


Q14118


Dystroglycan (Dystrophin-associated glycoprotein 1) [Cleaved into: Alpha-dystroglycan (Alpha-DG); Beta-dystroglycan (Beta-DG)]


895


P12838


Neutrophil defensin 4 (HNP-4) (HP-4) (Defensin, alpha 4)


97


P60022


Beta-defensin 1 (BD-1) (hBD-1) (Defensin, beta 1)


68


P20800


Endothelin-2 (ET-2) (Preproendothelin-2) (PPET2)


178


O75354


Ectonucleoside triphosphate diphosphohydrolase 6 (NTPDase 6) (EC 3.6.1.6) (CD39 antigen-like 2)


484


Q6BAA4


Fc receptor-like B (Fc receptor-like protein 2) (Fc receptor-like and mucin-like protein 2) (Fc receptor-related protein Y) (FcRY) (Fc receptor homolog


expressed in B cells protein 2) (FREB-2)


426


P31371


Glia-activating factor (GAF) (Fibroblast growth factor 9) (FGF-9) (HBGF-9)


208


Q06828


Fibromodulin (FM) (Collagen-binding 59 kDa protein) (Keratan sulfate proteoglycan fibromodulin) (KSPG fibromodulin)


376


P14207


Folate receptor beta (FR-beta) (Folate receptor 2) (Folate receptor, fetal/placental) (Placental folate-binding protein) (FBP)


255


P22466


Galanin [Cleaved into: Galanin; Galanin message-associated peptide (GMAP)]


123


Q99988


Growth/differentiation factor 15 (GDF-15) (Placental bone morphogenetic protein) (Placental TGF-beta) (Macrophage inhibitory cytokine 1) (MIC-1)


(Prostate differentiation factor) (NSAID-activated gene 1 protein) (NAG-1) (NSAID-regulated gene 1 protein) (NRG-1)


308


P07093


Glia-derived nexin (GDN) (Protease nexin I) (PN-1) (Protease inhibitor 7)


398


P01148


Progonadoliberin-1 (Progonadoliberin I) [Cleaved into: Gonadoliberin-1 (Gonadoliberin I) (Luteinizing hormone-releasing hormone I) (LH-RH I)


(Gonadotropin-releasing hormone I) (GnRH-I) (Luliberin I) (Gonadorelin); GnRH-associated peptide 1 (GnRH-associated peptide I)]


92


P49863


Granzyme K (EC 3.4.21.-) (Granzyme-3) (NK-tryptase-2) (NK-TRYP-2) (Fragmentin-3)


264


Q02747


Guanylin (Guanylate cyclase activator 2A) (Guanylate cyclase-activating protein 1) (Gap-I) [Cleaved into: HMW-guanylin; Guanylin]


115


Q16661


Guanylate cyclase activator 2B [Cleaved into: Guanylate cyclase C-activating peptide 2 (Guanylate cyclase C-activating peptide II) (GCAP-II);


Uroguanylin (UGN)]


112


Q9Y6W8


Inducible T-cell costimulator (Activation-inducible lymphocyte immunomediatory molecule) (CD antigen CD278)


199


P01571


Interferon alpha-17 (Interferon alpha-I') (LeIF I) (Interferon alpha-T) (Interferon alpha-88)


189


P01568


Interferon alpha-21 (Interferon alpha-F) (LeIF F)


189


P32881


Interferon alpha-8 (Interferon alpha-B2) (Interferon alpha-B) (LeIF B)


189


P05000


Interferon omega-1 (Interferon alpha-II-1)


195


P15814


Immunoglobulin lambda-like polypeptide 1 (Immunoglobulin-related protein 14.1) (Immunoglobulin omega polypeptide) (Ig lambda-5)


(CD179 antigen-like family member B) (CD antigen CD179b)


213


Q9HBE4


Interleukin-21 (IL-21) (Za11)


155


P08700


Interleukin-3 (IL-3) (Multipotential colony-stimulating factor) (Hematopoietic growth factor) (P-cell-stimulating factor) (Mast cell growth factor)


(MCGF)


152


P13232


Interleukin-7 (IL-7)


177


P05111


Inhibin alpha chain


366


Q14641


Early placenta insulin-like peptide (EPIL) (Placentin) (Insulin-like peptide 4) [Cleaved into: Early placenta insulin-like peptide B chain;


Early placenta insulin-like peptide A chain]


139


O60938


Keratocan (KTN) (Keratan sulfate proteoglycan keratocan)


352


Q16787


Laminin subunit alpha-3 (Epiligrin 170 kDa subunit) (E170) (Nicein subunit alpha)


3,333


P55268


Laminin subunit beta-2 (S-laminin) (Laminin B1s chain)


1,798


O00292


Left-right determination factor 2 (Protein lefty-2) (Left-right determination factor A) (Protein lefty-A) (Transforming growth factor beta-4)


(TGF-beta-4) (Endometrial bleeding-associated factor)


366


Q8N0V4


Leucine-rich repeat LGI family member 2 (Leucine-rich glioma-inactivated protein 2) (LGI1-like protein 2)


545


Q96JB6


Lysyl oxidase homolog 4 (EC 1.4.3.-) (Lysyl oxidase-like protein 4) (Lysyl oxidase-related protein C)


756


Q14767


Latent-transforming growth factor beta-binding protein 2 (LTBP-2)


1,821


P51884


Lumican (Keratan sulfate proteoglycan lumican) (KSPG lumican)


338


Q14703


Membrane-bound transcription factor site-1 protease (EC 3.4.21.112) (S1P endopeptidase) (Site-1 protease) (Subtilisin/kexin-isozyme 1) (SKI-1)


1,052


P24347


Stromelysin-3 (SL-3) (ST3) (EC 3.4.24.-) (Matrix metalloproteinase-11) (MMP-11)


488


P39900


Macrophage metalloelastase (HME) (EC 3.4.24.65) (Matrix metalloproteinase-12) (MMP-12) (Macrophage elastase) (ME)


470


O75900


Matrix metalloproteinase-23 (MMP-23) (EC 3.4.24.-) (Matrix metallopeptidase 21) (MMP-21) (Matrix metalloprotease 22) (MMP-22) (Femalysin)


(MIFR-1) [Cleaved into: Matrix metalloproteinase-23, soluble form]


390


P80303


Nucleobindin-2 (DNA-binding protein NEFA) (Gastric cancer antigen Zg4)


420


Q9UBM4


Opticin (Oculoglycan)


332


P04054


Phospholipase A2 (EC 3.1.1.4) (Phosphatidylcholine 2-acylhydrolase) (Group IB phospholipase A2)


148


P01298


Pancreatic prohormone (Pancreatic polypeptide) (PP) (Obinepitide) [Cleaved into: Pancreatic hormone (PH); Pancreatic icosapeptide (PI)]


95


P01213


Proenkephalin-B (Beta-neoendorphin-dynorphin) (Preprodynorphin) [Cleaved into: Alpha-neoendorphin; Beta-neoendorphin; Big dynorphin (Big Dyn);


Dynorphin A(1-17) (Dynorphin A) (Dyn-A17); Dynorphin A(1-13); Dynorphin A(1-8); Leu-enkephalin; Rimorphin (Dynorphin B) (Dyn-B)


(Dynorphin B(1-13)); Leumorphin (Dynorphin B-29)]


254


P01210


Proenkephalin-A [Cleaved into: Synenkephalin; Met-enkephalin (Opioid growth factor) (OGF); PENK(114-133); PENK(143-183);


Met-enkephalin-Arg-Gly-Leu; Leu-enkephalin; PENK(237-258); Met-enkephalin-Arg-Phe]


267


Q9Y646


Plasma glutamate carboxypeptidase (EC 3.4.17.-)


472


Q96LB8


Peptidoglycan recognition protein I-beta (PGRP-I-beta) (Peptidoglycan recognition protein intermediate beta) (PGLYRPIbeta) (Peptidoglycan


recognition protein 4)


373


Q16557


Pregnancy-specific beta-1-glycoprotein 3 (PSBG-3) (Carcinoembryonic antigen SG5)


428


Q00889


Pregnancy-specific beta-1-glycoprotein 6 (PSBG-6)


435


Q00887


Pregnancy-specific beta-1-glycoprotein 9 (Pregnancy-specific glycoprotein 9) (PSBG-9) (Pregnancy-specific beta-1 glycoprotein B) (PS-beta-B) (PS34)


(Pregnancy-specific glycoprotein 7) (PSG7)


426


P26022


Pentraxin-related protein PTX3 (Pentaxin-related protein PTX3) (Tumor necrosis factor-inducible gene 14 protein) (TSG-14)


381


P10082


Peptide YY (PYY) (PYY-I) (Peptide tyrosine tyrosine) [Cleaved into: Peptide YY(3-36) (PYY-II)]


97


P10745


Retinol-binding protein 3 (Interphotoreceptor retinoid-binding protein) (IRBP) (Interstitial retinol-binding protein)


1,247


Q68DV7


RING finger protein 43


783


Q6SPF0


Atherin (Sterile alpha motif domain-containing protein 1)


538


Q9UGP8


Translocation protein SEC63 homolog


760


Q02383


Semenogelin-2 (Semenogelin II) (SGII)


582


P21815


Bone sialoprotein 2 (Bone sialoprotein II) (BSP II) (Cell-binding sialoprotein) (Integrin-binding sialoprotein)


317


P10600


Transforming growth factor beta-3 (TGF-beta-3)


412


O14656


Torsin-1A (Torsin family 1 member A) (Dystonia 1 protein)


332


P11684


Uteroglobin (Secretoglobin family 1A member 1) (Clara cell phospholipid-binding protein) (CCPBP) (Clara cells 10 kDa secretory protein) (CC10)


(Urinary protein 1) (Urine protein 1) (UP1)


91


O00744


Protein Wnt-10b (Wnt-12)


389


P47992


Lymphotactin (C motif chemokine 1) (Cytokine SCM-1) (ATAC) (Lymphotaxin) (SCM-1-alpha) (Small-inducible cytokine C1)


(XC chemokine ligand 1)


114


Q9UQC9


Calcium-activated chloride channel regulator 2 (Calcium-activated chloride channel family member 2) (hCLCA2) (Calcium-activated chloride channel


protein 3) (CaCC-3) (hCaCC-3) [Cleaved into: Calcium-activated chloride channel regulator 2, 109 kDa form; Calcium-activated chloride channel


regulator 2, 35 kDa form]


943


P09341


Growth-regulated alpha protein (C-X-C motif chemokine 1) (Melanoma growth stimulatory activity) (MGSA) (Neutrophil-activating protein 3) (NAP-3) 


(GRO-alpha(1-73)) [Cleaved into: GRO-alpha(4-73); GRO-alpha(5-73); GRO-alpha(6-73)]


107


Q02505


Mucin-3A (MUC-3A) (Intestinal mucin-3A)


2,541


P10163


Basic salivary proline-rich protein 4 (Salivary proline-rich protein Po) (Parotid o protein) (Salivary proline-rich protein II-1) [Cleaved into: Protein N1; 


Glycosylated protein A; Peptide P-D (Proline-rich peptide IB-5)]


310


Q92626


Peroxidasin homolog (EC 1.11.1.7) (Vascular peroxidase 1) (Melanoma-associated antigen MG50) (p53-responsive gene 2 protein)


1,479


Q9UI42


Carboxypeptidase A4 (EC 3.4.17.-) (Carboxypeptidase A3)


421


Q6UX06


Olfactomedin-4 (OLM4) (G-CSF-stimulated clone 1 protein) (hGC-1) (hOLfD) (Antiapoptotic protein GW112)


510


Q13018


Secretory phospholipase A2 receptor (PLA2-R) (PLA2R) (180 kDa secretory phospholipase A2 receptor) (M-type receptor) [Cleaved into: Soluble


secretory phospholipase A2 receptor (Soluble PLA2-R) (Soluble PLA2R)]


1,463


Q6UXB2


VEGF co-regulated chemokine 1 (C-X-C motif chemokine 17) (Dendritic cell and monocyte chemokine-like protein) (DMC)


119


Q7Z4H4


ADM2 (Intermedin) [Cleaved into: Adrenomedullin-2 (Intermedin-long) (IMDL); Intermedin-short (IMDS)]


148


Q99218


Amelogenin, Y isoform


206


P54793


Arylsulfatase F (ASF) (EC 3.1.6.-)


590


Q6NT52


Choriogonadotropin subunit beta variant 2


195


Q8WTQ1


Beta-defensin 104 (Defensin, beta 104) (Beta-defensin 4) (BD-4) (hBD-4) (DEFB-4)


72


Q8NG35


Beta-defensin 105 (Defensin, beta 105) (Beta-defensin 5) (BD-5) (DEFB-5)


78


Q8IZN7


Beta-defensin 107 (Defensin, beta 107) (Beta-defensin 7) (BD-7) (DEFB-7)


70


Q7Z5J1


Hydroxysteroid 11-beta-dehydrogenase 1-like protein (EC 1.1.1.-) (Short-chain dehydrogenase/reductase 10) (11-beta-hydroxysteroid dehydrogenase 


type 3) (11-beta-HSD3)


315


O95925


Eppin (Epididymal protease inhibitor) (Serine protease inhibitor-like with Kunitz and WAP domains 1) (WAP four-disulfide core domain protein 7) 


(Protease inhibitor WAP7) (Cancer/testis antigen 71) (CT71)


133


O43555


Progonadoliberin-2 (Progonadoliberin II) [Cleaved into: Gonadoliberin-2 (Gonadoliberin II) (Luteinizing hormone-releasing hormone II) (LH-RH II)


(Gonadotropin-releasing hormone II) (GnRH II) (Luliberin II); GnRH-associated peptide 2 (GnRH-associated peptide II)]


120


Q7RTW8


Otoancorin


1,153


Q92752


Tenascin-R (TN-R) (Restrictin) (Janusin)


1,358


Q9NZC2


Triggering receptor expressed on myeloid cells 2 (Triggering receptor expressed on monocytes 2) (TREM-2)


230


Q8WU66


Protein TSPEAR (TSP-EAR)


669


Q8NEX6


Protein WFDC11


87


Q9BQY6


WAP four-disulfide core domain protein 6 (Putative protease inhibitor WAP6)


131


Q6UW88


Epigen (Epithelial mitogen) (EPG)


154


Q7Z5A7


Protein FAM19A5 (Chemokine-like protein TAFA-5)


132


Q9Y3Q7


Disintegrin and metalloproteinase domain-containing protein 18 (ADAM 18) (Transmembrane metalloproteinase-like, disintegrin-like, and cysteine-rich


protein III) (tMDC III)


739


Q13790


Apolipoprotein F (Apo-F) (Lipid transfer inhibitor protein) (LTIP)


308


Q6UXE8


Butyrophilin-like protein 3 (Butyrophilin-like receptor)


466


Q6UXG8


Butyrophilin-like protein 9


535


Q6P4E1


Protein CASC4 (Cancer susceptibility candidate gene 4 protein)


433


Q7Z692


Carcinoembryonic antigen-related cell adhesion molecule 19 (Carcinoembryonic antigen-like 1)


300


P56856


Claudin-18


261


P56747


Claudin-6 (Skullin 2)


220


P56748


Claudin-8


225


Q6UWF3


Transmembrane protein C17orf87


145


Q96MF6


Protein COQ10 A, mitochondrial


247


Q6UXV1


Uncharacterized protein C19orf41


221


P58658


Uncharacterized protein C21orf63 (SUE21)


441


Q6UY11


Protein delta homolog 2 (DLK-2) (EGF-like domain-containing protein 9) (Multiple EGF-like domain protein 9)


383


Q6UX65


DNA damage-regulated autophagy modulator protein 2 (Transmembrane protein 77)


266


Q9BXR6


Complement factor H-related protein 5 (FHR-5)


569


Q9Y680


FK506-binding protein 7 (EC 5.2.1.8) (Peptidyl-prolyl cis-trans isomerase) (PPIase) (Rotamase) (FKBP-23)


259


Q96PP8


Guanylate-binding protein 5 (Guanine nucleotide-binding protein 5) (GTP-binding protein 5) (GBP-5) (GBP-TA antigen)


586


P55259


Pancreatic secretory granule membrane major glycoprotein GP2 (Pancreatic zymogen granule membrane protein GP-2) (ZAP75)


537


Q86WI0


Lipoma HMGIC fusion partner-like 1 protein


220


Q9HAT1


Protein ERGIC-53-like (ERGIC53-like protein) (Lectin mannose-binding 1-like) (LMAN1-like protein)


526


Q8N456


Leucine-rich repeat-containing protein 18


261


Q8N386


Leucine-rich repeat-containing protein 25 (Monocyte and plasmacytoid-activated protein)


305


Q96PB8


Leucine-rich repeat-containing protein 3B (Leucine-rich repeat protein LRP15)


259


Q9BY71


Leucine-rich repeat-containing protein 3


257


Q6UWN0


Ly6/PLAUR domain-containing protein 4


246


Q9BZM2


Group IIF secretory phospholipase A2 (GIIF sPLA2) (EC 3.1.1.4) (Phosphatidylcholine 2-acylhydrolase GIIF) (sPLA(2)-IIF)


168


Q9HBH5


Retinol dehydrogenase 14 (EC 1.1.1.-) (Alcohol dehydrogenase PAN2)


336


Q96G75


Protein RMD5 homolog B


393


Q6ZMJ2


Scavenger receptor class A member 5 (Scavenger receptor hlg)


495


P62341


Selenoprotein T (SelT)


195


Q9H3T3


Semaphorin-6B (Semaphorin-Z) (Sema Z)


888


Q96SA4


Serine incorporator 2 (Tumor differentially expressed 2-like)


456


Q16842


CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase (Beta-galactoside alpha-2,3-sialyltransferase) (Alpha 2,3-ST) (EC 2.4.99.-) 


(Gal-NAc6S) (Gal-beta-1,3-GalNAc-alpha-2,3-sialyltransferase) (ST3GalA.2) (SIAT4-B) (ST3Gal II)


350


Q96RL6


Sialic acid-binding Ig-like lectin 11 (Sialic acid-binding lectin 11) (Siglec-11)


686


Q9Y343


Sorting nexin-24


169


Q96L08


Sushi domain-containing protein 3


255


Q6UXF1


Transmembrane protein 108


575


Q4V9L6


Transmembrane protein 119


283


Q6UXZ0


Transmembrane and immunoglobulin domain-containing protein 1


262


Q9Y2Y6


Transmembrane protein 98 (Protein TADA1)


226


Q9BY14


Testis-expressed protein 101 (Scleroderma-associated autoantigen) (Cell surface receptor NYD-SP8) (Spermatogenesis-related gene protein)


249


Q6UXZ4


Netrin receptor UNC5D (Protein unc-5 homolog D) (Unc-5 homolog 4)


953


Q9UKI3


Pre-B lymphocyte protein 3 (Protein VPreB3) (N27C7-2)


123


Q9H1B5


Xylosyltransferase 2 (EC 2.4.2.26) (Xylosyltransferase II) (XylT-II) (XT-II) (Peptide O-xylosyltransferase 1)


865


Q96TA2


ATP-dependent metalloprotease YME1L1 (EC 3.4.24.-) (YME1-like protein 1) (ATP-dependent metalloprotease FtsH1) (Meg-4) (Presenilin-associated 


metalloprotease) (PAMP)


773


Q9H3R2


Mucin-13 (MUC-13) (Down-regulated in colon cancer 1)


512


O57166


Growth factor (Secreted epidermal growth factor-like) (Vaccinia growth factor)


140


P20494


Growth factor (Secreted epidermal growth factor-like) (Vaccinia growth factor)


142


Q86607


Growth factor (Secreted epidermal growth factor-like) (Vaccinia growth factor)


140


Q9H336


Cysteine-rich secretory protein LCCL domain-containing 1 (LCCL domain-containing cysteine-rich secretory protein 1) (Cysteine-rich secretory 


protein 10) (CRISP-10) (Trypsin inhibitor Hl) (CocoaCrisp)


500


Q6UWT2


Adropin (Energy homeostasis-associated protein)


76


Q6UWR7


Ectonucleotide pyrophosphatase/phosphodiesterase family member 6 (E-NPP6) (NPP-6) (EC 3.1.-.-) [Cleaved into: Ectonucleotide 


pyrophosphatase/phosphodiesterase family member 6 soluble form]


440


Q96MK3


Protein FAM20A


541


P60985


Keratinocyte differentiation-associated protein


99


Q9BSG0


Protease-associated domain-containing protein of 21 kDa (hPAP21)


188


Q6UY27


Prostate and testis expressed protein 2 (PATE-like protein M) (PATE-M)


113


Q9BZM1


Group XIIA secretory phospholipase A2 (GXII sPLA2) (EC 3.1.1.4) (Phosphatidylcholine 2-acylhydrolase GXII) (sPLA2-XII)


189


Q9BX93


Group XIIB secretory phospholipase A2-like protein (Group XIII secretory phospholipase A2-like protein) (GXIII sPLA2-like) (sPLA2-GXIIB)


(GXIIB)


195


A8K2U0


Alpha-2-macroglobulin-like protein 1 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 9)


1,454


Q8TE60


A disintegrin and metalloproteinase with thrombospondin motifs 18 (ADAMTS-18) (ADAM-TS 18) (ADAM-TS18) (EC 3.4.24.-)


1,221


Q8TE59


A disintegrin and metalloproteinase with thrombospondin motifs 19 (ADAMTS-19) (ADAM-TS 19) (ADAM-TS19) (EC 3.4.24.-)


1,207


O15072


A disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS-3) (ADAM-TS 3) (ADAM-TS3) (EC 3.4.24.-) (Procollagen II amino 


propeptide-processing enzyme) (Procollagen II N-proteinase) (PC II-NP)


1,205


Q9UNA0


A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) (ADAM-TS 5) (ADAM-TS5) (EC 3.4.24.-) (Aggrecanase-2)


(ADMP-2) (A disintegrin and metalloproteinase with thrombospondin motifs 11) (ADAMTS-11) (ADAM-TS 11)


930


Q9UKP4


A disintegrin and metalloproteinase with thrombospondin motifs 7 (ADAMTS-7) (ADAM-TS 7) (ADAM-TS7) (EC 3.4.24.-) (COMPase)


1,686


Q9P2N4


A disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS-9) (ADAM-TS 9) (ADAM-TS9) (EC 3.4.24.-)


1,935


P12643


Bone morphogenetic protein 2 (BMP-2) (BMP-2A)


396


P22004


Bone morphogenetic protein 6 (BMP-6)


513


Q8N8U9


BMP-binding endothelial regulator protein (Bone morphogenetic protein-binding endothelial cell precursor-derived regulator) (Protein crossveinless-2)


(hCV2)


685


O95813


Cerberus (Cerberus-related protein) (DAN domain family member 4)


267


Q9H2A9


Carbohydrate sulfotransferase 8 (EC 2.8.2.-) (N-acetylgalactosamine-4-O-sulfotransferase 1) (GalNAc-4-O-sulfotransferase 1) (GalNAc-4-ST1)


(GalNAc4ST-1)


424


Q9BZ76


Contactin-associated protein-like 3 (Cell recognition molecule Caspr3)


1,288


P25940


Collagen alpha-3(V) chain


1,745


P06850


Corticoliberin (Corticotropin-releasing hormone) (Corticotropin-releasing factor) (CRF)


196


O75718


Cartilage-associated protein


401


Q969H8


UPF0556 protein C19orf10 (Stromal cell-derived growth factor SF20) (Interleukin-25) (IL-25)


173


P19876


C-X-C motif chemokine 3 (Macrophage inflammatory protein 2-beta) (MIP2-beta) (Growth-regulated protein gamma) (GRO-gamma) 


(GRO-gamma(1-73)) [Cleaved into: GRO-gamma(5-73)]


107


Q15828


Cystatin-M (Cystatin-6) (Cystatin-E)


149


Q01523


Defensin-5 (Defensin, alpha 5)


94


Q01524


Defensin-6 (Defensin, alpha 6)


100


Q07507


Dermatopontin (Tyrosine-rich acidic matrix protein) (TRAMP)


201


O43323


Desert hedgehog protein (DHH) (HHG-3) [Cleaved into: Desert hedgehog protein N-product; Desert hedgehog protein C-product]


396


Q9UBT3


Dickkopf-related protein 4 (Dickkopf-4) (Dkk-4) (hDkk-4) [Cleaved into: Dickkopf-related protein 4 short form]


224


O94769


Extracellular matrix protein 2 (Matrix glycoprotein SC1/ECM2)


699


Q9UM22


Mammalian ependymin-related protein 1 (MERP-1) (Upregulated in colorectal cancer gene 1 protein)


224


Q75N90


Fibrillin-3


2,809


Q9UGM5


Fetuin-B (Gugu) (IRL685) (16G2)


382


P10767


Fibroblast growth factor 6 (FGF-6) (Heparin-binding growth factor 6) (HBGF-6) (HST-2)


208


P21781


Keratinocyte growth factor (KGF) (Fibroblast growth factor 7) (FGF-7) (Heparin-binding growth factor 7) (HBGF-7)


194


O14793


Growth/differentiation factor 8 (GDF-8) (Myostatin)


375


P09681


Gastric inhibitory polypeptide (GIP) (Glucose-dependent insulinotropic polypeptide)


153


Q86YW7


Glycoprotein hormone beta-5 (Thyrostimulin subunit beta)


130


P51124


Granzyme M (EC 3.4.21.-) (Met-ase) (Natural killer cell granular protease) (HU-Met-1) (Met-1 serine protease)


257


P07492


Gastrin-releasing peptide (GRP) [Cleaved into: Neuromedin-C (GRP-10)]


148


Q92743


Serine protease HTRA1 (EC 3.4.21.-) (L56) (Serine protease 11)


480


P05014


Interferon alpha-4 (Interferon alpha-4B) (Interferon alpha-M1) (Interferon alpha-76)


189


P01569


Interferon alpha-5 (Interferon alpha-G) (LeIF G) (Interferon alpha-61)


189


P01567


Interferon alpha-7 (Interferon alpha-J1) (IFN-alpha-J1) (Interferon alpha-J) (LeIF J)


189


P27352


Gastric intrinsic factor (Intrinsic factor) (INF) (IF)


417


Q6WRI0


Immunoglobulin superfamily member 10 (Calvaria mechanical force protein 608) (CMF608)


2,623


Q9UHA7


Interleukin-1 family member 6 (IL-1F6) (Interleukin-1 epsilon) (IL-1 epsilon) (FIL1 epsilon)


158


Q14213


Interleukin-27 subunit beta (IL-27 subunit beta) (IL-27B) (Epstein-Barr virus-induced gene 3 protein) (EBV-induced gene 3 protein)


229


O95760


Interleukin-33 (IL-33) (Interleukin-1 family member 11) (IL-1F11) (Nuclear factor from high endothelial venules) (NF-HEV)


270


Q01113


Interleukin-9 receptor (IL-9R) (CD antigen CD129)


521


P15248


Interleukin-9 (IL-9) (T-cell growth factor P40) (P40 cytokine)


144


P09529


Inhibin beta B chain (Activin beta-B chain)


407


P20155


Serine protease inhibitor Kazal-type 2 (Acrosin-trypsin inhibitor) (HUSI-II)


84


A3KMH1


Uncharacterized protein KIAA0564


1,905


Q8N6L1


Keratinocyte-associated protein 2 (KCP-2)


162


P25391


Laminin subunit alpha-1 (Laminin A chain)


3,075


O14960


Leukocyte cell-derived chemotaxin-2 (hLECT2)


151


P07098


Gastric triacylglycerol lipase (Gastric lipase) (GL) (EC 3.1.1.3)


398


P54317


Pancreatic lipase-related protein 2 (PL-RP2) (EC 3.1.1.3)


469


Q96II8


Leucine-rich repeat and calponin homology domain-containing protein 3


777


Q9Y2E5


Epididymis-specific alpha-mannosidase (EC 3.2.1.24) (Mannosidase alpha class 2B member 2)


1,009


P20382


Pro-MCH [Cleaved into: Neuropeptide-glycine-glutamic acid (Neuropeptide G-E) (NGE); Neuropeptide-glutamic acid-isoleucine (Neuropeptide E-I)


(NEI); Melanin-concentrating hormone (MCH)]


165


Q13361


Microfibrillar-associated protein 5 (MFAP-5) (Microfibril-associated glycoprotein 2) (MAGP-2) (MP25)


173


P03971


Muellerian-inhibiting factor (MIS) (Anti-Muellerian hormone) (AMH) (Muellerian-inhibiting substance)


560


P09238


Stromelysin-2 (SL-2) (EC 3.4.24.22) (Matrix metalloproteinase-10) (MMP-10) (Transin-2)


476


Q9ULZ9


Matrix metalloproteinase-17 (MMP-17) (EC 3.4.24.-) (Membrane-type matrix metalloproteinase 4) (MT-MMP 4) (Membrane-type-4 matrix


metalloproteinase) (MT4-MMP)


606


O60882


Matrix metalloproteinase-20 (MMP-20) (EC 3.4.24.-) (Enamel metalloproteinase) (Enamelysin)


483


Q8N119


Matrix metalloproteinase-21 (MMP-21) (EC 3.4.24.-)


569


Q9H8L6


Multimerin-2 (EMILIN-3) (Elastin microfibril interface located protein 3) (Elastin microfibril interfacer 3) (EndoGlyx-1 p125/p140 subunit)


949


P12872


Promotilin [Cleaved into: Motilin; Motilin-associated peptide (MAP)]


115


Q8TAX7


Mucin-7 (MUC-7) (Salivary mucin-7) (Apo-MG2)


377


Q92832


Protein kinase C-binding protein NELL1 (NEL-like protein 1) (Nel-related protein 1)


810


Q99435


Protein kinase C-binding protein NELL2 (NEL-like protein 2) (Nel-related protein 2)


816


P56730


Neurotrypsin (EC 3.4.21.-) (Serine protease 12) (Motopsin) (Leydin)


875


Q99574


Neuroserpin (Serpin I1) (Protease inhibitor 12)


410


Q14112


Nidogen-2 (NID-2) (Osteonidogen)


1,375


P48745


Protein NOV homolog (NovH) (Nephroblastoma overexpressed gene protein homolog)


357


P34130


Neurotrophin-4 (NT-4) (Neutrophic factor 4) (Neurotrophin-5) (NT-5)


210


Q9UNK4


Group IID secretory phospholipase A2 (GIID sPLA2) (EC 3.1.1.4) (Phosphatidylcholine 2-acylhydrolase GIID) (PLA2IID) (sPLA(2)-IID)


(Secretory-type PLA, stroma-associated homolog)


145


O15496


Group 10 secretory phospholipase A2 (EC 3.1.1.4) (Group X secretory phospholipase A2) (GX sPLA2) (Phosphatidylcholine 2-acylhydrolase GX)


(sPLA2-X)


165


Q9BXP8


Pappalysin-2 (EC 3.4.24.-) (Pregnancy-associated plasma protein A2) (PAPP-A2) (Pregnancy-associated plasma protein E1) (PAPP-E)


1,791


P22079


Lactoperoxidase (LPO) (EC 1.11.1.7) (Salivary peroxidase) (SPO)


712


Q96FX8


p53 apoptosis effector related to PMP-22 (Keratinocyte-associated protein 1) (KCP-1) (P53-induced protein PIGPC1) (Transmembrane protein THW)


193


Q9HBJ0


Placenta-specific protein 1


212


Q15166


Serum paraoxonase/lactonase 3 (EC 3.1.1.-)


354


P51888


Prolargin (Proline-arginine-rich end leucine-rich repeat protein)


382


P13521


Secretogranin-2 (Secretogranin II) (SgII) (Chromogranin-C) [Cleaved into: Secretoneurin (SN)]


617


O76038


Secretagogin


276


Q13214


Semaphorin-3B (Semaphorin V) (Sema V) (Sema A(V))


749


P61278


Somatostatin (Growth hormone release-inhibiting factor) [Cleaved into: Somatostatin-28; Somatostatin-14]


116


O76061


Stanniocalcin-2 (STC-2) (Stanniocalcin-related protein) (STCRP) (STC-related protein)


302


P20061


Transcobalamin-1 (TC-1) (Transcobalamin I) (TC I) (TCI)


433


Q03403


Trefoil factor 2 (Spasmolytic polypeptide) (SP) (Spasmolysin)


129


Q08629


Testican-1 (Protein SPOCK)


439


Q96A98


Tuberoinfundibular peptide of 39 residues (TIP39) (Parathyroid hormone 2)


100


Q9Y6L7


Tolloid-like protein 2 (EC 3.4.24.-)


1,015


P07478


Trypsin-2 (EC 3.4.21.4) (Trypsin II) (Anionic trypsinogen) (Serine protease 2)


247


P55089


Urocortin


124


O96014


Protein Wnt-11


354


P09544


Protein Wnt-2 (Int-1-related protein) (IRP protein)


360


O00755


Protein Wnt-7a


349


Q93098


Protein Wnt-8b


351


P78423


Fractalkine (C-X3-C motif chemokine 1) (Neurotactin) (CX3C membrane-anchored chemokine) (Small-inducible cytokine D1) [Cleaved into:


Processed fractalkine]


397


Q96A84


EMI domain-containing protein 1 (Emilin and multimerin domain-containing protein 1) (Emu1)


441


Q6UXA7


Uncharacterized protein C6orf15 (Protein STG)


325


Q9Y6Z7


Collectin-10 (Collectin liver protein 1) (CL-L1) (Collectin-34)


277


Q6UWX4


HHIP-like protein 2


724


Q8WWZ8


Oncoprotein-induced transcript 3 protein (Liver-specific zona pellucida domain-containing protein)


545


O43692


Peptidase inhibitor 15 (Cysteine-rich secretory protein 8) (CRISP-8) (25 kDa trypsin inhibitor) (p25TI) (SugarCrisp)


258


Q96NZ9


Proline-rich acidic protein 1 (Uterine-specific proline-rich acidic protein)


151


Q96DX4


RING finger and SPRY domain-containing protein 1


576


Q8IYP2


Trypsin-X3 (EC 3.4.21.4)


241


Q5FVE4


Long-chain-fatty-acid--CoA ligase ACSBG2 (EC 6.2.1.3) (Acyl-CoA synthetase bubblegum family member 2) (Bubblegum-related protein)


(PRTD-NY3)


666


Q9C0J1


UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 4 (Beta-1,3-N-acetylglucosaminyltransferase-4) (Beta3Gn-T4) (BGnT-4) (EC 2.4.1.-)


378


Q9P1Z2


Calcium-binding and coiled-coil domain-containing protein 1 (Calphoglin) (Sarcoma antigen NY-SAR-3) (Coiled-coil coactivator protein)


691


Q5MY95


Ectonucleoside triphosphate diphosphohydrolase 8 (E-NTPDase 8) (NTPDase 8) (NTPDase8) (EC 3.6.1.5)


495


Q86WN1


FCH and double SH3 domains protein 1


690


Q7Z4V5


Hepatoma-derived growth factor-related protein 2 (HRP-2) (Hepatoma-derived growth factor 2)


671


P29459


Interleukin-12 subunit alpha (IL-12A) (IL-12 subunit p35) (Cytotoxic lymphocyte maturation factor 35 kDa subunit) (CLMF p35) (NK cell 


stimulatory factor chain 1) (NKSF1)


219


Q6UXG2


UPF0577 protein KIAA1324 (Estrogen-induced gene 121 protein)


1,013


Q6UVY6


DBH-like monooxygenase protein 1 (EC 1.14.17.-) (Monooxygenase X)


613


Q685J3


Mucin-17 (MUC-17) (Small intestinal mucin-3) (MUC-3)


4,493


Q8WWB7


Lysosomal protein NCU-G1


406


Q7Z4N8


Prolyl 4-hydroxylase subunit alpha-3 (EC 1.14.11.2) (4-PH alpha-3) (Procollagen-proline,2-oxoglutarate-4-dioxygenase subunit alpha-3)


544


Q75T13


GPI inositol-deacylase (EC 3.1.-.-) (Post-GPI attachment to proteins factor 1) (hPGAP1)


922


Q6UXB8


Peptidase inhibitor 16 (Cysteine-rich secretory protein 9) (CRISP-9) (PSP94-binding protein)


463


Q96NY8


Poliovirus receptor-related protein 4 (Nectin-4) (Ig superfamily receptor LNIR) [Cleaved into: Processed poliovirus receptor-related protein 4]


510


Q8IZD6


Solute carrier family 22 member 15 (Fly-like putative transporter 1) (Flipt 1)


547


Q6UX04


Peptidyl-prolyl cis-trans isomerase SDCCAG10 (EC 5.2.1.8) (Serologically defined colon cancer antigen 10) (Antigen NY-CO-10)


472


Q9BTV4


Transmembrane protein 43 (Protein LUMA)


400


Q9UKU9


Angiopoietin-related protein 2 (Angiopoietin-like 2)


493


Q8NI99


Angiopoietin-related protein 6 (Angiopoietin-like 6) (Angiopoietin-related growth factor) (Angiopoietin-related protein 5)


470


Q6UWY0


Arylsulfatase K (ASK) (EC 3.1.6.-) (Telethon sulfatase)


536


Q9H1Z8


Augurin (Esophageal cancer-related gene 4 protein)


148


Q9GZN4


Brain-specific serine protease 4 (BSSP-4) (EC 3.4.21.-) (Serine protease 22) (Serine protease 26) (Tryptase epsilon)


317


P0C862


Complement C1q tumor necrosis factor-related protein 9


333


Q9BUN1


Uncharacterized protein C1orf56


341


Q6UW01


Cerebellin-3


205


Q9NTU7


Cerebellin-4 (Cerebellin-like glycoprotein 1)


201


Q6UWE3


Colipase-like protein C6orf126


100


Q6UW78


Uncharacterized protein C11orf83


93


Q6UX73


Uncharacterized protein C16orf89


402


Q8N436


Carboxypeptidase-like protein X2


756


Q9H4G1


Cystatin-9-like


147


Q9NRR1


Cytokine-like protein 1 (Protein C17)


136


Q8N688


Beta-defensin 123 (Defensin, beta 123) (Beta-defensin 23) (DEFB-23)


67


Q7Z7B7


Beta-defensin 132 (Defensin, beta 132) (Beta-defensin 32) (BD-32) (Defensin HEL-75) (DEFB-32)


95


Q6UX07


Dehydrogenase/reductase SDR family member 13 (EC 1.1.-.-)


377


Q9UBU2


Dickkopf-related protein 2 (Dickkopf-2) (Dkk-2) (hDkk-2)


259


Q9UK85


Dickkopf-like protein 1 (Protein soggy-1) (SGY-1) (Cancer/testis antigen 34) (CT34)


242


P56851


Epididymal secretory protein E3-beta (Human epididymis-specific protein 3-beta) (HE3 beta)


147


Q969Y0


Protein FAM55C


559


Q6UWF7


Protein FAM55D


544


O60258


Fibroblast growth factor 17 (FGF-17)


216


Q9HCT0


Fibroblast growth factor 22 (FGF-22)


170


Q9BYJ0


Fibroblast growth factor-binding protein 2 (FGF-binding protein 2) (FGF-BP2) (FGFBP-2) (37 kDa killer-specific secretory protein) (Ksp37)


(HBp17-related protein) (HBp17-RP)


223


Q9NR23


Growth/differentiation factor 3 (GDF-3)


364


Q6UWM5


GLIPR1-like protein 1


242


Q96S86


Hyaluronan and proteoglycan link protein 3


360


Q9UHF5


Interleukin-17B (IL-17B) (Cytokine-like protein Zcyto7) (Neuronal interleukin-17-related factor) (Interleukin-20) (IL-20)


180


Q9P0M4


Interleukin-17C (IL-17C) (Cytokine CX2)


197


Q8TAD2


Interleukin-17D (IL-17D) (Interleukin-27) (IL-27)


202


Q6UWN8


Serine protease inhibitor Kazal-type 6


80


P58062


Serine protease inhibitor Kazal-type 7 (Esophagus cancer-related gene 2 protein) (ECRG-2)


85


Q53RY4


Keratinocyte-associated protein 3 (KCP-3)


240


Q6UWQ5


Lysozyme-like protein 1 (EC 3.2.1.17)


148


Q9H239


Matrix metalloproteinase-28 (MMP-28) (EC 3.4.24.-) (Epilysin)


520


Q6UXI9


Nephronectin (Preosteoblast EGF-like repeat protein with MAM domain) (Protein EGFL6-like)


565


O95158


Neurexophilin-4


308


Q6UWY5


Olfactomedin-like protein 1


402


Q68BL7


Olfactomedin-like protein 2A (Photomedin-1)


652


Q9BQR3


Serine protease 27 (EC 3.4.21.-) (Marapsin) (Pancreasin) (Channel-activating protease 2) (CAPH2)


290


Q8WXD2


Secretogranin-3 (Secretogranin III) (SgIII)


468


O75711


Scrapie-responsive protein 1 (ScRG-1)


98


O95025


Semaphorin-3D


777


Q96PL1


Secretoglobin family 3A member 2 (Uteroglobin-related protein 1) (Pneumo secretory protein 1) (PnSP-1)


93


O60235


Transmembrane protease, serine 11D (EC 3.4.21.-) (Airway trypsin-like protease) [Cleaved into: Transmembrane protease, serine 11D


non-catalytic chain; Transmembrane protease, serine 11D catalytic chain]


418


Q7Z5L0


Vitelline membrane outer layer protein 1 homolog


202


Q8WWY7


WAP four-disulfide core domain protein 12 (Putative protease inhibitor WAP12) (Whey acidic protein 2)


111


O14905


Protein Wnt-9b (Wnt-15) (Wnt-14b)


357


Q96DA0


Zymogen granule protein 16 homolog B


208


Q9UKQ2


Disintegrin and metalloproteinase domain-containing protein 28 (ADAM 28) (EC 3.4.24.-) (Metalloproteinase-like, disintegrin-like, and


cysteine-rich protein L) (MDC-L) (Epididymial metalloproteinase-like, disintegrin-like, and cysteine-rich protein II) (eMDC II)


775


Q9BPW4


Apolipoprotein L4 (Apolipoprotein L-IV) (ApoL-IV)


351


Q92485


Acid sphingomyelinase-like phosphodiesterase 3b (ASM-like phosphodiesterase 3b) (EC 3.1.4.-)


455


O95450


A disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2) (ADAM-TS 2) (ADAM-TS2) (EC 3.4.24.14) (Procollagen


I/II amino propeptide-processing enzyme) (Procollagen I N-proteinase) (PC I-NP) (Procollagen N-endopeptidase) (pNPI)


1,211


A6NMY6


Putative annexin A2-like protein (Annexin A2 pseudogene 2) (Lipocortin II pseudogene)


339


O95393


Bone morphogenetic protein 10 (BMP-10)


424


Q8NFQ6


Bactericidal/permeability-increasing protein-like 2


507


Q9BXJ3


Complement C1q tumor necrosis factor-related protein 4


329


Q8WWF1


Uncharacterized protein C1orf54


131


Q8N4T0


Carboxypeptidase A6 (EC 3.4.17.1) (Carboxypeptidase B)


437


Q99731


C-C motif chemokine 19 (Small-inducible cytokine A19) (Macrophage inflammatory protein 3 beta) (MIP-3-beta) (EBI1-ligand chemokine) (ELC)


(Beta chemokine exodus-3) (CK beta-11)


98


O15444


C-C motif chemokine 25 (Small-inducible cytokine A25) (Thymus-expressed chemokine) (Chemokine TECK)


150


Q86SI9


Protein CEI (Coordinated expression to IRXA2 protein)


138


P08218


Chymotrypsin-like elastase family member 2B (EC 3.4.21.71) (Elastase-2B)


269


Q86T20


Uncharacterized protein C6orf1 (Protein LBH)


159


Q96L11


Uncharacterized protein C7orf34 (MSSP-binding protein CTM-1)


122


A6NKQ9


Choriogonadotropin subunit beta variant 1


187


Q6ZRZ4


Uncharacterized protein C9orf47


202


P27658


Collagen alpha-1(VIII) chain (Endothelial collagen)


744


Q8IYD9


Uncharacterized protein C18orf54


372


Q9H114


Cystatin-like 1 (RCET11)


145


Q9Y426


C2 domain-containing protein 2


696


Q96HY6


DDRGK domain-containing protein 1


314


O43854


EGF-like repeat and discoidin I-like domain-containing protein 3 (Developmentally-regulated endothelial cell locus 1 protein)


(Integrin-binding protein DEL1)


480


Q96A83


Collagen alpha-1(XXVI) chain (EMI domain-containing protein 2) (Emilin and multimerin domain-containing protein 2) (Emu2)


441


Q8N3H0


Protein FAM19A2 (Chemokine-like protein TAFA-2)


131


Q9C0B6


Protein FAM5B (BMP/retinoic acid-inducible neural-specific protein 2) (DBCCR1-like protein 2)


783


P12034


Fibroblast growth factor 5 (FGF-5) (Heparin-binding growth factor 5) (HBGF-5) (Smag-82)


268


P83110


Probable serine protease HTRA3 (EC 3.4.21.-) (High-temperature requirement factor A3) (Pregnancy-related serine protease)


453


Q9NZH7


Interleukin-1 family member 8 (IL-1F8) (Interleukin-1 eta) (IL-1 eta) (FIL1 eta) (Interleukin-1 homolog 2) (IL-1H2)


164


O60575


Serine protease inhibitor Kazal-type 4 (Peptide PEC-60 homolog)


86


Q6ISS4


Leukocyte-associated immunoglobulin-like receptor 2 (LAIR-2) (CD antigen CD306)


152


Q969E1


Liver-expressed antimicrobial peptide 2 (LEAP-2)


77


Q08397


Lysyl oxidase homolog 1 (EC 1.4.3.-) (Lysyl oxidase-like protein 1) (LOL)


574


Q9Y4K0


Lysyl oxidase homolog 2 (EC 1.4.3.-) (Lysyl oxidase-like protein 2) (Lysyl oxidase-related protein 2) (Lysyl oxidase-related protein WS9-14)


774


P59827


Long palate, lung and nasal epithelium carcinoma-associated protein 4 (Ligand-binding protein RY2G5)


614


Q86SG7


Lysozyme g-like protein 2 (EC 3.2.1.-)


212


Q9ULC0


Endomucin (Endomucin-2) (Mucin-14) (MUC-14) (Gastric cancer antigen Ga34)


261


Q8NG41


Neuropeptide B (Preproprotein L7) (hPPL7) [Cleaved into: Neuropeptide B-23 (NPB23) (hL7); Neuropeptide B-29 (NPB29) (hL7C)]


125


Q8WWG1


Pro-neuregulin-4, membrane-bound isoform (Pro-NRG4) [Cleaved into: Neuregulin-4 (NRG-4)]


115


Q8NHW6


Otospiralin


89


P39877


Calcium-dependent phospholipase A2 (EC 3.1.1.4) (Phosphatidylcholine 2-acylhydrolase) (PLA2-10) (Group V phospholipase A2)


138


Q8NB37


Parkinson disease 7 domain-containing protein 1


220


Q15198


Platelet-derived growth factor receptor-like protein (PDGF receptor beta-like tumor suppressor)


375


Q9UIG4


Psoriasis susceptibility 1 candidate gene 2 protein (SPR1 protein)


136


P49223


Kunitz-type protease inhibitor 3 (HKIB9)


89


Q4W5P6


Protein TMEM155


130


Q8N2E6


Prosalusin (Torsin-2A) (Torsin family 2 member A) [Cleaved into: Salusin-alpha; Salusin-beta]


242


A6NCI4


von Willebrand factor A domain-containing protein 3A


1,184


Q8IUB3


Protein WFDC10B


73


Q8IUA0


WAP four-disulfide core domain protein 8 (Putative protease inhibitor WAP8)


241


Q9H1J7


Protein Wnt-5b


359


P56706


Protein Wnt-7b


349


Q6X784


Zona pellucida-binding protein 2 (ZPBP-like protein)


338


Q7L8J4


SH3 domain-binding protein 5-like


393


Q9H6B4


Adipocyte adhesion molecule (Coxsackie- and adenovirus receptor-like membrane protein) (CAR-like membrane protein)


373


Q96CM8


Acyl-CoA synthetase family member 2, mitochondrial (EC 6.2.1.-)


615


Q8NCW5


Apolipoprotein A-I-binding protein (AI-BP) (YjeF N-terminal domain-containing protein 1)


288


Q4VNC1


Probable cation-transporting ATPase 13A4 (EC 3.6.3.-) (P5-ATPase isoform 4)


1,196


Q6UXS9


Inactive caspase-12 (CASP-12)


341


A4D0V7


Uncharacterized protein C7orf58


1,026


Q2UY09


Collagen alpha-1(XXVIII) chain


1,125


Q8IXL6


Dentin matrix protein 4 (DMP-4) (Protein FAM20C)


570


Q96DN0


Endoplasmic reticulum resident protein ERp27


273


Q6UWW8


Carboxylesterase 3 (EC 3.1.1.1) (Liver carboxylesterase 31 homolog)


571


Q9BVA6


Adenosine monophosphate-protein transferase FICD (EC 2.7.7.n1) (AMPylator FICD) (FIC domain-containing protein) (Huntingtin-interacting


protein E) (Huntingtin yeast partner E) (Huntingtin-interacting protein 13)


458


Q9UHW5


GPN-loop GTPase 3 (ATP-binding domain 1 family member C)


284


Q3KR37


GRAM domain-containing protein 1B


738


Q96D96


Voltage-gated hydrogen channel 1 (Hydrogen voltage-gated channel 1) (HV1) (Voltage sensor domain-only protein)


273


Q8NEV9


Interleukin-27 subunit alpha (IL-27 subunit alpha) (IL27-A) (p28)


243


Q2M1P5


Kinesin-like protein KIF7


1,343


Q9H3W5


Leucine-rich repeat neuronal protein 3 (Neuronal leucine-rich repeat protein 3) (NLRR-3)


708


Q659A1


NMDA receptor-regulated protein 2


982


Q9UHQ9


NADH-cytochrome b5 reductase 1 (b5R.1) (EC 1.6.2.2) (NAD(P)H:quinone oxidoreductase type 3 polypeptide A2) (Humb5R2)


305


Q8WU39


Proapoptotic caspase adapter protein (PACAP)


189


Q96FC7


Phytanoyl-CoA hydroxylase-interacting protein-like


376


Q9BS91


Probable UDP-sugar transporter protein SLC35A5 (Solute carrier family 35 member A5)


424


Q8WV83


Solute carrier family 35 member F5 (Hepatitis C virus NS5A-transactivated protein 3) (HCV NS5A-transactivated protein 3)


523


Q96PQ1


Sialic acid-binding Ig-like lectin 12 (Siglec-12) (Sialic acid-binding Ig-like lectin-like 1) (Siglec-L1)


595


Q96J42


Thioredoxin domain-containing protein 15


360


Q6UXN9


WD repeat-containing protein 82 (Swd2)


313


Q8IUX7


Adipocyte enhancer-binding protein 1 (AE-binding protein 1) (Aortic carboxypeptidase-like protein)


1,158


P82987


ADAMTS-like protein 3 (ADAMTSL-3) (Punctin-2)


1,691


Q76M96


Coiled-coil domain-containing protein 80 (Down-regulated by oncogenes protein 1)


950


Q8TC92


Ecto-NOX disulfide-thiol exchanger 1 (Constitutive Ecto-NOX) (cNOX) (Candidate growth-related and time keeping constitutive hydroquinone


[NADH]oxidase) (cCNOX) (Cell proliferation-inducing gene 38 protein) [Includes: Hydroquinone [NADH]oxidase (EC 1.-.-.-);


Protein disulfide-thiol oxidoreductase (EC 1.-.-.-)]


643


Q4ZHG4


Fibronectin type III domain-containing protein 1 (Expressed in synovial lining protein) (Activation-associated cDNA protein)


1,888


Q17R60


Interphotoreceptor matrix proteoglycan 1 (Interphotoreceptor matrix proteoglycan of 150 kDa) (IPM-150) (Sialoprotein associated with cones and rods)


797


O95965


Integrin beta-like protein 1 (Ten integrin EGF-like repeat domain-containing protein) (Osteoblast-specific cysteine-rich protein)


494


Q8WWY8


Lipase member H (EC 3.1.1.-) (Membrane-associated phosphatidic acid-selective phospholipase A1-alpha) (mPA-PLA1 alpha) (LPD lipase-related


protein) (Phospholipase A1 member B)


451


Q7Z5P9


Mucin-19 (MUC-19)


6,254


O43897


Tolloid-like protein 1 (EC 3.4.24.-)


1,013


Q96NZ8


WAP, kazal, immunoglobulin, kunitz and NTR domain-containing protein 1 (WAP, follistatin, immunoglobulin, kunitz and NTR domain-containing


protein) (Growth and differentiation factor-associated serum protein 2) (GASP-2) (hGASP-2)


548


Q9BUJ0


Abhydrolase domain-containing protein 14A (EC 3.-.-.-)


271


Q9BXJ7


Protein amnionless


453


Q6UX02


Ankyrin repeat domain-containing protein 36


163


Q6UXY1


Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 (BAI1-associated protein 2-like protein 2)


529


Q6UWJ8


CD164 sialomucin-like 2 protein


174


O75309


Cadherin-16 (Kidney-specific cadherin) (Ksp-cadherin)


829


Q9H159


Cadherin-19


772


Q8IUK8


Cerebellin-2


224


Q9NPL8


Transmembrane protein C3orf1 (Protein M5-14)


285


Q96G28


Coiled-coil domain-containing protein 104


342


Q9NU53


Uncharacterized protein C6orf72


330


Q96F05


Uncharacterized protein C11orf24 (Protein DM4E3)


449


Q4KMG9


Uncharacterized protein C12orf59


183


Q2HXU8


C-type lectin domain family 12 member B (Macrophage antigen H)


276


Q8NC01


C-type lectin domain family 1 member A (C-type lectin-like receptor 1) (CLEC-1)


280


O75596


C-type lectin domain family 3 member A (C-type lectin superfamily member 1) (Cartilage-derived C-type lectin)


197


Q9ULY5


C-type lectin domain family 4 member E (C-type lectin superfamily member 9) (Macrophage-inducible C-type lectin)


219


Q6UXB4


C-type lectin domain family 4 member G


293


P56750


Claudin-17


224


Q9NPA0


UPF0480 protein C15orf24


242


P83436


Conserved oligomeric Golgi complex subunit 7 (COG complex subunit 7) (Component of oligomeric Golgi complex 7)


770


Q9H0I2


Uncharacterized protein C16orf48


346


Q6UWD8


Transmembrane protein C16orf54


224


Q9HCS2


Cytochrome P450 4F12 (EC 1.14.14.1) (CYPIVF12)


524


Q8N118


Cytochrome P450 4X1 (EC 1.14.14.1) (CYPIVX1)


509


Q86W10


Cytochrome P450 4Z1 (EC 1.14.14.1) (CYPIVZ1)


505


Q8IUH2


Protein CREG2


290


Q9UBS3


DnaJ homolog subfamily B member 9 (Microvascular endothelial differentiation gene 1 protein) (Mdg-1)


223


Q9H4B8


Dipeptidase 3 (EC 3.4.13.19)


488


Q8TBP5


Membrane protein FAM174A (Transmembrane protein 157) (Hepatitis C virus NS5A-transactivated protein 6) (HCV NS5A-transactivated protein 6)


190


Q7Z5A8


Protein FAM19A3 (Chemokine-like protein TAFA-3)


133


Q96LR4


Protein FAM19A4 (Chemokine-like protein TAFA-4)


140


O75063


Protein FAM20B


409


Q8N539


Fibrinogen C domain-containing protein 1


461


Q9NYL4


FK506-binding protein 11 (EC 5.2.1.8) (Peptidyl-prolyl cis-trans isomerase) (PPIase) (Rotamase) (19 kDa FK506-binding protein) (FKBP-19)


201


P59646


FXYD domain-containing ion transport regulator 4


89


O95390


Growth/differentiation factor 11 (GDF-11) (Bone morphogenetic protein 11)


407


Q9HCC8


Glycerophosphodiester phosphodiesterase domain-containing protein 2 (EC 3.1.-.-) (Glycerophosphodiester phosphodiesterase 3)


(Osteoblast differentiation promoting factor)


539


Q9H3K2


Growth hormone-inducible transmembrane protein (Dermal papilla-derived protein 2) (Transmembrane BAX inhibitor motif-containing protein 5)


345


Q9H9Y4


GPN-loop GTPase 2 (ATP-binding domain 1 family member B)


310


Q9Y278


Heparan sulfate glucosamine 3-O-sulfotransferase 2 (EC 2.8.2.29) (Heparan sulfate D-glucosaminyl 3-O-sulfotransferase 2) (Heparan sulfate


3-O-sulfotransferase 2) (h3-OST-2)


367


O43736


Integral membrane protein 2A (E25 protein)


263


Q6UW63


KDEL motif-containing protein 1 (Endoplasmic reticulum resident protein 58) (ER protein 58)


502


Q15012


Lysosomal-associated transmembrane protein 4A (Golgi 4-transmembrane spanning transporter MTP)


233


Q6UWM7


Lactase-like protein (Klotho/lactase-phlorizin hydrolase-related protein)


567


O75829


Chondromodulin-1 (Chondromodulin-I) (ChM-I) (Leukocyte cell-derived chemotaxin 1) [Cleaved into: Chondrosurfactant protein (CH-SP)]


334


O95214


Leptin receptor overlapping transcript-like 1


131


Q86YC3


Leucine-rich repeat-containing protein 33


692


Q9H8J5


MANSC domain-containing protein 1 (Loss of heterozygosity 12 chromosomal region 3 protein)


431


Q7Z553


MAM domain-containing glycosylphosphatidylinositol anchor protein 2 (MAM domain-containing protein 1)


956


Q9H1U4


Multiple epidermal growth factor-like domains 9 (EGF-like domain-containing protein 5) (Multiple EGF-like domain protein 5)


602


Q14696


Mesoderm development candidate 2 (Renal carcinoma antigen NY-REN-61)


234


Q9H8H3


Methyltransferase-like protein 7A (EC 2.1.1.-) (Protein AAM-B)


244


Q9UJH8


Meteorin


293


Q6N075


Major facilitator superfamily domain-containing protein 5


450


O60487


Myelin protein zero-like protein 2 (Epithelial V-like antigen 1)


215


Q9BV20


Methylthioribose-1-phosphate isomerase (MTR-1-P isomerase) (M1Pi) (EC 5.3.1.23) (S-methyl-5-thioribose-1-phosphate isomerase)


(Translation initiation factor eIF-2B subunit alpha/beta/delta-like protein)


369


Q9H7X0


N-acetyltransferase 15 (EC 2.3.1.-)


242


Q7Z3B1


Neuronal growth regulator 1


354


Q9P121


Neurotrimin (hNT)


344


Q8NGF6


Olfactory receptor 10W1 (Olfactory receptor OR11-236)


305


Q6UWI2


Protein PARM-1


310


O15018


PDZ domain-containing protein 2 (PDZ domain-containing protein 3) (Activated in prostate cancer protein) [Cleaved into: Processed PDZ


domain-containing protein 2]


2,839


Q96S96


Phosphatidylethanolamine-binding protein 4 (PEBP-4) (hPEBP4) (Protein cousin-of-RKIP 1)


227


Q8TDX9


Polycystic kidney disease protein 1-like 1 (Polycystin-1L1) (PC1-like 1 protein)


2,849


Q9NRZ5


1-acyl-sn-glycerol-3-phosphate acyltransferase delta (EC 2.3.1.51) (1-acylglycerol-3-phosphate O-acyltransferase 4) (1-AGP acyltransferase 4)


(1-AGPAT 4) (Lysophosphatidic acid acyltransferase delta) (LPAAT-delta)


378


Q9Y2Y8


Proteoglycan 3 (Eosinophil major basic protein homolog) (Prepro-major basic protein homolog) (Prepro-MBPH)


225


Q8NBN7


Retinol dehydrogenase 13 (EC 1.1.1.-)


331


Q8NC24


RELT-like protein 2


303


Q7Z769


Solute carrier family 35 member E3 (Bladder cancer overexpressed gene 1 protein)


313


Q9NUM3


Zinc transporter ZIP9 (Zrt- and Irt-like protein 9) (ZIP-9) (Solute carrier family 39 member 9)


307


Q8TDM5


Sperm acrosome membrane-associated protein 4 (Sperm acrosomal membrane-associated protein 14)


124


Q6UXD5


Seizure 6-like protein 2


910


Q14563


Semaphorin-3A (Semaphorin III) (Sema III)


771


Q9C0C4


Semaphorin-4C


833


O95562


Vesicle transport protein SFT2B (SFT2 domain-containing protein 2)


160


Q6UWI4


Protein shisa-2 homolog (Transmembrane protein 46)


295


Q96DD7


Protein shisa-4 (Transmembrane protein 58)


197


Q9NSC7


Alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 (EC 2.4.99.3) (GalNAc alpha-2,6-sialyltransferase I) (ST6GalNAc I) 


(Sialyltransferase 7A)


600


Q8NDV1


Alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 3 (EC 2.4.99.-) (GalNAc alpha-2,6-sialyltransferase III) (ST6GalNAc III)


(Sialyltransferase 7C) (STY)


305


O43760


Synaptogyrin-2 (Cellugyrin)


224


P61009


Signal peptidase complex subunit 3 (EC 3.4.-.-) (Microsomal signal peptidase 23 kDa subunit) (SPase 22 kDa subunit) (SPC22/23)


180


Q6UW49


Sperm equatorial segment protein 1 (SP-ESP) (Equatorial segment protein) (ESP) (Glycosylated 38 kDa sperm protein C-7/8)


350


Q53R12


Transmembrane 4 L6 family member 20


229


Q6UX40


Transmembrane protein 107


140


Q96AN5


Transmembrane protein 143


459


Q8NBL3


Transmembrane protein 178


297


Q6UW68


Transmembrane protein 205


189


Q96HV5


Transmembrane protein 41A


264


O95807


Transmembrane protein 50A (Small membrane protein 1)


157


P56557


Transmembrane protein 50B (HCV p7-trans-regulated protein 3)


158


Q9Y3Q3


Transmembrane emp24 domain-containing protein 3 (Membrane protein p24B)


217


Q9Y3A6


Transmembrane emp24 domain-containing protein 5


229


Q9HBJ8


Collectrin (Transmembrane protein 27)


222


Q6UXU6


Transmembrane protein 92


159


Q3KNT9


Transmembrane protein 95


176


Q9NQ34


Transmembrane protein 9B


198


Q9UKU6


Thyrotropin-releasing hormone-degrading ectoenzyme (TRH-degrading ectoenzyme) (TRH-DE) (EC 3.4.19.6) (TRH-specific aminopeptidase) 


(Thyroliberinase) (Pyroglutamyl-peptidase II) (PAP-II)


1,024


O95859


Tetraspanin-12 (Tspan-12) (Transmembrane 4 superfamily member 12) (Tetraspan NET-2)


305


O95857


Tetraspanin-13 (Tspan-13) (Transmembrane 4 superfamily member 13) (Tetraspan NET-6)


204


O95858


Tetraspanin-15 (Tspan-15) (Transmembrane 4 superfamily member 15) (Tetraspan NET-7)


294


Q9H6L2


UPF0513 transmembrane protein


316


O95847


Mitochondrial uncoupling protein 4 (UCP 4) (Solute carrier family 25 member 27)


323


Q6UX27


V-set and transmembrane domain-containing protein 1


236


Q6UX98


Probable palmitoyltransferase ZDHHC24 (EC 2.3.1.-) (Zinc finger DHHC domain-containing protein 24) (DHHC-24)


284


P60852


Zona pellucida sperm-binding protein 1 (Zona pellucida glycoprotein 1) (Zp-1) [Cleaved into: Processed zona pellucida sperm-binding protein 1]


638


Q05996


Zona pellucida sperm-binding protein 2 (Zona pellucida glycoprotein ZP2) (Zona pellucida protein A) [Cleaved into: Processed zona pellucida 


sperm-binding protein 2]


745


Q12836


Zona pellucida sperm-binding protein 4 (Zona pellucida protein B) [Cleaved into: Processed zona pellucida sperm-binding protein 4]


540


Q86V24


Adiponectin receptor protein 2 (Progestin and adipoQ receptor family member II)


386


P55103


Inhibin beta C chain (Activin beta-C chain)


352


Q2TAL6


Brorin (Brain-specific chordin-like protein) (von Willebrand factor C domain-containing protein 2)


325


Q9NZP8


Complement C1r subcomponent-like protein (C1r-like protein) (C1r-LP) (EC 3.4.21.-) (C1r-like serine protease analog protein) (CLSPa)


487


Q49AH0


Cerebral dopamine neurotrophic factor (Conserved dopamine neurotrophic factor) (ARMET-like protein 1)


187


A6NMZ7


Collagen alpha-6(VI) chain


2,263


Q8IZJ3


C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8


1,885


Q53RD9


Fibulin-7 (FIBL-7)


439


Q9ULI3


Protein HEG homolog 1


1,381


Q8NBM8


Prenylcysteine oxidase-like (EC 1.8.3.-)


494


Q53H76


Phospholipase A1 member A (EC 3.1.1.-) (Phosphatidylserine-specific phospholipase A1) (PS-PLA1)


456


P02812


Basic salivary proline-rich protein 2 (Salivary proline-rich protein) (Con1 glycoprotein) [Cleaved into: Basic proline-rich peptide IB-7; Basic


proline-rich peptide IB-8c (Basic peptide P-F); Basic proline-rich peptide IB-4]


416


Q9NWH7


Spermatogenesis-associated protein 6


488


O60687


Sushi repeat-containing protein SRPX2


465


Q9GZX9


Twisted gastrulation protein homolog 1


223


Q96DN2


von Willebrand factor C and EGF domain-containing protein (HBV X protein up-regulated gene 11 protein) (HBxAg up-regulated gene 11 protein)


955


P41221


Protein Wnt-5a


380


Q8NEB7


Acrosin-binding protein (Proacrosin-binding protein sp32) (Cancer/testis antigen OY-TES-1) (Cancer/testis antigen 23) (CT23)


543


Q86TW2


Uncharacterized aarF domain-containing protein kinase 1 (EC 2.7.11.-)


530


Q99541


Adipophilin (Adipose differentiation-related protein) (ADRP)


437


Q5NDL2


Uncharacterized glycosyltransferase AER61 (EC 2.4.-.-)


527


Q9NP70


Ameloblastin


447


Q9H4A4


Aminopeptidase B (Ap-B) (EC 3.4.11.6) (Arginine aminopeptidase) (Arginyl aminopeptidase)


650


Q15389


Angiopoietin-1 (ANG-1)


498


Q9Y264


Angiopoietin-4 (ANG-4) (ANG-3)


503


Q5FYB1


Arylsulfatase I (ASI) (EC 3.1.6.-)


569


Q92484


Acid sphingomyelinase-like phosphodiesterase 3a (ASM-like phosphodiesterase 3a) (EC 3.1.4.-)


453


Q6ZMM2


ADAMTS-like protein 5 (ADAMTSL-5) (Thrombospondin type-1 domain-containing protein 6)


471


Q8TE58


A disintegrin and metalloproteinase with thrombospondin motifs 15 (ADAMTS-15) (ADAM-TS 15) (ADAM-TS15) (EC 3.4.24.-)


950


Q8TE57


A disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS-16) (ADAM-TS 16) (ADAM-TS16) (EC 3.4.24.-)


1,224


Q8TE56


A disintegrin and metalloproteinase with thrombospondin motifs 17 (ADAMTS-17) (ADAM-TS 17) (ADAM-TS17) (EC 3.4.24.-)


1,095


P55107


Bone morphogenetic protein 3b (BMP-3b) (Growth/differentiation factor 10) (GDF-10) (Bone-inducing protein) (BIP)


478


P22003


Bone morphogenetic protein 5 (BMP-5)


454


P34820


Bone morphogenetic protein 8B (BMP-8B) (BMP-8) (Osteogenic protein 2) (OP-2)


402


Q8NFQ5


Bactericidal/permeability-increasing protein-like 3


453


O75973


C1q-related factor (Complement component 1 Q subcomponent-like 1)


258


B2RNN3


Complement C1q tumor necrosis factor-related protein 9-like


333


P23435


Cerebellin-1 (Precerebellin) [Cleaved into: Cerebellin (CER); [des-Ser1]-cerebellin]


193


Q8IVL8


Carboxypeptidase O (CPO) (EC 3.4.17.-)


374


Q8NDZ4


UPF0672 protein C3orf58


430


Q9H6E4


Coiled-coil domain-containing protein 134


229


Q6NSX1


Coiled-coil domain-containing protein 70


233


Q9NRJ3


C-C motif chemokine 28 (Small-inducible cytokine A28) (Mucosae-associated epithelial chemokine) (MEC) (Protein CCK1)


127


Q0P651


Uncharacterized protein C4orf29


414


Q5VXM1


CUB domain-containing protein 2


449


Q9UNI1


Chymotrypsin-like elastase family member 1 (EC 3.4.21.36) (Elastase-1)


258


Q6P5S2


Uncharacterized protein C6orf58


330


O15335


Chondroadherin (Cartilage leucine-rich protein)


359


Q8IUL8


Cartilage intermediate layer protein 2 (CILP-2) [Cleaved into: Cartilage intermediate layer protein 2 C1; Cartilage intermediate layer protein 2 C2]


1,156


Q5T742


Uncharacterized protein C10orf25


122


Q14406


Chorionic somatomammotropin hormone-like 1 (Chorionic somatomammotropin-like) (Lactogen-like)


199


O60676


Cystatin-8 (Cystatin-related epididymal spermatogenic protein)


142


Q16619


Cardiotrophin-1 (CT-1)


201


P17538


Chymotrypsinogen B (EC 3.4.21.1) [Cleaved into: Chymotrypsin B chain A; Chymotrypsin B chain B; Chymotrypsin B chain C]


263


Q07325


C-X-C motif chemokine 9 (Small-inducible cytokine B9) (Gamma-interferon-induced monokine) (MIG)


125


O43927


C-X-C motif chemokine 13 (Small-inducible cytokine B13) (B lymphocyte chemoattractant) (CXC chemokine BLC) (B cell-attracting chemokine 1)


(BCA-1) (ANGIE)


109


Q8NET1


Beta-defensin 108B (Defensin, beta 108B) (Defensin, beta 108) (Beta-defensin 8) (BD-8) (hBD-8) (DEFB-8)


73


Q96PH6


Beta-defensin 118 (Defensin, beta 118) (Beta-defensin 18) (DEFB-18) (Epididymal secretory protein 13.6) (ESP13.6)


123


Q8NES8


Beta-defensin 124 (Defensin, beta 124) (Beta-defensin 24) (DEFB-24)


71


Q8N687


Beta-defensin 125 (Defensin, beta 125) (Beta-defensin 25) (DEFB-25)


156


Q9BYW3


Beta-defensin 126 (Beta-defensin 26) (DEFB-26) (Epididymal secretory protein 13.2) (ESP13.2)


111


Q92874


Deoxyribonuclease-1-like 2 (EC 3.1.21.-) (Deoxyribonuclease I-like 2) (DNase I-like 2) (DNase I homolog protein DHP1)


299


Q6PKH6


Dehydrogenase/reductase SDR family member 4-like 2 (EC 1.1.-.-)


230


Q9NT22


EMILIN-3 (Elastin microfibril interface-located protein 3) (Elastin microfibril interfacer 3) (EMILIN-5) (Elastin microfibril interface-located protein 5)


(Elastin microfibril interfacer 5)


766


O94919


Endonuclease domain-containing 1 protein (EC 3.1.30.-)


500


Q14507


Epididymal secretory protein E3-alpha (Human epididymis-specific protein 3-alpha) (HE3 alpha)


147


Q99645


Epiphycan (Dermatan sulfate proteoglycan 3) (Small chondroitin/dermatan sulfate proteoglycan) (Proteoglycan-Lb) (PG-Lb)


322


O14944


Proepiregulin [Cleaved into: Epiregulin (EPR)]


169


Q9NQ30


Endothelial cell-specific molecule 1 (ESM-1 secretory protein) (ESM-1)


184


Q6NT32


Carboxylesterase 7 (EC 3.1.1.1) (Carboxylesterase-like urinary excreted protein homolog) (Cauxin)


575


Q5VST6


Abhydrolase domain-containing protein FAM108B1 (EC 3.-.-.-)


288


Q8WUF8


UPF0528 protein FAM172A


416


Q96GS6


Abhydrolase domain-containing protein FAM108A1 (EC 3.-.-.-)


310


Q92520


Protein FAM3C


227


Q76B58


Protein FAM5C (DBCCR1-like protein 1)


766


Q9NP95


Fibroblast growth factor 20 (FGF-20)


211


Q8TAT2


Fibroblast growth factor-binding protein 3 (FGF-binding protein 3) (FGF-BP3) (FGFBP-3)


258


Q5VTL7


Fibronectin type III domain-containing protein 7


734P41439


Folate receptor gamma (FR-gamma) (Folate receptor 3)


243


P0C091


FRAS1-related extracellular matrix protein 3


2,135


Q9UBC7


Galanin-like peptide


116


P27539


Embryonic growth/differentiation factor 1 (GDF-1)


372


Q9UK05


Growth/differentiation factor 2 (GDF-2) (Bone morphogenetic protein 9) (BMP-9)


429


Q9GZZ7


GDNF family receptor alpha-4 (GFR-alpha-4) (Persephin receptor)


299


Q9UJJ9


N-acetylglucosamine-1-phosphotransferase subunit gamma (GlcNAc-1-phosphotransferase subunit gamma)


(UDP-N-acetylglucosamine-1-phosphotransferase subunit gamma)


305


O14626


Probable G-protein coupled receptor 171 (G-protein coupled receptor H963)


319


O75715


Epididymal secretory glutathione peroxidase (EC 1.11.1.9) (Epididymis-specific glutathione peroxidase-like protein) (EGLP)


221


Q9H772


Gremlin-2 (Cysteine knot superfamily 1, BMP antagonist 2) (Protein related to DAN and cerberus) (DAN domain family member 3)


168


Q9GZV7


Hyaluronan and proteoglycan link protein 2 (Brain link protein 1)


340


Q86UW8


Hyaluronan and proteoglycan link protein 4 (Brain link protein 2)


402


P01566


Interferon alpha-10 (Interferon alpha-C) (LeIF C) (Interferon alpha-6L)


189


P05015


Interferon alpha-16 (Interferon alpha-WA)


189


P05013


Interferon alpha-6 (Interferon alpha-K) (LeIF K) (Interferon alpha-54)


189


Q96ID5


Immunoglobulin superfamily member 21


467


P20809


Interleukin-11 (IL-11) (Adipogenesis inhibitory factor) (AGIF) (Oprelvekin)


199


Q16552


Interleukin-17A (IL-17A) (IL-17) (Cytotoxic T-lymphocyte-associated antigen 8) (CTLA-8)


155


Q14116


Interleukin-18 (IL-18) (Interferon-gamma-inducing factor) (IFN-gamma-inducing factor) (Interleukin-1 gamma) (IL-1 gamma) (Iboctadekin)


193


Q9NPH9


Interleukin-26 (AK155 protein)


171


Q8IZJ0


Interleukin-28A (IL-28A) (Interferon lambda-2) (IFN-lambda-2) (Cytokine ZCYTO20)


200


Q8IZI9


Interleukin-28B (IL-28B) (IL-28C) (Interferon lambda-3) (IFN-lambda-3) (Interferon lambda-4) (IFN-lambda-4) (Cytokine ZCYTO22)


200


Q8IU54


Interleukin-29 (IL-29) (Interferon lambda-1) (IFN-lambda-1) (Cytokine ZCYTO21)


200


Q9Y581


Insulin-like peptide INSL6 (Insulin-like peptide 6) (Relaxin/insulin-like factor 1) [Cleaved into: Insulin-like peptide INSL6 B chain; Insulin-like


peptide INSL6 A chain]


213


Q8NC54


Keratinocyte-associated transmembrane protein 2


265


Q9H7L2


Putative killer cell immunoglobulin-like receptor-like protein KIR3DX1 (Leukocyte receptor cluster member 12)


352


Q9NVR0


Kelch-like protein 11


708


Q9UKR3


Kallikrein-13 (EC 3.4.21.-) (Kallikrein-like protein 4) (KLK-L4)


277


Q9UKQ9


Kallikrein-9 (EC 3.4.21.-) (Kallikrein-like protein 3) (KLK-L3)


250


Q9GZZ8


Extracellular glycoprotein lacritin


138


A4D0S4


Laminin subunit beta-4 (Laminin beta-1-related protein)


1,761


Q9Y6N6


Laminin subunit gamma-3 (Laminin 12 gamma 3 subunit)


1,587


P58215


Lysyl oxidase homolog 3 (EC 1.4.3.-) (Lysyl oxidase-like protein 3)


753


Q16609


Apolipoprotein(a)-like protein 2 (Apo(a)-like protein 2) (Lp(a)-liker protein 2) (Apolipoprotein a-related gene C protein) (Apo(a)rg-C)


132


Q7Z4W2


Lysozyme-like protein 2 (Lysozyme-2) (EC 3.2.1.17)


148


Q96KX0


Lysozyme-like protein 4


146


Q7Z304


MAM domain-containing protein 2 (MAM domain-containing proteoglycan) (Mamcan)


686


P55001


Microfibrillar-associated protein 2 (MFAP-2) (Microfibril-associated glycoprotein 1) (MAGP-1) (MAGP)


183


Q96PC5


Melanoma inhibitory activity protein 2


541


Q9Y5R2


Matrix metalloproteinase-24 (MMP-24) (EC 3.4.24.-) (Membrane-type matrix metalloproteinase 5) (MT-MMP 5) (Membrane-type-5 matrix


metalloproteinase) (MT5-MMP) [Cleaved into: Processed matrix metalloproteinase-24]


645


Q9NPA2


Matrix metalloproteinase-25 (MMP-25) (EC 3.4.24.-) (Membrane-type matrix metalloproteinase 6) (MT-MMP 6) (Membrane-type-6 matrix


metalloproteinase) (MT6-MMP) (Leukolysin)


562


O95631


Netrin-1


604


O00634


Netrin-3 (Netrin-2-like protein)


580


P30990


Neurotensin/neuromedin N [Cleaved into: Large neuromedin N (NmN-125); Neuromedin N (NmN) (NN); Neurotensin (NT); Tail peptide]


170


P48645


Neuromedin-U [Cleaved into: Neuromedin-U-25 (NmU-25)]


174


Q96S42


Nodal homolog


347


O95897


Noelin-2 (Olfactomedin-2)


454


O15130


FMRFamide-related peptides [Cleaved into: Neuropeptide SF (NPSF); Neuropeptide FF (NPFF); Neuropeptide AF (NPAF)]


113


P47972


Neuronal pentraxin-2 (NP2) (Neuronal pentraxin II) (NP-II)


431


Q8N729


Neuropeptide W (Preproprotein L8) (hPPL8) [Cleaved into: Neuropeptide W-23 (NPW23) (hL8); Neuropeptide W-30 (NPW30) (hL8C)]


165


Q99748


Neurturin


197


P58417


Neurexophilin-1


271


O95156


Neurexophilin-2


264


Q02509


Otoconin-90 (Oc90) (Phospholipase A2 homolog)


493


Q6GTS8


Probable carboxypeptidase PM20D1 (EC 3.4.17.-) (Peptidase M20 domain-containing protein 1)


502


Q9NZK7


Group IIE secretory phospholipase A2 (GIIE sPLA2) (EC 3.1.1.4) (Phosphatidylcholine 2-acylhydrolase GIIE) (sPLA(2)-IIE)


142


P10720


Platelet factor 4 variant (PF4var1) (PF4alt) (C-X-C motif chemokine 4 variant) (CXCL4L1) [Cleaved into: Platelet factor 4 variant(4-74);


PPlatelet factor 4 variant(5-74); latelet factor 4 variant(6-74)]


104


Q13519


Nociceptin [Cleaved into: Neuropeptide 1; Nociceptin (Orphanin FQ) (PPNOC); Neuropeptide 2]


176


Q5K4E3


Polyserase-2 (EC 3.4.21.-) (Polyserine protease 2) (Serine protease 36)


855


Q6PEZ8


Podocan-like protein 1


512


Q16378


Proline-rich protein 4 (Lacrimal proline-rich protein) (Nasopharyngeal carcinoma-associated proline-rich protein 4)


134


P81277


Prolactin-releasing peptide (PrRP) (Prolactin-releasing hormone) [Cleaved into: Prolactin-releasing peptide PrRP31; Prolactin-releasing peptide


PrRP20]


87


Q8NF86


Serine protease 33 (EC 3.4.21.-) (Serine protease EOS)


280


Q9UQ74


Pregnancy-specific beta-1-glycoprotein 8 (PSBG-8)


426


Q99969


Retinoic acid receptor responder protein 2 (Tazarotene-induced gene 2 protein) (RAR-responsive protein TIG2)


163


Q9HCQ7


FMRFamide-related peptides (Neuropeptide VF) [Cleaved into: Neuropeptide NPSF (Neuropeptide RFRP-1); Neuropeptide RFRP-2;


Neuropeptide NPVF (Neuropeptide RFRP-3)]


196


Q93091


Ribonuclease K6 (RNase K6) (EC 3.1.27.-)


150


O00584


Ribonuclease T2 (EC 3.1.27.-) (Ribonuclease 6)


256


Q6XPR3


Repetin


784


Q9BSG5


Retbindin


229


Q99470


Stromal cell-derived factor 2 (SDF-2)


211


P09683


Secretin


121


Q99985


Semaphorin-3C (Semaphorin-E) (Sema E)


751


O15041


Semaphorin-3E


775


Q9NS98


Semaphorin-3G (Semaphorin sem2)


782


Q8TD33


Secretoglobin family 1C member 1 (Secretoglobin RYD5)


95


O95968


Secretoglobin family 1D member 1 (Lipophilin-A)


90


O95969


Secretoglobin family 1D member 2 (Lipophilin-B)


90


Q8IW75


Serpin A12 (Visceral adipose tissue-derived serine protease inhibitor) (Vaspin) (Visceral adipose-specific serpin) (OL-64)


414


O75830


Serpin I2 (Myoepithelium-derived serine protease inhibitor) (Pancpin) (Protease inhibitor 14) (TSA2004)


405


Q14515


SPARC-like protein 1 (High endothelial venule protein) (Hevin) (MAST 9)


664


Q9BT56


Spexin (NPQ)


116


Q8WTU2


Scavenger receptor cysteine-rich domain-containing group B protein (S4-SRCRB)


575


P52823


Stanniocalcin-1 (STC-1)


247


Q96PL2


Beta-tectorin


329


Q99727


Metalloproteinase inhibitor 4 (Tissue inhibitor of metalloproteinases 4) (TIMP-4)


224


Q8TB96


T-cell immunomodulatory protein (Protein TIP) (Integrin-alpha FG-GAP repeat-containing protein 1)


612


O14657


Torsin-1B (Torsin family 1 member B)


336


P20396


Prothyroliberin [Cleaved into: Thyroliberin (Thyrotropin-releasing hormone) (TRH) (Thyrotropin-releasing factor) (TRF) (TSH-releasing factor) 


(Protirelin)]


242


Q8NHM4


Putative trypsin-6 (EC 3.4.21.4) (Trypsinogen C)


247


Q969D9


Thymic stromal lymphopoietin


159


Q9P2K2


Thioredoxin domain-containing protein 16


825


Q8WVF2


Unique cartilage matrix-associated protein [Cleaved into: Unique cartilage matrix-associated protein C-terminal fragment (Ucma-C) (Gla-rich protein)


(GRP)]


138


Q96RP3


Urocortin-2 (Urocortin II) (Ucn II) (Stresscopin-related peptide) (Urocortin-related peptide)


112


Q969E3


Urocortin-3 (Urocortin III) (Ucn III) (Stresscopin)


161


Q6PCB0


von Willebrand factor A domain-containing protein 1


445


Q8N2E2


Von Willebrand factor D and EGF domain-containing protein


1,590


Q9HC57


WAP four-disulfide core domain protein 1 (Prostate stromal protein ps20) (ps20 growth inhibitor)


220


Q8IUB2


WAP four-disulfide core domain protein 3 (Putative protease inhibitor WAP14)


231


Q8NEX5


Protein WFDC9


89


Q9GZT5


Protein Wnt-10a


417


P04628


Proto-oncogene protein Wnt-1


370


P56704


Protein Wnt-3a


352


P56703


Proto-oncogene protein Wnt-3


355


Q9Y6F9


Protein Wnt-6


365


O14904


Protein Wnt-9a (Wnt-14)


365


Q9UBD3


Cytokine SCM-1 beta (C motif chemokine 2) (XC chemokine ligand 2)


114


O60844


Zymogen granule membrane protein 16 (Zymogen granule protein 16) (Secretory lectin ZG16)


167


Q9BS86


Zona pellucida-binding protein 1 (Sp38)


351


Q8TD06


Anterior gradient protein 3 homolog (hAG-3) (AG-3) (AG3) (Breast cancer membrane protein 11)


166


Q6UX39


Amelotin


209


Q86XS5


Angiopoietin-related protein 5 (Angiopoietin-like 5)


388


Q5FYB0


Arylsulfatase J (ASJ) (EC 3.1.6.-)


599


Q8TB73


Fibronectin type-III domain-containing protein C4orf31


568


Q6UWT4


Uncharacterized protein C5orf46


87


Q6UXF7


C-type lectin domain family 18 member B (Mannose receptor-like protein 1)


455


Q8N129


Protein canopy homolog 4


248


Q8NBI3


Draxin (Dorsal repulsive axon guidance protein)


349


Q6UWF9


Protein FAM180A


173


Q3B7J2


Glucose-fructose oxidoreductase domain-containing protein 2 (EC 1.-.-.-)


385


Q86WN2


Interferon epsilon (Interferon epsilon-1)


208


Q6UXX5


Inter-alpha-trypsin inhibitor heavy chain H5-like protein (Inter-alpha inhibitor H5-like protein)


1,313


Q8WWU7


Intelectin-2 (Endothelial lectin HL-2)


325


Q6UWW0


Lipocalin-15


184


P62502


Epididymal-specific lipocalin-6 (Lipocalin-5)


163


Q9H306


Matrix metalloproteinase-27 (MMP-27) (EC 3.4.24.-)


513


Q8N3Z0


Inactive serine protease 35


413


Q6UWY2


Serine protease 1-like protein 1 (EC 3.4.21.-)


283


Q6UWP8


Suprabasin


247


Q6XE38


Secretoglobin family 1D member 4 (IFN-gamma-inducible secretoglobin) (IIS)


83


Q6UXN2


Trem-like transcript 4 protein (TLT-4) (Triggering receptor expressed on myeloid cells-like protein 4)


200


Q8TEU8


WAP, kazal, immunoglobulin, kunitz and NTR domain-containing protein 2 (WAP, follistatin, immunoglobulin, kunitz and 


NTR domain-containing-related protein) (WFIKKN-related protein) (Growth and differentiation factor-associated serum protein 1) (GASP-1)


(hGASP-1)


576


Q8TAG5


V-set and transmembrane domain-containing protein 2A


243


Q6UX34


Uncharacterized protein C2orf82


121


A8MWS1


Putative killer cell immunoglobulin-like receptor like protein KIR3DP1 (CD antigen CD158c)


328


Q6UW32


Insulin growth factor-like family member 1


110


A8K3U3


cDNA FLJ77863, highly similar to Homo sapiens secreted and transmembrane 1 (SECTM1), mRNA


248


Q4VNC0


Probable cation-transporting ATPase 13A5 (EC 3.6.3.-) (P5-ATPase isoform 5)


1,218


A6H8M9


Cadherin-like protein 29


788


Q6UY09


Carcinoembryonic antigen-related cell adhesion molecule 20


585


Q3KPI0


Carcinoembryonic antigen-related cell adhesion molecule 21


293


Q9H741


UPF0454 protein C12orf49


205


Q6UW02


Cytochrome P450 20A1 (EC 1.14.-.-)


462


Q6NUT2


Protein dpy-19 homolog 2 (Dpy-19-like protein 2)


758


Q9BVC3


Sister chromatid cohesion protein DCC1 (Defective in sister chromatid cohesion protein 1 homolog)


393


Q96M86


Dynein heavy chain domain-containing protein 1


1,021


Q6IAN0


Dehydrogenase/reductase SDR family member 7B (EC 1.1.-.-)


325


Q7Z5A9


Protein FAM19A1 (Chemokine-like protein TAFA-1)


133


Q5JW98


Protein FAM26D


314


Q8WVX9


Fatty acyl-CoA reductase 1 (EC 1.2.1.n2) (Male sterility domain-containing protein 2)


515


Q8TAL6


Fin bud initiation factor homolog


211


Q9H6D8


Fibronectin type III domain-containing protein 4 (Fibronectin type III repeat-containing protein 1)


234


Q6UXV0


GDNF family receptor alpha-like


394


Q9H1C3


Glycosyltransferase 8 domain-containing protein 2 (EC 2.4.1.-)


349


Q8TED1


Probable glutathione peroxidase 8 (EC 1.11.1.9)


209


Q8IYS0


GRAM domain-containing protein 1C


662


B1AKI9


Isthmin-1


464


Q6UY18


Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 4 (Leucine-rich repeat neuronal protein 6D)


593


Q86X40


Leucine-rich repeat-containing protein 28


367


Q8ND94


LRRN4 C-terminal-like protein


238


Q6UXB3


Ly6/PLAUR domain-containing protein 2


125


Q8N468


Major facilitator superfamily domain-containing protein 4


514


Q6UWV2


Myelin protein zero-like protein 3


235


Q6P988


Protein notum homolog (EC 3.-.-.-)


496


A1E959


Odontogenic ameloblast-associated protein (Apin)


279


Q9Y5I3


Protocadherin alpha-1 (PCDH-alpha-1)


950


Q96BZ4


Phospholipase D4 (PLD 4) (EC 3.1.4.4) (Choline phosphatase 4) (Phosphatidylcholine-hydrolyzing phospholipase D4)


506


Q8IZV5


Retinol dehydrogenase 10 (EC 1.1.1.n2)


341


Q08ET2


Sialic acid-binding Ig-like lectin 14 (Siglec-14)


396


Q9NX61


Transmembrane protein 161A


479


Q8NDZ6


Transmembrane protein 161B


487


Q6ZP80


Transmembrane protein 182


229


Q96BF3


Transmembrane and immunoglobulin domain-containing protein 2


282


Q8NDY8


Transmembrane protein 52


209


Q6P7N7


Transmembrane protein 81


255


Q6UWM9


UDP-glucuronosyltransferase 2A3 (UDPGT 2A3) (EC 2.4.1.17)


527


Q3SY77


UDP-glucuronosyltransferase 3A2 (UDPGT 3A2) (EC 2.4.1.17)


523


O15240


Neurosecretory protein VGF


615


A4QMV3


Secreted phosphoprotein 2, 24kDa (Fragment)


211


Q6UX46


Protein FAM150B


152


Q6UXS0


C-type lectin domain-containing protein UNQ5810/PRO19627


136


Q15846


Clusterin-like protein 1 (Retinal-specific clusterin-like protein)


466


Q8N5W8


Protein FAM24B


94


O60383


Growth/differentiation factor 9 (GDF-9)


454


Q6JVE6


Epididymal-specific lipocalin-10


187


Q6JVE5


Epididymal-specific lipocalin-12


192


Q6JVE9


Epididymal-specific lipocalin-8


175


P02811


Basic proline-rich peptide P-E (IB-9)


61


Q6UWK7


Putative uncharacterized protein C10orf99


81


Q96MU5


Uncharacterized protein C17orf77


206


Q6P093


Arylacetamide deacetylase-like 2 (EC 3.1.1.-)


401


Q9UP79


A disintegrin and metalloproteinase with thrombospondin motifs 8 (ADAMTS-8) (ADAM-TS 8) (ADAM-TS8) (EC 3.4.24.-) (METH-2) (METH-8)


890


Q86Y30


B melanoma antigen 2 (Cancer/testis antigen 2.2) (CT2.2)


109


Q86Y29


B melanoma antigen 3 (Cancer/testis antigen 2.3) (CT2.3)


109


Q075Z2


Bovine seminal plasma protein homolog 1


132


Q5VWW1


Complement C1q-like protein 3


255


Q8N8R5


UPF0565 protein C2orf69


385


Q7Z4R8


UPF0669 protein C6orf120


191


A2RUU4


Colipase-like protein C6orf127


121


Q9H7B7


Uncharacterized protein C7orf69


122


Q8WUY1


UPF0670 protein C8orf55 (Mesenchymal stem cell protein DSCD75)


208


Q6NUI6


Chondroadherin-like protein


762


Q8NCF0


C-type lectin domain family 18 member C (Mannose receptor-like protein 3)


446


Q96KW9


Putative uncharacterized protein C13orf28


195


Q8NFW1


Collagen alpha-1(XXII) chain


1,626


Q2VPA4


Complement component receptor 1-like protein (Complement C4b-binding protein CR-1-like protein)


569


Q9H3Y0


Peptidase inhibitor R3HDML (Cysteine-rich secretory protein R3HDML)


253


Q5W186


Cystatin-9 (Cystatin-like molecule)


159


Q8N907


DAN domain family member 5 (Cerberus-like protein 2) (Cerl-2) (Gremlin-3) (Cysteine knot superfamily 1, BMP antagonist 3)


189


Q7Z7B8


Beta-defensin 128 (Defensin, beta 128) (Beta-defensin 28) (DEFB-28)


93


Q96BH3


Epididymal sperm-binding protein 1 (Epididymal secretory protein 12) (hE12)


223


Q7RTY5


Epidermis-specific serine protease-like protein (EC 3.4.21.-)


336


Q8N323


Protein FAM55A


547


Q6KF10


Growth/differentiation factor 6 (GDF-6) (Growth/differentiation factor 16)


455


Q9NXC2


Glucose-fructose oxidoreductase domain-containing protein 1 (EC 1.-.-.-)


390


Q96MS3


Glycosyltransferase 1 domain-containing protein 1 (EC 2.4.-.-)


346


P59796


Glutathione peroxidase 6 (EC 1.11.1.9)


221


A8MTL9


Serpin-like protein HMSD (Minor histocompatibility protein HMSD) (Minor histocompatibility serpin domain-containing protein)


139


Q8WX77


Insulin-like growth factor-binding protein-like 1 (Insulin-like growth factor-binding-related protein 4) (IGFBP-rP4) (IGFBP-related protein 10)


278


Q6EBC2


Interleukin-31 (IL-31)


164


Q6ZMJ4


Interleukin-34 (IL-34)


242


Q6PEW0


Plasma kallikrein-like protein 4 (Cancer/testis antigen 67) (CT67)


395


Q8WX39


Epididymal-specific lipocalin-9 (MUP-like lipocalin)


190


Q5VXJ0


Lipase member K (EC 3.1.1.-) (Lipase-like abhydrolase domain-containing protein 2)


399


Q5VYY2


Lipase member M (EC 3.1.1.-) (Lipase-like abhydrolase domain-containing protein 3)


423


P59826


Long palate, lung and nasal epithelium carcinoma-associated protein 3 (Ligand-binding protein RYA3)


476


Q17RY6


Lymphocyte antigen 6K (Ly-6K)


165


A1L453


Serine protease MPN2 (EC 3.4.21.-) (Marapsin-2)


326


Q8WTR8


Netrin-5 (Netrin-1-like protein)


489


Q5JS37


NHL repeat-containing protein 3


347


Q68BL8


Olfactomedin-like protein 2B (Photomedin-2)


750


Q6ZRI0


Otogelin


2,925


Q7RTM1


Otopetrin-1


612


Q7RTY7


Ovochymase-1 (EC 3.4.21.-)


1,134


Q7RTZ1


Ovochymase-2 (EC 3.4.21.-) (Oviductin)


564


Q6IE36


Ovostatin homolog 2


1,432


P83859


Orexigenic neuropeptide QRFP (P518) [Cleaved into: QRF-amide (Pyroglutamylated arginine-phenylalanine-amide peptide) (Neuropeptide RF-amide)]


136


Q5R387


Putative inactive group IIC secretory phospholipase A2 (Phosphatidylcholine 2-acylhydrolase GIIC)


150


Q8WXA2


Prostate and testis expressed protein 1


126


Q6P4A8


Putative phospholipase B-like 1 (EC 3.1.1.-) (Phospholipase B domain-containing protein 1) (Lamina ancestor homolog 1) (LAMA-like protein 1)


552


Q86SH4


Putative testis-specific prion protein (Protein M8)


94


Q13046


Putative pregnancy-specific beta-1-glycoprotein 7 (PSBG-7)


419


Q8TDE3


Ribonuclease 8 (RNase 8) (EC 3.1.27.-)


154


Q8IVN8


RPE-spondin


264


Q6NUJ1


Proactivator polypeptide-like 1 [Cleaved into: Saposin A-like; Saposin B-Val-like; Saposin B-like; Saposin C-like; Saposin D-like]


521


Q8IVW8


Protein spinster homolog 2


549


Q765I0


Urotensin-2B (Urotensin-IIB) (U-IIB) (UIIB) (Urotensin II-related peptide) (Urotensin 2 domain-containing protein)


119


B2RUY7


von Willebrand factor C domain-containing protein 2-like


222


Q9BT30


Alkylated DNA repair protein alkB homolog 7 (Spermatogenesis-associated protein 11) (Spermatogenesis cell proliferation-related protein)


221


Q6UWV7


UPF0514 membrane protein FAM159A


190


Q86YQ2


Latherin (Breast cancer and salivary gland-expressed protein)


179


Q6UX53


Methyltransferase-like protein 7B (EC 2.1.1.-)


244


Q8WW62


Transmembrane emp24 domain-containing protein 6


240


Q96SJ8


Tetraspanin-18 (Tspan-18)


248


Q6UWH6


Protein TEX261


196


Q6UX68


XK-related protein 5


686


Q6UWQ7


Insulin growth factor-like family member 2


119


Q6UXB1


Insulin growth factor-like family member 3


125


Q9ULZ1


Apelin (APJ endogenous ligand) [Cleaved into: Apelin-36; Apelin-31; Apelin-28; Apelin-13]


77


Q567T5


Secreted phosphoprotein 1 (cDNA FLJ52507, highly similar to Osteopontin) (cDNA FLJ77801) (Secreted phosphoprotein 1 (Osteopontin,


bone sialoprotein I, early T-lymphocyte activation 1), isoform CRA_a)


287


A8TX70


Collagen alpha-5(VI) chain (Collagen alpha-1(XXIX) chain) (von Willebrand factor A domain-containing protein 4)


2,615


Q86Y27


B melanoma antigen 5 (Cancer/testis antigen 2.5) (CT2.5)


43


Q7Z5Y6


Bone morphogenetic protein 8A (BMP-8A)


402


Q5VUM1


UPF0369 protein C6orf57


108


Q8N7Q2


Putative uncharacterized protein C10orf31


184


Q8TAV5


Putative uncharacterized protein C11orf45


145


Q96LU7


Uncharacterized protein C12orf28


275


Q0P5P2


Uncharacterized protein C17orf67


114


Q5J5C9


Beta-defensin 121 (Defensin, beta 121) (Beta-defensin 21) (DEFB-21)


76


Q30KQ2


Beta-defensin 130 (Defensin, beta 130) (Beta-defensin 30) (DEFB-30)


79


Q9BX68


Histidine triad nucleotide-binding protein 2 (HINT-2) (EC 3.-.-.-) (HINT-3) (HIT-17kDa) (PKCI-1-related HIT protein)


163


O75200


Nuclear pore complex-interacting protein-like 1


221


Q5JTB6


Placenta-specific protein 9


97


Q86WS3


Placenta-specific 1-like protein


158


O60542


Persephin (PSP)


156


Q5W5W9


Regulated endocrine-specific protein 18 [Cleaved into: Regulated endocrine-specific protein 18; Triskadecapeptide]


173


P60153


Ribonuclease-like protein 9


205


Q9H1F0


WAP four-disulfide core domain protein 10A (Putative protease inhibitor WAP10A)


79


Q8IUB5


Protein WFDC13


93


Q9H1J5


Protein Wnt-8a (Wnt-8d)


355


A6NGN9


Ig-like domain-containing protein ENSP00000270642


336


Q6UXT9


Abhydrolase domain-containing protein 15 (EC 3.1.1.-)


468


P60827


Complement C1q tumor necrosis factor-related protein 8


262


Q6UXQ4


Uncharacterized protein C2orf66


117


Q6UX52


Uncharacterized protein C17orf99


265


Q6UXT8


Protein FAM150A


129


Q6UXH0


Hepatocellular carcinoma-associated protein TD26


198


Q7Z4B0


Uncharacterized protein C18orf20


112


Q30KR0


Beta-defensin 110 (Defensin, beta 110) (Beta-defensin 10) (DEFB-10)


62


Q5RGM9


Abhydrolase domain-containing protein FAM108A2/A3 (EC 3.-.-.-)


310


Q99954


Submaxillary gland androgen-regulated protein 3A (Proline-rich protein 5) (Proline-rich protein PBI)


134


Q6UVW9


C-type lectin domain family 2 member A (Proliferation-induced lymphocyte-associated receptor) (PILAR)


174


Q7Z2Q7


Leucine-rich repeat-containing protein 70 (Synleurin)


622


Q6UXR4


Serpin A13


307


Q6UXU0


Putative uncharacterized protein UNQ9165/PRO28630


137


Q8IYJ0


Uncharacterized protein C12orf53


282


Q6UXN8


C-type lectin domain family 9 member A


241


Q9Y6N3


Calcium-activated chloride channel regulator family member 3 (Calcium-activated chloride channel family member 3) (hCLCA3)


262


Q6UXZ3


CMRF35-like molecule 4 (CLM-4) (CMRF35-A4) (CD300 antigen-like family member D) (CD antigen CD300d)


194


Q6UXP7


Protein FAM151B


276


Q496H8


Neuritin-like protein


165


P61366


Osteocrin (Musclin)


133


A6NL71


Transmembrane protease, serine 11E2 (EC 3.4.21.-) [Cleaved into: Transmembrane protease, serine 11E2 non-catalytic chain; Transmembrane


protease, serine 11E2 catalytic chain]


423


Q6UXP3


Transmembrane protein 14E


125


Q6UWW9


Transmembrane protein 207


146


Q6UXN7


TOMM20-like protein 1


152


Q6UWB4


Probable serine protease UNQ9391/PRO34284 (EC 3.4.21.-)


352


Q6UXD1


Histidine-rich carboxyl terminus protein 1


115


Q13072


B melanoma antigen 1 (B melanoma antigen) (Antigen MZ2-BA) (Cancer/testis antigen 2.1) (CT2.1)


43


Q9UKY3


Inactive carboxylesterase 4 (Placental carboxylesterase 3) (PCE-3)


287


Q86VR8


Four-jointed box protein 1 (Four-jointed protein homolog)


437


Q6IFS5


Protein HSN2


434


Q8IVG9


Humanin


24


Q6ZWJ8


Kielin/chordin-like protein (Kielin/chordin-like protein 1) (Cysteine-rich motor neuron 2 protein) (CRIM-2) (Cysteine-rich BMP regulator 2)


1,503


Q2TV78


Putative macrophage-stimulating protein MSTP9 (Brain rescue factor 1) (BRF-1) (Hepatocyte growth factor-like protein homolog)


715


P0C859


Putative neurofibromin 1-like protein 4/6


116


A1KZ92


Peroxidasin-like protein (EC 1.11.1.7) (Vascular peroxidase 2) (Cardiac peroxidase)


1,463


A2VEC9


SCO-spondin


5,147


A6NE02


BTB/POZ domain-containing protein 17 (Galectin-3-binding protein-like)


478


Q86Z23


Complement C1q-like protein 4


238


Q9UFP1


Uncharacterized protein C3orf41


575


Q17RF5


Uncharacterized protein C4orf26


130


Q6MZM9


Uncharacterized protein C4orf40


219


Q8N2X6


Uncharacterized protein C5orf55


119


Q5JXM2


UPF0624 protein C6orf186


366


A6NNL5


Uncharacterized protein C15orf61


157


Q6GPI1


Chymotrypsinogen B2 (EC 3.4.21.1) [Cleaved into: Chymotrypsin B2 chain A; Chymotrypsin B2 chain B; Chymotrypsin B2 chain C]


263


A8MXU0


Beta-defensin 108A (Defensin, beta 108A) (Putative beta-defensin 108B pseudogene 1)


73


Q30KQ9


Beta-defensin 111 (Defensin, beta 111) (Beta-defensin 11) (DEFB-11)


67


Q30KQ8


Beta-defensin 112 (Defensin, beta 112) (Beta-defensin 12) (DEFB-12)


113


Q30KQ6


Beta-defensin 114 (Defensin, beta 114) (Beta-defensin 14) (DEFB-14)


69


Q30KQ5


Beta-defensin 115 (Defensin, beta 115) (Beta-defensin 15) (DEFB-15)


88


Q30KQ4


Beta-defensin 116 (Defensin, beta 116) (Beta-defensin 16) (DEFB-16)


102


P59861


Beta-defensin 131 (Defensin, beta 131) (Beta-defensin 31) (DEFB-31)


70


Q4QY38


Beta-defensin 134 (Defensin, beta 134)


66


Q30KP8


Beta-defensin 136 (Defensin, beta 136)


78


A6NNS2


Dehydrogenase/reductase SDR family member 7C (EC 1.1.-.-)


312


Q5T7M4


Protein FAM132A


302


Q4G0M1


Protein FAM132B


354


Q6IE38


Putative serine protease inhibitor Kazal-type 5-like 2


97


Q1W4C9


Serine protease inhibitor Kazal-type 5-like 3 (Hepatitis B virus DNA polymerase transactivated serine protease inhibitor) (Hespintor)


94


Q5DT21


Serine protease inhibitor Kazal-type 9 (Lymphoepithelial Kazal-type-related inhibitor 2)


86


Q5VXI9


Lipase member N (EC 3.1.1.-) (Lipase-like abhydrolase domain-containing protein 4)


398


Q17RR3


Pancreatic lipase-related protein 3 (PL-RP3) (EC 3.1.1.3)


467


Q641Q3


Meteorin-like protein


311


Q5H8A3


Neuromedin-S


153


P0C0P6


Neuropeptide S


89


Q96A99


Neuronal pentraxin-like protein C16orf38


478


A6NHN0


Otolin-1


477


Q6IE37


Ovostatin homolog 1


1,185


Q6ZNF0


Iron/zinc purple acid phosphatase-like protein (EC 3.1.3.2)


438


Q15195


Plasminogen-related protein A (Plasminogen-like protein A) (Plasminogen-like protein A1)


96


Q2L4Q9


Polyserase-3 (EC 3.4.21.-) (Polyserine protease 3)


553


Q9NRI6


Putative peptide YY-2 (Putative peptide YY2)


33


Q5JQD4


Putative peptide YY-3 (Putative peptide YY3) (PYY-III)


70


Q5GAN6


Ribonuclease-like protein 10


216


Q5GAN4


Ribonuclease-like protein 12


147


Q5GAN3


Ribonuclease-like protein 13


156


Q86U17


Serpin A11


422


Q6UDR6


Kunitz-type protease inhibitor 4


99


Q6URK8


Testis, prostate and placenta-expressed protein


271


Q7Z5A4


Putative testis serine protease 2 (EC 3.4.21.-)


293


A6NJ16


Putative V-set and immunoglobulin domain-containing protein 6


123


Q2M2E5


Uncharacterized protein FLJ37543


130


A4D1T9


Peptidase S1 domain-containing protein LOC136242


235


Q1ZYW2


Secreted frizzled-related protein 4


346


Q7Z5L3


Complement C1q-like protein 2


287


Q96LR1


Putative uncharacterized protein C17orf69


157


Q4G179


Putative cystatin-13


69


Q30KR1


Beta-defensin 109 (Defensin, beta 109)


87


Q30KQ7


Beta-defensin 113 (Defensin, beta 113) (Beta-defensin 13) (DEFB-13)


82


Q30KP9


Beta-defensin 135 (Defensin, beta 135)


77


A6NFZ4


Protein FAM24A


105


Q7Z4P5


Growth/differentiation factor 7 (GDF-7)


450


A6NJ69


IgA-inducing protein homolog


53


Q5VSP4


Putative lipocalin 1-like protein 1


162


A6NIE9


Putative serine protease 29 (Implantation serine proteinase 2-like protein) (ISP2-like protein)


313


Q4G0G5


Secretoglobin-like protein


96


Q4G0T1


Putative scavenger receptor cysteine-rich domain-containing protein LOC619207


232


A6NGW2


Putative stereocilin-like protein


1,772


A6NDP1


Putative V-set and immunoglobulin domain-containing protein 7


120


A6NJS3


Putative V-set and immunoglobulin domain-containing-like protein ENSP00000303034


120


Q6ZRU5


Putative uncharacterized protein FLJ46089


148


Q6B9Z1


Insulin growth factor-like family member 4


124


Q6UX72


UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 9 (Beta-1,3-N-acetylglucosaminyltransferase-9) (Beta3Gn-T9) (BGnT-9)


(EC 2.4.1.-)


402


O15150


Replication initiation-like protein (Myelin transcription factor 2) (MyT2) (Cerebrin-50)


435


B3GLJ2


Prostate and testis expressed protein 3 (PATE-like protein DJ) (PATE-DJ) (Acrosomal vesicle protein HEL-127)


98


Q86Y28


B melanoma antigen 4 (Cancer/testis antigen 2.4) (CT2.4)


39


Q7Z2R9


Putative uncharacterized protein C1orf191


100


A6NNL9


Putative abhydrolase domain-containing protein FAM108A5 (EC 3.-.-.-)


308


Q6UY13


Putative uncharacterized protein UNQ5830/PRO19650/PRO19816


95


B3KQR2


Secreted frizzled-related protein 2 (cDNA PSEC0060 fis, clone NT2RP2000638, highly similar to Secreted frizzled-related protein 2)


295


Q6UXP8


Putative uncharacterized protein UNQ6975/PRO21958


91


O43320


Fibroblast growth factor 16 (FGF-16)


207


P0C7L1


Serine protease inhibitor Kazal-type 8


97


Q9UF72


Uncharacterized protein KIAA0495


201


Q13862


Platelet basic protein-like 2 (DNA-binding protein amplifying expression of surfactant protein B) (DNA-binding protein SPBPBP)


106


A8MV23


Serpin E3


424


A1L4H1


Scavenger receptor cysteine-rich domain-containing protein LOC284297


1,096


A6XMV6


Secreted phosphoprotein 1


314


A4ZYV1


Stress induced secreted protein 1


311


P0C854


Putative cat eye syndrome critical region protein 9


216


A6NG13


Glycosyltransferase 54 domain-containing protein (EC 2.4.1.-)


371


A8MTI9


Putative serine protease LOC138652 (EC 3.4.21.-)


375


A6NC86


Uncharacterized protein ENSP00000244321


204


A8MT79


Putative zinc-alpha-2-glycoprotein-like 1


204


Q5TEV5


Putative uncharacterized protein C1orf134


83


A6NCS6


Uncharacterized protein C2orf72


168


Q8N8P7


Uncharacterized protein C11orf44


122


Q69YU5


Uncharacterized protein C12orf73


71


Q5W188


Putative cystatin-9-like 2


147


A6NDD2


Beta-defensin 108B-like


73


Q30KQ1


Beta-defensin 133 (Defensin, beta 133)


61


A6NEC5


Putative abhydrolase domain-containing protein FAM108A6 (EC 3.-.-.-)


299


P62706


Fibrosin-1


177


A6ND01


Probable folate receptor delta (FR-delta) (Folate receptor 4)


244


A6NF02


NPIP-like protein ENSP00000346774


221


A6NHN6


Nuclear pore complex-interacting protein-like 2


382


Q99935


Proline-rich protein 1 (PRL1) (Basic proline-rich lacrimal protein)


201


Q71RG6


Putative uncharacterized protein FP248


208


Q6ZVS6


Putative uncharacterized protein FLJ42147


177


A8MXB1


Putative zinc-alpha-2-glycoprotein-like 2


111


Q6IBK4


SPARC protein (Secreted protein, acidic, cysteine-rich (Osteonectin), isoform CRA_b)


303


Q6UWF6


Putative uncharacterized protein UNQ3029/PRO9830


113


B4DYC1


cDNA FLJ60957, highly similar to Secreted frizzled-related protein 4


368


B4DRV4


cDNA FLJ55667, highly similar to Secreted protein acidic and rich in cysteine


212


Q6UXU1


Putative uncharacterized protein UNQ6490/PRO21339


168


Q6UXQ6


Putative uncharacterized protein UNQ6125/PRO20090


108


Q6UXR6


Putative uncharacterized protein UNQ6494/PRO21346


183


Q6UXQ8


Putative uncharacterized protein UNQ6190/PRO20217


127


Q6UXV3


Uncharacterized protein UNQ6126/PRO20091


157


B3KSM5


cDNA FLJ36603 fis, clone TRACH2015180, highly similar to Secreted frizzled-related protein 2


238


P0C8F1


Prostate and testis expressed protein 4 (PATE-like protein B) (PATE-B)


95


Q6UX82


Uncharacterized protein UNQ511/PRO1026


237


Q6UWF5


Putative uncharacterized protein UNQ5815/PRO19632


114


Q6UXR8


Putative uncharacterized protein UNQ6493/PRO21345


122


Q5U0B9


Stem cell growth factor; lymphocyte secreted C-type lectin


323


B4E1T4


cDNA FLJ53955, highly similar to Secreted frizzled-related protein 4


343


Q6UXP9


Putative uncharacterized protein UNQ9370/PRO34162


181


Q9P1C3


Putative uncharacterized protein PRO2829


46


Q96I85


Putative uncharacterized protein C14orf144


54


P0C876


Uncharacterized protein FLJ90687


127


P04281


Basic proline-rich peptide IB-1


96


A8MTW9


Putative uncharacterized protein ENSP00000380674


85


A8MUN3


Putative uncharacterized protein ENSP00000381830


132


Q53G63


Secreted and transmembrane 1 precusor variant (Fragment)


248


Q53G27


Secreted and transmembrane 1 precusor variant (Fragment)


248


Q16521


CR1 receptor (CR1 receptor SCR9) (Fragment)


33


P61109


Kidney androgen-regulated protein (KAP) (ARP)


121


P85047


Opiorphin


5


Q6ZS96


Protein Wnt


329


Q4VAJ4


Protein Wnt


191


Q9UNH2


MC51L-53L-54L homolog (Fragment)


184


O60748


COBW-like placental protein (Fragment)


106


B8A597


Protein Wnt


205


B8A595


Protein Wnt


333


Q3SY79


Protein Wnt


385


Q96H90


Protein Wnt


349


Q5TEH9


Protein Wnt


391


Q6DK41


Protein Wnt (Fragment)


381


Q05BQ6


Protein Wnt (Fragment)


379


Q5TEH8


Protein Wnt


299


A0N6Y5


Fibroblast growth factor receptor FGFR-1 secreted form protein (Fragment)


92


Q6UXM4


Ficolin (Collagen/fibrinogen domain containing) 3 (Hakata antigen) (NL3) (Ficolin (Collagen/fibrinogen domain containing) 3 (Hakata antigen),


isoform CRA_b)


288


Q6UW50


TOM1


209


Q6UW36


ECE2


736


Q6UWM0


EPA6


285


Q53S44


Protein Wnt


417


Q5TEI2


Protein Wnt


372


A8K315


Protein Wnt


359


Q5IHW6


KIR2DL4 (Fragment)


367


A4D0W7


Protein Wnt


355


B8A596


Protein Wnt


349


Q53S45


Protein Wnt (Fragment)


338


Q86YL8


Putative soluble interleukin 18 receptor 1


170


Q5JYX2


Protein Wnt (Fragment)


149


Q6ZSP0


Protein Wnt


351


A6NE61


Protein Wnt (Fragment)


239


Q6UY50


NL3


218


Q5U0N2


Protein Wnt


370


A4D0V1


Protein Wnt


360


B2R7A5


Protein Wnt


389


A0FKD1


WNT1 induced secreted protein 1 splice variant x (Fragment)


43


B5MCC8


Protein Wnt


173


B7Z1Y5


Protein Wnt


153


A4D0W8


Protein Wnt


365


A8K0G1


Protein Wnt


353


Q5U0K5


Protein Wnt


354


Q9BTP0


Protein Wnt (Fragment)


293


Q8IUM6


Protein Wnt


351


B4DJF9


Protein Wnt


296


Q8N2E5


Protein Wnt


365


Q59G81


Protein Wnt (Fragment)


284


Q2TQ40


B cell maturation antigen transcript variant 4 (Tumor necrosis factor receptor superfamily member 17)


135


Q8IWS1


Keratinocytes associated transmembrane protein 1


178


B3KQX9


Protein Wnt


291


Q6UWZ0


WLPL514


86


Q6UWF4


GLGQ5807


153


Q6UWW7


TUFT1


199


Q6UWC1


DRLV8200


456


Q6UVX0


IDLW5808


80


Q6UWR4


UBAP2


363


B7U178


C1q/TNF-related protein 8


262


A3R3E3


KIR2DL4 (Fragment)


367


Q5I2A4


Chemokine-like factor super family 2 transcript variant 2


195


Q6UWX0


PLA2G2D


116


Q6UWT8


GKGM353


67


Q6UWM6


MATL2963


88


Q6UXV6


NINP6167


142


Q6UW61


POM121-like


428


Q6UXS7


RTFV9368 (SLE-dependent upregulation 1)


120


Q9UK79


Herstatin (V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (Avian), isoform CRA_a)


419


Q8N2D3


Protein Wnt


253


Q6UXQ9


ELCV5929


189


Q6UWM4


KVVM3106


93


Q6UXR9


ISPF6484


88


Q6UXP6


LKHP9428


218


Q6UXT6


VNFT9373


308


Q6UY24


ACAH3104


115


Q6UXU7


RVLA1944


88


Q6UY31


Wpep3002


85


Q6UWR9


ZDHHC11


142


Q6UWV9


AGLW2560


178


Q6UWH1


TSSP3028


102


Q6UXX8


RFVG5814


206


Q6UWJ0


SHSS3124


97


Q6UWI1


MMP19


105


Q6UXW7


GSQS6193


108


Q6UW46


VGPW2523


315


Q6UXR2


LMNE6487


150


Q6UWG3


ALLA2487


243


Q6UWR0


GALI1870


121


Q6UWG4


FRSS1829


73


Q6UXQ5


MRSS6228


114


Q6UWK3


GRPR5811


159


Q6UWH2


AVLL5809


159


Q6UXV8


KCNQ2


95


Q6UY29


PIKR2786


93


Q5M770


S100 calcium binding protein A7-like 3


96


Q6Y2K9


GTWW5826 (LP5085 protein)


148


Q6UXP2


KTIS8219 (HCG2020043)


101


Q6UWC4


PPIF


128


Q6UXQ0


Micronovel


115


Q6UX30


SAMK3000


100


Q6UY40


VFLL3057


126


Q6UXY5


CVWG5837


125


Q6UWI7


VGSA5840


90


Q6UXY7


GHPS3125


140


Q6UXS6


GRTR3118


119


Q6UXQ7


PAMP6501


173


Q6UXR7


LTLL9335


104


Q6UXT5


VCEW9374


127


Q6UXP0


AHPA9419


99


Q6UWJ2


MDHV1887


108


Q6UWG9


HSAL5836


89


Q6UXS3


LHLC1946


108


Q6UWR2


CLECSF12


90


Q6UWQ9


LPPA601


120


Q6UW64


PINK1


109


Q6UY30


SERH2790


109


Q6UXP4


FLFF9364


118


Q6UWT3


APELIN


73


Q6UXV5


GLSH6409


118


Q6UWE9


SFVP2550


103


Q6UXT2


RRLF9220


250


Q6UXW0


PTML5838


95


Q6UWG0


VLGN1945


125


Q6UXS4


AVPC1948


83


Q6UWR5


AWQG2491


83


Q6UXR5


PSVL6168


90


Q6UY26


LCII3035


78


Q6UXW9


PPRR6495


112


Q6UWB8


RLSC6348


110


Q6UWS5


CSRP2BP


81


Q6UX66


GLLV3061


139


Q6UXR0


GWSI6489


91


Q6UXU5


C8orf2


86


O76106


CR1 C3b/C4b receptor SCR9 (or 16) C-term. exon SCR = short consensus repeat


28


Q6UWF8


VSSW1971


120


Q6UXR3


KLIA6249


102


Q6UWG2


ALLW1950


84


Q6UWS8


GVEI466


52


Q6UXV9


ESFI5812


134


Q6UWM8


GNNC2999


100


Q6UXR1


AAGG6488


85


Q6UWP5


HHSL751


143

## Claims

1. A method of screening for a candidate kinetic modulating monoclonal antibody that modulates binding between a first protein component and a second protein component of a signaling complex, comprising the steps of:
(a) (i) measuring a binding affinity or binding rate parameter of a test antibody for said first component in the presence of said second component, and (ii) measuring a binding affinity or binding rate parameter of said test antibody for said first component in the absence of said second component; and
(b) (i) measuring a binding affinity or binding rate parameter of a test antibody for said second component in the presence of said first component and (ii) measuring a binding affinity or binding rate parameter of said test antibody for said second component in the absence of said first component; and
(c) identifying said test antibody as a candidate kinetic modulating antibody when said test antibody exhibits a 1.5-fold to 1000-fold difference in the binding affinity or binding rate parameter measured in steps (a) and (b),
wherein the test antibody is identified as a candidate positive modulating antibody if the binding affinity or binding rate parameter measured in step (a)(i) or (b)(i) is about 1.5-fold to 1000-fold stronger than the binding affinity or binding rate parameter measured in step (a)(ii) or (b)(ii),
and wherein at least one of the components or the test antibody is at a sub-saturating concentration relative to the other component or test antibody.

2. The method of claim 1 wherein the binding affinity or binding rate parameter measured in step (a)(i) or (b)(i) is about 2-fold to 200-fold stronger than the binding affinity or binding rate parameter measured in step (a)(ii) or (b)(ii).

3. The method of claim 1 wherein the binding affinity or binding rate parameter of the test antibody for the first component but not for the second component is measured.

4. The method of claim 3 wherein the binding affinity or binding rate parameter of the test antibody for the second component but not for the first component is measured.

5. The method of claim 4 wherein the binding affinity KD of the test antibody for a complex comprising the first and second components is about 10−5 M−1 or less, and the test antibody does not detectably bind to either the first component alone or the second component alone.

6. The method of claim 4 wherein the test antibody (M) is identified as a candidate positive modulating antibody if a binding affinity or binding rate parameter selected from the group consisting of (A) the binding affinity or binding rate parameter of the test antibody for a complex comprising the first component (C1) and the second component (C2), optionally K[C1C2]M, (B) the binding affinity or binding rate parameter of the first component for a complex comprising the antibody and the second component, optionally K[MC2]C1, or (C) the binding affinity or binding rate parameter of the second component for a complex comprising the antibody and the first component, optionally K[MC1]C2, is about 1.5-fold to 1000-fold stronger than a binding affinity or binding rate parameter selected from the group consisting of (1) the binding affinity or binding rate parameter of the test antibody for the second component alone, optionally KMC2 or (2) the binding affinity or binding rate parameter of the test antibody for the first component alone, optionally KMC1.

7. The method of claim 6 wherein the binding affinity or binding rate parameter of any one or more of (A), (B) or (C) is about 2-fold or 200-fold stronger than the binding affinity or binding rate parameter of any one or more of (1) or (2).

8. The method of claim 6 wherein the binding affinity or binding rate parameter of any one or more of (A), (B) or (C) is about 1.5-fold to 1000-fold stronger than the binding affinity or binding rate parameter of both (1) and (2).

9. The method of claim 6 wherein the binding affinity or binding rate parameter of any one or more of (A), (B) or (C) is about 2-fold to 200-fold stronger than the binding affinity or binding rate parameter of both (1) and (2).

10. The method of claim 6 wherein the binding affinity or binding rate parameter of (1) is stronger than the binding affinity of or binding rate parameter of (2).

11. The method of claim 6 wherein the binding affinity or binding rate parameter of (2) is stronger than the binding affinity or binding rate parameter of (1).

12. The method of claim 6 wherein the binding affinity is the equilibrium dissociation constant KD, and any one or more of K[C1C2]M, K[MC2]C1, or K[MC1]C2 is about 1.5-fold to 1000-fold lower than any one or more of KMC2 or KMC1.

13. The method of claim 12 wherein K[C1C2]M is about 1.5-fold to 1000-fold lower than KMC2.

14. The method of claim 12 wherein K[MC2]C1 is about 1.5-fold to 1000-fold lower than KMC2.

15. The method of claim 12 wherein K[MC1]C2 is about 1.5-fold to 1000-fold lower than KMC2.

16. The method of claim 12 wherein K[C1C2]M is about 1.5-fold to 1000-fold lower than KMC1.

17. The method of claim 12 wherein K[MC2]C1 is about 1.5-fold to 1000-fold lower than KMC1.

18. The method of claim 12 wherein K[MC1]C2 is about 1.5-fold to 1000-fold lower than KMC1.

19. The method of claim 6 wherein the binding affinity is the equilibrium association constant KA, and any one or more of K[C1C2]M, K[MC2]C1, or K[MC1]C2 is about 1.5-fold to 1000-fold higher than any one or more of KMC2 or KMC1.

20. The method of claim 1 wherein the antigen to which the test antibody binds is the first component and the test antibody is at a saturating concentration compared to the concentration of the first component.

21. The method of claim 1 wherein the antigen to which the test antibody binds is the second component and the test antibody is at a saturating concentration compared to the concentration of the second component.

22. The method of claim 20 wherein the concentration of the test antibody is greater than or equal to the KD of the test antibody for a complex comprising the first component and the second component.

23. The method of claim 22 wherein the concentration of the second component is less than the KD of the test antibody for the first component.

24. The method of claim 23 wherein the concentration of the first component is at a subsaturating concentration for the binding of first component to second component.

25. The method of claim 24 wherein the concentration of the first component is within the range of about EC20 to EC80 for the interaction of the first component with the second component.

26. The method of claim 1 wherein one or more concentrations of the test antibody is contacted with multiple different concentrations of the first component in the presence of one or more concentrations of the second component.

27. The method of claim 1 wherein one or more concentrations of the test antibody is contacted with multiple different concentrations of the second component in the presence of one or more concentrations of the first component.

28. The method of claim 2 wherein the test antibody is at a saturating concentration for a complex comprising the first component and the second component.

29. The method of claim 28 wherein the concentration of test antibody is greater than or equal to the KD of the test antibody for a complex comprising the first component and the second component.

30. The method of claim 29 wherein the concentration of the second component is greater than the KD of the second component for the first component.

31. The method of claim 30 wherein the concentration of the first component is a saturating concentration for the second component.

32. The method of claim 2 wherein the test antibody is at a subsaturating concentration for a complex comprising the first component and the second component.

33. The method of claim 32 wherein the concentration of the antibody is within the range of about EC20 to EC80 for the interaction of the first component with the second component.

34. The method of claim 33 wherein the concentration of the second component is greater than the KD of the second component for the first component.

35. The method of claim 34 wherein the concentration of the first component is a saturating concentration for the second component.

36. The method of claim 1 further comprising, prior to step (a), assaying a plurality of test antibodies for binding affinity to said first component, optionally with an equilibrium dissociation constant KD of 10−5M or stronger binding affinity.

37. The method of claim 1 further comprising, prior to step (b), assaying a plurality of test antibodies for binding affinity to said second component, optionally with an equilibrium dissociation constant KD of 10−5M or stronger binding affinity.

38. The method of claim 1 further comprising measuring a binding affinity or binding rate parameter of said first component for a binding partner wherein the binding partner is a decoy receptor, clearance receptor, or alternate signal pathway component, wherein the binding partner is not said second component, in the presence and absence of said test antibody.

39. The method of claim 38 comprising identifying a test antibody that does not significantly change the binding affinity or binding rate parameter of said first component for said binding partner.

40. The method of claim 1 wherein said test antibody is selected from the group consisting of antibody fragments, scFv, Fab, CDRs, rodent antibodies, mammalian antibodies, human antibodies, chimeric antibodies and humanized antibodies.

41. The method of claim 1 wherein said binding affinity or binding rate parameter is selected from the group consisting of equilibrium association constant KA, equilibrium dissociation constant KD, on-rate, off-rate and surrogate parameters for any of the foregoing.

42. The method of claim 41 wherein said surrogate parameter is the quantity or level of binding of said first component to said second component at a subsaturating concentration of either said first component or said second component.

43. The method of claim 1 wherein all of said test antibody, said first component, and said second component are in solution.

44. The method of claim 1 wherein one of said test antibody, said first component, and said second component is linked to a solid phase.

45. The method of claim 44 wherein the linkage is noncovalent.

46. The method of claim 44 wherein one of said test antibody, said first component, and said second component is coated on a bead.

47. The method of claim 1 wherein at least one of said first component or second component is expressed on a cell surface.

48. The method of claim 1 wherein said first component is expressed on a cell surface and said second component is expressed on a different cell surface.

49. The method of claim 1 wherein said first component is a soluble ligand and said second component is a membrane-bound receptor.

50. The method of claim 1 wherein said first component is a membrane-bound receptor and said second component is a soluble ligand.

51. The method of claim 1 wherein said first component is a membrane-bound ligand and said second component is a membrane-bound receptor.

52. The method of claim 50 or 51 wherein said membrane-bound receptor is selected from the group consisting of 7-transmembrane receptors, G-protein coupled receptors (GPCRs), adrenergic receptors, neurotransmitter receptors, olfactory receptors, opioid receptors, chemokine receptors, rhodopsin, receptor tyrosine kinases, growth factor receptors, integrins, and toll-like receptors.

53. The method of claim 1 wherein said first component is an enzyme and said second component is a substrate for said enzyme.

54. The method of claim 1 wherein said first component is a cytokine or chemokine and said second component is a receptor for said first component.

55. The method of claim 1 wherein said first component is a growth factor and said second component is a receptor for said first component.

56. The method of claim 1, wherein said first component is IL-1β and said second component is IL-1 receptor type I (IL-1RI).

57. The method of claim 1, wherein said first component is GCSF and said second component is GCSFR.

58. The method of claim 1, wherein said first component is GCSFR and said second component is GCSF.

59. The method of claim 1, wherein said first component is TNFα and said second component is TNFR1 or 2.

60. The method of claim 1, wherein said first component is TNFR1 or 2 and said second component is TNFα.

61. The method of claim 1 further comprising recloning the antibody identified in step (c) into an expression vector and expressing the antibody.

62. The method of claim 1 further comprising purifying the antibody identified in step (c).

63. The method of claim 1 further comprising sequencing the antibody identified in step (c).

64. The method of claim 1 further comprising adding or replacing an Fc region or fragment thereof of the test antibody.

65. The method of claim 1 further comprising formulating an antibody comprising at least six CDRs of the test antibody identified in step (c) in a sterile composition with a sterile pharmaceutically acceptable diluent.

66. The method of claim 1 further comprising administering an antibody comprising at least six CDRs of the test antibody identified in step (c) to an animal.

67. The method of claim 1 further comprising measuring the level of signaling mediated by said signaling complex in the presence and absence of the test antibody.

68. The method of claim 67 wherein the level of signaling mediated by the signaling complex is measured in a phosphorylation assay, ion flux assay, molecular transport assay, or gene expression assay.

69. The method of any of claims 67-68 wherein said test antibody increases the EC50 of the first component of said signaling complex by about 1.5-fold to about 1000-fold.

70. The method of any of claims 67-68 wherein said test antibody does not significantly change the maximal agonist response of the signaling produced by said first component.

71. The method of any of claims 67-68 wherein said test antibody reduces the maximal agonist response of the signaling produced by said signaling complex by about 1.5-fold to 1000-fold.

72. The method of any of claims 67-68 wherein said test antibody decreases the EC50 of the signaling produced by said signaling complex by about 1.5-fold to about 1000-fold.

73. The method of any of claims 67-68 wherein said candidate antibody increases the maximal agonist response of the signaling produced by said first component by at least 10%.

74. The method of claim 1 wherein said candidate antibody does not significantly decrease clearance of said first component, said second component, or said signaling complex comprising said first and second components.

75. The method of claim 38 wherein the first component is IL-1 beta, the second component is IL-1R1, and the binding partner is either IL-1R2 or IL-1 accessory protein.

76. The method of claim 38 wherein the first component is TNF alpha, the second component is TNFR1, and the binding partner is TNFR2.

77. The method of claim 38 wherein the first component is TNF alpha, the second component is TNFR2, and the binding partner is TNFR1.