# Patent

## Title
Heterodimeric proteins and uses thereof

## Abstract

The present invention relates, inter alia, to compositions and methods, including heterodimeric proteins that find use in the treatment of disease, such as immunotherapies for cancer and autoimmunity.

## Background
PRIORITY
This application is a continuation of U.S. application Ser. No. 16/571,853, filed Sep. 16, 2019, now U.S. Pat. No. 10,995,127, which is a continuation of International Application No. PCT/US19/38451, filed Jun. 21, 2019, which claims the benefit of U.S. Provisional Application No. 62/688,167, filed Jun. 21, 2018 and U.S. Provisional Application No. 62/703,248, filed Jul. 25, 2018, the contents of each of which are incorporated herein by reference in their entirety.


TECHNICAL FIELD
The present invention relates to heterodimeric proteins that find use in the treatment of diseases, such as immunotherapies for cancer and autoimmunity.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: SHK-00402_116981-5004_ST25; date created on: Mar. 24, 2021; file size: 114,059 bytes).
BACKGROUND
Protein-protein interactions are critical for the normal physiological functions of cells and multicellular organisms. For example, cytokines act as ligands which bind to their cognate receptors so as to regulate essential biological processes such as inflammation and immunity. In this regard, many natural cytokines, cytokine receptors, integrins, and other proteins exist or function as multimeric protein complexes. Some multimers, such as those within the tumor necrosis factor superfamily function as homotrimers, other ligands including the IL-12 family of cytokines (e.g., IL12, IL23, IL27, or IL-35) form heterodimers. Similarly, cytokine receptors may also function as heterodimeric complexes. For example, many interleukin receptors form heterodimers for signal transduction.
The modulation of protein-protein interactions is a useful mechanism for therapeutic intervention in various diseases and pathologies. Soluble binding proteins which interact with ligands can potentially sequester the ligand away from the receptor, thereby reducing the activation of that particular receptor pathway. Alternatively, sequestration of the ligand may delay its elimination or degradation, thereby increasing its duration of effect and biological activity. Additionally, soluble ligands may be utilized to either activate or inhibit specific receptors. Nevertheless, the synthesis and manufacturing of soluble proteins may be hampered where it is desirable to produce heterodimeric proteins. Particularly, the efficiency of synthesis may be greatly compromised by the formation of mixtures of homodimers and heterodimers.
Accordingly, there remains a need for novel methods for the efficient synthesis and manufacturing of heterodimeric proteins for therapeutic use.
SUMMARY
In various embodiments, the present invention provides a heterodimeric protein comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first subunit of a first protein at the amino terminus linked by a first charge polarized core domain to a first subunit of a second protein at the carboxy terminus; and the second polypeptide chain comprises a second subunit of the first protein at the amino terminus linked by a second charge polarized core domain to a second subunit of the second protein at the carboxy terminus.
In various embodiments, the first polypeptide chain and the second polypeptide chain heterodimers through electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second charge polarized core domains. In some embodiments, the positively charged amino acid residues may include one or more of amino acids selected from His, Lys, and Arg. In some embodiments, the negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu.
Accordingly, in various embodiments, each of the first and/or second charge polarized core domains comprises peptides having positively or negatively charged amino acid residues at the amino and carboxy terminus of the core domain. In an exemplary embodiment, the first charge polarized core domain may comprise a peptide having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a peptide having negatively charged amino acid residues at the carboxy terminus. In such an embodiment, the second charge polarized core domain may comprise a peptide having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a peptide having positively charged amino acid residues at the carboxy terminus. In another exemplary embodiment, the first charge polarized core domain may comprise a peptide having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a peptide having positively charged amino acid residues at the carboxy terminus. In such an embodiment, the second charge polarized core domain may comprise peptides having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a peptide having negatively charged amino acid residues at the carboxy terminus.
In various embodiments, each of the first and/or second charge polarized core domains further comprise a linker (e.g., a stabilizing domain) which adjoins the peptides having positively or negatively charged amino acids. In some embodiments, the linker (e.g., a stabilizing domain) is optionally selected from a flexible amino acid sequence, IgG hinge region, or antibody sequence. In an embodiment, the linker (e.g., a stabilizing domain) comprises the hinge-CH2-CH3 Fc domain derived from IgG1, optionally human IgG1. In another embodiment, the linker (e.g., a stabilizing domain) comprises the hinge-CH2-CH3 Fc domain derived from IgG4, optionally human IgG4.
In some embodiments, the first and/or second protein is selected from a cytokine, a growth factor, and/or a hormone. In some embodiments, the first and/or second protein is selected from a receptor for a cytokine, a growth factor, and/or a hormone.
In embodiments, in a heterodimeric protein, the first protein is selected from Table 1 and/or the second protein is selected from Table 1.
In embodiments, the first and/or second protein is an interleukin. In embodiments, the first and/or second protein is IL-35 comprising the IL12α and IL27β subunits.
In embodiments, the first and/or second protein is selected from a receptor for a cytokine, a growth factor, and/or a hormone. In embodiments, the first and/or second protein is a receptor for an interleukin.
In embodiments, the first and/or second protein is IL6 receptor comprising the IL6Rα and gp130 subunits.
In embodiments, the first and/or second protein is IL21 receptor comprising the IL21r and IL2rg subunits.
In embodiments, the first and/or second protein is IL21 receptor comprising the IFNgR and IFNGR2 subunits.
In embodiments, the protein on the amino- or carboxy-terminus is natively heterodimeric, and wherein the protein on the opposite terminus is not natively heterodimeric.
Also in various aspects, the present heterodimeric protein is used in a method for treating autoimmune diseases comprising administering an effective amount of a pharmaceutical composition comprising the heterodimeric protein to a patient in need thereof. In further aspects, the present heterodimeric protein is used in a method for treating infections, including without limitation, viral infections or other intracellular pathogens. In still further aspects, the present heterodimeric protein is used in a method for treating cancers.
Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment as disclosed herein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides illustrations protein engineering embodiments of the invention, showing an exemplary heterodimeric protein of the invention comprising the IL6 receptor (which includes the IL6Rα and gp130 subunits) and the IL-35 cytokine (which includes the IL12α and IL27β subunits). The heterodimeric protein is preferentially formed through electrostatic interactions between the charge polarized core domains.
FIG. 2 provides Western blot analysis of a production run of gp130-Fc-IL12a and IL6RA-Fc-IL27β from a transient transfection culture. The secreted protein was captured using affinity chromatography and eluted from the column to obtain a protein which contains a domain recognized by a human Fc specific antibody. Because the protein was run under denaturing conditions, individual bands are visualized for the alpha and beta strands.
FIG. 3 provides Western blot analysis of the purified gp130-Fc-IL12α and IL6RA-Fc-IL27β heterodimeric protein. The SDS-PAGE gels (left two gels) indicated the presence of a single band at approximately 300 kDa under non-denaturing conditions (left lane beside the molecular weight ladder in each blot). This band could be separated to the constituent alpha and beta strands following incubation with beta-mercaptoethanol (middle lane in each gel), which ran at an apparent molecular weight which was higher than predicted, and potentially indicative of post-translational modifications including glycosylation. This was confirmed in the right-most lane in each gel, which indicated that the molecular weight of the alpha and beta strands decreased to the predicted molecular weight following removal of N- and O-linked glycosylations. For the gel on the right, the native PAGE gel was used to further investigate the proportion of the purified protein which existed in the alpha/beta heterodimer form as compared to the alpha/alpha or beta/beta homodimer form. The gel indicated an enrichment of the alpha/beta heterodimer to approximately 60% of the total protein in the preparation as compared to approximately 30% of the alpha/alpha homodimer and 10% of the beta/beta homodimer.
FIG. 4 depicts quantification of captured heterodimeric IL-6R-Fc-IL-35 protein using spectrophotometry.
FIG. 5 provides a size-exclusion chromatography (SEC) chromatogram of the IL-6R-Fc-IL-35 construct following dual transfection of the gp130-alpha-IL12A and IL6RA-beta-IL27B constructs in CHO cells followed by purification of the secreted protein using protein A.
FIG. 6 provides a schematic of an ELISA assay that was developed to demonstrate that the IL-6R-Fc-IL-35 protein was capable of binding to immobilized human IL-6. Only the intended species (shown in the upper middle diagram) was expected to bind IL-6 in this assay, which could be specifically detected with an antibody against the IL-27a (EBI3) domain of the heterodimer.
FIG. 7 provides another schematic of an ELISA assay that was developed to demonstrate that the IL-6R-Fc-IL-35 protein was capable of binding to immobilized human IL-6. The bound protein was detected using the IL-6RA domain.
FIG. 8 provides a schematic of an ELISA assay that was developed which specifically captured an exemplary heterodimeric protein of the invention using an anti-human gp130 antibody and detected the bound protein with an anti-human IL-27a (EBI3) antibody.
FIG. 9 provides a schematic of an ELISA assay that was developed which specifically captured an exemplary heterodimeric protein of the invention using an anti-human gp130 antibody and detected the bound protein using the IL-6RA domain.
FIG. 10 provides a schematic of an ELISA assay that was developed which specifically captured an exemplary heterodimeric protein of the invention using the IL-6RA domain and detected the bound protein with an anti-human IL-27a (EBI3) antibody.
FIG. 11 provides a schematic of an ELISA assay that was developed which specifically captured an exemplary heterodimeric protein of the invention using the IL-6RA domain and detected the bound protein using the IL-6RA domain.
FIG. 12 provides a schematic of an ELISA assay that was developed which specifically captured an exemplary heterodimeric protein of the invention using the IL-12a p35 and detected the bound protein with an anti-human IL-27a (EBI3) antibody.
FIG. 13 provides a schematic of an ELISA assay that was developed which specifically captured an exemplary heterodimeric protein of the invention using the IL-12a p35 domain and detected the bound protein with the IL-6RA domain.
FIG. 14 provides a schematic of an ELISA assay that was developed which specifically captured an exemplary heterodimeric protein of the invention using an anti-human IL-27a (EBI3) antibody and detected the bound protein with the anti-human IL-27a (EBI3) antibody.
FIG. 15 provides a schematic of an ELISA assay that was developed which specifically captured an exemplary heterodimeric protein of the invention using an anti-human IL-27a (EBI3) antibody and detected the bound protein with the IL-6RA domain.
FIG. 16A and FIG. 16B provide size-exclusion chromatography (SEC) chromatogram of the IL-6R-Fc-IL-35 heterodimeric protein IL-6R-Fc-IL-35. In FIG. 16A, the absorbance wavelength was 210 nm and in FIG. 16B, the absorbance wavelength was 280 nm.
FIG. 17 is a graph showing the results of an IL-6 SINK Assay using the IL-6R-Fc-IL-35 heterodimeric protein.
FIG. 18 includes graph showing the ability of the IL-6R-Fc-IL-35 heterodimeric protein (identified as HdA '00) to induce at least IL-35. The condition “Halycon” refers to treatments with a control chimeric protein.
FIG. 19A shows a schematic of the IL-21R-Fc-IL-35 heterodimeric protein comprising a IL-21r-Fc(alpha)-IL12a chain and a IL2rg-Fc(beta)-IL27B chain. FIG. 19B shows an SDS-PAGE gel indicating the presence of two single bands at approximately 84.4 kDa and 78.1 kDa under reduced deglycosylated conditions (right-most lane).
FIG. 20 provides a size-exclusion chromatography (SEC) chromatogram of the IL-21R-Fc-IL-35 IFNγR-Fc-IL-35 heterodimeric protein following dual transfection of the IL-21r-Fc(alpha)-IL12a and IL2rg-Fc(beta)-IL27B constructs in CHO cells followed by purification of the secreted protein using protein A.
FIG. 21 are western blot analyses of the IFNγR-Fc-IL-35 heterodimeric protein comprising the IFNgR-Alpha-IL12a chain and the IFNGR2-Beta-IL27B chain probed with an antibody indicated below each blot. The proteins were run under non-denaturing conditions (left lane beside the molecular weight ladder in each blot), denaturing conditions with beta-mercaptoethanol treatment (middle lane in each gel), and both denaturing and deglycosylation treatments.
FIG. 22 provides a size-exclusion chromatography (SEC) chromatogram of the IFNγR-Fc-IL-35 heterodimeric protein following dual transfection of the IFNgR-Alpha-IL12a and IFNGR2-Beta-IL27B chain constructs in CHO cells followed by purification of the secreted protein using protein A.



DETAILED DESCRIPTION
The present invention is directed to a protein engineering platform for synthesizing and manufacturing heterodimeric proteins. The method of the invention allows for the efficient production of heterodimeric proteins for use in modulating immune signals for the treatment of various diseases, including, without limitation, autoimmune diseases.
Charge Polarized Core Domains
In one aspect, the present invention relates to heterodimeric proteins. In various embodiments, the heterodimeric protein of the invention comprises two polypeptide chains. The first polypeptide chain comprises a first subunit of a first protein at the amino terminus linked by a first charge polarized core domain to a first subunit of a second protein at the carboxy terminus. The second polypeptide chain comprises a second subunit of the first protein at the amino terminus linked by a second charge polarized core domain to a second subunit of the second protein at the carboxy terminus. In various embodiments, the first polypeptide chain and the second polypeptide chain forms a heterodimer through electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second polarized core domains.
In various embodiments, each of the first and second charge polarized core domains comprises peptides having positively or negatively charged amino acid residues at the amino and carboxy terminus of the core domain. In an exemplary embodiment, the first charge polarized core domain may comprise a peptide having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a peptide having negatively charged amino acid residues at the carboxy terminus. The second charge polarized core domain may comprise a peptide having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a peptide having positively charged amino acid residues at the carboxy terminus.
In another exemplary embodiment, the first charge polarized core domain may comprise a peptide having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a peptide having positively charged amino acid residues at the carboxy terminus. The second charge polarized core domain may comprise peptides having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a peptide having negatively charged amino acid residues at the carboxy terminus.
In various embodiments, formation of heterodimeric proteins is driven by electrostatic interactions between the positively charged and negatively charged amino acid residues located at the amino and carboxy termini of the first and second charge polarized core domains. Further, formation of homodimeric proteins is prevented by the repulsion between the positively charged amino acid residues or negatively charged amino acid residues located at the amino and carboxy termini of the first and second charge polarized core domains.
In various embodiments, the peptide comprising positively and/or negatively charged amino acid residues at the amino or carboxy terminus of the charge polarized core domains is about 2 to about 50 amino acids long. For example, the peptide comprising positively and/or negatively charged amino acid residues at either terminus of the charge polarized core domain may be about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long.
In various embodiments, the peptide comprising positively charged amino acid residues may include one or more of amino acids selected from His, Lys, and Arg. In various embodiments, the peptide comprising negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu.
In various embodiments, each of the first and/or second charge polarized core domains may comprise a peptide comprising an amino acid sequence as provided in the Table below or an amino acid sequence having at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto.











SEQ ID NO.
Sequence
















1
YnXnYnXnYn (where X is a positively charged amino acid such as arginine, histidine or lysine



and Y is a spacer amino acid such as serine or glycine)


 


2
YnZnYnZnYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic



acid and Y is a spacer amino acid such as serine or glycine)


 


3
YYnXXnYYnXXnYYn (where X is a positively charged amino acid such as arginine, histidine



or lysine and Y is a spacer amino acid such as serine or glycine)


 


4
YYnZZnYYnZZnYYn (where Z is a negatively charged amino acid such as aspartic acid or



glutamic acid and Y is a spacer amino acid such as serine or glycine)


 


5
YnXnCYnXnYn (where X is a positively charged amino acid such as arginine, histidine or lysine



and Y is a spacer amino acid such as serine or glycine)


 


6
YnZnCYnZnYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic



acid and Y is a spacer amino acid such as serine or glycine)


 


7
GSGSRKGGKRGS


 


8
GSGSRKCGKRGS


 


9
GSGSDEGGEDGS


 


10
GSGSDECGEDGS









For example, in an embodiment, each of the first and second charge polarized core domains may comprise a peptide comprising the sequence YYnXXnYYnXXnYYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine; SEQ ID NO: 3). Exemplary peptide sequences include, but are not limited to, RKGGKR (SEQ ID NO: 11) or GSGSRKGGKRGS (SEQ ID NO: 12).
In another exemplary embodiment, each of the first and second charge polarized core domains may comprise a peptide comprising the sequence YYnZZnYYnZZnYYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine). Exemplary peptide sequences include, but are not limited to, DEGGED (SEQ ID NO: 13) or GSGSDEGGEDGS (SEQ ID NO: 14).
In embodiments, a charge polarized core domain (negative-positive, also referred herein as an “alpha core domain”) is provided below:







(SEQ ID NO: 16)


GSGSRKGGKRGSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPE


 


VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV


 


LHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEM


 


TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS


 


RLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKDEGGEDGSGS






In embodiments, a heterodimeric protein comprises a variant alpha core domain. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 16.
In embodiments, a charge polarized core domain (positive-negative, also referred herein as a “beta core domain”) is provided below:







(SEQ ID NO: 17)


GSGSDEGGEDGSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPE


 


VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV


 


LHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEM


 


TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS


 


RLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKRKGGKRGSGS






In embodiments, a heterodimeric protein comprises a variant beta core domain. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 17.
In various embodiments, the peptide comprising the charged amino acid residues may further comprise one or more cysteine residues to facilitate disulfide bonding between the electrostatically charged core domains as an additional method to stabilize the heterodimer.
In various embodiments, each of the first and second charge polarized core domains comprises a linker sequence which may optionally function as a stabilizing domain. In various embodiments, the linker may be derived from naturally-occurring multi-domain proteins or are empirical linkers as described, for example, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contents of which are hereby incorporated by reference. In some embodiments, the linker may be designed using linker designing databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369 and Crasto et. al., (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference.
In some embodiments, the linker (e.g., a stabilizing domain) is a synthetic linker such as PEG.
In other embodiments, the linker (e.g., a stabilizing domain) is a polypeptide. In some embodiments, the linker (e.g., a stabilizing domain) is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long. For example, the linker (e.g., a stabilizing domain) may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long.
In various embodiments, the linker (e.g., a stabilizing domain) is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycines and serines).
In various embodiments, the linker (e.g., a stabilizing domain) is a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of IgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks a glycine residue, is relatively short, and contains a rigid poly-proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the IgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In IgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of IgG4 is shorter than that of IgG1 and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge regions reportedly decreases in the order IgG3>IgG1>IgG4>IgG2. In other embodiments, the linker may be derived from human IgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding.
According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. Id. The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. In various embodiments, the present linker (e.g., a stabilizing domain) comprises, one, or two, or three of the upper hinge region, the core region, and the lower hinge region of any antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin. In various embodiments, the linker (e.g., a stabilizing domain) of the present invention comprises one or more glycosylation sites.
In various embodiments, the linker (e.g., a stabilizing domain) comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human IgG4 antibody. In various embodiments, the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human IgG1 antibody. In some embodiments, the Fc domain exhibits increased affinity for and enhanced binding to the neonatal Fc receptor (FcRn). In some embodiments, the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the present heterodimeric proteins.
In some embodiments, the Fc domain contains one or more amino acid substitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311, 428, 433 or 434 (in accordance with Kabat numbering), or equivalents thereof. In an embodiment, the amino acid substitution at amino acid residue 250 is a substitution with glutamine. In an embodiment, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan or threonine. In an embodiment, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In an embodiment, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In an embodiment, the amino acid substitution at amino acid residue 308 is a substitution with threonine. In an embodiment, the amino acid substitution at amino acid residue 309 is a substitution with proline. In an embodiment, the amino acid substitution at amino acid residue 311 is a substitution with serine. In an embodiment, the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine. In an embodiment, the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In an embodiment, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In an embodiment, the amino acid substitution at amino acid residue 389 is a substitution with proline, serine or asparagine. In an embodiment, the amino acid substitution at amino acid residue 428 is a substitution with leucine. In an embodiment, the amino acid substitution at amino acid residue 433 is a substitution with arginine, serine, isoleucine, proline, or glutamine. In an embodiment, the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine, or tyrosine.
In some embodiments, the Fc domain (e.g., comprising an IgG constant region) comprises one or more mutations such as substitutions at amino acid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabat numbering). In an embodiment, the IgG constant region includes a triple M252Y/S254T/T256E mutation or YTE mutation. In another embodiment, the IgG constant region includes a triple H433K/N434F/Y436H mutation or KFH mutation. In a further embodiment, the IgG constant region includes an YTE and KFH mutation in combination.
In some embodiments, the modified humanized antibodies of the invention comprise an IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435. Illustrative mutations include T250Q, M428, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A. In an embodiment, the IgG constant region comprises a M428L/N434S mutation or LS mutation. In another embodiment, the IgG constant region comprises a T250Q/M428L mutation or QL mutation. In another embodiment, the IgG constant region comprises an N434A mutation. In another embodiment, the IgG constant region comprises a T307A/E380A/N434A mutation or MA mutation. In another embodiment, the IgG constant region comprises an I253A/H310A/H435A mutation or IHH mutation. In another embodiment, the IgG constant region comprises a H433K/N434F mutation. In another embodiment, the IgG constant region comprises a M252Y/S254T/T256E and a H433K/N434F mutation in combination.
In various embodiments, mutations are introduced to increase stability and/or half-life of the Fc domain. An illustrative Fc stabilizing mutant is S228P. Additional illustrative Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311S and the present linkers (e.g., stabilizing domains) may comprise 1, or 2, or 3, or 4, or 5 of these mutants.
In embodiments, a core domain, which lacks charge polarization, has the following sequence:







(SEQ ID NO: 15)


SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQE


 


DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEY


 


KCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLV


 


KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE


 


GNVFSCSVLHEALHNHYTQKSLSLSLGKIEGRMD






Additional exemplary mutations in the IgG constant region are described, for example, in Robbie, et al., Antimicrobial Agents and Chemotherapy (2013), 57(12):6147-6153, Dall'Acqua et al., JBC (2006), 281(33):23514-24, Dall'Acqua et al., Journal of Immunology (2002), 169:5171-80, Ko et al. Nature (2014) 514:642-645, Grevys et al. Journal of Immunology. (2015), 194(11):5497-508, and U.S. Pat. No. 7,083,784, the entire contents of which are hereby incorporated by reference.
In various embodiments, the linker may be flexible, including without limitation highly flexible. In various embodiments, the linker may be rigid, including without limitation a rigid alpha helix.
In various embodiments, the linker may be functional. For example, without limitation, the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, and/or improve the bioactivity of the present heterodimeric protein. In another example, the linker may function to target the heterodimeric protein to a particular cell type or location.
In embodiments, a core domain comprises one or more “Knobs-into-holes” amino acid changes. A “Knobs-into-holes” amino acid change is a rational design strategy previously used in antibody engineering for the heterodimerization of their heavy chains. See, e.g., Ridgway, J. B. et al. “Knobs-into-holes” engineering of antibody CH3 domains for heavy chain heterodimerization,” Protein Eng. 9(7):617-2 (1996) and Carter, “Bispecific human IgG by design.” Immunol. Methods, 248(1-2):7-15 (2001), the contents of each of which is incorporated herein by reference in its entirety. Here, amino acid changes are engineered in order to create a “knob” in the CH3 domain of an “alpha” heavy chain and a “hole” in the CH3 of the “beta” heavy chain; alternately, Here, amino acid changes are engineered in order to create a “knob” in the CH3 domain of an “beta” heavy chain and a “hole” in the CH3 of the “alpha” heavy chain. In one example, the “knob” is represented by a tyrosine (Y) that belongs to the “very large” IMGT volume class of amino acids, whereas the “hole” is represented by a threonine (T) that belongs to the “small” IMGT volume class. Characterizations of the IMGT classes of amino acids is described at Pommié, C. et al., “IMGT standardized criteria for statistical analysis of immunoglobulin V-REGION amino acid properties.” J. Mol. Recognit., 17, 17-32 (2004), the contents of which is incorporated herein by reference in its entirety. In the interface between two CH3 domains on separate heavy chains, the threonine (T) T22 in the beta heavy chain is within hydrogen-bonding distance of tyrosine (Y) Y86 in the alpha heavy chain. The Y86 is the principal interdomain contact of T22 and these amino acids are involved in a hydrogen bond. However, Y86 also makes numerous van der Waals contacts with Y86 and with Lysine (K) K88 on its opposite heavy chain.
Below are illustrative hinge-CH2-CH3 comprising “knobs-into-holes” amino acid changes and useful in the present invention. The below illustrative sequences are based on IgG1 and further comprise additional effector and complement silencing substitutions: L234A and L235A (LALA) and optionally, P329G; and half-life extension mutations: M252Y, S254T, T256E.
An illustrative human IGHG1 Knob In Hole “alpha core domain” (T22Y) is shown below:







(SEQ ID NO: 24)


EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVD


 


VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN


 


GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL


 


YCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS


 


RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK






An illustrative human IGHG1 Knob In Hole “beta core domain” (Y86T)







(SEQ ID NO: 25)


EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVD


 


VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN


 


GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL


 


TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKS


 


RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK






Any core domain useful in the present invention may comprise one or more “knob in holes” mutation.
Protein Subunits
In various embodiments, the heterodimeric protein of the invention comprises two polypeptide chains. In various embodiments, each polypeptide chain comprises a subunit of a first protein linked by a charge polarized core domain to a subunit of a second protein. Upon electrostatic interactions between the charge polarized core domains, the subunits are heterodimerized to form a functional dimeric first protein and a functional dimeric second protein. In some embodiments, the polypeptide chains form a functional two-sided heterodimeric protein linked via the charge polarized core domains, which optionally include a linker (e.g., a stabilizing domain) such as an Fc region.
In various embodiments, the first and second proteins may be any multimeric protein having two or more subunits. In some embodiments, the first protein and second protein are selected from cytokines, growth factors, and/or hormones. Illustrative examples of such cytokines, growth factors, and hormones include, but are not limited to, lymphokines, monokines, traditional polypeptide hormones, including, but not limited to, colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as, without limitation, IL-18, IL-27, and IL-35; interleukin receptors such as, without limitation, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-15R, IL-17R, IL-18R, IL-20R, IL-21R, IL-22R, IL-23R, IL-27R, IL-35R; and other polypeptide factors including, without limitation, EGFR, integrins, neuropilins, and somatostatin receptors. As used herein, cytokines, growth factors, and hormones include proteins obtained from natural sources or produced from recombinant bacterial, eukaryotic or mammalian cell culture systems and biologically active equivalents of the native sequence cytokines.
In some embodiments, the first and/or second protein is an immune-modulating agent, e.g., one or more of an interleukin and interferon.
In some embodiments, the first and/or second protein is an interleukin, including for example IL-18, IL-27, and IL-35, or a fragment, variant, analogue, or family-member thereof. Interleukins are a group of multi-functional cytokines synthesized by lymphocytes, monocytes, and macrophages. Known functions include stimulating proliferation of immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferons: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20.
In some embodiments, the first and/or second protein is a hormone such as somatostatin.
In various embodiments, the first and/or second protein is a receptor for a cytokine, growth factor, and/or hormone. In some embodiments, the first and/or second protein is a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, TGF-beta Receptors, a receptor in the immunoglobulin (Ig) superfamily, and/or a receptor in the tyrosine kinase superfamily.
In some embodiments, the first and/or second protein is a Type I cytokine receptor. Type I cytokine receptors are known in the art and include, but are not limited to receptors for IL2 (beta-subunit), IL3, IL4, IL5, IL6, IL7, IL9, 11_11, IL12, GM-CSF, G-CSF, LIF, CNTF, and also the receptors for Thrombopoietin (TPO), Prolactin, and Growth hormone. Illustrative type I cytokine receptors include, but are not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptors.
In some embodiments, the first and/or second protein is a Type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterologous subunits, and are receptors mainly for interferons. This family of receptors includes, but is not limited to, receptors for interferon-α, interferon-β and interferon-γ, IL10, IL22, and tissue factor. Illustrative type II cytokine receptors include, but are not limited to, IFN-α receptor (e.g., IFNAR1 and IFNAR2), IFN-β receptor, IFN-γ receptor (e.g., IFNGR1 and IFNGR2), and type II IL receptors.
In some embodiments, the first and/or second protein is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors with seven transmembrane structure and coupled to G-protein for signal transduction. Chemokine receptors include, but are not limited to, CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptor (XCR1). Exemplary chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.
In some embodiments, the first and/or second protein is a TGF-beta receptor. TGF-beta receptors are single pass serine/threonine kinase receptors. TGF-beta receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR3.
In some embodiments, the first and/or second protein is an Ig superfamily receptor. Receptors in the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Receptors in the Ig superfamily include, but are not limited to, interleukin-1 receptors, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.
In some embodiments, the first and/or second protein is a B7 superfamily member. Members of the B7 superfamily share structural homology with one another. Members of this family include, but are not limited to, CD28, CD80, CD86, ICOS, ICOSL, B7-H3, B7-H4, PD-1, PD-L1, PD-L2, etc.
In some embodiments, the first and/or second protein is a tyrosine kinase superfamily receptor. Receptors in the tyrosine kinase superfamily are well known in the art. There are about 58 known receptor tyrosine kinases (RTKs), grouped into 20 subfamilies. Receptors in the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various isoforms such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.
In an exemplary embodiment, the first and/or second protein is an IFN-α/β receptor (IFNAR) comprising IFNAR1 and/or IFNAR2 subunits.
In an exemplary embodiment, the first and/or second protein is an interferon-gamma receptor (IFNGR) comprising IFNGR1 (also known as IFNgR) and IFNGR2 subunits.
In an exemplary embodiment, the first and/or second protein is a VEGF receptor including VEGFR-1, VEGFR-2, and VEGFR-3.
In any heterodimeric protein disclosed herein, the amino- or carboxy-terminus is natively heterodimeric, and wherein the protein on the opposite terminus is not natively heterodimeric.
In an exemplary embodiment, the first and/or second protein is a receptor for IL-1 such as IL-1R1 and/or IL-1RAcP.
In an exemplary embodiment, the first and/or second protein is a receptor for IL-2 such as IL-2Rα or IL-2Rβ or IL-2Rγ.
In an exemplary embodiment, the first and/or second protein is an IL-3 receptor, which is a heterodimer with a unique alpha chain paired with the common beta (beta c or CD131) subunit.
In an exemplary embodiment, the first and/or second protein is a receptor for IL-4 such as a type 1 or type 2 IL-4 receptor.
In an exemplary embodiment, the first and/or second protein is a receptor for IL-6, which is a cell-surface type I cytokine receptor complex including the ligand-binding IL-6R chain (CD126 or IL-6Rα) and the signal-transducing component gp130.
In an exemplary embodiment, the first and/or second protein is a receptor for IL-10, such as IL-10 receptor-1 and IL-10 receptor-2.
In an exemplary embodiment, the first and/or second protein is a receptor for IL-11, such as IL-11Rα or IL-11Rβ or gp130.
In an exemplary embodiment, the first and/or second protein is a receptor for IL-12, such as IL-12Rβ1 and/or IL-12Rβ2.
In an exemplary embodiment, the first and/or second protein is a receptor for IL-13, such as the IL-4 receptor (IL-4Rα) or IL-13Rα1.
In an exemplary embodiment, the first and/or second protein is IL-18. In another exemplary embodiment, the first and/or second protein is a receptor for IL-18, such as IL-18Rα and/or IL-18Rβ.
In an exemplary embodiment, the first and/or second protein is a receptor for IL-21, which is a cell-surface type I cytokine receptor complex including the ligand-binding IL-21R chain comprising IL-21r and IL-2rg.
In an exemplary embodiment, the first and/or second protein is a receptor for IL-33, such as the ST-2 receptor or IL-1RAcP.
In an exemplary embodiment, the first and/or second protein is IL-35 (e.g., comprising the IL12α and IL27β subunits). In another exemplary embodiment, the first and/or second protein is a receptor for IL-35, such as an IL-35 receptor comprising IL6Rα and gp130 subunits.
In an exemplary embodiment, the first and/or second protein is a receptor for EGP such as EGFR (ErbB1), ErbB2, ErbB3 and ErbB4.
In an exemplary embodiment, the first and/or second protein is a receptor for insulin or an insulin analog such as the insulin receptor and/or IGF1 or IGF2 receptor.
In an exemplary embodiment, the first and/or second protein is a receptor for EPO such as the EPO receptor (EPOR) receptor and/or the ephrin receptor (EphR)
In various embodiments, the first and second proteins may comprise a domain of a soluble (e.g., non-membrane associated) protein. In various embodiments, the first and second proteins may comprise a fragment of the soluble protein which is involved in signaling (e.g., a portion of the soluble protein which interacts with a receptor).
In various embodiments, the first and second proteins may comprise the extracellular domain of a transmembrane protein. In various embodiments, one of the extracellular domains transduces an immune inhibitory signal and one of the extracellular domains transduces an immune stimulatory signal.
In some embodiments, an extracellular domain refers to a portion of a transmembrane protein which is capable of interacting with the extracellular environment. In various embodiments, an extracellular domain refers to a portion of a transmembrane protein which is sufficient to bind to a ligand or receptor and effective transmit a signal to a cell. In various embodiments, an extracellular domain is the entire amino acid sequence of a transmembrane protein which is external of a cell or the cell membrane. In various embodiments, an extracellular domain is the that portion of an amino acid sequence of a transmembrane protein which is external of a cell or the cell membrane and is needed for signal transduction and/or ligand binding as may be assayed using methods know in the art (e.g., in vitro ligand binding and/or cellular activation assays).
In some embodiments, an immune inhibitory signal refers to a signal that diminishes or eliminates an immune response. For example, in the context of oncology, such signals may diminish or eliminate antitumor immunity. Under normal physiological conditions, inhibitory signal are useful in the maintenance of self-tolerance (e.g., prevention of autoimmunity) and also to protect tissues from damage when the immune system is responding to pathogenic infection. For instance, without limitation, immune inhibitory signal may be identified by detecting an increase in cellular proliferation, cytokine production, cell killing activity or phagocytic activity when such an inhibitory signal is blocked.
In some embodiments, an immune stimulatory signal refers to a signal that enhances an immune response. For example, in the context of oncology, such signals may enhance antitumor immunity. For instance, without limitation, immune stimulatory signal may be identified by directly stimulating proliferation, cytokine production, killing activity or phagocytic activity of leukocytes. Specific examples include direct stimulation of cytokine receptors such as IL-2R, IL-7R, IL-15R, IL-17R or IL-21R using fusion proteins encoding the ligands for such receptors (IL-2, IL-7, IL-15, IL-17 or IL-21, respectively). Stimulation from any one of these receptors may directly stimulate the proliferation and cytokine production of individual T cell subsets.
In some embodiments, the extracellular domain may be used to produce a soluble protein to competitively inhibit signaling by that receptor's ligand. For instance, without limitation, competitive inhibition of PD-L1 or PD-L2 could be achieved using PD-1, or competitive inhibition of PVR could be achieved using TIGIT. In some embodiments, the extracellular domain may be used to provide artificial signaling.
In some embodiments, the present heterodimeric proteins deliver or mask an immune inhibitory signal. In some embodiments, the present heterodimeric proteins deliver or mask an immune stimulatory signal.
In various embodiments, the present heterodimeric proteins comprise two independent binding domains, each from one subunit of a heterodimeric human protein. Exemplary proteins that may be formed as part of the heterodimeric protein of the invention are provided in Table 1. In various embodiments, the present heterodimeric proteins have one of the exemplary proteins provided in Table 1. In various embodiments, the present heterodimeric proteins have two of the exemplary proteins provided in Table 1.







TABLE 1







Illustrative proteins which may be incorporated into the present compositions and methods include the following (as used herein,


“Entry” refers to the protein entry in the Uniprot database and “Entry name” refers to the protein entry in the Uniprot database):










Entry
Entry name
Protein names
Gene names





P00533
EGFR_HUMAN
Epidermal growth factor receptor (EC 2.7.10.1) (Proto-oncogene c-ErbB-1)
EGFR




(Receptor tyrosine-protein kinase erbB-1)
ERBB





ERBB1





HER1


P49768
PSN1_HUMAN
Presenilin-1 (PS-1) (EC 3.4.23.-) (Protein S182) [Cleaved into: Presenilin-1 NTF
PSEN1




subunit; Presenilin-1 CTF subunit; Presenilin-1 CTF12 (PS1-CTF12)]
AD3





PS1





PSNL1


P13569
CFTR_HUMAN
Cystic fibrosis transmembrane conductance regulator (CFTR) (ATP-binding
CFTR




cassette sub-family C member 7) (Channel conductance-controlling ATPase) (EC
ABCC7




3.6.3.49) (cAMP-dependent chloride channel)



P04626
ERBB2_HUMAN
Receptor tyrosine-protein kinase erbB-2 (EC 2.7.10.1) (Metastatic lymph node
ERBB2




gene 19 protein) (MLN 19) (Proto-oncogene Neu) (Proto-oncogene c-ErbB-2)
HER2




(Tyrosine kinase-type cell surface receptor HER2) (p185erbB2) (CD antigen
MLN19




CD340)
NEU





NGL


P08581
MET_HUMAN
Hepatocyte growth factor receptor (HGF receptor) (EC 2.7.10.1) (HGF/SF
MET




receptor) (Proto-oncogene c-Met) (Scatter factor receptor) (SF receptor)





(Tyrosine-protein kinase Met)



P17861
XBP1_HUMAN
X-box-binding protein 1 (XBP-1) (Tax-responsive element-binding protein 5)
XBP1




(TREB-5) [Cleaved into: X-box-binding protein 1, cytoplasmic form; X-box-binding
TREB5




protein 1, luminal form]
XBP2


P05106
ITB3_HUMAN
Integrin beta-3 (Platelet membrane glycoprotein IIIa) (GPIIIa) (CD antigen CD61)
ITGB3





GP3A


P05556
ITB1_HUMAN
Integrin beta-1 (Fibronectin receptor subunit beta) (Glycoprotein IIa) (GPIIA)
ITGB1




(VLA-4 subunit beta) (CD antigen CD29)
FNRB





MDF2





MSK12


P46531
NOTC1_HUMAN
Neurogenic locus notch homolog protein 1 (Notch 1) (hN1) (Translocation-
NOTCH1




associated notch protein TAN-1) [Cleaved into: Notch 1 extracellular truncation
TAN1




(NEXT); Notch 1 intracellular domain (NICD)]



P16671
CD36_HUMAN
Platelet glycoprotein 4 (Fatty acid translocase) (FAT) (Glycoprotein IIIb) (GPIIIB)
CD36




(Leukocyte differentiation antigen CD36) (PAS IV) (PAS-4) (Platelet collagen
GP3B




receptor) (Platelet glycoprotein IV) (GPIV) (Thrombospondin receptor) (CD
GP4




antigen CD36)



Q15303
ERBB4_HUMAN
Receptor tyrosine-protein kinase erbB-4 (EC 2.7.10.1) (Proto-oncogene-like
ERBB4




protein c-ErbB-4) (Tyrosine kinase-type cell surface receptor HER4) (p180erbB4)
HER4




[Cleaved into: ERBB4 intracellular domain (4ICD) (E4ICD) (s80HER4)]



Q02763
TIE2_HUMAN
Angiopoietin-1 receptor (EC 2.7.10.1) (Endothelial tyrosine kinase) (Tunica
TEK




interna endothelial cell kinase) (Tyrosine kinase with Ig and EGF homology
TIE2




domains-2) (Tyrosine-protein kinase receptor TEK) (Tyrosine-protein kinase
VMCM




receptor TIE-2) (hTIE2) (p140 TEK) (CD antigen CD202b)
VMCM1


O00206
TLR4_HUMAN
Toll-like receptor 4 (hToll) (CD antigen CD284)
TLR4


Q14118
DAG1_HUMAN
Dystroglycan (Dystrophin-associated glycoprotein 1) [Cleaved into: Alpha-
DAG1




dystroglycan (Alpha-DG); Beta-dystroglycan (Beta-DG)]



P16473
TSHR_HUMAN
Thyrotropin receptor (Thyroid-stimulating hormone receptor) (TSH-R)
TSHR





LGR3


Q99527
GPER1_HUMAN
G-protein coupled estrogen receptor 1 (Chemoattractant receptor-like 2) (Flow-
GPER1




induced endothelial G-protein coupled receptor 1) (FEG-1) (G protein-coupled
CEPR




estrogen receptor 1) (G-protein coupled receptor 30) (GPCR-Br) (IL8-related
CMKRL2




receptor DRY12) (Lymphocyte-derived G-protein coupled receptor) (LYGPR)
DRY12




(Membrane estrogen receptor) (mER)
GPER





GPR30


P17948
VGFR1_HUMAN
Vascular endothelial growth factor receptor 1 (VEGFR-1) (EC 2.7.10.1) (Fms-like
FLT1




tyrosine kinase 1) (FLT-1) (Tyrosine-protein kinase FRT) (Tyrosine-protein kinase
FLT




receptor FLT) (FLT) (Vascular permeability factor receptor)
FRT





VEGFR1


Q9UM47
NOTC3_HUMAN
Neurogenic locus notch homolog protein 3 (Notch 3) [Cleaved into: Notch 3
NOTCH3




extracellular truncation; Notch 3 intracellular domain]



P21860
ERBB3_HUMAN
Receptor tyrosine-protein kinase erbB-3 (EC 2.7.10.1) (Proto-oncogene-like
ERBB3




protein c-ErbB-3) (Tyrosine kinase-type cell surface receptor HER3)
HER3


P01920
DQB1_HUMAN
HLA class II histocompatibility antigen, DQ beta 1 chain (MHC class II antigen
HLA-DQB1




DQB1)
HLA-DQB


P06756
ITAV_HUMAN
Integrin alpha-V (Vitronectin receptor) (Vitronectin receptor subunit alpha) (CD
ITGAV




antigen CD51) [Cleaved into: Integrin alpha-V heavy chain; Integrin alpha-V light
MSK8




chain]
VNRA





VTNR


Q9H251
CAD23_HUMAN
Cadherin-23 (Otocadherin)
CDH23





KIAA1774





KIAA1812





UNQ1894/





PRO4340


Q07954
LRP1_HUMAN
Prolow-density lipoprotein receptor-related protein 1 (LRP-1) (Alpha-2-
LRP1




macroglobulin receptor) (A2MR) (Apolipoprotein E receptor) (APOER) (CD
A2MR




antigen CD91) [Cleaved into: Low-density lipoprotein receptor-related protein 1
APR




85 kDa subunit (LRP-85); Low-density lipoprotein receptor-related protein 1 515





kDa subunit (LRP-515); Low-density lipoprotein receptor-related protein 1





intracellular domain (LRPICD)]



O60603
TLR2_HUMAN
Toll-like receptor 2 (Toll/interleukin-1 receptor-like protein 4) (CD antigen CD282)
TLR2





TIL4


P04839
CY24B_HUMAN
Cytochrome b-245 heavy chain (EC 1.-.-.-) (CGD91-phox) (Cytochrome b(558)
CYBB




subunit beta) (Cytochrome b558 subunit beta) (Heme-binding membrane
NOX2




glycoprotein gp91phox) (NADPH oxidase 2) (Neutrophil cytochrome b 91 kDa





polypeptide) (Superoxide-generating NADPH oxidase heavy chain subunit)





(gp91-1) (gp91-phox) (p22 phagocyte B-cytochrome)



P04233
HG2A_HUMAN
HLA class II histocompatibility antigen gamma chain (HLA-DR antigens-
CD74




associated invariant chain) (Ia antigen-associated invariant chain) (Ii) (p33) (CD
DHLAG




antigen CD74)



P13746
1A11_HUMAN
HLA class I histocompatibility antigen, A-11 alpha chain (MHC class I antigen
HLA-A




A*11)
HLAA


P18462
1A25_HUMAN
HLA class I histocompatibility antigen, A-25 alpha chain (HLA class I
HLA-A




histocompatibility antigen, A-10 alpha chain) (MHC class I antigen A*25)
HLAA


P16188
1A30_HUMAN
HLA class I histocompatibility antigen, A-30 alpha chain (MHC class I antigen
HLA-A




A*30)
HLAA


P30457
1A66_HUMAN
HLA class I histocompatibility antigen, A-66 alpha chain (Aw-66) (HLA class I
HLA-A




histocompatibility antigen, A-10 alpha chain) (MHC class I antigen A*66)
HLAA


P10316
1A69_HUMAN
HLA class I histocompatibility antigen, A-69 alpha chain (Aw-69) (HLA class I
HLA-A




histocompatibility antigen, A-28 alpha chain) (MHC class I antigen A*69)
HLAA


Q09160
1A80_HUMAN
HLA class I histocompatibility antigen, A-80 alpha chain (Aw-80) (HLA class I
HLA-A




histocompatibility antigen, A-1 alpha chain) (MHC class I antigen A*80)
HLAA


P30460
1B08_HUMAN
HLA class I histocompatibility antigen, B-8 alpha chain (MHC class I antigen B*8)
HLA-B





HLAB


P30685
1B35_HUMAN
HLA class I histocompatibility antigen, B-35 alpha chain (MHC class I antigen
HLA-B




B*35)
HLAB


P08195
4F2_HUMAN
4F2 cell-surface antigen heavy chain (4F2hc) (4F2 heavy chain antigen)
SLC3A2




(Lymphocyte activation antigen 4F2 large subunit) (Solute carrier family 3
MDU1




member 2) (CD antigen CD98)



P28222
5HT1B_HUMAN
5-hydroxytryptamine receptor 1B (5-HT-1B) (5-HT1B) (S12) (Serotonin 1D beta
HTR1B




receptor) (5-HT-1D-beta) (Serotonin receptor 1B)
HTR1DB


P28221
5HT1D_HUMAN
5-hydroxytryptamine receptor 1D (5-HT-1D) (5-HT1D) (Serotonin 1D alpha
HTR1D




receptor) (5-HT-1D-alpha) (Serotonin receptor 1D)
HTR1DA





HTRL


O14678
ABCD4_HUMAN
ATP-binding cassette sub-family D member 4 (PMP70-related protein) (P70R)
ABCD4




(Peroxisomal membrane protein 1-like) (PXMP1-L) (Peroxisomal membrane
PXMP1L




protein 69) (PMP69)



P30462
1B14_HUMAN
HLA class I histocompatibility antigen, B-14 alpha chain (MHC class I antigen
HLA-B




B*14)
HLAB


P30466
1B18_HUMAN
HLA class I histocompatibility antigen, B-18 alpha chain (MHC class I antigen
HLA-B




B*18)
HLAB


Q04826
1B40_HUMAN
HLA class I histocompatibility antigen, B-40 alpha chain (Bw-60) (MHC class I
HLA-B




antigen B*40)
HLAB


P30480
1B42_HUMAN
HLA class I histocompatibility antigen, B-42 alpha chain (MHC class I antigen
HLA-B




B*42)
HLAB


P30484
1B46_HUMAN
HLA class I histocompatibility antigen, B-46 alpha chain (Bw-46) (MHC class I
HLA-B




antigen B*46)
HLAB


P30487
1B49_HUMAN
HLA class I histocompatibility antigen, B-49 alpha chain (HLA class I
HLA-B




histocompatibility antigen, B-21 alpha chain) (MHC class I antigen B*49)
HLAB


P18464
1B51_HUMAN
HLA class I histocompatibility antigen, B-51 alpha chain (MHC class I antigen
HLA-B




B*51)
HLAB


P30495
1B56_HUMAN
HLA class I histocompatibility antigen, B-56 alpha chain (Bw-22) (Bw-56) (MHC
HLA-B




class I antigen B*56)
HLAB


P30498
1B78_HUMAN
HLA class I histocompatibility antigen, B-78 alpha chain (MHC class I antigen
HLA-B




B*78)
HLAB


Q29718
1B82_HUMAN
HLA class I histocompatibility antigen, B-82 alpha chain (MHC class I antigen
HLA-B




B*82)
HLAB


P30501
1C02_HUMAN
HLA class I histocompatibility antigen, Cw-2 alpha chain (MHC class I antigen
HLA-C




Cw*2)
HLAC


P30504
1C04_HUMAN
HLA class I histocompatibility antigen, Cw-4 alpha chain (MHC class I antigen
HLA-C




Cw*4)
HLAC


P01912
2B13_HUMAN
HLA class II histocompatibility antigen, DRB1-3 chain (Clone P2-beta-3) (MHC
HLA-DRB1




class II antigen DRB1*3)



P13760
2B14_HUMAN
HLA class II histocompatibility antigen, DRB1-4 beta chain (MHC class II antigen
HLA-DRB1




DRB1*4) (DR-4) (DR4)



P01911
2B1F_HUMAN
HLA class II histocompatibility antigen, DRB1-15 beta chain (DW2.2/DR2.2)
HLA-DRB1




(MHC class II antigen DRB1*15)
HLA-DRB2


P01892
1A02_HUMAN
HLA class I histocompatibility antigen, A-2 alpha chain (MHC class I antigen A*2)
HLA-A





HLAA


P03989
1B27_HUMAN
HLA class I histocompatibility antigen, B-27 alpha chain (MHC class I antigen
HLA-B




B*27)
HLAB


P13761
2B17_HUMAN
HLA class II histocompatibility antigen, DRB1-7 beta chain (MHC class II antigen
HLA-DRB1




DRB1*7) (DR-7) (DR7)



Q9TQE0
2B19_HUMAN
HLA class II histocompatibility antigen, DRB1-9 beta chain (MHC class II antigen
HLA-DRB1




DRB1*9) (DR-9) (DR9)



P20039
2B1B_HUMAN
HLA class II histocompatibility antigen, DRB1-11 beta chain (DR-5) (DR5)
HLA-DRB1




(DRw11) (MHC class II antigen DRB1*11)



O75027
ABCB7_HUMAN
ATP-binding cassette sub-family B member 7, mitochondrial (ATP-binding
ABCB7




cassette transporter 7) (ABC transporter 7 protein)
ABC7


P16189
1A31_HUMAN
HLA class I histocompatibility antigen, A-31 alpha chain (MHC class I antigen
HLA-A




A*31)
HLAA


P30456
1A43_HUMAN
HLA class I histocompatibility antigen, A-43 alpha chain (Aw-43) (MHC class I
HLA-A




antigen A*43)
HLAA


P30459
1A74_HUMAN
HLA class I histocompatibility antigen, A-74 alpha chain (Aw-19) (Aw-74) (MHC
HLA-A




class I antigen A*74)
HLAA


P18463
1B37_HUMAN
HLA class I histocompatibility antigen, B-37 alpha chain (MHC class I antigen
HLA-B




B*37)
HLAB


P30483
1B45_HUMAN
HLA class I histocompatibility antigen, B-45 alpha chain (Bw-45) (MHC class I
HLA-B




antigen B*45)
HLAB


P30485
1B47_HUMAN
HLA class I histocompatibility antigen, B-47 alpha chain (Bw-47) (MHC class I
HLA-B




antigen B*47)
HLAB


P30486
1B48_HUMAN
HLA class I histocompatibility antigen, B-48 alpha chain (Bw-48) (MHC class I
HLA-B




antigen B*48)
HLAB


P30491
1B53_HUMAN
HLA class I histocompatibility antigen, B-53 alpha chain (Bw-53) (MHC class I
HLA-B




antigen B*53)
HLAB


Q29940
1B59_HUMAN
HLA class I histocompatibility antigen, B-59 alpha chain (MHC class I antigen
HLA-B




B*59)
HLAB


Q31612
1B73_HUMAN
HLA class I histocompatibility antigen, B-73 alpha chain (MHC class I antigen
HLA-B




B*73)
HLAB


Q31610
1B81_HUMAN
HLA class I histocompatibility antigen, B-81 alpha chain (B′DT) (MHC class I
HLA-B




antigen B*81)
HLAB


P30499
1C01_HUMAN
HLA class I histocompatibility antigen, Cw-1 alpha chain (MHC class I antigen
HLA-C




Cw*1)
HLAC


Q9TNN7
1C05_HUMAN
HLA class I histocompatibility antigen, Cw-5 alpha chain (MHC class I antigen
HLA-C




Cw*5)
HLAC


P30508
1C12_HUMAN
HLA class I histocompatibility antigen, Cw-12 alpha chain (MHC class I antigen
HLA-C




Cw*12)
HLAC


Q29865
1C18_HUMAN
HLA class I histocompatibility antigen, Cw-18 alpha chain (MHC class I antigen
HLA-C




Cw*18)
HLAC


Q5Y7A7
2B1D_HUMAN
HLA class II histocompatibility antigen, DRB1-13 beta chain (MHC class II antigen
HLA-DRB1




DRB1*13) (DR-13) (DR13)



Q29974
2B1G_HUMAN
HLA class II histocompatibility antigen, DRB1-16 beta chain (MHC class II antigen
HLA-DRB1




DRB1*16) (DR-16) (DR16)



Q9NS82
AAA1_HUMAN
Asc-type amino acid transporter 1 (Asc-1) (Solute carrier family 7 member 10)
SLC7A10





ASC1


P30447
1A23_HUMAN
HLA class I histocompatibility antigen, A-23 alpha chain (HLA class I
HLA-A




histocompatibility antigen, A-9 alpha chain) (MHC class I antigen A*23)
HLAA


P30450
1A26_HUMAN
HLA class I histocompatibility antigen, A-26 alpha chain (MHC class I antigen
HLA-A




A*26)
HLAA


P10314
1A32_HUMAN
HLA class I histocompatibility antigen, A-32 alpha chain (MHC class I antigen
HLA-A




A*32)
HLAA


P30455
1A36_HUMAN
HLA class I histocompatibility antigen, A-36 alpha chain (Aw-36) (MHC class I
HLA-A




antigen A*36)
HLAA


P30461
1B13_HUMAN
HLA class I histocompatibility antigen, B-13 alpha chain (MHC class I antigen
HLA-B




B*13)
HLAB


P30464
1B15_HUMAN
HLA class I histocompatibility antigen, B-15 alpha chain (MHC class I antigen
HLA-B




B*15)
HLAB


P30475
1B39_HUMAN
HLA class I histocompatibility antigen, B-39 alpha chain (MHC class I antigen
HLA-B




B*39)
HLAB


P30479
1B41_HUMAN
HLA class I histocompatibility antigen, B-41 alpha chain (Bw-41) (MHC class I
HLA-B




antigen B*41)
HLAB


P30481
1B44_HUMAN
HLA class I histocompatibility antigen, B-44 alpha chain (Bw-44) (MHC class I
HLA-B




antigen B*44)
HLAB


P30488
1B50_HUMAN
HLA class I histocompatibility antigen, B-50 alpha chain (Bw-50) (HLA class I
HLA-B




histocompatibility antigen, B-21 alpha chain) (MHC class I antigen B*50)
HLAB


P30490
1B52_HUMAN
HLA class I histocompatibility antigen, B-52 alpha chain (Bw-52) (HLA class I
HLA-B




histocompatibility antigen, B-5 alpha chain) (MHC class I antigen B*52)
HLAB


P30493
1B55_HUMAN
HLA class I histocompatibility antigen, B-55 alpha chain (Bw-55) (HLA class I
HLA-B




histocompatibility antigen, B-12 alpha chain) (MHC class I antigen B*55)
HLAB





CDABP0067


P18465
1B57_HUMAN
HLA class I histocompatibility antigen, B-57 alpha chain (Bw-57) (MHC class I
HLA-B




antigen B*57)
HLAB


Q29836
1B67_HUMAN
HLA class I histocompatibility antigen, B-67 alpha chain (MHC class I antigen
HLA-B




B*67)
HLAB


P10319
1B58_HUMAN
HLA class I histocompatibility antigen, B-58 alpha chain (Bw-58) (MHC class I
HLA-B




antigen B*58)
HLAB


P04222
1C03_HUMAN
HLA class I histocompatibility antigen, Cw-3 alpha chain (MHC class I antigen
HLA-C




Cw*3)
HLAC


Q29963
1C06_HUMAN
HLA class I histocompatibility antigen, Cw-6 alpha chain (MHC class I antigen
HLA-C




Cw*6)
HLAC


P30505
1C08_HUMAN
HLA class I histocompatibility antigen, Cw-8 alpha chain (MHC class I antigen
HLA-C




Cw*8)
HLAC


Q07000
1C15_HUMAN
HLA class I histocompatibility antigen, Cw-15 alpha chain (MHC class I antigen
HLA-C




Cw*15)
HLAC


Q95604
1C17_HUMAN
HLA class I histocompatibility antigen, Cw-17 alpha chain (MHC class I antigen
HLA-C




Cw*17)
D6S204





HLA-JY3





HLAC


Q95365
1B38_HUMAN
HLA class I histocompatibility antigen, B-38 alpha chain (Bw-4) (MHC class I
HLA-B




antigen B*38)
HLAB


P30492
1B54_HUMAN
HLA class I histocompatibility antigen, B-54 alpha chain (Bw-22) (Bw-54) (MHC
HLA-B




class I antigen B*54)
HLAB


P10321
1C07_HUMAN
HLA class I histocompatibility antigen, Cw-7 alpha chain (MHC class I antigen
HLA-C




Cw*7)
HLAC


P30510
1C14_HUMAN
HLA class I histocompatibility antigen, Cw-14 alpha chain (MHC class I antigen
HLA-C




Cw*14)
HLAC


Q29960
1C16_HUMAN
HLA class I histocompatibility antigen, Cw-16 alpha chain (MHC class I antigen
HLA-C




Cw*16)
HLAC


P04229
2B11_HUMAN
HLA class II histocompatibility antigen, DRB1-1 beta chain (MHC class II antigen
HLA-DRB1




DRB1*1) (DR-1) (DR1)



Q30167
2B1A_HUMAN
HLA class II histocompatibility antigen, DRB1-10 beta chain (DRw10) (MHC class
HLA-DRB1




II antigen DRB1*10)



Q9GIY3
2B1E_HUMAN
HLA class II histocompatibility antigen, DRB1-14 beta chain (MHC class II antigen
HLA-DRB1




DRB1*14) (DR-14) (DR14)



P08908
5HT1A_HUMAN
5-hydroxytryptamine receptor 1A (5-HT-1A) (5-HT1A) (G-21) (Serotonin receptor
HTR1A




1A)
ADRB2RL1





ADRBRL1


Q96PE1
AGRA2_HUMAN
Adhesion G protein-coupled receptor A2 (G-protein coupled receptor 124) (Tumor
ADGRA2




endothelial marker 5)
GPR124





KIAA1531





TEM5


P05534
1A24_HUMAN
HLA class I histocompatibility antigen, A-24 alpha chain (Aw-24) (HLA class I
HLA-A




histocompatibility antigen, A-9 alpha chain) (MHC class I antigen A*24)
HLAA


P30512
1A29_HUMAN
HLA class I histocompatibility antigen, A-29 alpha chain (Aw-19) (MHC class I
HLA-A




antigen A*29)
HLAA


P16190
1A33_HUMAN
HLA class I histocompatibility antigen, A-33 alpha chain (Aw-19) (Aw-33) (MHC
HLA-A




class I antigen A*33)
HLAA


P30453
1A34_HUMAN
HLA class I histocompatibility antigen, A-34 alpha chain (Aw-34) (HLA class I
HLA-A




histocompatibility antigen, A-10 alpha chain) (MHC class I antigen A*34)
HLAA


P01891
1A68_HUMAN
HLA class I histocompatibility antigen, A-68 alpha chain (Aw-68) (HLA class I
HLA-A




histocompatibility antigen, A-28 alpha chain) (MHC class I antigen A*68)
HLAA


P01889
1B07_HUMAN
HLA class I histocompatibility antigen, B-7 alpha chain (MHC class I antigen B*7)
HLA-B





HLAB


Q95IE3
2B1C_HUMAN
HLA class II histocompatibility antigen, DRB1-12 beta chain (MHC class II antigen
HLA-DRB1




DRB1*12) (DR-12) (DR12)



Q99965
ADAM2_HUMAN
Disintegrin and metalloproteinase domain-containing protein 2 (ADAM 2)
ADAM2




(Cancer/testis antigen 15) (CT15) (Fertilin subunit beta) (PH-30) (PH30) (PH30-
FTNB




beta)



Q30134
2B18_HUMAN
HLA class II histocompatibility antigen, DRB1-8 beta chain (MHC class II antigen
HLA-DRB1




DRB1*8) (DR-8) (DR8) (DRw8)



Q9UHX3
AGRE2_HUMAN
Adhesion G protein-coupled receptor E2 (EGF-like module receptor 2) (EGF-like
ADGRE2




module-containing mucin-like hormone receptor-like 2) (CD antigen CD312)
EMR2


O60242
AGRB3_HUMAN
Adhesion G protein-coupled receptor B3 (Brain-specific angiogenesis inhibitor 3)
ADGRB3





BAI3





KIAA0550


Q96F25
ALG14_HUMAN
UDP-N-acetylglucosamine transferase subunit ALG14 homolog
ALG14


Q9Y653
AGRG1_HUMAN
Adhesion G-protein coupled receptor G1 (G-protein coupled receptor 56) (Protein
ADGRG1




TM7XN1) [Cleaved into: ADGRG1 N-terminal fragment (ADGRG1 NT) (GPR56
GPR56




N-terminal fragment) (GPR56 NT) (GPR56(N)) (GPR56 extracellular subunit)
TM7LN4




(GPR56 subunit alpha); ADGRG1 C-terminal fragment (ADGRG1 CT) (GPR56 C-
TM7XN1




terminal fragment) (GPR56 CT) (GPR56(C)) (GPR56 seven-transmembrane
UNQ540/




subunit) (GPR56 7TM) (GPR56 subunit beta)]
PRO1083


Q8IZF2
AGRF5_HUMAN
Adhesion G protein-coupled receptor F5 (G-protein coupled receptor 116)
ADGRF5





GPR116





KIAA0758


O60241
AGRB2_HUMAN
Adhesion G protein-coupled receptor B2 (Brain-specific angiogenesis inhibitor 2)
ADGRB2





BAI2


Q86SQ3
AGRE4_HUMAN
Putative adhesion G protein-coupled receptor E4P (EGF-like module receptor 4)
ADGRE4P




(EGF-like module-containing mucin-like hormone receptor-like 4) (G-protein
EMR4




coupled receptor 127) (G-protein coupled receptor PGR16)
EMR4P





GPR127





PGR16


Q8IZP9
AGRG2_HUMAN
Adhesion G-protein coupled receptor G2 (G-protein coupled receptor 64) (Human
ADGRG2




epididymis-specific protein 6) (He6)
GPR64





HE6





TM7LN2


Q9HBW9
AGRL4_HUMAN
Adhesion G protein-coupled receptor L4 (EGF, latrophilin and seven
ADGRL4




transmembrane domain-containing protein 1) (EGF-TM7-latrophilin-related
ELTD1




protein) (ETL protein)
ETL





UNQ202/





PRO228


Q16853
AOC3_HUMAN
Membrane primary amine oxidase (EC 1.4.3.21) (Copper amine oxidase) (HPAO)
AOC3




(Semicarbazide-sensitive amine oxidase) (SSAO) (Vascular adhesion protein 1)
VAP1




(VAP-1)



Q9BY15
AGRE3_HUMAN
Adhesion G protein-coupled receptor E3 (EGF-like module receptor 3) (EGF-like
ADGRE3




module-containing mucin-like hormone receptor-like 3)
EMR3





UNQ683/





PRO1562


O94910
AGRL1_HUMAN
Adhesion G protein-coupled receptor L1 (Calcium-independent alpha-latrotoxin
ADGRL1




receptor 1) (CIRL-1) (Latrophilin-1) (Lectomedin-2)
KIAA0821





LEC2





LPHN1


O95490
AGRL2_HUMAN
Adhesion G protein-coupled receptor L2 (Calcium-independent alpha-latrotoxin
ADGRL2




receptor 2) (CIRL-2) (Latrophilin homolog 1) (Latrophilin-2) (Lectomedin-1)
KIAA0786





LEC1





LPHH1





LPHN2


Q86WK6
AMGO1_HUMAN
Amphoterin-induced protein 1 (AMIGO-1) (Alivin-2)
AMIGO1





ALI2





AMIGO





KIAA1163


P21397
AOFA_HUMAN
Amine oxidase [flavin-containing] A (EC 1.4.3.4) (Monoamine oxidase type A)
MAOA




(MAO-A)



P27338
AOFB_HUMAN
Amine oxidase [flavin-containing] B (EC 1.4.3.4) (Monoamine oxidase type B)
MAOB




(MAO-B)



Q99941
ATF6B_HUMAN
Cyclic AMP-dependent transcription factor ATF-6 beta (cAMP-dependent
ATF6B




transcription factor ATF-6 beta) (Activating transcription factor 6 beta) (ATF6-
CREBL1




beta) (Protein G13) (cAMP response element-binding protein-related protein)
G13




(Creb-rp) (cAMP-responsive element-binding protein-like 1) [Cleaved into:





Processed cyclic AMP-dependent transcription factor ATF-6 beta]



Q15041
AR6P1_HUMAN
ADP-ribosylation factor-like protein 6-interacting protein 1 (ARL-6-interacting
ARL6IP1




protein 1) (Aip-1) (Apoptotic regulator in the membrane of the endoplasmic
ARL6IP




reticulum)
ARMER





KIAA0069


P18850
ATF6A_HUMAN
Cyclic AMP-dependent transcription factor ATF-6 alpha (cAMP-dependent
ATF6




transcription factor ATF-6 alpha) (Activating transcription factor 6 alpha) (ATF6-





alpha) [Cleaved into: Processed cyclic AMP-dependent transcription factor ATF-





6 alpha]



Q9UHQ4
BAP29_HUMAN
B-cell receptor-associated protein 29 (BCR-associated protein 29) (Bap29)
BCAP29





BAP29


P82251
BAT1_HUMAN
b(0, +)-type amino acid transporter 1 (b(0, +)AT1) (Glycoprotein-associated amino
SLC7A9




acid transporter b0, +AT1) (Solute carrier family 7 member 9)
BAT1


P51572
BAP31_HUMAN
B-cell receptor-associated protein 31 (BCR-associated protein 31) (Bap31) (6C6-
BCAP31




AG tumor-associated antigen) (Protein CDM) (p28)
BAP31





DXS1357E


O60238
BNI3L_HUMAN
BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (Adenovirus
BNIP3L




E1B19K-binding protein B5) (BCL2/adenovirus E1B 19 kDa protein-interacting
BNIP3A




protein 3A) (NIP3-like protein X) (NIP3L)
BNIP3H





NIX


Q9UMX3
BOK_HUMAN
Bcl-2-related ovarian killer protein (hBOK) (Bcl-2-like protein 9) (Bcl2-L-9)
BOK





BCL2L9


P15291
B4GT1_HUMAN
Beta-1,4-galactosyltransferase 1 (Beta-1,4-GalTase 1) (Beta4Gal-T1) (b4Gal-T1)
B4GALT1




(EC 2.4.1.-) (UDP-Gal: beta-GlcNAc beta-1,4-galactosyltransferase 1) (UDP-
GGTB2




galactose: beta-N-acetylglucosamine 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





synthase); Beta-N-acetylglucosaminylglycopeptide beta-1,4-





galactosyltransferase (EC 2.4.1.38); Beta-N-acetylglucosaminyl-glycolipid beta-





1,4-galactosyltransferase (EC 2.4.1.-)]



Q12983
BNIP3_HUMAN
BCL2/adenovirus E1B 19 kDa protein-interacting protein 3
BNIP3





NIP3


Q16602
CALRL_HUMAN
Calcitonin gene-related peptide type 1 receptor (CGRP type 1 receptor)
CALCRL




(Calcitonin receptor-like receptor)
CGRPR


P10966
CD8B_HUMAN
T-cell surface glycoprotein CD8 beta chain (CD antigen CD8b)
CD8B





CD8B1


P09693
CD3G_HUMAN
T-cell surface glycoprotein CD3 gamma chain (T-cell receptor T3 gamma chain)
CD3G




(CD antigen CD3g)
T3G


P60033
CD81_HUMAN
CD81 antigen (26 kDa cell surface protein TAPA-1) (Target of the antiproliferative
CD81




antibody 1) (Tetraspanin-28) (Tspan-28) (CD antigen CD81)
TAPA1





TSPAN28


P01732
CD8A_HUMAN
T-cell surface glycoprotein CD8 alpha chain (T-lymphocyte differentiation antigen
CD8A




T8/Leu-2) (CD antigen CD8a)
MAL


P06126
CD1A_HUMAN
T-cell surface glycoprotein CD1a (T-cell surface antigen T6/Leu-6) (hTa1
CD1A




thymocyte antigen) (CD antigen CD1a)



P40259
CD79B_HUMAN
B-cell antigen receptor complex-associated protein beta chain (B-cell-specific
CD79B




glycoprotein B29) (Ig-beta) (Immunoglobulin-associated B29 protein) (CD antigen
B29




CD79b)
IGB


P11912
CD79A_HUMAN
B-cell antigen receptor complex-associated protein alpha chain (Ig-alpha) (MB-1
CD79A




membrane glycoprotein) (Membrane-bound immunoglobulin-associated protein)
IGA




(Surface IgM-associated protein) (CD antigen CD79a)
MB1


P15812
CD1E_HUMAN
T-cell surface glycoprotein CD1e, membrane-associated (hCD1e) (R2G1) (CD
CD1E




antigen CD1e) [Cleaved into: T-cell surface glycoprotein CD1e, soluble (sCD1e)]



P20963
CD3Z_HUMAN
T-cell surface glycoprotein CD3 zeta chain (T-cell receptor T3 zeta chain) (CD
CD247




antigen CD247)
CD3Z





T3Z





TCRZ


A6NJW9
CD8B2_HUMAN
Putative T-cell surface glycoprotein CD8 beta-2 chain (CD8b pseudogene)
CD8B2





CD8BP


P29017
CD1C_HUMAN
T-cell surface glycoprotein CD1c (CD antigen CD1c)
CD1C


P13688
CEAM1_HUMAN
Carcinoembryonic antigen-related cell adhesion molecule 1 (Biliary glycoprotein
CEACAM1




1) (BGP-1) (CD antigen CD66a)
BGP





BGP1


P29016
CD1B_HUMAN
T-cell surface glycoprotein CD1b (CD antigen CD1b)
CD1B


P15813
CD1D_HUMAN
Antigen-presenting glycoprotein CD1d (R3G1) (CD antigen CD1d)
CD1D


P20273
CD22_HUMAN
B-cell receptor CD22 (B-lymphocyte cell adhesion molecule) (BL-CAM) (Sialic
CD22




acid-binding Ig-like lectin 2) (Siglec-2) (T-cell surface antigen Leu-14) (CD antigen
SIGLEC2




CD22)



P04234
CD3D_HUMAN
T-cell surface glycoprotein CD3 delta chain (T-cell receptor T3 delta chain) (CD
CD3D




antigen CD3d)
T3D


P07766
CD3E_HUMAN
T-cell surface glycoprotein CD3 epsilon chain (T-cell surface antigen T3/Leu-4
CD3E




epsilon chain) (CD antigen CD3e)
T3E


P48960
CD97_HUMAN
CD97 antigen (Leukocyte antigen CD97) (CD antigen CD97) [Cleaved into: CD97
CD97




antigen subunit alpha; CD97 antigen subunit beta]



Q9UHP7
CLC2D_HUMAN
C-type lectin domain family 2 member D (Lectin-like NK cell receptor) (Lectin-like
CLEC2D




transcript 1) (LLT-1) (Osteoclast inhibitory lectin)
CLAX





LLT1





OCIL


P51790
CLCN3_HUMAN
H(+)/CI(−) exchange transporter 3 (Chloride channel protein 3) (CIC-3) (Chloride
CLCN3




transporter CIC-3)



Q68CJ9
CR3L3_HUMAN
Cyclic AMP-responsive element-binding protein 3-like protein 3 (cAMP-
CREB3L3




responsive element-binding protein 3-like protein 3) (Transcription factor CREB-
CREBH




H) [Cleaved into: Processed cyclic AMP-responsive element-binding protein 3-
HYST1481




like protein 3]



P34998
CRFR1_HUMAN
Corticotropin-releasing factor receptor 1 (CRF-R-1) (CRF-R1) (CRFR-1)
CRHR1




(Corticotropin-releasing hormone receptor 1) (CRH-R-1) (CRH-R1)
CRFR





CRFR1





CRHR


Q9HC73
CRLF2_HUMAN
Cytokine receptor-like factor 2 (Cytokine receptor-like 2) (IL-XR) (Thymic stromal
CRLF2




lymphopoietin protein receptor) (TSLP receptor)
CRL2





ILXR





TSLPR


P09603
CSF1_HUMAN
Macrophage colony-stimulating factor 1 (CSF-1) (M-CSF) (MCSF) (Lanimostim)
CSF1




[Cleaved into: Processed macrophage colony-stimulating factor 1]



P15509
CSF2R_HUMAN
Granulocyte-macrophage colony-stimulating factor receptor subunit alpha (GM-
CSF2RA




CSF-R-alpha) (GMCSFR-alpha) (GMR-alpha) (CDw116) (CD antigen CD116)
CSF2R





CSF2RY


P06340
DOA_HUMAN
HLA class II histocompatibility antigen, DO alpha chain (MHC DN-alpha) (MHC
HLA-DOA




DZ alpha) (MHC class II antigen DOA)
HLA-DNA





HLA-DZA


P01906
DQA2_HUMAN
HLA class II histocompatibility antigen, DQ alpha 2 chain (DX alpha chain) (HLA
HLA-DQA2




class II histocompatibility antigen, DQ(6) alpha chain) (HLA-DQA1) (MHC class II
HLA-DXA




DQA2)



P13762
DRB4_HUMAN
HLA class II histocompatibility antigen, DR beta 4 chain (MHC class II antigen
HLA-DRB4




DRB4)



P13765
DOB_HUMAN
HLA class II histocompatibility antigen, DO beta chain (MHC class II antigen DOB)
HLA-DOB


P04440
DPB1_HUMAN
HLA class II histocompatibility antigen, DP beta 1 chain (HLA class II
HLA-DPB1




histocompatibility antigen, DP(W4) beta chain) (MHC class II antigen DPB1)
HLA-DP1B


P01909
DQA1_HUMAN
HLA class II histocompatibility antigen, DQ alpha 1 chain (DC-1 alpha chain) (DC-
HLA-DQA1




alpha) (HLA-DCA) (MHC class II DQA1)



P28067
DMA_HUMAN
HLA class II histocompatibility antigen, DM alpha chain (MHC class II antigen
HLA-DMA




DMA) (Really interesting new gene 6 protein)
DMA





RING6


P79483
DRB3_HUMAN
HLA class II histocompatibility antigen, DR beta 3 chain (MHC class II antigen
HLA-DRB3




DRB3)



P28068
DMB_HUMAN
HLA class II histocompatibility antigen, DM beta chain (MHC class II antigen
HLA-DMB




DMB) (Really interesting new gene 7 protein)
DMB





RING7


P01903
DRA_HUMAN
HLA class II histocompatibility antigen, DR alpha chain (MHC class II antigen
HLA-DRA




DRA)
HLA-DRA1


P20036
DPA1_HUMAN
HLA class II histocompatibility antigen, DP alpha 1 chain (DP(W3)) (DP(W4))
HLA-DPA1




(HLA-SB alpha chain) (MHC class II DP3-alpha) (MHC class II DPA1)
HLA-DP1A





HLASB


P05538
DQB2_HUMAN
HLA class II histocompatibility antigen, DQ beta 2 chain (HLA class II
HLA-DQB2




histocompatibility antigen, DX beta chain) (MHC class II antigen DQB2)
HLA-DXB


P27487
DPP4_HUMAN
Dipeptidyl peptidase 4 (EC 3.4.14.5) (ADABP) (Adenosine deaminase
DPP4




complexing protein 2) (ADCP-2) (Dipeptidyl peptidase IV) (DPP IV) (T-cell
ADCP2




activation antigen CD26) (TP103) (CD antigen CD26) [Cleaved into: Dipeptidyl
CD26




peptidase 4 membrane form (Dipeptidyl peptidase IV membrane form); Dipeptidyl





peptidase 4 soluble form (Dipeptidyl peptidase IV soluble form)]



Q30154
DRB5_HUMAN
HLA class II histocompatibility antigen, DR beta 5 chain (DR beta-5) (DR2-beta-
HLA-DRB5




2) (Dw2) (MHC class II antigen DRB5)



P61565
ENK21_HUMAN
Endogenous retrovirus group K member 21 Env polyprotein (EnvK1 protein)
ERVK-21




(Envelope polyprotein) (HERV-K_12q14.1 provirus ancestral Env polyprotein)





[Cleaved into: Surface protein (SU); Transmembrane protein (TM)]



Q902F9
EN113_HUMAN
Endogenous retrovirus group K member 113 Env polyprotein (EnvK5 protein)
HERVK_113




(Envelope polyprotein) (HERV-K113 envelope protein) (HERV-K_19p13.11





provirus ancestral Env polyprotein) [Cleaved into: Surface protein (SU);





Transmembrane protein (TM)]



P60507
EFC1_HUMAN
Endogenous retrovirus group FC1 Env polyprotein (Envelope polyprotein)
ERVFC1




(Fc1env) (HERV-F(c)1_Xq21.33 provirus ancestral Env polyprotein) (HERV-





Fc1env) [Cleaved into: Surface protein (SU); Transmembrane protein (TM)]



P13224
GP1BB_HUMAN
Platelet glycoprotein Ib beta chain (GP-Ib beta) (GPIb-beta) (GPIbB) (Antigen
GP1BB




CD42b-beta) (CD antigen CD42c)



Q9HB15
KCNKC_HUMAN
Potassium channel subfamily K member 12 (Tandem pore domain halothane-
KCNK12




inhibited potassium channel 2) (THIK-2)



P23276
KELL_HUMAN
Kell blood group glycoprotein (EC 3.4.24.-) (CD antigen CD238)
KEL


Q13241
KLRD1_HUMAN
Natural killer cells antigen CD94 (KP43) (Killer cell lectin-like receptor subfamily
KLRD1




D member 1) (NK cell receptor) (CD antigen CD94)
CD94


O14649
KCNK3_HUMAN
Potassium channel subfamily K member 3 (Acid-sensitive potassium channel
KCNK3




protein TASK-1) (TWIK-related acid-sensitive K(+) channel 1) (Two pore
TASK




potassium channel KT3.1) (Two pore K(+) channel KT3.1)
TASK1


Q09470
KCNA1_HUMAN
Potassium voltage-gated channel subfamily A member 1 (Voltage-gated K(+)
KCNA1




channel HuKI) (Voltage-gated potassium channel HBK1) (Voltage-gated





potassium channel subunit Kv1.1)



O00180
KCNK1_HUMAN
Potassium channel subfamily K member 1 (Inward rectifying potassium channel
KCNK1




protein TWIK-1) (Potassium channel K2P1) (Potassium channel KCNO1)
HOHO1





KCNO1





TWIK1


Q9NPC2
KCNK9_HUMAN
Potassium channel subfamily K member 9 (Acid-sensitive potassium channel
KCNK9




protein TASK-3) (TWIK-related acid-sensitive K(+) channel 3) (Two pore
TASK3




potassium channel KT3.2) (Two pore K(+) channel KT3.2)



P54851
EMP2_HUMAN
Epithelial membrane protein 2 (EMP-2) (Protein XMP)
EMP2





XMP


O71037
ENK19_HUMAN
Endogenous retrovirus group K member 19 Env polyprotein (EnvK3 protein)
ERVK-19




(Envelope polyprotein) (HERV-K(C19) envelope protein) (HERV-K_19q11





provirus ancestral Env polyprotein) [Cleaved into: Surface protein (SU);





Transmembrane protein (TM)]



P61567
ENK7_HUMAN
Endogenous retrovirus group K member 7 Env polyprotein (Envelope polyprotein)
ERVK-7




(HERV-K(III) envelope protein) (HERV-K102 envelope protein) (HERV-K_1q22





provirus ancestral Env polyprotein) [Cleaved into: Surface protein (SU);





Transmembrane protein (TM)]



Q9UKH3
ENK9_HUMAN
Endogenous retrovirus group K member 9 Env polyprotein (EnvK4 protein)
ERVK-9




(Envelope polyprotein) (HERV-K(C6) envelope protein) (HERV-K109 envelope





protein) (HERV-K_6q14.1 provirus ancestral Env polyprotein) [Cleaved into:





Surface protein (SU); Transmembrane protein (TM)]



Q9NZ08
ERAP1_HUMAN
Endoplasmic reticulum aminopeptidase 1 (EC 3.4.11.-) (ARTS-1) (Adipocyte-
ERAP1




derived leucine aminopeptidase) (A-LAP) (Aminopeptidase PILS) (Puromycin-
APPILS




insensitive leucyl-specific aminopeptidase) (PILS-AP) (Type 1 tumor necrosis
ARTS1




factor receptor shedding aminopeptidase regulator)
KIAA0525





UNQ584/





PRO1154


Q9N2K0
ENH1_HUMAN
HERV-H_2q24.3 provirus ancestral Env polyprotein (Env protein HERV-H/p62)





(Env protein HERV-H19) (Env protein HERV-Hcl.3) (Envelope polyprotein)





(HERV-H/env62) [Cleaved into: Surface protein (SU); Transmembrane protein





(TM)]



Q9NX77
ENK13_HUMAN
Endogenous retrovirus group K member 13-1 Env polyprotein (Envelope
ERVK13-1




polyprotein) (HERV-K_16p13.3 provirus ancestral Env polyprotein) [Cleaved into:





Surface protein (SU); Transmembrane protein (TM)]



P61566
ENK24_HUMAN
Endogenous retrovirus group K member 24 Env polyprotein (Envelope
ERVK-24




polyprotein) (HERV-K101 envelope protein) (HERV-K_22q11.21 provirus





ancestral Env polyprotein) [Cleaved into: Surface protein (SU); Transmembrane





protein (TM)]



Q69384
ENK6_HUMAN
Endogenous retrovirus group K member 6 Env polyprotein (EnvK2 protein)
ERVK-6




(Envelope polyprotein) (HERV-K(C7) envelope protein) (HERV-K(HML-2.HOM)
ERVK6




envelope protein) (HERV-K108 envelope protein) (HERV-K_7p22.1 provirus





ancestral Env polyprotein) [Cleaved into: Surface protein (SU); Transmembrane





protein (TM)]



Q9N2J8
ENH3_HUMAN
HERV-H_2q24.1 provirus ancestral Env polyprotein (Env protein HERV-H/p59)





(Envelope polyprotein) (HERV-H/env59) [Cleaved into: Surface protein (SU);





Transmembrane protein (TM)]



O42043
ENK18_HUMAN
Endogenous retrovirus group K member 18 Env polyprotein (Envelope
ERVK-18




polyprotein) (HERV-K(C1a) envelope protein) (HERV-K110 envelope protein)





(HERV-K18 envelope protein) (HERV-K18 superantigen) (HERV-K_1q23.3





provirus ancestral Env polyprotein) (IDDMK1,2 22 envelope protein) (IDDMK1,2





22 superantigen) [Cleaved into: Surface protein (SU); Transmembrane protein





(TM)]



Q902F8
ENK8_HUMAN
Endogenous retrovirus group K member 8 Env polyprotein (EnvK6 protein)
ERVK-8




(Envelope polyprotein) (HERV-K115 envelope protein) (HERV-K_8p23.1 provirus





ancestral Env polyprotein) [Cleaved into: Surface protein (SU); Transmembrane





protein (TM)]



P29317
EPHA2_HUMAN
Ephrin type-A receptor 2 (EC 2.7.10.1) (Epithelial cell kinase) (Tyrosine-protein
EPHA2




kinase receptor ECK)
ECK


P61570
ENK25_HUMAN
Endogenous retrovirus group K member 25 Env polyprotein (Envelope
ERVK-25




polyprotein) (HERV-K_11q22.1 provirus ancestral Env polyprotein) [Cleaved into:





Surface protein (SU); Transmembrane protein (TM)]



P98073
ENTK_HUMAN
Enteropeptidase (EC 3.4.21.9) (Enterokinase) (Serine protease 7)
TMPRSS15




(Transmembrane protease serine 15) [Cleaved into: Enteropeptidase non-
ENTK




catalytic heavy chain; Enteropeptidase catalytic light chain]
PRSS7


Q6P179
ERAP2_HUMAN
Endoplasmic reticulum aminopeptidase 2 (EC 3.4.11.-) (Leukocyte-derived
ERAP2




arginine aminopeptidase) (L-RAP)
LRAP


P55899
FCGRN_HUMAN
IgG receptor FcRn large subunit p51 (FcRn) (IgG Fc fragment receptor
FCGRT




transporter alpha chain) (Neonatal Fc receptor)
FCRN


O75899
GABR2_HUMAN
Gamma-aminobutyric acid type B receptor subunit 2 (GABA-B receptor 2)
GABBR2




(GABA-B-R2) (GABA-BR2) (GABABR2) (Gb2) (G-protein coupled receptor 51)
GPR51




(HG20)
GPRC3B


Q14318
FKBP8_HUMAN
Peptidyl-prolyl cis-trans isomerase FKBP8 (PPIase FKBP8) (EC 5.2.1.8) (38 kDa
FKBP8




FK506-binding protein) (38 kDa FKBP) (FKBP-38) (hFKBP38) (FK506-binding
FKBP38




protein 8) (FKBP-8) (FKBPR38) (Rotamase)



Q9UBS5
GABR1_HUMAN
Gamma-aminobutyric acid type B receptor subunit 1 (GABA-B receptor 1)
GABBR1




(GABA-B-R1) (GABA-BR1) (GABABR1) (Gb1)
GPRC3A


Q9UG22
GIMA2_HUMAN
GTPase IMAP family member 2 (Immunity-associated protein 2) (hIMAP2)
GIMAP2





IMAP2


Q6P531
GGT6_HUMAN
Gamma-glutamyltransferase 6 (GGT 6) (EC 2.3.2.2) (Gamma-
GGT6




glutamyltranspeptidase 6) (Glutathione hydrolase 6) (EC 3.4.19.13) [Cleaved into:





Gamma-glutamyltransferase 6 heavy chain; Gamma-glutamyltransferase 6 light





chain]



Q9UJ14
GGT7_HUMAN
Gamma-glutamyltransferase 7 (GGT 7) (EC 2.3.2.2) (Gamma-
GGT7




glutamyltransferase-like 3) (Gamma-glutamyltransferase-like 5) (Gamma-
GGTL3




glutamyltranspeptidase 7) (Glutathione hydrolase 7) (EC 3.4.19.13) [Cleaved into:
GGTL5




Gamma-glutamyltransferase 7 heavy chain; Gamma-glutamyltransferase 7 light





chain]



P14770
GPIX_HUMAN
Platelet glycoprotein IX (GP-IX) (GPIX) (Glycoprotein 9) (CD antigen CD42a)
GP9


P19440
GGT1_HUMAN
Gamma-glutamyltranspeptidase 1 (GGT 1) (EC 2.3.2.2) (Gamma-
GGT1




glutamyltransferase 1) (Glutathione hydrolase 1) (EC 3.4.19.13) (Leukotriene-C4
GGT




hydrolase) (EC 3.4.19.14) (CD antigen CD224) [Cleaved into: Gamma-





glutamyltranspeptidase 1 heavy chain; Gamma-glutamyltranspeptidase 1 light





chain]



P36269
GGT5_HUMAN
Gamma-glutamyltransferase 5 (GGT 5) (EC 2.3.2.2) (Gamma-glutamyl
GGT5




transpeptidase-related enzyme) (GGT-rel) (Gamma-glutamyltransferase-like
GGTLA1




activity 1) (Gamma-glutamyltranspeptidase 5) (Glutathione hydrolase 5) (EC





3.4.19.13) (Leukotriene-C4 hydrolase) (EC 3.4.19.14) [Cleaved into: Gamma-





glutamyltransferase 5 heavy chain; Gamma-glutamyltransferase 5 light chain]



P07359
GP1BA_HUMAN
Platelet glycoprotein Ib alpha chain (GP-Ib alpha) (GPIb-alpha) (GPIbA)
GP1BA




(Glycoprotein Ibalpha) (Antigen CD42b-alpha) (CD antigen CD42b) [Cleaved into:





Glycocalicin]



P32249
GP183_HUMAN
G-protein coupled receptor 183 (Epstein-Barr virus-induced G-protein coupled
GPR183




receptor 2) (EBI2) (EBV-induced G-protein coupled receptor 2) (hEBI2)
EBI2


P17693
HLAG_HUMAN
HLA class I histocompatibility antigen, alpha chain G (HLA G antigen) (MHC class
HLA-G




I antigen G)
HLA-6.0





HLAG


P01893
HLAH_HUMAN
Putative HLA class I histocompatibility antigen, alpha chain H (HLA-12.4) (HLA-
HLA-H




AR) (MHC class I antigen H)
HLAH


P13747
HLAE_HUMAN
HLA class I histocompatibility antigen, alpha chain E (MHC class I antigen E)
HLA-E





HLA-6.2





HLAE


Q99665
I12R2_HUMAN
Interleukin-12 receptor subunit beta-2 (IL-12 receptor subunit beta-2) (IL-12R
IL12RB2




subunit beta-2) (IL-12R-beta-2) (IL-12RB2)



P30511
HLAF_HUMAN
HLA class I histocompatibility antigen, alpha chain F (CDA12) (HLA F antigen)
HLA-F




(Leukocyte antigen F) (MHC class I antigen F)
HLA-5.4





HLAF


P42701
I12R1_HUMAN
Interleukin-12 receptor subunit beta-1 (IL-12 receptor subunit beta-1) (IL-12R
IL12RB1




subunit beta-1) (IL-12R-beta-1) (IL-12RB1) (IL-12 receptor beta component) (CD
IL12R




antigen CD212)
IL12RB


Q96F46
I17RA_HUMAN
Interleukin-17 receptor A (IL-17 receptor A) (IL-17RA) (CDw217) (CD antigen
IL17RA




CD217)
IL17R


Q9NPH3
IL1AP_HUMAN
Interleukin-1 receptor accessory protein (IL-1 receptor accessory protein) (IL-
IL1RAP




1RAcP) (Interleukin-1 receptor 3) (IL-1R-3) (IL-1R3)
C3orf13





IL1R3


P32927
IL3RB_HUMAN
Cytokine receptor common subunit beta (CDw131) (GM-CSF/IL-3/IL-5 receptor
CSF2RB




common beta subunit) (CD antigen CD131)
IL3RB





IL5RB


Q08334
I10R2_HUMAN
Interleukin-10 receptor subunit beta (IL-10 receptor subunit beta) (IL-10R subunit
IL10RB




beta) (IL-10RB) (Cytokine receptor class-II member 4) (Cytokine receptor family
CRFB4




2 member 4) (CRF2-4) (Interleukin-10 receptor subunit 2) (IL-10R subunit 2) (IL-
D21S58




10R2) (CD antigen CDw210b)
D21S66


Q8NAC3
I17RC_HUMAN
Interleukin-17 receptor C (IL-17 receptor C) (IL-17RC) (Interleukin-17 receptor
IL17RC




homolog) (IL17Rhom) (Interleukin-17 receptor-like protein) (IL-17RL) (ZcytoR14)
UNQ6118/





PR020040/





PRO38901


Q8NFR9
I17RE_HUMAN
Interleukin-17 receptor E (IL-17 receptor E) (IL-17RE)
IL17RE





UNQ3056/





PRO9877


O95256
I18RA_HUMAN
Interleukin-18 receptor accessory protein (IL-18 receptor accessory protein) (IL-
IL18RAP




18RAcP) (Accessory protein-like) (AcPL) (CD218 antigen-like family member B)
IL1R7




(CDw218b) (IL-1R accessory protein-like) (IL-1RAcPL) (Interleukin-1 receptor 7)





(IL-1R-7) (IL-1R7) (Interleukin-18 receptor accessory protein-like) (Interleukin-18





receptor beta) (IL-18R-beta) (IL-18Rbeta) (CD antigen CD218b)



Q6UXL0
I20RB_HUMAN
Interleukin-20 receptor subunit beta (IL-20 receptor subunit beta) (IL-20R-beta)
IL20RB




(IL-20RB) (Fibronectin type III domain containing 6) (FNDC6) (IL-20R2)
DIRS1





UNQ557/P





RO1114


Q9HBE5
IL21R_HUMAN
Interleukin-21 receptor (IL-21 receptor) (IL-21R) (Novel interleukin receptor) (CD
IL21R




antigen CD360)
NILR





UNQ3121/





PRO10273


Q9UHF4
I20RA_HUMAN
Interleukin-20 receptor subunit alpha (IL-20 receptor subunit alpha) (IL-20R-
IL20RA




alpha) (IL-20RA) (Cytokine receptor class-II member 8) (Cytokine receptor family
UNQ681/




2 member 8) (CRF2-8) (IL-20R1) (ZcytoR7)
PRO1315


P14778
IL1R1_HUMAN
Interleukin-1 receptor type 1 (IL-1R-1) (IL-1RT-1) (IL-1RT1) (CD121 antigen-like
IL1R1




family member A) (Interleukin-1 receptor alpha) (IL-1R-alpha) (Interleukin-1
IL1R




receptor type I) (p80) (CD antigen CD121a) [Cleaved into: Interleukin-1 receptor
IL1RA




type 1, membrane form (mIL-1R1) (mIL-1RI); Interleukin-1 receptor type 1,
IL1RT1




soluble form (sIL-1RI) (sIL-1RI)]



Q8N6P7
I22R1_HUMAN
Interleukin-22 receptor subunit alpha-1 (IL-22 receptor subunit alpha-1) (IL-22R-
IL22RA1




alpha-1) (IL-22RA1) (Cytokine receptor class-II member 9) (Cytokine receptor
IL22R




family 2 member 9) (CRF2-9) (ZcytoR11)



Q13478
IL18R_HUMAN
Interleukin-18 receptor 1 (IL-18R-1) (IL-18R1) (CD218 antigen-like family member
IL18R1




A) (CDw218a) (IL1 receptor-related protein) (IL-1Rrp) (IL1R-rp) (CD antigen
IL1RRP




CD218a)



P26951
IL3RA_HUMAN
Interleukin-3 receptor subunit alpha (IL-3 receptor subunit alpha) (IL-3R subunit
IL3RA




alpha) (IL-3R-alpha) (IL-3RA) (CD antigen CD123)
IL3R


Q01344
IL5RA_HUMAN
Interleukin-5 receptor subunit alpha (IL-5 receptor subunit alpha) (IL-5R subunit
IL5RA




alpha) (IL-5R-alpha) (IL-5RA) (CDw125) (CD antigen CD125)
IL5R


Q8NI17
IL31R_HUMAN
Interleukin-31 receptor subunit alpha (IL-31 receptor subunit alpha) (IL-31R
IL31RA




subunit alpha) (IL-31R-alpha) (IL-31RA) (Cytokine receptor-like 3) (GLM-R)
CRL3




(hGLM-R) (Gp130-like monocyte receptor) (Gp130-like receptor) (ZcytoR17)
GPL





UNQ6368/





PRO21073/





PRO21384


Q9UKX5
ITA11_HUMAN
Integrin alpha-11
ITGA11





MSTP018


P17301
ITA2_HUMAN
Integrin alpha-2 (CD49 antigen-like family member B) (Collagen receptor)
ITGA2




(Platelet membrane glycoprotein Ia) (GPIa) (VLA-2 subunit alpha) (CD antigen
CD49B




CD49b)



Q13683
ITA7_HUMAN
Integrin alpha-7 [Cleaved into: Integrin alpha-7 heavy chain; Integrin alpha-7 light
ITGA7




chain; Integrin alpha-7 70 kDa form]
UNQ406/





PRO768


P53708
ITA8_HUMAN
Integrin alpha-8 [Cleaved into: Integrin alpha-8 heavy chain; Integrin alpha-8 light
ITGA8




chain]



P38570
ITAE_HUMAN
Integrin alpha-E (HML-1 antigen) (Integrin alpha-IEL) (Mucosal lymphocyte 1
ITGAE




antigen) (CD antigen CD103) [Cleaved into: Integrin alpha-E light chain; Integrin





alpha-E heavy chain]



P05107
ITB2_HUMAN
Integrin beta-2 (Cell surface adhesion glycoproteins LFA-1/CR3/p150, 95 subunit
ITGB2




beta) (Complement receptor C3 subunit beta) (CD antigen CD18)
CD18





MFI7


P18564
ITB6_HUMAN
Integrin beta-6
ITGB6


Q8IU57
INLR1_HUMAN
Interferon lambda receptor 1 (IFN-lambda receptor 1) (IFN-lambda-R1) (Cytokine
IFNLR1




receptor class-II member 12) (Cytokine receptor family 2 member 12) (CRF2-12)
IL28RA




(Interleukin-28 receptor subunit alpha) (IL-28 receptor subunit alpha) (IL-28R-
LICR2




alpha) (IL-28RA) (Likely interleukin or cytokine receptor 2) (LICR2)



Q5VWK5
IL23R_HUMAN
Interleukin-23 receptor (IL-23 receptor) (IL-23R)
IL23R


P17181
INAR1_HUMAN
Interferon alpha/beta receptor 1 (IFN-R-1) (IFN-alpha/beta receptor 1) (Cytokine
IFNAR1




receptor class-II member 1) (Cytokine receptor family 2 member 1) (CRF2-1)
IFNAR




(Type I interferon receptor 1)



P16871
IL7RA_HUMAN
Interleukin-7 receptor subunit alpha (IL-7 receptor subunit alpha) (IL-7R subunit
IL7R




alpha) (IL-7R-alpha) (IL-7RA) (CDw127) (CD antigen CD127)



Q96T52
IMP2L_HUMAN
Mitochondrial inner membrane protease subunit 2 (EC 3.4.21.-) (IMP2-like
IMMP2L




protein)



P13612
ITA4_HUMAN
Integrin alpha-4 (CD49 antigen-like family member D) (Integrin alpha-IV) (VLA-4
ITGA4




subunit alpha) (CD antigen CD49d)
CD49D


Q13349
ITAD_HUMAN
Integrin alpha-D (ADB2) (CD11 antigen-like family member D) (Leukointegrin
ITGAD




alpha D) (CD antigen CD11d)



P78508
KCJ10_HUMAN
ATP-sensitive inward rectifier potassium channel 10 (ATP-dependent inwardly
KCNJ10




rectifying potassium channel Kir4.1) (Inward rectifier K(+) channel Kir1.2)





(Potassium channel, inwardly rectifying subfamily J member 10)



P11215
ITAM_HUMAN
Integrin alpha-M (CD11 antigen-like family member B) (CR-3 alpha chain) (Cell
ITGAM




surface glycoprotein MAC-1 subunit alpha) (Leukocyte adhesion receptor MO1)
CD11B




(Neutrophil adherence receptor) (CD antigen CD11b)
CR3A


P16144
ITB4_HUMAN
Integrin beta-4 (GP150) (CD antigen CD104)
ITGB4


P26012
ITB8_HUMAN
Integrin beta-8
ITGB8


Q9NPI9
KCJ16_HUMAN
Inward rectifier potassium channel 16 (Inward rectifier K(+) channel Kir5.1)
KCNJ16




(Potassium channel, inwardly rectifying subfamily J member 16)



P38484
INGR2_HUMAN
Interferon gamma receptor 2 (IFN-gamma receptor 2) (IFN-gamma-R2)
IFNGR2




(Interferon gamma receptor accessory factor 1) (AF-1) (Interferon gamma
IFNGT1




receptor beta-chain) (IFN-gamma-R-beta) (Interferon gamma transducer 1)



P56199
ITA1_HUMAN
Integrin alpha-1 (CD49 antigen-like family member A) (Laminin and collagen
ITGA1




receptor) (VLA-1) (CD antigen CD49a)



P26006
ITA3_HUMAN
Integrin alpha-3 (CD49 antigen-like family member C) (FRP-2) (Galactoprotein
ITGA3




B3) (GAPB3) (VLA-3 subunit alpha) (CD antigen CD49c) [Cleaved into: Integrin
MSK18




alpha-3 heavy chain; Integrin alpha-3 light chain]



P20702
ITAX_HUMAN
Integrin alpha-X (CD11 antigen-like family member C) (Leu M5) (Leukocyte
ITGAX




adhesion glycoprotein p150, 95 alpha chain) (Leukocyte adhesion receptor
CD11C




p150, 95) (CD antigen CD11c)



P18084
ITB5_HUMAN
Integrin beta-5
ITGB5


Q9UJ96
KCNG2_HUMAN
Potassium voltage-gated channel subfamily G member 2 (Cardiac potassium
KCNG2




channel subunit) (Voltage-gated potassium channel subunit Kv6.2)
KCNF2


O95069
KCNK2_HUMAN
Potassium channel subfamily K member 2 (Outward rectifying potassium channel
KCNK2




protein TREK-1) (TREK-1 K(+) channel subunit) (Two pore domain potassium
TREK




channel TREK-1) (Two pore potassium channel TPKC1)
TREK1


Q9H427
KCNKF_HUMAN
Potassium channel subfamily K member 15 (Acid-sensitive potassium channel
KCNK15




protein TASK-5) (TWIK-related acid-sensitive K(+) channel 5) (Two pore
TASK5




potassium channel KT3.3) (Two pore K(+) channel KT3.3)



O75578
ITA10_HUMAN
Integrin alpha-10
ITGA10





UNQ468/





PRO827


P08514
ITA2B_HUMAN
Integrin alpha-IIb (GPalpha IIb) (GPIIb) (Platelet membrane glycoprotein IIb) (CD
ITGA2B




antigen CD41) [Cleaved into: Integrin alpha-IIb heavy chain; Integrin alpha-IIb
GP2B




light chain, form 1; Integrin alpha-IIb light chain, form 2]
ITGAB


P23229
ITA6_HUMAN
Integrin alpha-6 (CD49 antigen-like family member F) (VLA-6) (CD antigen
ITGA6




CD49f) [Cleaved into: Integrin alpha-6 heavy chain; Integrin alpha-6 light chain;





Processed integrin alpha-6 (Alpha6p)]



P08648
ITA5_HUMAN
Integrin alpha-5 (CD49 antigen-like family member E) (Fibronectin receptor
ITGA5




subunit alpha) (Integrin alpha-F) (VLA-5) (CD antigen CD49e) [Cleaved into:
FNRA




Integrin alpha-5 heavy chain; Integrin alpha-5 light chain]



Q13797
ITA9_HUMAN
Integrin alpha-9 (Integrin alpha-RLC)
ITGA9


P20701
ITAL_HUMAN
Integrin alpha-L (CD11 antigen-like family member A) (Leukocyte adhesion
ITGAL




glycoprotein LFA-1 alpha chain) (LFA-1A) (Leukocyte function-associated
CD11A




molecule 1 alpha chain) (CD antigen CD11a)



P26010
ITB7_HUMAN
Integrin beta-7 (Gut homing receptor beta subunit)
ITGB7


P48551
INAR2_HUMAN
Interferon alpha/beta receptor 2 (IFN-R-2) (IFN-alpha binding protein) (IFN-
IFNAR2




alpha/beta receptor 2) (Interferon alpha binding protein) (Type I interferon
IFNABR




receptor 2)
IFNARB


P15260
INGR1_HUMAN
Interferon gamma receptor 1 (IFN-gamma receptor 1) (IFN-gamma-R1)
IFNGR1




(CDw119) (Interferon gamma receptor alpha-chain) (IFN-gamma-R-alpha) (CD





antigen CD119)



Q01650
LAT1_HUMAN
Large neutral amino acids transporter small subunit 1 (4F2 light chain) (4F2 LC)
SLC7A5




(4F2LC) (CD98 light chain) (Integral membrane protein E16) (L-type amino acid
CD98LC




transporter 1) (hLAT1) (Solute carrier family 7 member 5) (y + system cationic
LAT1




amino acid transporter)
MPE16


Q9UHI5
LAT2_HUMAN
Large neutral amino acids transporter small subunit 2 (L-type amino acid
SLC7A8




transporter 2) (hLAT2) (Solute carrier family 7 member 8)
LAT2


P42702
LIFR_HUMAN
Leukemia inhibitory factor receptor (LIF receptor) (LIF-R) (CD antigen CD118)
LIFR


O75096
LRP4_HUMAN
Low-density lipoprotein receptor-related protein 4 (LRP-4) (Multiple epidermal
LRP4




growth factor-like domains 7)
KIAA0816





LRP10





MEGF7


Q07820
MCL1_HUMAN
Induced myeloid leukemia cell differentiation protein Mcl-1 (Bcl-2-like protein 3)
MCL1




(Bcl2-L-3) (Bcl-2-related protein EAT/mcl1) (mcl1/EAT)
BCL2L3


Q29980
MICB_HUMAN
MHC class I polypeptide-related sequence B (MIC-B)
MICB





PERB11.2


Q99650
OSMR_HUMAN
Oncostatin-M-specific receptor subunit beta (Interleukin-31 receptor subunit beta)
OSMR




(IL-31 receptor subunit beta) (IL-31R subunit beta) (IL-31R-beta) (IL-31RB)
OSMRB


Q86UW1
OSTA_HUMAN
Organic solute transporter subunit alpha (OST-alpha) (Solute carrier family 51
SLC51A




subunit alpha)
OSTA


P41143
OPRD_HUMAN
Delta-type opioid receptor (D-OR-1) (DOR-1)
OPRD1





OPRD


Q96QU1
PCD15_HUMAN
Protocadherin-15
PCDH15





USH1F


P07202
PERT_HUMAN
Thyroid peroxidase (TPO) (EC 1.11.1.8)
TPO


Q29983
MICA_HUMAN
MHC class I polypeptide-related sequence A (MIC-A)
MICA





PERB11.1


Q7L4E1
MIGA2_HUMAN
Mitoguardin 2 (Protein FAM73B)
MIGA2





C9orf54





FAM73B





PSEC0112


Q9BPX6
MICU1_HUMAN
Calcium uptake protein 1, mitochondrial (Atopy-related autoantigen CALC) (ara
MICU1




CALC) (Calcium-binding atopy-related autoantigen 1) (allergen Hom s 4)
CALC





CBARA1


Q8TCY5
MRAP_HUMAN
Melanocortin-2 receptor accessory protein (B27) (Fat cell-specific low molecular
MRAP




weight protein) (Fat tissue-specific low MW protein)
C21orf61





FALP


Q8NAN2
MIGA1_HUMAN
Mitoguardin 1 (Protein FAM73A)
MIGA1





FAM73A


Q13585
MTR1L_HUMAN
Melatonin-related receptor (G protein-coupled receptor 50) (H9)
GPR50


O15146
MUSK_HUMAN
Muscle, skeletal receptor tyrosine-protein kinase (EC 2.7.10.1) (Muscle-specific
MUSK




tyrosine-protein kinase receptor) (MuSK) (Muscle-specific kinase receptor)



Q96G30
MRAP2_HUMAN
Melanocortin-2 receptor accessory protein 2 (MC2R accessory protein 2)
MRAP2





C6orf117


O43908
NKG2F_HUMAN
NKG2-F type II integral membrane protein (NK cell receptor F) (NKG2-F-
KLRC4




activating NK receptor)
NKG2F


Q9Y2A7
NCKP1_HUMAN
Nck-associated protein 1 (NAP 1) (Membrane-associated protein HEM-2)
NCKAP1




(p125Nap1)
HEM2





KIAA0587





NAP1


O00533
NCHL1_HUMAN
Neural cell adhesion molecule L1-like protein (Close homolog of L1) [Cleaved
CHL1




into: Processed neural cell adhesion molecule L1-like protein]
CALL


P26715
NKG2A_HUMAN
NKG2-A/NKG2-B type II integral membrane protein (CD159 antigen-like family
KLRC1




member A) (NK cell receptor A) (NKG2-A/B-activating NK receptor) (CD antigen
NKG2A




CD159a)



Q9NZ94
NLGN3_HUMAN
Neuroligin-3 (Gliotactin homolog)
NLGN3





KIAA1480





NL3


P26717
NKG2C_HUMAN
NKG2-C type II integral membrane protein (CD159 antigen-like family member C)
KLRC2




(NK cell receptor C) (NKG2-C-activating NK receptor) (CD antigen CD159c)
NKG2C


Q07444
NKG2E_HUMAN
NKG2-E type II integral membrane protein (NK cell receptor E) (NKG2-E-
KLRC3




activating NK receptor)
NKG2E


Q99466
NOTC4_HUMAN
Neurogenic locus notch homolog protein 4 (Notch 4) (hNotch4) [Cleaved into:
NOTCH4




Notch 4 extracellular truncation; Notch 4 intracellular domain]
INT3


O14786
NRP1_HUMAN
Neuropilin-1 (Vascular endothelial cell growth factor 165 receptor) (CD antigen
NRP1




CD304)
NRP





VEGF165R


Q04721
NOTC2_HUMAN
Neurogenic locus notch homolog protein 2 (Notch 2) (hN2) [Cleaved into: Notch
NOTCH2




2 extracellular truncation (N2ECD); Notch 2 intracellular domain (N2ICD)]



O60462
NRP2_HUMAN
Neuropilin-2 (Vascular endothelial cell growth factor 165 receptor 2)
NRP2





VEGF165R2


Q86UW2
OSTB_HUMAN
Organic solute transporter subunit beta (OST-beta) (Solute carrier family 51
SLC51B




subunit beta)
OSTB


Q9P0L9
PK2L1_HUMAN
Polycystic kidney disease 2-like 1 protein (Polycystin-2 homolog) (Polycystin-2L1)
PKD2L1




(Polycystin-L) (Polycystin-L1)
PKD2L





PKDL





TRPP3


Q8TDX9
PK1L1_HUMAN
Polycystic kidney disease protein 1-like 1 (PC1-like 1 protein) (Polycystin-1L1)
PKD1L1





UNQ5785/





PRO19563


Q16651
PRSS8_HUMAN
Prostasin (EC 3.4.21.- (Channel-activating protease 1) (CAP1) (Serine protease
PRSS8




8) [Cleaved into: Prostasin light chain; Prostasin heavy chain]



Q96HA9
PX11C_HUMAN
Peroxisomal membrane protein 11C (Peroxin-11C) (Peroxisomal biogenesis
PEX11G




factor 11C) (Protein PEX11 homolog gamma) (PEX11-gamma)
PEX11C


O60896
RAMP3_HUMAN
Receptor activity-modifying protein 3 (Calcitonin-receptor-like receptor activity-
RAMP3




modifying protein 3) (CRLR activity-modifying protein 3)



O60895
RAMP2_HUMAN
Receptor activity-modifying protein 2 (Calcitonin-receptor-like receptor activity-
RAMP2




modifying protein 2) (CRLR activity-modifying protein 2)



O60894
RAMP1_HUMAN
Receptor activity-modifying protein 1 (Calcitonin-receptor-like receptor activity-
RAMP1




modifying protein 1) (CRLR activity-modifying protein 1)



O43157
PLXB1_HUMAN
Plexin-B1 (Semaphorin receptor SEP)
PLXNB1





KIAA0407





PLXN5





SEP


O15031
PLXB2_HUMAN
Plexin-B2 (MM1)
PLXNB2





KIAA0315


Q9UG56
PISD_HUMAN
Phosphatidylserine decarboxylase proenzyme, mitochondrial (EC 4.1.1.65)
PISD




[Cleaved into: Phosphatidylserine decarboxylase beta chain; Phosphatidylserine





decarboxylase alpha chain]



O75915
PRAF3_HUMAN
PRA1 family protein 3 (ADP-ribosylation factor-like protein 6-interacting protein 5)
ARL6IP5




(ARL-6-interacting protein 5) (Aip-5) (Cytoskeleton-related vitamin A-responsive
DERP11




protein) (Dermal papilla-derived protein 11) (GTRAP3-18) (Glutamate transporter
JWA




EAAC1-interacting protein) (JM5) (Prenylated Rab acceptor protein 2) (Protein
PRA2




JWa) (Putative MAPK-activating protein PM27)
PRAF3





HSPC127


O96011
PX11B_HUMAN
Peroxisomal membrane protein 11B (Peroxin-11B) (Peroxisomal biogenesis
PEX11B




factor 11B) (Protein PEX11 homolog beta) (PEX11-beta)



O75192
PX11A_HUMAN
Peroxisomal membrane protein 11A (HsPEX11p) (28 kDa peroxisomal integral
PEX11A




membrane protein) (PMP28) (Peroxin-11A) (Peroxisomal biogenesis factor 11A)
PEX11




(Protein PEX11 homolog alpha) (PEX11-alpha)



Q6ISU1
PTCRA_HUMAN
Pre T-cell antigen receptor alpha (pT-alpha) (pTa) (pT-alpha-TCR)
PTCRA


Q68DV7
RNF43_HUMAN
E3 ubiquitin-protein ligase RNF43 (EC 2.3.2.27) (RING finger protein 43) (RING-
RNF43




type E3 ubiquitin transferase RNF43)



P51811
XK_HUMAN
Membrane transport protein XK (Kell complex 37 kDa component) (Kx antigen)
XK




(XK-related protein 1)
XKR1





XRG1


Q04912
RON_HUMAN
Macrophage-stimulating protein receptor (MSP receptor) (EC 2.7.10.1) (CDw136)
MST1R




(Protein-tyrosine kinase 8) (p185-Ron) (CD antigen CD136) [Cleaved into:
PTK8




Macrophage-stimulating protein receptor alpha chain; Macrophage-stimulating
RON




protein receptor beta chain]



Q96DX8
RTP4_HUMAN
Receptor-transporting protein 4 (28 kDa interferon-responsive protein) (3CxxC-
RTP4




type zinc finger protein 4)
IFRG28





Z3CXXC4


Q8WTV0
SCRB1_HUMAN
Scavenger receptor class B member 1 (SRB1) (CD36 and LIMPII analogous 1)
SCARB1




(CLA-1) (CD36 antigen-like 1) (Collagen type I receptor, thrombospondin
CD36L1




receptor-like 1) (SR-BI) (CD antigen CD36)
CLA1


Q12884
SEPR_HUMAN
Prolyl endopeptidase FAP (EC 3.4.21.26) (170 kDa melanoma membrane-bound
FAP




gelatinase) (Dipeptidyl peptidase FAP) (EC 3.4.14.5) (Fibroblast activation protein





alpha) (FAPalpha) (Gelatine degradation protease FAP) (EC 3.4.21.- (Integral





membrane serine protease) (Post-proline cleaving enzyme) (Serine integral





membrane protease) (SIMP) (Surface-expressed protease) (Seprase) [Cleaved





into: Antiplasmin-cleaving enzyme FAP, soluble form (APCE) (EC 3.4.14.5) (EC





3.4.21.-) (EC 3.4.21.26)]



Q9Y5M8
SRPRB_HUMAN
Signal recognition particle receptor subunit beta (SR-beta) (Protein APMCF1)
SRPRB





PSEC0230


O15270
SPTC2_HUMAN
Serine palmitoyltransferase 2 (EC 2.3.1.50) (Long chain base biosynthesis
SPTLC2




protein 2) (LCB 2) (Long chain base biosynthesis protein 2a) (LCB2a) (Serine-
KIAA0526




palmitoyl-CoA transferase 2) (SPT 2)
LCB2


Q07837
SLC31_HUMAN
Neutral and basic amino acid transport protein rBAT (NBAT) (D2h) (Solute carrier
SLC3A1




family 3 member 1) (b(0, +)-type amino acid transport protein)
RBAT


O15269
SPTC1_HUMAN
Serine palmitoyltransferase 1 (EC 2.3.1.50) (Long chain base biosynthesis
SPTLC1




protein 1) (LCB 1) (Serine-palmitoyl-CoA transferase 1) (SPT 1) (SPT1)
LCB1


P30874
SSR2_HUMAN
Somatostatin receptor type 2 (SS-2-R) (SS2-R) (SS2R) (SRIF-1)
SSTR2


Q9NUV7
SPTC3_HUMAN
Serine palmitoyltransferase 3 (EC 2.3.1.50) (Long chain base biosynthesis
SPTLC3




protein 2b) (LCB2b) (Long chain base biosynthesis protein 3) (LCB 3) (Serine-
C20orf38




palmitoyl-CoA transferase 3) (SPT 3)
SPTLC2L


P32745
SSR3_HUMAN
Somatostatin receptor type 3 (SS-3-R) (SS3-R) (SS3R) (SSR-28)
SSTR3


P35346
SSR5_HUMAN
Somatostatin receptor type 5 (SS-5-R) (SS5-R) (SS5R)
SSTR5


P60508
SYCY2_HUMAN
Syncytin-2 (Endogenous retrovirus group FRD member 1) (Envelope polyprotein)
ERVFRD-1




(HERV-FRD) (HERV-FRD_6p24.1 provirus ancestral Env polyprotein) [Cleaved
ERVFRDE1




into: Surface protein (SU); Transmembrane protein (TM)]
UNQ6191/





PRO20218


Q86SS6
SYT9_HUMAN
Synaptotagmin-9 (Synaptotagmin IX) (SytIX)
SYT9


Q9BQG1
SYT3_HUMAN
Synaptotagmin-3 (Synaptotagmin III) (SytIII)
SYT3


Q5T7P8
SYT6_HUMAN
Synaptotagmin-6 (Synaptotagmin VI) (SytVI)
SYT6


Q6XYQ8
SYT10_HUMAN
Synaptotagmin-10 (Synaptotagmin X) (SytX)
SYT10


Q03518
TAP1_HUMAN
Antigen peptide transporter 1 (APT1) (ATP-binding cassette sub-family B member
TAP1




2) (Peptide supply factor 1) (Peptide transporter PSF1) (PSF-1) (Peptide
ABCB2




transporter TAP1) (Peptide transporter involved in antigen processing 1) (Really
PSF1




interesting new gene 4 protein)
RING4





Y3


Q03519
TAP2_HUMAN
Antigen peptide transporter 2 (APT2) (ATP-binding cassette sub-family B member
TAP2




3) (Peptide supply factor 2) (Peptide transporter PSF2) (PSF-2) (Peptide
ABCB3




transporter TAP2) (Peptide transporter involved in antigen processing 2) (Really
PSF2




interesting new gene 11 protein)
RING11





Y1


P01848
TCA_HUMAN
T-cell receptor alpha chain C region
TRAC





TCRA


Q9UKZ4
TEN1_HUMAN
Teneurin-1 (Ten-1) (Protein Odd Oz/ten-m homolog 1) (Tenascin-M1) (Ten-m1)
TENM1




(Teneurin transmembrane protein 1) [Cleaved into: Ten-1 intracellular domain
ODZ1




(IDten-1) (Ten-1 ICD); Teneurin C-terminal-associated peptide (TCPA-1) (Ten-1
TNM1




extracellular domain) (Ten-1 ECD)]



Q9UL52
TM11E_HUMAN
Transmembrane protease serine 11E (EC 3.4.21.-) (Serine protease DESC1)
TMPRSS11E




(Transmembrane protease serine 11E2) [Cleaved into: Transmembrane protease
DESC1




serine 11E non-catalytic chain; Transmembrane protease serine 11E catalytic
TMPRSS11E2




chain]
UNQ742/





PRO1461


P35590
TIE1_HUMAN
Tyrosine-protein kinase receptor Tie-1 (EC 2.7.10.1)
TIE1





TIE


Q9Y2C9
TLR6_HUMAN
Toll-like receptor 6 (CD antigen CD286)
TLR6


Q9NT68
TEN2_HUMAN
Teneurin-2 (Ten-2) (Protein Odd Oz/ten-m homolog 2) (Tenascin-M2) (Ten-m2)
TENM2




(Teneurin transmembrane protein 2) [Cleaved into: Ten-2, soluble form; Ten-2
KIAA1127




intracellular domain (Ten-2 ICD)]
ODZ2





TNM2


Q6N022
TEN4_HUMAN
Teneurin-4 (Ten-4) (Protein Odd Oz/ten-m homolog 4) (Tenascin-M4) (Ten-m4)
TENM4




(Teneurin transmembrane protein 4)
KIAA1302





ODZ4





TNM4


Q15399
TLR1_HUMAN
Toll-like receptor 1 (Toll/interleukin-1 receptor-like protein) (TIL) (CD antigen
TLR1




CD281)
KIAA0012


Q7RTY8
TMPS7_HUMAN
Transmembrane protease serine 7 (EC 3.4.21.-) (Matriptase-3)
TMPRSS7


P01850
TRBC1_HUMAN
T-cell receptor beta-1 chain C region
TRBC1


O15533
TPSN_HUMAN
Tapasin (TPN) (TPSN) (NGS-17) (TAP-associated protein) (TAP-binding protein)
TAPBP





NGS17





TAPA


Q9BX84
TRPM6_HUMAN
Transient receptor potential cation channel subfamily M member 6 (EC 2.7.11.1)
TRPM6




(Channel kinase 2) (Melastatin-related TRP cation channel 6)
CHAK2


P30530
UFO_HUMAN
Tyrosine-protein kinase receptor UFO (EC 2.7.10.1) (AXL oncogene)
AXL





UFO


O00526
UPK2_HUMAN
Uroplakin-2 (UP2) (Uroplakin II) (UPII)
UPK2


Q92536
YLAT2_HUMAN
Y + L amino acid transporter 2 (Cationic amino acid transporter, y + system) (Solute
SLC7A6




carrier family 7 member 6) (y(+)L-type amino acid transporter 2) (Y + LAT2)
KIAA0245




(y + LAT-2)



Q9UM01
YLAT1_HUMAN
Y + L amino acid transporter 1 (Monocyte amino acid permease 2) (MOP-2)
SLC7A7




(Solute carrier family 7 member 7) (y(+)L-type amino acid transporter 1) (Y + LAT1)





(y + LAT-1)



O75631
UPK3A_HUMAN
Uroplakin-3a (UP3a) (Uroplakin III) (UPIII)
UPK3A





UPK3


Q9BT76
UPK3B_HUMAN
Uroplakin-3b (UP3b) (Uroplakin IIIb) (UPIIIb) (p35)
UPK3B


P35916
VGFR3_HUMAN
Vascular endothelial growth factor receptor 3 (VEGFR-3) (EC 2.7.10.1) (Fms-like
FLT4




tyrosine kinase 4) (FLT-4) (Tyrosine-protein kinase receptor FLT4)
VEGFR3


O75841
UPK1B_HUMAN
Uroplakin-1b (UP1b) (Tetraspanin-20) (Tspan-20) (Uroplakin Ib) (UPIb)
UPK1B





TSPAN20


Q9P0L0
VAPA_HUMAN
Vesicle-associated membrane protein-associated protein A (VAMP-A) (VAMP-
VAPA




associated protein A) (VAP-A) (33 kDa VAMP-associated protein) (VAP-33)
VAP33


Q9ULK5
VANG2_HUMAN
Vang-like protein 2 (Loop-tail protein 1 homolog) (Strabismus 1) (Van Gogh-like
VANGL2




protein 2)
KIAA1215





STB1


Q8TAA9
VANG1_HUMAN
Vang-like protein 1 (Loop-tail protein 2 homolog) (LPP2) (Strabismus 2) (Van
VANGL1




Gogh-like protein 1)
STB2


O95292
VAPB_HUMAN
Vesicle-associated membrane protein-associated protein B/C (VAMP-B/VAMP-
VAPB




C) (VAMP-associated protein B/C) (VAP-B/VAP-C)
UNQ484/





PRO983


Q9UPY5
XCT_HUMAN
Cystine/glutamate transporter (Amino acid transport system xc-) (Calcium
SLC7A11




channel blocker resistance protein CCBR1) (Solute carrier family 7 member 11)





(xCT)



Q6XR72
ZNT10_HUMAN
Zinc transporter 10 (ZnT-10) (Manganese transporter SLC30A10) (Solute carrier
SLC30A10




family 30 member 10)
ZNT10





ZNT8


A0A024
A0A024R0A1_HUMAN
Macrophage colony-stimulating factor 1 (CSF-1) (MCSF)
CSF1


R0A1


hCG_40247


P31785
IL2RG_HUMAN
Cytokine receptor common subunit gamma (Interleukin-2 receptor subunit
IL2RG




gamma) (IL-2 receptor subunit gamma) (IL-2R subunit gamma) (IL-2RG)





(gammaC) (p64) (CD antigen CD132)



P32927
IL3RB_HUMAN
Cytokine receptor common subunit beta (CDw131) (GM-CSF/IL-3/IL-5 receptor
CSF2RB




common beta subunit) (CD antigen CD131)
IL3RB





IL5RB


P26951
IL3RA_HUMAN
Interleukin-3 receptor subunit alpha (IL-3 receptor subunit alpha) (IL-3R subunit
IL3RA




alpha) (IL-3R-alpha) (IL-3RA) (CD antigen CD123)
IL3R


P15509
CSF2R_HUMAN
Granulocyte-macrophage colony-stimulating factor receptor subunit alpha (GM-
CSF2RA




CSF-R-alpha) (GMCSFR-alpha) (GMR-alpha) (CDw116) (CD antigen CD116)
CSF2R





CSF2RY


Q01344
IL5RA_HUMAN
Interleukin-5 receptor subunit alpha (IL-5 receptor subunit alpha) (IL-5R subunit
IL5RA




alpha) (IL-5R-alpha) (IL-5RA) (CDw125) (CD antigen CD125)
IL5R


P08887
IL6RA_HUMAN
Interleukin-6 receptor subunit alpha (IL-6 receptor subunit alpha) (IL-6R subunit
IL6R




alpha) (IL-6R-alpha) (IL-6RA) (IL-6R 1) (Membrane glycoprotein 80) (gp80) (CD





antigen CD126)



P40189
IL6RB_HUMAN
Interleukin-6 receptor subunit beta (IL-6 receptor subunit beta) (IL-6R subunit
IL6ST




beta) (IL-6R-beta) (IL-6RB) (CDw130) (Interleukin-6 signal transducer)





(Membrane glycoprotein 130) (gp130) (Oncostatin-M receptor subunit alpha) (CD





antigen CD130)



Q14626
I11RA_HUMAN
Interleukin-11 receptor subunit alpha (IL-11 receptor subunit alpha) (IL-11R
IL11RA




subunit alpha) (IL-11R-alpha) (IL-11RA)



P42702
LIFR_HUMAN
Leukemia inhibitory factor receptor (LIF receptor) (LIF-R) (CD antigen CD118)
LIFR


Q99650
OSMR_HUMAN
Oncostatin-M-specific receptor subunit beta (Interleukin-31 receptor subunit beta)
OSMR




(IL-31 receptor subunit beta) (IL-31R subunit beta) (IL-31R-beta) (IL-31RB)
OSMRB


P31785
IL2RG_HUMAN
Cytokine receptor common subunit gamma (Interleukin-2 receptor subunit
IL2RG




gamma) (IL-2 receptor subunit gamma) (IL-2R subunit gamma) (IL-2RG)





(gammaC) (p64) (CD antigen CD132)



P01589
IL2RA_HUMAN
Interleukin-2 receptor subunit alpha (IL-2 receptor subunit alpha) (IL-2-RA) (IL-2R
IL2RA




subunit alpha) (IL2-RA) (TAC antigen) (p55) (CD antigen CD25)



P14784
IL2RB_HUMAN
Interleukin-2 receptor subunit beta (IL-2 receptor subunit beta) (IL-2R subunit
IL2RB




beta) (IL-2RB) (High affinity IL-2 receptor subunit beta) (Interleukin-15 receptor
IL15RB




subunit beta) (p70-75) (p75) (CD antigen CD122)



P24394
IL4RA_HUMAN
Interleukin-4 receptor subunit alpha (IL-4 receptor subunit alpha) (IL-4R subunit
IL4R




alpha) (IL-4R-alpha) (IL-4RA) (CD antigen CD124) [Cleaved into: Soluble
IL4RA




interleukin-4 receptor subunit alpha (Soluble IL-4 receptor subunit alpha) (Soluble
582J2.1




IL-4R-alpha) (sIL4Ralpha/prot) (IL-4-binding protein) (IL4-BP)]



P16871
IL7RA_HUMAN
Interleukin-7 receptor subunit alpha (IL-7 receptor subunit alpha) (IL-7R subunit
IL7R




alpha) (IL-7R-alpha) (IL-7RA) (CDw127) (CD antigen CD127)



Q01113
IL9R_HUMAN
Interleukin-9 receptor (IL-9 receptor) (IL-9R) (CD antigen CD129)
IL9R


P78552
I13R1_HUMAN
Interleukin-13 receptor subunit alpha-1 (IL-13 receptor subunit alpha-1) (IL-13R
IL13RA1




subunit alpha-1) (IL-13 R-alpha-1) (IL-13RA1) (Cancer/testis antigen 19) (CT19)
IL13R




(CD antigen CD213a1)
IL13RA


Q14627
I13R2_HUMAN
Interleukin-13 receptor subunit alpha-2 (IL-13 receptor subunit alpha-2) (IL-13R
IL13RA2




subunit alpha-2) (IL-13R-alpha-2) (IL-13RA2) (Interleukin-13-binding protein) (CD
IL13R




antigen CD213a2)



Q13651
I10R1_HUMAN
Interleukin-10 receptor subunit alpha (IL-10 receptor subunit alpha) (IL-10R
IL10RA




subunit alpha) (IL-10RA) (CDw210a) (Interleukin-10 receptor subunit 1) (IL-10R
IL10R




subunit 1) (IL-10R1) (CD antigen CD210)



P26992
CNTFR_HUMAN
Ciliary neurotrophic factor receptor subunit alpha (CNTF receptor subunit alpha)
CNTFR




(CNTFR-alpha)



Q9NPF7
IL23A_HUMAN
Interleukin-23 subunit alpha (IL-23 subunit alpha) (IL-23-A) (Interleukin-23 subunit
IL23A




p19) (IL-23p19)
SGRF





UNQ2498/





PRO5798


P29459
IL12A_HUMAN
Interleukin-12 subunit alpha (IL-12A) (Cytotoxic lymphocyte maturation factor 35
IL12A




kDa subunit) (CLMF p35) (IL-12 subunit p35) (NK cell stimulatory factor chain 1)
NKSF1




(NKSF1)



Q8NEV9
IL27A_HUMAN
Interleukin-27 subunit alpha (IL-27 subunit alpha) (IL-27-A) (IL27-A) (Interleukin-
IL27




30) (p28)
IL27A





IL30


Q14213
IL27B_HUMAN
Interleukin-27 subunit beta (IL-27 subunit beta) (IL-27B) (Epstein-Barr virus-
EBI3




induced gene 3 protein) (EBV-induced gene 3 protein)
IL27B









In various embodiments, the present heterodimeric proteins may be engineered to target one or more molecules that reside on human leukocytes including, without limitation, the extracellular domains (where applicable) of SLAMF4, IL-2Rα, IL-2 R β, ALCAM, B7-1, IL-4 R, B7-H3, BLAME/SLAMFS, CEACAM1, IL-6 R, IL-7 Rα, IL-10R α, IL-I0 R β, IL-12 R β 1, IL-12 R β 2, CD2, IL-13 R α 1, IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a, lutegrin α 4/CD49d, CDS, Integrin α E/CD103, CD6, Integrin α M/CD 11 b, CDS, Integrin α X/CD11c, Integrin β 2/CDIS, KIR/CD15S, KIR2DL1, CD2S, KIR2DL3, KIR2DL4/CD15Sd, CD31/PECAM-1, KIR2DS4, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, Leukotriene B4-R1, CDS4/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common γ Chain/IL-2 R γ, Osteopontin, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, CX3CR1, CX3CL1, L-Selectin, SIRP β 1, SLAM, TCCR/WSX-1, DNAM-1, Thymopoietin, EMMPRIN/CD147, TIM-1, EphB6, TIM-2, TIM-3, TIM-4, Fcγ RIII/CD16, TIM-6, Granulysin, ICAM-1/CD54, ICAM-2/CD102, IFN-γR1, IFN-γ R2, TSLP, IL-1 R1 and TSLP R.
In some embodiments, the present heterodimeric proteins may be engineered to target one or more molecules involved in immune inhibition, including for example: CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTANSIG8, KIR, 2B4, TIGIT, CD160 (also referred to as BY55), CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), and various B-7 family ligands (including, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7).
In some embodiments, the present heterodimeric proteins comprise an extracellular domain of an immune inhibitory agent.
In some embodiments, the present heterodimeric proteins comprise an extracellular domain of a soluble or membrane protein which has immune inhibitory properties.
In some embodiments, the present heterodimeric proteins simulate binding of an inhibitory signal ligand to its cognate receptor but inhibit the inhibitory signal transmission to an immune cell (e.g., a T cell, macrophage or other leukocyte).
In various embodiments, the heterodimeric protein comprises an immune inhibitory receptor extracellular domain and an immune stimulatory ligand extracellular domain which can, without limitation, deliver an immune stimulation to a T cell while masking a tumor cell's immune inhibitory signals. In various embodiments, the heterodimeric protein delivers a signal that has the net result of T cell activation.
In some embodiments, the present heterodimeric proteins comprise an extracellular domain of a soluble or membrane protein which has immune stimulatory properties.
In embodiments, a heterodimeric protein useful in the present invention comprises the extracellular domain of Gp130. Gp130 (also known as Interleukin-6 receptor subunit beta, IL-6R-beta, IL-6RB, and IL-6ST) is a signal-transducing molecule. The receptor systems for IL6, LIF, OSM, CNTF, IL11, CTF1 and BSF3 can utilize Gp130 for initiating signal transmission. Binding of IL6 to IL6R induces IL6ST homodimerization and formation of a high-affinity receptor complex, which activates Janus kinases. That causes phosphorylation of Gp130 tyrosine residues which in turn activates STAT3. Gp130 mediates signals which regulate immune response, hematopoiesis, pain control and bone metabolism (By similarity).
In embodiments, a heterodimeric protein useful in the present invention comprises a variant of the extracellular domain of Gp130. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known amino acid sequence of Gp130, e.g., human Gp130.
In embodiments, the extracellular domain of Gp130 has the following amino acid sequence:







(SEQ ID NO: 18)


ELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHF


 


TIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIIS


 


GLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADC


 


KAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKP


 


NPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQI


 


PPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITY


 


EDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTR


 


WKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQ


 


ATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQ


 


EDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKG


 


PTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDS


 


SHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIE






In embodiments, a heterodimeric protein comprises a variant of the extracellular domain of Gp130. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 18.
In embodiments, one chain of the heterodimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 18.
One of ordinary skill may select variants of the known amino acid sequence of Gp130 by consulting the literature, e.g., Hibi et al, “Molecular cloning and expression of an IL-6 signal transducer, gp130” Cell 63 (6), 1149-1157 (1990); Waetzig et al., “N-linked glycosylation is essential for the stability but not the signaling function of the interleukin-6 signal transducer glycoprotein 130”, J. Biol. Chem. 285 (3), 1781-1789 (2010); Schutt et al., “gp130 activation is regulated by D2-D3 interdomain connectivity”, Biochem. J. 450 (3), 487-496 (2013); Bravo et al., “Crystal structure of a cytokine-binding region of gp130”, EMBO J. 17 (6), 1665-1674 (1998); Chow et al., “Structure of an extracellular gp130 cytokine receptor signaling complex”, Science 291 (5511), 2150-2155 (2001); Boulanger et al., “Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp130 complex”, Science 300 (5628), 2101-2104 (2003); Xu et al., “Crystal structure of the entire ectodomain of gp130: insights into the molecular assembly of the tall cytokine receptor complexes”, J. Biol. Chem. 285 (28), 21214-21218 (2010), each of which is incorporated by reference in its entirety.
In embodiments, a heterodimeric protein useful in the present invention comprises the extracellular domain of IL-6RA. IL-6RA (also known as Interleukin-6 receptor subunit alpha, IL-6 receptor subunit alpha, IL-6R subunit alpha, and IL-6R-alpha) is part of the receptor for interleukin 6. Binds to IL6 with low affinity, but does not transduce a signal. Signal activation necessitate an association with gp130. Activation may lead to the regulation of the immune response, acute-phase reactions and hematopoiesis. Low concentration of a soluble form of IL6 receptor acts as an agonist of IL6 activity. Dysregulated production of IL6 and this receptor are implicated in the pathogenesis of many diseases, such as multiple myeloma, autoimmune diseases and prostate cancer.
In embodiments, a heterodimeric protein useful in the present invention comprises a variant of the extracellular domain of IL-6RA. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known amino acid sequence of IL-6RA, e.g., human IL-6RA.
In embodiments, the extracellular domain of IL-6RA has the following amino acid sequence:







(SEQ ID NO: 19)


LAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATVHWVLRKPAAGS


 


HPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVHLLVDVPPEEPQL


 


SCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQ


 


ESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPP


 


ANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWM


 


VKDLQHHCVIHDAWSGLRHWQLRAQEEFGQGEWSEWSPEAMGTPWTESRS


 


PPAENEVSTPMQALTTNKDDDNILFRDSANATSLPVQDSSSVPLP






In embodiments, a heterodimeric protein comprises a variant of the extracellular domain of IL-6RA. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 19.
In embodiments, one chain of the heterodimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 19.
One of ordinary skill may select variants of the known amino acid sequence of IL-6RA by consulting the literature, e.g., Yamasaki et al., “Cloning and expression of the human interleukin-6 (BSF-2/IFN beta 2) receptor” Science 241 (4867), 825-828 (1988); Buk et al., “Increased association with detergent-resistant membranes/lipid rafts of apically targeted mutants of the interleukin-6 receptor gp80” Eur. J. Cell Biol. 84 (10), 819-831 (2005); Yawata et al., Structure-function analysis of human IL-6 receptor: dissociation of amino acid residues required for IL-6-binding and for IL-6 signal transduction through gp130′′ EMBO J. 12 (4), 1705-1712 (1993); Horiuchi et al., “Soluble interleukin-6 receptors released from T cell or granulocyte/macrophage cell lines and human peripheral blood mononuclear cells are generated through an alternative splicing mechanism” Eur. J. Immunol. 24 (8), 1945-1948 (1994); Boulanger et al., “Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp130 complex”, Science 300 (5628), 2101-2104 (2003), each of which is incorporated by reference in its entirety.
In embodiments, a heterodimeric protein useful in the present invention comprises the extracellular domain of IL-12A. IL-12A (also known as Interleukin-12 subunit alpha and IL-12 subunit p35) is a cytokine that can act as a growth factor for activated T and NK cells, enhance the lytic activity of NK/lymphokine-activated killer cells, and stimulate the production of IFN-gamma by resting PBMC. The cytokine is a disulfide-linked heterodimer composed of the 35-kD subunit encoded by this gene, and a 40-kD subunit that is a member of the cytokine receptor family. This cytokine is required for the T-cell-dependent induction of interferon gamma (INF-γ), and is important for the differentiation of both Th1 and Th2 cells. The responses of lymphocytes to this cytokine are mediated by the activator of transcription protein STAT4.
In embodiments, a heterodimeric protein useful in the present invention comprises a variant of the extracellular domain of IL-12A. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known amino acid sequence of IL-12A, e.g., human IL-12A.
In embodiments, the extracellular domain of IL-12A has the following amino acid sequence:







(SEQ ID NO: 20)


RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHE


 


DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMAL


 


CLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNF


 


NSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS






In embodiments, a heterodimeric protein comprises a variant of the extracellular domain of IL-12A. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 20.
In embodiments, one chain of the heterodimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 20.
One of ordinary skill may select variants of the known amino acid sequence of IL-12A by consulting the literature, e.g., Wolf et al., “Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells”, J. Immunol. 146 (9), 3074-3081 (1991); Devergne et al., “Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin”, Proc. Natl. Acad. Sci. U.S.A. 94 (22), 12041-12046 (1997); Yoon et al., “Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12”, EMBO J. 19 (14), 3530-3541 (2000), each of which is incorporated by reference in its entirety.
In embodiments, a heterodimeric protein useful in the present invention comprises the extracellular domain of IL-27B. IL-27b (also known as Interleukin-27 subunit beta, IL-27 subunit beta, and IL-27B), together with IL-12a forms Interleukin 35 (IL-35). IL-35 is a dimeric protein composed of IL-12a and IL-273 chains, which are encoded by two separate genes called IL12A and EBI3, respectively. IL-27 has pro- and anti-inflammatory properties, that can regulate T-helper cell development, suppress T-cell proliferation, stimulate cytotoxic T-cell activity, induce isotype switching in B-cells, and that has diverse effects on innate immune cells. Its gene was identified by its induced expression in B lymphocytes in response Epstein-Barr virus infection. IL-27 regulates T cell and inflammatory responses, in part by activating the Jak/STAT pathway of CD4+ T cells.
In embodiments, a heterodimeric protein useful in the present invention comprises a variant of the extracellular domain of IL-27B. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known amino acid sequence of IL-27B, e.g., human IL-27B.
In embodiments, the extracellular domain of IL-27B has the following amino acid sequence:







(SEQ ID NO: 21)


RKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGM


 


AARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVP


 


FITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYK


 


RQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLP


 


ATATMSLGK






In embodiments, a heterodimeric protein comprises a variant of the extracellular domain of IL-27B. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 21.
In embodiments, one chain of the heterodimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21.
One of ordinary skill may select variants of the known amino acid sequence of IL-27B by consulting the literature, e.g., Devergne “Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin”, Proc. Natl. Acad. Sci. U.S.A. 94 (22), 12041-12046 (1997); Pflanz et al., “IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4+ T cells”, Immunity 16 (6), 779-790 (2002); Batten and Ghilardi “The biology and therapeutic potential of interleukin 27”, J. Mol. Med. 85 (7), 661-672 (2007), each of which is incorporated by reference in its entirety.
In embodiments, an alpha chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16 or SEQ ID NO: 24. Such an alpha chain may be referred to as “Gp130-Alpha-IL12A”.
In embodiments, a Gp130-Alpha-IL12A chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 22)


ELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHF


 


TIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIIS


 


GLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADC


 


KAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKP


 


NPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQI


 


PPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITY


 


EDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTR


 


WKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQ


 


ATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQ


 


EDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKG


 


PTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDS


 


SHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEGSG


 


SRKGGKRGSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTC


 


VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ


 


DWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKN


 


QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT


 


VDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKDEGGEDGSGSRNLP


 


VATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITK


 


DKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSS


 


IYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSET


 


VPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS*






In embodiments, an beta chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17 or SEQ ID NO: 25. Such an alpha chain may be referred to as “IL6RA-Beta-IL27B”. In embodiments, an IL6RA-Beta-IL27B chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 23)


LAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATVHWVLRKPAAGS


 


HPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVHLLVDVPPEEPQL


 


SCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQ


 


ESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPP


 


ANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWM


 


VKDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESR


 


SPPAENEVSTPMQALTTNKDDDNILFRDSANATSLPVQDSSSVPLPGSGS


 


DEGGEDGSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCV


 


VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD


 


WLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQ


 


VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV


 


DKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKRKGGKRGSGSRKGPP


 


AALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGH


 


SWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEH


 


IIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAA


 


RFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATM


 


SLGK*






When a Gp130-Alpha-IL12A chain and an IL6RA-Beta-IL27B chain are combined (within a cell or in vitro), they form a heterodimeric protein referred to herein as IL-6R-Fc-IL-35.
In embodiments, a heterodimeric protein useful in the present invention comprises the extracellular domain of IL-21r. Interleukin-21 receptor (also known as IL-21 receptor and IL-21R) is a receptor for interleukin-21 belongs to the type I cytokine receptors, and has been shown to form a heterodimeric receptor complex with the common gamma-chain, a receptor subunit also shared by the receptors for interleukin 2, 4, 7, 9, and 15. This receptor transduces the growth-promoting signal of IL21, and is important for the proliferation and differentiation of T cells, B cells, and natural killer (NK) cells. The ligand binding of this receptor leads to the activation of multiple downstream signaling molecules, including JAK1, JAK3, STAT1, and STAT3. Knockout studies of a similar gene in mouse suggest a role for this gene in regulating immunoglobulin production.
In embodiments, a heterodimeric protein useful in the present invention comprises a variant of the extracellular domain of IL-21r. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known amino acid sequence of IL-21r, e.g., human IL-21r.
In embodiments, the extracellular domain of IL-21r has the following amino acid sequence:







(SEQ ID NO: 26)


CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHR


 


SAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIK


 


PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAV


 


SPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSD


 


PVIFQTQSEELKE






In embodiments, a heterodimeric protein comprises a variant of the extracellular domain of IL-21r. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 26.
In embodiments, one chain of the heterodimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 26.
One of ordinary skill may select variants of the known amino acid sequence of IL-21r by consulting the literature, e.g., Ozaki et al., “Cloning of a type I cytokine receptor most related to the IL-2 receptor beta chain”, Proc. Natl. Acad. Sci. U.S.A. 97 (21), 11439-11444 (2000); Kotlarz et al., “Loss-of-function mutations in the IL-21 receptor gene cause a primary immunodeficiency syndrome” J. Exp. Med. 210 (3), 433-443 (2013); Hamming et al., “Crystal structure of interleukin-21 receptor (IL-21R) bound to IL-21 reveals that sugar chain interacting with WSXWS motif is integral part of IL-21R” J. Biol. Chem. 287 (12), 9454-9460 (2012), each of which is incorporated by reference in its entirety.
In embodiments, a heterodimeric protein useful in the present invention comprises the extracellular domain of IL2RG. Interleukin-2 receptor subunit gamma (also known as Cytokine receptor common subunit gamma, IL-2 receptor subunit gamma, IL-2R subunit gamma, and IL-2RG) is a common subunit for the receptors for a variety of interleukins, including those of interleukin-2, -4, -7 and -21, and is thus referred to as the common gamma chain. Mutations in this gene cause X-linked severe combined immunodeficiency (XSCID), as well as X-linked combined immunodeficiency (XCID), a less severe immunodeficiency disorder.
In embodiments, a heterodimeric protein useful in the present invention comprises a variant of the extracellular domain of IL2RG. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known amino acid sequence of IL2RG, e.g., human IL2RG.
In embodiments, the extracellular domain of IL2RG has the following amino acid sequence:







(SEQ ID NO: 27)


LNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNC


 


TWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEI


 


HLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLEL


 


NWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFR


 


VRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEA






In embodiments, a heterodimeric protein comprises a variant of the extracellular domain of IL2RG. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 27.
In embodiments, one chain of the heterodimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 27.
One of ordinary skill may select variants of the known amino acid sequence of IL2RG by consulting the literature, e.g., Takeshita et al., “Cloning of the gamma chain of the human IL-2 receptor”, Science 257 (5068), 379-382 (1992); Ratthe et al., “Interleukin-15 enhances human neutrophil phagocytosis by a Syk-dependent mechanism: importance of the IL-15Ralpha chain”, J. Leukoc. Biol. 76 (1), 162-168 (2004); Bamborough et al., “The interleukin-2 and interleukin-4 receptors studied by molecular modelling”, Structure 2 (9), 839-851 (1994); Wang et al., “Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gammac receptors” Science 310 (5751), 1159-1163 (2005); Stauber et al., “Crystal structure of the IL-2 signaling complex: paradigm for a heterotrimeric cytokine receptor”, Proc. Natl. Acad. Sci. U.S.A. 103 (8), 2788-2793 (2006), each of which is incorporated by reference in its entirety.
In embodiments, an alpha chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16 or SEQ ID NO: 24. Such an alpha chain may be referred to as “IL21r-Alpha-IL12a”.
In embodiments, an IL21r-Alpha-IL12α chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 28)


CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHR


 


SAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIK


 


PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAV


 


SPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSD


 


PVIFQTQSEELKEGSGSRKGGKRGSKYGPPCPPCPAPEFLGGPSVFLFPP


 


KPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ


 


FNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPRE


 


PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP


 


PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSL


 


GKDEGGEDGSGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLE


 


FYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSC


 


LASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNML


 


AVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTID


 


RVMSYLNAS






In embodiments, an beta chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17 or SEQ ID NO: 25. Such a beta chain may be referred to as “IL2rg-Beta-IL27B”.
In embodiments, an IL2rg-Beta-IL27B chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 29)


LNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNC


 


TWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEI


 


HLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLEL


 


NWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFR


 


VRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAGSGSDEGGED


 


GSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQ


 


EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKE


 


YKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ


 


EGNVFSCSVLHEALHNHYTQKSLSLSLGKRKGGKRGSGSRKGPPAALTLP


 


RVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQ


 


QTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDP


 


PEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVG


 


PIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK






When an IL21r-Alpha-IL12α chain and an IL2rg-Beta-IL27B chain are combined (within a cell or in vitro), they form a heterodimeric protein referred to herein as IL-21R-Fc-IL-35.
In embodiments, an alpha chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17 or SEQ ID NO: 25. Such an alpha chain may be referred to as “IL21r-Beta-IL12a”.
In embodiments, an IL21r-Beta-IL12a chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 37)


CPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHR


 


SAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIK


 


PAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAV


 


SPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSD


 


PVIFQTQSEELKEGSGSDEGGEDGSKYGPPCPPCPAPEFLGGPSVFLFPP


 


KPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ


 


FNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPRE


 


PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP


 


PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSL


 


GKRKGGKRGSGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLE


 


FYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSC


 


LASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNML


 


AVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTID


 


RVMSYLNAS






In embodiments, an beta chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16 or SEQ ID NO: 24. Such a beta chain may be referred to as “IL2rg-Alpha-IL27B”.
In embodiments, an IL2rg-Alpha-IL27B chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 36)


LNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNC


 


TWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEI


 


HLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLEL


 


NWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFR


 


VRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAGSGSRKGGKR


 


GSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQ


 


EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKE


 


YKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCL


 


VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ


 


EGNVFSCSVLHEALHNHYTQKSLSLSLGKDEGGEDGSGSRKGPPAALTLP


 


RVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQ


 


QTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDP


 


PEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVG


 


PIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK






When an IL21r-Beta-IL12α chain and an IL2rg-Alpha-IL27B chain are combined (within a cell or in vitro), they form a heterodimeric protein may also be referred to herein as IL-21R-Fc-IL-35.
In embodiments, a heterodimeric protein useful in the present invention comprises the extracellular domain of IFNgR. IFNgR (also known as Interferon gamma receptor 1, IFN-gamma receptor 1, IFN-gamma-R1, IFN-gamma-R-alpha, IFNgR, and IFNGR1) associates with IFNGR2 to form a receptor for the cytokine interferon gamma (IFNG). Ligand binding stimulates activation of the JAK/STAT signaling pathway. It plays an essential role in the IFN-gamma pathway that is required for the cellular response to infectious agents. A genetic variation in IFNGR1 is associated with susceptibility to Helicobacter pylori infection. In addition, defects in IFNGR1 are a cause of Mendelian susceptibility to mycobacterial disease, also known as familial disseminated atypical mycobacterial infection.
In embodiments, a heterodimeric protein useful in the present invention comprises a variant of the extracellular domain of IFNgR. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known amino acid sequence of IFNgR, e.g., human IFNgR.
In embodiments, the extracellular domain of IFNgR has the following amino acid sequence:







(SEQ ID NO: 30)


EMGTADLGPSSVPTPTNVTIESYNMNPIVYWEYQIMPQVPVFTVEVKNYG


 


VKNSEWIDACINISHHYCNISDHVGDPSNSLWVRVKARVGQKESAYAKSE


 


EFAVCRDGKIGPPKLDIRKEEKQIMIDIFHPSVFVNGDEQEVDYDPETTC


 


YIRVYNVYVRMNGSEIQYKILTQKEDDCDEIQCQLAIPVSSLNSQYCVSA


 


EGVLHVWGVTTEKSKEVCITIFNSSIKG






In embodiments, a heterodimeric protein comprises a variant of the extracellular domain of IFNgR. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 30.
In embodiments, one chain of the heterodimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 30.
One of ordinary skill may select variants of the known amino acid sequence of IFNgR by consulting the literature, e.g., Aguet et al., “Molecular cloning and expression of the human interferon-gamma receptor” Cell 55 (2), 273-280 (1988); Stuber et al., “Alignment of disulfide bonds of the extracellular domain of the interferon gamma receptor and investigation of their role in biological activity”, Biochemistry 32 (9), 2423-2430 (1993); Sakatsume et al., “The Jak kinases differentially associate with the alpha and beta (accessory factor) chains of the interferon gamma receptor to form a functional receptor unit capable of activating STAT transcription factors”, J. Biol. Chem. 270 (29), 17528-17534 (1995); Walter et al., “Crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor”, Nature 376 (6537), 230-235 (1995); Sogabe et al., “Neutralizing epitopes on the extracellular interferon gamma receptor (IFNgammaR) alpha-chain characterized by homolog scanning mutagenesis and X-ray crystal structure of the A6 fab-IFNgammaR1-108 complex”, J. Mol. Biol. 273 (4), 882-897 (1997); Thiel et al., “Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex”, Structure 8 (9), 927-936 (2000); van de Wetering et al., “Functional analysis of naturally occurring amino acid substitutions in human IFN-gammaR1.” Mol. Immunol. 47:1023-1030(2010), each of which is incorporated by reference in its entirety.
In embodiments, a heterodimeric protein useful in the present invention comprises the extracellular domain of IFNGR2. IFNGR2 (also known as Interferon gamma receptor 2, also known as IFN-gamma receptor 2, and IFN-gamma-R2) is the non-ligand-binding beta chain of the gamma interferon receptor. Human interferon-gamma receptor is a heterodimer of IFNGR1 and IFNGR2. Ligand binding stimulates activation of the JAK/STAT signaling pathway. IFNGR2 is required for signal transduction in contrast to other receptor subunit responsible for ligand binding. Defects in IFNGR2 are a cause of Mendelian susceptibility to mycobacterial disease (MSMD), also known as familial disseminated atypical mycobacterial infection. MSMD is a genetically heterogeneous disease with autosomal recessive, autosomal dominant or X-linked inheritance.
In embodiments, a heterodimeric protein useful in the present invention comprises a variant of the extracellular domain of IFNGR2. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the known amino acid sequence of IFNGR2, e.g., human IFNGR2.
In embodiments, the extracellular domain of IFNGR2 has the following amino acid sequence:







(SEQ ID NO: 31)


SQLPAPQHPKIRLYNAEQVLSWEPVALSNSTRPVVYQVQFKYTDSKWFTA


 


DIMSIGVNCTQITATECDFTAASPSAGFPMDFNVTLRLRAELGALHSAWV


 


TMPWFQHYRNVTVGPPENIEVTPGEGSLIIRFSSPFDIADTSTAFFCYYV


 


HYWEKGGIQQVKGPFRSNSISLDNLKPSRVYCLQVQAQLLWNKSNIFRVG


 


HLSNISCYETMADASTELQQ






In embodiments, a heterodimeric protein comprises a variant of the extracellular domain of IFNGR2. As examples, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 31.
In embodiments, one chain of the heterodimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31.
One of ordinary skill may select variants of the known amino acid sequence of IFNGR2 by consulting the literature, e.g., Soh et al., “Identification and sequence of an accessory factor required for activation of the human interferon gamma receptor”, Cell 76 (5), 793-802 (1994); Sakatsume et al., “The Jak kinases differentially associate with the alpha and beta (accessory factor) chains of the interferon gamma receptor to form a functional receptor unit capable of activating STAT transcription factors”, J. Biol. Chem. 270 (29), 17528-17534 (1995); Rosenzweig et al., “Characterization of a dipeptide motif regulating IFN-gamma receptor 2 plasma membrane accumulation and IFN-gamma responsiveness”, J. Immunol. 173 (6), 3991-3999 (2004); Mikulecky et al., “Crystal structure of human interferon-gamma receptor 2 reveals the structural basis for receptor specificity”, Acta Crystallogr. D 75, 1017-1024 (2016); Kotenko et al., “Interaction between the components of the interferon gamma receptor complex.” J. Biol. Chem. 270:20915-20921(1995), each of which is incorporated by reference in its entirety.
In embodiments, an alpha chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16 or SEQ ID NO: 24. Such an alpha chain may be referred to as “IFNgR-Alpha-IL12a”.
In embodiments, an IFNgR-Alpha-IL12α chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 32)


EMGTADLGPSSVPTPTNVTIESYNMNPIVYWEYQIMPQVPVFTVEVKNYG


 


VKNSEWIDACINISHHYCNISDHVGDPSNSLWVRVKARVGQKESAYAKSE


 


EFAVCRDGKIGPPKLDIRKEEKQIMIDIFHPSVFVNGDEQEVDYDPETTC


 


YIRVYNVYVRMNGSEIQYKILTQKEDDCDEIQCQLAIPVSSLNSQYCVSA


 


EGVLHVWGVTTEKSKEVCITIFNSSIKGGSGSRKGGKRGSKYGPPCPPCP


 


APEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVD


 


GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPS


 


SIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE


 


WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHE


 


ALHNHYTQKSLSLSLGKDEGGEDGSGSRNLPVATPDPGMFPCLHHSQNLL


 


RAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNES


 


CLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKL


 


LMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKL


 


CILLHAFRIRAVTIDRVMSYLNAS






In embodiments, an beta chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17 or SEQ ID NO: 25. Such a beta chain may be referred to as “IFNGR2-Beta-IL27B”.
In embodiments, an IFNGR2-Beta-IL27B chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 33)


SQLPAPQHPKIRLYNAEQVLSWEPVALSNSTRPVVYQVQFKYTDSKWFTA


 


DIMSIGVNCTQITATECDFTAASPSAGFPMDFNVTLRLRAELGALHSAWV


 


TMPWFQHYRNVTVGPPENIEVTPGEGSLIIRFSSPFDIADTSTAFFCYYV


 


HYWEKGGIQQVKGPFRSNSISLDNLKPSRVYCLQVQAQLLWNKSNIFRVG


 


HLSNISCYETMADASTELQQGSGSDEGGEDGSKYGPPCPPCPAPEFLGGP


 


SVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK


 


TKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISN


 


ATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE


 


NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQ


 


KSLSLSLGKRKGGKRGSGSRKGPPAALTLPRVQCRASRYPIAVDCSWTLP


 


PAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAP


 


YVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEP


 


PGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYV


 


QVAAQDLTDYGELSDWSLPATATMSLGK






When an IFNgR-Alpha-IL12α chain and an IFNGR2-Beta-IL27B chain are combined (within a cell or in vitro), they form a heterodimeric protein referred to herein as IFNγR-Fc-IL-35.
In embodiments, an alpha chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17 or SEQ ID NO: 25. Such an alpha chain may be referred to as “IFNgR-Beta-IL12a”.
In embodiments, an IFNgR-Beta-IL12α chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 38)


EMGTADLGPSSVPTPTNVTIESYNMNPIVYWEYQIMPQVPVFTVEVKNYG


 


VKNSEWIDACINISHHYCNISDHVGDPSNSLWVRVKARVGQKESAYAKSE


 


EFAVCRDGKIGPPKLDIRKEEKQIMIDIFHPSVFVNGDEQEVDYDPETTC


 


YIRVYNVYVRMNGSEIQYKILTQKEDDCDEIQCQLAIPVSSLNSQYCVSA


 


EGVLHVWGVTTEKSKEVCITIFNSSIKGGSGSDEGGEDGSKYGPPCPPCP


 


APEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVD


 


GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPS


 


SIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE


 


WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHE


 


ALHNHYTQKSLSLSLGKRKGGKRGSGSRNLPVATPDPGMFPCLHHSQNLL


 


RAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNES


 


CLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKL


 


LMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKL


 


CILLHAFRIRAVTIDRVMSYLNAS






In embodiments, an beta chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16 or SEQ ID NO: 24. Such a beta chain may be referred to as “IFNGR2-Alpha-IL27B”.
In embodiments, an IFNGR2-Alpha-IL27B chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 39)


SQLPAPQHPKIRLYNAEQVLSWEPVALSNSTRPVVYQVQFKYTDSKWFTA


 


DIMSIGVNCTQITATECDFTAASPSAGFPMDFNVTLRLRAELGALHSAWV


 


TMPWFQHYRNVTVGPPENIEVTPGEGSLIIRFSSPFDIADTSTAFFCYYV


 


HYWEKGGIQQVKGPFRSNSISLDNLKPSRVYCLQVQAQLLWNKSNIFRVG


 


HLSNISCYETMADASTELQQGSGSRKGGKRGSKYGPPCPPCPAPEFLGGP


 


SVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK


 


TKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISN


 


ATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE


 


NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQ


 


KSLSLSLGKDEGGEDGSGSRKGPPAALTLPRVQCRASRYPIAVDCSWTLP


 


PAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAP


 


YVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEP


 


PGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYV


 


QVAAQDLTDYGELSDWSLPATATMSLGK






When an IFNgR-Beta-IL12α chain and an IFNGR2-Alpha-IL27B chain are combined (within a cell or in vitro), they form a heterodimeric protein may also be referred to herein as IFNγR-Fc-IL-35.
One embodiment of the IL-6R-Fc-IL-35 heterodimeric protein is disclosed above, i.e., comprising a Gp130-Alpha-IL12A chain and an IL6RA-Beta-IL27B chain. In alternate embodiment, an IL-6R-Fc-IL-35 heterodimeric protein can comprise an IL6RA-Alpha-IL12a chain and a Gp130-Beta-IL27b.
In embodiments, an alpha chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16 or SEQ ID NO: 24. Such an alpha chain may be referred to as “IL6RA-Alpha-IL12a”.
In embodiments, an IL6RA-Alpha-IL12α chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 34)


LAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATVHWVLRKPAAGS


 


HPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVHLLVDVPPEEPQL


 


CFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQE


 


SQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPA


 


NITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMV


 


KDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRS


 


PPAENEVSTPMQALTTNKDDDNILFRDSANATSLPVQDSSSVPLPGSGSR


 


KGGKRGSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVV


 


VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW


 


LSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQV


 


SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD


 


KSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKDEGGEDGSGSRNLPVA


 


TPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDK


 


TSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIY


 


EDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVP


 


QKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS






In embodiments, an beta chain useful in a heterodimeric chimeric protein of the present invention comprises: (1) a first domain comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof, (b) a second domain comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain, or variant thereof, which comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17 or SEQ ID NO: 25. Such a beta chain may be referred to as “Gp130-Beta-IL27b”.
In embodiments, a Gp130-Beta-IL27b chain used in the present invention and has the following amino acid sequence:







(SEQ ID NO: 35)


ELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHF


 


TIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIIS


 


GLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADC


 


KAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKP


 


NPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQI


 


PPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITY


 


EDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTR


 


WKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQ


 


ATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQ


 


EDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKG


 


PTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDS


 


SHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIE






When an IL6RA-Alpha-IL12a chain and a Gp130-Beta-IL27b chain are combined (within a cell or in vitro), they form a heterodimeric protein may also be referred to herein as IL-6R-Fc-IL-35.
In various embodiments, the present heterodimeric protein may comprise variants of any of the known cytokines, growth factors, and/or hormones. In various embodiments, the present heterodimeric proteins may comprise variants of any of the known receptors for cytokines, growth factors, and/or hormones. In various embodiments, the present heterodimeric proteins may comprises variants of any of the known extracellular domains, for instance, a sequence having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) sequence identity with the known amino acid or nucleic acid sequences.
In various embodiments, the present heterodimeric protein may comprise an amino acid sequence having one or more amino acid mutations relative to any of the known protein sequences. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.
“Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt α-helices.
As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
In various embodiments, the substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine β-alanine, GABA and 6-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β methyl amino acids, C α-methyl amino acids, N α-methyl amino acids, and amino acid analogs in general).
Mutations may also be made to the nucleotide sequences of the heterodimeric proteins by reference to the genetic code, including taking into account codon degeneracy.
In various embodiments, the present heterodimeric proteins are capable of, and can be used in methods comprising, promoting immune activation (e.g., against tumors). In various embodiments, the present heterodimeric proteins are capable of, and can be used in methods comprising, suppressing immune inhibition (e.g., that allows tumors to survive). In various embodiments, the present heterodimeric protein provides improved immune activation and/or improved suppression of immune inhibition.
In various embodiments, the present heterodimeric proteins are capable of, or can be used in methods comprising, modulating the amplitude of an immune response, e.g., modulating the level of effector output. In some embodiments, e.g., when used for the treatment of cancer, the present heterodimeric protein alters the extent of immune stimulation as compared to immune inhibition to increase the amplitude of a T cell response, including, without limitation, stimulating increased levels of cytokine production, proliferation or target killing potential.
In various embodiments, the present heterodimeric proteins, in some embodiments are capable of, or find use in methods involving, masking an inhibitory ligand on the surface of a tumor cell and replacing that immune inhibitory ligand with an immune stimulatory ligand. Accordingly, the present heterodimeric proteins, in some embodiments are capable of, or find use in methods involving, reducing or eliminating an inhibitory immune signal and/or increasing or activating an immune stimulatory signal. For example, a tumor cell bearing an inhibitory signal (and thus evading an immune response) may be substituted for a positive signal binding on a T cell that can then attack a tumor cell. Accordingly, in some embodiments, an inhibitory immune signal is masked by the present heterodimeric proteins and a stimulatory immune signal is activated. Such beneficial properties are enhanced by the single construct approach of the present heterodimeric proteins. For instance, the signal replacement can be effected nearly simultaneously and the signal replacement is tailored to be local at a site of clinical importance (e.g., the tumor microenvironment).
In various embodiments, the present heterodimeric proteins are capable of, or find use in methods comprising, stimulating or enhancing the binding of immune stimulatory receptor/ligand pairs.
In other embodiments, the present heterodimeric proteins are capable of, or find use in methods involving, enhancing, restoring, promoting and/or stimulating immune modulation. In some embodiments, the present heterodimeric proteins described herein, restore, promote and/or stimulate the activity or activation of one or more immune cells against tumor cells including, but not limited to: T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendritic cells. In some embodiments, the present heterodimeric proteins enhance, restore, promote and/or stimulate the activity and/or activation of T cells, including, by way of a non-limiting example, activating and/or stimulating one or more T-cell intrinsic signals, including a pro-survival signal; an autocrine or paracrine growth signal; a p38 MAPK-, ERK-, STAT-, JAK-, AKT- or PI3K-mediated signal; an anti-apoptotic signal; and/or a signal promoting and/or necessary for one or more of: proinflammatory cytokine production or T cell migration or T cell tumor infiltration.
In some embodiments, the present heterodimeric proteins are capable of, or find use in methods involving, causing an increase of one or more of T cells (including without limitation cytotoxic T lymphocytes, T helper cells, natural killer T (NKT) cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, and macrophages (e.g., one or more of M1 and M2) into a tumor or the tumor microenvironment. In some embodiments, the present heterodimeric proteins are capable of, or find use in methods involving, inhibiting and/or causing a decrease in recruitment of immunosuppressive cells (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs)) to the tumor and/or tumor microenvironment (TME). In some embodiments, the present therapies may alter the ratio of M1 versus M2 macrophages in the tumor site and/or TME to favor M1 macrophages.
In various embodiments, the present heterodimeric proteins are capable of, and can be used in methods comprising, inhibiting and/or reducing T cell inactivation and/or immune tolerance to a tumor, comprising administering an effective amount of a heterodimeric protein described herein to a subject. In some embodiments, the present heterodimeric proteins are able to increase the serum levels of various cytokines including, but not limited to, one or more of IFNγ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, and IL-22. In some embodiments, the present heterodimeric proteins are capable of enhancing IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, or IFNγ in the serum of a treated subject.
In various embodiments, the present heterodimeric proteins inhibit, block and/or reduce cell death of an anti-tumor CD8+ and/or CD4+ T cell; or stimulate, induce, and/or increase cell death of a pro-tumor T cell. T cell exhaustion is a state of T cell dysfunction characterized by progressive loss of proliferative and effector functions, culminating in clonal deletion. Accordingly, a pro-tumor T cell refers to a state of T cell dysfunction that arises during many chronic infections and cancer. This dysfunction is defined by poor proliferative and/or effector functions, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. In addition, an anti-tumor CD8+ and/or CD4+ T cell refers to T cells that can mount an immune response to a tumor. Illustrative pro-tumor T cells include, but are not limited to, Tregs, CD4+ and/or CD8+ T cells expressing one or more checkpoint inhibitory receptors, Th2 cells and Th17 cells. Checkpoint inhibitory receptors refers to receptors (e.g., CTLA-4, B7-H3, B7-H4, TIM-3) expressed on immune cells that prevent or inhibit uncontrolled immune responses.
In various embodiments, the present heterodimeric proteins are capable of, and can be used in methods comprising, increasing a ratio of effector T cells to regulatory T cells. Illustrative effector T cells include ICOS+ effector T cells; cytotoxic T cells (e.g., αβ TCR, CD3+, CD8+, CD45RO+); CD4+ effector T cells (e.g., αβ TCR, CD3+, CD4+, CCR7+, CD62Lhi, IL−7R/CD127+); CD8+ effector T cells (e.g., αβ TCR, CD3+, CD8+, CCR7+, CD62Lhi, IL−7 R/CD127+); effector memory T cells (e.g., CD62Llow, CD44+, TCR, CD3+, IL7R/CD127+, IL-15R+, CCR7low); central memory T cells (e.g., CCR7+, CD62L+, CD27+; or CCR7hi, CD44+, CD62Lhi, TCR, CD3+, IL-7R/CD127+, IL-15 R+); CD62L+ effector T cells; CD8+ effector memory T cells (TEM) including early effector memory T cells (CD27+CD62L−) and late effector memory T cells (CD27− CD62L−) (TemE and TemL, respectively); CD127(+)CD25(low/−) effector T cells; CD127(−)CD250 effector T cells; CD8+ stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca(+)); TH1 effector T-cells (e.g., CXCR3+, CXCR6+ and CCR5+; or αβ TCR, CD3+, CD4+, IL-12R+, IFNγR+, CXCR3+), TH2 effector T cells (e.g., CCR3+, CCR4+ and CCR8+; or αβ TCR, CD3+, CD4+, IL-4R+, IL-33R+, CCR4+, IL-17RB+, CRTH2+); TH9 effector T cells (e.g., αβ TCR, CD3+, CD4+); TH17 effector T cells (e.g., αβ TCR, CD3+, CD4+, IL-23R+, CCR6+, IL-1R+); CD4+CD45RO+CCR7+ effector T cells, CD4+CD45RO+CCR7(−) effector T cells; and effector T cells secreting IL-2, IL-4 and/or IFN-γ. Illustrative regulatory T cells include ICOS+ regulatory T cells, CD4+CD25+FOXP3+ regulatory T cells, CD4+CD25+ regulatory T cells, CD4+CD25− regulatory T cells, CD4+CD25high regulatory T cells, TIM-3+PD-1+ regulatory T cells, lymphocyte activation gene-3 (LAG-3)+ regulatory T cells, CTLA-4/CD152+ regulatory T cells, neuropilin-1 (Nrp-1)+ regulatory T cells, CCR4+CCR8+ regulatory T cells, CD62L (L-selectin)+ regulatory T cells, CD45RBlow regulatory T cells, CD127low regulatory T cells, LRRC32/GARP+ regulatory T cells, CD39+ regulatory T cells, GITR+ regulatory T cells, LAP+ regulatory T cells, 1B11+ regulatory T cells, BTLA+ regulatory T cells, type 1 regulatory T cells (Tr1 cells), T helper type 3 (Th3) cells, regulatory cell of natural killer T cell phenotype (NKTregs), CD8+ regulatory T cells, CD8+CD28− regulatory T cells and/or regulatory T-cells secreting IL-10, IL-35, TGF-β, TNF-α, Galectin-1, IFN-γ and/or MCP1.
In various embodiments, the present heterodimeric proteins are capable of, and can be used in methods comprising, transiently stimulating effector T cells for no longer than about 12 hours, about 24 hours, about 48 hours, about 72 hours or about 96 hours or about 1 week or about 2 weeks. In various embodiments, the present heterodimeric proteins are capable of, and can be used in methods comprising, transiently depleting or inhibiting regulatory T cells for no longer than about 12 hours, about 24 hours, about 48 hours, about 72 hours or about 96 hours or about 1 week or about 2 weeks. In various embodiments, the transient stimulation of effector T cells and/or transient depletion or inhibition of regulatory T cells occurs substantially in a patient's bloodstream or in a particular tissue/location including lymphoid tissues such as for example, the bone marrow, lymph-node, spleen, thymus, mucosa-associated lymphoid tissue (MALT), non-lymphoid tissues, or in the tumor microenvironment.
In various embodiments, the present heterodimeric proteins provide advantages including, without limitation, ease of use and ease of production. This is because two distinct immunotherapy agents are combined into a single product which allows for a single manufacturing process instead of two independent manufacturing processes. In addition, administration of a single agent instead of two separate agents allows for easier administration and greater patient compliance. Further, in contrast to, for example, monoclonal antibodies, which are large multimeric proteins containing numerous disulfide bonds and post-translational modifications such as glycosylation, the present heterodimeric proteins are easier and more cost effective to manufacture.
In various embodiments, the present heterodimeric proteins provide synergistic therapeutic effects as it allows for improved site-specific interplay of two immunotherapy agents. In some embodiments, the present heterodimeric proteins provide the potential for reducing off-site and/or systemic toxicity.
Diseases; Methods of Treatment, and Patient Selections
In various embodiments, the present invention pertains to the use of the heterodimeric proteins for the treatment of one or more autoimmune diseases or disorders. In various embodiments, the treatment of an autoimmune disease or disorder may involve modulating the immune system with the present heterodimeric proteins to favor immune inhibition over immune stimulation. Illustrative autoimmune diseases or disorders treatable with the present heterodimeric proteins include those in which the body's own antigens become targets for an immune response, such as, for example, rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), and vasculitis.
Exemplary autoimmune diseases or conditions that may be treated or prevented using the heterodimeric protein of the invention include, but are not limited to, multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome; transplantation rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjögren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, and other autoimmune diseases.
In various embodiments, the present invention pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors. As described elsewhere herein, the treatment of cancer may involve in various embodiments, modulating the immune system with the present heterodimeric proteins to favor immune stimulation over immune inhibition.
Cancers or tumors refer to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Also, included are cells having abnormal proliferation that is not impeded by the immune system (e.g., virus infected cells). The cancer may be a primary cancer or a metastatic cancer. The primary cancer may be an area of cancer cells at an originating site that becomes clinically detectable, and may be a primary tumor. In contrast, the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part. The metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis. The cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body. The cancer may be due to a process such as lymphatic or hematogeneous spread. The cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor. The cancer may be this new tumor, which may be a metastatic (or secondary) tumor.
The cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor. The cells of the tumor may be like those in the original tumor. As an example, if a breast cancer or colon cancer metastasizes to the liver, the secondary tumor, while present in the liver, is made up of abnormal breast or colon cells, not of abnormal liver cells. The tumor in the liver may thus be a metastatic breast cancer or a metastatic colon cancer, not liver cancer.
The cancer may have an origin from any tissue. The cancer may originate from melanoma, colon, breast, or prostate, and thus may be made up of cells that were originally skin, colon, breast, or prostate, respectively. The cancer may also be a hematological malignancy, which may be leukemia or lymphoma. The cancer may invade a tissue such as liver, lung, bladder, or intestinal.
Representative cancers and/or tumors of the present invention include, but are not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
In some embodiments, the heterodimeric protein is used to treat a subject that has a treatment-refractory cancer. In some embodiments, the heterodimeric protein is used to treat a subject that is refractory to one or more immune-modulating agents. For example, in some embodiments, the heterodimeric protein is used to treat a subject that presents no response to treatment, or even progress, after 12 weeks or so of treatment. For instance, in some embodiments, the subject is refractory to a PD-1 and/or PD-L1 and/or PD-L2 agent, including, for example, nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), Ibrutinib (PHARMACYCLICS/ABBVIE), atezolizumab (TECENTRIQ, GENENTECH), and/or MPDL328OA (ROCHE)-refractory patients. For instance, in some embodiments, the subject is refractory to an anti-CTLA-4 agent, e.g., ipilimumab (YERVOY)-refractory patients (e.g., melanoma patients). Accordingly, in various embodiments, the present invention provides methods of cancer treatment that rescue patients that are non-responsive to various therapies, including monotherapy of one or more immune-modulating agents.
In various embodiments, the present invention provides heterodimeric proteins which target a cell or tissue within the tumor microenviroment. In some embodiments, the cell or tissue within the tumor microenvironment expresses one or more targets or binding partners of the heterodimeric protein. The tumor microenvironment refers to the cellular milieu, including cells, secreted proteins, physiological small molecules, and blood vessels in which the tumor exists. In some embodiments, the cells or tissue within the tumor microenvironment are one or more of: tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor. In various embodiments, the present heterodimeric protein targets a cancer cell. In some embodiments, the cancer cell expresses one or more of targets or binding partners of the heterodimeric protein.
In various embodiments, the heterodimeric protein of the invention may target a cell (e.g., cancer cell or immune cell) that expresses any of the receptors as described herein. For example, the heterodimeric protein of the invention may target a cell that expresses any of the receptors for a cytokine, growth factor, and/or hormone as described herein.
In some embodiments, the present methods provide treatment with the heterodimeric protein in a patient who is refractory to an additional agent, such “additional agents” being described elsewhere herein, inclusive, without limitation, of the various chemotherapeutic agents described herein.
In some aspects, the present chimeric agents are used to eliminate intracellular pathogens. In some aspects, the present chimeric agents are used to treat one or more infections. In some embodiments, the present heterodimeric proteins are used in methods of treating viral infections (including, for example, HIV and HCV), parasitic infections (including, for example, malaria), and bacterial infections. In various embodiments, the infections induce immunosuppression. For example, HIV infections often result in immunosuppression in the infected subjects. Accordingly, as described elsewhere herein, the treatment of such infections may involve, in various embodiments, modulating the immune system with the present heterodimeric proteins to favor immune stimulation over immune inhibition. Alternatively, the present invention provides methods for treating infections that induce immunoactivation. For example, intestinal helminth infections have been associated with chronic immune activation. In these embodiments, the treatment of such infections may involve modulating the immune system with the present heterodimeric proteins to favor immune inhibition over immune stimulation.
In various embodiments, the present invention provides methods of treating viral infections including, without limitation, acute or chronic viral infections, for example, of the respiratory tract, of papilloma virus infections, of herpes simplex virus (HSV) infection, of human immunodeficiency virus (HIV) infection, and of viral infection of internal organs such as infection with hepatitis viruses. In some embodiments, the viral infection is caused by a virus of family Flaviviridae. In some embodiments, the virus of family Flaviviridae is selected from Yellow Fever Virus, West Nile virus, Dengue virus, Japanese Encephalitis Virus, St. Louis Encephalitis Virus, and Hepatitis C Virus. In other embodiments, the viral infection is caused by a virus of family Picornaviridae, e.g., poliovirus, rhinovirus, coxsackievirus. In other embodiments, the viral infection is caused by a member of Orthomyxoviridae, e.g., an influenza virus. In other embodiments, the viral infection is caused by a member of Retroviridae, e.g., a lentivirus. In other embodiments, the viral infection is caused by a member of Paramyxoviridae, e.g., respiratory syncytial virus, a human parainfluenza virus, rubulavirus (e.g., mumps virus), measles virus, and human metapneumovirus. In other embodiments, the viral infection is caused by a member of Bunyaviridae, e.g., hantavirus. In other embodiments, the viral infection is caused by a member of Reoviridae, e.g., a rotavirus.
In various embodiments, the present invention provides methods of treating parasitic infections such as protozoan or helminths infections. In some embodiments, the parasitic infection is by a protozoan parasite. In some embodiments, the oritiziab parasite is selected from intestinal protozoa, tissue protozoa, or blood protozoa. Illustrative protozoan parasites include, but are not limited to, Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonas meleagridis. In some embodiments, the parasitic infection is by a helminthic parasite such as nematodes (e.g., Adenophorea). In some embodiments, the parasite is selected from Secementea (e.g., Trichuris trichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, Dracunculus medinensis). In some embodiments, the parasite is selected from trematodes (e.g., blood flukes, liver flukes, intestinal flukes, and lung flukes). In some embodiments, the parasite is selected from: Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyes heterophyes, Paragonimus westermani. In some embodiments, the parasite is selected from cestodes (e.g., Taenia solium, Taenia saginata, Hymenolepis nana, Echinococcus granulosus).
In various embodiments, the present invention provides methods of treating bacterial infections. In various embodiments, the bacterial infection is by gram-positive bacteria, gram-negative bacteria, aerobic and/or anaerobic bacteria. In various embodiments, the bacteria are selected from, but not limited to, Staphylococcus, Lactobacillus, Streptococcus, Sarcina, Escherichia, Enterobacter, Klebsiella, Pseudomonas, Acinetobacter, Mycobacterium, Proteus, Campylobacter, Citrobacter, Neisseria, Bacillus, Bacteroides, Peptococcus, Clostridium, Salmonella, Shigella, Serratia, Haemophilus, Brucella and other organisms. In some embodiments, the bacteria is selected from, but not limited to, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, or Staphylococcus saccharolyticus. 
In still another other aspect, the present invention is directed toward methods of treating and preventing T cell-mediated diseases and disorders, such as, but not limited to diseases or disorders described elsewhere herein and inflammatory disease or disorder, graft-versus-host disease (GVHD), transplant rejection, and T cell proliferative disorder.
In some aspects, the present chimeric agents are used in methods of activating a T cell, e.g., via the extracellular domain having an immune stimulatory signal.
In some aspects, the present chimeric agents are used in methods of preventing the cellular transmission of an immunosuppressive signal.
Combination Therapies and Conjugation
In some embodiments, the invention provides for heterodimeric proteins and methods that further comprise administering an additional agent to a subject. In some embodiments, the invention pertains to co-administration and/or co-formulation. Any of the compositions described herein may be co-formulated and/or co-administered.
In some embodiments, any heterodimeric protein described herein acts synergistically when co-administered with another agent and is administered at doses that are lower than the doses commonly employed when such agents are used as monotherapy. In various embodiments, any agent referenced herein may be used in combination with any of the heterodimeric proteins described herein.
In various embodiments, any of the heterodimeric proteins disclosed herein may be co-administered with another heterodimeric protein disclosed herein. Without wishing to be bound by theory, it is believed that a combined regimen involving the administration of one or more heterodimeric proteins which induce an innate immune response and one or more heterodimeric proteins which induce an adaptive immune response may provide synergistic effects (e.g., synergistic anti-tumor effects).
In various embodiments, any heterodimeric protein which induces an innate immune response may be utilized in the present invention. In various embodiments, any heterodimeric protein which induces an adaptive immune response may be utilized in the present invention.
In some embodiments, inclusive of, without limitation, cancer applications, the present invention pertains to chemotherapeutic agents as additional agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as minoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2′′-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111), and TAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with κ-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation. In addition, the methods of treatment can further include the use of photodynamic therapy.
In various embodiments, inclusive of, without limitation, cancer applications, the present additional agent is one or more immune-modulating agents selected from an agent that blocks, reduces and/or inhibits PD-1 and PD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way of non-limiting example, one or more of nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), atezolizumab (TECENTRIQ, GENENTECH), MPDL328OA (ROCHE)), an agent that increases and/or stimulates CD137 (4-1BB) and/or the binding of CD137 (4-1BB) with one or more of 4-1BB ligand (by way of non-limiting example, urelumab (BMS-663513 and anti-4-1BB antibody), and an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A and/or the binding of OX40 with OX40L (by way of non-limiting example GBR 830 (GLENMARK), MED16469 (MEDIMMUNE).
In some embodiments, inclusive of, without limitation, infectious disease applications, the present invention pertains to anti-infectives as additional agents. In some embodiments, the anti-infective is an anti-viral agent including, but not limited to, Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, and Foscarnet. In some embodiments, the anti-infective is an anti-bacterial agent including, but not limited to, cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem). In some embodiments, the anti-infectives include anti-malarial agents (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.
In some embodiments, inclusive, without limitation, of autoimmune applications, the additional agent is an immunosuppressive agent. In some embodiments, the immunosuppressive agent is an anti-inflammatory agent such as a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent (NSAID). Steroids, particularly the adrenal corticosteroids and their synthetic analogues, are well known in the art. Examples of corticosteroids useful in the present invention include, without limitation, hydroxyltriamcinolone, alpha-methyl dexamethasone, beta-methyl betamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, clobetasol valerate, desonide, desoxymethasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, clocortelone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate. (NSAIDS) that may be used in the present invention, include but are not limited to, salicylic acid, acetyl salicylic acid, methyl salicylate, glycol salicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin. In some embodiments, the immunosupressive agent may be cytostatics such as alkylating agents, antimetabolites (e.g., azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g., cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF binding proteins, mycophenolates, and small biological agents (e.g., fingolimod, myriocin).
In some embodiments, the heterodimeric proteins (and/or additional agents) described herein, include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition. For example, but not by way of limitation, derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of turicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids. In still other embodiments, the heterodimeric proteins (and/or additional agents) described herein further comprise a cytotoxic agent, comprising, in illustrative embodiments, a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death. Such agents may be conjugated to a composition described herein.
The heterodimeric proteins (and/or additional agents) described herein may thus be modified post-translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
Formulations
The heterodimeric proteins (and/or additional agents) described herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.
In some embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt.
Further, any heterodimeric protein (and/or additional agents) described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration. Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.
In some embodiments, the compositions described herein are resuspended in a saline buffer (including, without limitation TBS, PBS, and the like).
In various embodiments, the heterodimeric proteins may by conjugated and/or fused with another agent to extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties. In some embodiments, the heterodimeric proteins may be fused or conjugated with one or more of PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, and the like. In various embodiments, each of the individual heterodimeric proteins is fused to one or more of the agents described in BioDrugs (2015) 29:215-239, the entire contents of which are hereby incorporated by reference.
Administration, Dosing, and Treatment Regimens
The present invention includes the described heterodimeric protein (and/or additional agents) in various formulations. Any heterodimeric protein (and/or additional agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. DNA or RNA constructs encoding the protein sequences may also be used. In one embodiment, the composition is in the form of a capsule (see, e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.
Where necessary, the formulations comprising the heterodimeric protein (and/or additional agents) can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.
The formulations comprising the heterodimeric protein (and/or additional agents) of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).
In one embodiment, any heterodimeric protein (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.
Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In some embodiments, the administering is effected orally or by parenteral injection. In most instances, administration results in the release of any agent described herein into the bloodstream.
Any heterodimeric protein (and/or additional agents) described herein can be administered orally. Such heterodimeric proteins (and/or additional agents) can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer.
In specific embodiments, it may be desirable to administer locally to the area in need of treatment. In one embodiment, for instance in the treatment of cancer, the heterodimeric protein (and/or additional agents) are administered in the tumor microenvironment (e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell, inclusive of, for example, tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor) or lymph node and/or targeted to the tumor microenvironment or lymph node. In various embodiments, for instance in the treatment of cancer, the heterodimeric protein (and/or additional agents) are administered intratumorally.
In the various embodiments, the present heterodimeric protein allows for a dual effect that provides less side effects than are seen in conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ). For example, the present heterodimeric proteins reduce or prevent commonly observed immune-related adverse events that affect various tissues and organs including the skin, the gastrointestinal tract, the kidneys, peripheral and central nervous system, liver, lymph nodes, eyes, pancreas, and the endocrine system; such as hypophysitis, colitis, hepatitis, pneumonitis, rash, and rheumatic disease. Further, the present local administration, e.g., intratumorally, obviate adverse event seen with standard systemic administration, e.g., IV infusions, as are used with conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ).
Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
The dosage of any heterodimeric protein (and/or additional agents) described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject's general health, and the administering physician's discretion. Any heterodimeric protein described herein, can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional agent, to a subject in need thereof. In various embodiments any heterodimeric protein and additional agent described herein are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.
In various embodiments, the present invention relates to the co-administration of a heterodimeric protein which induces an innate immune response and another heterodimeric protein which induces an adaptive immune response. In such embodiments, the heterodimeric protein which induces an innate immune response may be administered before, concurrently with, or subsequent to administration of the heterodimeric protein which induces an adaptive immune response. For example, the heterodimeric proteins may be administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart. In an exemplary embodiment, the heterodimeric protein which induces an innate immune response and the heterodimeric protein which induces an adaptive response are administered 1 week apart, or administered on alternate weeks (i.e., administration of the heterodimeric protein inducing an innate immune response is followed 1 week later with administration of the heterodimeric protein which induces an adaptive immune response and so forth).
The dosage of any heterodimeric protein (and/or additional agents) described herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.
For administration of any heterodimeric protein (and/or additional agents) described herein by parenteral injection, the dosage may be about 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day, or about 3 mg to about 5 mg per day. Generally, when orally or parenterally administered, the dosage of any agent described herein may be about 0.1 mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mg to about 5 mg per day, or about 200 to about 1,200 mg per day (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about 1,200 mg per day).
In some embodiments, administration of the heterodimeric protein (and/or additional agents) described herein is by parenteral injection at a dosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mg to about 10 mg per treatment, or about 0.5 mg to about 5 mg per treatment, or about 200 to about 1,200 mg per treatment (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about 1,200 mg per treatment).
In some embodiments, a suitable dosage of the heterodimeric protein (and/or additional agents) is in a range of about 0.01 mg/kg to about 100 mg/kg of body weight, or about 0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg body weight, inclusive of all values and ranges therebetween.
In another embodiment, delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).
Any heterodimeric protein (and/or additional agents) described herein can be administered by controlled-release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.
In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).
In another embodiment, a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer, 1990, Science 249:1527-1533) may be used.
Administration of any heterodimeric protein (and/or additional agents) described herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject.
The dosage regimen utilizing any heterodimeric protein (and/or additional agents) described herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the invention employed. Any heterodimeric protein (and/or additional agents) described herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. Furthermore, any heterodimeric protein (and/or additional agents) described herein can be administered continuously rather than intermittently throughout the dosage regimen.
Cells and Nucleic Acids
In various embodiments, the present invention provides an expression vector, comprising a nucleic acid encoding the heterodimeric protein (e.g., a heterodimeric protein comprising a first and second polypeptide chains) described herein. In various embodiments, the expression vector comprises DNA or RNA. In various embodiments, the expression vector is a mammalian expression vector.
Both prokaryotic and eukaryotic vectors can be used for expression of the heterodimeric protein. Prokaryotic vectors include constructs based on E. coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538). Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, lpp, phoA, recA, tac, T3, T7 and λPL. Non-limiting examples of prokaryotic expression vectors may include the λgt vector series such as λgt11 (Huynh et al., in “DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studier et al., Methods Enzymol 1990, 185:60-89). Prokaryotic host-vector systems cannot perform much of the post-translational processing of mammalian cells, however. Thus, eukaryotic host-vector systems may be particularly useful. A variety of regulatory regions can be used for expression of the heterodimeric proteins in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used. Inducible promoters that may be useful in mammalian cells include, without limitation, promoters associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the β-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters also may be advantageous for driving expression of the fusion proteins in recombinant host cells.
In some embodiments, expression vectors of the invention comprise a nucleic acid encoding at least the first and/or second polypeptide chains of the heterodimeric proteins (and/or additional agents), or a complement thereof, operably linked to an expression control region, or complement thereof, that is functional in a mammalian cell. The expression control region is capable of driving expression of the operably linked blocking and/or stimulating agent encoding nucleic acid such that the blocking and/or stimulating agent is produced in a human cell transformed with the expression vector.
Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that influence expression of an operably linked nucleic acid. An expression control region of an expression vector of the invention is capable of expressing operably linked encoding nucleic acid in a human cell. In an embodiment, the cell is a tumor cell. In another embodiment, the cell is a non-tumor cell. In an embodiment, the expression control region confers regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.
In an embodiment, the present invention contemplates the use of inducible promoters capable of effecting high level of expression transiently in response to a cue. For example, when in the proximity of a tumor cell, a cell transformed with an expression vector for the heterodimeric protein (and/or additional agents) comprising such an expression control sequence is induced to transiently produce a high level of the agent by exposing the transformed cell to an appropriate cue. Illustrative inducible expression control regions include those comprising an inducible promoter that is stimulated with a cue such as a small molecule chemical compound. Particular examples can be found, for example, in U.S. Pat. Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941, each of which is incorporated herein by reference in its entirety.
Expression control regions and locus control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants which retain all or part of full-length or non-variant function. As used herein, the term “functional” and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence).
As used herein, “operable linkage” refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner. In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. Typically, an expression control region that modulates transcription is juxtaposed near the 5′ end of the transcribed nucleic acid (i.e., “upstream”). Expression control regions can also be located at the 3′ end of the transcribed sequence (i.e., “downstream”) or within the transcript (e.g., in an intron). Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is usually located 5′ of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5′ or 3′ of the transcribed sequence, or within the transcribed sequence.
Expression systems functional in human cells are well known in the art, and include viral systems. Generally, a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3′ terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived from SV40. Introns may also be included in expression constructs.
There are a variety of techniques available for introducing nucleic acids into viable cells. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc. For in vivo gene transfer, a number of techniques and reagents may also be used, including liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction. In some situations, it is desirable to provide a targeting agent, such as an antibody or ligand specific for a tumor cell surface membrane protein. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990).
Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), Flp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty, transposases of the mariner family (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). In addition, direct and targeted genetic integration strategies may be used to insert nucleic acid sequences encoding the chimeric fusion proteins including CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editing technologies.
In one aspect, the invention provides expression vectors for the expression of the heterodimeric proteins (and/or additional agents) that are viral vectors. Many viral vectors useful for gene therapy are known (see, e.g., Lundstrom, Trends Biotechnol., 21: 1 17, 122, 2003. Illustrative viral vectors include those selected from Antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (MV), and a viruses, though other viral vectors may also be used. For in vivo uses, viral vectors that do not integrate into the host genome are suitable for use, such as a viruses and adenoviruses. Illustrative types of a viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV). For in vitro uses, viral vectors that integrate into the host genome are suitable, such as retroviruses, AAV, and Antiviruses. In one embodiment, the invention provides methods of transducing a human cell in vivo, comprising contacting a solid tumor in vivo with a viral vector of the invention.
In various embodiments, the present invention provides a host cell, comprising the expression vector comprising the heterodimeric protein described herein.
Expression vectors can be introduced into host cells for producing the present heterodimeric proteins. Cells may be cultured in vitro or genetically engineered, for example. Useful mammalian host cells include, without limitation, cells derived from humans, monkeys, and rodents (see, for example, Kriegler in “Gene Transfer and Expression: A Laboratory Manual,” 1990, New York, Freeman & Co.). These include monkey kidney cell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651); human embryonic kidney lines (e.g., 293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol 1977, 36:59); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse sertoli cells (Mather, Biol Reprod 1980, 23:243-251); mouse fibroblast cells (e.g., NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); African green monkey kidney cells. (e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells (e.g., Hep G2, HB 8065); and mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51). Illustrative cancer cell types for expressing the fusion proteins described herein include mouse fibroblast cell line, NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC #2 and SCLC #7.
Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients with an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers.
Cells that can be used for production of the present heterodimeric proteins in vitro, ex vivo, and/or in vivo include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells (e.g., as obtained from bone marrow), umbilical cord blood, peripheral blood, fetal liver, etc. The choice of cell type depends on the type of tumor or infectious disease being treated or prevented, and can be determined by one of skill in the art.
Production and purification of Fc-containing macromolecules (such as Fc fusion proteins) has become a standardized process, with minor modifications between products. For example, many Fc containing macromolecules are produced by human embryonic kidney (HEK) cells (or variants thereof) or Chinese Hamster Ovary (CHO) cells (or variants thereof) or in some cases by bacterial or synthetic methods. Following production, the Fc containing macromolecules that are secreted by HEK or CHO cells are purified through binding to Protein A columns and subsequently ‘polished’ using various methods. Generally speaking, purified Fc containing macromolecules are stored in liquid form for some period of time, frozen for extended periods of time or in some cases lyophilized. In various embodiments, production of the heterodimeric proteins contemplated herein may have unique characteristics as compared to traditional Fc containing macromolecules. In certain examples, the heterodimeric proteins may be purified using specific chromatography resins, or using chromatography methods that do not depend upon Protein A capture. In other embodiments, the heterodimeric proteins may be purified in an oligomeric state, or in multiple oligomeric states, and enriched for a specific oligomeric state using specific methods. Without being bound by theory, these methods could include treatment with specific buffers including specified salt concentrations, pH and additive compositions. In other examples, such methods could include treatments that favor one oligomeric state over another. The heterodimeric proteins obtained herein may be additionally ‘polished’ using methods that are specified in the art. In some embodiments, the heterodimeric proteins are highly stable and able to tolerate a wide range of pH exposure (between pH 3-12), are able to tolerate a large number of freeze/thaw stresses (greater than 3 freeze/thaw cycles) and are able to tolerate extended incubation at high temperatures (longer than 2 weeks at 40 degrees C.). In other embodiments, the heterodimeric proteins are shown to remain intact, without evidence of degradation, deamidation, etc. under such stress conditions.
Subjects and/or Animals
In some embodiments, the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In other embodiments, the subject and/or animal is a non-mammal, such, for example, a zebrafish. In some embodiments, the subject and/or animal may comprise fluorescently-tagged cells (with e.g., GFP). In some embodiments, the subject and/or animal is a transgenic animal comprising a fluorescent cell.
In some embodiments, the subject and/or animal is a human. In some embodiments, the human is a pediatric human. In other embodiments, the human is an adult human. In other embodiments, the human is a geriatric human. In other embodiments, the human may be referred to as a patient.
In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
In other embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the non-human animal is a livestock animal.
Kits
The invention provides kits that can simplify the administration of any agent described herein. An illustrative kit of the invention comprises any composition described herein in unit dosage form. In one embodiment, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent described herein. The kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent described herein. In one embodiment, the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those described herein.
Definitions
As used in this Specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About is understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
A stated range is understood to be any value between and at the limits of the stated range. As examples, a range between 1 and 5 includes 1, 2, 3, 4, and 5; a range between 1 and 10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and a range between 1 and 100 includes 1, 2, 3, 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, 100.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although other probes, compositions, methods, and kits similar, or equivalent, to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.
The invention will be further described in the following example, which does not limit the scope of the invention described in the claims.
EXAMPLES
Example 1: Construction and Characterization of the IL-6R-Fc-IL-35 Heterodimeric Protein
A heterodimeric protein comprising the IL6 receptor (IL6R) linked by a charge polarized core domain to IL-35 was constructed (see, e.g., FIG. 1). Specifically, the heterodimeric protein comprises two polypeptide chains. The first polypeptide chain comprises the IL6R subunit Gp130 linked by a charge polarized core domain to the IL-35 subunit IL12α. The second polypeptide chain comprises the IL6R subunit IL6Rα linked by a charge polarized core domain to the IL-35 subunit IL276. The IL-6R-Fc-IL-35 heterodimeric protein was expressed in mammalian cells by a dual transient transfection with both the IL6RA-Beta-IL27β and gp130-Alpha-IL12α constructs. Coomassie staining indicated the presence of expressed proteins, which were confirmed using an anti-human IgG Western blot to include proteins corresponding to the approximate molecular weights of the Alpha and Beta constructs (see, e.g., FIG. 2).
Additional analysis of the purified protein under non-reduced, reduced and reduced and deglycosylated conditions provided further evidence for assembly of the heterodimeric construct. Specifically, anti-human Fc and anti-human IL-6R staining by Western blot demonstrated the presence of a single high-molecular weight band corresponding to the approximate molecular weight of the alpha/beta heterodimer comprising IL-6R-Fc-IL-35. This heterodimer could be disassembled into the constituent alpha and beta strands under reducing conditions, which showed an apparent molecular weight higher than the predicted molecular weight based on amino acid content alone. This was expected due to the known presence of glycosylation sites, and deglycosylation of the alpha and beta strands led to those individual proteins appearing at their predicted molecular weights by Western analysis (see, e.g., FIG. 3).
Additionally, because the presence of SDS may disrupt any charge interactions which contribute to protein multimerization, the IL-6R-Fc-IL-35 construct was further analyzed by Blue Native PAGE. These data indicate that a majority of the secreted protein (estimated at 60%) represents the alpha/beta heterodimer (FIG. 3). The concentration of the purified IL-6R-Fc-IL-35 protein was confirmed by spectrophotometric analysis (see, e.g., FIG. 4).
Size-exclusion chromatography (SEC) was performed of the IL-6R-Fc-IL-35 heterodimeric protein following dual transfection of the gp130-Fc(alpha)-IL12A and IL6RA-Fc(beta)-IL27B constructs in CHO cells followed by purification of the secreted protein using protein A. The appearance of a single peak by SEC indicated that there is likely only a single species of heterodimeric protein present, which was intended from using charge polarized linker domains (Fc-alpha and Fc-beta) in the two constructs (FIG. 5).
To confirm that the assembled IL-6R-Fc-IL-35 heterodimer retained the ability to engage with the cognate ligand (e.g., IL-6) and be recognized by specific antibodies against each constituent protein of the assembled heterodimer (i.e., IL-6RA, gp130, IL27(3/EBI3 and IL12α), a series of ELISA assays were performed to demonstrate the specific presence of the IL-6R-Fc-IL-35 heterodimer. In FIG. 6 to FIG. 15, the schematic of the ELISA assays is illustrated in the top portion of each figure. In the schematic, the capture and detection strategy is illustrated. In each case, the presence of the IL-6R-Fc-IL-35 heterodimer was observed through capture with recombinant IL-6 and detection with anti-IL-27B/EBI3 (FIG. 6), capture with recombinant IL-6 and detection with anti-human IL-6RA (FIG. 7), capture with anti-human gp130 and detection with anti-IL27B/EBI3 (FIG. 8), capture with anti-human gp130 and detection with anti-human IL-6RA (FIG. 9), capture with anti-IL-6RA and detection with anti-IL27B/EBI3 (FIG. 10), capture with anti-IL-6RA detection and with anti-IL-6RA (FIG. 11), capture with anti-human p35 and detection with anti-IL-27B/EBI3 (FIG. 12), capture with anti-human p35 and detection with anti-human IL-6RA (FIG. 13), capture with anti-human p35 and detection with anti-IL27B/EBI3 (FIG. 14), and capture with anti-IL27B/EBI3 and detection with anti-human IL-6RA (FIG. 15).
The sequence of an illustrative charge polarized core domain (negative-positive, i.e., “alpha core domain”) is provided by SEQ ID NO: 16 and an illustrative alpha core domain comprising knob in hole mutations is provided in SEQ ID NO: 24.
The sequence of an illustrative charge polarized core domain (positive-negative, i.e., “beta core domain”) is provided by SEQ ID NO: 17 and an illustrative beta core domain comprising knob in hole mutations is provided in SEQ ID NO: 25.
The sequences of the components of illustrative polypeptide chains are set forth in SEQ ID NO: 18 for the Gp130 ECD (Type 1), in SEQ ID NO: 19 for the IL-6RA ECD (Type 1), in SEQ ID NO: 20 for the IL-12a (Type 2, first part of IL-35), and in SEQ ID NO: 21 for the IL-27b (Type 2, second part of IL-35).
An illustrative Gp130-Alpha-IL12A chain had the sequence set forth in SEQ ID NO: 22, and an illustrative IL6RA-Beta-IL27B chain had the sequence set forth in SEQ ID NO: 23.
In alternate embodiment, an IL-6R-Fc-IL-35 heterodimeric protein can comprise an IL6RA-Alpha-IL12α chain (SEQ ID NO: 34) and a Gp130-Beta-IL27b chain (SEQ ID NO: 35).
Example 2: Further Characterization of the IL-6R-Fc-IL-35 Heterodimeric Protein
Size-exclusion chromatography (SEC) was performed with the IL-6R-Fc-IL-35 heterodimeric protein. The appearance of a single peak by SEC, with an absorbance wavelength of 210 nm, indicated that there is likely only a single species of protein present, which was intended from using charge polarized linker domains (Fc-alpha and Fc-beta) in the two constructs (FIG. 16A). Interestingly, SEC with an absorbance wavelength of 280 nm showed a second, lower molecular weight band (FIG. 16B).
IL-6R-Fc-IL-35 heterodimeric protein was then used in an IL-6 SINK Assay. Here, the ability of the IL-6R-Fc-IL-35 heterodimeric protein to sequester IL6 was tested. Cultures of DS-1 cells, a B cell line that is dependent on exogenous IL6 for survival, was incubated with the IL-6R-Fc-IL-35 heterodimeric protein and in the presence of exogenous IL6. When the DS-1 cells are not exposed to IL6, cell death results. Thus, these experiments were conducted to determine if the IL-6R-Fc-IL-35 heterodimeric protein can sequester IL6 and lead to DS-1 cell death.
DS-1 cells were cultured in the presence of IL-6R-Fc-IL-35 at increasing molar ratios to IL-6 for 24 hours. Cell death was measured by caspase 3/7 activity (with a luciferase readout).
FIG. 17 shows that IL-6R-Fc-IL-35 heterodimeric protein (identified as Lot '00 and Lot '48) are able to induce cell death in DS-1 cells. Indeed, depending on the lot used, the heterodimeric protein showed between 7 and 281 times greater sequestering of IL-6 than Tocilizumab (an anti-human IL-6 receptor monoclonal antibody which blocks DS-1's binding to IL6) depending on the lot used.
The functionality of IL-6R-Fc-IL-35 heterodimeric protein was then tested. IL-35 has been reported to induce an atypical regulatory phenotype in CD4 T Cells, which is characterized by little or no FoxP3 production coupled with production of IL-35. Additionally, IL-35 is known to turn off TGF-β and IL-10 production.
Here, magnetically enriched human naïve CD4 T cells were isolated from a single donor and activated with αCD3/αCD28 beads and cultured for 5 days in the presence of the indicated agent (shown in FIG. 18). Total mRNA was isolated and RT-qPCR performed.
FIG. 18 shows that the IL-6R-Fc-IL-35 heterodimeric protein (identified as HdA '00) induces IL-35 (which is a dimer of EBI3 and IL12A) production. Surprisingly, the heterodimeric protein also increases production of FoxP3. Moreover, the IL-6R-Fc-IL-35 heterodimeric protein was permissive for cell proliferation, unlike the other treated agents. Although, IL-35 is known to turn off TGF-β and IL-10 production, the IL-6R-Fc-IL-35 heterodimeric protein resulted in detectable levels of IL-10 production (2× over control). Finally, the heterodimeric protein has no notable impact on IL-6 production.
Example 3: Construction and Characterization of the IL-21R-Fc-IL-35 Heterodimeric Protein
Constructs encoding an IL21r-Alpha-IL12a chain and an IL2rg-Beta-IL27B chain were dual transfected into CHO cells, followed by purification of the secreted protein using protein A. When an IL21r-Alpha-IL12a chain and an IL2rg-Beta-IL27B chain are combined (within a cell or in vitro), they form a heterodimeric protein referred to herein as IL-21R-Fc-IL-35. (FIG. 19A).
Western blots were performed on expressed heterodimeric proteins. These revealed, under denaturing and deglycosylated conditions, bands corresponding to the predicted molecular weights of the IL21r-Alpha-IL12α chain and the IL2rg-Beta-IL27B chain (FIG. 19B).
Size-exclusion chromatography (SEC) was performed with the IL-21R-Fc-IL-35 heterodimeric protein. The appearance of a single peak by SEC indicated that there is likely only a single species of protein present, which was intended from using charge polarized linker domains (Fc-alpha and Fc-beta) in the two constructs (FIG. 20).
In these experiments, an illustrative charge polarized core domain (negative-positive, i.e., “alpha core domain”) sequence is provided by SEQ ID NO: 16 and an illustrative alpha core domain comprising knob in hole mutations is provided in SEQ ID NO: 24. An illustrative charge polarized core domain (positive-negative, i.e., “beta core domain”) sequence is provided by SEQ ID NO: 17 and an illustrative beta core domain comprising knob in hole mutations is provided in SEQ ID NO: 25.
The sequences of the components of illustrative polypeptide chains used in this example are set forth in SEQ ID NO: 26 for the extracellular domain of IL-21r, in SEQ ID NO: 27 for the extracellular domain of IL2RG, in SEQ ID NO: 20 for the IL-12a, and in SEQ ID NO: 21 for the IL-27b. An illustrative IL21r-Alpha-IL12a chain had the sequence set forth in SEQ ID NO: 28, and an illustrative IL2rg-Beta-IL27B chain had the sequence set forth in SEQ ID NO: 29.
In alternate embodiment, an IL-21R-Fc-IL-35 heterodimeric protein can comprise an IL2rg-Alpha-IL27B chain (SEQ ID NO: 36) and an IL21r-Beta-IL12a chain (SEQ ID NO: 37).
Example 4: Construction and Characterization of the IFNγR-Fc-IL-35 Heterodimeric Protein
Constructs encoding an IFNgR-Alpha-IL12a chain and an IFNGR2-Beta-IL27B chain were dual transfected into CHO cells, followed by purification of the secreted protein using protein A. When an IFNgR-Alpha-IL12α chain and an IFNGR2-Beta-IL27B chain are combined (within a cell or in vitro), they form a heterodimeric protein referred to herein as IFNγR-Fc-IL-35.
Western blots were performed on the IFNγR-Fc-IL-35 heterodimeric protein comprising the IFNgR-Alpha-IL12α chains and the hIFNGR2-Beta-IL27B chain probed with an antibody indicated below each blot. These revealed, under denaturing and deglycosylated conditions, bands corresponding to the predicted molecular weights of the IFNgR-Alpha-IL12α and the hIFNGR2-Beta-IL27B (FIG. 21). Bands noted in yellow highlighting are non-specific bands.
Size-exclusion chromatography (SEC) was performed with the IFNγR-Fc-IL-35 heterodimeric protein. The appearance of a single peak by SEC indicated that there is likely only a single species of protein present, which was intended from using charge polarized linker domains (Fc-alpha and Fc-beta) in the two constructs (FIG. 22).
In these experiments, an illustrative charge polarized core domain (negative-positive, i.e., “alpha core domain”) sequence is provided by SEQ ID NO: 16 and an illustrative alpha core domain comprising knob in hole mutations is provided in SEQ ID NO: 24. An illustrative charge polarized core domain (positive-negative, i.e., “beta core domain”) sequence is provided by SEQ ID NO: 17 and an illustrative beta core domain comprising knob in hole mutations is provided in SEQ ID NO: 25.
The sequences of the components of illustrative polypeptide chains used in this example are set forth in SEQ ID NO: 30 for the extracellular domain of IFNgR, in SEQ ID NO: 31 for the extracellular domain of IFNGR2, in SEQ ID NO: 20 for the IL-12a, and in SEQ ID NO: 21 for the IL-27b. An illustrative IFNgR-Alpha-IL12α chain had the sequence set forth in SEQ ID NO: 32, and an illustrative IFNGR2-Beta-IL27B chain had the sequence set forth in SEQ ID NO: 33.
In alternate embodiment, an IFNγR-Fc-IL-35 heterodimeric protein can comprise an IFNGR2-Alpha-IL27B chain (SEQ ID NO: 39) and an IFNgR-Beta-IL12α chain (SEQ ID NO: 38).
EQUIVALENTS
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
INCORPORATION BY REFERENCE
All patents and publications referenced herein are hereby incorporated by reference in their entireties.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

## Claims

1. A heterodimeric protein comprising a first polypeptide chain and a second polypeptide chain, wherein:
the first polypeptide chain comprises a first subunit of a first protein at the amino terminus linked by a first charge polarized core domain to a first subunit of a second protein at the carboxy terminus; and
the second polypeptide chain comprises a second subunit of the first protein at the amino terminus linked by a second charge polarized core domain to a second subunit of the second protein at the carboxy terminus; and
the first polypeptide chain and the second polypeptide chain form a heterodimer through electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second charge polarized core domains,
the first and second charge polarized core domains comprise a hinge-CH2-CH3 Fc domain,
one of the first protein and the second protein is interleukin-35 (IL-35) and
the other of the first protein and the second protein is selected from interleukin-2 receptor (IL-2R), interleukin-3 receptor (IL-3R), interleukin-4 receptor (IL-4R), interleukin-5 receptor (IL-5R), interleukin-6 receptor (IL-6R), interleukin-7 receptor (IL-7R), interleukin-9 receptor (IL-9R), interleukin-10 receptor (IL-10R), interleukin-11 receptor (IL-11R), interleukin-12 receptor (IL-12R), interleukin-13 receptor (IL-13R), interleukin-15 receptor (IL-15R), interleukin-17 receptor (IL-17R), interleukin-18 receptor (IL-18R), interleukin-20 receptor (IL-20R), interleukin-21 receptor (IL-21R), interleukin-22 receptor (IL-22R), interleukin-23 receptor (IL-23R), interleukin-27 receptor (IL-27R), interferon alpha receptor (IFN-αR), interferon beta receptor (IFN-βR), and interferon gamma receptor (IFN-γR).

2. The heterodimeric protein of claim 1, wherein the linker comprises:
the hinge-CH2-CH3 Fc domain derived from IgG1; or
the hinge-CH2-CH3 Fc domain derived from IgG4.

3. The heterodimeric protein of claim 1, wherein the first and/or second charge polarized core domain further comprise peptides having positively and/or negatively charged amino acid residues at the amino and carboxy terminus of the charge polarized core domain.

4. The heterodimeric protein of claim 3, wherein the peptide comprising positively charged amino acid residues include one or more of amino acids selected from histidine (His), lysine (Lys), and arginine (Arg).

5. The heterodimeric protein of claim 4, wherein the peptide comprising positively charged amino acid residues comprises the sequence RKGGKR (SEQ ID NO: 11) or GSGSRKGGKRGS (SEQ ID NO: 12).

6. The heterodimeric protein of claim 3, wherein the peptide comprising negatively charged amino acid residues include one or more amino acids selected from aspartic acid (Asp) and glutamic acid (Glu).

7. The heterodimeric protein of claim 6, wherein the peptide comprising positively charged amino acid residues comprises the sequence DEGGED (SEQ ID NO: 13) or GSGSDEGGEDGS (SEQ ID NO: 14).

8. The heterodimeric protein of claim 1, wherein the first and/or second charge polarized core domain comprises one or more amino acid changes for promoting heterodimerization via increased hydrogen bonding and/or van der Waals forces.

9. The heterodimeric protein of claim 8, wherein the one or more amino acid changes creates a knob in hole motif, wherein:
the knob in hole motif is formed by one or more amino acid changes that replaces one or more tyrosine (Y) residues with one or more threonine (T) residues in the first charge polarized core domain and/or is formed by one or more amino acid changes that replaces one or more threonine (T) residues with one or more tyrosine (Y) residues in the second charge polarized core domain, and/or
the knob in hole motif is formed by one or more amino acid changes that replaces one or more tyrosine (Y) residues with one or more threonine (T) residues in the second charge polarized core domain and/or is formed by one or more amino acid changes that replaces one or more threonine (T) residues with one or more tyrosine (Y) residues in the first charge polarized core domain.

10. The heterodimeric protein of claim 8, wherein one or both of the charge polarized core domains comprise:
one or more effector and complement silencing substitutions selected from L234A, L235A (LALA), and P329G; and/or
one or more half-life extension mutations selected from M252Y, S254T, and T256E.

11. The heterodimeric protein of claim 1, wherein the first protein is IL-35.

12. The heterodimeric protein of claim 1, wherein the second protein is IL-35.

13. The heterodimeric protein of claim 1, wherein the first protein and the second protein, or the second protein and the first protein are (i) IL-35 and IL-6R, (ii) IL-21R and IL-35, or (iii) and IL-35, respectively.

14. The heterodimeric protein of claim 1, wherein
the first or second protein is IL-35 comprising the IL-12α and IL-27β subunits,
the first or second protein is IL-6R comprising the IL-6Rα and gp130 subunits,
the first or second protein is IL-21R comprising the IL-21r and IL-2rg subunits, or
the first or second protein is IFN-γR comprising the IFN-gR and IFNGR2 subunits.

15. The heterodimeric protein of claim 1, wherein the first or second protein is IL-6R and comprises the IL-6RA and gp130 subunits, and the other protein is IL-35 and comprises the IL12α and IL-27β subunits.

16. The heterodimeric protein of claim 1, wherein:
the heterodimeric protein is capable of both (i) delivering an immune inhibitory signal and (ii) masking immune stimulatory signal.

17. A nucleic acid encoding the first and/or second polypeptide chains of the heterodimeric protein of claim 1.

18. An expression vector comprising the nucleic acid of claim 17.

19. The heterodimeric protein of claim 14, wherein the first or second protein is IL-21R comprising the IL-21r and IL-2rg subunits.

20. The heterodimeric protein of claim 14, wherein the first or second protein is IFN-γR comprising the IFN-gR and IFNGR2 subunits.