# Patent

## Title
MAdCAM targeted immunotolerance

## Abstract

Methods and compounds for conferring site-specific or local immune privilege, such as targeting to a cell expressing MAdCAM.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 62/850,172, filed May 20, 2019, which is hereby incorporated by reference in its entirety.
This application is also related to U.S. Provisional Application No. 62/721,644, filed Aug. 23, 2018, U.S. provisional Application No. 62/675,972 filed May 24, 2018, U.S. provisional Application No. 62/595,357 filed Dec. 6, 2017, U.S. Provisional Application No. 62/595,348, filed Dec. 6, 2017, U.S. Non-Provisional application Ser. No. 16/109,875, filed Aug. 23, 2018, U.S. Non-Provisional application Ser. No. 16/109,897, filed Aug. 23, 2018, U.S. Non-Provisional application Ser. No. 15/988,311, filed May 24, 2018, PCT Application No. PCT/US2018/034334, filed May 24, 2018, and, PCT/US2018/062780, filed Nov. 28, 2018, each of which are hereby incorporated by reference in their entirety.


FIELD
The embodiments provided herein relate to, for example, methods and compositions for local or targeted immune-privilege.
BACKGROUND
Instances of unwanted immune responses, e.g., as in the rejection of transplanted tissue or in autoimmune disorders, constitute a major health problem for millions of people across the world. Long-term outcomes for organ transplantation are frequently characterized by chronic rejection, and eventual failure of the transplanted organ. More than twenty autoimmune disorders are known, affecting essentially every organ of the body, and affecting over fifty million people in North America alone. The broadly active immunosuppressive medications used to combat the pathogenic immune response in both scenarios have serious side effects.
SUMMARY
Disclosed herein are methods and therapeutic compounds that provide site-specific immune privilege. Embodiments disclosed herein are incorporated by reference into this section.
In some embodiments, the therapeutic compound comprises an engineered multi-specific compound, e.g., an engineered bi-specific molecule, e.g., an engineered bi-specific antibody molecule, comprising:
1) a specific targeting moiety selected from:
a) a donor specific targeting moiety which, e.g., preferentially binds a donor target (preferentially as compared with binding to a recipient antigen), and is useful for providing site-specific immune privilege for a transplant tissue, e.g., an organ, from a donor; or
b) a tissue specific targeting moiety which, e.g., preferentially binds a subject target tissue (preferentially as compared with subject non-target tissue), and is useful for providing site-specific immune privilege for a subject tissue undergoing unwanted immune attack, e.g., in an autoimmune disorder); and
2) an effector binding/modulating moiety selected from:
(a) an immune cell inhibitory molecule binding/modulating moiety (referred to herein as an ICIM binding/modulating moiety);
(b) an immunosuppressive immune cell binding/modulating moiety (referred to herein as an IIC binding/modulating moiety);
(c) an effector binding/modulating moiety that, as part of a therapeutic compound, promotes an immuno-suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target (referred to herein as an SM binding/modulating moiety); or
(d) an immune cell stimulatory molecule binding/modulating moiety (referred to herein as an ICSM binding/modulating moiety), wherein the ICSM inhibits immune activation by, for example, blocking the interaction between a costimulatory molecule and its counterstructure.
An effector binding/modulating moiety can fall into more than one of classes a, b and c. E.g., as is shown below, a CTLA4 binding molecule falls into both of categories a and b.
In some embodiments, the therapeutic compound comprises an ICIM binding/modulating moiety. In some embodiments, an ICIM binding/modulating molecule and binds, and agonizes, an inhibitory molecule, e.g., an inhibitory immune checkpoint molecule, or otherwise inhibits or reduces the activity of an immune cell, e.g., a cytotoxic T cell, a B cell, NK cell, or a myeloid cell, e.g., a neutrophil or macrophage.
In some embodiments, the therapeutic compound comprises an engineered multi-specific compound, e.g., an engineered bi-specific molecule, e.g., an engineered bi-specific antibody molecule, comprising:
1) a specific targeting moiety, e.g., a donor specific targeting moiety (which binds a donor target and is useful for providing site-specific immune privilege for a transplant tissue, e.g., an organ, from a donor) or a tissue specific targeting moiety (which binds a subject tissue target and is useful for providing site-specific immune privilege for a subject tissue undergoing unwanted immune attack, e.g., in an autoimmune disorder); and
2) an effector binding/modulating moiety comprising an ICIM binding/modulating moiety that binds to an effector molecule on an immune cell, e.g., an inhibitory receptor, e.g., PD-1, wherein, upon binding of the specific targeting moiety to its target, and binding of the ICIM binding/modulating moiety to an effector molecule on the immune cell, an immune cell activity, e.g., the ability of the immune cell to mount an immune attack, is down regulated, e.g., through an inhibitory signal dependent on the clustering of effector molecules on the immune cell. In some embodiments, the engineered multi-specific compound comprises additional binding moieties so that it binds more than two specific molecules, such as, but not limited to, 3 or 4.
In some embodiments, the therapeutic compound comprises an ICIM binding/modulating moiety and has one or both of the following properties: (a) the level of down regulation of an immune cell is greater when the therapeutic compound is bound to its target than when the therapeutic compound is not bound to its target; and (b) the therapeutic compound, when engaged with a cell surface inhibitory receptor, e.g., PD-1, on an immune cell, does not inhibit, or does not substantially inhibit the ability of the cell surface inhibitory receptor to bind an endogenous ligand.
In some embodiments, the level of down regulation of an immune cell is greater when the therapeutic compound is bound to its target than when the therapeutic compound is not bound to its target. In embodiments, the level of down regulation by target bound therapeutic compound is equal to or greater than 1.5-fold, 2-fold, 4-fold, 8-fold or 10-fold greater than what is seen when it is not bound to its target. In embodiments, therapeutic compound does not, or does not significantly down regulate immune cells when it is not bound to target. Thus, indiscriminant or unwanted agonism of an inhibitory receptor, e.g., PD-1, is minimized or eliminated. E.g., when the therapeutic compound is bound to an immune cell, but not bound to the targeted moiety, engagement of a inhibitory immune checkpoint molecule by the therapeutic compound does not result in down regulation or does not result in substantia down regulation, e.g., the inhibitory receptor on the immune cell to which the therapeutic compound is bound, is not clustered or not clustered sufficiently to result in an inhibitory signal sufficient to give down regulation or substantial inhibition of the immune cell.
In embodiments, the therapeutic compound, when engaged with a cell surface inhibitory receptor, e.g., PD-1, on an immune cell, does not inhibit, or does not substantially inhibit the ability of the cell surface inhibitory receptor to bind an endogenous ligand. In some embodiments, the therapeutic compound can bind to the PD-L1/2 binding site on PD-1. Thus, indiscriminant or unwanted antagonism of an inhibitory receptor, e.g., PD-1, is minimized or eliminated. In embodiments, binding of the therapeutic compound to an inhibitory receptor, e.g. PD-1, on an immune cell does not impede, or substantially impede, the ability of the inhibitory receptor to bind a natural ligand, e.g., PD-L1. In embodiments, binding of the therapeutic compound to an inhibitory receptor, e.g. PD-1, on an immune reduces binding of a natural ligand, e.g., PD-L1, by less than 50, 40, 30, 20, 10, or 5% of what is seen in the absence of therapeutic compound.
In some embodiments, the therapeutic compound comprises an ICIM binding/modulating moiety and, when administered to a subject at a therapeutically effective dose, does not result in unacceptable levels of systemic immune suppression, as would be possible if indiscriminant agonism of the inhibitory receptor in all immune cells of a type, e.g., all T cells, occurred, or unacceptable levels of systemic immune activation, as would be possible if the therapeutic compound antagonized the interaction of the inhibitory receptor with its natural ligand.
While not wishing to be bound by theory, it is believed that, upon administration to a subject, a therapeutic compound comprising an ICIM binding/modulating moiety can exist in any one of four states: i) unbound and in free solution; ii) bound to only an inhibitory receptor expressed on the surface of an immune cell, e.g., a T cell, through the ICIM binding/modulating moiety; iii) bound to only the surface of the target transplant or subject tissue through the targeting moiety; and iv) bound to both the surface of target transplant or subject tissue through the targeting moiety and to an inhibitory receptor expressed by an immune cell, e.g., a T cell, through the ICIM binding/modulating moiety. When the therapeutic compound is bound only to the target transplant or subject tissue (iii) through the targeting moiety, it has no, or no substantial, effect on the target transplant or tissue. When the therapeutic compound is bound to the target transplant or tissue through the targeting moiety and bound to an inhibitory receptor expressed by an immune cell, e.g., a T cell, through the ICIM binding/modulating moiety (iv), it creates immune privilege at the target organ or tissue. While not wishing to be bound by theory, is believed that this is achieved by the target transplant or donor tissue multimerizing the therapeutic compound molecules on its surface, e.g., by immobilizing a plurality of therapeutic compound molecules at a high density and valency. The multimerization of the therapeutic compound molecules allows the ICIM binding/modulating moieties of the therapeutic compounds to promote clustering of inhibitory receptors expressed on the surface of the immune cell, e.g., a pathogenic T cell, and transmission of an inhibitory signal functioning to silence or down-regulate the immune cell. E.g., in the case of T cells, a therapeutic compound comprising an ICIM binding/modulating moiety comprising a PD-L1 molecule, or an anti-PD-1 Ab, can be used. Binding of a plurality of the therapeutic compound molecules to the target results in multimerization of the therapeutic compound molecules, which in turn, by virtue of the PD-L1 molecule, or a functional anti-PD-1 antibody molecule, leads to clustering of PD-1 on the T cell. If that clustering occurs in the context of antigen presentation by the target MHC, to T cell receptor on the T cell, a negative signal is generated and the T cell will be inactivated. In embodiments the ICIM binding/modulating moiety, e.g., a functional antibody molecule, binds the effector molecule but does not inhibit, or substantially inhibit, interaction of the effector molecule with its native ligand(s).
In some embodiments, the therapeutic compound comprises an IIC binding/modulating moiety, which binds and recruits an immune suppressive immune cell, e.g., a Treg, e.g., a Foxp3+CD25+ Treg, to the proximity of the target tissue.
In some embodiments, the therapeutic compound comprises a SM binding/modulating moiety, which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP.
In some embodiments, the therapeutic compound comprises a targeting moiety that is specific for a target on an immune cell. In some embodiments, the target is as described herein. In some embodiments, the target is MAdCAM. In some embodiments, the targeting moiety is an antibody that binds to MAdCAM.
In some embodiments the therapeutic compound comprises an ICSM binding/modulating moiety, which binds a stimulatory molecule, e.g., a costimulatory molecule. In some embodiments, the ICSM inhibits the costimulatory molecule counterstructure by. Binding/modulating either the costimulatory molecule or the costimulatory molecule counterstructure can serve to down regulate the ability of an immune cell to mount an immune response. In some embodiments, the ICSM binding/modulating moiety can bind a stimulatory, e.g., costimulatory molecule on an immune cell, e.g., OX40 on T cells, or the counter member of the stimulatory molecule e.g. OX40L on another cell, such as, but not limited to, immune cells such as NK cells, mast cells, dendritic cells, or, for example, non-immune cells such as endothelial cells, or smooth muscle cells.
In some embodiments, the therapeutic compound comprises a donor specific targeting moiety and provides site-specific immune privilege for donor transplant tissue implanted in a subject. In some embodiments, the therapeutic compound comprises a tissue specific targeting moiety and provides site-specific immune privilege for a tissue of a subject, e.g., a tissue afflicted with an unwanted immune response in an autoimmune disorder.
The targeting moiety is specific for the donor transplant or subject tissue to be protected from the immune system. In some embodiments, the effector molecule binding moiety comprises a de novo generated binding domain, e.g. a functional antibody molecule. In some embodiments, the effector binding/modulating moiety comprises amino acid sequence deriving from the natural ligand that recognizes an inhibitory receptor expressed on the surface of an immune cell, e.g., a T cell.
In some embodiments, the therapeutic compound silences immune cells, e.g., T cells, proximal to the transplant or donor tissue to be protected but does not silence immune cells, e.g., T cells, not proximal to the target, as the therapeutic compound requires the presence of the target transplant or donor tissue for function. This in contrast to when the therapeutic compound binds only to the inhibitory receptor expressed by the immune cell, e.g., T cell, in which case there is no functional consequence.
Methods and therapeutic compounds described here are based at least in part on providing site-specific immune-privilege. Therapeutic compounds and method of using them described herein allow the minimization, e.g., the reduction or elimination of, non-site specific systemic administration of immune-suppressive therapeutic agents in clinical settings, e.g., where reversal and suppression of an immune response is desired, such as in autoimmune diseases or tissue, e.g., organ, transplant. While capable of clinically meaningful response when the underlying pathophysiology driven by an aberrant immune system is impacted, broadly acting immunosuppressants have the undesirable effect of reducing the patient's systemic immune system function. As the role of a normally functioning immune system is to combat the constant barrage of pathogenic and opportunistic organisms existing in the surrounding environment and to constantly purge healthy individuals of cancerous cells, patients undergoing chronic immunosuppression are at an increased risk to develop infections and cancer. Methods and therapeutic compounds described herein provide therapies that selectively target and attenuate, reduce, or extinguish only the pathogenic immune response at the site of pathology while having minimal inhibition of normal systemic immune system function elsewhere.
In some embodiments, a therapeutic compound is provided as provided herein. In some embodiments, the compound comprises a i) a specific targeting moiety selected from: a) a donor specific targeting moiety which, e.g., preferentially binds a donor target; or b) a tissue specific targeting moiety which, e.g., preferentially binds target tissue of a subject; and ii) an effector binding/modulating moiety selected from: (a) an immune cell inhibitory molecule binding/modulating moiety (ICIM binding/modulating moiety); (b) an immunosuppressive immune cell binding/modulating moiety (IIC binding/modulating moiety); or (c) an effector binding/modulating moiety that, as part of a therapeutic compound, promotes an immuno-suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target (SM binding/modulating moiety).
In some embodiments, the effector binding/modulating moiety comprises an ICIM binding/modulating moiety. In some embodiments, the effector binding/modulating moiety comprises an ICIM binding/modulating moiety comprising an inhibitory immune checkpoint molecule ligand molecule. In some embodiments, the inhibitory immune molecule counter-ligand molecule comprises a PD-L1 molecule. In some embodiments, the ICIM is wherein the inhibitory immune molecule counter ligand molecule engages a cognate inhibitory immune checkpoint molecule selected from PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4. In some embodiments, the ICIM is an antibody. In some embodiments, the ICIM comprises an antibody that binds to PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4. In some embodiments, the ICIM binding/modulating moiety which comprises a functional antibody molecule to a cell surface inhibitory molecule.
In some embodiments, the cell surface inhibitory molecule is an inhibitory immune checkpoint molecule. In some embodiments, the inhibitory immune checkpoint molecule is selected from PD-1, KIR2DL4, LILRB1, LILRB2, CTLA-4, or selected from Table 1.
In some embodiments, the effector binding/modulating moiety comprises an IIC binding/modulating moiety.
In some embodiments, the compound has the formula from N-terminus to C-terminus:
R1---Linker Region A-R2 or R3-Linker Region B-R4,
wherein, R1, R2, R3, and R4, each independently comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety; a specific targeting moiety; or is absent; provided that an effector binding/modulating moiety and a specific targeting moiety are present.
In some embodiments, polypeptides comprising a targeting moiety that binds to a target cell and an effector binding/modulating moiety, wherein the effector binding/modulating moiety is a IL-2 mutein polypeptide (IL-2 mutein), which is a mutant IL-2 protein, are provided. In some embodiments, the targeting moiety comprises an antibody that binds to a target protein on the surface of a target cell. In some embodiments, the polypeptide comprises two polypeptide chains as provided for herein. In some embodiments, the first chain comprises a VH domain and the second chain comprises a VL domain of an antibody that binds to the target cell or a protein that is expressed on the target cell, such as, but not limited to, MAdCAM. In some embodiments, the targeting moiety is an antibody that binds to MAdCAM. In some embodiments, the targeting moiety binds to OAT1 (SLC22A6) and OCT2 (SLC22A2). In some embodiments, the targeting moiety is an antibody that binds to OAT1 (SLC22A6) and OCT2 (SLC22A2). In some embodiments, the targeting moiety does not bind to OAT1 (SLC22A6) and OCT2 (SLC22A2). For the avoidance of doubt, the OCT2 referenced herein is not the transcription factor, but rather is the surface protein expressed in kidney tissue. In some embodiments, the targeting moiety is a moiety that specifically binds to a protein found in the pancreas. In some embodiments, the targeting moiety binds to FXYD2, TSPAN7, DPP6, HEPACAM2, TMEM27, or GPR119. In some embodiments, the targeting moiety does not bind to FXYD2, TSPAN7, DPP6, HEPACAM2, TMEM27, or GPR119. In some embodiments, the targeting moiety is antibody that binds to FXYD2, TSPAN7, DPP6, HEPACAM2, TMEM27, or GPR119.
In some embodiments, the polypeptide comprises a first chain and a second chain that form the polypeptide or therapeutic compound, wherein
the first chain comprises:
VH-Hc-Linker-C1, wherein VH is a variable heavy domain that binds to the target cell with a VL domain of the second chain; Hc is a heavy chain of antibody comprising CH1-CH2-CH3 domain, the Linker is a glycine/serine amino acid sequence as provided herein or is absent, and C1 is a IL-2 mutein that can be fused to a Fc protein in either the N-terminal or C-terminal orientation as provided for herein, wherein there can be a glycine/serine linker linking the IL-2 mutein to the Fc protein; and
the second chain comprises:
VL-Lc, wherein VL is a variable light chain domain that binds to the target cell with the VH domain of the first chain, and the Lc domain is a light chain CK domain. In some embodiments, the first chain comprises C1-Linker-VH-Hc, with the variables as defined above.
In some embodiments, the polypeptide comprises the formula of C1-linker-CH2-CH3-Linker-scFv, wherein C1 and the Linker are as defined above and herein, the CH2 and CH3 are heavy chain domains and the scFv is a single chain antibody like fragment that acts as the targeting moiety to bind to tissue targets as provided for herein. In some embodiments, the mutein is fused to the Fc region as provided herein and one or more of the linkers are absent. In some embodiments, the Linker is a glycine/serine linker as provided for herein. In some embodiments, the linker is a peptide sequence.
In some embodiments, methods of treating auto-immune diseases or conditions are provided herein, the methods comprising administering one or more of the therapeutic compounds or polypeptides provided herein.
In some embodiments, methods of treating diseases or conditions described herein are provided herein, the methods comprising administering one or more of the therapeutic compounds or polypeptides provided herein.
In some embodiments, methods of treating a subject with inflammatory bowel disease are provided, the methods comprising administering a therapeutic compound or polypeptides provided herein to the subject to treat the inflammatory bowel disease. In some embodiments, the subject has Crohn's disease and or ulcerative colitis.
In some embodiments, methods of treating a subject with auto-immune hepatitis are provided, the methods comprising administering a therapeutic compound or polypeptides as provided herein to the subject to treat the auto-immune hepatitis.
In some embodiments, methods of treating primary sclerosing cholangitis are provided, the methods comprising administering a therapeutic compound or polypeptides as provided herein to the subject to treat the primary sclerosing cholangitis.
In some embodiments, methods of treating (e.g., reducing) inflammation in the intestine are provided, the methods comprising administering a therapeutic compound or polypeptides as provided herein to the subject to treat the inflammation in the intestine. In some embodiments, the inflammation is in the small intestine. In some embodiments, the inflammation is in the large intestine. In some embodiments, the inflammation is in the bowel or colon.
In some embodiments, methods of treating (e.g., reducing) inflammation in the pancreas are provided, the methods comprising administering a therapeutic compound or polypeptides as provided herein to the subject to treat the inflammation in the pancreas. In some embodiments, the methods treat pancreatitis.
In some embodiments, methods of treating Type 1 diabetes are provided, the methods comprising administering a therapeutic compound or polypeptides as provided herein to the subject to treat the Type 1 diabetes.
In some embodiments, methods of treating a transplant subject are provided, the methods comprising administering a therapeutically effective amount of a therapeutic compound or polypeptides as provided herein to the subject, thereby treating a transplant (recipient) subject.
In some embodiments, methods of treating GVHD in a subject having a transplanted a donor tissue are provided, the methods comprising administering a therapeutically effective amount of a therapeutic compound or polypeptides as provided herein to the subject.
In some embodiments, methods of treating a subject having, or at risk, or elevated risk, for having, an autoimmune disorder are provided, the methods comprising administering a therapeutically effective amount of a therapeutic compound or polypeptides as provided herein, thereby treating the subject.
In some embodiments, the compound has the formula from N-terminus to C-terminus:
A1---Linker A---A2---Linker B---A3 or A3---Linker A---A2---Linker B---A1,
wherein, A1 and A3, are each, independently, an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety; or a specific targeting moiety, wherein A2 comprises an Fc region or is absent; and Linker A and Linker B, are linkers, but do not have to be the same.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts non-limiting embodiments of the therapeutic compounds provided herein.
FIG. 2 depicts a non-limiting illustration of how a therapeutic compound provided herein could function.
FIG. 3 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 3A depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 4 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 5 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 6 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 7 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 8 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 9 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 10 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 11 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 12 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 13 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 14 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 15 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 16 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 17 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 18 depicts a non-limiting illustration of the therapeutic compounds provided herein.
FIG. 19 depicts a non-limiting illustration of the therapeutic compounds provided herein.



DETAILED DESCRIPTION
This application incorporates by reference each of the following in its entirety: U.S. application Ser. No. 15/922,592 filed Mar. 15, 2018 and PCT Application No. PCT/US2018/022675, filed Mar. 15, 2018. This application also incorporate by reference, each of the following in their entirety: U.S. Provisional Application No. 62/721,644, filed Aug. 23, 2018, U.S. provisional Application No. 62/675,972 filed May 24, 2018, U.S. provisional Application No. 62/595,357 filed Dec. 6, 2017, U.S. Provisional Application No. 62/595,348, filed Dec. 6, 2017, U.S. Non-Provisional application Ser. No. 16/109,875, filed Aug. 23, 2018, U.S. Non-Provisional application Ser. No. 16/109,897, filed Aug. 23, 2018, U.S. Non-Provisional application Ser. No. 15/988,311, filed May 24, 2018, PCT Application No. PCT/US2018/034334, filed May 24, 2018, and, PCT/US2018/062780, filed Nov. 28, 2018.
As used herein and unless otherwise indicated, the term “about” is intended to mean±5% of the value it modifies. Thus, about 100 means 95 to 105.
As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.
As used herein, the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments.
As used herein, the term “animal” includes, but is not limited to, humans and non-human vertebrates such as wild, domestic, and farm animals.
As used herein, the term “contacting” means bringing together of two elements in an in vitro system or an in vivo system. For example, “contacting” a therapeutic compound with an individual or patient or cell includes the administration of the compound to an individual or patient, such as a human, as well as, for example, introducing a compound into a sample containing a cellular or purified preparation containing target.
As used herein, the terms “comprising” (and any form of comprising, such as “comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any composition or method that recites the term “comprising” should also be understood to also describe such compositions as consisting, consisting of, or consisting essentially of the recited components or elements.
As used herein, the term “fused” or “linked” when used in reference to a protein having different domains or heterologous sequences means that the protein domains are part of the same peptide chain that are connected to one another with either peptide bonds or other covalent bonding. The domains or section can be linked or fused directly to one another or another domain or peptide sequence can be between the two domains or sequences and such sequences would still be considered to be fused or linked to one another. In some embodiments, the various domains or proteins provided for herein are linked or fused directly to one another or a linker sequences, such as the glycine/serine sequences described herein link the two domains together. Two peptide sequences are linked directly if they are directly connected to one another or indirectly if there is a linker or other structure that links the two regions. A linker can be directly linked to two different peptide sequences or domains.
As used herein, the term “individual,” “subject,” or “patient,” used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.
As used herein, the term “inhibit” refers to a result, symptom, or activity being reduced as compared to the activity or result in the absence of the compound that is inhibiting the result, symptom, or activity. In some embodiments, the result, symptom, or activity, is inhibited by about, or, at least, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. An result, symptom, or activity can also be inhibited if it is completely elimination or extinguished.
As used herein, the phrase “in need thereof” means that the subject has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the subject can be in need thereof. In some embodiments, the subject is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent.
As used herein, the phrase “integer from X to Y” means any integer that includes the endpoints. For example, the phrase “integer from X to Y” means 1, 2, 3, 4, or 5.
As used herein, the term “mammal” means a rodent (i.e., a mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or a human. In some embodiments, the mammal is a human.
In some embodiments, therapeutic compounds are provided herein. In some embodiments, the therapeutic compound is a protein or a polypeptide, that has multiple chains that interact with one another. The polypeptides can interact with one another through non-covalent interactions or covalent interactions, such as through disulfide bonds or other covalent bonds. Therefore, if an embodiment refers to a therapeutic compound it can also be said to refer to a protein or polypeptide as provided for herein and vice versa as the context dictates.
As used herein, the phrase “ophthalmically acceptable” means having no persistent detrimental effect on the treated eye or the functioning thereof, or on the general health of the subject being treated. However, it will be recognized that transient effects such as minor irritation or a “stinging” sensation are common with topical ophthalmic administration of drugs and the existence of such transient effects is not inconsistent with the composition, formulation, or ingredient (e.g., excipient) in question being “ophthalmically acceptable” as herein defined. In some embodiments, the pharmaceutical compositions can be ophthalmically acceptable or suitable for ophthalmic administration.
“Specific binding” or “specifically binds to” or is “specific for” a particular antigen, target, or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.
Specific binding for a particular antigen, target, or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10−4M, at least about 10−5M, at least about 10−6 M, at least about 10−7M, at least about 10−8M, at least about 10−9M, alternatively at least about 10−10 M, at least about 10−11M at least about 10−12M, or greater, where KD refers to a dissociation rate of a particular antibody-target interaction. Typically, an antibody that specifically binds an antigen or target will have a KD that is, or at least, 2-, 4-, 5-, 10-, 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000-, or more times greater for a control molecule relative to the antigen or epitope.
In some embodiments, specific binding for a particular antigen, target, or an epitope can be exhibited, for example, by an antibody having a KA or Ka for a target, antigen, or epitope of at least 2-, 4-, 5-, 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the target, antigen, or epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.
As provided herein, the therapeutic compounds and compositions can be used in methods of treatment as provided herein. As used herein, the terms “treat,” “treated,” or “treating” mean both therapeutic treatment and prophylactic measures wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results. For purposes of these embodiments, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
Provided herein are therapeutic compounds, e.g., therapeutic protein molecules, e.g., fusion proteins, including a targeting moiety and an effector binding/modulating moiety, typically as separate domains. Also provided are methods of using and making the therapeutic compounds. The targeting moiety serves to localize the therapeutic compound, and thus the effector binding/modulating moiety, to a site at which immune-privilege is desired. The effector binding/modulating moiety comprises one or more of: (a) an immune cell inhibitory molecule binding/modulating moiety (an ICIM binding/modulating moiety): (b) an immunosuppressive immune cell binding/modulating moiety (an IIC binding/modulating moiety); (c) a soluble molecule binding/modulating moiety (a SM binding/modulating moiety) or (d) a molecule that blocks or inhibits immune cell stimulatory molecule binding/modulating moiety (referred to herein as an ICSM binding/modulating moiety). In some embodiments, the ICSM inhibits immune activation by, for example, blocking the interaction between a costimulatory molecule and its counterstructure. In some embodiments, a therapeutic compound comprises: (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); or (a), (b), (c), and (d). The present disclosure provides, for example, molecules that can act as PD-1 agonists.
Without being bound to any particular theory, agonism of PD-1 inhibits T cell activation/signaling and can be accomplished by different mechanisms. For example crosslinking can lead to agonism, bead-bound, functional PD-1 agonists have been described (Akkaya. Ph.D. Thesis: Modulation of the PD-1 pathway by inhibitory antibody superagonists. Christ Church College, Oxford, U K, 2012), which is hereby incorporated by reference. Crosslinking of PD-1 with two mAbs that bind non-overlapping epitopes induces PD-1 signaling (Davis, US 2011/0171220), which is hereby incorporated by reference. Another example is illustrated through the use of a goat anti-PD-1 antiserum (e.g. AF1086, R&D Systems) which is hereby incorporated by reference, which acts as an agonist when soluble (Said et al., 2010, Nat Med) which is hereby incorporated by reference. Non-limiting examples of PD-1 agonists that can be used in the present embodiments include, but are not limited to, UCB clone 19 or clone 10, PD1AB-1, PD1AB-2, PD1AB-3, PD1AB-4 and PD1AB-5, PD1AB-6 (Anaptys/Celgene), PD1-17, PD1-28, PD1-33 and PD1-35 (Collins et al, US 2008/0311117 A1 Antibodies against PD-1 and uses therefor, which is incorporated by reference), or can be a bi-specific, monovalent anti-PD-1/anti-CD3 (Ono), and the like. In some embodiments, the PD-1 agonist antibodies can be antibodies that block binding of PD-L1 to PD-1. In some embodiments, the PD-1 agonist antibodies can be antibodies that do not block binding of PD-L1 to PD-1.
PD-1 agonism can be measured by any method, such as the methods described in the examples. For example, cells can be constructed that express, including stably express, constructs that include a human PD-1 polypeptide fused to a b-galactosidase “Enzyme donor” and 2) a SHP-2 polypeptide fused to a b-galactosidase “Enzyme acceptor.” Without being bound by any theory, when PD-1 is engaged, SHP-2 is recruited to PD-1. The enzyme acceptor and enzyme donor form a fully active b-galactosidase enzyme that can be assayed. Although, the assay does not directly show PD-1 agonism, but shows activation of PD-1 signaling. PD-1 agonism can also be measured by measuring inhibition of T cell activation because, without being bound to any theory, PD-1 agonism inhibits anti-CD3-induced T cell activation. For example, PD-1 agonism can be measured by preactivating T cells with PHA (for human T cells) or ConA (for mouse T cells) so that they express PD-1. The cells can then be reactivated with anti-CD3 in the presence of anti-PD-1 (or PD-L1) for the PD-1 agonism assay. T cells that receive a PD-1 agonist signal in the presence of anti-CD3 will show decreased activation, relative to anti-CD3 stimulation alone. Activation can be readout by proliferation or cytokine production (IL-2, IFNg, IL-17) or other markers, such as CD69 activation marker. Thus, PD-1 agonism can be measured by either cytokine production or cell proliferation. Other methods can also be used to measure PD-1 agonism.
PD-1 is Ig superfamily member expressed on activated T cells and other immune cells. The natural ligands for PD-1 appear to be PD-L1 and PD-L2. Without being bound to any particular theory, when PD-L1 or PD-L2 bind to PD-1 on an activated T cell, an inhibitory signaling cascade is initiated, resulting in attenuation of the activated T effector cell function. Thus, blocking the interaction between PD-1 on a T cell, and PD-L1/2 on another cell (for example, a tumor cell) with a PD-1 antagonist is known as checkpoint inhibition, and releases the T cells from inhibition. In contrast, PD-1 agonist antibodies can bind to PD-1 and send an inhibitory signal and attenuate the function of a T cell. Thus, PD-1 agonist antibodies can be incorporated into various embodiments described herein as an effector molecule binding/modulating moiety, which can accomplish localized tissue-specific immunomodulation when paired with a targeting moiety.
The effector molecule binding/modulating moiety can provide an immunosuppressive signal or environment in a variety of ways. In some embodiments, the effector binding/modulating moiety comprises an ICIM binding/modulating moiety that directly binds and (under the appropriate conditions as described herein) activates an inhibitory receptor expressed by immune cells responsible for driving disease pathology. In another embodiment the effector binding/modulating moiety comprises and IIC binding/modulating moiety and binds and accumulates immunosuppressive immune cells. In some embodiments, the accumulated immune suppressive cells promote immune privilege. In another embodiment the effector binding/modulating moiety comprises an SM binding/modulating moiety which manipulates the surrounding microenvironment to make it less permissible for the function of immune cells, e.g., immune cells driving disease pathology. In some embodiments, the SM binding/modulating moiety depletes an entity that promotes immune attack or activation. In some embodiments the effector binding/modulating moiety comprises an ICSM binding/modulating moiety that binds a member of a pair of stimulatory molecules, e.g., costimulatory molecules, and inhibits the interaction between the costimulatory molecule and the costimulatory molecule counterstructure, such as, but not limited to, OX40 or CD30 or CD40 and OX40L, or CD30L or CD40L and inhibits the immune stimulation of a cell, such as, but not limited to, a T cell, B cell, NK cell, or other immune cell comprising a member of the pair.
The targeting moiety and effector binding/modulating moiety are physically tethered, covalently or non-covalently, directly or through a linker entity, to one another, e.g., as a member of the same protein molecule in a therapeutic protein molecule. In some embodiments, the targeting and effector moieties are provided in a therapeutic protein molecule, e.g., a fusion protein, typically as separate domains. In some embodiments, the targeting moiety, the effector binding/modulating moiety, or both each comprises a single domain antibody molecule, e.g., a camelid antibody VHH molecule or human soluble VH domain. It may also contain a single-chain fragment variable (scFv) or a Fab domain. In some embodiments, the therapeutic protein molecule, or a nucleic acid, e.g., an mRNA or DNA, encoding the therapeutic protein molecule, can be administered to a subject. In some embodiments, the targeting and effector molecule binding/modulating moieties are linked to a third entity, e.g., a carrier, e.g., a polymeric carrier, a dendrimer, or a particle, e.g., a nanoparticle. The therapeutic compounds can be used to down regulate an immune response at or in a tissue at a selected target or site while having no or substantially less immunosuppressive function systemically. The target or site can comprise donor tissue or autologous tissue.
Provided herein are methods of providing site-specific immune privilege for a transplanted donor tissue, e.g., an allograft tissue, e.g., a tissue described herein, e.g., an allograft liver, an allograft kidney, an allograft heart, an allograft pancreas, an allograft thymus or thymic tissue, allograft skin, or an allograft lung, with therapeutic compounds disclosed herein. In embodiments the treatment minimizes rejection of, minimizes immune effector cell mediated damage to, prolongs acceptance of, or prolongs the functional life of, donor transplant tissue.
Also provided herein are methods of inhibiting graft versus host disease (GVHD) by minimizing the ability of donor immune cells, e.g., donor T cells, to mediate immune attack of recipient tissue, with therapeutic compounds disclosed herein.
Also provided herein are methods of treating, e.g., therapeutically treating or prophylactically treating (or preventing), an auto-immune disorder or response in a subject by administration of a therapeutic compound disclosed herein, e.g., to provide site or tissue specific modulation of the immune system. In some embodiments, the method provides tolerance to, minimization of the rejection of, minimization of immune effector cell mediated damage to, or prolonging a function of, subject tissue. In some embodiments, the therapeutic compound includes a targeting moiety that targets, e.g., specifically targets, the tissue under, or at risk for, autoimmune attack. Non-limiting exemplary tissues include, but are not limited to, the pancreas, myelin, salivary glands, synoviocytes, and myocytes.
As used herein, the terms “treat,” “treated,” or “treating” in regards to therapeutic treatment wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results. For example, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. Thus, “treatment of an auto-immune disease/disorder” means an activity that alleviates or ameliorates any of the primary phenomena or secondary symptoms associated with the auto-immune disease/disorder or other condition described herein. The various disease or conditions are provided herein. The therapeutic treatment can also be administered prophylactically to preventing or reduce the disease or condition before the onset.
In some embodiments, administration of the therapeutic compound begins after the disorder is apparent. In some embodiments, administration of the therapeutic compound, begins prior to onset, or full onset, of the disorder. In some embodiments, administration of the therapeutic compound, begins prior to onset, or full onset, of the disorder, e.g., in a subject having the disorder, a high-risk subject, a subject having a biomarker for risk or presence of the disorder, a subject having a family history of the disorder, or other indicator of risk of, or asymptomatic presence of, the disorder. For example, In some embodiments, a subject having islet cell damage but which is not yet diabetic, is treated.
While not wishing to be bound by theory, it is believed that the targeting moiety functions to bind and accumulate the therapeutic to a target selectively expressed at the anatomical site where immune privilege is desired. In some embodiments, e.g., in the context of donor tissue transplantation, the target moiety binds to a target, e.g., an allelic product, present in the donor tissue but not the recipient. For treatment of autoimmune disorders, the targeting moiety binds a target preferentially expressed at the anatomical site where immune privilege is desired, e.g., in the pancreas. For treatment of GVHD, the targeting moiety targets the host tissue, and protects the host against attack from transplanted immune effector cells derived from transplanted tissue.
Again, while not wishing to be bound by theory it is believed that the effector binding/modulating moiety serves to deliver an immunosuppressive signal or otherwise create an immune privileged environment.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these embodiments belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Headings, sub-headings or numbered or lettered elements, e.g., (a), (b), (i) etc., are presented merely for ease of reading. The use of headings or numbered or lettered elements in this document does not require the steps or elements be performed in alphabetical order or that the steps or elements are necessarily discrete from one another. Other features, objects, and advantages of the embodiments will be apparent from the description and drawings, and from the claims.
Additional Definitions
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 embodiments pertains. In describing and claiming the present embodiments, the following terminology and terminology otherwise referenced throughout the present application will be used according to how it is defined, where a definition is provided.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Antibody molecule, as that term is used herein, refers to a polypeptide, e.g., an immunoglobulin chain or fragment thereof, comprising at least one functional immunoglobulin variable domain sequence. An antibody molecule encompasses antibodies (e.g., full-length antibodies) and antibody fragments. In some embodiments, an antibody molecule comprises an antigen binding or functional fragment of a full length antibody, or a full length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that is naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes). In embodiments, an antibody molecule refers to an immunologically active, antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment, e.g., functional fragment, comprises a portion of an antibody, e.g., Fab, Fab′, F(ab′)2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single chain variable fragment (scFv). A functional antibody fragment binds to the same antigen as that recognized by the intact (e.g., full-length) antibody. The terms “antibody fragment” or “functional fragment” also include isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”). In some embodiments, an antibody fragment does not include portions of antibodies without antigen binding activity, such as Fc fragments or single amino acid residues. Exemplary antibody molecules include full length antibodies and antibody fragments, e.g., dAb (domain antibody), single chain, Fab, Fab′, and F(ab′)2 fragments, and single chain variable fragments (scFvs).
The term “antibody molecule” also encompasses whole or antigen binding fragments of domain, or single domain, antibodies, which can also be referred to as “sdAb” or “VHH.” Domain antibodies comprise either VH or VL that can act as stand-alone, antibody fragments. Additionally, domain antibodies include heavy-chain-only antibodies (HCAbs). Domain antibodies also include a CH2 domain of an IgG as the base scaffold into which CDR loops are grafted. It can also be generally defined as a polypeptide or protein comprising an amino acid sequence that is comprised of four framework regions interrupted by three complementarity determining regions. This is represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. sdAbs can be produced in camelids such as llamas, but can also be synthetically generated using techniques that are well known in the art. The numbering of the amino acid residues of a sdAb or polypeptide is according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest,” US Public Health Services, NIH Bethesda, Md., Publication No. 91, which is hereby incorporated by reference). According to this numbering, FR1 of a sdAb comprises the amino acid residues at positions 1-30, CDR1 of a sdAb comprises the amino acid residues at positions 31-36, FR2 of a sdAb comprises the amino acids at positions 36-49, CDR2 of a sdAb comprises the amino acid residues at positions 50-65, FR3 of a sdAb comprises the amino acid residues at positions 66-94, CDR3 of a sdAb comprises the amino acid residues at positions 95-102, and FR4 of a sdAb comprises the amino acid residues at positions 103-113. Domain antibodies are also described in WO2004041862 and WO2016065323, each of which is hereby incorporated by reference. The domain antibodies can be a targeting moiety as described herein.
Antibody molecules can be monospecific (e.g., monovalent or bivalent), bispecific (e.g., bivalent, trivalent, tetravalent, pentavalent, or hexavalent), trispecific (e.g., trivalent, tetravalent, pentavalent, hexavalent), or with higher orders of specificity (e.g., tetraspecific) and/or higher orders of valency beyond hexavalency. An antibody molecule can comprise a functional fragment of a light chain variable region and a functional fragment of a heavy chain variable region, or heavy and light chains may be fused together into a single polypeptide.
Examples of formats for multispecific therapeutic compounds, e.g., bispecific antibody molecules are shown in the following non-limiting examples. Although illustrated with antibody molecules, they can be used as platforms for therapeutic molecules that include other non-antibody moieties as specific binding or effector moieties. In some embodiments, these non-limiting examples are based upon either a symmetrical or asymmetrical Fc formats.
For example, the figures illustrate non-limiting and varied symmetric homodimer approach. In some embodiments, the dimerization interface centers around human IgG1 CH2-CH3 domains, which dimerize via a contact interface spanning both CH2/CH2 and CH3/CH3. The resulting bispecific antibodies shown have a total valence comprised of four binding units with two identical binding units at the N-terminus on each side of the dimer and two identical units at the C-terminus on each side of the dimer. In each case the binding units at the N-terminus of the homo-dimer are different from those at the C-terminus of the homo-dimer.
Using this type of bivalency for both an inhibitory T cell receptor at either terminus of the molecule and bivalency for a tissue tethering antigen can be achieved at either end of the molecule.
For example, in FIG. 3, a non-limiting embodiment is illustrated. The N-terminus of the homodimer contains two identical Fab domains comprised of two identical light chains, which are separate polypeptides, interfaced with the n-terminal VH-CH1 domains of each heavy chain via the VH/VL interaction and Ckappa or Clambda interaction with CH1. The native disulphide bond between the Ckappa or Clambda with CH1 is present providing a covalent anchor between the light and heavy chains. At the c-terminus of this design are two identical scFv units where by (in this example) the c-terminus of the CH3 domain of the Fc, is followed by a flexible, hydrophilic linker typically comprised of (but not limited to) serine, glycine, alanine, and/or threonine residues, which is followed by the VH domain of each scFv unit, which is followed by a glycine/serine rich linker, followed by a VL domain. These tandem VH and VL domains associate to form a single chain fragment variable (scFv) appended at the c-terminus of the Fc. Two such units exist at the c-terminus of this molecule owing to the homodimeric nature centered at the Fc. The domain order of scFvs may be configured to be from N to C terminus either VH-Linker-VL or VL-Linker-VH.
A non-limiting example of a molecule that has different binding regions on the different ends is where, one end is a PD-1 agonist and the antibody that provides target specificity is an anti-MAdCAM-1 antibody. This can be illustrated as shown, for example, in FIG. 3A, which illustrates the molecules in different orientations.
In some embodiments, the MAdCAM antibody is a blocking or non-blocking antibody as described elsewhere herein. Without being bound to any theory, MAdCAM has been shown to interact with the headpiece of the integrin α4β7 expressed on lymphocytes via multiple residues within its two Ig superfamily I-set domains and the atomic level structural basis for that interaction has been described (Viney J L et al. (1996). J Immunol. 157, 2488-2497; Yu Y et al (2013). J Biol Chem. 288, 6284-6294; Yu Y et al (2012). J Cell Biol. 196, 131-146, each of which is incorporated by reference in its entirety). It has been shown in great structural, mechanistic and functional detail in both the human (Chen J et al (2003). Nat Struct Biol. 10, 995-1001; de Chateau M et al (2001). Biochemistry. 40, 13972-13979) and mouse (Day E S et al (2002). Cell Commun Adhes. 9, 205-219; Hoshino H et al (2011). J Histochem Cytochem. 59, 572-583) molecular systems that any interaction of MAdCAM with α4β7 is dependent on three dication binding sites present in the integrin beta 7 sub unit I-like domain and that these metal binding sites can coordinate with Ca2+, Mn2+, and Mg2+. Using cell adhesion assays, flow cytometry, and/or flow chamber assays in the presence of high levels of Ca2+ with or without Mg2+ or Mn2+, the MAdCAM/α4β7 interaction is shown to be of a lower functional affinity and permits rolling adhesion of lymphocytes, whereas in low Ca2+ but higher Mg2+ or Mn2+ which activates the integrin, the MAdCAM/α4β7 interaction is of a higher functional affinity and mediates firm lymphocyte adhesion (Chen J et al (2003). Nat Struct Biol. 10, 995-1001). A number of groups have shown that various cell:cell, cell:membrane prep, and/or cell:protein based adhesion/interaction assays can be utilized, with FACS, cell flow chamber based counts, or IHC based read-outs to monitor the impact of anti-MAdCAM or anti-α4β7 antibodies upon the interaction of MAdCAM with α4β7, allowing one to identify blocking or non-blocking antibodies (Nakache, M et al (1989). Nature. 337, 179-181; Streeter, P R et al (1988). Nature. 331. 41-46; Yang Y et al (1995). Scand J Immunol. 42. 235-247; Leung E et al (2004). Immunol Cell Biol. 82. 400-409; Pullen N et al (2009). B J Pharmacol. 157. 281-293; Soler D et al (2009). J Pharmacol Exp Ther. 330. 864-875; Qi J et al (2012). J Biol Chem. 287. 15749-15759).
This has been exemplified in the mouse system setting with the identification of anti-mouse MAdCAM antibodies such as MECA-89 (non-blocking) and MECA-367 (blocking)) Nakache, M et al (1989). Nature. 337, 179-181; Streeter, P R et al (1988). Nature. 331. 41-46; Yang Y et al (1995). Scand J Immunol. 42. 235-247). In a human system, antibodies have been identified that block the interaction of human MAdCAM with human α4β7 such as anti-human MAdCAM PF-00547659 (Pullen N et al (2009). B J Pharmacol. 157. 281-293) and anti-human α4β7 vedolizumab (Soler D et al (2009). J Pharmacol Exp Ther. 330. 864-875), as well as antibodies that do not block the interaction such as anti-human MAdCAM clone 17F5 (Soler D et al (2009). J Pharmacol Exp Ther. 330. 864-875), and anti-human α4β7 clone J19 (Qi J et al (2012). J Biol Chem. 287. 15749-15759). Thus, the antibody can either be blocking or non-blocking based upon the desired effect. In some embodiments, the antibody is a non-blocking MAdCAM antibody. In some embodiments, the antibody is a blocking MAdCAM antibody. One non-limiting example of demonstrating whether an antibody is blocking or non-blocking can be found in Example 6, but any method can be used. Each of the references described herein are incorporated by reference in its entirety. In some embodiments, the PD-1 Agonist is replaced with an IL-2 mutein, such as, but not limited to, the ones described herein.
In another example, and as depicted in FIG. 4, the N-terminus of the homodimer contains two identical Fab domains comprised of two identical light chains, which are separate polypeptides, interfaced with the n-terminal VH-CH1 domains of each heavy chain via the VH/VL interaction and Ckappa or Clambda interaction with CH1. The native disulphide bond between the Ckappa or Clambda with CH1 is present providing a covalent anchor between the light and heavy chains. At the c-terminus of this design are two identical VH units (though non-antibody moieties could also be substituted here or at any of the four terminal attachment/fusion points) where by (in this example) the c-terminus of the CH3 domain of the Fc, is followed by a flexible, hydrophilic linker typically comprised of (but not limited to) serine, glycine, alanine, and/or threonine residues, which is followed by a soluble independent VH3 germline family based VH domain. Two such units exist at the c-terminus of this molecule owing to the homodimeric nature centered at the Fc.
In another non-limiting example, as depicted in FIG. 5, the N-terminus of the homodimer contains two identical Fab domains comprised of two identical light chains, which, unlike FIG. 3 and FIG. 4, are physically conjoined with the heavy chain at the N-terminus via a linker between the c-terminus of Ckappa or Clambda and the N-terminus of the VH. The linker may be 36-80 amino acids in length and comprised of serine, glycine, alanine and threonine residues. The physically conjoined n-terminal light chains interface with the n-terminal VH-CH1 domains of each heavy chain via the VH/VL interaction and Ckappa or Clambda interaction with CH1. The native disulphide bond between the Ckappa or Clambda with CH1 is present providing additional stability between the light and heavy chains. At the c-terminus of this design are two identical Fab units where by (in this example) the c-terminus of the CH3 domain of the Fc, is followed by a flexible, hydrophilic linker typically comprised of (but not limited to) serine, glycine, alanine, and/or threonine residues, which is followed by a CH1 domain, followed by a VH domain at the c-terminus. The light chain that is designed to pair with the c-terminal CH1/VH domains is expressed as a separate polypeptide, unlike the N-terminal light chain which is conjoined to the n-terminal VH/CH1 domains as described. The C-terminal light chains form an interface at between VH/VL and Ckappa or Clambda with CH1. The native disulphide anchors this light chain to the heavy chain. Again, any of the antibody moieties at any of the four attachment/fusion points can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
The bispecific antibodies can also be asymmetric as shown in the following non-limiting examples. Non-limiting example are also depicted in FIG. 6, FIG. 7, and FIG. 8, which illustrate an asymmetric/heterodimer approach. Again, in any of these formats, any of the antibody moieties at any of the four attachment/fusion points can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule. In some embodiments, the dimerization interface centers around the human IgG1 CH2-CH3 domains, which dimerize via a contact interface spanning both CH2/CH2 and CH3/CH3. However, in order to achieve heterodimerization instead of homodimerization of each heavy chain, mutations are introduced in each CH3 domain. The heterodimerizing mutations include T366W mutation (kabat) in one CH3 domain and T366S, L368A, and Y407V (kabat) mutations in the other CH3 domain. The heterodimerizing interface may be further stabilized with de novo disulphide bonds via mutation of native residues to cysteine residues such as 5354 and Y349 on opposite sides of the CH3/CH3 interface. The resulting bispecific antibodies shown have a total valence comprised of four binding units. With this approach, the overall molecule can be designed to have bispecificity at just one terminus and monospecificity at the other terminus (trispecificity overall) or bispecificity at either terminus with an overall molecular specificity of 2 or 4. In the illustrative examples below, the C-terminus comprises two identical binding domains which could, for example, provide bivalent monospecificity for a tissue tethering target. At the N-terminus of all three of the illustrative examples, both binding domains comprise different recognition elements/paratopes and which could achieve recognition of two different epitopes on the same effector moiety target, or could recognize for examples a T cell inhibitory receptor and CD3. In some embodiments, the N-terminal binding moieties may be interchanged with other single polypeptide formats such as scFv, single chain Fab, tandem scFv, VH or VHH domain antibody configurations for example. Other types of recognition element may be used also, such as linear or cyclic peptides.
An example of an asymmetric molecule is depicted in FIG. 6. Referring to FIG. 6, the N-terminus of the molecule is comprised of a first light chain paired with a first heavy chain via VH/VL and Ckappa or Clambda/CH1 interactions and a covalent tether comprised of the native heavy/light chain disulphide bond. On the opposite side of this heterodimeric molecule at the N-terminus is a second light chain and a second heavy chain which are physically conjoined via a linker between the c-terminus of Ckappa or Clambda and the N-terminus of the VH. The linker may be 36-80 amino acids in length and comprised of serine, glycine, alanine and threonine residues. The physically conjoined n-terminal light chains interface with the n-terminal VH-CH1 domains of each heavy chain via the VH/VL interaction and Ckappa or Clambda interaction with CH1. The native disulphide bond between the Ckappa or Clambda with CH1 is present providing additional stability between the light and heavy chains. At the c-terminus of the molecule are two identical soluble VH3 germline family VH domains joined via an N-terminal glycine/serine/alanine/threonine based linker to the c-terminus of the CH3 domain of both heavy chain 1 and heavy chain 2.
In some embodiments, an asymmetric molecule can be as illustrated as depicted in FIG. 7. For example, the N-terminus of the molecule is comprised of two different VH3 germlined based soluble VH domains linked to the human IgG1 hinge region via a glycine/serine/alanine/threonine based linker. The VH domain connected to the first heavy chain is different to the VH domain connected to the second heavy chain. At the c-terminus of each heavy chain is an additional soluble VH3 germline based VH domain, which is identical on each of the two heavy chains. The heavy chain heterodimerizes via the previously described knobs into holes mutations present at the CH3 interface of the Fc module.
In some embodiments, an asymmetric molecule can be as illustrated in FIG. 8. This example is similar to the molecule shown in FIG. 7, except both N-terminal Fab units are configured in a way that light chain 1 and light chain 2 are physically conjoined with heavy chain 1 and heavy chain 2 via a linker between the c-terminus of Ckappa or Clambda and the N-terminus of each respective VH. The linker in each case may be 36-80 amino acids in length and comprised of serine, glycine, alanine and threonine residues. The physically conjoined n-terminal light chains interface with the n-terminal VH-CH1 domains of each heavy chain via the VH/VL interaction and Ckappa or Clambda interaction with CH1. The native disulphide bond between the Ckappa or Clambda with CH1 is present providing additional stability between the light and heavy chains.
Bi-specific molecules can also have a mixed format. This is illustrated, for example, in FIG. 9, FIG. 10, and FIG. 11.
For example, as illustrated in FIG. 9, illustrates a homodimer Fc based approach (see FIGS. 3, 4, and 5), combined with the moiety format selection of FIG. 7, whereby the total molecular valency is four, but specificity is restricted to two specificities. The N-terminus is comprised of two identical soluble VH3 germline based VH domains and the c-terminus is comprised of two identical soluble VH3 germlined based VH domains of different specificity to the N-terminal domains. Therefore, each specificity has a valence of two. Again, in this format, any of the antibody moieties at any of the four attachment/fusion points can be substituted with a non-antibody moiety, e.g., an effector binding/modulating moiety that does not comprise an antibody molecule.
FIG. 10 illustrates another example. In this example, the molecule is comprised of four VH3 germline based soluble VH domains. The first two domains have the same specificity (for example an inhibitory receptor), the 3rd domain from the N-terminus may have specificity for a tissue antigen and the fourth domain from the N-terminus may have specificity for human serum albumin (HSA), thereby granting the molecule extended half-life in the absence of an Ig Fc domain. Three glycine, serine, alanine and/or threonine rich linkers exists between domains 1 and 2, domains 2 and 3, and domains 3 and 4. This format may be configured with up to tetraspecificity, but monovalent in each case, or to have bispecificity with bivalency in each case. The order of domains can be changed. Again, in this format, any of the antibody moieties can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
FIG. 11 illustrates yet another approach. This example is similar to FIGS. 3 and 4, in that it is Fc homodimer based with two identical Fab units (bivalent mono specificity) at the N-terminus of the molecule. This example differs in that the C-terminus of each heavy chain is appended with a tandem-scFv. Thus, in each case the c-terminus of the CH3 domain of the Fc is linked via a glycine/serine/alanine/threonine based linker to the N-terminus of a first VH domain, which is linked via the C-terminus by a 12-15 amino acid glycine/serine rich linker to the N-terminus of a first VL domain, which linked via a 25-35 amino acid glycine/serine/alanine/threonine based linker at the c-terminus to the N-terminus of a second VH domain, which is linked via the c-terminus with a 12-15 amino acid glycine/serine based linker to the N-terminus of a 2nd VL domain. In this Fc homodimer based molecule there are therefore two identical tandem scFvs at the c-terminus of the molecule offering either tetravalency for a single tissue antigen for example or bivalency to two different molecules. This format could also be adapted with a heterodimer Fc core allowing two different tandem-scFvs at the c-terminus of the Fc allowing for monovalent tetraspecificity at the c-terminus while retaining either bivalent monospecificity at the N-terminus or monovalent bispecificity at the N-terminal via usage of single chain Fab configurations as in FIGS. 5, 6, and 7. This molecule can therefore be configured to have 2, 3, 4, 5, or 6 specificities. The domain order of scFvs within the tandem—scFv units may be configured to be from N to C terminus either VH-Linker-VL or VL-Linker-VH. Again, in this format, any of the antibody moieties at any of the four attachment/fusion points can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
Bi-specific antibodies can also be constructed to have, for example, shorter systemic PK while having increased tissue penetration. These types of antibodies can be based upon, for example, a human VH3 based domain antibody format. These are illustrated, for example, in FIGS. 12, 13, and 14. FIGS. 12, 13, and 14 each comprised a soluble VH3 germline family based VH domain modules. Each domain is approximately 12.5 kDa allowing for a small overall MW, which, without being bound to any particular theory, should be beneficial for enhanced tissue penetration. In these examples, none of the VH domains recognize any half-life extending targets such as FcRn or HSA. As illustrated in FIG. 12, the molecule is comprised of two VH domains joined with a flexible hydrophilic glycine/serine based linker between the C-terminus of the first domain and N-terminus of the second domain. In this example one domain may recognize a T cell co-stimulatory receptor and the second may recognize a tissue tethering antigen. As illustrated in FIG. 13, the molecule is comprised of three VH domains with N-C terminal linkages of hydrophilic glycine/serine based linkers. The molecule may be configured to be trispecific but monovalent for each target. It may be bispecific with bivalency for one target and monovalency for another. As illustrated in FIG. 14, the molecule is comprised of four VH domains with N-C terminal Glycine/Serine rich linkers between each domain. This molecule may be configured to be tetraspecific, trispecific, or bispecific with varying antigenic valencies in each case. Again, in this format, any of the antibody moieties at can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
Other embodiments of bi-specific antibodies are illustrated in FIGS. 15 and 16. FIGS. 15 and 16 are comprised of the naturally heterodimerizing core of the human IgG CH1/Ckappa interface, including the c-terminal heavy/light disulphide bond which covalently anchors the interaction. This format does not contain an Fc or any moieties for half-life extension. As illustrated in FIG. 15, the molecule, at the N-terminus of the constant kappa domain is appended with an scFv fragment consisting of an N-terminal VH domain, linked at its C-terminus to the N-terminus of a VL domain via a 12-15 amino acid gly/ser based linker, which is linked by its C-terminus to the N-terminus of the constant kappa domain via the native VL-Ckappa elbow sequence. The CH1 domain is appended at the N-terminus with an scFv fragment consisting of an N-terminal VL domain linked at its c-terminus via a 12-15 amino acid gly/ser linker to the N-terminus of a VH domain, which is linked at its c-terminus to the N-terminus of the CH1 domains via the natural VH-CH1 elbow sequence. As illustrated in FIG. 16, the molecule has the same N-terminal configuration to Example 13. However the C-terminus of the constant kappa and CH1 domains are appended with scFv modules which may be in either the VH-VL or VL-VH configuration and may be either specific for the same antigen or specific for two different antigens. The VH/VL inter-domain linkers may be 12-15 amino acids in length and consisting of gly/ser residues. The scFv binding sub-units may be swapped for soluble VH domains, or peptide recognition elements, or even tandem-scFv elements. This approach can also be configured to use variable lambda and/or constant lambda domains. Again, in this format, any of the antibody moieties at any of the attachment/fusion points can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
FIG. 17 illustrates another embodiment. FIG. 17 represents a tandem scFv format consisting of a first N-terminal VL domain linked at its C-terminus to the N-terminus of a first VH domain with a 12-15 amino acid gly/ser rich linker, followed at the first VH c-terminus by a 25-30 amino acid gly/ser/ala/thr based linker to the N-terminus of a second VL domain. The second VL domain is linked at the C-terminus to the N-terminus of a 2nd VH domain by a 12-15 amino acid gly/ser linker. Each scFv recognizes a different target antigen such as a co-stimulatory T cell molecule and a tissue tethering target. Again, in this format, any of the antibody moieties can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
FIG. 18 illustrates another embodiment. FIG. 18 is a F(ab′)2 scFv fusion. This consists of two identical Fab components joined via two disulphide bonds in the native human IgG1 hinge region c-terminal of the human IgG CH1 domain. The human IgG1 CH2 and CH3 domains are absent. At the c-terminus of heavy chains 1 and 2 are two identical scFv fragments linked via a gly/ser/ala/thr rich linker to the c-terminus of the huIgG1 hinge region. In the configuration shown, the VH is N-terminal in each scFv unit and linked via a 12-15 amino acid gly/ser rich linker to the N-terminus of a VL domain. An alternative configuration would be N-term-VL-Linker-VH-C-term. In this design, the construct is bispecific with bivalency for reach target. Again, in this format, any of the antibody moieties at any of the four attachment/fusion points can be substituted with a non-antibody moiety, e.g., a effector binding/modulating moiety that does not comprise an antibody molecule.
CD39 molecule, as that term as used herein, refers to a polypeptide having sufficient CD39 sequence that, as part of a therapeutic compound, it phosphohydrolyzes ATP to AMP. In some embodiments, a CD39 molecule phosphohydrolizes ATP to AMP equivalent to, or at least, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the rate of a naturally occurring CD39, e.g., the CD39 from which the CD39 molecule was derived. In some embodiments, a CD39 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring CD39.
Any functional isoform can be used (with CD39 or other proteins discussed herein). Exemplary CD39 sequence include Genbank accession # NP_001767.3 or a mature form from the following sequence:









(SEQ ID NO: 1)



MEDTKESNVKTFCSKNILAILGFSSIIAVIALLAVGLTQNKALPE



 



NVKYGIVLDAGSSHTSLYIYKWPAEKENDTGVVHQVEECRVKGPG



 



ISKFVQKVNEIGIYLTDCMERAREVIPRSQHQETPVYLGATAGMR



 



LLRMESEELADRVLDVVERSLSNYPFDFQGARIITGQEEGAYGWI



 



TINYLLGKFSQKTRWFSIVPYETNNQETFGALDLGGASTQVTFVP



 



QNQTIESPDNALQFRLYGKDYNVYTHSFLCYGKDQALWQKLAKDI



 



QVASNEILRDPCFHPGYKKVVNVSDLYKTPCTKRFEMTLPFQQFE



 



IQGIGNYQQCHQSILELFNTSYCPYSQCAFNGIFLPPLQGDFGAF



 



SAFYFVMKFLNLTSEKVSQEKVTEMMKKFCAQPWEEIKTSYAGVK



 



EKYLSEYCFSGTYILSLLLQGYHFTADSWEHIHFIGKIQGSDAGW



 



TLGYMLNLTNMIPAEQPLSTPLSHSTYVFLMVLFSLVLFTVAIIG



 



LLIFHKPSYFWKDMV.






In some embodiments, a CD39 molecule comprises a soluble catalytically active form of CD39 found to circulate in human or murine serum, see, e.g., Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-1 and NTPDase1/CD39 activities, Yegutkin et al. FASEB J. 2012 September; 26(9):3875-83. A soluble recombinant CD39 fragment is also described in Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39, Gayle, et al., J Clin Invest. 1998 May 1; 101(9): 1851-1859.
CD73 molecule, as that term as used herein, refers to a polypeptide having sufficient CD73 sequence that, as part of a therapeutic compound, it dephosphorylates extracellular AMP to adenosine. In some embodiments, a CD73 molecule dephosphorylates extracellular AMP to adenosine equivalent to, or at least, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the rate of a naturally occurring CD73, e.g., the CD73 from which the CD73 molecule was derived. In some embodiments, a CD73 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring CD73. Exemplary CD73 sequences include GenBank AAH65937.1 5′-nucleotidase, ecto (CD73) [Homo sapiens] or a mature form from the following sequence,









(SEQ ID NO: 2)



MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTS



 



EDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDACiDQYQG



 



TIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKE



 



AKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSK



 



ETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFE



 



MDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTS



 



DDGRKVPVVQAYAFGKYLGYLKIEFDERGNV1SSHGNPILLNSSI



 



PEDPS1KADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECN



 



MGNLICDAMINNNLRHADETFWNHVSMCILNGGGIRSPIDERNNG



 



TITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEF



 



LQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYK



 



VILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIY



 



PAVEGRIKFSTGSHCHGSFSLIFLSLWAVIFVLYQ.






In some embodiments, a CD73 molecule comprises a soluble form of CD73 which can be shed from the membrane of endothelial cells by proteolytic cleavage or hydrolysis of the GPI anchor by shear stress see, e.g., Reference: Yegutkin G, Bodin P, Burnstock G. Effect of shear stress on the release of soluble ecto-enzymes ATPase and 5′-nucleotidase along with endogenous ATP from vascular endothelial cells. Br J Pharmacol 2000; 129: 921-6. For CD73 function see Colgan et al., Physiological roles for ecto-5′-nucleotidase (CD73), Purinergic Signalling, June 2006, 2:351.
Cell surface molecule binder, as that term is used herein, refers to a molecule, typically a polypeptide, that binds, e.g., specifically, to a cell surface molecule on a cell, e.g., an immunosuppressive immune cell, e.g., a Treg. In some embodiments, the cell surface binder has sufficient sequence from a naturally occurring ligand of the cell surface molecule, that it can specifically bind the cell surface molecule (a cell surface molecule ligand). In some embodiments, the cell surface binding is an antibody molecule that binds, e.g., specifically binds, the cell surface molecule.
Donor specific targeting moiety, as that term is used herein, refers to a moiety, e.g., an antibody molecule, that as a component of a therapeutic compound, localizes the therapeutic compound preferentially to an implanted donor tissue, as opposed to tissue of a recipient. As a component of a therapeutic compound, the donor specific targeting moiety provides site-specific immune privilege for a transplant tissue, e.g., an organ, from a donor.
In some embodiments, a donor specific targeting moiety it binds to the product, e.g., a polypeptide product, of an allele present at a locus, which allele is not present at the locus in the (recipient) subject. In some embodiments, a donor specific targeting moiety binds to an epitope on product, which epitope is not present in the (recipient) subject.
In some embodiments, a donor specific targeting moiety, as a component of a therapeutic compound, preferentially binds to a donor target or antigen, e.g., has a binding affinity for the donor target that is greater for donor antigen or tissue, e.g., at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 fold greater, than its affinity for than for subject antigen or tissue. In some embodiments, a donor specific targeting moiety, has a binding affinity for a product of an allele of a locus present in donor tissue (but not present in the subject) at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 fold greater, than its affinity for the product of the allele of the locus present in the subject (which allele is not present in donor tissue). Affinity of a therapeutic compound of which the donor specific moiety is a component, can be measured in a cell suspension, e.g., the affinity for suspended cells having the allele is compared with its affinity for suspended cells not having the allele. In some embodiments, the binding affinity for the donor allele cells is below 10 nM. In some embodiments, the binding affinity for the donor allele cells is below 100 pM, 50 pM, or 10 pM.
In some embodiments, the specificity for a product of a donor allele is sufficient that when the donor specific targeting moiety is coupled to an immune-down regulating effector: i) immune attack of the implanted tissue, e.g., as measured by histological inflammatory response, infiltrating T effector cells, or organ function, in the clinical setting—e.g. creatinine for the kidney, is substantially reduced, e.g., as compared to what would be seen in an otherwise similar implant but lacking the donor specific targeting moiety is coupled to an immune-down regulating effector; and/or ii) immune function in the recipient, outside or away from the implanted tissue, is substantially maintained. In some embodiments, one or more of the following is seen: at therapeutic levels of therapeutic compound, peripheral blood lymphocyte counts are not substantially impacted, e.g., the level of T cells is within 25, 50, 75, 85, 90, or 95% of normal, the level of B cells is within 25, 50, 75, 85, 90, or 95% of normal, and/or the level of granuloctyes (PMNs) cells is within 25, 50, 75, 85, 90, or 95% of normal, or the level of monocytes is within 25, 50, 75, 85, 90, or 95% of normal; at therapeutic levels of therapeutic compound, the ex vivo proliferative function of PBMCs (peripheral blood mononuclear cells) against non-disease relevant antigens is substantially normal or is within 70, 80, or 90% of normal; at therapeutic levels of therapeutic compound, the incidence or risk of risk of opportunistic infections and cancers associated with immunosuppression is not substantially increased over normal; or at therapeutic levels of therapeutic compound, the incidence or risk of risk of opportunistic infections and cancers associated with immunosuppression is substantially less than would be seen with standard of care, or non-targeted, immunosuppression. In some embodiments, the donor specific targeting moiety comprises an antibody molecule, a target specific binding polypeptide, or a target ligand binding molecule.
Effector, as that term is used herein, refers to an entity, e.g., a cell or molecule, e.g., a soluble or cell surface molecule, which mediates an immune response.
Effector ligand binding molecule, as used herein, refers to a polypeptide that has sufficient sequence from a naturally occurring counter-ligand of an effector, that it can bind the effector with sufficient specificity that it can serve as an effector binding/modulating molecule. In some embodiments, it binds to effector with at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the affinity of the naturally occurring counter-ligand. In some embodiments, it has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring counter-ligand for the effector.
Effector specific binding polypeptide, as used herein, refers to a polypeptide that can bind with sufficient specificity that it can serve as an effector binding/modulating moiety. In some embodiments, a specific binding polypeptide comprises a effector ligand binding molecule.
Elevated risk, as used herein, refers to the risk of a disorder in a subject, wherein the subject has one or more of a medical history of the disorder or a symptom of the disorder, a biomarker associated with the disorder or a symptom of the disorder, or a family history of the disorder or a symptom of the disorder.
Functional antibody molecule to an effector or inhibitory immune checkpoint molecule, as that term is used herein, refers to an antibody molecule that when present as the ICIM binding/modulating moiety of a multimerized therapeutic compound, can bind and agonize the effector or inhibitory immune checkpoint molecule. In some embodiments, the anti-effector or inhibitory immune checkpoint molecule antibody molecule, when binding as a monomer (or binding when the therapeutic compound is not multimerized), to the effector or inhibitory immune checkpoint molecule, does not antagonize, substantially antagonize, prevent binding, or prevent substantial binding, of an endogenous counter ligand of the inhibitory immune checkpoint molecule to inhibitory immune checkpoint molecule. In some embodiments, the anti-effector or inhibitory immune checkpoint molecule antibody molecule when binding as a monomer (or binding when the therapeutic compound is not multimerized), to the inhibitory immune checkpoint molecule, does not agonize or substantially agonize, the effector or inhibitory molecule.
ICIM binding/modulating moiety, as that term is used herein, refers to an effector binding/modulating moiety that, as part of a therapeutic compound, binds and agonizes a cell surface inhibitory molecule, e.g., an inhibitory immune checkpoint molecule, e.g., PD-1, or binds or modulates cell signaling, e.g., binds a FCRL, e.g., FCRL1-6, or binds and antagonizes a molecule that promotes immune function.
IIC binding/modulating moiety, as that term is used herein, refers to an effector binding/modulating moiety that, as part of a therapeutic compound, binds an immunosuppressive immune cell. In some embodiments, the IIC binding/modulating moiety increases the number or concentration of an immunosuppressive immune cell at the binding site.
ICSM binding/modulating moiety, as that term is used herein, refers to an effector binding/modulating moiety that antagonizes an immune stimulatory effect of a stimulatory, e.g., co-stimulatory, binding pair. A stimulatory or co-stimulatory binding pair, as that term is used herein, comprises two members, 1) a molecule on the surface of an immune cell; and 2) the binding partner for that cell molecule, which may be an additional immune cell, or a non-immune cell. Ordinarily, upon binding of one member to the other, assuming other requirements are met, the member on the immune cell surfaces stimulates the immune cell, e.g., a costimulatory molecule, and an immune response is promoted. In situations where the costimulatory molecule and the costimulatory molecule counterstructure are both expressed on immune cells, bi-directional activation of both cells may occur. In an embodiment an ICSM binding/modulating moiety binds and antagonizes the immune cell expressed member of a binding pair. For example, it binds and antagonizes OX40. In another embodiment, an ICSM binding/modulating moiety binds and antagonizes the member of the binding pair that itself binds the immune cell expressed member, e.g., it binds and antagonizes OX40L. In either case, inhibition of stimulation or co-stimulation of an immune cell is achieved. In an embodiment the ICSM binding/modulating moiety decreases the number or the activity of an immunostimulating immune cell at the binding site.
IL-2 mutein molecule, as that term is used herein, refers to an IL2 variant that binds with high affinity to the CD25 (IL-2R alpha chain) and with low affinity to the other IL-2R signaling components CD122 (IL-2R beta) and CD132 (IL-2R gamma). Such an IL-2 mutein molecule preferentially activates Treg cells, In embodiments, either alone, or as a component of a therapeutic compound, an IL-2 mutein activates Tregs at least 2, 5, 10, or 100 fold more than cytotoxic or effector T cells. Exemplary IL-2 mutein molecules are described in WO2010085495, WO2016/164937, US2014/0286898A1, WO2014153111A2, WO2010/085495, cytotoxic WO2016014428A2, WO2016025385A1, and US20060269515. Muteins disclosed in these references that include additional domains, e.g., an Fc domain, or other domain for extension of half-life can be used in the therapeutic compounds and methods described herein without such additional domains. In another embodiment an IIC binding/modulating moiety comprises an IL-2 mutein, or active fragment thereof, coupled, e.g., fused, to another polypeptide, e.g., a polypeptide that extends in vivo half-life, e.g., an immunoglobulin constant region, or a multimer or dimer thereof, e.g., AMG 592. In an embodiment the therapeutic compound comprises the IL-2 portion of AMG 592. In an embodiment the therapeutic compound comprises the IL-2 portion but not the immunoglobulin portion of AMG 592. In some embodiments, the mutein does not comprise a Fc region. For some IL-2 muteins, the muteins are engineered to contain a Fc region because such region has been shown to increase the half-life of the mutein. In some embodiments, the extended half-life is not necessary for the methods described and embodied herein. In some embodiments, the Fc region that is fused with the IL-2 mutein comprises a N297 mutations, such as, but not limited to, N297A. In some embodiments, the Fc region that is fused with the IL-2 mutein does not comprise a N297 mutation, such as, but not limited to, N297A.
An “inhibitory immune checkpoint molecule ligand molecule,” as that term is used herein, refers to a polypeptide having sufficient inhibitory immune checkpoint molecule ligand sequence, e.g., in the case of a PD-L1 molecule, sufficient PD-L1 sequence, that when present as an ICIM binding/modulating moiety of a multimerized therapeutic compound, can bind and agonize its cognate inhibitory immune checkpoint molecule, e.g., again in the case of a PD-L1 molecule, PD-1.
In some embodiments, the inhibitory immune checkpoint molecule ligand molecule, e.g., a PD-L1 molecule, when binding as a monomer (or binding when the therapeutic compound is not multimerized), to its cognate ligand, e.g., PD-1, does not antagonize or substantially antagonize, or prevent binding, or prevent substantial binding, of an endogenous inhibitory immune checkpoint molecule ligand to the inhibitory immune checkpoint molecule. E.g., in the case of a PD-L1 molecule, the PD-L1 molecule does not antagonize binding of endogenous PD-L1 to PD-1.
In some embodiments, the inhibitory immune checkpoint molecule ligand when binding as a monomer, to its cognate inhibitory immune checkpoint molecule does not agonize or substantially agonize the inhibitory immune checkpoint molecule. By way of example, e.g., a PD-L1 molecule when binding to PD-1, does not agonize or substantially agonize PD-1.
In some embodiments, an inhibitory immune checkpoint molecule ligand molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring inhibitory immune checkpoint molecule ligand.
Exemplary inhibitory immune checkpoint molecule ligand molecules include: a PD-L1 molecule, which binds to inhibitory immune checkpoint molecule PD-1, and in embodiments has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring PD-L1, e.g., the PD-L1 molecule comprising the sequence of









(SEQ ID NO: 3)



MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVE



 



KQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKD



 



QLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYN



 



KINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTT



 



TTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELV



 



IPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVK



 



KCGIQDTNSKKQSDTHLEET,






or an active fragment thereof; in some embodiments, the active fragment comprises residues 19 to 290 of the PD-L1 sequence; a HLA-G molecule, which binds to any of inhibitory immune checkpoint molecules KIR2DL4, LILRB1, and LILRB2, and in embodiments has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring HLA-G. Exemplary HLA-G sequences include, e.g., a mature form found in the sequence at GenBank P17693.1 RecName: Full=HLA class I histocompatibility antigen, alpha chain G; AltName: Full=HLA G antigen; AltName: Full=MHC class I antigen G; Flags: Precursor, or in the sequence








(SEQ ID NO: 4)


MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMG


 


YVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRM


 


NLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLAL


 


NEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGK


 


EMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQ


 


DVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQ


 


SSLPTIPIMGIVA.






Inhibitory molecule counter ligand molecule, as that term is used herein, refers to a polypeptide having sufficient inhibitory molecule counter ligand sequence such that when present as the ICIM binding/modulating moiety of a multimerized therapeutic compound, can bind and agonize a cognate inhibitory molecule. In some embodiments, the inhibitory molecule counter ligand molecule, when binding as a monomer (or binding when the therapeutic compound is not multimerized), to the inhibitory molecule, does not antagonize, substantially antagonize, prevent binding, or prevent substantial binding, of an endogenous counter ligand of the inhibitory molecule to the inhibitory molecule. In some embodiments, the inhibitory molecule counter ligand molecule when binding as a monomer (or binding when the therapeutic compound is not multimerized), to the inhibitory molecule, does not agonize or substantially agonize, the inhibitory molecule.
Sequence identity, percentage identity, and related terms, as those terms are used herein, refer to the relatedness of two sequences, e.g., two nucleic acid sequences or two amino acid or polypeptide sequences. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.
In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.
The term “functional variant” refers to polypeptides that have a substantially identical amino acid sequence to the naturally-occurring sequence, or are encoded by a substantially identical nucleotide sequence, and are capable of having one or more activities of the naturally-occurring sequence.
Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).
The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to for example any a nucleic acid sequence provided herein. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules provided herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.
It is understood that the molecules and compounds of the present embodiments may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.
The term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. As used herein the term “amino acid” includes both the D- or L-optical isomers and peptidomimetics. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). CD39 molecule, a CD73 molecule, a Cell surface molecule binder, Donor specific targeting moiety Effector ligand binding molecule, ICIM binding/modulating moiety IIC binding/modulating moiety, an inhibitory immune checkpoint molecule ligand molecule, Inhibitory molecule counter ligand molecule, SM binding/modulating moiety, or ICSM binding/modulating moiety.
SM binding/modulating moiety, as that term is used herein, refers to an effector binding/modulating moiety that, as part of a therapeutic compound, promotes an immuno-suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target. In some embodiments, the SM binding/modulating moiety comprises, or binds, a molecule that inhibits or minimizes attack by the immune system of the target. In some embodiments, a therapeutic compound comprises an SM binding/modulating moiety that binds and accumulates a soluble substance, e.g., an endogenous or exogenous substance, having immunosuppressive function. In some embodiments, a therapeutic compound comprises an SM binding/modulating moiety that binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble substance, typically and endogenous soluble substance, that promotes immune attack. In some embodiments, a therapeutic compound comprises an SM binding/modulating moiety that comprises an immune-suppressive substance, e.g. a fragment of protein known to be immunosuppressive. By way of example, an effector molecule binding moiety that binds, or comprises, a substance e.g., a CD39 molecule or a CD73 molecule, that depletes a component, that promotes immune effector cell function, e.g., ATP or AMP.
Specific targeting moiety, as that term is used herein, refers to donor specific targeting moiety or a tissue specific targeting moiety.
Subject, as that term is used herein, refers to a mammalian subject, e.g., a human subject. In some embodiments, the subject is a non-human mammal, e.g., a horse, dog, cat, cow, goat, or pig.
Target ligand binding molecule, as used herein, refers to a polypeptide that has sufficient sequence from a naturally occurring counter-ligand of a target ligand that it can bind the target ligand on a target tissue (e.g., donor tissue or subject target tissue) with sufficient specificity that it can serve as a specific targeting moiety. In some embodiments, it binds to target tissue or cells with at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the affinity of the naturally occurring counter-ligand. In some embodiments, it has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring counter-ligand for the target ligand.
Target site, as that term is used herein, refers to a site which contains the entity, e.g., epitope, bound by a targeting moiety. In some embodiments, the target site is the site at which immune privilege is established.
Tissue specific targeting moiety, as that term is used herein, refers to a moiety, e.g., an antibody molecule, that as a component of a therapeutic molecule, localizes the therapeutic molecule preferentially to a target tissue, as opposed to other tissue of a subject. As a component of a therapeutic compound, the tissue specific targeting moiety provides site-specific immune privilege for a target tissue, e.g., an organ or tissue undergoing or at risk for autoimmune attack. In some embodiments, a tissue specific targeting moiety binds to a product, e.g., a polypeptide product, which is not present outside the target tissue, or is present at sufficiently low levels that, at therapeutic concentrations of therapeutic molecule, unacceptable levels of immune suppression are absent or substantially absent. In some embodiments, a tissue specific targeting moiety binds to an epitope, which epitope is not present outside, or not substantially present outside, the target site.
In some embodiments, a tissue specific targeting moiety, as a component of a therapeutic compound, preferentially binds to a target tissue or target tissue antigen, e.g., has a binding affinity for the target tissue or antigen that is greater for target antigen or tissue, e.g., at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 fold greater, than its affinity for than for non-target tissue or antigen present outside the target tissue. Affinity of a therapeutic compound of which the tissue specific moiety is a component, can be measured in a cell suspension, e.g., the affinity for suspended cells having the target antigen is compared with its affinity for suspended cells not having the target antigen. In some embodiments, the binding affinity for the target antigen bearing cells is below 10 nM.
In some embodiments, the binding affinity for the target antigen bearing cells is below 100 pM, 50 pM, or 10 pM. In some embodiments, the specificity for a target antigen is sufficient, that when the tissue specific targeting moiety is coupled to an immune-down regulating effector: i) immune attack of the target tissue, e.g., as measured by histological inflammatory response, infiltrating T effector cells, or organ function, in the clinical setting—e.g. creatinine for kidney, is substantially reduced, e.g., as compared to what would be seen in an otherwise similar implant but lacking the tissue specific targeting moiety is coupled to an immune-down regulating effector; and/or ii) immune function in the recipient, outside or away from the target tissue, is substantially maintained.
In some embodiments, one or more of the following is seen: at therapeutic levels of therapeutic compound, peripheral blood lymphocyte counts are not substantially impacted, e.g., the level of T cells is within 25, 50, 75, 85, 90, or 95% of normal, the level of B cells is within 25, 50, 75, 85, 90, or 95% of normal, and/or the level of granulocytes (PMNs) cells is within 25, 50, 75, 85, 90, or 95% of normal, or the level of monocytes is within 25, 50, 75, 85, 90, or 95% of normal l; at therapeutic levels of therapeutic compound, the ex vivo proliferative function of PBMCs (peripheral blood mononuclear cells) against non-disease relevant antigens is substantially normal or is within 70, 80, or 90% of normal; at therapeutic levels of therapeutic compound, the incidence or risk of risk of opportunistic infections and cancers associated with immunosuppression is not substantially increased over normal; or at therapeutic levels of therapeutic compound, the incidence or risk of risk of opportunistic infections and cancers associated with immunosuppression is substantially less than would be seen with standard of care, or non-targeted, immunosuppression. In some embodiments, the tissue specific targeting moiety comprises an antibody molecule. In some embodiments, the donor specific targeting moiety comprises an antibody molecule, a target specific binding polypeptide, or a target ligand binding molecule. In some embodiments, the tissue specific targeting moiety binds a product, or a site on a product, that is present or expressed exclusively, or substantially exclusively, on target tissue.
ICIM Binding/Modulating Moieties: Effector Binding/Modulating Moieties that Bind Inhibitory Receptors
Methods and compounds described herein provide for a therapeutic compound having an effector binding/modulating moiety comprising an ICIM binding/modulating moiety, that directly binds and activates an inhibitory receptor on the surface of an immune cell, e.g., to reduce or eliminate, or substantially eliminate, the ability of the immune cell to mediate immune attack. Coupling of the ICIM binding/modulating moiety to a targeting entity, promotes site-specific or local down regulation of the immune cell response, e.g., confined substantially to the locations having binding sites for the targeting moiety. Thus, normal systemic immune function is substantially retained. In some embodiments, an ICIM binding/modulating moiety comprises an inhibitory immune checkpoint molecule counter ligand molecule, e.g., a natural ligand, or fragment of a natural ligand (e.g., PD-L1 or HLA-G) of the inhibitory immune checkpoint molecule. In some embodiments, an ICIM binding/modulating moiety comprises a functional antibody molecule, e.g., a functional antibody molecule comprising an scFv binding domain, that engages inhibitory immune checkpoint molecule.
In some embodiments, the ICIM binding/modulating moiety, comprising, e.g., a functional antibody molecule, or inhibitory immune checkpoint molecule ligand molecule, binds the inhibitory receptor but does not prevent binding of a natural ligand of the inhibitory receptor to the inhibitory receptor. In embodiments a format is used wherein a targeting moiety is coupled, e.g., fused, to an ICIM binding/modulating moiety, comprising, e.g., an scFv domain, and configured so that upon binding of an inhibitory receptor while in solution (e.g., in blood or lymph) (and presumably in a monomeric format), the therapeutic molecule: i) fails to agonize, or fails to substantially agonize (e.g., agonizes at less than 30, 20, 15, 10, or 5% of the level seen with a full agonizing molecule) the inhibitory receptor on the immune cell; and/or ii) fails to antagonize, or fails to substantially antagonize (e.g., antagonizes at less than 30, 20, 15, 10, or 5% of the level seen with a full antagonizing molecule) the inhibitory receptor on the immune cell. A candidate molecule can be evaluated for its ability to agonize or not agonize by its ability to either increase or decrease the immune response in an in vitro cell based assay wherein the target is not expressed, e.g., using an MLR-based assay (mixed lymphocyte reaction).
In some embodiments, candidate ICIM binding/modulating moieties can reduce, completely or substantially eliminate systemic immunosuppression and systemic immune activation. In some embodiments, the targeting domain of the therapeutic compound, when bound to target, will serve to cluster or multimerize the therapeutic compound on the surface of the tissue desiring immune protection. In some embodiments, the ICIM binding/modulating moiety, e.g., an ICIM binding/modulating moiety comprising a scFv domain, requires a clustered or multimeric state to be able to deliver an agonistic and immunosuppressive signal, or substantial levels of such signal, to local immune cells. This type of therapeutic can, for example, provide to a local immune suppression whilst leaving the systemic immune system unperturbed or substantially unperturbed. That is, the immune suppression is localized to where the suppression is needed as opposed to being systemic and not localized to a particular area or tissue type.
In some embodiments, upon binding to the target e.g., a target organ, tissue or cell type, the therapeutic compound coats the target, e.g., target organ, tissue or cell type. When circulating lymphocytes attempt to engage and destroy the target, this therapeutic will provide an ‘off’ signal only at, or to a greater extent at, the site of therapeutic compound accumulation.
A candidate therapeutic compound can be evaluated for the ability to bind, e.g., specifically bind, its target, e.g., by ELISA, a cell based assay, or surface plasmon resonance. This property should generally be maximized, as it mediates the site-specificity and local nature of the immune privilege. A candidate therapeutic compound can be evaluated for the ability to down regulate an immune cell when bound to target, e.g., by a cell based activity assay. This property should generally be maximized, as it mediates the site-specificity and local nature of the immune privilege. The level of down regulation effected by a candidate therapeutic compound in monomeric (or non-bound) form can be evaluated, e.g., by a cell based activity assay. This property should generally be minimized, as could mediate systemic down regulation of the immune system. The level of antagonism of a cell surface inhibitory molecule, e.g., an inhibitory immune checkpoint molecule, effected by a candidate therapeutic compound in monomeric (or non-bound) form can be evaluated, e.g., by, e.g., by a cell based activity assay. This property should generally be minimized, as could mediate systemic unwanted activation of the immune system. Generally, the properties should be selected and balanced to produce a sufficiently robust site specific immune privilege without unacceptable levels of non-site specific agonism or antagonism of the inhibitory immune checkpoint molecule.
Exemplary Inhibitory Immune Checkpoint Molecules
Exemplary inhibitory molecules (e.g., an inhibitory immune checkpoint molecule) (together with their counter ligands) can be found in Table 1. This table lists molecules to which exemplary ICIM binding moieties can bind.







TABLE 1







Cell surface inhibitory molecules, e.g., 


inhibitory immune checkpoint molecules


(column A), counter ligands (column B)


and cell types affected (column C).









A
B
C





PD-1
PD-L1, PD-L2
T cells, B cells


Alkaline




phosphatase




B7-H3
Unknown
T cells


B7-H4
Neuropilin 1,
T cells



neuropilin 2,




Plexin4A



BTLA
HVEM
T cells, B cells


CTLA-4
CD80, CD86
T cells


IDO1
Tryptophan
Lymphocytes


TDO2
Tryptophan
Lymphocytes


KIR2DL1,
HLA MHC class I
NK cells


KIR2DL2/3,




KIR3DL1,




KIR3DL2




LAG3
HLA MHC class II
T cells


TIM-3
Galectin-9
T cells


VISTA
Unknown
T cells, myeloid cells


TIGIT
CD155
T cells


KIR2DL4
HLA-G
NK cells


LILRB1
HLA-G
T cells, NK cells, B cells, 




monocytes, dendritic cells


LILRB2
HLA-G
Monocytes, dendritic cells, 




neutrophils, some tumor cells


NKG2A
nonclassical MHC
T cells, NK cells



glycoproteins class I



FCRL1-6
FCRL1—2 not
B cells



known




FCRL4 = IgA




FCRL5 = IgG




FCRL6 = MHC Class II




BUTYROPHILINS,
Modulation of immune cells



for example




BTN1A1, BTN2A2,




BTNL2, BTNL1,




BTNL8









The PD-L1/PD-1 Pathway
Programmed cell death protein 1, (often referred to as PD-1) is a cell surface receptor that belongs to the immunoglobulin superfamily. PD-1 is expressed on T cells and other cell types including, but not limited to, B cells, myeloid cells, dendritic cells, monocytes, T regulatory cells, iNK T cells. PD-1 binds two ligands, PD-L1 and PD-L2, and is an inhibitory immune checkpoint molecule. Engagement with a cognate ligand, PD-L1 or PD-L2, in the context of engagement of antigen loaded MCH with the T Cell Receptor on a T cell minimizes or prevents the activation and function of T cells. The inhibitory effect of PD-1 can include both promoting apoptosis (programmed cell death) in antigen specific T-cells in lymph nodes and reducing apoptosis in regulatory T cells (suppressor T cells).
In some embodiments, a therapeutic compound comprises an ICIM binding/modulating moiety which agonizes PD-1 inhibition. An ICIM binding/modulating moiety can include an inhibitory molecule counter ligand molecule, e.g., comprising a fragment of a ligand of PD-1 (e.g., a fragment of PD-L1 or PD-L2) or another moiety, e.g., a functional antibody molecule, comprising, e.g., an scFv domain that binds PD-1.
In some embodiments, a therapeutic compound comprises a targeting moiety that is preferentially binds a donor antigen not present in, present in substantially lower levels in the subject, e.g., a donor antigen from Table 2, and is localized to donor graft tissue in a subject. In some embodiments, it does not bind, or does not substantially bind, other tissues. In some embodiments, a therapeutic compound can include a targeting moiety that is specific for HLA-A2 and specifically binds donor allograft tissue but does not bind, or does not substantially bind, host tissues. In some embodiments, the therapeutic compound comprises an ICIM binding/modulating moiety, e.g., an inhibitory molecule counter ligand molecule, e.g., comprising a fragment of a ligand of PD-1 (e.g., a fragment of PD-L1 or PD-L2) or another moiety, e.g., a functional antibody molecule, comprising, e.g., an scFv domain that binds PD-1, such that the therapeutic compound, e.g., when bound to target, activates PD-1. The therapeutic compound targets an allograft and provides local immune privilege to the allograft.
In some embodiments, a therapeutic compound comprises a targeting moiety that is preferentially binds to an antigen of Table 3, and is localized to the target in a subject, e.g., a subject having an autoimmune disorder, e.g., an autoimmune disorder of Table 3. In some embodiments, it does not bind, or does not substantially bind, other tissues. In some embodiments, the therapeutic compound comprises an ICIM binding/modulating moiety, e.g., an inhibitory molecule counter ligand molecule, e.g., comprising a fragment of a ligand of PD-1 (e.g., a fragment of PD-L1 or PD-L2) or another moiety, e.g., a functional antibody molecule, comprising, e.g., an scFv domain that binds PD-1, such that the therapeutic compound, e.g., when bound to target, activates PD-1. The therapeutic compound targets a tissue subject to autoimmune attack and provides local immune privilege to the tissue.
PD-L1 and PDL2, or polypeptides derived therefrom, can provide candidate ICIM binding moieties. However, in monomer form, e.g., when the therapeutic compound is circulating in blood or lymph, this molecule could have an undesired effect of antagonizing the PD-L1/PD-1 pathway, and may only agonize the PD-1 pathway when clustered or multimerized on the surface of a target, e.g., a target organ. In some embodiments, a therapeutic compound comprises an ICIM binding/modulating moiety comprising a functional antibody molecule, e.g., a scFv domain, that is inert, or substantially inert, to the PD-1 pathway in a soluble form but which agonizes and drives an inhibitory signal when multimerized (by the targeting moiety) on the surface of a tissue.
The HLA-G: KIR2DL4/LILRB1/LILRB2 Pathway
KIR2DL4, LILRB1, and LILRB2 are inhibitory molecules found on T cells, NK cells, and myeloid cells. HLA-G is a counter ligand for each.
KIR2DL4 is also known as CD158D, G9P, KIR-103AS, KIR103, KIR103AS, KIR, KIR-2DL4, killer cell immunoglobulin like receptor, and two Ig domains and long cytoplasmic tail 4. LILRB1 is also known as LILRB1, CD85J, ILT-2, ILT2, LIR-1, LIR1, MIR-7, MIR7, PIR-B, PIRB, leukocyte immunoglobulin like receptor B1. LILRB2 is also known as CD85D, ILT-4, LIR-2, LIR2, MIR-10, MIR10, and ILT4.
A therapeutic compound comprising an HLA-G molecule can be used to provide inhibitory signals to an immune cell comprising any of KIR2DL4, LILRB1, and LILRB2, e.g., with multimerized therapeutic compound molecules comprising an HLA-G molecule and thus provide site-specific immune privilege.
A therapeutic compound comprising an agonistic anti-KIR2DL4, anti-LILRB1, or anti-LILRB2 antibody molecule can be used to provide inhibitory signals to an immune cell comprising any of KIR2DL4, LILRB1, and LILRB2.
HLA-G only delivers an inhibitory signal when multimerized, for example, when expressed on the surface of a cell or when conjugated to the surface of a bead. In embodiments, a therapeutic compound comprising an HLA-G molecule which therapeutic compound does not multimerize in solution (or does not multimerize sufficiently to result in significant levels of inhibitory molecule agonization), is provided. The use of HLA-G molecules that minimize mulitmerization in solution will minimize systemic agonization of immune cells and unwanted immune suppression.
While not wishing to be bound by theory it is believed that HLA-G is not effective in down regulation unless multimerized, that binding of the therapeutic compound to target, through the targeting moiety, multimerizes the ICIM binding entity, and that the multimerized ICIM binding entity, binds and clusters inhibitory molecules on the surface of an immune cell, thus mediating a negative signal that down regulates the immune cell. Thus, infiltrating immune cells attempting to damage the target tissue, including antigen presenting cells and other myeloid cells, NK cells and T cells, are down regulated.
While HLA-G molecules minimize antagonism when in monomeric form are desirable, the redundancy of LILRB1 and LILRB2 will minimize, the impact on systemic even with some monomeric antagonism.
In some embodiments, the therapeutic compound comprises an ICIM binding/modulating moiety that comprises a HLA-G molecule, e.g., an B2M-free isoform (e.g., HLA-G5), see Carosella et al., Advances in Immunology, 2015, 127:33. In a B2M-free format, HLA-G preferentially binds LILRB2.
Suitable sequences for the construction of HLA-G molecules include GenBank P17693.1 RecName: Full=HLA class I histocompatibility antigen, alpha chain G; AltName: Full=HLA G antigen; AltName: Full=MHC class I antigen G; Flags: Precursor, or







(SEQ ID NO: 5)


MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMG


 


YVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRM


 


NLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLAL


 


NEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGK


 


EMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQ


 


DVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQ


 


SSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD.






A candidate HLA-G molecule can be tested for suitability for use in methods and compounds, e.g., by methods analogous to those described in “Synthetic HLA-G proteins for therapeutic use in transplantation,” LeMaoult et al., 2013 The FASEB Journal 27:3643.

In some embodiments, a therapeutic compound comprises a targeting moiety that is preferentially binds a donor antigen not present in, present in substantially lower levels in the subject, e.g., a donor antigen from Table 2, and is localized to donor graft tissue in a subject. In some embodiments, it does not bind, or does not substantially bind, other tissues. In some embodiments, a therapeutic compound can include a targeting moiety that is specific for HLA-A2 and specifically binds a donor allograft but does not bind host tissues and is combined with an ICIM binding/modulating moiety that comprises a HLA-G molecule that binds KIR2DL4, LILRB1, or LILRB2, such that the therapeutic compound, e.g., when bound to target, activates KIR2DL4, LILRB1, or LILRB2. The therapeutic compound targets an allograft and provides local immune privilege to the allograft.
In some embodiments, a therapeutic compound comprises a targeting moiety that is preferentially binds a tissue specific antigen, e.g., an antigen from Table 3, and is localized to the target site in a subject, e.g., a subject having an autoimmune disorder, e.g., an autoimmune disorder from Table 3. In some embodiments, it does not bind, or does not substantially bind, other tissues. In embodiments the therapeutic compound comprises an ICIM binding/modulating moiety that comprises a HLA-G molecule binds KIR2DL4, LILRB1, or LILRB2, such that the therapeutic compound, e.g., when bound to target, activates KIR2DL4, LILRB1, or LILRB2. The therapeutic compound targets an tissue subject to autoimmune attack and provides local immune privilege to the tissue.
It is likely possible to engineer a stable and soluble HLA-G-B2M fusion protein that can also bind LILRB1. For example, the crystal structure of HLA-G was determined using HLA-G/B2M monomers (Clements et al. 2005 PNAS 102:3360)
FCRL Family
FCRL1-6 generally inhibit B cell activation or function. These type 1 transmembrane glycoproteins are composed of different combinations of 5 types of immunoglobulin-like domains, with each protein consisting of 3 to 9 domains, and no individual domain type conserved throughout all of the FCRL proteins. In general, FCRL expression is restricted to lymphocytes, with the primary expression in B-lymphocytes. Generally, FCRLs function to repress B-cell activation.
An ICIM binding/modulating moiety can comprise an agonistic anti-BCMA antibody molecule. In some embodiments, the therapeutic compound comprises an anti-FCRL antibody molecule and an anti-B cell receptor (BCR) antibody molecule. While not wishing to be bound be theory is believed that a therapeutic compound comprising anti-body molecules of both specificities will bring the FCRL into close proximity with the BCR and inhibit BCR signaling.
Butyrophilins and Butyrophilin-Like Molecules
Effector binding/modulating moiety can comprise an agonist or antagonist of a butyrophilin. In some embodiments, an effector binding/modulating moiety an agonistic or functional BTN1A1 molecule, BTN2A2 molecule, BTNL2 molecule, or BTNL1 molecule.
A functional BTNX1 molecule (where Xi=1A1, 2A2, L2 or L1), as that term as used herein, refers to a polypeptide having sufficient BTNX1 sequence that, as part of a therapeutic compound, it inhibits T cells. In some embodiments, a BTNX1 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring butyrophilin or butyrophilin-like molecule.
In some embodiments, an effector binding/modulating moiety an antagonistic BTNL8 molecule.
An antagonistic BTNL8 molecule, as that term as used herein, refers to a polypeptide having sufficient BTNL8 sequence that, as part of a therapeutic compound, it inhibits the activation, proliferation, or secretion of cytokine by a resting T cell. In some embodiments, a BTNL8 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring butyrophilin.
IIC Binding/Modulating Moieties: Effector Binding/Modulating Moieties that Recruit Immunosuppressive T Cells
In some embodiments, a therapeutic compound comprises an effector binding/modulating moiety, e.g., an IIC binding/modulating moiety, that binds, activates, or retains immunosuppressive cells, e.g., immunosuppressive T cells, at the site mediated by the targeting moiety, providing site-specific immune privilege. The IIC binding/modulating moiety, e.g., an IIC binding/modulating moiety comprising an antibody molecule, comprising, e.g., an scFv binding domain, binds immunosuppressive cell types, e.g., Tregs, e.g., Foxp3+CD25+ Tregs. Organ, tissue or specific cell type tolerance is associated with an overwhelming increase of Tregs proximal and infiltrating the target organ; in embodiments, the methods and compounds described herein synthetically re-create and mimic this physiological state. Upon accumulation of Tregs, an immunosuppressive microenvironment is created that serves to protect the organ of interest from the immune system.
GARP-Binders as a Treg and TGFB Targeting Molecule
GARP is a membrane protein receptor for latent TGF-beta expressed on the surface of activated Tregs (Tran et al. 2009 PNAS 106:13445 and Wang et al. 2009 PNAS 106:13439). In some embodiments, a therapeutic compound comprises an IIC binding entity that binds one or both of soluble GARP and GARP-expressing cells, such as activated human Tregs, and a targeting moiety that targets the therapeutic compound to the target tissue of interest. IIC binding/modulating moieties that comprises a GARP-Binder include, e.g., an IIC binding/modulating moiety that comprises an anti-GARP antibody molecule, e.g., an anti-GARP scFv domain. While not wishing to be bound by theory, it is believed that the therapeutic compound that comprises a GARP binder effects accumulation of GARP-expressing Tregs at the site targeted by the targeting moiety of the therapeutic compound, e.g., a transplant or site of organ injury. Again, while not wishing to be bound by theory, it is believed that a therapeutic compound that comprises a GARP binder effects can also effect accumulation of soluble GARP at site of organ injury, which will serve to bind and activate TGFB1, an immuno-suppressive cytokine, in a local manner (Fridrich et al. 2016 PLoS One 11:e0153290; doi: 10.1371/journal.pone.0153290 and Hahn et al. 2013 Blood 15:1182). Thus, an effector binding/modulating moiety that comprises a GARP binder can act as either a IIC binding/modulating moiety or an SM binding/modulating moiety.
CTLA4 as a Treg Targeting and T Effector Cell Silencing Molecule
In some embodiments, an effector binding/modulating moiety, e.g., comprises an antibody molecule, e.g., an scFv domain, that binds CTLA4 expressed on the surface of Tregs. The therapeutic molecule accumulates or retains CTLA4+ Tregs at the target site, with local immunosuppression the consequence.
Though expressed more highly on Tregs, CTLA4 is also expressed on activated T cells. A therapeutic compound comprising an effector binding/modulating moiety, e.g., an anti-CTLA4 antibody, or a functional anti-CTLA4 antibody, can down regulate the CTLA4 expressing T cell. Thus, in a therapeutic compound comprising an effector binding/modulating moiety that binds CTLA4, the effector moiety can also act as an ICIM binding/modulating moiety.
In some embodiments, the anti-CTLA4 binder is neither antagonizing or agonizing when in monomeric format, and is only agonizing when clustered or multimerized upon binding to the target.
While not wishing to be bound by theory it is believed that the binding of the therapeutic compound, via the targeting moiety, to the target, effects multimerization of therapeutic compound. In the case of memory and activated T cells, CTLA4 bound by the effector binding/modulating moiety of the therapeutic compound, is clustered, and an inhibitory signal by engagement of CTLA4 expressed by memory and activated T cells
In some embodiments, the anti-CTLA4 binder is neither antagonizing or agonizing when in monomeric format, and is only agonizing when clustered or multimerized upon binding to the target.
IL-2 Mutein Molecules: IL2 Receptor Binders that Activate Tregs
IL-2 mutein molecules that preferentially expand or stimulate Treg cells (over cytotoxic T cells) can be used as an IIC binding/modulating moiety.
In some embodiments, IIC binding/modulating moiety comprises a IL-2 mutein molecule. As used herein, the term “IL-2 mutein molecule” or “IL-2 mutein” refers to an IL-2 variant that preferentially activates Treg cells. In some embodiments, either alone, or as a component of a therapeutic compound, an IL-2 mutein molecule activates Tregs at least 2, 5, 10, or 100 fold more than cytotoxic T cells. A suitable assay for evaluating preferential activation of Treg cells can be found in U.S. Pat. No. 9,580,486 at, for example, Examples 2 and 3, or in WO2016014428 at, for example, Examples 3, 4, and 5, each of which is incorporated by reference in its entirety. The sequence of mature IL-2 is







(SEQ ID NO: 6)


APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA


 


TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE


 


TTFMCEYADETATIVEFLNRWITFCQSIISTLT


 


(mature IL-2 sequence)






The immature sequence of IL-2 can be represented by








(SEQ ID NO: 15)


MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINN


 


YKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHL


 


RPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIS


 


TLT.






In some embodiments, an IIC binding/modulating moiety comprises an IL-2 mutein, or active fragment thereof, coupled, e.g., fused, to another polypeptide, e.g., a polypeptide that extends in vivo half-life, e.g., an immunoglobulin constant region, or a multimer or dimer thereof.

An IL-2 mutein molecule can be prepared by mutating one or more of the residues of IL-2. Non-limiting examples of IL-2-muteins can be found in WO2016/164937, U.S. Pat. Nos. 9,580,486, 7,105,653, 9,616,105, 9,428,567, US2017/0051029, US2014/0286898A1, WO2014153111A2, WO2010/085495, WO2016014428A2, WO2016025385A1, and US20060269515, each of which are incorporated by reference in its entirety.
In some embodiments, the alanine at position 1 of the sequence above is deleted. In some embodiments, the IL-2 mutein molecule comprises a serine substituted for cysteine at position 125 of the mature IL-2 sequence. Other combinations of mutations and substitutions that are IL-2 mutein molecules are described in US20060269515, which is incorporated by reference in its entirety. In some embodiments, the cysteine at position 125 is also substituted with a valine or alanine. In some embodiments, the IL-2 mutein molecule comprises a V91K substitution. In some embodiments, the IL-2 mutein molecule comprises a N88D substitution. In some embodiments, the IL-2 mutein molecule comprises a N88R substitution. In some embodiments, the IL-2 mutein molecule comprises a substitution of H16E, D84K, V91N, N88D, V91K, or V91R, any combinations thereof. In some embodiments, these IL-2 mutein molecules also comprise a substitution at position 125 as described herein. In some embodiments, the IL-2 mutein molecule comprises one or more substitutions selected from the group consisting of: T3N, T3A, L12G, L12K, L12Q, L12S, Q13G, EISA, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H165, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20H, D201, D20Y, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88I, N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91S, I92K, I92R, E95G, and Q126. In some embodiments, the amino acid sequence of the IL-2 mutein molecule differs from the amino acid sequence set forth in mature IL-2 sequence with a C125A or C125S substitution and with one substitution selected from T3N, T3A, L12G, L12K, L12Q L12S, Q13G, EISA, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88I, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91S, I92K, I92R, E95G, Q1261, Q126L, and Q126F. In some embodiments, the IL-2 mutein molecule differs from the amino acid sequence set forth in mature IL-2 sequence with a C125A or C125S substitution and with one substitution selected from D20H, D201, D20Y, D20E, D20G, D20W, D84A, D84S, H16D, H16G, H16K, H16R, H16T, H16V, I92K, I92R, L12K, L19D, L19N, L19T, N88D, N88R, N88S, V91D, V91G, V91K, and V91S. In some embodiments, the IL-2 mutein comprises N88R and/or D20H mutations.
In some embodiments, the IL-2 mutein molecule comprises a mutation in the polypeptide sequence at a position selected from the group consisting of amino acid 30, amino acid 31, amino acid 35, amino acid 69, and amino acid 74. In some embodiments, the mutation at position 30 is N30S. In some embodiments, the mutation at position 31 is Y31H. In some embodiments, the mutation at position 35 is K35R. In some embodiments, the mutation at position 69 is V69A. In some embodiments, the mutation at position 74 is Q74P. In some embodiments, the mutein comprises a V69A mutation, a Q74P mutation, a N88D or N88R mutation, and one or more of L53I, L56I, L80I, or L118I mutations. In some embodiments, the mutein comprises a V69A mutation, a Q74P mutation, a N88D or N88R mutation, and a L to I mutation selected from the group consisting of: L53I, L56I, L80I, and L118I mutation. In some embodiments, the IL-2 mutein comprises a V69A, a Q74P, a N88D or N88R mutation, and a L53I mutation. In some embodiments, the IL-2 mutein comprises a V69A, a Q74P, a N88D or N88R mutation, and a L56I mutation. In some embodiments, the IL-2 mutein comprises a V69A, a Q74P, a N88D or N88R mutation, and a L80I mutation. In some embodiments, the IL-2 mutein comprises a V69A, a Q74P, a N88D or N88R mutation, and a L118I mutation. As provided for herein, the muteins can also comprise a C125A or C125S mutation.
In some embodiments, the mutein comprises a T3A mutation. The full length IL-2 muteins provided herein may not be illustrated with a T3A or other mutations provided for herein, but such mutations can be added into the muteins provided herein as is the case for any of the other mutations illustrated herein. Accordingly, In some embodiments, the mutein comprises a T3N mutation. In some embodiments, the mutein comprises a T3A mutation. In some embodiments, the mutein comprises a L12G mutation. In some embodiments, the mutein comprises a L12K mutation. In some embodiments, the mutein comprises a L12Q mutation. In some embodiments, the mutein comprises a L12S mutation. In some embodiments, the mutein comprises a Q13G mutation. In some embodiments, the mutein comprises a EISA mutation. In some embodiments, the mutein comprises a E15G mutation. In some embodiments, the mutein comprises a E15S mutation. In some embodiments, the mutein comprises a H16A mutation. In some embodiments, the mutein comprises a H16D mutation. In some embodiments, the mutein comprises a H16G mutation. In some embodiments, the mutein comprises a H16K mutation. In some embodiments, the mutein comprises a H16M mutation. In some embodiments, the mutein comprises a H16N mutation. In some embodiments, the mutein comprises a H16R mutation. In some embodiments, the mutein comprises a H16S mutation. In some embodiments, the mutein comprises a H16T mutation. In some embodiments, the mutein comprises a H16V mutation. In some embodiments, the mutein comprises a H16Y mutation. In some embodiments, the mutein comprises a L19A mutation. In some embodiments, the mutein comprises a L19D mutation. In some embodiments, the mutein comprises a L19E mutation. In some embodiments, the mutein comprises a L19G mutation. In some embodiments, the mutein comprises a L19N mutation. In some embodiments, the mutein comprises a L19R mutation. In some embodiments, the mutein comprises a L19S mutation. In some embodiments, the mutein comprises a L19T mutation. In some embodiments, the mutein comprises a L19V mutation. In some embodiments, the mutein comprises a D20A mutation. In some embodiments, the mutein comprises a D20E mutation. In some embodiments, the mutein comprises a D20H mutation. In some embodiments, the mutein comprises a D201 mutation. In some embodiments, the mutein comprises a D20Y mutation. In some embodiments, the mutein comprises a D20F mutation. In some embodiments, the mutein comprises a D20G mutation. In some embodiments, the mutein comprises a D20T mutation. In some embodiments, the mutein comprises a D20W mutation. In some embodiments, the mutein comprises a M23R mutation. In some embodiments, the mutein comprises a R81A mutation. In some embodiments, the mutein comprises a R81G mutation. In some embodiments, the mutein comprises a R81S mutation. In some embodiments, the mutein comprises a R81T mutation. In some embodiments, the mutein comprises a D84A mutation. In some embodiments, the mutein comprises a D84E mutation. In some embodiments, the mutein comprises a D84G mutation. In some embodiments, the mutein comprises a D84I mutation. In some embodiments, the mutein comprises a D84M mutation. In some embodiments, the mutein comprises a D84Q mutation. In some embodiments, the mutein comprises a D84R mutation. In some embodiments, the mutein comprises a D84S mutation. In some embodiments, the mutein comprises a D84T mutation. In some embodiments, the mutein comprises a S87R mutation. In some embodiments, the mutein comprises a N88A mutation. In some embodiments, the mutein comprises a N88D mutation. In some embodiments, the mutein comprises a N88E mutation. In some embodiments, the mutein comprises a N88I mutation. In some embodiments, the mutein comprises a N88F mutation. In some embodiments, the mutein comprises a N88G mutation. In some embodiments, the mutein comprises a N88M mutation. In some embodiments, the mutein comprises a N88R mutation. In some embodiments, the mutein comprises a N88S mutation. In some embodiments, the mutein comprises a N88V mutation. In some embodiments, the mutein comprises a N88W mutation. In some embodiments, the mutein comprises a V91D mutation. In some embodiments, the mutein comprises a V91E mutation. In some embodiments, the mutein comprises a V91G mutation. In some embodiments, the mutein comprises a V91S mutation. In some embodiments, the mutein comprises a I92K mutation. In some embodiments, the mutein comprises a I92R mutation. In some embodiments, the mutein comprises a E95G mutation. In some embodiments, the mutein comprises a Q126 mutation.
Although the mutations are illustrated in list form, this is simply for convenience and the muteins may have one or more of the substitutions provided herein.
In some embodiments, the IL-2 mutein molecule comprises a substitution selected from the group consisting of: N88R, N88I, N88G, D20H, D109C, Q126L, Q126F, D84G, or D84I relative to mature human IL-2 sequence provided above. In some embodiments, the IL-2 mutein molecule comprises a substitution of D109C and one or both of a N88R substitution and a C125S substitution. In some embodiments, the cysteine that is in the IL-2 mutein molecule at position 109 is linked to a polyethylene glycol moiety, wherein the polyethylene glycol moiety has a molecular weight of between 5 and 40 kDa.
In some embodiments, any of the substitutions described herein are combined with a substitution at position 125. The substitution can be a C125S, C125A, or C125V substitution.
In addition to the substitutions or mutations described herein, in some embodiments, the IL-2 mutein has a substitution/mutation at one or more of positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 15 or positions at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a mutation at positions 73 and 76; 73 and 100; 73 and 138; 76 and 100; 76 and 138; 100 and 138; 73, 76, and 100; 73, 76, and 138; 73, 100, and 138; 76, 100 and 138; or at each of 73, 76, 100, and 138 that correspond to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a mutation at positions 53 and 56; 53 and 80; 53 and 118; 56 and 80; 56 and 118; 80 and 118; 53, 56, and 80; 53, 56, and 118; 53, 80, and 118; 56, 80 and 118; or at each of 53, 56, 80, and 118 that correspond to SEQ ID NO: 6. As the IL-2 can be fused or tethered to other proteins, as used herein, the term corresponds to as reference to a SEQ ID NOs: 6 or 15 refer to how the sequences would align with default settings for alignment software, such as can be used with the NCBI website. In some embodiments, the mutation is leucine to isoleucine. Thus, the IL-2 mutein can comprise one more isoleucines at positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 15 or positions at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 6. In some embodiments, the mutein comprises a mutation at L53 that correspond to SEQ ID NO: 6. In some embodiments, the mutein comprises a mutation at L56 that correspond to SEQ ID NO: 6. In some embodiments, the mutein comprises a mutation at L80 that correspond to SEQ ID NO: 6. In some embodiments, the mutein comprises a mutation at L118 that correspond to SEQ ID NO: 6. In some embodiments, the mutation is leucine to isoleucine. In some embodiments, the mutein also comprises a mutation as position 69, 74, 88, 125, or any combination thereof in these muteins that correspond to SEQ ID NO: 6. In some embodiments, the mutation is a V69A mutation. In some embodiments, the mutation is a Q74P mutation. In some embodiments, the mutation is a N88D or N88R mutation. In some embodiments, the mutation is a C125A or C125S mutation.
In some embodiments, the IL-2 mutein comprises a mutation at one or more of positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145 that correspond to SEQ ID NO: 15 or one or more positions 29, 31, 35, 37, 48, 69, 71, 74, 88, and 125 that correspond to SEQ ID NO: 6. The substitutions can be used alone or in combination with one another. In some embodiments, the IL-2 mutein comprises substitutions at 2, 3, 4, 5, 6, 7, 8, 9, or each of positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145. Non-limiting examples such combinations include, but are not limited to, a mutation at positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145; 49, 51, 55, 57, 68, 89, 91, 94, and 108; 49, 51, 55, 57, 68, 89, 91, and 94; 49, 51, 55, 57, 68, 89, and 91; 49, 51, 55, 57, 68, and 89; 49, 51, 55, 57, and 68; 49, 51, 55, and 57; 49, 51, and 55; 49 and 51; 51, 55, 57, 68, 89, 91, 94, 108, and 145; 51, 55, 57, 68, 89, 91, 94, and 108; 51, 55, 57, 68, 89, 91, and 94; 51, 55, 57, 68, 89, and 91; 51, 55, 57, 68, and 89; 55, 57, and 68; 55 and 57; 55, 57, 68, 89, 91, 94, 108, and 145; 55, 57, 68, 89, 91, 94, and 108; 55, 57, 68, 89, 91, and 94; 55, 57, 68, 89, 91, and 94; 55, 57, 68, 89, and 91; 55, 57, 68, and 89; 55, 57, and 68; 55 and 57; 57, 68, 89, 91, 94, 108, and 145; 57, 68, 89, 91, 94, and 108; 57, 68, 89, 91, and 94; 57, 68, 89, and 91; 57, 68, and 89; 57 and 68; 68, 89, 91, 94, 108, and 145; 68, 89, 91, 94, and 108; 68, 89, 91, and 94; 68, 89, and 91; 68 and 89; 89, 91, 94, 108, and 145; 89, 91, 94, and 108; 89, 91, and 94; 89 and 91; 91, 94, 108, and 145; 91, 94, and 108; 91, and 94; or 94 and 108. Each mutation can be combined with one another. The same substitutions can be made in SEQ ID NO: 6, but the numbering would adjusted appropriately as is clear from the present disclosure (20 less than the numbering for SEQ ID NO: 15 corresponds to the positions in SEQ ID NO: 6).
In some embodiments, the IL-2 mutein comprises a mutation at one or more positions of 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95, or 126). These mutations can be combined with the other leucine to isoleucine mutations described herein or the mutation at positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 15 or at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 6. In some embodiments, the mutation is a E35Q, H36N, Q42E, D104N, E115Q, or Q146E, or any combination thereof. In some embodiments, one or more of these substitutions is wild type. In some embodiments, the mutein comprises a wild-type residue at one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95, and 126).
The mutations at these positions can be combined with any of the other mutations described herein, including, but not limited to substitutions at positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 15 or positions at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 6 described herein and above. In some embodiments, the IL-2 mutein comprises a N49S mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a Y51S or a Y51H mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a K55R mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a T57A mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a K68E mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a V89A mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a N91R mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a Q94P mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a N108D or a N108R mutation that corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a C145A or C145S mutation that corresponds to SEQ ID NO: 15. These substitutions can be used alone or in combination with one another. In some embodiments, the mutein comprises each of these substitutions. In some embodiments, the mutein comprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein comprises a wild-type residue at one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95, and 126).
In some embodiments, the IL-2 mutein comprises a N29S mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a Y31S or a Y31H mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a K35R mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a T37A mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a K48E mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a V69A mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a N71R mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a Q74P mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a N88D or a N88R mutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a C125A or C125S mutation that corresponds to SEQ ID NO: 6. These substitutions can be used alone or in combination with one another. In some embodiments, the mutein comprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein comprises each of these substitutions. In some embodiments, the mutein comprises a wild-type residue at one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95, and 126).
For any of the IL-2 muteins described herein, in some embodiments, one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95, or 126) are wild-type (e.g. are as shown in SEQ ID NOs: 6 or 15). In some embodiments, 2, 3, 4, 5, 6, or each of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95, and 126) are wild-type.
In some embodiments, the IL-2 mutein comprises a sequence of:







(SEQ ID NO: 16)


MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISN


 


HKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHL


 


RPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIS


 


TLT






In some embodiments, the IL-2 mutein comprises a sequence of:







(SEQ ID NO: 17)


MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISN


 


HKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHL


 


RPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIS


 


TLT






In some embodiments, the IL-2 mutein comprises a sequence of:







(SEQ ID NO: 18)


MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISN


 


HKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHI


 


RPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIS


 


TLT






In some embodiments, the IL-2 mutein comprises a sequence of:







(SEQ ID NO: 19)


MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISN


 


HKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHL


 


RPRDLISDINVIVLELKGSETTFMCEYADETATIVEFINRWITFSQSIIS


 


TLT






In some embodiments, the IL-2 mutein sequences described herein do not comprise the IL-2 leader sequence. The IL-2 leader sequence can be represented by the sequence of MYRMQLLSCIALSLALVTNS (SEQ ID NO: 20). Therefore, in some embodiments, the sequences illustrated above can also encompass peptides without the leader sequence. Although SEQ ID NOs; 16-20 are illustrated with only mutation at one of positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 15 or positions at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 6, the peptides can comprises one, two, three or 4 of the mutations at these positions. In some embodiments, the substitution at each position is isoleucine or other type of conservative amino acid substitution. In some embodiments, the leucine at the recited positions are substituted with, independently, isoleucine, valine, methionine, or phenylalanine.

In some embodiments, the IL-2 mutein molecule is fused to a Fc Region or other linker region as described herein. Examples of such fusion proteins can be found in U.S. Pat. Nos. 9,580,486, 7,105,653, 9,616,105, 9,428,567, US2017/0051029, WO2016/164937, US2014/0286898A1, WO2014153111A2, WO2010/085495, WO2016014428A2, WO2016025385A1, US2017/0037102, and US2006/0269515, each of which are incorporated by reference in its entirety.
In some embodiments, the Fc Region comprises what is known as the LALA mutation. Using the Kabat numbering of the Fc region, this would correspond to L247A, L248A, and G250A. In some embodiments, using the EU numbering of the Fc region, the Fc region comprises a L234A mutation, a L235A mutation, and/or a G237A mutation. Regardless of the numbering system used, in some embodiments, the Fc portion can comprise mutations that correspond to these residues. In some embodiments, the Fc Region comprises N297G or N297A (kabat numbering) mutations. The Kabat numbering is based upon a full-length sequence, but would be used in a fragment based upon a traditional alignment used by one of skill in the art for the Fc region.
In some embodiments, the Fc Region comprises a sequence of:







(SEQ ID NO: 21)


DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED


 


PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK


 


CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG


 


NVFSCSVMHEALHNHYTQKSLSLSPG.


or


 


(SEQ ID NO: 28)


DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED


 


PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK


 


CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK


 


GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG


 


NVFSCSVMHEALHNHYTQKSLSLSPG.






In some embodiments, the IL-2 mutein is linked to the Fc Region. Non-limiting examples of linkers are glycine/serine linkers. For example, a glycine/serine linkers can be a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) or GGGGSGGGGSGGGGS (SEQ ID NO: 30). This is simply a non-limiting example and the linker can have varying number of GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29). In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29) repeats. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 30 amino acids in length. In some embodiments, the linker is 35 amino acids in length. In some embodiments, the linker is from 5-50 amino acids in length.
Thus, the IL-2/Fc Fusion can be represented by the formula of ZIL-2M-Lgs-ZFc, wherein ZIL-2M is a IL-2 mutein as described herein, Lgs is a linker sequence as described herein (e.g. glycine/serine linker) and ZFc is a Fc region described herein or known to one of skill in the art. In some embodiments, the formula can be in the reverse orientation ZFc-Lgs-ZIL-2M.
In some embodiments, the IL-2/Fc fusion comprises a sequence of







(SEQ ID NO: 24)


MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISN


 


HKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHL


 


RPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIS


 


TLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN


 


STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ


 


VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV


 


LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG


 


(SEQ ID NO: 25)


MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISN


 


HKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHL


 


RPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIS


 


TLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN


 


STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ


 


VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV


 


LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG


 


(SEQ ID NO: 26)


MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISN


 


HKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHI


 


RPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIIS


 


TLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN


 


STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ


 


VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV


 


LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG


 


(SEQ ID NO: 27)


MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISN


 


HKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHL


 


RPRDLISDINVIVLELKGSETTFMCEYADETATIVEFINRWITFSQSIIS


 


TLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKP


 


KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN


 


STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ


 


VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV


 


LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.






In some embodiments, the IL-2/Fc Fusion comprises a sequence selected from the following table, Table 3:







TABLE 3







IL-2/Fc Fusion Protein Amino Acid Sequences








Sequence



Identification
Sequence





SEQ ID NO: 7
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE



ELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWI



TFSQSIISTLTGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC



VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCK



VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES



NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS



PGK


 


SEQ ID NO: 8
APTSSSTKKTQLQLEHLLLHLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE



ELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWI



TFSQSIISTLTVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ



FNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT



ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP



MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK


 


SEQ ID NO: 9
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE



ELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWI



TFSQSIISTLTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED



PEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP



IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK



TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG


 


SEQ ID NO: 10
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE



ELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWI



TFSQSIISTLTGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD



VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNK



ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP



ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG


 


SEQ ID NO: 11
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE



ELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWI



TFSQSIISTLTGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT



CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKC



KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE



SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL



SPG


 


SEQ ID NO: 12
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE



ELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWI



TFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNG



KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI



AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNYHTQ



KSLSLSPG


 


SEQ ID NO: 13
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE



ELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWI



TFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT



LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQ



DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF



YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH



NHYTQKSLSLSPG


 


SEQ ID NO: 14
APTSSSTKKTQLQLEHLLLHLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE



ELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWI



TFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR



TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYPVVSVLTVLHQDWLNG



KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI



AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ



KSLSLSPG









In some embodiments, the IL-2 muteins comprises one or more of the sequences provided in the following table, which, in some embodiments, shows the IL-2 mutein fused with other proteins or linkers. The table also provides sequences for a variety of Fc domains or variants that the IL-2 can be fused with:












SEQ
Brief



ID NO:
Description
Amino Acid Sequence







31
Human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with C1255
TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE



mutation
TTFMCEYADETATIVEFLNRWITFSQSIISTLT


 


32
Human IL-2
APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with C125S
TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE



and T3A
TTFMCEYADETATIVEFLNRWITFSQSIISTLT



mutations



 


33
Human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with N88R and
TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSE



C125S
TTFMCEYADETATIVEFLNRWITFSQSIISTLT


 


34
Human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with V69A,
TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISNINVIVLELKGSE



Q74Pand
TTFMCEYADETATIVEFLNRWITFSQSIISTLT



C125S




mutations



 


35
Human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with V69A,
TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE



Q74P, N88D
TTFMCEYADETATIVEFLNRWITFSQSIISTLT



and C125S




mutations



 


36
Human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with V69A,
TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISRINVIVLELKGSE



Q74P, N88R
TTFMCEYADETATIVEFLNRWITFSQSIISTLT



and C125S




mutations



 


37
Human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with N88D and
TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSE



C125S
TTFMCEYADETATIVEFLNRWITFSQSIISTLT


 


38
Human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with L53I,
TEIKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE



V69A, Q74P,
TTFMCEYADETATIVEFLNRWITFSQSIISTLT



N88D and




C125S




mutations



 


39
Human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with L56I,
TELKHIQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE



V69A, Q74P,
TTFMCEYADETATIVEFLNRWITFSQSIISTLT



N88D and




C125S




mutations



 


40
Human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with V69A,
TELKHLQCLEEELKPLEEALNLAPSKNFHIRPRDLISDINVIVLELKGSE



Q74P, L80I,
TTFMCEYADETATIVEFLNRWITFSQSIISTLT



N88D and




C125S




mutations



 


41
Human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



with V69A,
TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE



Q74P, N88D,
TTFMCEYADETATIVEFINRWITFSQSIISTLT



L1181, and




C125S




mutations



 


21
Human IgG1 Fc
DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED



(N-terminal
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK



fusions) with
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK



L234A, L235A,
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG



and G237A
NVFSCSVMHEALHNHYTQKSLSLSPG



mutations



 


30
GGGGSGGGGSGGG
GGGGSGGGGSGGGGS



GS linker (15




amino acids)



 


22
GGGGSGGGGSGGG
GGGGSGGGGSGGGGSGGGGS



GSGGGGS




linker (20




amino acids)



 


23
GGGGS linker
GGGGS



(5 amino




acids)



 


43
Human IgG1 Fc
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED



(truncated)
PEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYK



with N297G
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK



mutation
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG




NVFSCSVMHEALHNHYTQKSLSLSPG


 


44
Antibody
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV



Heavy Chain
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP



CH1-CH2-CH3
KSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS



domains
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK



(human IgG1
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC



with L234A,
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW



L235A, and
QQGNVFSCSVMHEALHNHYTQKSLSLSPG



G237A)



 


45
Antibody
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG



Kappa
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK



Constant
SFNRGEC



Domain




(human)



 


46
IL-2-G4Sx3-Fc
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA




TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE




TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDK




THTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE




VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK




VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF




YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV




FSCSVMHEALHNHYTQKSLSLSPG


 


47
IL-2 T3A-
APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



G4Sx3-Fc
TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE




TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDK




THTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE




VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK




VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF




YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV




FSCSVMHEALHNHYTQKSLSLSPG


 


48
IL-2 N88R-
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



G4Sx3-Fc
TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSE




TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDK




THTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE




VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK




VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF




YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV




FSCSVMHEALHNHYTQKSLSLSPG


 


49
IL-2 V69A,
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



Q74P,
TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISNINVIVLELKGSE



-G4Sx3-Fc
TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDK




THTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE




VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK




VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF




YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV




FSCSVMHEALHNHYTQKSLSLSPG


 


50
IL-2 N88D
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



V69A, Q74P-
TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE



G4Sx3-Fc
TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDK




THTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE




VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK




VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF




YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV




FSCSVMHEALHNHYTQKSLSLSPG


 


51
IL-2 N88R
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



V69A, Q74P-
TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISRINVIVLELKGSE



G4Sx3-Fc
TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDK




THTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE




VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK




VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF




YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV




FSCSVMHEALHNHYTQKSLSLSPG


 


52
IL-2 N88D-
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



G4Sx3-Fc
TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSE




TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDK




THTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE




VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK




VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF




YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV




FSCSVMHEALHNHYTQKSLSLSPG


 


53
IL-2 L53I
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



N88D V69A,
TEIKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE



Q74P, C1255-
TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGG



G4Sx4-Fc
GGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS




HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK




EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC




LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW




QQGNVFSCSVMHEALHNHYTQKSLSLSPG


 


54
IL-2 L56I
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



N88D V69A,
TELKHIQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE



Q74P, C1255-
TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGG



G4Sx4-Fc
GGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS




HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK




EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC




LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW




QQGNVFSCSVMHEALHNHYTQKSLSLSPG


 


55
IL-2 L801
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



N88D V69A,
TELKHLQCLEEELKPLEEALNLAPSKNFHIRPRDLISDINVIVLELKGSE



C125S Q74P-
TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGG



G4Sx4-Fc
GGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS




HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK




EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC




LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW




QQGNVFSCSVMHEALHNHYTQKSLSLSPG


 


56
IL-2 L118I
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



N88D V69A,
TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE



Q74P, C1255-
TTFMCEYADETATIVEFINRWITFSQSIISTLTGGGGSGGGGSGGGGSGG



G4Sx4-Fc
GGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS




HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK




EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC




LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW




QQGNVFSCSVMHEALHNHYTQKSLSLSPG


 


57
IL-2 N88D
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



V69A, Q74P-
TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE



G4Sx4-Fc
TTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGG




GGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS




HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK




EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC




LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW




QQGNVFSCSVMHEALHNHYTQKSLSLSPG


 


58
Fc-G4S-IL-2
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED



N88D V69A,
PEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYK



Q74P
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK




GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG




NVFSCSVMHEALHNHYTQKSLSLSPGGGGGSAPTSSSTKKTQLQLEHLLL




DLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEA




LNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLN




RWITFAQSIISTLT


 


59
IL-2 N88D
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



V69A, Q74P,
TEX1KHX2QCLEEELKPLEEALNLAPSKNFHX3RPRDLISDINVIVLELKG



C1255-G4Sx4-
SETTFMCEYADETATIVEFX4NRWITFSQSIISTLTGGGGSGGGGSGGGGS



Fc, wherein
GGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVD



at least one
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN



of X1, X2, X3,
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL



and X4 is I
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS



and the
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG



remainder are




Lor I.



 


60
IL-2 N88D
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



V69A, Q74P,
TEX1KHX2QCLEEELKPLEEALNLAPSKNFHX3RPRDLISDINVIVLELKG



C1255,
SETTFMCEYADETATIVEFX4NRWITFSQSIISTLT



wherein at




least one of




X1, X2, X3,




and X4 is I




and the




remainder are




Lor I.









In some embodiments, the sequences shown in the table or throughout comprise or don't comprise one or more mutations that correspond to positions L53, L56, L80, and L118. In some embodiments, the sequences shown in the table or throughout the present application comprise or don't comprise one or more mutations that correspond to positions L59I, L63I, I24L, L94I, L96I or L1321 or other substitutions at the same positions. In some embodiments, the mutation is leucine to isoleucine. In some embodiments, the mutein does not comprise another mutation other than as shown or described herein. In some embodiments, the peptide comprises a sequence of SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.
In some embodiments, the protein comprises a IL-2 mutein as provided for herein. In some embodiments, a polypeptide is provided comprising SEQ ID NO: 59 or SEQ ID NO: 60, wherein at least one of X1, X2, X3, and X4 is I and the remainder are L or I. In some embodiments, X1, X2, and X3 are L and X4 is I. In some embodiments, X1, X2, and X4 are L and X3 is I. In some embodiments, X2, X3, and X4 are L and X1 is I. In some embodiments, X1, X3, and X4 are L and X2 is I. In some embodiments, X1 and X2 are L and X3 and X4 are I. In some embodiments, X1 and X3 are L and X2 and X4 are I. In some embodiments, X1 and X4 are L and X2 and X3 are I. In some embodiments, X2 and X3 are L and X1 and X4 are I. In some embodiments, X2 and X4 are L and X1 and X3 are I. In some embodiments, X3 and X4 are L and X1 and X2 are I. In some embodiments, X1, X2, and X3 are L and X4 is I. In some embodiments, X2, X3, and X4 are L and X1 is I. In some embodiments, X1, X3, and X4 are L and X2 is I. In some embodiments, X1, X2, and X4 are L and X3 is I.
In some embodiments, the Fc portion of the fusion is not included. In some embodiments, the peptide consists essentially of a IL-2 mutein provided for herein. In some embodiments, the protein is free of a Fc portion.
For illustrative purposes only, embodiments of IL-2 mutein fused with a Fc and with a targeting moiety are illustrated in FIG. 19.
The sequences are for illustrative purposes only and are not intended to be limiting. In some embodiments, the compound comprises an amino acid sequence of SEQ ID NO: 53, 54, 55, or 56. In some embodiments, the compound comprises an amino acid sequence of SEQ ID NO: 53, 54, 55, or 56 with or without a C125A or C125S mutation. In some embodiments, the residue at position 125 is C, S, or A. In some embodiments, the compound comprises an amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 60, wherein at least one of X1, X2, X3, and X4 is I and the remainder are L or I. In some embodiments, the protein comprises a IL-2 mutein as provided for herein. In some embodiments, a polypeptide is provided comprising SEQ ID NO: 59 or SEQ ID NO: 60, wherein at least one of X1, X2, X3, and X4 is I and the remainder are L or I. In some embodiments, X1, X2, and X3 are L and X4 is I. In some embodiments, X1, X2, and X4 are L and X3 is I. In some embodiments, X2, X3, and X4 are L and X1 is I. In some embodiments, X1, X3, and X4 are L and X2 is I. In some embodiments, X1 and X2 are L and X3 and X4 are I. In some embodiments, X1 and X3 are L and X2 and X4 are I. In some embodiments, X1 and X4 are L and X2 and X3 are I. In some embodiments, X2 and X3 are L and X1 and X4 are I. In some embodiments, X2 and X4 are L and X1 and X3 are I. In some embodiments, X3 and X4 are L and X1 and X2 are I. In some embodiments, X1, X2, and X3 are L and X4 is I. In some embodiments, X2, X3, and X4 are L and X1 is I. In some embodiments, X1, X3, and X4 are L and X2 is I. In some embodiments, X1, X2, and X4 are L and X3 is I.
Each of the proteins may also be considered to have the C125S and the LALA and/or G237A mutations as provided for herein. The C125 substitution can also be C125A as described throughout the present application.
In an embodiment, an IL-2 mutein molecule comprises at least 60, 70, 80, 85, 90, 95, or 97% sequence identity or homology with a naturally occurring human IL-2 molecule, e.g., a naturally occurring IL-2 sequence disclosed herein or those that incorporated by reference.
As described herein the IL-2 muteins can be part of a bi-specific molecule with a tethering moiety, such as a MAdCAM antibody that will target the IL-2 mutein to a MAdCAM expressing cell. As described herein, the bispecific molecule can be produced from two polypeptide chains. In some embodiments, the following can be used:










Table of MAdCAM-IL-2 Mutein Bispecific Compounds 










Chain 1 N-terminal to C-terminal Molecule 
Chain 2 N-terminal 



Component Sequence IDs 
to C-terminal 














Antibody Heavy


Molecule Component 




Chain 


Sequence IDs 














Antibody VH 
CH1—CH2—CH3 

C-terminal 
Light Chain 
Light Chain 


Detail 
Domain 
Domains 
Linker 1 
Moiety 
VK Domain 
CK Domain





1. Anti- 
Rat Anti- 
SEQ ID 
SEQ ID 
SEQ ID 
Rat Anti- 
SEQ ID 


MAdCam-Fc- 
MAdCam-VH1 
NO: 44 
NO: 23 
NO: 35 
MAdCam- 
NO: 45 


IL-2 N88D 




VK1 



V69A, Q74P 








2. Anti- 
Rat Anti- 
SEQ ID 
SEQ ID 
SEQ ID 
Rat Anti- 
SEQ ID 


MAdCam- 
MAdCam-VH2 
NO: 44 
NO: 23 
NO: 35 
MAdCam-VK2 
NO: 45 


Fc-IL-2 








N88D V69A, 








Q74P 








3. Anti- 
Rat Anti- 
SEQ ID 
SEQ ID 
SEQ ID 
Rat Anti- 
SEQ ID 


MAdCam- 
MAdCam-VH1 
NO: 44 
NO: 23 
NO: 41 
MAdCam-VK3 
NO: 45 


Fc-IL-2 








L118I N88D 








V69A, Q74P 








4. TTJ2- 
Human 
SEQ ID 
SEQ ID 
SEQ ID 
Human TTJ2- 
SEQ ID 


Fc-IL-2 
TTJ2-VH 
NO: 44 
NO: 23 
NO: 41 
VK 
NO: 45 


L118I N88D 








V69A, Q74P 








5. anti 
Anti-MAdCam 
SEQ ID 
SEQ ID 
SEQ ID 
Anti-MAdCam 
SEQ ID 


hu.MAdCAM- 
Human VH3 
NO: 44 
NO: 23 
NO: 41 
Human VK3 
NO: 45 


Fc-IL-2 








L118I N88D 








V69A, Q74P 








6. anti 
Anti-MAdCam 
SEQ ID 
SEQ ID 
SEQ ID 
Anti-MAdCam 
SEQ ID 


hu.MAdCAM- 
Human VH4 
NO: 44 
NO: 23 
NO: 41 
Human VK4 
NO: 45 


Fc-IL-2 








L118I N88D 








V69A, Q74P 








7. anti 
Anti-MAdCam 
SEQ ID 
SEQ ID 
SEQ ID 
Anti-MAdCam 
SEQ ID 


hu.MAdCAM- 
Human VH5 
NO: 44 
NO: 23 
NO: 41 
Human VK5 
NO: 45 


Fc-IL-2 








L118I N88D 








V69A, Q74P









The proteins can be produced with or without a C125A or C125S mutation in the IL-2 mutein. Examples of IL-2 muteins that can be included are illustrated herein, such as, but not limited to, a sequence of SEQ ID NO: 59 or SEQ ID NO: 60.
In some embodiments, the constant kappa domain in any of the light chains can be replaced with a constant lambda domain.
GITR-Binders
GITR(CD357) is a cell surface marker present on Tregs. Blockade of the GITR-GITRL interaction maintains Treg function. In some embodiments, a therapeutic compound comprises an IIC binding entity that binds GITR-expressing Treg cells and a targeting moiety that targets the therapeutic compound to the target tissue of interest.
In some embodiments, a therapeutic compound comprises an anti-GITR antibody molecule, e.g., anti-GITR antibody molecule that inhibit binding of GITR to GITRL.
In some embodiments, a therapeutic compound comprises an anti-GITR antibody molecule, anti-GITR antibody molecule that inhibit binding of GITR to GITRL, and PD-1 agonist, IL-2 mutein molecule, or other effector described herein.
While not wishing to be bound by theory, it is believed that the therapeutic compound that comprises a GITR binder effects accumulation of GITR-expressing Tregs at the site targeted by the targeting moiety of the therapeutic compound, e.g., a transplant or site of organ injury.
Butyrophilins/Butyrophilin-Like Molecules
Effector binding/modulating moiety can comprise an agonistic BTNL2 molecule. While not wishing to be bound by theory it is believed that agonistic BTNL2 molecules induce Treg cells.
An agonistic BTNL2 molecule as that term as used herein, refers to a polypeptide having sufficient BTNL2 sequence that, as part of a therapeutic compound, it induces Treg cells. In some embodiments, a BTNL2 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantial sequence identity, with a naturally occurring butyrophilin.
In some embodiments, an effector binding/modulating moiety an antagonistic BTNL8 molecule.
Therapeutic Compounds Comprising an SM Binding/Modulating Moiety: Manipulation of Local Microenvironment
A therapeutic compound can comprise an effector binding/modulating moiety that promotes an immuno-suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target, referred to herein a SM binding/modulating moiety.
In some embodiments, the SM binding/modulating moiety comprises a molecule that inhibits or minimizes attack by the immune system of the target (referred to herein as an SM binding/modulating moiety). In some embodiments, a therapeutic compound comprises an SM binding/modulating moiety that binds and accumulates a soluble substance, e.g., an endogenous or exogenous substance having immunosuppressive function. In some embodiments, a therapeutic compound comprises an SM binding/modulating moiety, e.g., a CD39 molecule or a CD73 molecule or alkaline phosphatase molecule, that binds, inhibits, sequesters, degrades or otherwise neutralizes a soluble substance, typically and endogenous soluble substance, e.g., ATP in the case of a CD39 molecule or alkaline phosphatase molecule, or AMP in the case of a CD73 molecule, that promotes immune attack. In some embodiments, a therapeutic compound comprises an SM binding/modulating moiety that comprises an immune-suppressive substance, e.g. a fragment of protein that is immunosuppressive.
Therapeutic Compounds Comprising an ICSM Binding/Modulating Moiety: Inhibition of Stimulation, e.g., Inhibition of Co-Stimulation of Immune Cells
A therapeutic compound can comprise an ICSM binding/modulating moiety that inhibits or antagonizes a stimulatory, e.g., co-stimulatory binding pair, e.g., OX40 and OX40L. The ICSM binding/modulating moiety can bind and antagonize either member of the pair.
In an embodiment, the ICSM binding/modulating moiety comprises an antibody molecule that binds and antagonizes either member of a stimulatory, e.g., co-stimulatory binding pair. In an embodiment the ICSM binding/modulating moiety comprises antagonistic analog of one of the members of the binding pair. In such embodiments the ICSM binding/modulating moiety can comprise a soluble fragment of one of the members that binds the other. Typically the analog will have at least 50, 60, 70, 80, 90, 95, or 98% homology or sequence identity with a naturally occurring member that binds the target member of the pair. In the case of an ICSM binding/modulating moiety that binds the member present on the surface of an immune cell, the ICSM binding/modulating moiety typically binds but does not activate, or allow endogenous counter member to bind and activate.
Thus, in the case of the binding pair that includes, for example, the OX40 immune cell member and the OX40L counter member, an ICSM binding/modulating member can comprise any of the following:
a) an antibody molecule that binds the OX40 immune cell member and antagonizes stimulation, e.g., by blocking binding of endogenous OX40L counter member;
b) an antibody molecule that binds OX40L counter member and antagonizes stimulation, e.g., by blocking effective binding of the endogenous OX40L counter member to the OX40 immune cell member;
c) a soluble fragment or analog of OX40L counter member which binds OX40 immune cell member and antagonizes stimulation; and
c) a soluble fragment or analog of OX40 immune cell member which binds OX40L counter member and antagonizes stimulation.
For example, the ICSM binding/modulating moiety, e.g., an antibody molecule or an antagonistic analog or of the counter member, can bind to CD2, ICOS, CD40L, CD28, LFA1, SLAM, TIM1, CD30, OX40 (CD134), 41BB (CD137), CD27, HVEM, DR3, GITR, BAFFR, TACI, BCMA, or CD30, CD40. In another embodiment, the ICSM binding/modulating moiety, e.g., an antibody molecule or an antagonistic analog or of the counter member, can bind to B7.1, B7.2, ICOSL (B7-H2, B7RP1), LFA3, CD48, CD58, ICAM1, SLAM, TIM4, CD40, CD30L, OX40L (CD252), 41BBL (CD137L), CD70, LIGHT, TL1A, GITRL, BAFF, APRIL, or CD30, CD40L.
In some embodiments, the ICSM binding/modulating molecule binds, and antagonizes, an activating or costimulatory molecule, e.g., a costimulatory molecule, present on an immune cell, or binds the counter member preventing the counter member from activating the costimulatory molecule present on the immune cell. In some embodiments, the ICSM comprises an antagonistic antibody molecule e.g., an antibody molecule that binds the costimulatory molecule on an immune cell or binds the counter member of the ICSM, preventing the counter member from activating the costimulatory molecule on the immune cell, and results in inhibiting the activity of the costimulatory molecule. In some embodiments, the ICSM comprises an antagonistic counterpart molecule, e.g., a fragment of a molecule that binds the costimulatory molecule, and results in the inhibition of the costimulatory molecule activity.
In some embodiments, one member of the binding pair will be on the surface of an immune cell, e.g., a T, B, or NK cell or dendritic cell, while the counter member will be on another immune cell, or an APC such as a dendritic cell or on non-immune cells such as smooth cells, or endothelial cells.
The following table provides non-limiting examples of costimulatory molecule and counterstructure pairs







TABLE 4







Costimulatory molecule 


and counterstructure pairs










Costimulatory




Molecule (e.g. 




on T cells)
Counterstructure







CD28
B7.1 or B7.2



ICOS
ICOSL (B7H-2,




B7RP1)



CD2
LFA3, CD48, CD58



LFA1
ICAM1



SLAM
SLAM



TIM1
TIM4



CD40L
CD40



CD30
CD3OL



OX40/CD134
OX40L (CD252)  



41BB/CD137
41BBL (CD137L)



CD27
CD70



HVEM
LIGHT



DR3
TL1A



GITR
GITRL



Costimulatory
Counterstructure



Molecule (e.g. 




on B cells)




BAFFR
BAFF



TACI
BAFF and APRIL



BCMA
BAFF and APRIL



CD40
CD40L



CD30L
CD30










Donor Tissue

Therapeutic compounds and methods described herein can be used in conjunction with a transplantation of donor tissue into a subject and can minimizes rejection of, minimizes immune effector cell mediated damage to, prolongs acceptance of, or prolongs the functional life of, donor transplant tissue. The tissue can be xenograft or allograft tissue. Transplanted tissue can comprise all or part of an organ, e.g., a liver, kidney, heart, pancreas, thymus, skin or lung. In embodiments, therapeutic compounds described herein reduce, or eliminate the need for systemic immune suppression. Therapeutic compounds and methods described herein can also be used to treat GVHD. In some embodiments, host cells are coated with a therapeutic compound that comprises, as an effector binding/modulating moiety, a PD-L1 molecule.
Table 2 provides target molecules for transplant indications. A target molecule is the target to which a targeting moiety binds. As discussed elsewhere herein, In some embodiments, a targeting moiety is selected that binds a product of an allele present on donor tissue and which is not expressed by the subject (recipient) or at expressed at a different level (e.g. reduced or substantially reduced).







TABLE 5







Target Molecules for Transplant Indications









Indication
Organ/cell type
Target





Allograft transplant 
All
HLA-A, HLA-B, HLA-C,


tissue, e.g., allograft solid 

HLA-DP, HLA-DQ or HLA-


organ transplant, GvHD

DR


Transplant
Kidney
Antigens expressed in the




kidney where immune cells




infiltrate, for example




including but not limited to




the tubular interstitial




region e.g. Uromodulin,




SLC22A2, SLC22A6, FXYD4,




SLC5A10, SLC6A13, AQP6,




SLC13A3, TMEM72, BSND,




NPR3, and the proximal and




distal tubular epithelium,




such as OAT1, OCT2









Auto-Immune Disorders

Therapeutic compounds and methods described herein can be used to treat a subject having or at risk for having an unwanted autoimmune response, e.g., an auto immune response in Type 1 Diabetes, Multiple Sclerosis, Cardiomyositis, vitiligo, alopecia, inflammatory bowel disease (IBD, e.g. Crohn's disease or ulcerative colitis), Sjogren's syndrome, focal segmented glomerular sclerosis (FSGS), scleroderma/systemic sclerosis (SSc) or rheumatoid arthritis. In some embodiments, the treatment minimizes rejection of, minimizes immune effector cell mediated damage to, prolongs the survival of subject tissue undergoing, or a risk for, autoimmune attack. Table 3 provides target molecules for several autoimmune indications and organ/cell types. A target molecule is the target to which a targeting moiety binds.







TABLE 2







Target Molecules for autoimmune indications









Indication
Organ/cell type
Target Molecule





Type 1 Diabetes 
Pancreas/Pancreatic 
SEZ6L2, LRP11, DISP2,


and Transplant
islets, beta cells
SLC30A8, FXYD2 TSPAN7




TMEM27 (reference Hald et




al. 2012 Diabetelogia




55:154); FXYD2; GPR119;




HEPACAM2,




DPP6, or MAdCAM


Multiple Sclerosis
CNS/myelin sheath of
MOG, PLP, MBP



oligodendrocytes



Cardiomyositis, 
Cardiomyocytes, monocytes,
SIRPA (CD172a)


rheumatoid arthritis
macrophages, myeloid cells



Inflammatory bowel disease
Intestine
MAdCAM


(ulcerative colitis, Crohn's




disease) or GVHD; 




Celiac disease




Autoimmune hepatitis 
liver
MAdCAM


(AIH); Primary Sclerosing




Cholangitis (PSC);




Primary Biliary 




Sclerosis; (PBC);




transplant




Focal Segmented Glomerular
Kidney, podocytes, tubules,
COL1A1, Cadherin 2,


Sclerosis (FSGS) and other
epithelial cells
VCAM-1, Thy1, Podocin,


diseases that can affect

KIM1 (Hodgin et al, Am J


kidney for example lupus

Pathol 177:1675 2010);


nephritis, systemic

PLA2R; OAT1; OCT2; K-


scleroderma, membranous

cadherin 6


glomerular nephropathy




(MGN); Membranous




nephropathy (MN); Minimal




Change Disease (MCD); IgA




nephropathy; ANCA-




associated vasculitis (AAV)




Sjogren's syndrome
Salivary glands, epithelial
FCGR3B, HLAB, KIM1 (Hu



cells, kidney
et al Arth and Rheum




56:3588 2007


Scleroderma, systemic
skin, kidney, lung,
Collagen I, III, VI, VII,


sclerosis (SSc)
Fibroblasts, connective tissue
fibronectin (Wang et al Arth




and Rheum 54:2271 2006)


vitiligo
Skin, epidermis, Langerhans
COL17A1, CD1A, CD207,



cells, keratinocytes,
desmoglein 1-4, keratin 1



melanocytes



Alopecia areata
Skin, Hair follicle/hair bulb,
CD133 (Yang and Cotsarelis,



dermis
J Dermatol Sci 57:2 2010)









Other examples of autoimmune disorders and diseases that can be treated with the compounds described herein include, but are not limited to, Myocarditis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Subacute bacterial endocarditis, Anti-Glomerular Basement Membrane nephritis, Interstitial cystitis, Lupus nephritis, membranous glomerulonephropathy, Chronic Kidney Disease (“CKD”), Autoimmune hepatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Antisynthetase syndrome, alopecia areata, autoimmune angioedema, autoimmune progesterone dermatitis, autoimmune urticaria, bullous pemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, discoid lupus erythematosus, epidermolysis bullosa acquisita, erythema nodosum, gestational pemphigoid, hidradenitis suppurativa, lichen planus, lichen sclerosus, linear iga disease (lad), morphea, pemphigus vulgaris, pityriasis lichenoides et varioliformis acuta, mucha-habermann disease, psoriasis, systemic scleroderma, vitiligo, Addison's disease, Autoimmune polyendocrine syndrome (APS) type 1, Autoimmune polyendocrine syndrome (APS) type 2, Autoimmune polyendocrine syndrome (APS) type 3, Autoimmune pancreatitis (AIP), Diabetes mellitus type 1, Autoimmune thyroiditis, Ord's thyroiditis, Graves' disease, Autoimmune Oophoritis, Endometriosis, Autoimmune orchitis, Sjogren's syndrome, Autoimmune enteropathy, Coeliac disease, Crohn's disease, Microscopic colitis, Ulcerative colitis, thrombocytopenia, Adiposis, dolorosa, Adult-onset Still's, disease, Ankylosing, Spondylitis, CREST syndrome, Drug-induced lupus, Enthesitis-related arthritis, Eosinophilic fasciitis, Felty syndrome, IgG4-related disease, Juvenile, Arthritis, Lyme disease (Chronic), Mixed connective tissue disease (MCTD), Palindromic rheumatism, Parry Romberg syndrome, Parsonage-Turner syndrome, Psoriatic arthritis, Reactive arthritis, Relapsing polychondritis, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schnitzler syndrome, Systemic Lupus Erythematosus (SLE), Undifferentiated connective tissue disease (UCTD), Dermatomyositis, Fibromyalgia, Inclusion body myositis, Myositis, Myasthenia gravis, Neuromyotonia, Paraneoplastic cerebellar degeneration, Polymyositis, Acute disseminated encephalomyelitis (ADEM), Acute motor axonal neuropathy, Anti-N-Methyl-D-Aspartate (anti-NMDA) Receptor Encephalitis, Balo concentric sclerosis, Bickerstaff's encephalitis, Chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Hashimoto's encephalopathy, Idiopathic inflammatory demyelinating diseases, Lambert-Eaton myasthenic syndrome, Multiple sclerosis, Oshtoran syndrome, Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus (PANDAS), Progressive inflammatory neuropathy, Restless leg syndrome, Stiff person syndrome, Sydenham chorea, Transverse myelitis, Autoimmune retinopathy, Autoimmune uveitis, Cogan syndrome, Graves ophthalmopathy, Intermediate uveitis, Ligneous conjunctivitis, Mooren's ulcer, Neuromyelitis optica, Opsoclonus myoclonus syndrome, Optic neuritis, Scleritis, Susac's syndrome, Sympathetic ophthalmia, Tolosa-Hunt syndrome, Autoimmune inner ear disease (AIED), Meniere's disease, Behcet's disease, Eosinophilic granulomatosis with polyangiitis (EGPA), Giant cell arteritis, Granulmatosis with polyangiitis (GPA), IgA vasculitis (IgAV), Kawasaki's disease, Leukocytoclastic vasculitis, Lupus vasculitis, Rheumatoid vasculitis, Microscopic polyangiitis (MPA), Polyarteritis nodosa (PAN), Polymyalgia rheumaticia, Vasculitis, Primary Immune Deficiency, and the like.
Other examples of potential autoimmune disorders and diseases, as well as autoimmune comorbidities that can be treated with the compounds described herein include, but are not limited to, Chronic fatigue syndrome, Complex regional pain syndrome, Eosinophilic esophagitis, Gastirtis, Interstitial lung disease, POEMS syndrome, Raynaud's phenomenon, Primary immunodeficiency, Pyoderma gangrenosum, Agammaglobulinemia, Anyloidosis, Anyotrophic lateral sclerosis, Anti-tubular basement membrane nephritis, Atopic allergy, Atopic dermatitis, Autoimmune peripheral neuropathy, Blau syndrome, Castleman's disease, Chagas disease, Chronic obstructive pulmonary disease, Chronic recurrent multifocal osteomyelitis, Complement component 2 deficiency, Contact dermatitis, Cushing's syndrome, Cutaneous leukocytoclastic angiitis, Dego' deiase, Eczema, Eosinophilic gastroenteritis, Eosinophilic pneumonia, Erythroblastosis fetalsis, Fibrodysplasia ossificans progressive, Gastrointestinal pemphigoid, Hypogammaglobulinemia, Idiopathic giant-cell myocarditis, Idiopathic pulmonary fibrosis, IgA nephropathy, Immunregulatory lipoproteins, IPEX syndrome, Ligenous conjunctivitis, Majeed syndrome, Narcolepsy, Rasmussen's encephalitis, Schizophrenia, Serum sickness, Spondyloathropathy, Sweet's syndrome, Takayasu's arteritis, and the like.
In some embodiments, the autoimmune disorder does not comprise pemphigus Vulgaris, pemphigus. In some embodiments, the autoimmune disorder does not comprise pemphigus foliaceus. In some embodiments, the autoimmune disorder does not comprise bullous pemphigoid. In some embodiments, the autoimmune disorder does not comprise Goodpasture's Disease. In some embodiments, the autoimmune disorder does not comprise psoriasis. In some embodiments, the autoimmune disorder does not comprise a skin disorder. In some embodiments, the disorder does not comprise a neoplastic disorder, e.g., cancer.
Therapeutic Compounds
A therapeutic compound comprises a specific targeting moiety functionally associated with an effector binding/modulating moiety. In some embodiments, the specific targeting moiety and effector binding/modulating moiety are linked to one another by a covalent or noncovalent bond, e.g., a covalent or non-covalent bond directly linking the one to the other. In other embodiments, a specific targeting moiety and effector binding/modulating moiety are linked, e.g., covalently or noncovalently, through a linker moiety. E.g., in the case of a fusion polypeptide, a polypeptide sequence comprising the specific targeting moiety and a polypeptide sequence can be directly linked to one another or linked through one or more linker sequences. In some embodiments, the linker moiety comprises a polypeptide. Linkers are not, however, limited to polypeptides. In some embodiments, a linker moiety comprises other backbones, e.g., a non-peptide polymer, e.g., a PEG polymer. In some embodiments, a linker moiety can comprise a particle, e.g., a nanoparticle, e.g., a polymeric nanoparticle. In some embodiments, a linker moiety can comprise a branched molecule, or a dendrimer. However, in embodiments where the effector binding/modulating moiety comprises an ICIM binding/modulating moiety (which binds an effector like PD-1) structures that result in clustering in the absence of target binding should be avoided as they may cause clustering in the absence of target binding. Thus in embodiments, the therapeutic compound has a structure, e.g., the copies of an ICIM are sufficiently limited, such that clustering in the absence of target binding is minimized or substantially eliminated, or eliminated, or is sufficiently minimized that substantial systemic immune suppression does not occur.
In some embodiments, a therapeutic compound comprises a polypeptide comprising a specific targeting moiety covalently or non-covalently conjugated to an effector binding/modulating moiety. In some embodiments, a therapeutic molecule comprises a fusion protein having comprising a specific targeting moiety fused, e.g., directly or through a linking moiety comprising one or more amino acid residues, to an effector binding/modulating moiety. In some embodiments, a therapeutic molecule comprises a polypeptide comprising a specific targeting moiety linked by a non-covalent bond or a covalent bond, e.g., a covalent bond other than a peptide bond, e.g., a sulfhydryl bond, to an effector binding/modulating moiety.
In some embodiments, a therapeutic compound comprises polypeptide, e.g., a fusion polypeptide, comprising:
1.a) a specific targeting moiety comprising a target specific binding polypeptide;
1.b) a specific targeting moiety comprising a target ligand binding molecule;
1.c) a specific targeting moiety comprising an antibody molecule;
1.d) a specific targeting moiety comprising a single chain antibody molecule, e.g., a scFv domain; or
1.e) a specific targeting moiety comprising a first of the light or heavy chain variable region of an antibody molecule, and wherein the other variable region is covalently or non covalently associated with the first;
and
2.a) an effector binding/modulating moiety comprising an effector specific binding polypeptide;
2.b) an effector binding/modulating moiety comprising an effector ligand binding molecule;
2.c) an effector binding/modulating moiety comprising an antibody molecule;
2.d) an effector binding/modulating moiety comprising a single chain antibody molecule, e.g., a scFv domain; or
2.e) an effector binding/modulating moiety comprising a first of the light or heavy chain variable region of an antibody molecule, and wherein the other variable region is covalently or non covalently associated with the first.
In some embodiments, a therapeutic compound comprises 1.a and 2.a.
In some embodiments, a therapeutic compound comprises 1.a and 2.b.
In some embodiments, a therapeutic compound comprises 1.a and 2.c.
In some embodiments, a therapeutic compound comprises 1.a and 2.d.
In some embodiments, a therapeutic compound comprises 1.a and 2.e.
In some embodiments, a therapeutic compound comprises 1.b and 2.a.
In some embodiments, a therapeutic compound comprises 1.b and 2.b.
In some embodiments, a therapeutic compound comprises 1.b and 2.c.
In some embodiments, a therapeutic compound comprises 1.b and 2.d.
In some embodiments, a therapeutic compound comprises 1.b and 2.e.
In some embodiments, a therapeutic compound comprises 1.c and 2.a.
In some embodiments, a therapeutic compound comprises 1.c and 2.b.
In some embodiments, a therapeutic compound comprises 1.c and 2.c.
In some embodiments, a therapeutic compound comprises 1.c and 2.d.
In some embodiments, a therapeutic compound comprises 1.c and 2.e.
In some embodiments, a therapeutic compound comprises 1.d and 2.a.
In some embodiments, a therapeutic compound comprises 1.d and 2.b.
In some embodiments, a therapeutic compound comprises 1.d and 2.c.
In some embodiments, a therapeutic compound comprises 1.d and 2.d.
In some embodiments, a therapeutic compound comprises 1.d and 2.e.
In some embodiments, a therapeutic compound comprises 1.e and 2.a.
In some embodiments, a therapeutic compound comprises 1.e and 2.b.
In some embodiments, a therapeutic compound comprises 1.e and 2.c.
In some embodiments, a therapeutic compound comprises 1.e and 2.d.
In some embodiments, a therapeutic compound comprises 1.e and 2.e.
Therapeutic compounds disclosed herein can, for example, comprise a plurality of effector binding/modulating and specific targeting moieties. Any suitable linker or platform can be used to present the plurality of moieties. The linker is typically coupled or fused to one or more effector binding/modulating and targeting moieties.
In some embodiments, two (or more) linkers associate, either covalently or noncovelaently, e.g., to form a hetero or homo-dimeric therapeutic compound. E.g., the linker can comprise an Fc region and two Fc regions associate with one another. In some embodiments of a therapeutic compound comprising two linker regions, the linker regions can self-associate, e.g., as two identical Fc regions. In some embodiments of a therapeutic compound comprising two linker regions, the linker regions are not capable of, or not capable of substantial, self-association, e.g., the two Fc regions can be members of a knob and hole pair.
Non-limiting exemplary configurations of therapeutic compounds comprise the following (e.g., in N to C terminal order):
R1---Linker Region A-R2
R3---Linker Region B---R4,
wherein,
R1, R2, R3, and R4, each independently comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety; a specific targeting moiety; or is absent;
Linker Region A and Linker B comprise moieties that can associate with one another, e.g., Linker A and Linker B each comprises an Fc moiety provided that an effector binding/modulating moiety and a specific targeting moiety are present.
In some embodiments:
R1 comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
R2 comprises a specific targeting moiety, or is absent;
R3 comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
R4 comprises a specific targeting moiety, or is absent;
Linker Region A and Linker B comprise moieties that can associate with one another, e.g., Linker A and Linker B each comprises an Fc moiety, provided that one of R1 or R3 is present and one of R2 or R4 is present.
In some embodiments:
R1 comprises a specific targeting moiety, or is absent;
R2 comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
R3 comprises a specific targeting moiety, or is absent;
R4 comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, or is absent;
Linker Region A and Linker B comprise moieties that can associate with one another, e.g., Linker A and Linker B each comprises an Fc moiety, provided that one of R1 or R3 is present and one of R2 or R4 is present.
Non-limiting examples include, but are not limited to:

















Linker 


Linker 





Region 


Region 




R1 
A 
R2 
R3 
B 
R4 
Other







HCVR 
Fc 
fcFv 
HCVR 
Fc 
scFv 
Self-Pairing 


and 
Region 

and 
Region 

Linker 


LCVR 


LCVR 


Regions 


HCVR 
Fc 
fcFv 
HCVR 
Fc 
scFv 
Non-Self 


and 
Region 

and 
Region 

Pairing 


LCVR 


LCVR 


linker 








regions 


HCVR 
Fc 
fcFv 
HCVR 
Fc 
scFv 
Self-Pairing 


and 
Region 

and 
Region 

Linker 


LCVR 


LCVR 


Regions 


(or 


(or 


One of R1 


absent)


absent)


or R3 is 








absent. 


HCVR 
Fc 
fcFv 
HCVR 
Fc 
scFv 
Non-Self 


and 
Region 

and 
Region 

Pairing 


LCVR 


LCVR 


Linker 


(or 


(or 


Regions 


absent)


absent)


One of R1 








or R3 is 








absent. 


HCVR 
Fc 
fcFv 
HCVR 
Fc 
scFv 
Self-Pairing 


and 
Region 
(or 
and 
Region 
(or 
linker 


LCVR 

absent) 
LCVR 

absent) 
regions 








One of R2 








or R4 is 








absent. 


HCVR 
Fc 
fcFv 
HCVR 
Fc 
scFv 
Non-Self 


and 
Region 
(or 
and 
Region 
(or 
Pairing 


LCVR 

absent) 
LCVR 

absent) 
linker 








regions 








One of R2 








or R4 is 








absent. 


HCVR 
Fc 
fcFv 
HCVR 
Fc 
scFv 
Self-Pairing 


and 
Region 

and 
Region 

Linker 


LCVR 


LCVR 


Regions 








R1 and R3 








are the 








same 


HCVR 
Fc 
fcFv 
HCVR 
Fc 
scFv 
Non-Self 


and 
Region 

and 
Region 

Pairing 


LCVR 


LCVR 


linker 








regions 








R1 and R3 








are 








different 


HCVR 
Fc 
fcFv 
HCVR 
Fc 
scFv 
Self-Pairing 


and 
Region 

and 
Region 

Linker 


LCVR 


LCVR 


Regions 








R2 and R4 








are the 








same 


HCVR 
Fc 
fcFv 
HCVR 
Fc 
scFv 
Non-Self 


and 
Region 

and 
Region 

Pairing 


LCVR 


LCVR 


linker 








regions 








R2 and R4 








are 








different





HCVR and LCVR: refers to an moiety comprising an antigen binding portion of a heavy and light chain variable region, typically with the heavy chain fused to the Linker region.


Self-pairing: wherein a liker region can pair with itself, e.g., an Fc region that can pair a copy of itself.


Non-Self Pairing: wherein a Linker Region does not pair with itself, or does not substantially pair with itself, e.g., an Fc region does not or does not significantly pair with itself, e.g., wherein Linker Region A and Linker Region B are members of a knob and hole pair.






In some embodiments:

R1, R2, R3 and R4 each independently comprise: an effector binding modulating moiety that activates an inhibitory receptor on an immune cell, e.g., a T cell or a B cell, e.g., a PD-L1 molecule or a functional anti-PD-1 antibody molecule (an agonist of PD-1); a specific targeting moiety; or is absent;

provided that an effector binding moiety and a specific targeting moiety are present.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties).

In some embodiments:

R1 and R3 independently comprise an effector binding modulating moiety that activates an inhibitory receptor on an immune cell, e.g., a T cell or a B cell, e.g., a PD-L1 molecule or an functional anti-PD-1 antibody molecule (an agonist of PD-1); and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties).

In some embodiments:

R1 and R3 independently comprise a functional anti-PD-1 antibody molecule (an agonist of PD-1); and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties).

In some embodiments:

R1 and R3 independently comprise specific targeting moieties, e.g., an anti-tissue antigen antibody; and

R2 and R4 independently comprise a functional anti-PD-1 antibody molecule (an agonist of PD-1), e.g., an scFv molecule.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties).

In some embodiments:

R1 and R3 independently comprise a PD-L1 molecule (an agonist of PD-1); and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen; and

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties).

In some embodiments:

R1 and R3 independently comprise specific targeting moieties, e.g., an anti-tissue antigen antibody; and

R2 and R4 independently comprise a PD-L1 molecule (an agonist of PD-1).

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties).

In some embodiments:

R1, R2, R3 and R4 each independently comprise: an SM binding/modulating moiety which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP, e.g., a CD39 molecule or a CD73 molecule; a specific targeting moiety; or is absent;

provided that an SM binding/modulating moiety and a specific targeting moiety are present.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

R1 and R3 independently comprise an SM binding/modulating moiety which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP, e.g., a CD39 molecule or a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

R1 and R3 independently comprise a CD39 molecule or a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

R1 and R3 each comprises a CD39 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen; and

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

R1 and R3 each comprises a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

One of R1 and R3 comprises a CD39 molecule and the other comprises a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

R1, R2, R3 and R4 each independently comprise: an HLA-G molecule; a specific targeting moiety; or is absent;

provided that an HLA-G molecule and a specific targeting moiety are present.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

R1 and R3 each comprise an HLG-A molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

R1 and R3 each comprise an agonistic anti-LILRB1 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

R1 and R3 each comprise an agonistic anti-KIR2DL4 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

R1 and R3 each comprise an agonistic anti-LILRB2 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

R1 and R3 each comprise an agonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

one of R1 and R3 comprises a first moiety chosen from, and the other comprises a different moiety chosen from: an antagonistic anti-LILRB1 antibody molecule, an agonistic anti-KR2DL4 antibody molecule, and an agonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

one of R1 and R3 comprises an antagonistic anti-LILRB1 antibody molecule and the other comprises an agonistic anti-KR2DL4 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In some embodiments:

one of R1 and R3 comprises an antagonistic anti-LILRB1 antibody molecule and the other comprises an agonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In an embodiment:
R1, R2, R3 and R4 each independently comprise: an IL-2 mutein molecule; a specific targeting moiety; or is absent;
provided that an IL-2 mutein molecule and a specific targeting moiety are present.
In an embodiment Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).
One of R1, R2, R3 and R4 comprises an IL-2 mutein molecule, one comprises an anti-GITR antibody molecule, e.g., an anti-GITR antibody molecule that inhibits binding of GITRL to GITR, and one comprises a specific targeting moiety;
In an embodiment Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).
In an embodiment:
R1 and R3 each comprise an IL-2 mutein molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
In an embodiment Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).
In an embodiment:
one of R1 and R3 comprises a GARP binding molecule, e.g., an anti-GARP antibody molecule or a GITR binding molecule, e.g., an anti-GITR antibody molecule and the other comprises an IL-2 mutein molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
In an embodiment Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).
In an embodiment:
one of R1 and R3 comprises a GARP binding molecule, e.g., an anti-GARP antibody molecule and the other comprises an IL-2 mutein molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
In an embodiment Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).
In an embodiment:
one of R1 and R3 comprises a GITR binding molecule, e.g., an anti-GITR antibody molecule, and the other comprises an IL-2 mutein molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
In an embodiment Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).
In some embodiments:
R1, R2, R3 and R4 each independently comprise: an effector binding modulating moiety that activates an inhibitory receptor on a B cell, e.g., an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule; a specific targeting moiety; or is absent; provided that an effector binding moiety and a specific targeting moiety are present.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

In embodiment the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule, directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
In some embodiments:
R1 and R3 each comprises an agonistic anti-FCRL antibody molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).
In embodiment the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
In some embodiments:
R1 and R3 independently comprise specific targeting moieties, e.g., antibody molecules against a tissue antigen; and
R2 and R4 each comprises an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule, e.g., an scFv molecule.
In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).
In embodiment the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
In some embodiments:
One of R1, R2, R3 and R4 comprises an anti-BCR antibody molecule, e.g., an antagonistic anti-BCR antibody molecule, one comprises an anti FCRL antibody molecule, and one comprises a specific targeting moiety.
In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).
In some embodiments, the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
In some embodiments:
One of R1, R2, R3 and R4 comprises a bispecific antibody molecule comprising an anti-BCR antibody molecule, e.g., an antagonistic anti-BCR antibody molecule, and an anti FCRL antibody molecule, and one comprises a specific targeting moiety;
In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).
In embodiment the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.
In some embodiments:
R1, R2, R3 and R4 each independently comprise:
i) an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, that minimizes or inhibits T cell activity, expansion, or function (a T cell effector binding/modulating moiety);

ii) an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety, that minimizes or inhibits B cell activity, expansion, or function (a B cell effector binding/modulating moiety);

iii) a specific targeting moiety; or

iv) is absent;

provided that, a T cell effector binding/modulating moiety, a B cell effector binding/modulating moiety, and a specific targeting moiety are present.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties).

In some embodiments, one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody and one comprises an HLA-G molecule.

In some embodiments, one of R1, R2, R3, and R4 comprises an SM binding/modulating moiety, e.g., a CD39 molecule or a CD73 molecule. In some embodiments, one of R1, R2, R3, and R4 comprises an entity that binds, activates, or maintains, a regulatory immune cell, e.g., a Treg cell or a Breg cell, for example, an IL-2 mutein molecule.
In some embodiments, one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, or one comprises an HLA-G molecule, and one comprises an IL-2 mutein molecule. In some embodiments, the PD-1 antibody is replaced with a IL-2 mutein molecule. In some embodiments, one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, one comprises an HLA-G molecule, and one comprises CD39 molecule or a CD73 molecule. In some embodiments, the PD-1 antibody is replaced with a IL-2 mutein molecule.
Linker Regions
As discussed elsewhere herein specific targeting and effector binding/modulating moieties can be linked by linker regions. Any linker region described herein can be used as a linker. For example, linker Regions A and B can comprise Fc regions. In some embodiments, a therapeutic compound comprises a Linker Region that can self-associate. In some embodiments, a therapeutic compound comprises a Linker Region that has a moiety that minimizes self-association, and typically Linker Region A and Linker Region B are heterodimers. Linkers also include glycine/serine linkers. In some embodiments, the linker can comprise one or more repeats of GGGGS (SEQ ID NO: 23). In some embodiments, the linker comprises 1, 2, 3, 4, or 5 repeats of SEQ ID NO: 23. In some embodiments, the linker comprises or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) GGGGSGGGGSGGGGS (SEQ ID NO: 30). These linkers can be used in any of the therapeutic compounds or compositions provided herein.
The linker region can comprise a Fc region that has been modified (e.g. mutated) to produce a heterodimer. In some embodiments, the CH3 domain of the Fc region can be mutated. Examples of such Fc regions can be found in, for example, U.S. Pat. No. 9,574,010, which is hereby incorporated by reference in its entirety. The Fc region as defined herein comprises a CH3 domain or fragment thereof, and may additionally comprise one or more addition constant region domains, or fragments thereof, including hinge, CH1, or CH2. It will be understood that the numbering of the Fc amino acid residues is that of the EU index as in Kabat et al., 1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va. The “EU index as set forth in Kabat” refers to the EU index numbering of the human IgG1 Kabat antibody. For convenience, Table B of U.S. Pat. No. 9,574,010 provides the amino acids numbered according to the EU index as set forth in Kabat of the CH2 and CH3 domain from human IgG1, which is hereby incorporated by reference. Table 1.1 of U.S. Pat. No. 9,574,010 provides mutations of variant Fc heterodimers that can be used as linker regions. Table 1.1 of U.S. Pat. No. 9,574,010 is hereby incorporated by reference.
In some embodiments, the Linker Region A comprises a first CH3 domain polypeptide and a the Linker Region B comprises a second CH3 domain polypeptide, the first and second CH3 domain polypeptides independently comprising amino acid modifications as compared to a wild-type CH3 domain polypeptide, wherein the first CH3 domain polypeptide comprises amino acid modifications at positions T350, L351, F405, and Y407, and the second CH3 domain polypeptide comprises amino acid modifications at positions T350, T366, K392 and T394, wherein the amino acid modification at position T350 is T350V, T3501, T350L or T350M; the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405T or F405S; the amino acid modification at position Y407 is Y407V, Y407A or Y4071; the amino acid modification at position T366 is T366L, T366I, T366V, or T366M, the amino acid modification at position K392 is K392F, K392L or K392M, and the amino acid modification at position T394 is T394W, and wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.
In some embodiments, the amino acid modification at position K392 is K392M or K392L. In some embodiments, the amino acid modification at position T350 is T350V. In some embodiments, the first CH3 domain polypeptide further comprises one or more amino acid modifications selected from Q347R and one of 5400R or S400E. In some embodiments, the second CH3 domain polypeptide further comprises one or more amino acid modifications selected from L351Y, K360E, and one of N390R, N390D or N390E. In some embodiments, the first CH3 domain polypeptide further comprises one or more amino acid modifications selected from Q347R and one of 5400R or S400E, and the second CH3 domain polypeptide further comprises one or more amino acid modifications selected from L351Y, K360E, and one of N390R, N390D or N390E. In some embodiments, the amino acid modification at position T350 is T350V. In some embodiments, the amino acid modification at position F405 is F405A. In some embodiments, the amino acid modification at position Y407 is Y407V. In some embodiments, the amino acid modification at position T366 is T366L or T366I. In some embodiments, the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is and Y407V, the amino acid modification at position T366 is T366L or T366I, and the amino acid modification at position K392 is K392M or K392L. In some embodiments, the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405V and Y407V, and the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392M and T394W. In some embodiments, the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405T and Y407V, and the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392M and T394W. In some embodiments, the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405S and Y407V, and the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392M and T394W. In some embodiments, the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A and Y407V, and the second CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, T366L, N390R, K392M and T394W. In some embodiments, the first CH3 domain polypeptide comprises the amino acid modifications Q347R, T350V, L351Y, S400E, F405A and Y407V, and the second CH3 domain polypeptide comprises the amino acid modifications T350V, K360E, T366L, N390R, K392M and T394W. In some embodiments, the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400R, F405A and Y407V, and the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390D, K392M and T394W. In some embodiments, the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400R, F405A and Y407V, and the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390E, K392M and T394W. In some embodiments, the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A and Y407V, and the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392L and T394W. In some embodiments, the first CH3 domain polypeptide comprises the amino acid modifications T350V, L351Y, S400E, F405A and Y407V, and the second CH3 domain polypeptide comprises the amino acid modifications T350V, T366L, N390R, K392F and T394W.
In some embodiments, an isolated heteromultimer comprising a heterodimeric CH3 domain comprising a first CH3 domain polypeptide and a second CH3 domain polypeptide, the first CH3 domain polypeptide comprising amino acid modifications at positions F405 and Y407, and the second CH3 domain polypeptide comprising amino acid modifications at positions T366 and T394, wherein: (i) the first CH3 domain polypeptide further comprises an amino acid modification at position L351, and (ii) the second CH3 domain polypeptide further comprises an amino acid modification at position K392, wherein the amino acid modification at position F405 is F405A, F405T, F405S or F405V; and the amino acid modification at position Y407 is Y407V, Y407A, Y407L or Y4071; the amino acid modification at position T394 is T394W; the amino acid modification at position L351 is L351Y; the amino acid modification at position K392 is K392L, K392M, K392V or K392F, and the amino acid modification at position T366 is T366I, T366L, T366M or T366V, wherein the heterodimeric CH3 domain has a melting temperature (Tm) of about 70.degree. C. or greater and a purity greater than about 90%, and wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.
In some embodiments, the Linker Region A comprises a first CH3 domain polypeptide and a t Linker Region B comprises a second CH3 domain polypeptide, wherein the first CH3 domain polypeptide comprising amino acid modifications at positions F405 and Y407, and the second CH3 domain polypeptide comprising amino acid modifications at positions T366 and T394, wherein: (i) the first CH3 domain polypeptide further comprises an amino acid modification at position L351, and (ii) the second CH3 domain polypeptide further comprises an amino acid modification at position K392, wherein the amino acid modification at position F405 is F405A, F405T, F405S or F405V; and the amino acid modification at position Y407 is Y407V, Y407A, Y407L or Y4071; the amino acid modification at position T394 is T394W; the amino acid modification at position L351 is L351Y; the amino acid modification at position K392 is K392L, K392M, K392V or K392F, and the amino acid modification at position T366 is T366I, T366L, T366M or T366V, wherein the heterodimeric CH3 domain has a melting temperature (Tm) of about 70 C. or greater and a purity greater than about 90%, and wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat. In some embodiments, the amino acid modification at position F405 is F405A. In some embodiments, the amino acid modification at position T366 is T366I or T366L. In some embodiments, the amino acid modification at position Y407 is Y407V. In some embodiments, the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366I or T366L, and the amino acid modification at position K392 is K392L or K392M. In some embodiments, the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366L, and the amino acid modification at position K392 is K392M. In some embodiments, the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366L, and the amino acid modification at position K392 is K392L. In some embodiments, the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366I, and the amino acid modification at position K392 is K392M. In some embodiments, the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366I, and the amino acid modification at position K392 is K392L. In some embodiments, the first CH3 domain polypeptide further comprises an amino acid modification at position 5400 selected from S400D and S400E, and the second CH3 domain polypeptide further comprises the amino acid modification N390R. In some embodiments, the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y405V, the amino acid modification at position 5400 is S400E, the amino acid modification at position T366 is T366L, and the amino acid modification at position K392 is K392M.
In some embodiments, the modified first and second CH3 domains are comprised by an Fc construct based on a type G immunoglobulin (IgG). The IgG can be an IgG1, IgG2, IgG3 or IgG4.
Other Linker Region A and Linger Region B comprising variant CH3 domains are described in U.S. Pat. Nos. 9,499,634 and 9,562,109, each of which is incorporated by reference in its entirety.
A Linker Region A and Linker Region B can be complementary fragments of a protein, e.g., a naturally occurring protein such as human serum albumin. In embodiments, one of Linker Region A and Linker Region B comprises a first, e.g., an N terminal fragment of the protein, e.g., hSA, and the other comprises a second, e.g., a C terminal fragment of the protein, e.g., has. In an embodiment the fragments comprise an N terminal and a C terminal fragment. In an embodiment the fragments comprise two internal fragments. Typically the fragments do not overlap. In an embodiment the First and second fragment, together, provide the entire sequence of the original protein, e.g., hSA. The first fragment provides a N terminus and a C terminus for linking, e.g., fusing, to other sequences, e.g., sequences of R1, R2, R3, or R4 (as defined herein).
The Linker Region A and the Linker Region B can be derived from albumin polypeptide. In some embodiments, the albumin polypeptide is selected from native human serum albumin polypeptide and human alloalbumin polypeptide. The albumin polypeptide can be modified such that the Linker Region A and Linker Region B interact with one another to form heterodimers. Examples of modified albumin polypeptides are described in U.S. Pat. Nos. 9,388,231 and 9,499,605, each of which is hereby incorporated by reference in its entirety. Accordingly, provided herein are multifunctional heteromultimer proteins of the formula R1---Linker Region A-R2 and R3---Linker Region B---R4, wherein the Linker Region A and Linker Region B form a heteromultimer. In some embodiments, the Linker Region A comprises a first polypeptide and the Linker Region B comprises a second polypeptide; wherein each of said first and second polypeptides comprises an amino acid sequence comprising a segment of an albumin polypeptide selected from native human serum albumin polypeptide and human alloalbumin polypeptide; wherein said first and second polypeptides are obtained by segmentation of said albumin polypeptide at a segmentation site, such that the segmentation results in a deletion of zero to 3 amino acid residues at the segmentation site; wherein said first polypeptide comprises at least one mutation selected from A194C, L198C, W214C, A217C, L331C and A335C, and said second polypeptide comprises at least one mutation selected from L331C, A335C, V343C, L346C, A350C, V455C, and N458C; and wherein said first and second polypeptides self-assemble to form a quasi-native structure of the monomeric form of the albumin polypeptide.
In some embodiments, the segmentation site resides on a loop of the albumin polypeptide that has a high solvent accessible surface area (SASA) and limited contact with the rest of the albumin structure, b) the segmentation results in a complementary interface between the transporter polypeptides. These segmentation sites are described, for example, in U.S. Pat. No. 9,388,231, which is hereby incorporated by reference in its entirety.
In some embodiments, the first polypeptide comprises residues 1-337 or residues 1-293 of the albumin polypeptide with one or more of the mutations described herein. In some embodiments, the second polypeptide comprises residues of 342-585 or 304-585 of the albumin polypeptide with one or more of the mutations described herein. In some embodiments, the first polypeptide comprises residues 1-339, 1-300, 1-364, 1-441, 1-83, 1-171, 1-281, 1-293, 1-114, 1-337, or 1-336 of the albumin protein. In some embodiments, the second polypeptide comprises residues 301-585, 365-585, 442-585, 85-585, 172-585, 282-585, or 115-585, 304-585, 340-585, or 342-585 of the albumin protein.
In some embodiments, the first and second polypeptide comprise the residues of the albumin protein as shown in the table below. The sequence of the albumin protein is described below.














First Polypeptide Residues
Second Polypeptide Residues









1-300
301-585



1-364
365-585



1-441
442-585



1-83
85-585



1-171
172-585



1-281
282-585



1-114
115-585



1-339
340-585



1-337
342-585



1-293
304-585



1-336
342-585










In some embodiments, the first and second polypeptides comprise a linker that can form a covalent bond with one another, such as a disulfide bond. A non-limiting example of the linker is a peptide linker. In some embodiments, the peptide linker comprises GGGGS (SEQ ID NO: 23). The linker can be fused to the C-terminus of the first polypeptide and the N-terminus of the second polypeptide. The linker can also be used to attach the moieties described herein without abrogating the ability of the linkers to form a disulfide bond. In some embodiments, the first and second polypeptides do not comprise a linker that can form a covalent bond. In some embodiments, the first and second polypeptides have the following substitutions.














First Polypeptide Substitution
Second Polypeptide Substitution









A217C
V343C



L331C
A350C



A217C
L346C



W214C
V343C



A335C
L346C



L198C
V455C



A217C
A335C



A217C
L331C



L198C
N458C



A194C
V455C










The sequence of the albumin polypeptide can be The sequence of human albumin is as shown, in the post-protein form with the N-terminal signaling residues removed







(human albumin, SEQ ID NO: 42)


(MKWVTFISLLFLFSSAYSRGVFRR)DAHKSEVAHRFKDLGEENFKALVL


 


IAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKL


 


CTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMC


 


TAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK


 


AACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFP


 


KAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSK


 


LKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD


 


VFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVF


 


DEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLV


 


EVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVT


 


KCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIK


 


KQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKK


 


LVAASQAALGL






In some embodiments, the Linker Region A and the Linker Region B form a heterodimer as described herein.
In some embodiments, the polypeptide comprises at the N-terminus an antibody comprised of F(ab′)2 on an IgG1 Fc backbone fused with scFvs on the C-terminus of the IgG Fc backbone. In some embodiments, the IgG Fc backbone is a IgG1 Fc backbone. In some embodiments, the IgG1 backbone is replaced with a IgG4 backbone, IgG2 backbone, or other similar IgG backbone. The IgG backbones described in this paragraph can be used throughout this application where a Fc region is referred to as part of the therapeutic compound. Thus, in some embodiments, the antibody comprised of F(ab′)2 on an IgG1 Fc backbone can be an anti-MAdCAM antibody or an anti-PD-1 antibody on an IgG1 Fc or any other targeting moiety or effector binding/modulating moiety provided herein. In some embodiments, the scFV segments fused to the C-terminus could be an anti-PD-1 antibody, if the N-terminus region is an anti-MAdCAM antibody, or anti-MAdCAM antibody, if the N-terminus region is an anti-PD-1 antibody. In this non-limiting example, the N-terminus can be the targeting moiety, such as any one of the ones provided for herein, and the C-terminus can be the effector binding/modulating moiety, such as any of the ones provided for herein. Alternatively, in some embodiments, the N-terminus can be the effector binding/modulating moiety, such as any one of the ones provided for herein, and the C-terminus can be the targeting moiety, such as any of the ones provided for herein.
In some embodiments, the N-terminus can be the targeting moiety, such as any one of the ones provided for herein, and the C-terminus can be the effector binding/modulating moiety, such as any of the ones provided for herein.
In some embodiments, the therapeutic compound comprises two polypeptides that homodimerize. In some embodiments, the N-terminus of the polypeptide comprises an effector binding/modulating moiety that is fused to a human IgG1 Fc domain (e.g. CH2 and/or CH3 domains). In some embodiments, the C-terminus of the Fc domain is another linker that is fused to the targeting moiety. Thus, in some embodiments, the molecule could be represented using the formula of R1-Linker A-Fc Region-Linker B-R2, wherein R1 can be an effector binding/modulating moiety, R2 is a targeting moiety, Linker A and Linker B are independently linkers as provided for herein. In some embodiments, Linker 1 and Linker 2 are different.
In some embodiments, the molecule could be represented using the formula of R1-Linker A-Fc Region-Linker B-R2, wherein R1 can be a targeting moiety, R2 is an effector binding/modulating moiety, Linker A and Linker B are independently linkers as provided for herein. In some embodiments, Linker A and Linker B are different. The linkers can be chosen from the non-limiting examples provided for herein. In some embodiments, R1 and R2 are independently selected from F(ab′)2 and scFV antibody domains. In some embodiments, R1 and R2 are different antibody domains. In some embodiments, the scFV is in the VL-VH domain orientation.
In some embodiments, the therapeutic compound is a bispecific antibody. In some embodiments, the bispecific antibodies are comprised of four polypeptide chains comprising the following:
Chain 1: nt-VH1-CH1-CH2-CH3-Linker A-scFv[VL2-Linker B-VH2]-ct
Chain 2: nt-VH1-CH1-CH2-CH3-Linker A-scFv[VL2-Linker B-VH2]-ct
Chain 3: nt-VL1-CL-ct
Chain 4: nt-VL1-CL-ct,
wherein chains 1 and 2 are identical to each other, and chains 3 and 4 are identical to each other,
wherein chain 1 forms a homodimer with chain 2; and chain 3 and 4 associate with chain 1 and chain 2. That is, when each light chain associates with each heavy chain, VL1 associates with VH1 and CL associates with CH1 to form two functional Fab units. Without being bound to any particular theory, each scFv unit is intrinsically functional since VL2 and VH2 are covalently linked in tandem with a linker as provided herein (e.g. GGGGS (SEQ ID NO: 23), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), or GGGGSGGGGSGGGGS (SEQ ID NO: 30). The sequences of Linker A and Linker B, which are independent of one another can be the same or different and as otherwise described throughout the present application. Thus, in some embodiments, Linker A comprises GGGGS (SEQ ID NO: 23), or two repeats thereof, GGGGSGGGGSGGGGS (SEQ ID NO: 30), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22). In some embodiments, Linker B comprises GGGGS (SEQ ID NO: 23), or two repeats thereof, GGGGSGGGGSGGGGS (SEQ ID NO: 30), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22). The scFv may be arranged in the NT-VH2-VL2-CT or NT-VL2-VH2-CT orientation. NT or nt stands for N-terminus and CT or ct stands for C-terminus of the protein. CH1, CH2, and CH3 are the domains from the IgG Fc region, and CL stands for Constant Light chain, which can be either kappa or lambda family light chains. The other definitions stand for the way they are normally used in the art.
In some embodiments, the VH1 and VL1 domains are derived from the effector molecule and the VH2 and VL2 domains are derived from the targeting moiety. In some embodiments the VH1 and VL1 domains are derived from a targeting moiety and the VH2 and VL2 domains are derived from an effector binding/modulating moiety.
In some embodiments, the VH1 and VL1 domains are derived from an anti-PD-1 antibody, and the VH2 and VL2 domains are derived from an anti-MAdCAM antibody. In some embodiments the VH1 and VL1 domains are derived from an anti-MAdCAM antibody and the VH2 and VL2 domains are derived from an anti-PD-1 antibody.
In some embodiments, Linker A comprises 1, 2, 3, 4, or 5 GGGGS (SEQ ID NO: 23) repeats. In some embodiments, Linker B comprises 1, 2, 3, 4, or 5 GGGGS (SEQ ID NO: 23) repeats. For the avoidance of doubt, the sequences of Linker A and Linker B, which are used throughout this application, are independent of one another. Therefore, in some embodiments, Linker A and Linker B can be the same or different. In some embodiments, Linker A comprises GGGGS (SEQ ID NO: 23), or two repeats thereof, GGGGSGGGGSGGGGS (SEQ ID NO: 30), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22). In some embodiments, Linker B comprises GGGGS (SEQ ID NO: 23), or two repeats thereof, GGGGSGGGGSGGGGS (SEQ ID NO: 30), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22).
In some embodiments, the therapeutic compound comprises a light chain and a heavy chain. In some embodiments, the light and heavy chain begin at the N-terminus with the VH domain of a targeting moiety followed by the CH1 domain of a human IgG1, which is fused to a Fc region (e.g. CH2-CH3) of human IgG1. In some embodiments, at the c-terminus of the Fc region is fused to a linker as provided herein, such as but not limited to, GGGGS (SEQ ID NO: 23), or two or three repeats thereof, or GGGGSGGGGSGGGGS (SEQ ID NO: 30). The linker can then be fused to an effector binding/modulating moiety, such as any one of the effector moieties provided for herein. The polypeptides can homodimerize because through the heavy chain homodimerization, which results in a therapeutic compound having two effector moieties, such as two anti-PD-1 antibodies. In this orientation, the targeting moiety is an IgG format, there are two Fab arms that each recognize binding partner of the targeting moiety, for example, MAdCAM being bound by the anti-MAdCAM targeting moiety.
In some embodiments, the therapeutic or polypeptide comprises a formula of: An antibody (targeting moiety) with a variable heavy chain and a variable light chain, in an IgG isotype, for example, with an effector molecule, such as an IL-2 mutein. In some embodiments, the IL-2 mutein is fused at the c-terminus of the variable heavy chain. This can be represented by the formula of VL and VH-IgGConstantDomain-L1-E, wherein L1 is a linker, such as a glycine/serine linker as provided herein, E is an effector molecule, such as an IL-2 mutein and VL and VH are the variable light and heavy chains. The VL domain can be a kappa domain. In some embodiments, the IgG Constant domain comprises the sequence of:







(SEQ ID NO: 44)


ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV


 


HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP


 


KSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS


 


HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK


 


EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC


 


LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW


 


QQGNVFSCSVMHEALHNHYTQKSLSLSPG






In some embodiments, the linker comprises GGGGS. In some embodiments, the IL-2 mutein comprises the IL-2 muteins provided herein, such as one of SEQ ID NOs: 31-41, which can also have a Fc molecule appended to the N- or C-terminus of the IL-2 mutein. The Fc domain can comprise SEQ ID NO: 21 or 43. In some embodiments, the IL-2 mutein comprises one of SEQ ID NO: 47-60. In some embodiments, the IL-2 mutein comprises SEQ ID NO: 41 or SEQ ID NO: 56. In some embodiments, the IL-2 mutein comprises SEQ ID NO: 40 or SEQ ID NO: 55.

In some embodiments, the targeting moiety is a MAdCAM antibody.
In some embodiments, the MAdCAM antibody is selected from the following table














TABLE 6





Clone ID 
HCDR1
HCDR2
HCDR3
LCDR1
LCDR2
LCDR3
scFv







 1.
FTFSSYG
AVISDDG
CTTSKYY
QASQDIS
AASSLQS
CQQGYST
EVQLLESGGGLVQPGGSLRLSCAA



MH
SDKYYA
YYYGMDV
KSLN
(SEQ ID
PLTF
SGFTFSSYGMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
W
(SEQ ID
NO: 65)
(SEQ ID
AVISDDGSDKYYADSVKGRFTISR



NO: 61)
NO: 62)
(SEQ ID
NO: 64)

NO: 66)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 63)



TTSKYYYYYGMDVWGQGTTVTVSS









GGGGSGGGGSGGGGSGGGGSDIQM









TQSPSSLSASVGDRVTITCQASQD









ISKSLNWYQQKPGKAPKLLIYAAS









SLQSGVPSRFSGSGSGTDFTLTIS









SLQPEDFATYYCQQGYSTPLTFGG









GTKVEIK









(SEQ ID NO: 67)


 


 2.
YPFIGYY
GIINPSG
CAREGRL
RASQSIS
GASTLES
CQQTWGP
QVQLVQSGAEVKKPGASVKVSCKA



LH
GSTSYA
SYGMDAW
SYLA
(SEQ ID
PFTF
SGYPFIGYYLHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 72)
(SEQ ID
GIINPSGGSTSYAQKFQGRVTMTR



NO: 68)
NO: 69)
NO: 70)
NO: 71)

NO: 73)
DTSTSTVYMELSSLRSEDTAVYYC









AREGRLSYGMDAWGQGTLVTVSSG









GGGSGGGGSGGGGSGGGGSDIQMT









QSPSSLSASVGDRVTITCRASQSI









SSYLAWYQQKPGKAPKLLIYGAST









LESGVPSRFSGSGSGTDFTLTISS









LQPEDFATYYCQQTWGPPFTFGQG









TKLEIK









(SEQ ID NO: 74)


 


 3.
YFPIGQY
GIINPSG
CAREGRL
RASQSIS
GASTLE
CQQTWGP
QVQLVQSGAEVKKPGASVKVSCKA



LH
GSTSYA
SYGMDAW
SYLA
(SEQ ID
PFTF
SGYPFIGQYLHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 72
(SEQ ID
GIINPSGGSTSYAQKFQGRVTMTR



NO: 75)
NO: 69)
NO: 70)
NO: 71)

NO: 73)
DTSTSTVYMELSSLRSEDTAVYYC









AREGRLSYGMDAWGQGTLVTVSSG









GGGSGGGGSGGGGSGGGGSDIQMT









QSPSSLSASVGDRVTITCRASQSI









SSYLAWYQQKPGKAPKLLIYGAST









LESGVPSRFSGSGSGTDFTLTISS









LQPEDFATYYCQQTWGPPFTFGQG









TKLEIK









(SEQ ID NO: 76)


 


 4.
GTFSSYA
GSINPSG
CAKDKAQ
QASQDIS
AASSLQS
CQQSYSS
QVQLVQSGAEVKKPGASVKVSCKA



IS
DTTSYA
WLVGYFD
NSLN
(SEQ ID
VITF
SGGTFSSYAISWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
YW
(SEQ ID
NO: 65)
(SEQ ID
GSINPSGDTTSYAQKFQGRVTMTR



NO: 77)
NO: 78)
(SEQ ID
NO: 80)

NO: 81)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 79)



AKDKAQWLVGYFDYWGQGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCQASQ









DISNSLNWYQQKPGKAPKLLIYAA









SSLQSGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQSYSSVITFG









QGTKVEIK









(SEQ ID NO: 82)


 


 5.
FTFSSYW
SSISPGG
CAREVQL
RASQGIS
GASSLQS
CQQANSF
EVQLLESGGGLVQPGGSLRLSCAA



MH
SNIDYA
SHYDYW
NSLA
(SEQ ID
PFTF
SGFTFSSYWMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 87)
(SEQ ID
SSISPGGSNIDYADSVKGRFTISR



NO: 83)
NO: 84)
NO: 85)
NO: 86)

NO: 88)
DNSKNTLYLQMNSLRAEDTAVYYC









AREVQLSHYDYWGQGTLVTVSSGG









GGSGGGGSGGGGSGGGGSDIQMTQ









SPSSLSASVGDRVTITCRASQGIS









NSLAWYQQKPGKAPKLLIYGASSL









QSGVPSRFSGSGSGTDFTLTISSL









QPEDFATYYCQQANSFPFTFGQGT









KVEIK









(SEQ ID NO: 89)


 


 6.
FTFNNYA
SRINSYG
CAREGPV
RASQIIG
GASSLQS
CQQSYRL
EVQLLESGGGLVQPGGSLRLSCAA



FH
TSTTYA
AGYWYFD
TNLA
(SEQ ID
PFTF
SGFTFNNYAFHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
LW
(SEQ ID
NO: 87)
(SEQ ID
SRINSYGTSTTYADSVKGRFTISR



NO: 90)
NO: 91)
(SEQ ID
NO: 93)

NO: 94)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 92)



AREGPVAGYWYFDLWGQGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









IIGTNLAWYQQKPGKAPKLLIYGA









SSLQSGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQSYRLPFTFG









QGTKVEIK









(SEQ ID NO: 95)


 


 7.
YTFTGYH
GIINPSG
CAKDWSS
RASQNIS
AASSLQS
CQQSYTT
QVQLVQSGAEVKKPGASVKVSCKA



IH
GSTIYA
WYLGPFD
SSLN
(SEQ ID
PYTF
SGYTFTGYHIHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
YW
(SEQ ID
NO: 65)
(SEQ ID
GIINPSGGSTIYAQKFQGRVTMTR



NO: 96)
NO: 97)
(SEQ ID
NO: 99)

NO: 100)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 98)



AKDWSSWYLGPFDYWGQGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









NISSSLNWYQQKPGKAPKLLIYAA









SSLQSGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQSYTTPYTFG









QGTKVEIK









(SEQ ID NO: 101)


 


 8.
FMFGDYA
SAISGSG
CAKDLVV
RASQGIS
DASSLES
CQQTHSF
EVQLLESGGGLVQPGGSLRLSCAA



MH
GSTYYA
AGIWYFD
NNLN
(SEQ ID
PSTF
SGFMFGDYAMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
LW
(SEQ ID
NO: 106)
(SEQ ID
SAISGSGGSTYYADSVKGRFTISR



NO: 102)
NO: 103)
(SEQ ID
NO: 105)

NO: 107)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 104)



AKDLVVAGIWYFDLWGRGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









GISNNLNWYQQKPGKAPKLLIYDA









SSLESGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQTHSFPSTFG









QGTKLEIK









(SEQ ID NO: 108)


 


 9.
FTFSDYY
SVIGESG
CAADPVS
RASQGIS
AASTLQS
CQQSYST
EVQLLESGGGLVQPGGSLRLSCAA



MN
GSTYYA
RWPKHGG
SSLA
(SEQ ID
PWTF
SGFTFSDYYMNWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
GDYW
(SEQ ID
NO: 113)
(SEQ ID
SVIGESGGSTYYADSVKGRFTISR



NO: 109)
NO: 110)
(SEQ ID
NO: 112)

NO: 114)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 111)



AADPVSRWPKHGGGDYWGQGTLVT









VSSGGGGSGGGGSGGGGSGGGGSD









IQMTQSPSSLSASVGDRVTITCRA









SQGISSSLAWYQQKPGKAPKLLIY









AASTLQSGVPSRFSGSGSGTDFTL









TISSLQPEDFATYYCQQSYSTPWT









FGQGTKVEIK









(SEQ ID NO: 115)


 


10.
YTLTTWY
GWINPNR
CAKGDLW
RASDNIG
AASSLQS
CQQGYST
QVQLVQSGAEVKKPGASVKVSCKA



MY
GATNYA
GAMDVW
SWLA
(SEQ ID
PPTF
SGYTLTTWYMYWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 65)
(SEQ ID
GWINPNRGATNYAQKFQGRVTMTR



NO: 116)
NO: 117)
NO: 118)
NO: 119)

NO: 120)
DTSTSTVYMELSSLRSEDTAVYYC









AKGDLWGAMDVWGQGTLVTVSSGG









GGSGGGGSGGGGSGGGGSDIQMTQ









SPSSLSASVGDRVTITCRASDNIG









SWLAWYQQKPGKAPKLLIYAASSL









QSGVPSRFSGSGSGTDFTLTISSL









QPEDFATYYCQQGYSTPPTFGQGT









KVEIK









(SEQ ID NO: 121)


 


11.
YTFTTYY
GGFDPED
CARHAVA
RASESIS
AASTLQS
CQQSYSV
QVQLVQSGAEVKKPGASVKVSCKA



MH
GETIYA
GAVAGYY
NWLA
(SEQ ID
PFTF
SGYTFTTYYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
GMDVW
(SEQ ID
NO: 113)
(SEQ ID
GGFDPEDGETIYAQKFQGRVTMTR



NO: 122)
NO: 123)
(SEQ ID
NO: 125)

NO: 126)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 124)



ARHAVAGAVGAGYYYYGMDVWGQG









TMVTVSSGGGGSGGGGSGGGGSGG









GGSDIQMTQSPSSLSASVGDRVTI









TCRASESISNWLAWYQQKPGKAPK









LLIYAASTLQSGVPSRFSGSGSGT









DFTLTISSLQPEDFATYYCQQSYS









VPFTFGPGTKVDIK









(SEQ ID NO: 127)


 


12.
YTFTGYY
GWIGPNS
CARDLNH
RSSQSLL
SSSNRAP
CMQALHI
QVQLVQSGAEVKKPGASVKVSCKA



MH
GDTNYA
DWYFDLW
HSNGYNY
(SEQ ID
PLTF
SGYTFTGYYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
LD
NO: 132)
(SEQ ID
GWIGPNSGDTNYAQKFQGRVTMTR



NO: 128)
NO: 129)
NO: 130)
(SEQ ID

NO: 133)
DTSTSTVYMELSSLRSEDTAVYYC






NO: 131)


ARDLDHNWYFDLWGRGTLVTVSSG









GGGSGGGGSGGGGSGGGGSDIVMT









QSPLSLPVTPGEPASISCRSSQSL









LHSNGYNYLDWYLQKPGQSPQLLI









YSSSNRAPGVPDRFSGSGSGTDFT









LKISRVEAEDVGVYYCMQALHIPL









TFGGGTKVEIK









(SEQ ID NO: 134)


 


13.
FTFDDYA
SYIDASG
CAKDQAA
QASQDIS
KASTLES
CQQSYST
EVQLLESGGGLVQPGGSLRLSCAA



MH
TTIYYA
AGYWYFD
NYLN
(SEQ ID
PITF
SGFTFDDYAMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
LW
(SEQ ID
NO: 139)
(SEQ ID
SYIDASGTTIYYADSVKGRFTISR



NO: 135)
NO: 136)
(SEQ ID
NO: 138)

NO: 140)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 137)



AKDQAAAGYWYFDLWGRGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCQASQ









DISNYLNWYQQKPGKAPKLLIYKA









STLESGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQSYSTPITFG









QGTRLEIK









(SEQ ID NO: 141)


 


14.
YTFTDYH
GGIVPRS
CAKDESS
RSSQSLL
SAYNRAS
CMQALQT
QVQLVQSGAEVKKPGSSVKVSCKA



IH
GSTTYA
GWYYFDY
HSNGYNY
(SEQ ID
PLTF
SGYTFTDYHIHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
W
LD
NO: 145)
(SEQ ID
GGIVPRSGSTTYAQKFQGRVTITA



NO: 142)
NO: 143)
(SEQ ID
(SEQ ID

NO: 146)
DESTSTAYMELSSLRSEDTAVYYC





NO: 144)
NO: 131)


AKDESSGWYYFDYWGQGTLVTVSS









GGGGSGGGGSGGGGSGGGGSDIVM









TQSPLSLPVTPGEPASISCRSSQS









LLHSNGYNYLDWYLQKPGQSPQLL









IYSAYNRASGVPDRFSGSGSGTDF









TLKISRVEAEDVGVYYCMQALQTP









LTFGQGTKVEIK









(SEQ ID NO: 147)


 


15.
YTFTNYY
GGIIPIV
CAKGRYT
QANQDIS
RASKLEA
CQQSSEI
QVQLVQSGAEVKKPGSSVKVSCKA



MH
DRVKYA
VNYYYGM
NYLN
(SEQ ID
PYSF
SGYTFTNYYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
DVW
(SEQ ID
NO: 152)
(SEQ ID
GGIIPIVDRVKYAQKFQGRVTITA



NO: 148)
NO: 149)
(SEQ ID
NO: 151)

NO: 153)
DESTSTAYMELSSLRSEDTAVYYC





NO: 150)



AKGRYTVNYYYGMDVWGQGTTVTV









SSGGGGSGGGGSGGGGSGGGGSDI









QMTQSPSSLSASVGDRVTITCQAN









QDISNYLNWYQQKPGKAPKLLIYR









ASKLEAGVPSRFSGSGSGTDFTLT









ISSLQPEDFATYYCQQSSEIPYSF









GQGTKLEIK









(SEQ ID NO: 154)


 


16.
FTFEDYA
SYLNSDG
CAKDYCT
RASQSIS
DASNLET
CQQSYTI
EVQLLESGGGLVQPGGSLRLSCAA



MH
GSTSYA
NGVCAFD
TYLN
(SEQ ID
PITF
SGFTFEDYAMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
YW
(SEQ ID
NO: 159)
(SEQ ID
SYLNSDGGSTSYADSVKGRFTISR



NO: 155)
NO: 156)
(SEQ ID
NO: 158)

NO: 160)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 157)



AKDYCTNGVCAFDYWGQGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









SISTYLNWYQQKPGKAPKLLIYDA









SNLETGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQSYTIPITFG









QGTRLEIK









(SEQ ID NO: 161)


 


17.
FTFSDSA
SAISGSG
CVSDIAV
RASQSIS
AASRLEG
CQQANSF
EVQLLESGGGLVQPGGSLRLSCAA



MH
STIYYA
AGHWYFD
TFLN
(SEQ ID
PLTF
SGFTFSDSAMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
LW
(SEQ ID
NO: 166)
(SEQ ID
SAISGSGSTIYYADSVKGRFTISR



NO: 162)
NO: 163)
(SEQ ID
NO: 165)

NO: 167)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 164)



VSDIAVAGHWYFDLWGRGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









SISTFLNWYQQKPGKAPKLLIYAA









SRLEGGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQANSFPLTFG









PGTKVDIK









(SEQ ID NO: 168)


 


18.
FTFSSYW
SYISGDS
CARANSS
RASQSIS
AASSLQS
CQQSYST
EVQLVESGGGLVKPGGSLRLSCAA



MS
GYTNYA
GWYDWYF
SYLN
(SEQ ID
PLTF
SGFTFSSYWMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
DLW
(SEQ ID
NO: 65)
(SEQ ID
SYISGDSGYTNYAAPVKGRFTISR



NO: 169)
NO: 170)
(SEQ ID
NO: 172)

NO: 173)
DDSKNTLYLQMNSLKTEDTAVYYC





NO: 171)



ARANSSGWYDWYFDLWGRGTLVTV









SSGGGGSGGGGSGGGGSGGGGSDI









QMTQSPSSLSASVGDRVTITCRAS









QSISSYLNWYQQKPGKAPKLLIYA









ASSLQSGVPSRFSGSGSGTDFTLT









ISSLQPEDFATYYCQQSYSTPLTF









GGGTKVEIK









(SEQ ID NO: 174)


 


19.
FTFDDYA
SGISWNS
CAKDIVA
QASQDIS
DASNLET
CQQSYST
EVQLLESGGGLVQPGGSLRLSCAA



MH
GSIGYA
AGHYYYG
NYLN
(SEQ ID
PLTF
SGFTFDDYAMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
MDVW
(SEQ ID
NO: 159)
(SEQ ID
SGISWNSGSIGYADSVKGRFTISR



NO: 135)
NO: 175)
(SEQ ID
NO: 138)

NO: 173)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 176)



AKDIVAAGHYYYGMDVWGQGTTVT









VSSGGGGSGGGGSGGGGSGGGGSD









IQMTQSPSSLSASVGDRVTITCQA









SQDISNYLNWYQQKPGKAPKLLIY









DASNLETGVPSRFSGSGSGTDFTL









TISSLQPEDFATYYCQQSYSTPLT









FGGGTKVEIK









(SEQ ID NO: 177)


 


20.
FTFDDYA
SYIDTSS
CARDEAA
QAGQDIS
DASNLET
CQQTYST
EVQLLESGGGLVQPGGSLRLSCAA



MH
SHLYYA
AGYYGMD
NYLN
(SEQ ID
PITF
SGFTFDDYAMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
VW
(SEQ ID
NO: 159)
(SEQ ID
SYIDTSSSHLYYADSVKGRFTISR



NO: 135)
NO: 178)
(SEQ ID
NO: 180)

NO: 181)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 179)



ARDEAAAGYYGMDVWGQGTTVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCQAGQ









DISNYLNWYQQKPGKAPKLLIYDA









SNLETGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQTYSTPITFG









QGTKLEIK









(SEQ ID NO: 182)


 


21.
FTFSNAW
STIVGNG
CARDNPL
RASQDIS
AASSLQS
CQQSYSI
EVQLLESGGGLVQPGGSLRLSCAA



MS
GATYYA
RWQGMDV
NYLN
(SEQ ID
PPTF
SGFTFSNAWMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
W
(SEQ ID
NO: 65)
(SEQ ID
STIVGNGGATYYADSVKGRFTISR



NO: 183)
NO: 184)
(SEQ ID
NO: 186)

NO: 187)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 185)



ARDNPLRWQGMDVWGQGTLVTVSS









GGGGSGGGGSGGGGSGGGGSDIQM









TQSPSSLSASVGDRVTITCRASQD









ISNYLNWYQQKPGKAPKLLIYAAS









SLQSGVPSRFSGSGSGTDFTLTIS









SLQPEDFATYYCQQSYSIPPTFGP









GTKVDIK









(SEQ ID NO: 188)


 


22.
FTFSSYQ
SYISSSS
CARANSS
RASQSIS
AASSLQS
CQQSYST
EVQLLESGGGLVQPGGSLRLSCAA



MS
TYTNYA
SWYDWYF
SYLN
(SEQ ID
PLTF
SGFTFSSYQMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
DLW
(SEQ ID
NO: 65)
(SEQ ID
SYISSSSTYTNYADSVKGRFTISR



NO: 189)
NO: 190)
(SEQ ID
NO: 172)

NO: 173)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 191)



ARANSSSWYDWYFDLWGQGTLVTV









SSGGGGSGGGGSGGGGSGGGGSDI









QMTQSPSSLSASVGDRVTITCRAS









QSISSYLNWYQQKPGKAPKLLIYA









ASSLQSGVPSRFSGSGSGTDFTLT









ISSLQPEDFATYYCQQSYSTPLTF









GGGTKVEIK









(SEQ ID NO: 192)


 


23.
FTFSSYA
SGISGSG
CATSQAP
RASQSIS
AASNLQR
CQQSYSI
EVQLLESGGGLVQPGGSLRLSCAA



MH
GSAYYA
VDYYYYG
SWLA
(SEQ ID
PITF
SGFTFSSYAMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
MDVW
(SEQ ID
NO: 197)
(SEQ ID
SGISGSGGSAYYADSVKGRFTISR



NO: 193)
NO: 194)
(SEQ ID
NO: 196)

NO: 198)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 195)



ATSQAPVDYYYYGMDVWGQGTTVT









VSSGGGGSGGGGSGGGGSGGGGSD









IQMTQSPSSLSASVGDRVTITCRA









SQSISSWLAWYQQKPGKAPKLLIY









AASNLQRGVPSRFSGSGSGTDFTL









TISSLQPEDFATYYCQQSYSIPIT









FGQGTKVEIK









(SEQ ID NO: 199)


 


24.
FTFSSYW
SYISGSS
CARVGSS
RASQSIS
AASSLQS
CQQSYST
EVQLVESGGGLVKPGGSLRLSCAA



MS
SYTNYA
GWYDWYF
SYLN
(SEQ ID
PLTF
SGFTFSSYWMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
DLW
(SEQ ID
NO: 65)
(SEQ ID
SYISGSSSYTNYAAPVKGRFTISR



NO: 169)
NO: 200)
(SEQ ID
NO: 172)

NO: 173)
DDSKNTLYLQMNSLKTEDTAVYYC





NO: 201)



ARVGSSGWYDWYFDLWGRGTLVTV









SSGGGGSGGGGSGGGGSGGGGSDI









QMTQSPSSLSASVGDRVTITCRAS









QSISSYLNWYQQKPGKAPKLLIYA









ASSLQSGVPSRFSGSGSGTDFTLT









ISSLQPEDFATYYCQQSYSTPLTF









GQGTKVEIK









(SEQ ID NO: 202)


 


25.
YTLTTWY
GWINPNR
CAKGDLW
RASDNIG
AASSLQS
CQQGYST
QVQLVQSGAEVKKPGASVKVSCKA



MY
GATNYA
GAMDVW
SWLA
(SEQ ID
PPTF
SGYTLTTWYMYWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 65)
(SEQ ID
GWINPNRGATNYAQKFQGRVTMTR



NO: 116)
NO: 117)
NO: 118)
NO: 119)

NO: 120)
DTSTSTVYMELSSLRSEDTAVYYC









AKGDLWGAMDVWGQGTLVTVSSGG









GGSGGGGSGGGGSGGGGSDIQMTQ









SPSSLSASVGDRVTITCRASDNIG









SWLAWYQQKPGKAPKLLIYAASSL









QSGVPSRFSGSGSGTDFTLTISSL









QPEDFATYYCQQGYSTPPTFGQGT









KVEIK









(SEQ ID NO: 121)


 


26.
YTLTTWY
GWINPNR
CAKGDLW
RASDNIG
AASSLQS
CQQGYST
QVQLVQSGAEVKKPGASVKVSCKA



MY
GATNYA
GAMDVW
SWLA
(SEQ ID
PPTF
SGYTLTTWYMYWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 65)
(SEQ ID
GWINPNRGATNYAQKFQGRVTMTR



NO: 116)
NO: 117)
NO: 118)
NO: 119)

NO: 120)
DTSTSTVYMELSSLRSEDTAVYYC









AKGDLWGAMDVWGQGTTVTVSSGG









GGSGGGGSGGGGSGGGGSDIQMTQ









SPSSLSASVGDRVTITCRASDNIG









SWLAWYQQKPGKAPKLLIYAASSL









QSGVPSRFSGSGSGTDFTLTISSL









QPEDFATYYCQQGYSTPPTFGQGT









KVEIK









(SEQ ID NO: 203)


 


27.
YTFTGYY
GWMNPNS
CARDPGF
RASQSIS
AASSLQS
CQQSYTA
QVQLVQSGAEVKKPGASVKVSCKA



IH
GNTGYA
LGYCSGG
SYLH
(SEQ ID
PYTF
SGYTFTGYYIHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
SCYDGW
(SEQ ID
NO: 65)
(SEQ ID
GWMNPNSGNTGYAQKFQGRVTMTR



NO: 204)
NO: 205)
(SEQ ID
NO: 207)

NO: 208)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 206)



ARDPGFLGYCSGGSCYDGWFDPWG









QGTLVTVSSGGGGSGGGGSGGGGS









GGGGSDIQMTQSPSSLSASVGDRV









TITCRASQSISSYLHWYQQKPGKA









PKLLIYAASSLQSGVPSRFSGSGS









GTDFTLTISSLQPEDFATYYCQQS









YTAPYTFGQGTKLEIK









(SEQ ID NO: 209)


 


28.
YTFTDYF
GWMNPTS
CAREGEG
RASQGIN
DASNLET
CQQSYST
QVQLVQSGAEVKKPGASVKVSCKA



LH
GNTGYA
SGFDYW
SWLA
(SEQ ID
PLTF
SGYTFTDYFLHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 159)
(SEQ ID
GWMNPTSGNTGYAQKFQGRVTMTR



NO: 210)
NO: 211)
NO: 212)
NO: 213)

NO: 173)
DTSTSTVYMELSSLRSEDTAVYYC









AREGEGSGFDYWGQGTLVTVSSGG









GGSGGGGSGGGGSGGGGSDIQMTQ









SPSSLSASVGDRVTITCRASQGIN









SWLAWYQQKPGKAPKLLIYDASNL









ETGVPSRFSGSGSGTDFTLTISSL









QPEDFATYYCQQSYSTPLTFGGGT









KVEIK









(SEQ ID NO: 214)


 


29.
YTFTSYY
AWMNPNS
CARDYDF
RASQGIS
AASSLQS
CQQSYST
QVQLVQSGAEVKKPGASVKVSCKA



NH
GNTGYA
WSGSLGY
NYLA
(SEQ ID
PWTF
SGYTFTSYYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
W
(SEQ ID
NO: 65)
(SEQ ID
AWMNPNSGNTGYAQKFQGRVTMTR



NO: 215)
NO: 216)
(SEQ ID
NO: 218)

NO: 114)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 217)



ARDYDFWSGSLGYWGQGTLVTVSS









GGGGSGGGGSGGGGSGGGGSDIQM









TQSPSSLSASVGDRVTITCRASQG









ISNYLAWYQQKPGKAPKLLIYAAS









SLQSGVPSRFSGSGSGTDFTLTIS









SLQPEDFATYYCQQSYSTPWTFGQ









GTKVEIK









(SEQ ID NO: 219)


 


30.
YTLTTWY
GWINPNR
CAKGDLW
RASDNIG
AASSLQS
CQQGYST
QVQLVQSGAEVKKPGASVKVSCKA



MY
GATNYA
GAMDVW
SWLA
(SEQ ID
PPTF
SGYTLTTWYMYWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 65)
(SEQ ID
GWINPNRGATNYAQKFQGRVTMTR



NO: 116)
NO: 117)
NO: 118)
NO: 119)

NO: 120)
DTSTSTVYMELSSLRSEDTAVYYC









AKGDLWGAMDVWGQGTLVTVSSGG









GGSGGGGSGGGGSGGGGSDIQMTQ









SPSSLSASVGDRVTITCRASDNIG









SWLAWYQQKPGKAPKLLIYAASSL









QSGVPSRFSGSGSGTDFTLTISSL









QPEDFATYYCQQGYSTPPTFGQGT









KVEIK









(SEQ ID NO: 121)


 


31.
YTFTSYY
GIINPSG
CARDTGY
RASQSIG
DASNLQS
CQQSYSI
QVQLVQSGAEVKKPGASVKVSCKA



NH
GSTSYA
SYGRYYY
RWLA
(SEQ ID
PITF
SGYTFTSYYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
YGMDVW
(SEQ ID
NO: 222)
(SEQ ID
GIINPSGGSTSYAQKFQGRVTMTR



NO: 215)
NO: 69)
(SEQ ID
NO: 221)

NO: 198)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 220)



ARDTGYSYGRYYYYGMDVWGQGTL









VTVSSGGGGSGGGGSGGGGSGGGG









SDIQMTQSPSSLSASVGDRVTITC









RASQSIGRWLAWYQQKPGKAPKLL









IYDASNLQSGVPSRFSGSGSGTDF









TLTISSLQPEDFATYYCQQSYSIP









ITFGQGTKVEIK









(SEQ ID NO: 223)


 


32.
YTLTDYY
GIINPSG
CAREEYS
RASQGIS
AASSLQS
CQQSYST
QVQLVQSGAEVKKPGASVKVSCKA



MH
GSTSYA
SSSGYFD
SWLA
(SEQ ID
PLTF
SGYTLTDYYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
YW
(SEQ ID
NO: 65)
(SEQ ID
GIINPSGGSTSYAQKFQGRVTMTR



NO: 224)
NO: 69)
(SEQ ID
NO: 226)

NO: 173)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 225)



AREEYSSSSGYFDYWGQGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









GISSWLAWYQQKPGKAPKLLIYAA









SSLQSGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQSYSTPLTFG









QGTKVEIK









(SEQ ID NO: 227)


 


33.
YTFTSYG
GWMHPKS
CARDTPY
RASQSIS
AASSLQS
CQQSYSV
QVQLVQSGAEVKKPGASVKVSCKA



IS
GDTGLT
YYYGMDV
SWLA
(SEQ ID
PITF
SGYTFTSYGISWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
W
(SEQ ID
NO: 65)
(SEQ ID
GWMHPKSGDTGLTQKFQGRVTMTR



NO: 228)
NO: 229)
(SEQ ID
NO: 196)

NO: 231)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 230)



ARDTPYYYYGMDVWGQGTTVTVSS









GGGGSGGGGSGGGGSGGGGSDIQM









TQSPSSLSASVGDRVTITCRASQS









ISSWLAWYQQKPGKAPKLLIYAAS









SLQSGVPSRFSGSGSGTDFTLTIS









SLQPEDFATYYCQQSYSVPITFGQ









GTKVEIK









(SEQ ID NO: 232)


 


34.
FTFGDYA
SYISGDI
CARDVAA
RASQSIS
AASSLQS
CQQSYST
EVQLVESGGGLVKPGGSLRLSCAA



MS
GYTNYA
TGNWYFD
SYLN
(SEQ ID
PLTF
SGFTFGDYAMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
LW
(SEQ ID
NO: 65)
(SEQ ID
SYISGDIGYTNYAAPVKGRFTISR



NO: 233)
NO: 234)
(SEQ ID
NO: 172)

NO: 173)
DDSKNTLYLQMNSLKTEDTAVYYC





NO: 235)



ARDVAATGNWYFDLWGRGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









SISSYLNWYQQKPGKAPKLLIYAA









SSLQSGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQSYSTPLTFG









GGTKVEIK









(SEQ ID NO: 236)


 


35.
FSFSSYT
SFITSSS
CARDRRG
RASQSVR
GASTRAT
CQQYGSS
EVQLLESGGGLVQPGGSLRLSCAA



MN
RTIYYA
DYGDSWY
NYLA

PLTF
SGFSFSSYTMNWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
FDLW
(SEQ ID
(SEQ ID
(SEQ ID
SFITSSSRTIYYADSVKGRFTISR



NO: 237)
NO: 238)
(SEQ ID
NO: 240)
NO: 241)
NO: 242)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 239)



ARDRRGDYGDSWYFDLWGRGTLVT









VSSGGGGSGGGGSGGGGSGGGGSE









IVMTQSPATLSVSPGERATLSCRA









SQSVRNYLAWYQQKPGQAPRLLIY









GASTRATGIPARFSGSGSGTEFTL









TISSLQSEDFAVYYCQQYGSSPLT









FGGGTKVEIK









(SEQ ID NO: 243)


 


36.
YTFTGHY
GIINPSG
CARDTGY
RASQSIG
DASNLQS
CQQSYSI
QVQLVQSGAEVKKPGASVKVSCKA



MH
GSTSYA
SYGRYYY
RWLA
(SEQ ID
PITF
SGYTFTGHYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
YGMDVW
(SEQ ID
NO: 222)
(SEQ ID
GIINPSGGSTSYAQKFQGRVTMTR



NO: 244)
NO: 69)
(SEQ ID
NO: 221)

NO: 198)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 220)



ARDTGYSYGRYYYYGMDVWGQGTT









VTVSSGGGGSGGGGSGGGGSGGGG









SDIQMTQSPSSLSASVGDRVTITC









RASQSIGRWLAWYQQKPGKAPKLL









IYDASNLQSGVPSRFSGSGSGTDF









TLTISSLQPEDFATYYCQQSYSIP









ITFGGGTKVEIK









(SEQ ID NO: 245)


 


37.
YTFSKHF
GWMNPNS
CARGEGG
RASQSIS
AASTLQS
CQQSYST
QVQLVQSGAEVKKPGASVKVSCKA



VH
GNSGYA
YYYYGMD
SWLA
(SEQ ID
PWTF
SGYTFSKHFVHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
VW
(SEQ ID
NO: 113)
(SEQ ID
GWMNPNSGNSGYAQKFQGRVTMTR



NO: 246)
NO: 247)
(SEQ ID
NO: 196)

NO: 114)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 248)



ARGEGGYYYYGMDVWGQGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









SISSWLAWYQQKPGKAPKLLIYAA









STLQSGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQSYSTPWTFG









QGTKVEIK









(SEQ ID NO: 249)


 


38.
FTFGSYS
SAIGTGG
CAKGTPY
RASQPLS
AASSLQS
CQQAISF
EVQLLESGGGLVQPGGSLRLSCAA



MS
GTYYA
YYYYGMD
NWLA
(SEQ ID
PLTF
SGFTFGSYSMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
VW
(SEQ ID
NO: 65)
(SEQ ID
SAIGTGGGTYYADSVKGRFTISRD



NO: 250)
NO: 251)
(SEQ ID
NO: 253)

NO: 254)
NSKNTLYLQMNSLRAEDTAVYYCA





NO: 252)



KGTPYYYYYGMDVWGQGTMVTVSS









GGGGSGGGGSGGGGSGGGGSDIQM









TQSPSSLSASVGDRVTITCRASQP









LSNWLAWYQQKPGKAPKLLIYAAS









SLQSGVPSRFSGSGSGTDFTLTIS









SLQPEDFATYYCQQAISFPLTFGG









GTKVEIK









(SEQ ID NO: 255)


 


39.
YTFTSYY
GWMNPNS
CARDLGY
QSSEDIS
AASSLQI
CQQTYST
QVQLVQSGAEVKKPGASVKVSCKA



MH
GNTGYA
YDSSGYF
SSLN
(SEQ ID
PYTF
SGYTFTSYYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
GAFDIW
(SEQ ID
NO: 258)
(SEQ ID
GWMNPNSGNTGYAQKFQGRVTMTR



NO: 215)
NO: 205)
(SEQ ID
NO: 257)

NO: 259)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 256)



ARDLGYYDSSGYFGAFDIWGQGTT









VTVSSGGGGSGGGGSGGGGSGGGG









SDIQMTQSPSSLSASVGDRVTITC









QSSEDISSSLNWYQQKPGKAPKLL









IYAASSLQIGVPSRFSGSGSGTDF









TLTISSLQPEDFATYYCQQTYSTP









YTFGQGTKVEIK









(SEQ ID NO: 260)


 


40.
YTFTSYG
GIINPRG
CARGTRS
RASQGIG
AASNLET
CQQIHSY
QVQLVQSGAEVKKPGASVKVSCKA



IS
GSTIFA
SGWYGWF
NWLA
(SEQ ID
PLTF
SGYTFTSYGISWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
DPW
(SEQ ID
NO: 264)
(SEQ ID
GIINPRGGSTIFAQKFQGRVTMTR



NO: 228)
NO: 261)
(SEQ ID
NO: 263)

NO: 265)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 262)



ARGTRSSGWYGWFDPWGQGTLVTV









SSGGGGSGGGGSGGGGSGGGGSDI









QMTQSPSSLSASVGDRVTITCRAS









QGIGNWLAWYQQKPGKAPKLLIYA









ASNLETGVPSRFSGSGSGTDFTLT









ISSLQPEDFATYYCQQIHSYPLTF









GGGTKVEIK









(SEQ ID NO: 266)


 


41.
FTFDDYG
SYISSSS
CAREIAA
RASQSIS
AASSLQS
CQQSYST
EVQLLESGGGLVQPGGSLRLSCAA



MS
SYIYYA
AGFYGMD
SYLN
(SEQ ID
PLTF
SGFTFDDYGMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
VW
(SEQ ID
NO: 65)
(SEQ ID
SYISSSSSYIYYADSVKGRFTISR



NO: 267)
NO: 268)
(SEQ ID
NO: 172)

NO: 173)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 269)



AREIAAAGFYGMDVWGQGTTVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









SISSYLNWYQQKPGKAPKLLIYAA









SSLQSGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQSYSTPLTFG









GGTKVEIK









(SEQ ID NO: 270)


 


42.
GTLSRYG
GGIIPIF
CARDRVY
RASQSVS
GASTRAT
CQQYGSS
QVQLVQSGAEVKKPGSSVKVSCKA



VS
GTTNYA
YDSSGYP
SSYLA
(SEQ ID
PITF
SGGTLSRYGVSWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
TWYFDLW
(SEQ ID
NO: 241)
(SEQ ID
GGIIPIFGTTNYAQKFQGRVTITA



NO: 271)
NO: 272)
(SEQ ID
NO: 274)

NO: 275)
DESTSTAYMELSSLRSEDTAVYYC





NO: 273)



ARDRVYYDSSGYPTWYFDLWGRGT









LVTVSSGGGGSGGGGSGGGGSGGG









GSEIVMTQSPATLSVSPGERATLS









CRASQSVSSSYLAWYQQKPGQAPR









LLIYGASTRATGIPARFSGSGSGT









EFTLTISSLQSEDFAVYYCQQYGS









SPITFGQGTKVEIK









(SEQ ID NO: 276)


 


43.
FTFDDFA
SGISGNG
CARDASY
QASQDIR
KASTLES
CQQANSF
EVQLLESGGGLVQPGGSLRLSCAA



MH
DSRYYA
GGNYGMD
NYLN
(SEQ ID
PLTF
SGFTFDDFAMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
VW
(SEQ ID
NO: 139)
(SEQ ID
SGISGNGDSRYYADSVKGRFTISR



NO: 277)
NO: 278)
(SEQ ID
NO: 280)

NO: 167)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 279)



ARDASYGGNYGMDVWGQGTTVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCQASQ









DIRNYLNWYQQKPGKAPKLLIYKA









STLESGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQANSFPLTFG









PGTKVDIK









(SEQ ID NO: 281)


 


44.
FTFSSYW
SAIGTGG
CAREWLV
RASQSIS
GASNLQS
CQQSYST
EVQLVESGGGLVKPGGSLRLSCAA



MS
GTYYA
PYYGMDV
RWLA
(SEQ ID
PWTF
SGFTFSSYWMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
W
(SEQ ID
NO: 284)
(SEQ ID
SAIGTGGGTYYAAPVKGRFTISRD



NO: 169)
NO: 251)
(SEQ ID
NO: 283)

NO: 114)
DSKNTLYLQMNSLKTEDTAVYYCA





NO: 282)



REWLVPYYGMDVWGQGTTVTVSSG









GGGSGGGGSGGGGSGGGGSDIQMT









QSPSSLSASVGDRVTITCRASQSI









SRWLAWYQQKPGKAPKLLIYGASN









LQSGVPSRFSGSGSGTDFTLTISS









LQPEDFATYYCQQSYSTPWTFGQG









TKVEIK









(SEQ ID NO: 285)


 


45.
FSVSSNY
AGISYDG
CARSRGI
KSSQSVL
WASTRQS
CHQYYGH
EVQLLESGGGLVQPGGSLRLSCAA



MS
SSKPYA
AARPLQH
YSSNNKN
(SEQ ID
PPTF
SGFSVSSNYMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
W
YLA
NO: 290)
(SEQ ID
AGISYDGSSKPYADSVKGRFTISR



NO: 286)
NO: 287)
(SEQ ID
(SEQ ID

NO: 291)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 288)
NO: 289)


ARSRGIAARPLQHWGQGTLVTVSS









GGGGSGGGGSGGGGSGGGGSDIVM









TQSPDSLAVSLGERATINCKSSQS









VLYSSNNKNYLAWYQQKPGQPPKL









LIYWASTRQSGVPDRFSGSGSGTD









FTLTISSLQAEDVAVYYCHQYYGH









PPTFGGGTKVEIK









(SEQ ID NO: 292)


 


46.
FSVSSNY
AGISYDG
CARSRGI
KSSQSVL
QASTRQS
CHQYYGH
EVQLLESGGGLVQPGGSLRLSCAA



MS
SSKPYA
AARPLQH
YSSNNKN
(SEQ ID
PPTF
SGFSVSSNYMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
W
YLA
NO: 293)
(SEQ ID
AGISYDGSSKPYADSVKGRFTISR



NO: 286)
NO: 287)
(SEQ ID
(SEQ ID

NO: 291)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 288)
NO: 289)


ARSRGIAARPLQHWGQGTLVTVSS









GGGGSGGGGSGGGGSGGGGSDIVM









TQSPDSLAVSLGERATINCKSSQS









VLYSSNNKNYLAWYQQKPGQPPKL









LIYQASTRQSGVPDRFSGSGSGTD









FTLTISSLQAEDVAVYYCHQYYGH









PPTFGGGTKVEIK









(SEQ ID NO: 294)


 


47.
FSFSDYG
SAISGSG
CARDGGW
RASQGIS
DASNLET
CQQSYST
EVQLLESGGGLVQPGGSLRLSCAA



MH
GSTYYA
QPAQILD
NNLN
(SEQ ID
PLTF
SGFSFSDYGMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
YW
(SEQ ID
NO: 159)
(SEQ ID
SAISGSGGSTYYADSVKGRFTISR



NO: 295)
NO: 103)
(SEQ ID
NO: 105)

NO: 173)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 296)



ARDGGWQPAAILDYWGQGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









GISNNLNWYQQKPGKAPKLLIYDA









SNLETGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQSYSTPLTFG









GGTKVEIK









(SEQ ID NO: 297)


 


48.
FTFSDHG
SVIYGGE
CARDPAV
RASQGIS
DASNLET
CQQSYST
EVQLLESGGGLVQPGGSLRLSCAA



MH
STYYA
AGGGIFD
NYLA
(SEQ ID
CYTF
SGFTFSDHGMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
YW
(SEQ ID
NO: 159)
(SEQ ID
SVIYGGESTYYADSVKGRFTISRD



NO: 298)
NO: 299)
(SEQ ID
NO: 218)

NO: 301)
NSKNTLYLQMNSLRAEDTAVYYCA





NO: 300)



RDPAVAGGGIFDYWGQGTLVTVSS









GGGGSGGGGSGGGGSGGGGSDIQM









TQSPSSLSASVGDRVTITCRASQG









ISNYLAWYQQKPGKAPKLLIYDAS









NLETGVPSRFSGSGSGTDFTLTIS









SLQPEDFATYYCQQSYSTCYTFGQ









GTKLEIK









(SEQ ID NO: 302)


 


49.
DTFTGYY
GWINPNS
CARSGLW
RASQTIS
DASTLQS
CQQYSSY
QVQLVQSGAEVKKPGASVKVSCKA



IH
GGTNYA
LGSYYGM
IWLA
(SEQ ID
PLTF
SGDTFTGYYIHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
DVW
(SEQ ID
NO: 307)
(SEQ ID
GWINPNSGGTNYAQKFQGRVTMTR



NO: 303)
NO: 304)
(SEQ ID
NO: 306)

NO: 308)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 305)



ARSGLWLGSYYGMDVWGQGTLVTV









SSGGGGSGGGGSGGGGSGGGGSDI









QMTQSPSSLSASVGDRVTITCRAS









QTISIWLAWYQQKPGKAPKLLIYD









ASTLQSGVPSRFSGSGSGTDFTLT









ISSLQPEDFATYYCQQYSSYPLTF









GQGTKVEIK









(SEQ ID NO: 309)


 


50.
YTFTSYD
GWINPNS
CARSPYY
RASHFIS
AASTLQS
CQQSYSG
QVQLVQSGAEVKKPGASVKVSCKA



IN
GTTGYA
YYGMDVW
RWVA
(SEQ ID
ISF
SGYTFTSYDINWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 113)
(SEQ ID
GWINPNSGTTGYAQKFQGRVTMTR



NO: 310)
NO: 311)
NO: 312)
NO: 313)

NO: 314)
DTSTSTVYMELSSLRSEDTAVYYC









ARSPYYYYGMDVWGQGTTVTVSSG









GGGSGGGGSGGGGSGGGGSDIQMT









QSPSSLSASVGDRVTITCRASHFI









SRWVAWYQQKPGKAPKLLIYAAST









LQSGVPSRFSGSGSGTDFTLTISS









LQPEDFATYYCQQSYSGISFGPGT









KVDIK









(SEQ ID NO: 315)


 


51.
FTFNNYG
SRINSDG
CARGAYY
RASQSVS
ATSSRAS
CQQYYSG
EVQLLESGGGLVQPGGSLRLSCAA



MN
SSTSYA
YYYMDVW
GSYLA
(SEQ ID
LTF
SGFTFNNYGMNWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 320)
(SEQ ID
SRINSDGSSTSYADSVKGRFTISR



NO: 316)
NO: 317)
NO: 318)
NO: 319)

NO: 321)
DNSKNTLYLQMNSLRAEDTAVYYC









ARGAYYYYYMDVWGQGTLVTVSSG









GGGSGGGGSGGGGSGGGGSEIVMT









QSPATLSVSPGERATLSCRASQSV









SGSYLAWYQQKPGQAPRLLIYATS









SRASGIPARFSGSGSGTEFTLTIS









SLQSEDFAVYYCQQYYSGLTFGQG









TKVEIK









(SEQ ID NO: 322)


 


52.
FTFSNSD
AHIWNDG
CARDRTD
RASQDIR
DASSLET
CQQATSL
EVQLLESGGGLVQPGGSLRLSCAA



MN
SQKYYA
PGYSSAM
NYLG
(SEQ ID
PLTF
SGFTFSNSDMNWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
DVW
(SEQ ID
NO: 327)
(SEQ ID
AHIWNDGSQKYYADSVKGRFTISR



NO: 323)
NO: 324)
(SEQ ID
NO: 326)

NO: 328)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 325)



ARDRTDPGYSSAMDVWGQGTTVTV









SSGGGGSGGGGSGGGGSGGGGSDI









QMTQSPSSLSASVGDRVTITCRAS









QDIRNYLGWYQQKPGKAPKLLIYD









ASSLETGVPSRFSGSGSGTDFTLT









ISSLQPEDFATYYCQQATSLPLTF









GGGTKVEIK









(SEQ ID NO: 329)


 


53.
YTFTSYD
GWMNPNS
CAKDSDY
RASQDIT
QASSLES
CQQSYTI
QVQLVQSGAEVKKPGASVKVSCKA



IN
GNTGYA
SNLLWDY
NDLG
(SEQ ID
PLTF
SGYTFTSYDINWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
W
(SEQ ID
NO: 332)
(SEQ ID
GWMNPNSGNTGYAQKFQGRVTMTR



NO: 310)
NO: 205)
(SEQ ID
NO: 331)

NO: 333)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 330)



AKDSDYSNLLWDYWGQGTLVTVSS









GGGGSGGGGSGGGGSGGGGSDIQM









TQSPSSLSASVGDRVTITCRASQD









ITNDLGWYQQKPGKAPKLLIYQAS









SLESGVPSRFSGSGSGTDFTLTIS









SLQPEDFATYYCQQSYTIPLTFGQ









GTKVEIK









(SEQ ID NO: 334)


 


54.
FTFGDYA
AVVSYDG
CAKDICS
RASQNIN
DASNLET
CQQANSF
EVQLLESGGGLVQPGGSLRLSCAA



MS
TNKYYA
STSCYFD
NYVN
(SEQ ID
PPTF
SGFTFGDYAMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
LW
(SEQ ID
NO: 159)
(SEQ ID
AVVSYDGTNKYYADSVKGRFTISR



NO: 233)
NO: 335)
(SEQ ID
NO: 337)

NO: 338)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 336)



AKDICSSTSCYFDLWGRGTLVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCRASQ









NINNYVNWYQQKPGKAPKLLIYDA









SNLETGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQANSFPPTFG









QGTRLEIK









(SEQ ID NO: 339)


 


55.
YTFTSYY
GIIDPSG
CAREEWS
RASQGIS
ATSSLQT
CQQTYSI
QVQLVQSGAEVKKPGASVKVSCKA



MH
GSTSYA
SGGVGYF
SYLA
(SEQ ID
PITF
SGYTFTSYYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
DYW
(SEQ ID
NO: 343)
(SEQ ID
GIIDPSGGSTSYAQKFQGRVTMTR



NO: 213)
NO: 340)
(SEQ ID
NO: 342)

NO: 344)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 341)



AREEWSSGGVGYFDYWGQGTLVTV









SSGGGGSGGGGSGGGGSGGGGSDI









QMTQSPSSLSASVGDRVTITCRAS









QGISSYLAWYQQKPGKAPKLLIYA









TSSLQTGVPSRFSGSGSGTDFTLT









ISSLQPEDFATYYCQQTYSIPITF









GQGTRLEIK









(SEQ ID NO: 345)


 


56.
FTFDDYA
SAISGGG
CARDASY
QASQDIR
KASSLES
CQQANSV
EVQLLESGGGLVQPGGSLRLSCAA



MH
EDTYYA
GGNYGMD
NYLN
(SEQ ID
PVTF
SGFTFDDYAMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
VW
(SEQ ID
NO: 347)
(SEQ ID
SAISGGGEDTYYADSVKGRFTISR



NO: 135)
NO: 346)
(SEQ ID
NO: 280)

NO: 348)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 279)



ARDASYGGNYGMDVWGQGTTVTVS









SGGGGSGGGGSGGGGSGGGGSDIQ









MTQSPSSLSASVGDRVTITCQASQ









DIRNYLNWYQQKPGKAPKLLIYKA









SSLESGVPSRFSGSGSGTDFTLTI









SSLQPEDFATYYCQQANSYPVTFG









GGTKVEIK









(SEQ ID NO: 349)


 


57.
YTFTSYY
GIINPSG
VARDSVA
RASQGIS
AASSLQG
CQQSYSL
QVQLVQSGAEVKKPGASVKVSCKA



MH
GSTSYA
GTGGRYY
NYFA
(SEQ ID
PYTF
SGYTFTSYYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
GMDVW
(SEQ ID
NO: 352)
(SEQ ID
GIINPSGGSTSYAQKFQGRVTMTR



NO: 215)
NO: 69)
(SEQ ID
NO: 351)

NO: 353)
DTSTSTVYMELSSLRSEDTAVYYC





NO: 350)



ARDSVAGTGGRYYGMDVWGQGTLV









TVSSGGGGSGGGGSGGGGSGGGGS









DIQMTQSPSSLSASVGDRVTITCR









ASQGISNYFAWYQQKPGKAPKLLI









YAASSLQGGVPSRFSGSGSGTDFT









LTISSLQPEDFATYYCQQSYSLPY









TFGQGTKLEIK









(SEQ ID NO: 354)


 


58.
YTFTGYY
GIINPSG
CTTADYY
RASQGIS
AASSLQS
CQQYYSN
QVQLVQSGAEVKKPGASVKVSCKA



MH
GNTKYA
YYMDVW
NYLA
(SEQ ID
ADF
SGYTFTGYYMHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 65)
(SEQ ID
GIINPSGGNTKYAQKFQGRVTMTR



NO: 128)
NO: 355)
NO: 356)
NO: 218)

NO: 357)
DTSTSTVYMELSSLRSEDTAVYYC









TTADYYYYMDVWGKGTTVTVSSGG









GGSGGGGSGGGGSGGGGSDIQMTQ









SPSSLSASVGDRVTITCRASQGIS









NYLAWYQQKPGKAPKLLIYAASSL









QSGVPSRFSGSGSGTDFTLTISSL









QPEDFATYYCQQYYSNADFGQGTK









VEIK









(SEQ ID NO: 358)


 


59.
FTFSDFW
SYISGDS
CARDRPY
RASQSVS
SSLQS
QQYKSYP
EVQLLESGGGLVQPGGSLRLSCAA



MH
GYTNYA
YYYMDVW
RSLA
(SEQ ID
VT
SGFTFSDFWMHWVRQAPGKGLEWI



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 362)
(SEQ ID
SYISGDSGYTNYADSVKGRFTISR



NO: 359)
NO: 170)
NO: 360)
NO: 361)

NO: 363)
DNSKNTLYLQMNSLRAEDTAVYYC









ARDRPYYYYMDVWGKGTTVTVSSG









GGGSGGGGSGGGGSGGGGSDIQMT









QSPSSLSASVGDRVTITCRASQSV









SRSLAWYQQKPGKAPKLLIYAASS









LQSGVPSRFSGSGSGTDFTLTISS









LQPEDFATYYCQQYKSYPVTFGQG









TKVEIK









(SEQ ID NO: 364)


 


60.
FTFDDYT
SDISGSG
CAKDVVV
QASQDIS
SYLQS
QQAHNYP
EVQLLESGGGLVQPGGSLRLSCAA



MH
GSTYYA
AGTPLHF
NYLN
(SEQ ID
IT
SGFTFDDYTMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
DYW
(SEQ ID
NO: 368)
(SEQ ID
SDISGSGGSTYYADSVKGRFTISR



NO: 365)
NO: 366)
(SEQ ID
NO: 138)

NO: 369)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 367)



AKDVVVAGTPLHFDYWGQGTLVTV









SSGGGGSGGGGSGGGGSGGGGSDI









QMTQSPSSLSASVGDRVTITCQAS









QDISNYLNWYQQKPGKAPKLLIYA









ASYLQ SGVPSRFSGSGSGTDFTLT









ISSLQPEDFATYYCQQAHNYPITF









GQGTRLEIK









(SEQ ID NO: 370)


 


61.
FTFSNAW
ASISSTS
VAREVVG
RASQSIS
SSLQS
QQANAFP
EVQLLESGGGLVQPGGSLRLSCAA



MS
AYIDYA
ATTFDYW
TWLA
(SEQ ID
PT
SEFTFSNAWMSWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 362)
(SEQ ID
ASISSTSAYIDYADSVKGRFTISR



NO: 183)
NO: 371)
NO: 372)
NO: 373)

NO: 374)
DNSKNTLYLQMNSLRAEDTAVYYC









AREVVGATTFDYWGQGTLVTVSSG









GGGSGGGGSGGGGSGGGGSDIQMT









QSPSSLSASVGDRVTITCRASQSI









STWLAWYQQKPGKAPKLLIYAASS









LQSGVPSRFSGSGSGTDFTLTISS









LQPEDFATYYCQQANAFPPTFGQG









TRLEIK









(SEQ ID NO: 375)


 


62.
GTFSSYA
GWMEPHT
CAKGGFS
KSSQSVL
STRES
QQYYSTP
QVQLVQSGAEVKKPGSSVKVSCKA



IS
GNTRYA
WFDPW
YSSNNKN
(SEQ ID
PT
SGGTFSSYAISWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
YLA
NO: 378)
(SEQ ID
GWMEPHTGNTRYAQKFQGRVTITA



NO: 77)
NO: 376)
NO: 377)
(SEQ ID

NO: 379)
DESTSTAYMELSSLRSEDTAVYYC






NO: 289)


AKGGFSWFDPWGQGTLVTVSSGGG









GSGGGGSGGGGSGGGGSDIVMTQS









PDSLAVSLGERATINCKSSQSVLY









SSNNKNYLAWYQQKPGQPPKLLIY









WASTRESGVPDRFSGSGSGTDFTL









TISSLQAEDVAVYYCQQYYSTPPT









FGQGTRLEIK









(SEQ ID NO: 380)


 


63.
FTFDDYA
ASITSSS
CARERVD
RASQGIS
STRAT
QQYYTYP
EVQLLESGGGLVKPGGSLRLSCAA



MH
AFIDYA
WNSYFDL
NSYLA
(SEQ ID
PT
SGFTFDDYAMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
W
(SEQ ID
NO: 384)
(SEQ ID
ASITSSSAFIDYAASVKGRFTISR



NO: 135)
NO: 381)
(SEQ ID
NO: 383)

NO: 385)
DDSKNTLYLQMNSLKTEDTAVYYC





NO: 382)



ARERVDWNSYFDLWGRGTLVTVSS









GGGGSGGGGSGGGGSGGGGSEIVM









TQSPATLSVSPGERATLSCRASQG









ISNSYLAWYQQKPGQAPRLLIYGA









STRATGIPARFSGSGSGTEFTLTI









SSLQSEDFAVYYCQQYYTYPPTFG









PGTKVDIK









(SEQ ID NO: 386)


 


64.
FAFSSHW
AGTSGSG
CARETYY
RASQGIS
ANLEG
QQSDIFP
EVQLLESGGGLVKPGGSLRLSCAA



MH
ESRDYA
YYYMDVW
NYLA
(SEQ ID
PT
SGFAFSSHWMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 390)
(SEQ ID
AGTSGSGESRDYADFVKGRFTISR



NO: 387)
NO: 388)
NO: 389)
NO: 218)

NO: 391)
DDSKNTLYLQMNSLKTEDTAVYYC









ARETYYYYYMDVWGKGTTVTVSSG









GGGSGGGGSGGGGSGGGGSDIQMT









QSPSSLSASVGDRVTITCRASQGI









SNYLAWYQQKPGKAPKLLIYDAAN









LEGGVPSRFSGSGSGTDFTLTISS









LQPEDFATYYCQQSDIFPPTFGQG









TKVEIK









(SEQ ID NO: 392)


 


65.
YTFTRHW
GWINVKT
CARESSG
RASQSIS
SSLQS
QQSNSFP
QVQLVQSGAEVKKPGASVKVSCKA



IH
GGAGYA
WYGTDVW
NYLA
(SEQ ID
LT
SGYTFTRHWIHWVRQAPGQGLEWM



(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 396)
(SEQ ID
GWINVKTGGAGYAQKFQGRVTMTR



NO: 393)
NO: 394)
NO: 395)
NO: 396)

NO: 397)
DTSTSTVYMELSSLRSEDTAVYYC









ARESSGWYGTDVWGQGTTVTVSSG









GGGSGGGGSGGGGSGGGGSDIQMT









QSPSSLSASVGDRATITCRASQSI









SNYLAWYQQKPGKAPKLLIYAASS









LQSGVPSRFSGSGSGTDFTLTISS









LQPEDFATYYCQQSNSFPLTFGGG









TKVEIK









(SEQ ID NO: 398)


 


66.
FTFSSYW
AAISYDG
CARENKQ
QASQDIS
NLRS
QQANSFP
EVQLLESGGGLVQPGGSLRLSCAA



MH
KYKDYE
WLASFDY
NFVN
(SEQ ID
VT
SGFTFSSYWMHWVRQAPGKGLEWV



(SEQ ID
(SEQ ID
W
(SEQ ID
NO: 402)
(SEQ ID
AAISYDGKYKDYEDSVKGRFTISR



NO: 83)
NO: 399)
(SEQ ID
NO:401 )

NO: 404)
DNSKNTLYLQMNSLRAEDTAVYYC





NO: 400)



ARENKQWLASFDYWGQGTLVTVSS









GGGGSGGGGSGGGGSGGGGSDIQM









TQSPSSLSASVGDRVTITCQASQD









ISNFVNWYQQKPGKAPKLLIYAAN









LRSGVPSRFSGSGSGTDFTLTISS









LQPEDFATYYCQQANSFPVTFGPG









TKVDIK









(SEQ ID NO: 404)









In some embodiments, the antibody comprises a CDR set as set forth in Table 6 or Table 7. In some embodiments, the antibody comprises the CDRs of Clone ID: 6, Clone ID: 59, or Clone ID: 63 of Table 6.

The antibodies, can be in a scFv format, which are also illustrated in a non-limiting embodiment in Table 6.
In some embodiments, the MAdCAM antibody is selected from the following table, which can be in a IgG format as illustrated in Table 7.















TABLE 7





Clone










ID
HCDR1
HCDR2
HCDR3
LCDR1
LCDR2
LCDR3
VH
VK







 1.
FTFS
AVISD
CTTS
QASQ
AASS
CQQGY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



SYGM
DGSDK
KYYY
DISK
LQS
STPLT
GSLRLSCAASGFTFS
TITCQASQDISKSLNWYQQ



H
YYA
YYGM
SLN
(SEQ
F
SYGMHWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
DVW
(SEQ
ID
(SEQ
EWVAVISDDGSDKYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQGYSTP



NO:
NO:
ID
NO:
65)
NO:
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



61)
62)
NO:
64)

66)
TAVYYCTTSKYYYYY
ID NO: 406)





63)



GMDVWGQGTTVTVSS










(SEQ ID NO:










405)



 


 2.
YPFI
GIINP
CARE
RASQ
GAST
CQQTW
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



GYYL
SGGST
GRLS
SISS
LES
GPPFT
ASVKVSCKASGYPFI
TITCRASQSISSYLAWYQQ



H
SYA
YGMD
YLA
(SEQ
F
GYYLHWVRQAPGQGL
KPGKAPKLLIYGASTLESG



(SEQ
(SEQ
AW
(SEQ
ID
(SEQ
EWMGIINPSGGSTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQTWGPP



NO:
NO:
ID
NO:
72)
NO:
TSTVYMELSSLRSED
FTFGQGTKLEIK (SEQ



68)
69)
NO:
71)

73)
TAVYYCAREGRLSYG
ID NO: 408)





70)



MDAWGQGTLVTVSS










(SEQ ID NO:










407)



 


 3.
YPFI
GIINP
CARE
RASQ
GAST
CQQTW
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



GQYL
SGGST
GRLS
SISS
LES
GPPFT
ASVKVSCKASGYPFI
TITCRASQSISSYLAWYQQ



H
SYA
YGMD
YLA
(SEQ
F
GQYLHWVRQAPGQGL
KPGKAPKLLIYGASTLESG



(SEQ
(SEQ
AW
(SEQ
ID
(SEQ
EWMGIINPSGGSTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQTWGPP



NO:
NO:
ID
NO:
72)
NO:
TSTVYMELSSLRSED
FTFGQGTKLEIK (SEQ



75
69)
NO:
71)

73)
TAVYYCAREGRLSYG
ID NO: 408)





70)



MDAWGQGTLVTVSS










(SEQ ID NO:










409)



 


 4. 
GTFS
GSINP
CAKD
QASQ
AASS
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYAI
SGDTT
KAQW
DISN
LQS
SSVIT
ASVKVSCKASGGTFS
TITCQASQDISNSLNWYQQ



S
SYA
LVGY
SLN
(SEQ
F
SYAISWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
FDYW
(SEQ
ID
(SEQ
EWMGSINPSGDTTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSSV



NO:
NO:
ID
NO:
65)
NO:
TSTVYMELSSLRSED
ITFGQGTKVEIK (SEQ



77)
78)
NO:
80)

81)
TAVYYCAKDKAQWLV
ID NO: 411)





79)



GYFDYWGQGTLVTVS










S (SEQ ID NO:










410)



 


 5.
FTFS
SSISP
CARE
RASQ
GASS
CQQAN
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



SYWM
GGSNI
VQLS
GISN
LQS
SFPFT
GSLRLSCAASGFTFS
TITCRASQGISNSLAWYX



H
DYA
HYDY
SLA
(SEQ
F
SYWMHWVRQAPGKGL
KPGKAPKLLIYGASSLQSG



(SEQ
(SEQ
W
(SEQ
ID
(SEQ
EWVSSISPGGSNIDY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQANSFP



NO:
NO:
ID
NO:
87)
NO:
KNTLYLQMNSLRAED
FTFGQGTKVEIK (SEQ



83)
84)
NO:
86)

88)
TAVYYCAREVQLSHY
ID NO: 413)





85)



DYWGQGTLVTVSS










(SEQ ID NO:










412)



 


 6.
FTFN
SRINS
CARE
RASQ
GASS
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



NYAF
YGTST
GPVA
IIGT
LQS
RLPFT
GSLRLSCAASGFTFN
TITCRASQIIGTNLAWYQQ



H
TYA
GYWY
NLA
(SEQ
F
NYAFHWVRQAPGKGL
KPGKAPKLLIYGASSLQSG



(SEQ
(SEQ
FDLW
(SEQ
ID
(SEQ
EWVSRINSYGTSTTY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYRLP



NO:
NO:
ID
NO:
87)
NO:
KNTLYLQMNSLRAED
FTFGQGTKVEIK (SEQ



90)
91)
NO:
93)

94)
TAVYYCAREGPVAGY
ID NO: 415)





92)



WYFDLWGQGTLVTVS










S (SEQ ID NO:










414)



 


 7.
FTFS
AIISH
CAKP
RASR
STLQ
QQAYS
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYQM
ADGGF
YSSG
GITN
S
FPWT
GSLRLSCAASGFTFS
TITCRASRGITNDLGWYQQ



S
KDYA
WSAV
DLG
(SEQ
(SEQ
DYQMSWVRQAPGKGL
KPGKAPKLLIYAASTLQSG



(SEQ
(SEQ
YYFD
(SEQ
ID
ID
EWVAIISHADGGFKD
VPSRFSGSGSGTDFTLTIS



ID
ID
YW
ID
NO:
NO:
YADSVKGRFTISRDN
SLQPEDFATYYCQQAYSFP



NO:
NO:
(SEQ
NO:
420)
421)
SKNTLYLQMNSLRAE
WTFGQGTKVEIK (SEQ



416)
417)
ID
419)


DTAVYYCAKPYSSGW
ID NO: 423)





NO:



SAVYYFDYWGQGTLV






418)



TVSS (SEQ ID










NO: 422)



 


 8.
YTFT
GIINP
CAKD
RASQ
AASS
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



GYHI
SGGST
WSSW
NISS
LQS
TTPYT
ASVKVSCKASGYTFT
TITCRASQNISSSLNWYQQ



H
IYA
YLGP
SLN
(SEQ
F
GYHIHWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
FDYW
(SEQ
ID
(SEQ
EWMGIINPSGGSTIY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYTTP



NO:
NO:
ID
NO:
65)
NO:
TSTVYMELSSLRSED
YTFGQGTKVEIK (SEQ



96)
97)
NO:
99)

100)
TAVYYCAKDWSSWYL
ID NO: 425)





98)



GPFDYWGQGTLVTVS










S (SEQ ID NO:










424)



 


 9.
YTFT
GIINH
CARP
RASQ
STLQ
QQSYS
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYYM
SGGST
YSGW
SISS
S
TPLT
ASVKVSCKASGYTFT
TITCRASQSISSSLNWYQQ



H
SYA
YFAF
SLN
(SEQ
(SEQ
SYYMHWVRQAPGQGL
KPGKAPKLLIYAASTLQSG



(SEQ
(SEQ
DIW
(SEQ
ID
ID
EWMGIINHSGGSTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
420)
429)
TSTVYMELSSLRSED
LTFGQGTKVEIK (SEQ



215)
426)
NO:
428)


TAVYYCARPYSGWYF
ID NO: 431)





427)



AFDIWGQGTLVTVSS










(SEQ ID NO:










430)



 


10
FMFG
SAISG
CAKD
RASQ
DASS
CQQTH
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYAM
SGGST
LVVA
GISN
LES
SFPST
GSLRLSCAASGFMFG
TITCRASQGISNNLNWYQQ



H
YYA
GIWY
NLN
(SEQ
F
DYAMHWVRQAPGKGL
KPGKAPKLLIYDASSLESG



(SEQ
(SEQ
FDLW
(SEQ
ID
(SEQ
EWVSAISGSGGSTYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQTHSFP



NO:
NO:
ID
NO:
106)
NO:
KNTLYLQMNSLRAED
STFGQGTKLEIK (SEQ



102)
103)
NO:
105)

107)
TAVYYCAKDLVVAGI
ID NO: 433)





104)



WYFDLWGRGTLVTVS










S (SEQ ID NO:










432)



 


11
FTFS
SVIGE
CAAD
RASQ
AAST
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYYM
SGGST
PVSR
GISS
LQS
STPWT
GSLRLSCAASGFTFS
TITCRASQGISSSLAWYQQ



N
YYA
WPKH
SLA
(SEQ
F
DYYMNWVRQAPGKGL
KPGKAPKLLIYAASTLQSG



(SEQ
(SEQ
GGGD
(SEQ
ID
(SEQ
EWVSVIGESGGSTYY
VPSRFSGSGSGTDFTLTIS



ID
ID
YW
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
(SEQ
NO:
113)
NO:
KNTLYLQMNSLRAED
WTFGQGTKVEIK (SEQ



109)
110)
ID
112)

114)
TAVYYCAADPVSRWP
ID NO: 435)





NO:



KHGGGDYWGQGTLVT






111)



VSS (SEQ ID NO:










434)



 


12
YTLT
GWINP
CAKG
RASD
AASS
CQQGY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



TWYM
NRGAT
DLWG
NIGS
LQS
STPPT
ASVKVSCKASGYTLT
TITCRASDNIGSWLAWYQQ



Y
NYA
AMDV
WLA
(SEQ
F
TWYMYWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
W
(SEQ
ID
(SEQ
EWMGWINPNRGATNY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQGYSTP



NO:
NO:
ID
NO:
65)
NO:
TSTVYMELSSLRSED
PTFGQGTKVEIK (SEQ



116)
117)
NO:
119)

120)
TAVYYCAKGDLWGAM
ID NO: 437)





118)



DVWGQGTLVTVSS










(SEQ ID NO:










436)



 


13
YTFT
GGFDP
CARH
RASE
AAST
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



TYYM
EDGET
AVAG
SISN
LQS
SVPFT
ASVKVSCKASGYTFT
TITCRASESISNWLAWYQQ



H
IYA
AVGA
WLA
(SEQ
F
TYYMHWVRQAPGQGL
KPGKAPKLLIYAASTLQSG



(SEQ
(SEQ
GYYY
(SEQ
ID
(SEQ
EWMGGFDPEDGETIY
VPSRFSGSGSGTDFTLTIS



ID
ID
YGMD
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSVP



NO:
NO:
VW
NO:
113)
NO:
TSTVYMELSSLRSED
FTFGPGTKVDIK (SEQ



122)
123)
(SEQ
125)

126)
TAVYYCARHAVAGAV
ID NO: 439)





ID



GAGYYYYGMDVWGQG






NO:



TMVTVSS (SEQ ID






124)



NO: 438)



 


14
YTFT
GGIIP
CAKG
QANQ
SKLE
QQSSE
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



NYYM
IVDGV
QFTG
DISN
A
IPYS
SSVKVSCKASGYTFT
TITCQANQDISNYLNWYQQ



H
KYA
NYYY
YLN
(SEQ
(SEQ
NYYMHWVRQAPGQGL
KPGKAPKLLIYRASKLEAG



(SEQ
(SEQ
GMDY
(SEQ
ID
ID
EWMGGIIPIVDGVKY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
NO:
AQKFQGRVTITADES
SLQPEDFATYYCQQSSEIP



NO:
NO:
(SEQ
NO:
442)
443)
TSTAYMELSSLRSED
YSFGQGTKVEIK (SEQ



148)
440)
ID
151)


TAVYYCAKGQFTGNY
ID NO: 445)





NO:



YYGMDYWGQGTLVTV






441)



SS (SEQ ID NO:










444)



 


15
YTFT
GWIGP
CARD
RSSQ
SSSN
CMQAL
QVQLVQSGAEVKKPG
DIVMTQSPLSLPVTPGEPA



GYYM
NSGDT
LDHN
SLLH
RAP
HIPLT
ASVKVSCKASGYTFT
SISCRSSQSLLHSNGYNYL



H
NYA
WYFD
SNGY
(SEQ
F
GYYMHWVRQAPGQGL
DWYLQKPGQSPQLLIYSSS



(SEQ
(SEQ
LW
NYLD
ID
(SEQ
EWMGWIGPNSGDTNY
NRAPGVPDRFSGSGSGTDF



ID
ID
(SEQ
(SEQ
NO:
ID
AQKFQGRVTMTRDTS
TLKISRVEAEDVGVYYCMQ



NO:
NO:
ID
ID
132)
NO:
TSTVYMELSSLRSED
ALHIPLTFGGGTKVEIK



128)
129)
NO:
NO:

133)
TAVYYCARDLDHNWY
(SEQ ID NO: 447)





130)
131)


FDLWGRGTLVTVSS










(SEQ ID NO:










446)



 


16
FTFD
SYIDA
CAKD
QASQ
KAST
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYAM
SGTTI
QAAA
DISN
LES
STPIT
GSLRLSCAASGFTFD
TITCQASQDISNYLNWYQQ



H
YYA
GYWY
YLN
(SEQ
F
DYAMHWVRQAPGKGL
KPGKAPKLLIYKASTLESG



(SEQ
(SEQ
FDLW
(SEQ
ID
(SEQ
EWVSYIDASGTTIYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
139)
NO:
KNTLYLQMNSLRAED
ITFGQGTRLEIK (SEQ



135)
136)
NO:
138)

140)
TAVYYCAKDQAAAGY
ID NO: 449)





137)



WYFDLWGRGTLVTVS










S (SEQ ID NO:










448)



 


17
YTFT
GGIVP
CAKD
RSSQ
SAYN
CMQAL
QVQLVQSGAEVKKPG
DIVMTQSPLSLPVTPGEPA



DYHI
RSGST
ESSG
SLLH
RAS
QTPLT
SSVKVSCKASGYTFT
SISCRSSQSLLHSNGYNYL



H
TYA
WYYF
SNGY
(SEQ
F
DYHIHWVRQAPGQGL
DWYLQKPGQSPQLLIYSAY



(SEQ
(SEQ
DYW
NYLD
ID
(SEQ
EWMGGIVPRSGSTTY
NRASGVPDRFSGSGSGTDF



ID
ID
(SEQ
(SEQ
NO:
ID
AQKFQGRVTITADES
TLKISRVEAEDVGVYYCMQ



NO:
NO:
ID
ID
145)
NO:
TSTAYMELSSLRSED
ALQTPLTFGQGTKVEIK



142)
143)
NO:
NO:

146)
TAVYYCAKDESSGWY
(SEQ ID NO: 451)





144)
131)


YFDYWGQGTLVTVSS










(SEQ ID NO:










450)



 


18
YTFT
GGIIP
CAKG
QANQ
RASK
CQQSS
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



NYYM
IVDRV
RYTV
DISN
LEA
EIPYS
SSVKVSCKASGYTFT
TITCQANQDISNYLNWYQQ



H
KYA
NYYY
YLN
(SEQ
F
NYYMHWVRQAPGQGL
KPGKAPKLLIYRASKLEAG



(SEQ
(SEQ
GMDV
(SEQ
ID
(SEQ
EWMGGIIPIVDRVKY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
ID
AQKFQGRVTITADES
SLQPEDFATYYCQQSSEIP



NO:
NO:
(SEQ
NO:
152)
NO:
TSTAYMELSSLRSED
YSFGQGTKLEIK (SEQ



148)
149)
ID
151)

153)
TAVYYCAKGRYTVNY
ID NO: 453)





NO:



YYGMDVWGQGTTVTV






150)



SS (SEQ ID NO:










452)



 


19
FIFE
SYLNS
CAKD
RASQ
DASN
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYAM
DGGST
YCTN
SISI
LET
TIPIT
GSLRLSCAASGFTFE
TITCRASQSISTYLNWYQQ



H
SYA
GVCA
YLN
(SEQ
F
DYAMHWVRQAPGKGL
KPGKAPKLLIYDASNLETG



(SEQ
(SEQ
FDYW
(SEQ
ID
(SEQ
EWVSYLNSDGGSTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYTIP



NO:
NO:
ID
NO:
159)
NO:
KNTLYLQMNSLRAED
ITFGQGTRLEIK (SEQ



155)
156)
NO:
158)

160)
TAVYYCAKDYCTNGV
ID NO: 455)





157)



CAFDYWGQGTLVTVS










S (SEQ ID NO:










454)



 


20
FTFS
SAISG
CVSD
RASQ
AASR
CQQAN
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DSAM
SGSTI
IAVA
SIST
LEG
SFPLT
GSLRLSCAASGFTFS
TITCRASQSISTFLNWYQQ



H
YYA
GHWY
FLN
(SEQ
F
DSAMHWVRQAPGKGL
KPGKAPKLLIYAASRLEGG



(SEQ
(SEQ
FDLW
(SEQ
ID
(SEQ
EWVSAISGSGSTIYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQANSFP



NO:
NO:
ID
NO:
166)
NO:
KNTLYLQMNSLRAED
LTFGPGTKVDIK (SEQ



162)
163)
NO:
165)

167)
TAVYYCVSDIAVAGH
ID NO: 457)





164)



WYFDLWGRGTLVTVS










S (SEQ ID NO:










456)



 


21
FTFS
SYISG
CARA
RASQ
AASS
CQQSY
EVQLVESGGGLVKPG
DIQMTQSPSSLSASVGDRV



SYWM
DSGYT
NSSG
SISS
LQS
STPLT
GSLRLSCAASGFTFS
TITCRASQSISSYLNWYQQ



S
NYA
WYDW
YLN
(SEQ
F
SYWMSWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
YFDL
(SEQ
ID
(SEQ
EWVSYISGDSGYTNY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
ID
AAPVKGRFTISRDDS
SLQPEDFATYYCQQSYSTP



NO:
NO:
(SEQ
NO:
65)
NO:
KNTLYLQMNSLKTED
LTFGGGTKVEIK (SEQ



169)
170)
ID
172)

173)
TAVYYCARANSSGWY
ID NO: 459)





NO:



DWYFDLWGRGTLVTV






171)



SS (SEQ ID NO:










458)



 


22
FTFD
SGISW
CAKD
QASQ
DASN
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYAM
NSGSI
IVAA
DISN
LET
STPLT
GSLRLSCAASGFTFD
TITCQASQDISNYLNWYQQ



H
GYA
GHYY
YLN
(SEQ
F
DYAMHWVRQAPGKGL
KPGKAPKLLIYDASNLETG



(SEQ
(SEQ
YGMD
(SEQ
ID
(SEQ
EWVSGISWNSGSIGY
VPSRFSGSGSGTDFTLTIS



ID
ID
VW
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
(SEQ
NO:
159)
NO:
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



135)
175)
ID
138)

173)
TAVYYCAKDIVAAGH
ID NO: 461)





NO:



YYYGMDVWGQGTTVT






176)



VSS (SEQ ID NO:










460)



 


23
FTFD
SYIDT
CARD
QAGQ
DASN
CQQTY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYAM
SSSHL
EAAA
DISN
LET
STPIT
GSLRLSCAASGFTFD
TITCQAGQDISNYLNWYQQ



H
YYA
GYYG
YLN
(SEQ
F
DYAMHWVRQAPGKGL
KPGKAPKLLIYDASNLETG



(SEQ
(SEQ
MDVW
(SEQ
ID
(SEQ
EWVSYIDTSSSHLYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQTYSTP



NO:
NO:
ID
NO:
159)
NO:
KNTLYLQMNSLRAED
ITFGQGTKLEIK (SEQ



135)
178)
NO:
180)

181)
TAVYYCARDEAAAGY
ID NO: 463)





179)



YGMDVWGQGTTVIVS










S (SEQ ID NO:










462)



 


24
FTFS
SRISS
CARG
RASQ
SNLQ
QQSYS
EVQLVESGGGLVKPG
DIQMTQSPSSLSASVGDRV



SYWM
DGRIT
TSYC
SIGR
S
IPLT
GSLRLSCAASGFTFS
TITCRASQSIGRNLNWYQQ



S
TYA
TGGV
NLN
(SEQ
(SEQ
SYWMSWVRQAPGKGL
KPGKAPKLLIYSASNLQSG



(SEQ
(SEQ
CDID
(SEQ
ID
ID
EWVSRISSDGRITTY
VPSRFSGSGSGTDFTLTIS



ID
ID
YW
ID
NO:
NO:
AAPVKGRFTISRDDS
SLQPEDFATYYCQQSYSIP



NO:
NO:
(SEQ
NO:
467)
468)
KNTLYLQMNSLKTED
LTFGPGTKVDIK (SEQ



169)
464)
ID
466)


TAVYYCARGTSYCTG
ID NO: 470)





NO:



GVCDIDYWGQGTLVT






465)



VSS (SEQ ID NO:










469)



 


25
FTFS
STIVG
CARD
RASQ
AASS
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



NAWM
NGGAT
NPLR
DISN
LQS
SIPPT
GSLRLSCAASGFTFS
TITCRASQDISNYLNWYQQ



S
YYA
WQGM
YLN
(SEQ
F
NAWMSWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
DVW
(SEQ
ID
(SEQ
EWVSTIVGNGGATYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSIP



NO:
NO:
ID
NO:
65)
NO:
KNTLYLQMNSLRAED
PTFGPGTKVDIK (SEQ



183)
184)
NO:
186)

187)
TAVYYCARDNPLRWQ
ID NO: 472)





185)



GMDVWGQGTLVTVSS










(SEQ ID NO:










471)



 


26
FTFS
SYISS
CARA
RASQ
SGLQ
QQSYS
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



SYAM
SSTYT
NSSS
SISS
S
TPLT
GSLRLSCAASGFTFS
TITCRASQSISSYLNWYQQ



S
NYA
WYDW
YLN
(SEQ
(SEQ
SYAMSWVRQAPGKGL
KPGKAPKLLIYAASGLQSG



(SEQ
(SEQ
YFDL
(SEQ
ID
ID
EWVSYISSSSTYTNY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
NO:
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
(SEQ
NO:
474)
429)
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



473)
190)
ID
172)


TAVYYCARANSSSWY
ID NO: 476)





NO:



DWYFDLWGQGTLVTV






191)



SS (SEQ ID NO:










475)



 


27
FTFS
SYISS
CARA
RASQ
SGLQ
QQSYS
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



SYQM
SSTYT
NSSS
SISS
S
TPLT
GSLRLSCAASGFTFS
TITCRASQSISSYLNWYQQ



S
NYA
WYDW
YLN
(SEQ
(SEQ
SYQMSWVRQAPGKGL
KPGKAPKLLIYAASGLQSG



(SEQ
(SEQ
YFDL
(SEQ
ID
ID
EWVSYISSSSTYTNY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
NO:
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
(SEQ
NO:
474)
429)
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



189)
190)
ID
172)


TAVYYCARANSSSWY
ID NO: 476)





NO:



DWYFDLWGQGTLVTV






191)



SS (SEQ ID NO:










477)



 


28
FTFS
SYISS
CARA
RASQ
SSLQ
QQSYS
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



SYAM
SSTYT
NSSS
SISS
S
TPLT
GSLRLSCAASGFTFS
TITCRASQSISSYLNWYQQ



S
NYA
WYDW
YLN
(SEQ
(SEQ
SYAMSWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
YFDL
(SEQ
ID
ID
EWVSYISSSSTYTNY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
NO:
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
(SEQ
NO:
362)
429)
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



473)
190)
ID
172)


TAVYYCARANSSSWY
ID NO: 459)





NO:



DWYFDLWGQGTLVTV






191)



SS (SEQ ID NO:










475)



 


29
FTFS
SYISS
CARA
RASQ
AASS
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



SYQM
SSTYT
NSSS
SISS
LQS
STPLT
GSLRLSCAASGFTFS
TITCRASQSISSYLNWYQQ



S
NYA
WYDW
YLN
(SEQ
F
SYQMSWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
YFDL
(SEQ
ID
(SEQ
EWVSYISSSSTYTNY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
(SEQ
NO:
65)
NO:
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



189)
190)
ID
172)

173)
TAVYYCARANSSSWY
ID NO: 459)





NO:



DWYFDLWGQGTLVTV






191)



SS (SEQ ID NO:










477)



 


30
FTFS
SGISG
CATS
RASQ
AASN
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



SYAM
SGGSA
QAPV
SISS
LQR
SIPIT
GSLRLSCAASGFTFS
TITCRASQSISSWLAWYQQ



H
YYA
DYYY
WLA
(SEQ
F
SYAMHWVRQAPGKGL
KPGKAPKLLIYAASNLQRG



(SEQ
(SEQ
YGMD
(SEQ
ID
(SEQ
EWVSGISGSGGSAYY
VPSRFSGSGSGTDFTLTIS



ID
ID
VW
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSIP



NO:
NO:
(SEQ
NO:
197)
NO:
KNTLYLQMNSLRAED
ITFGQGTKVEIK (SEQ



193)
194)
ID
196)

198)
TAVYYCATSQAPVDY
ID NO: 479)





NO:



YYYGMDVWGQGTTVT






195)



VSS (SEQ ID NO:










478)



 


31
FTFS
SYISG
CARV
RASQ
AASS
CQQSY
EVQLVESGGGLVKPG
DIQMTQSPSSLSASVGDRV



SYWM
SSSYT
GSSG
SISS
LQS
STPLT
GSLRLSCAASGFTFS
TITCRASQSISSYLNWYQQ



S
NYA
WYDW
YLN
(SEQ
F
SYWMSWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
YFDL
(SEQ
ID
(SEQ
EWVSYISGSSSYTNY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
ID
AAPVKGRFTISRDDS
SLQPEDFATYYCQQSYSTP



NO:
NO:
(SEQ
NO:
65)
NO:
KNTLYLQMNSLKTED
LTFGQGTKVEIK (SEQ



169)
200)
ID
172)

173)
TAVYYCARVGSSGWY
ID NO: 481)





NO:



DWYFDLWGRGTLVTV






201)



SS (SEQ ID NO:










480)



 


32
YTLT
GWINP
CAKG
RASD
AASS
CQQGY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



TWYM
NRGAT
DLWG
NIGS
LQS
STPPT
ASVKVSCKASGYTLT
TITCRASDNIGSWLAWYQQ



Y
NYA
AMDV
WLA
(SEQ
F
TWYMYWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
W
(SEQ
ID
(SEQ
EWMGWINPNRGATNY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQGYSTP



NO:
NO:
ID
NO:
65)
NO:
TSTVYMELSSLRSED
PTFGQGTKVEIK (SEQ



116)
117)
NO:
119)

120)
TAVYYCAKGDLWGAM
ID NO: 437)





118)



DVWGQGTLVTVSS










(SEQ ID NO:










436)



 


33
YTLT
GWINP
CAKG
RASD
AASS
CQQGY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



TWYM
NRGAT
DLWG
NIGS
LQS
STPPT
ASVKVSCKASGYTLT
TITCRASDNIGSWLAWYQQ



Y
NYA
AMDV
WLA
(SEQ
F
TWYMYWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
W
(SEQ
ID
(SEQ
EWMGWINPNRGATNY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQGYSTP



NO:
NO:
ID
NO:
65)
NO:
TSTVYMELSSLRSED
PTFGQGTKVEIK (SEQ



116)
117)
NO:
119)

120)
TAVYYCAKGDLWGAM
ID NO: 437)





118)



DVWGQGTTVTVSS










(SEQ ID NO:










482)



 


34
YTFT
GWMNP
CARD
RASQ
AASS
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



GYYI
NSGNT
PGFL
SISS
LQS
TAPYT
ASVKVSCKASGYTFT
TITCRASQSISSYLHWYQQ



H
GYA
GYCS
YLH
(SEQ
F
GYYIHWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
GGSC
(SEQ
ID
(SEQ
EWMGWMNPNSGNTGY
VPSRFSGSGSGTDFTLTIS



ID
ID
YDGW
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYTAP



NO:
NO:
FDPW
NO:
65)
NO:
TSTVYMELSSLRSED
YTFGQGTKLEIK (SEQ



204)
205)
(SEQ
207)

208)
TAVYYCARDPGFLGY
ID NO: 484)





ID



CSGGSCYDGWFDPWG






NO:



QGTLVTVSS (SEQ






206)



ID NO: 483)



 


35
YTFT
GWMNP
CARD
RASQ
AASS
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



GYYI
NSGNT
PGFL
SISS
LQS
TAPYT
ASVKVSCKASGYTFT
TITCRASQSISSYLHWYQQ



H
GYA
GYSS
YLH
(SEQ
F
GYYIHWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
GGSC
(SEQ
ID
(SEQ
EWMGWMNPNSGNTGY
VPSRFSGSGSGTDFTLTIS



ID
ID
YDGW
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYTAP



NO:
NO:
FDPW
NO:
65)
NO:
TSTVYMELSSLRSED
YTFGQGTKLEIK (SEQ



204)
205)
(SEQ
207)

208)
TAVYYCARDPGFLGY
ID NO: 484)





ID



SSGGSSYDGWFDPWG






NO:



QGTLVTVSS (SEQ






485)



ID NO: 486)



 


36
FTFD
SAISG
CARD
QASQ
SNLE
QQSYS
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYAL
DGRST
GTVN
DISK
T
IPFT
GSLRLSCAASGFTFD
TITCQASQDISKYLNWYQQ



H
TYA
GATG
YLN
(SEQ
(SEQ
DYALHWVRQAPGKGL
KPGKAPKLLIYDASNLETG



(SEQ
(SEQ
WFDP
(SEQ
ID
ID
EWVSAISGDGRSTTY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
NO:
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSIP



NO:
NO:
(SEQ
NO:
491)
492)
KNTLYLQMNSLRAED
FTFGPGTKVDIK (SEQ



487)
488)
ID
490)


TAVYYCARDGTVNGA
ID NO: 494)





NO:



TGWFDPWGQGTLVTV






489)



SS (SEQ ID NO:










493)



 


37
FTFS
SAISG
CARD
RASQ
SNLE
QQSYS
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYGM
SGGST
GGWQ
GISN
T
TPLT
GSLRLSCAASGFTFS
TITCRASQGISNNLNWYQQ



P
YYA
PAAI
NLN
(SEQ
(SEQ
DYGMPWVRQAPGKGL
KPGKAPKLLIYDASNLETG



(SEQ
(SEQ
LDYW
(SEQ
ID
ID
EWVSAISGSGGSTYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
491)
429)
KNTLYLQMNSLRAED
LTFGQGTKVEIK (SEQ



495)
103)
NO:
105)


TAVYYCARDGGWQPA
ID NO: 497)





296)



AILDYWGQGTLVTVS










S (SEQ ID NO:










496)



 


38
YTFT
GWMNP
CARE
RASQ
DASN
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



DYFL
TSGNT
GEGS
GINS
LET
STPLT
ASVKVSCKASGYTFT
TITCRASQGINSWLAWYQQ



H
GYA
GFDY
WLA
(SEQ
F
DYFLHWVRQAPGQGL
KPGKAPKLLIYDASNLETG



(SEQ
(SEQ
W
(SEQ
ID
(SEQ
EWMGWMNPTSGNTGY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
159)
NO:
TSTVYMELSSLRSED
LTFGGGTKVEIK (SEQ



210)
211)
NO:
213)

173)
TAVYYCAREGEGSGF
ID NO: 499)





212)



DYWGQGTLVTVSS










(SEQ ID NO:










498)



 


39
YTFT
AWMNP
CARD
RASQ
AASS
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYYM
NSGNT
YDFW
GISN
LQS
STPWT
ASVKVSCKASGYTFT
TITCRASQGISNYLAWYQQ



H
GYA
SGSL
YLA
(SEQ
F
SYYMHWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
GYW
(SEQ
ID
(SEQ
EWMAWMNPNSGNTGY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
65)
NO:
TSTVYMELSSLRSED
WTFGQGTKVEIK (SEQ



215)
216)
NO:
218)

114)
TAVYYCARDYDFWSG
ID NO: 501)





217)



SLGYWGQGTLVTVSS










(SEQ ID NO:










500)



 


40
YTLT
GWINP
CAKG
RASD
AASS
CQQGY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



TWYM
NRGAT
DLWG
NIGS
LQS
STPPT
ASVKVSCKASGYTLT
TITCRASDNIGSWLAWYQQ



Y
NYA
AMDV
WLA
(SEQ
F
TWYMYWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
W
(SEQ
ID
(SEQ
EWMGWINPNRGATNY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQGYSTP



NO:
NO:
ID
NO:
65)
NO:
TSTVYMELSSLRSED
PTFGQGTKVEIK (SEQ



116)
117)
NO:
119)

120)
TAVYYCAKGDLWGAM
ID NO: 437)





118)



DVWGQGTLVTVSS










(SEQ ID NO:










436)



 


41
YTFT
GIINP
CARD
RASQ
DASN
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYYM
SGGST
TGYS
SIGR
LQS
SIPIT
ASVKVSCKASGYTFT
TITCRASQSIGRWLAWYQQ



H
SYA
YGRY
WLA
(SEQ
F
SYYMHWVRQAPGQGL
KPGKAPKLLIYDASNLQSG



(SEQ
(SEQ
YYYG
(SEQ
ID
(SEQ
EWMGIINPSGGSTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
MDVW
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSIP



NO:
NO:
(SEQ
NO:
222)
NO:
TSTVYMELSSLRSED
ITFGQGTKVEIK (SEQ



215)
69)
ID
221)

198)
TAVYYCARDTGYSYG
ID NO: 503)





NO:



RYYYYGMDVWGQGTL






220)



VTVSS (SEQ ID










NO: 502)



 


42
YTLT
GIINP
CARE
RASQ
AASS
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



DYYM
SGGST
EYSS
GISS
LQS
STPLT
ASVKVSCKASGYTLT
TITCRASQGISSWLAWYQQ



H
SYA
SSGY
WLA
(SEQ
F
DYYMHWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
FDYW
(SEQ
ID
(SEQ
EWMGIINPSGGSTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
65)
NO:
TSTVYMELSSLRSED
LTFGQGTKVEIK (SEQ



224)
69)
NO:
226)

173)
TAVYYCAREEYSSSS
ID NO: 505)





225)



GYFDYWGQGTLVTVS










S (SEQ ID NO:










504)



 


43
YTFT
GWMHP
CARD
RASQ
AASS
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYGI
KSGDT
TPYY
SISS
LQS
SVPIT
ASVKVSCKASGYTFT
TITCRASQSISSWLAWYQQ



S
GLT
YYGM
WLA
(SEQ
F
SYGISWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
DVW
(SEQ
ID
(SEQ
EWMGWMHPKSGDTGL
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
TQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSVP



NO:
NO:
ID
NO:
65)
NO:
TSTVYMELSSLRSED
ITFGQGTKVEIK (SEQ



228)
229)
NO:
196)

231)
TAVYYCARDTPYYYY
ID NO: 507)





230)



GMDVWGQGTTVTVSS










(SEQ ID NO:










506)



 


44
FTFS
SAISG
CAKE
QASQ
SSLQ
QQTYS
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



SYAM
SGGST
RFID
DISN
S
GWT
GSLRLSCAASGFTFS
TITCQASQDISNYLNWYQQ



S
YYA
YGMD
YLN
(SEQ
(SEQ
SYAMSWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
VW
(SEQ
ID
ID
EWVSAISGSGGSTYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
ADSVKGRFTISRDNS
SLQPEDFATYYCQQTYSGW



NO:
NO:
ID
NO:
362)
509)
KNTLYLQMNSLRAED
TFGPGTKVDIK (SEQ ID



473)
103)
NO:
138)


TAVYYCAKERFIDYG
NO: 511)





508)



MDVWGQGTTVTVSS










(SEQ ID NO:










510)



 


45
FTFG
SYISG
CARD
RASQ
AASS
CQQSY
EVQLVESGGGLVKPG
DIQMTQSPSSLSASVGDRV



DYAM
DIGYT
VAAT
SISS
LQS
STPLT
GSLRLSCAASGFTFG
TITCRASQSISSYLNWYQQ



S
NYA
GNWY
YLN
(SEQ
F
DYAMSWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
FDLW
(SEQ
ID
(SEQ
EWVSYISGDIGYTNY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AAPVKGRFTISRDDS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
65)
NO:
KNTLYLQMNSLKTED
LTFGGGTKVEIK (SEQ



233)
234)
NO:
172)

173)
TAVYYCARDVAATGN
ID NO: 459)





235)



WYFDLWGRGTLVTVS










S (SEQ ID NO:










512)



 


46
FSFS
SFITS
CARD
RASQ
GAST
CQQYG
EVQLLESGGGLVQPG
EIVMTQSPATLSVSPGERA



SYTM
SSRTI
RRGD
SVRN
RAT
SSPLT
GSLRLSCAASGFSFS
TLSCRASQSVRNYLAWYQQ



N
YYA
YGDS
YLA
(SEQ
F
SYTMNWVRQAPGKGL
KPGQAPRLLIYGASTRATG



(SEQ
(SEQ
WYFD
(SEQ
ID
(SEQ
EWVSFITSSSRTIYY
IPARFSGSGSGTEFTLTIS



ID
ID
LW
ID
NO:
ID
ADSVKGRFTISRDNS
SLQSEDFAVYYCQQYGSSP



NO:
NO:
(SEQ
NO:
241)
NO:
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



237)
238)
ID
240)

242)
TAVYYCARDRRGDYG
ID NO: 514)





NO:



DSWYFDLWGRGTLVT






239)



VSS (SEQ ID NO:










513)



 


47
YTFT
GIINP
CARD
RASQ
DASN
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



GHYM
SGGST
TGYS
SIGR
LQS
SIPIT
ASVKVSCKASGYTFS
TITCRASQSISSWLAWYQQ



H
SYA
YGRY
WLA
(SEQ
F
KHFVHWVRQAPGQGL
KPGKAPKLLIYAASTLQSG



(SEQ
(SEQ
YYYG
(SEQ
ID
(SEQ
EWMGWMNPNSGNSGY
VPSRFSGSGSGTDFTLTIS



ID
ID
MDVW
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSTP



NO:
NO:
(SEQ
NO:
222)
NO:
TSTVYMELSSLRSED
WTFGQGTKVEIK (SEQ



244)
69)
ID
221)

198)
TAVYYCARGEGGYYY
ID NO: 516)





NO:



YGMDVWGQGTLVTVS






220)



S (SEQ ID NO:










515)



 


48
YTFS
GWMNP
CARG
RASQ
AAST
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



KHFV
NSGNS
EGGY
SISS
LQS
STPWT
GSLRLSCAASGFTFG
TITCRASQPLSNWLAWYQQ



H
GYA
YYYG
WLA
(SEQ
F
SYSMSWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
MDVW
(SEQ
ID
(SEQ
EWVSAIGTGGGTYYA
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
DSVKGRFTISRDNSK
SLQPEDFATYYCQQAISFP



NO:
NO:
ID
NO:
113)
NO:
NTLYLQMNSLRAEDT
LTFGGGTKVEIK (SEQ



246)
247)
NO:
196)

114)
AVYYCAKGTPYYYYY
ID NO: 518)





248)



GMDVWGQGTMVTVSS










(SEQ ID NO:










517)



 


49
FTFG
SAIGT
CAKG
RASQ
AASS
CQQAI
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYSM
GGGTY
TPYY
PLSN
LQS
SFPLT
ASVKVSCKASGYTFT
TITCQSSEDISSSLNWYQQ



S
YA
YYYG
WLA
(SEQ
F
SYYMHWVRQAPGQGL
KPGKAPKLLIYAASSLQIG



(SEQ
(SEQ
MDVW
(SEQ
ID
(SEQ
EWMGWMNPNSGNTGY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQTYSTP



NO:
NO:
ID
NO:
65)
NO:
TSTVYMELSSLRSED
YTFGQGTKVEIK (SEQ



250)
251)
NO:
253)

254)
TAVYYCARDLGYYDS
ID NO: 520)





252)



SGYFGAFDIWGQGTT










VTVSS (SEQ ID










NO: 519)



 


50
YTFT
GWMNP
CARD
QSSE
AASS
CQQTY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYYM
NSGNT
LGYY
DISS
LQI
STPYT
ASVKVSCKASGYTFT
TITCRASQGIGNWLAWYQQ



H
GYA
DSSG
SLN
(SEQ
F
SYGISWVRQAPGQGL
KPGKAPKLLIYAASNLETG



(SEQ
(SEQ
YFGA
(SEQ
ID
(SEQ
EWMGIINPRGGSTIF
VPSRFSGSGSGTDFTLTIS



ID
ID
FDIW
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQIHSYP



NO:
NO:
(SEQ
NO:
258)
NO:
TSTVYMELSSLRSED
LTFGGGTKVEIK (SEQ



215)
205)
ID
257)

259)
TAVYYCARGTRSSGW
ID NO: 522)





NO:



YGWFDPWGQGTLVTV






256)



SS (SEQ ID NO:










521)



 


51
YTFT
GIINP
CARG
RASQ
AASN
CQQIH
EVQLVESGGGLVKPG
DIVMTQSPLSLPVTPGEPA



SYGI
RGGST
TRSS
GIGN
LET
SYPLT
GSLRLSCAASGFIFQ
SISCRSSQSLLHSNGYNYL



S
IFA
GWYG
WLA
(SEQ
F
DSAIHWVRQAPGKGL
DWYLQKPGQSPQLLIYDAS



(SEQ
(SEQ
WFDP
(SEQ
ID
(SEQ
EWVSAIGTGGGTYYA
NLETGVPDRFSGSGSGTDF



ID
ID
W
ID
NO:
ID
APVKGRFTISRDDSK
TLKISRVEAEDVGVYYCMQ



NO:
NO:
(SEQ
NO:
264)
NO:
NTLYLQMNSLKTEDT
ALQTPLTFGQGTKVEIK



228)
261)
ID
263)

265)
AVYYCARSYCSGGSC
(SEQ ID NO: 524)





NO:



SLGSWGQGTLVTVSS






262)



(SEQ ID NO:










523)



 


52
FTFD
SYISS
CARE
RASQ
AASS
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYGM
SSSYI
IAAA
SISS
LQS
STPLT
GSLRLSCAASGFTFD
TITCRASQSISSYLNWYQQ



S
YYA
GFYG
YLN
(SEQ
F
DYGMSWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
MDVW
(SEQ
ID
(SEQ
EWVSYISSSSSYIYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
65)
NO:
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



267)
268)
NO:
172)

173)
TAVYYCAREIAAAGF
ID NO: 459)





269)



YGMDVWGQGTTVTVS










S (SEQ ID NO:










525)



 


53
YTFT
GWMNP
CARE
RASQ
SSLQ
QQSYS
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYYM
NSGNT
GLGY
GISS
S
TPYT
ASVKVSCKASGYTFT
TITCRASQGISSWLAWYQQ



H
GYA
CTNG
WLA
(SEQ
(SEQ
SYYMHWVRQAPGQGL
KPGKAPKLLIYGASSLQSG



(SEQ
(SEQ
VCWN
(SEQ
ID
ID
EWMGWMNPNSGNTGY
VPSRFSGSGSGTDFTLTIS



ID
ID
YYGM
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSTP



NO:
NO:
DVW
NO:
362)
527)
TSTVYMELSSLRSED
YTFGQGTKVEIK (SEQ



215)
205)
(SEQ
226)


TAVYYCAREGLGYCT
ID NO: 529)





ID



NGVCWNYYGMDVWGQ






NO:



GTLVTVSS (SEQ






526)



ID NO: 528)



 


54
GTLS
GGIIP
CARD
RASQ
GAST
CQQYG
QVQLVQSGAEVKKPG
EIVMTQSPATLSVSPGERA



RYGV
IFGTT
RVYY
SVSS
RAT
SSPIT
SSVKVSCKASGGTLS
TLSCRASQSVSSSYLAWYQ



S
NYA
DSSG
SYLA
(SEQ
F
RYGVSWVRQAPGQGL
QKPGQAPRLLIYGASTRAT



(SEQ
(SEQ
YPTW
(SEQ
ID
(SEQ
EWMGGIIPIFGTTNY
GIPARFSGSGSGTEFTLTI



ID
ID
YFDL
ID
NO:
ID
AQKFQGRVTITADES
SSLQSEDFAVYYCQQYGSS



NO:
NO:
W
NO:
241)
NO:
TSTAYMELSSLRSED
PITFGQGTKVEIK (SEQ



271)
272)
(SEQ
274)

275)
TAVYYCARDRVYYDS
ID NO: 531)





ID



SGYPTWYFDLWGRGT






NO:



LVTVSS (SEQ ID






273)



NO: 530)



 


55
FTFD
SGISG
CARD
QASQ
KAST
CQQAN
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DFAM
NGDSR
ASYG
DIRN
LES
SFPLT
GSLRLSCAASGFTFD
TITCQASQDIRNYLNWYQQ



H
YYA
GNYG
YLN
(SEQ
F
DFAMHWVRQAPGKGL
KPGKAPKLLIYKASTLESG



(SEQ
(SEQ
MDVW
(SEQ
ID
(SEQ
EWVSGISGNGDSRYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQANSFP



NO:
NO:
ID
NO:
139)
NO:
KNTLYLQMNSLRAED
LTFGPGTKVDIK (SEQ



277)
278)
NO:
280)

167)
TAVYYCARDASYGGN
ID NO: 533)





279)



YGMDVWGQGTTVTVS










S (SEQ ID NO:










532)



 


56
FTFS
SAIGT
CARE
RASQ
GASN
CQQSY
EVQLVESGGGLVKPG
DIQMTQSPSSLSASVGDRV



SYWM
GGGTY
WLVP
SISR
LQS
STPWT
GSLRLSCAASGFTFS
TITCRASQSISRWLAWYQQ



S
YA
YYGM
WLA
(SEQ
F
SYWMSWVRQAPGKGL
KPGKAPKLLIYGASNLQSG



(SEQ
(SEQ
DVW
(SEQ
ID
(SEQ
EWVSAIGTGGGTYYA
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
APVKGRFTISRDDSK
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
284)
NO:
NTLYLQMNSLKTEDT
WTFGQGTKVEIK (SEQ



169)
251)
NO:
283)

114)
AVYYCAREWLVPYYG
ID NO: 535)





282)



MDVWGQGTTVTVSS










(SEQ ID NO:










534)



 


57
FSVS
AGISY
CARS
KSSQ
WAST
CHQYY
EVQLLESGGGLVQPG
DIVMTQSPDSLAVSLGERA



SNYM
DGSSK
RGIA
SVLY
RQS
GHPPT
GSLRLSCAASGFSVS
TINCKSSQSVLYSSNNKNY



S
PYA
ARPL
SSNN
(SEQ
F
SNYMSWVRQAPGKGL
LAWYQQKPGQPPKLLIYWA



(SEQ
(SEQ
QHW
KNYL
ID
(SEQ
EWVAGISYDGSSKPY
STRQSGVPDRFSGSGSGTD



ID
ID
(SEQ
A
NO:
ID
ADSVKGRFTISRDNS
FTLTISSLQAEDVAVYYCH



NO:
NO:
ID
(SEQ
290)
NO:
KNTLYLQMNSLRAED
QYYGHPPTFGGGTKVEIK



286)
287)
NO:
ID

291)
TAVYYCARSRGIAAR
(SEQ ID NO: 537)





288)
NO:


PLQHWGQGTLVTVSS







289)


(SEQ ID NO:










536)



 


58
FSVS
AGISY
CARS
KSSQ
QAST
CHQYY
EVQLLESGGGLVQPG
DIVMTQSPDSLAVSLGERA



SNYM
DGSSK
RGIA
SVLY
RQS
GHPPT
GSLRLSCAASGFSVS
TINCKSSQSVLYSSNNKNY



S
PYA
ARPL
SSNN
(SEQ
F
SNYMSWVRQAPGKGL
LAWYQQKPGQPPKLLIYQA



(SEQ
(SEQ
QHW
KNYL
ID
(SEQ
EWVAGISYDGSSKPY
STRQSGVPDRFSGSGSGTD



ID
ID
(SEQ
A
NO:
ID
ADSVKGRFTISRDNS
FTLTISSLQAEDVAVYYCH



NO:
NO:
ID
(SEQ
293)
NO:
KNTLYLQMNSLRAED
QYYGHPPTFGGGTKVEIK



286)
287)
NO:
ID

291)
TAVYYCARSRGIAAR
(SEQ ID NO: 538)





288)
NO:


PLQHWGQGTLVTVSS







289)


(SEQ ID NO:










536)



 


59
FSFS
SAISG
CARD
RASQ
DASN
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYGM
SGGST
GGWQ
GISN
LET
STPLT
GSLRLSCAASGFSFS
TITCRASQGISNNLNWYQQ



H
YYA
PAAI
NLN
(SEQ
F
DYGMHWVRQAPGKGL
KPGKAPKLLIYDASNLETG



(SEQ
(SEQ
LDYW
(SEQ
ID
(SEQ
EWVSAISGSGGSTYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
159)
NO:
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



295)
103)
NO:
105)

173)
TAVYYCARDGGWQPA
ID NO: 540)





296)



AILDYWGQGTLVTVS










S (SEQ ID NO:










539)



 


60
FTFS
SVIYG
CARD
RASQ
DASN
CQQSY
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DHGM
GESTY
PAVA
GISN
LET
STCYT
GSLRLSCAASGFTFS
TITCRASQGISNYLAWYQQ



H
YA
GGGI
YLA
(SEQ
F
DHGMHWVRQAPGKGL
KPGKAPKLLIYDASNLETG



(SEQ
(SEQ
FDYW
(SEQ
ID
(SEQ
EWVSVIYGGESTYYA
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
DSVKGRFTISRDNSK
SLQPEDFATYYCQQSYSTC



NO:
NO:
ID
NO:
159)
NO:
NTLYLQMNSLRAEDT
YTFGQGTKLEIK (SEQ



298)
299)
NO:
218)

301)
AVYYCARDPAVAGGG
ID NO: 542)





300)



IFDYWGQGTLVTVSS










(SEQ ID NO:










541)



 


61
DTFT
GWINP
CARS
RASQ
DAST
CQQYS
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



GYYI
NSGGT
GLWL
TISI
LQS
SYPLT
ASVKVSCKASGDTFT
TITCRASQTISIWLAWYQQ



H
NYA
GSYY
WLA
(SEQ
F
GYYIHWVRQAPGQGL
KPGKAPKLLIYDASTLQSG



(SEQ
(SEQ
GMDV
(SEQ
ID
(SEQ
EWMGWINPNSGGTNY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQYSSYP



NO:
NO:
(SEQ
NO:
307)
NO:
TSTVYMELSSLRSED
LTFGQGTKVEIK (SEQ



303)
304)
ID
306)

308)
TAVYYCARSGLWLGS
ID NO: 544)





NO:



YYGMDVWGQGTLVTV






305)



SS (SEQ ID NO:










543)



 


62
YTFT
GWINP
CARS
RASH
AAST
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYDI
NSGTT
PYYY
FISR
LQS
SGISF
ASVKVSCKASGYTFT
TITCRASHFISRWVAWYQQ



N
GYA
YGMD
WVA
(SEQ
(SEQ
SYDINWVRQAPGQGL
KPGKAPKLLIYAASTLQSG



(SEQ
(SEQ
VW
(SEQ
ID
ID
EWMGWINPNSGTTGY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSGI



NO:
NO:
ID
NO:
113)
314)
TSTVYMELSSLRSED
SFGPGTKVDIK (SEQ ID



310)
311)
NO:
313)


TAVYYCARSPYYYYG
NO: 546)





312)



MDVWGQGTTVTVSS










(SEQ ID NO:










545)



 


63
FTFN
SRINS
CARG
RASQ
ATSS
CQQYY
EVQLLESGGGLVQPG
EIVMTQSPATLSVSPGERA



NYGM
DGSST
AYYY
SVSG
RAS
SGLTF
GSLRLSCAASGFTFN
TLSCRASQSVSGSYLAWYQ



N
SYA
YYMD
SYLA
(SEQ
(SEQ
NYGMNWVRQAPGKGL
QKPGQAPRLLIYATSSRAS



(SEQ
(SEQ
VW
(SEQ
ID
ID
EWVSRINSDGSSTSY
GIPARFSGSGSGTEFTLTI



ID
ID
(SEQ
ID
NO:
NO:
ADSVKGRFTISRDNS
SSLQSEDFAVYYCQQYYSG



NO:
NO:
ID
NO:
320)
321)
KNTLYLQMNSLRAED
LTFGQGTKVEIK (SEQ



316)
317)
NO:
319)


TAVYYCARGAYYYYY
ID NO: 548)





318)



MDVWGQGTLVTVSS










(SEQ ID NO:










547)



 


64
FTFS
AHIWN
CARD
RASQ
DASS
CQQAT
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



NSDM
DGSQK
RTDP
DIRN
LET
SLPLT
GSLRLSCAASGFTFS
TITCRASQDIRNYLGWYQQ



N
YYA
GYSS
YLG
(SEQ
F
NSDMNWVRQAPGKGL
KPGKAPKLLIYDASSLETG



(SEQ
(SEQ
AMDV
(SEQ
ID
(SEQ
EWVAHIWNDGSQKYY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQATSLP



NO:
NO:
(SEQ
NO:
327)
NO:
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



323)
324)
ID
326)

328)
TAVYYCARDRTDPGY
ID NO: 550)





NO:



SSAMDVWGQGTTVTV






325)



SS (SEQ ID NO:










549)



 


65
YTFT
GWMNP
CAKD
RASQ
QASS
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYDI
NSGNT
SDYS
DITN
LES
TIPLT
ASVKVSCKASGYTFT
TITCRASQDITNDLGWYQQ



N
GYA
NLLW
DLG
(SEQ
F
SYDINWVRQAPGQGL
KPGKAPKLLIYQASSLESG



(SEQ
(SEQ
DYW
(SEQ
ID
(SEQ
EWMGWMNPNSGNTGY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYTIP



NO:
NO:
ID
NO:
332)
NO:
TSTVYMELSSLRSED
LTFGQGTKVEIK (SEQ



310)
205)
NO:
331)

333)
TAVYYCAKDSDYSNL
ID NO: 552)





330)



LWDYWGQGTLVTVSS










(SEQ ID NO:










551)



 


66
YTFT
GIINP
CARD
RASQ
SNLE
QQYYS
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



GHYM
SGGST
GAWF
GISN
T
FPLYT
ASVKVSCKASGYTFT
TITCRASQGISNWLAWYQQ



H
SYA
GEEY
WLA
(SEQ
(SEQ
GHYMHWVRQAPGQGL
KPGKAPKLLIYDASNLETG



(SEQ
(SEQ
YYGM
(SEQ
ID
ID
EWMGIINPSGGSTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
DVW
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQYYSFP



NO:
NO:
(SEQ
NO:
491)
555)
TSTVYMELSSLRSED
LYTFGQGTKVEIK (SEQ



244)
69)
ID
554)


TAVYYCARDGAWFGE
ID NO: 557)





NO:



EYYYGMDVWGQGTTV






553)



TVSS (SEQ ID










NO: 556)



 


67
YTFT
GMIYP
CAMT
RASQ
STLQ
QQSYS
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



GYYM
RDGST
GWGY
GINN
S
APPT
ASVKVSCKASGYTFT
TITCRASQGINNYLAWYQQ



H
SYA
GMDV
YLA
(SEQ
(SEQ
GYYMHWVRQAPGQGL
KPGKAPKLLIYDASTLQSG



(SEQ
(SEQ
W
(SEQ
ID
ID
EWMGMIYPRDGSTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSAP



NO:
NO:
ID
NO:
420)
561)
TSTVYMELSSLRSED
PTFGQGTKLEIK (SEQ



128)
558)
NO:
560)


TAVYYCAMTGWGYGM
ID NO: 563)





559)



DVWGKGTTVTVSS










(SEQ ID NO:










562)



 


68
FTFG
AVVSY
CAKD
RASQ
DASN
CQQAN
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYAM
DGTNK
ICSS
NINN
LET
SFPPT
GSLRLSCAASGFTFG
TITCRASQNINNYVNWYQQ



S
YYA
TSCY
YVN
(SEQ
F
DYAMSWVRQAPGKGL
KPGKAPKLLIYDASNLETG



(SEQ
(SEQ
FDLW
(SEQ
ID
(SEQ
EWVAVVSYDGTNKYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
ID
ADSVKGRFTISRDNS
SLQPEDFATYYCQQANSFP



NO:
NO:
ID
NO:
159)
NO:
KNTLYLQMNSLRAED
PTFGQGTRLEIK (SEQ



233)
335)
NO:
337)

338)
TAVYYCAKDICSSTS
ID NO: 565)





336)



CYFDLWGRGTLVTVS










S (SEQ ID NO:










564)



 


69
YTFT
GIIDP
CARE
RASQ
ATSS
CQQTY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYYM
SGGST
EWSS
GISS
LQT
SIPIT
ASVKVSCKASGYTFT
TITCRASQGISSYLAWYQQ



H
SYA
GGVG
YLA
(SEQ
F
SYYMHWVRQAPGQGL
KPGKAPKLLIYATSSLQTG



(SEQ
(SEQ
YFDY
(SEQ
ID
(SEQ
EWMGIIDPSGGSTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQTYSIP



NO:
NO:
(SEQ
NO:
343)
NO:
TSTVYMELSSLRSED
ITFGQGTRLEIK (SEQ



215)
340)
ID
342)

344)
TAVYYCAREEWSSGG
ID NO: 567)





NO:



VGYFDYWGQGTLVTV






341)



SS (SEQ ID NO:










566)



 


70
YPFT
GWIKP
CARD
RASQ
SSLQ
QQSYD
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



DYYM
NSGDT
RFVG
SISV
S
TPYT
ASVKVSCKASGYPFT
TITCRASQSISVWLAWYQQ



H
EYA
KPDY
WLA
(SEQ
(SEQ
DYYMHWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
YYYG
(SEQ
ID
ID
EWMGWIKPNSGDTEY
VPSRFSGSGSGTDFTLTIS



ID
ID
MDVW
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYDTP



NO:
NO:
(SEQ
NO:
362)
572)
TSTVYMELSSLRSED
YTFGQGTKLEIK (SEQ



568)
569)
ID
571)


TAVYYCARDRFVGKP
ID NO: 574)





NO:



DYYYYGMDVWGQGTM






570)



VTVSS (SEQ ID










NO: 573)



 


71
YTFT
GIINP
CARD
RASQ
AASS
CQQSY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYYM
SGGST
SVAG
GISN
LQG
SLPYT
ASVKVSCKASGYTFT
TITCRASQGISNYFAWYQQ



H
SYA
TGGR
YFA
(SEQ
F
SYYMHWVRQAPGQGL
KPGKAPKLLIYAASSLQGG



(SEQ
(SEQ
YYGM
(SEQ
ID
(SEQ
EWMGIINPSGGSTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
DVW
ID
NO:
ID
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSLP



NO:
NO:
(SEQ
NO:
352)
NO:
TSTVYMELSSLRSED
YTFGQGTKLEIK (SEQ



215)
69)
ID
351)

353)
TAVYYCARDSVAGTG
ID NO: 576)





NO:



GRYYGMDVWGQGTLV






350)



TVSS (SEQ ID










NO: 575)



 


72
YTFT
GVINP
CASG
RASQ
SYLA
QQSYS
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SYYM
IGGTT
APSY
SISS
T
TPLT
ASVKVSCKASGYTFT
TITCRASQSISSYLNWYQQ



H
TYA
YYYG
YLN
(SEQ
(SEQ
SYYMHWVRQAPGQGL
KPGKAPKLLIYGTSYLATG



(SEQ
(SEQ
MDVW
(SEQ
ID
ID
EWMGVINPIGGTTTY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
579)
429)
TSTVYMELSSLRSED
LTFGQGTKVEIK (SEQ



215)
577)
NO:
172)


TAVYYCASGAPSYYY
ID NO: 581)





578)



YGMDVWGQGTLVTVS










S (SEQ ID NO:










580)



 


73
YTFT
GRINP
CARA
QASQ
TALR
QQSYS
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SNYV
HSGDT
GQLW
DIRN
T
HPLT
ASVKVSCKASGYTFT
TITCQASQDIRNYLNWYQQ



H
SYA
SDWY
YLN
(SEQ
(SEQ
SNYVHWVRQAPGQGL
KPGKAPKLLIYAATALRTG



(SEQ
(SEQ
FDLW
(SEQ
ID
ID
EWMGRINPHSGDTSY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSYSHP



NO:
NO:
ID
NO:
585)
586)
TSTVYMELSSLRSED
LTFGQGTKVEIK (SEQ



582)
583)
NO:
280)


TAVYYCARAGQLWSD
ID NO: 588)





584)



WYFDLWGRGTLVTVS










S (SEQ ID NO:










587)



 


74
YTFT
GIINP
CTTA
RASQ
AASS
CQQYY
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



GYYM
SGGNT
DYYY
GISN
LQS
SNADF
ASVKVSCKASGYTFT
TITCRASQGISNYLAWYQQ



H
KYA
YMDV
YLA
(SEQ
(SEQ
GYYMHWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
W
(SEQ
ID
ID
EWMGIINPSGGNTKY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQYYSNA



NO:
NO:
ID
NO:
65)
357)
TSTVYMELSSLRSED
DFGQGTKVEIK (SEQ ID



128)
355)
NO:
218)


TAVYYCTTADYYYYM
NO: 590)





356)



DVWGKGTTVTVSS










(SEQ ID NO:










589)



 


75
FTFS
SYISG
CARD
RASQ
SSLQ
QQYKS
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DFWM
DSGYT
RPYY
SVSR
S
YPVT
GSLRLSCAASGFTFS
TITCRASQSVSRSLAWYQQ



H
NYA
YYMD
SLA
(SEQ
(SEQ
DFWMHWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
VW
(SEQ
ID
ID
EWISYISGDSGYTNY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
ADSVKGRFTISRDNS
SLQPEDFATYYCQQYKSYP



NO:
NO:
ID
NO:
362)
363)
KNTLYLQMNSLRAED
VTFGQGTKVEIK (SEQ



359)
170)
NO:
361)


TAVYYCARDRPYYYY
ID NO: 592)





360)



MDVWGKGTTVTVSS










(SEQ ID NO:










591)



 


76
FTFD
SDISG
CAKD
QASQ
SYLQ
QQAHN
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYTM
SGGST
VVVA
DISN
S
YPIT
GSLRLSCAASGFTFD
TITCQASQDISNYLNWYQQ



H
YYA
GTPL
YLN
(SEQ
(SEQ
DYTMHWVRQAPGKGL
KPGKAPKLLIYAASYLQSG



(SEQ
(SEQ
HFDY
(SEQ
ID
ID
EWVSDISGSGGSTYY
VPSRFSGSGSGTDFTLTIS



ID
ID
W
ID
NO:
NO:
ADSVKGRFTISRDNS
SLQPEDFATYYCQQAHNYP



NO:
NO:
(SEQ
NO:
368)
369)
KNTLYLQMNSLRAED
ITFGQGTRLEIK (SEQ



365)
366)
ID
138)


TAVYYCAKDVVVAGT
ID NO: 594)





NO:



PLHFDYWGQGTLVTV






367)



SS (SEQ ID NO:










593)



 


77
FTFS
ASISS
CARE
RASQ
SSLQ
QQANA
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



NAWM
TSAYI
VVGA
SIST
S
FPPT
GSLRLSCAASEFTFS
TITCRASQSISTWLAWYQQ



S
DYA
TTFD
WLA
(SEQ
(SEQ
NAWMSWVRQAPGKGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
YW
(SEQ
ID
ID
EWVASISSTSAYIDY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
ADSVKGRFTISRDNS
SLQPEDFATYYCQQANAFP



NO:
NO:
ID
NO:
362)
374)
KNTLYLQMNSLRAED
PTFGQGTRLEIK (SEQ



183)
371)
NO:
373)


TAVYYCAREVVGATT
ID NO: 596)





372)



FDYWGQGTLVTVSS










(SEQ ID NO:










595)



 


78
GTFS
GWMEP
CAKG
KSSQ
SIRE
QQYYS
QVQLVQSGAEVKKPG
DIVMTQSPDSLAVSLGERA



SYAI
HTGNT
GFSW
SVLY
S
TPPT
SSVKVSCKASGGTFS
TINCKSSQSVLYSSNNKNY



S
RYA
FDPW
SSNN
(SEQ
(SEQ
SYAISWVRQAPGQGL
LAWYQQKPGQPPKLLIYWA



(SEQ
(SEQ
(SEQ
KNYL
ID
ID
EWMGWMEPHTGNTRY
STRESGVPDRFSGSGSGTD



ID
ID
ID
A
NO:
NO:
AQKFQGRVTITADES
FTLTISSLQAEDVAVYYCQ



NO:
NO:
NO:
(SEQ
378)
379)
TSTAYMELSSLRSED
QYYSTPPTFGQGTRLEIK



77)
376)
377)
ID


TAVYYCAKGGFSWFD
(SEQ ID NO: 598)






NO:


PWGQGTLVTVSS







289)


(SEQ ID NO:










597)



 


79
FTFD
ASITS
CARE
RASQ
STRA
QQYYT
EVQLLESGGGLVKPG
EIVMTQSPATLSVSPGERA



DYAM
SSAFI
RVDW
GISN
T
YPPT
GSLRLSCAASGFTFD
TLSCRASQGISNSYLAWYQ



H
DYA
NSYF
SYLA
(SEQ
(SEQ
DYAMHWVRQAPGKGL
QKPGQAPRLLIYGASTRAT



(SEQ
(SEQ
DLW
(SEQ
ID
ID
EWVASITSSSAFIDY
GIPARFSGSGSGTEFTLTI



ID
ID
(SEQ
ID
NO:
NO:
AASVKGRFTISRDDS
SSLQSEDFAVYYCQQYYTY



NO:
NO:
ID
NO:
384)
385)
KNTLYLQMNSLKTED
PPTFGPGTKVDIK (SEQ



135)
381)
NO:
383)


TAVYYCARERVDWNS
ID NO: 600)





382)



YFDLWGRGTLVTVSS










(SEQ ID NO:










599)



 


80
FTFD
SAISG
CAKD
QASQ
SNLE
QQSYS
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



DYAM
SGGST
LGVV
DISN
A
TPLT
GSLRLSCAASGFTFD
TITCQASQDISNHLNWYQQ



H
YYA
VPAA
HLN
(SEQ
(SEQ
DYAMHWVRQAPGKGL
KPGKAPKLLIYDASNLEAG



(SEQ
(SEQ
LDYW
(SEQ
ID
ID
EWVSAISGSGGSTYY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
ADSVKGRFTISRDNS
SLQPEDFATYYCQQSYSTP



NO:
NO:
ID
NO:
603)
429)
KNTLYLQMNSLRAED
LTFGGGTKVEIK (SEQ



135)
103)
NO:
602)


TAVYYCAKDLGVVVP
ID NO: 605)





601)



AALDYWGQGTTVTVS










S (SEQ ID NO:










604)



 


81
FAFS
AGTSG
CARE
RASQ
ANLE
QQSDI
EVQLLESGGGLVKPG
DIQMTQSPSSLSASVGDRV



SHWM
SGESR
TYYY
GISN
G
FPPT
GSLRLSCAASGFAFS
TITCRASQGISNYLAWYQQ



H
DYA
YYMD
YLA
(SEQ
(SEQ
SHWMHWVRQAPGKGL
KPGKAPKLLIYDAANLEGG



(SEQ
(SEQ
VW
(SEQ
ID
ID
EWVAGTSGSGESRDY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
ADFVKGRFTISRDDS
SLQPEDFATYYCQQSDIFP



NO:
NO:
ID
NO:
390)
391)
KNTLYLQMNSLKTED
PTFGQGTKVEIK (SEQ



387)
388)
NO:
218)


TAVYYCARETYYYYY
ID NO: 607)





389)



MDVWGKGTTVTVSS










(SEQ ID NO:










606)



 


82
YTFT
GWINV
CARE
RASQ
SSLQ
QQSNS
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRA



RHWI
KTGGA
SSGW
SISN
S
FPLT
ASVKVSCKASGYTFT
TITCRASQSISNYLAWYQQ



H
GYA
YGTD
YLA
(SEQ
(SEQ
RHWIHWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
VW
(SEQ
ID
ID
EWMGWINVKTGGAGY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQSNSFP



NO:
NO:
ID
NO:
362)
397)
TSTVYMELSSLRSED
LTFGGGTKVEIK (SEQ



393)
394)
NO:
396)


TAVYYCARESSGWYG
ID NO: 609)





395)



TDVWGQGTTVTVSS










(SEQ ID NO:










608)



 


83
FTFS
AAISY
CARE
QASQ
NLRS
QQANS
EVQLLESGGGLVQPG
DIQMTQSPSSLSASVGDRV



SYWM
DGKYK
NKQW
DISN
(SEQ
FPVT
GSLRLSCAASGFTFS
TITCQASQDISNFVNWYQQ



H
DYE
LASF
FVN
ID
(SEQ
SYWMHWVRQAPGKGL
KPGKAPKLLIYAANLRSGV



(SEQ
(SEQ
DYW
(SEQ
NO:
ID
EWVAAISYDGKYKDY
PSRFSGSGSGTDFTLTISS



ID
ID
(SEQ
ID
402)
NO:
EDSVKGRFTISRDNS
LQPEDFATYYCQQANSFPV



NO:
NO:
ID
NO:

403)
KNTLYLQMNSLRAED
TFGPGTKVDIK (SEQ ID



83)
399)
NO:
401)


TAVYYCARENKQWLA
NO: 611)





400)



SFDYWGQGTLVTVSS










(SEQ ID NO:










610)



 


84
GTFS
GWISA
CASR
QASE
SSLQ
QQTDS
QVQLVQSGAEVKKPG
DIQMTQSPSSLSASVGDRV



SSAI
YNGYT
VHSG
HIYN
S
IPIT
ASVKVSCKASGGTFS
TITCQASEHIYNYLNWYQQ



S
NYA
GSYP
YLN
(SEQ
(SEQ
SSAISWVRQAPGQGL
KPGKAPKLLIYAASSLQSG



(SEQ
(SEQ
DDYW
(SEQ
ID
ID
EWMGWISAYNGYTNY
VPSRFSGSGSGTDFTLTIS



ID
ID
(SEQ
ID
NO:
NO:
AQKFQGRVTMTRDTS
SLQPEDFATYYCQQTDSIP



NO:
NO:
ID
NO:
362)
616)
TSTVYMELSSLRSED
ITFGQGTKVEIK (SEQ



612)
613)
NO:
615)


TAVYYCASRVHSGGS
ID NO: 618)





614)



YPDDYWGQGTLVTVS










S (SEQ ID NO:










617)









In some embodiments, the antibody comprises the CDRs of Clone ID: 6, Clone ID: 75, or Clone ID: 79 of Table 7.
The IgG and scFv formats illustrated herein are simply non-limiting examples. The CDRs provided herein can be placed in different formats, including different VH and VL/VK formats and still be able to bind to MAdCAM.
Although the CDRs are illustrated in the tables provided herein, there are other ways to annotate or identify CDRs. For example, in some embodiments, the HCDR2 can have an extra amino acid at the N-terminus. For example, for the HCDR2 of Clone 6 the table indicates that it has a sequence of: SRINSYGTSTTYA (SEQ ID NO: 91) However, in some embodiments, the HCDR2 has a sequence of VSRINSYGTSTTYA (SEQ ID NO: 629), which is shown with an extra residue, a valine, at the N-terminus of the HCDR2. The valine is clearly illustrated in VH peptide of the tables provided herein. Therefore, in some embodiments, the HCDR2 comprises one additional amino acid immediately to the N-terminus of the HCDR2 listed in the table. The residue would be the residue that is immediately to the N-terminus of the HCDR2 found in the VH sequence provided for in the table in the same row. One of skill in the art with this information could immediately envisage the HCDR2 peptide sequence that has the additional amino acid residue immediately to the N-terminus of the HCDR2 listed in the table. These embodiments are sufficiently described and do not require application to list each of these different annotations and one of skill in the art with the guidance and description provided herein could write them out individually without any undue experimentation.
Similarly, the HCDR3 can exclude the cysteine residue. Each of the HCDR3 polypeptides provided for in Tables 6 and 7 begins with a cysteine residue. In some embodiments, the HCDR3 does not include the cysteine. Furthermore, in some embodiments, the HCDR3 does not have the last C-terminal residue illustrated in Table 6 and 7 provided for herein. Therefore, in some embodiments, the HCDR3 does not have the cysteine and/or the last C-terminal residue illustrated in the tables. One of skill in the art with this information could immediately envisage the HCDR3 peptide sequence that does not have the cysteine and/or the last C-terminal residue illustrated in the tables. These embodiments are sufficiently described and do not require application to list each of these different annotations and one of skill in the art with the guidance and description provided herein could write them out individually without any undue experimentation.
In some embodiments, the light chain CDR2 can have one or two extra amino acid residues at the N-terminus. These additional residues would be those that are immediately to the N-terminus of the light chain CDR2 (LCDR2) present in the VL/VK chain provided for herein, in the same row as the CDRs that are listed. For example, the LCDR2 of Clone 6 is provided as GASSLQS (SEQ ID NO: 87), but in some embodiments could be IYGASSLQS (SEQ ID NO: 630) or YGASSLQS (SEQ ID NO: 631). One of skill in the art with this information could immediately envisage the LCDR2 peptide sequence that has one or two extra amino acid residues at the N-terminus of the LCDR2 sequence provided for herein. These embodiments are sufficiently described and do not require application to list each of these different annotations and one of skill in the art with the guidance and description provided herein could write them out individually without any undue experimentation.
There are also alternative systems for annotating CDRs, all of which can be used. For example, CDRs can be chosen based on the Kabat systems, the IMGT system, or the CHOTHIA. Other proprietary systems can also be used, which may be based on the predicted 3-dimensional structure of the protein. Accordingly, in some embodiments, the CDRs of Clone ID: 6, Clone ID: 75, or Clone ID: 79 of Table 7 can also be characterized as shown in Table 8. These alternative CDRs can be substituted for these clone referenced in Table 7 or the equivalent clone numbering in Table 6, i.e., Clone 6, Clone 59, and Clone 63.







TABLE 8







Alterative CDRs for Certain Clones














Clone









No.









(Table
Annotation








7)
System
LCDR1
LCDR2
LCDR3
HCDR1
HCDR2
HCDR3

















6
Proprietary
RASQIIGTN
SSLQS
QQSYRLP
FTFNNYAF
SRINSYG
CAREGPVAG




LA (SEQ
(SEQ ID
FT (SEQ
H (SEQ
TSTTYA
YWYFDLW




ID NO:
NO:
ID NO:
ID NO:
(SEQ ID
(SEQ ID




93)
362)
632)
90)
NO: 91)
NO: 92)



Other
RASQIIGTN
GASSLQS
CQQSYRL
FTFNNYAF
SRINSYG
CAREGPVAG



Annotation
LA (SEQ
(SEQ ID
PFTF
H (SEQ
TSTTYA
YWYFDLW




ID NO:
NO: 87)
(SEQ ID
ID NO:
(SEQ ID
(SEQ ID




93)

NO: 94)
90
NO: 91)
NO: 92)



Kabat
RASQIIGTN
GASSLQS
QQSYRLP
NYAFH
RINSYGT
EGPVAGYWY




LA (SEQ
(SEQ ID
FT (SEQ
(SEQ ID
STTYADS
FDL (SEQ




ID NO:
NO: 87)
ID NO:
NO: 633)
VKG
ID NO:




93)

632)

(SEQ ID
635)








NO:









634)




IMGT
QIIGTN
GAS
QQSYRLP
GFTFNNYA
INSYGTS
AREGPVAGY




(SEQ ID

FT (SEQ
(SEQ ID
T (SEQ
WYFDL




NO: 636)

ID NO:
NO: 637)
ID NO:
(SEQ ID






632)

638)
NO: 639)



CHOTHIA
RASQIIGTN
GASSLQS
QQSYRLP
GFTFNNY
NSYGTS
EGPVAGYWY




LA (SEQ
(SEQ ID
FT (SEQ
(SEQ ID
(SEQ ID
FDL (SEQ




ID NO:
NO: 87)
ID NO:
NO: 640)
NO: 641)
ID NO:




93)

632)


635)


 


75
Proprietary
RASQSVSRS
SSLQS
QQYKSYP
FTFSDFWM
SYISGDS
CARDRPYYY




LA (SEQ
(SEQ ID
VT (SEQ
H (SEQ
GYTNYA
YMDVW




ID NO:
NO:
ID NO:
ID NO:
(SEQ ID
(SEQ ID




361)
362)
363)
359)
NO: 170)
NO: 360)



Other
RASQSVSRS
AASSLQS
CQQYKSY
FTFSDFWM
SYISGDS
CARDRPYYY



Annotation
LA (SEQ
(SEQ ID
PVTF
H (SEQ
GYTNYA
YMDVW




ID NO:
NO: 65)
(SEQ ID
ID NO:
(SEQ ID
(SEQ ID




361)

NO:
359)
NO: 170)
NO: 360)






642)






Kabat
RASQSVSRS
AASSLQS
QQYKSYP
DFWMH
YISGDSG
DRPYYYYMD




LA (SEQ
(SEQ ID
VT (SEQ
(SEQ ID
YTNYADS
V (SEQ ID




ID NO:
NO: 65)
ID NO:
NO: 643)
VKG
NO: 645)




361)

363)

(SEQ ID









NO: 644)




IMGT
QSVSRS
AAS
QQYKSYP
GFTFSDFW
ISGDSGY
ARDRPYYYY




(SEQ ID

VT (SEQ
(SEQ ID
T (SEQ
MDV (SEQ




NO: 646)

ID NO:
NO: 647)
ID NO:
ID NO:






363)

648)
649)



CHOTHIA
RASQSVSRS
AASSLQS
QQYKSYP
GFTFSDF
SGDSGY
DRPYYYYMD




LA (SEQ
(SEQ ID
VT (SEQ
(SEQ ID
(SEQ ID
V (SEQ ID




ID NO:
NO: 65)
ID NO:
NO: 650)
NO:
NO: 645)




361)

363)

651)



 


79
Proprietary
RASQGISNS
STRAT
QQYYTYP
FTFDDYAM
ASITSSS
CARERVDWN




YLA (SEQ
(SEQ ID
PT (SEQ
H (SEQ
AFIDYA
SYFDLW




ID NO:
NO:
ID NO:
ID NO:
(SEQ ID
(SEQ ID




383)
384)
385)
135)
NO: 381)
NO: 382)



Other
RASQGISNS
GASTRAT
CQQYYTY
FTFDDYAM
ASITSSS
CARERVDWN



Annotation
YLA (SEQ
(SEQ ID
PPTF
H (SEQ
AFIDYA
SYFDLW




ID NO:
NO: 241)
(SEQ ID
ID NO:
(SEQ ID
(SEQ ID




383)

NO: 652)
135)
NO: 381)
NO: 382)



Kabat
RASQGISNS
GASTRAT
QQYYTYP
DYAMH
SITSSSA
ERVDWNSYF




YLA (SEQ
(SEQ ID
PT (SEQ
(SEQ ID
FIDYAAS
DL (SEQ




ID NO:
NO:
ID NO:
NO: 653)
VKG
ID NO:




383)
241)
385)

(SEQ ID
655)








NO: 654)




IMGT
QGISNSY
GAS
QQYYTYP
GFTFDDYA
ITSSSAF
ARERVDWNS




(SEQ ID

PT (SEQ
(SEQ ID
I (SEQ
YFDL (SEQ




NO: 656)

ID NO:
NO: 657)
ID NO:
ID NO:






385)

658)
659)



CHOTHIA
RASQGISNS
GASTRAT
QQYYTYP
GFTFDDY
TSSSAF
ERVDWNSYF




YLA (SEQ
(SEQ ID
PT (SEQ
(SEQ ID
(SEQ ID
DL (SEQ




ID NO:
NO: 241)
ID NO:
NO: 660)
NO: 661)
ID NO:




383)

385)


655)









In some embodiments, the antibody is linked to another antibody or therapeutic. In some embodiments, the MAdCAM antibody is linked to a PD-1 antibody or a IL-2 mutein as provided herein or that is incorporated by reference.
In some embodiments, the MAdCAM antibody comprises a sequence as shown in Table 6. In some embodiments, the antibody is in a scFV format as illustrated Table 6. In some embodiments, the antibody comprises a CDR1 from any one of clones 1-66 of Table 6, a CDR2 from any one of clones 1-84, and a CDR3 from any one of clones 1-66 of Table 6. In some embodiments, the antibody comprises a LCDR1 from any one of clones 1-66 of Table 6, a LCDR2 from any one of clones 1-66 of Table 6, and a LCDR3 from any one of clones 1-66 of Table 6. In some embodiments, the amino acid residues of the CDRs shown above contain mutations. In some embodiments, the CDRs contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions or mutations. In some embodiments, the substitution is a conservative substitution.
In some embodiments, the MAdCAM antibody has a VH region selected from any one of clones 1-84 of Table 7 and a VL region selected from any one of clones 1-84 as set forth in of Table 7. In some embodiments, the antibody comprises a CDR1 from any one of clones 1-84 of Table 7, a CDR2 from any one of clones 1-84, and a CDR3 from any one of clones 1-84 of Table 7. In some embodiments, the antibody comprises a LCDR1 from any one of clones 1-84 of Table 7, a LCDR2 from any one of clones 1-84 of Table 7, and a LCDR3 from any one of clones 1-84 of Table 7. In some embodiments, the amino acid residues of the CDRs shown above contain mutations. In some embodiments, the CDRs contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions or mutations. In some embodiments, the substitution is a conservative substitution.
In some embodiments, the molecule comprises an antibody that binds to MAdCAM. In some embodiments, the antibody comprises (i) a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino acid sequence of any of the CDR1 sequences set forth in Table 6 or Table 7; the heavy chain CDR2 has the amino acid sequence of any of the CDR2 sequences set forth in Table 6 or Table 7, and the heavy chain CDR3 has the amino acid sequence of any of the CDR3 sequences set forth in Table 6 or Table 7; or variants of any of the foregoing; and (ii) a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain CDR1 sequence has the amino acid sequence of any of the LCDR1 sequences set forth in Table 6 or Table 7; the light chain LCDR2 has the amino acid sequence of any of the LCDR2 sequences set forth in Table 6 or Table 7, and the light chain CDR3 has the amino acid sequence of any of the LCDR3 sequences set forth in Table 6 or Table 7, or variants of any of the foregoing.
In some embodiments, the antibody comprises a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1, CDR2, and CDR3 sequences have the amino acid sequence as set forth in Antibody 6 of Table 6 or Antibody 6 of Table 7, or variants of any of the foregoing; and (ii) a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain CDR1, CDR2, and CDR3 sequences have the amino acid sequence as set forth sequence as set forth in Antibody 6 of Table 6 or Antibody 6 of Table 7, or variants of any of the foregoing.
In some embodiments, the antibody comprises a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1, CDR2, and CDR3 sequences have the amino acid sequence as set forth in Antibody 59 of Table 6 or Antibody 75 of Table 7, or variants of any of the foregoing; and (ii) a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain CDR1, CDR2, and CDR3 sequences have the amino acid sequence as set forth sequence as set forth in Antibody 59 of Table 6 or Antibody 75 of Table 7, or variants of any of the foregoing.
In some embodiments, the antibody comprises a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1, CDR2, and CDR3 sequences have the amino acid sequence as set forth in Antibody 63 of Table 6 or Antibody 79 of Table 7, or variants of any of the foregoing; and (ii) a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain CDR1, CDR2, and CDR3 sequences have the amino acid sequence as set forth sequence as set forth in Antibody 63 of Table 6 or Antibody 79 of Table 7, or variants of any of the foregoing.
These are non-limiting illustrative examples and the antibodies can have the CDRs as set forth in the tables provided herein and are explicitly referenced without writing out the previous paragraphs for each CDR set.
In some embodiments, the MAdCAM antibody comprises a VH and VL(VK) chain as provided herein, such as those listed in the Table 7. In some embodiments, the VH peptide comprises a sequence of SEQ ID NO: 414, 591, or 599. In some embodiments, the VK chain comprises a sequence of 415, 592, or 600. In some embodiments, the antibody comprises a VH of SEQ ID NO: 414 and a VK of SEQ ID NO: 415. In some embodiments, the antibody comprises a VH of SEQ ID NO: 591 and a VK of SEQ ID NO: 592. In some embodiments, the antibody comprises a VH of SEQ ID NO: 599 and a VK of SEQ ID NO: 600. The VH and VK can also be in a scFV format as illustrated in the Table 6.
In some embodiments, a therapeutic is provided comprising one or more of the following polypeptides:











SEQ ID



NO:
Sequence







620
EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYAFHWVRQAPGKGLEWVSRINSYGTSTTYADS



VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGPVAGYWYFDLWGQGTLVTVSSASTKG



PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV



VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPK



DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ



DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP



SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT



QKSLSLSPGGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK



ATELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETA



TIVEFINRWITFSQSIISTLT


 


621
DIQMTQSPSSLSASVGDRVTITCRASQIIGTNLAWYQQKPGKAPKLLIYGASSLQSGVPSRFS



GSGSGTDFTLTISSLQPEDFATYYCQQSYRLPFTFGQGTKVEIKRRTVAAPSVFIFPPSDEQL



KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK



HKVYACEVTHQGLSSPVTKSFNRGEC


 


622
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDFWMHWVRQAPGKGLEWISYISGDSGYTNYADS



VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPYYYYMDVWGKGTTVTVSSASTKGPS



VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT



VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDT



LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW



LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD



IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK



SLSLSPGGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT



ELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATI



VEFINRWITFSQSIISTLT


 


623
DIQMTQSPSSLSASVGDRVTITCRASQSVSRSLAWYQQKPGKAPKLLIYAASSLQSGVPSRFS



GSGSGTDFTLTISSLQPEDFATYYCQQYKSYPVTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK



SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH



KVYACEVTHQGLSSPVTKSFNRGEC


 


624
EVQLLESGGGLVKPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVASITSSSAFIDYAAS



VKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARERVDWNSYFDLWGRGTLVTVSSASTKGP



SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV



TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKD



TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD



WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS



DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ



KSLSLSPGGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA



TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETAT



IVEFINRWITFSQSIISTLT


 


625
EIVMTQSPATLSVSPGERATLSCRASQGISNSYLAWYQQKPGQAPRLLIYGASTRATGIPARF



SGSGSGTEFTLTISSLQSEDFAVYYCQQYYTYPPTFGPGTKVDIKRTVAAPSVFIFPPSDEQL



KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK



HKVYACEVTHQGLSSPVTKSFNRGEC









In some embodiments, the polypeptide comprises one peptide of SEQ ID NO: 620, 622, or 624 and a second peptide of SEQ ID NO: 621, 623, or 625. In some embodiments, a polypeptide is provided comprising a first peptide of SEQ ID NO: 620 and a second peptide comprising a sequence of SEQ ID NO: 621. In some embodiments, a polypeptide is provided comprising a first peptide of SEQ ID NO: 620 and a second peptide comprising a sequence of SEQ ID NO: 623. In some embodiments, a polypeptide is provided comprising a first peptide of SEQ ID NO: 620 and a second peptide comprising a sequence of SEQ ID NO: 625. In some embodiments, a polypeptide is provided comprising a first peptide of SEQ ID NO: 622 and a second peptide comprising a sequence of SEQ ID NO: 621. In some embodiments, a polypeptide is provided comprising a first peptide of SEQ ID NO: 622 and a second peptide comprising a sequence of SEQ ID NO: 623. In some embodiments, a polypeptide is provided comprising a first peptide of SEQ ID NO: 622 and a second peptide comprising a sequence of SEQ ID NO: 625. In some embodiments, a polypeptide is provided comprising a first peptide of SEQ ID NO: 624 and a second peptide comprising a sequence of SEQ ID NO: 621. In some embodiments, a polypeptide is provided comprising a first peptide of SEQ ID NO: 624 and a second peptide comprising a sequence of SEQ ID NO: 623. In some embodiments, a polypeptide is provided comprising a first peptide of SEQ ID NO: 624 and a second peptide comprising a sequence of SEQ ID NO: 625.
In some embodiments, the polypeptide is referred to as an antibody or antigen binding protein.
In some embodiments, as provided for herein, the MAdCAM antibody, or binding fragment thereof, is linked directly or indirectly to a PD-1 antibody or binding fragment thereof.
In some embodiments, as provided for herein, the MAdCAM antibody, or binding fragment thereof, is linked directly or indirectly to a IL-2 mutein or binding fragment thereof. The IL-2 mutein can be any mutein as provided for herein or other IL-2 muteins known to one of skill in the art.
In some embodiments, if the therapeutic compound comprises a Fc portion, the Fc domain, (portion) bears mutations to render the Fc region “effectorless,” that is unable to bind FcRs. The mutations that render Fc regions effectorless are known. In some embodiments, the mutations in the Fc region, which is according to the known numbering system, are selected from the group consisting of: K322A, L234A, L235A, G237A, L234F, L235E, N297, P331S, or any combination thereof. In some embodiments, the Fc mutations comprises a mutation at L234 and/or L235 and/or G237. In some embodiments, the Fc mutations comprise L234A and/or L235A mutations, which can be referred to as LALA mutations. In some embodiments, the Fc mutations comprise L234A, L235A, and G237A mutations.
Disclosed herein are Linker Region polypeptides, therapeutic peptides, and nucleic acids encoding the polypeptides (e.g. therapeutic compounds), vectors comprising the nucleic acid sequences, and cells comprising the nucleic acids or vectors
Therapeutic compounds can comprise a plurality of specific targeting moieties. In some embodiments, the therapeutic compound comprises a plurality one specific targeting moiety, a plurality of copies of a donor specific targeting moiety or a plurality of tissue specific targeting moieties. In some embodiments, a therapeutic compound comprises a first and a second donor specific targeting moiety, e.g., a first donor specific targeting moiety specific for a first donor target and a second donor specific targeting moiety specific for a second donor target, e.g., wherein the first and second target are found on the same donor tissue. In some embodiments, the therapeutic compound comprises e.g., a first specific targeting moiety for a tissue specific target and a second specific targeting moiety for a second target, e.g., wherein the first and second target are found on the same or different target tissue,
In some embodiments, a therapeutic compound comprises a plurality of effector binding/modulating moieties each comprising an ICIM binding/modulating moiety, the number of ICIM binding/modulating moieties is sufficiently low that clustering of the ICIM binding/modulating moiety's ligand on immune cells (in the absence of target binding) is minimized, e.g., to avoid systemic agonizing of immune cells in the absence of binding of the therapeutic compound to target.
In some embodiments, the therapeutic compound has the formula from N-terminus to C-terminus:
A1---Linker A---A2---Linker B---A3
A3---Linker A---A2---Linker B---A1,
wherein,
A1 and A3, each independently comprises an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, ICSM binding/modulating moiety, or an SM binding/modulating moiety; or a specific targeting moiety, A2 comprises an Fc region or is absent; and
Linker A and Linker B, each are independent linkers.
In some embodiments,
A1 comprises an IL-2 mutein molecule,
A3 comprises a specific targeting moiety, e.g. anti-human MAdCAM Ab, such as a scFv,
A2 comprises an Fc region, and
Linker A and Linker B, each are independent linkers further comprising glycine/serine linkers.
In some embodiments, a polypeptide is provided, wherein the polypeptide comprises a peptide of the formula
Ab-ConstantDomain-LinkerA-IL2Mutein-LinkerB-FcRegion, wherein the Ab is a variable heavy chain domain that binds to MAdCAM, the Constant domain is an Ig constant domain such as IgG1, IgG2, IgG3, or IgG4, Linker A is a linker, such as those provided herein, and the IL2Mutein is an IL-2 mutein, such as those provided for herein. In some embodiments, the variable heavy domain is a variable heavy chain domain as illustrated in Table 7. In some embodiments, the variable heavy chain domain comprises the variable heavy chain domain of Clone ID: 6, 75, or 79 of Table 7. In some embodiments, the variable heavy chain domain comprises the CDRs of the heavy domain of 6, 75, or 79 of Table 7. In some embodiments, the VH comprises a sequence of SEQ ID NO: 414, SEQ ID NO: 591, and SEQ ID NO: 599.
In some embodiments, the ConstantDomain comprises a IgG1 constant domain, such as those provided for herein. In some embodiments, the constant domain comprises mutations to render the constant region “effectorless,” that is unable to bind FcRs. The mutations that render constant regions effectorless are known. In some embodiments, the mutations in the constant region, which is according to the known numbering system, are selected from the group consisting of: K322A, L234A, L235A, G237A, L234F, L235E, N297, P331S, or any combination thereof. In some embodiments, the constant region mutations comprises a mutation at L234 and/or L235 and/or G237. In some embodiments, the constant region mutations comprise L234A and/or L235A mutations, which can be referred to as LALA mutations. In some embodiments, the constant region mutations comprise L234A, L235A, and G237A mutations. In some embodiments, the ConstantDomain comprises SEQ ID NO: 44.
In some embodiments, the variable heavy chain domain comprises a first CDR of SEQ ID NO: 90, a second CDR of SEQ ID NO: 91, and a third CDR of SEQ ID NO: 92. In some embodiments, the variable heavy chain domain comprises a first CDR of SEQ ID NO: 359, a second CDR of SEQ ID NO: 170, and a third CDR of SEQ ID NO: 360. In some embodiments, the variable heavy chain domain comprises a first CDR of SEQ ID NO: 135, a second CDR of SEQ ID NO: 381, and a third CDR of SEQ ID NO: 382. These are illustrative only and the CDR sets as set forth herein and in the tables are also provided.
In some embodiments, the LinkerA is a glycine/serine linker, which can be any glycine/serine linker provided for herein. In some embodiments, the linker is a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) or GGGGSGGGGSGGGGS (SEQ ID NO: 30). These are non-limiting examples and the linker can have varying number of GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29), or a mixture of the two. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 23) and/or GGGGA repeats (SEQ ID NO: 29) repeats. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 30 amino acids in length. In some embodiments, the linker is 35 amino acids in length. In some embodiments, the linker is from 5-50 amino acids in length.
In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 31. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 32. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 33. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 34. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 35. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 36. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 37. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 38. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 39. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 40. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 41. In some embodiments, the IL-2 mutein further comprises a T3A substitution (mutation). In some embodiments, the Fc Region comprises a peptide having a sequence of SEQ ID NO: 21. In some embodiments, the Fc Region comprises a peptide having a sequence of SEQ ID NO: 28. In some embodiments, the C-terminus of the Fc Region is linked to the N-terminus or the C-terminus of the variable heavy chain or IL-2 mutein. In some embodiments, the linker linking the Fc Region to the variable heavy chain or the IL-2 mutein is a glycine/serine or a glycine/alanine linker. In some embodiments, the linker linking the Fc region to the C- or N-terminus of the variable heavy chain or IL-2 mutein is a glycine/serine linker, which can be a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) or GGGGSGGGGSGGGGS (SEQ ID NO: 30). These are non-limiting examples and the linker can have varying number of GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29), or a mixture of the two. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 23) and/or GGGGA repeats (SEQ ID NO: 29) repeats. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 30 amino acids in length. In some embodiments, the linker is 35 amino acids in length. In some embodiments, the linker is from 5-50 amino acids in length.
In some embodiments, the polypeptide further comprises a polypeptide of formula VL-ConstantDomainLight, wherein VL is a variable light chain and ConstantDomainLight is a IgG light chain constant domain, wherein the polypeptide can be or is associated with the polypeptide having the formula of Ab-ConstantDomain-LinkerA-IL2Mutein-LinkerB-FcRegion. In some embodiments, the VL comprises a sequence of SEQ ID NO: 415, SEQ ID NO: 592 or SEQ ID NO: 600. These are illustrative only and the VL domain can be VL/VK sequence provided for herein, such as in Table 7. In some embodiments, the variable light chain domain comprises a first CDR of SEQ ID NO: 93, a second CDR of SEQ ID NO: 87, and a third CDR of SEQ ID NO: 94. In some embodiments, the variable light chain domain comprises a first CDR of SEQ ID NO: 361, a second CDR of SEQ ID NO: 362, and a third CDR of SEQ ID NO: 363. In some embodiments, the variable heavy chain domain comprises a first CDR of SEQ ID NO: 383, a second CDR of SEQ ID NO: 384, and a third CDR of SEQ ID NO: 385. These are illustrative only and the CDR sets as set forth herein and in the tables are also provided.
In some embodiments, the constant domain also comprises mutations to negate the effector function, such as those provided for herein. In some embodiments, the ConstantDomainLight comprises a sequence of:







(SEQ ID NO: 45)


RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN


 


SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF


 


NRGEC






The different polypeptides of formula IL2Mutein-LinkerA-FcRegion-LinkerB-Ab and VL-ConstantDomainLight can be interchanged with one another. In some embodiments, the polypeptide comprises a variable heavy chain comprising a first CDR of SEQ ID NO: 90, a second CDR of SEQ ID NO: 91, and a third CDR of SEQ ID NO: 92 and a variable light chain comprising a first CDR of SEQ ID NO: 93, a second CDR of SEQ ID NO: 87, and a third CDR of SEQ ID NO: 94. In some embodiments, the polypeptide comprises a variable heavy chain comprising a first CDR of SEQ ID NO: 359, a second CDR of SEQ ID NO: 170, and a third CDR of SEQ ID NO: 360 and a variable light chain comprising a first CDR of SEQ ID NO: 361, a second CDR of SEQ ID NO: 362, and a third CDR of SEQ ID NO: 363. In some embodiments, the polypeptide comprises a variable heavy chain comprising a first CDR of SEQ ID NO: 135, a second CDR of SEQ ID NO: 381, and a third CDR of SEQ ID NO: 382 and a variable light chain comprising a first CDR of SEQ ID NO: 383, a second CDR of SEQ ID NO: 384, and a third CDR of SEQ ID NO: 385. These are non-limiting examples and the CDR combinations as illustrated in the MAdCAM Antibody Tables 1 and 2 can be also be used and are provided for herein.
In some embodiments, compounds are provided comprising the following formula, from N-terminus to C-terminus:
IL2Mutein-LinkerA-FcRegion-LinkerB-Ab, wherein the IL2Mutein is any IL-2 mutein that can, for example, preferentially activate Tregs; the LinkerA and Linker B are, each, independently, a linker as provided herein, the Fc Region can any one of such as provided herein, and the Ab is a tissue targeting moiety, such as those provided herein. In some embodiments, the Ab is an antibody that binds to MAdCAM or another cell surface target as provided herein. In some embodiments, the antibody is in a scFV format. In some embodiments, the antibody in scFV format is an antibody as provided in the Table 6. In some embodiments, the antibody in scFV format is an antibody that comprises the CDRs as set forth in Table 6 or Table 7.
In some embodiments, the C-terminus of the IL-2 mutein is linked to the N-terminus of the Fc region. In some embodiments, the linkage is direct or through a linker, such as those described herein. In some embodiments, the linker is a glycine/serine linker. In some embodiments, the linker linking the IL-2 mutein to the Fc region is a glycine/serine linker, which can be a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) or GGGGSGGGGSGGGGS (SEQ ID NO: 30). These are non-limiting examples and the linker can have varying number of GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29), or a mixture of the two. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 23) and/or GGGGA repeats (SEQ ID NO: 29) repeats. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 30 amino acids in length. In some embodiments, the linker is 35 amino acids in length. In some embodiments, the linker is from 5-50 amino acids in length.
In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 31. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 32. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 33. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 34. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 35. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 36. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 37. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 38. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 39. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 40. In some embodiments, the IL-2 mutein comprises a sequence of SEQ ID NO: 41. In some embodiments, the IL-2 mutein further comprises a T3A substitution (mutation). In some embodiments, the Fc Region comprises a peptide having a sequence of SEQ ID NO: 21. In some embodiments, the Fc Region comprises a peptide having a sequence of SEQ ID NO: 28. In some embodiments, the C-terminus of the Fc Region is linked to the N-terminus of the variable heavy chain. In some embodiments, the linker linking the Fc Region to the variable heavy chain is a glycine/serine or a glycine/alanine linker. In some embodiments, the linker linking the Fc region to the N-terminus of the variable heavy chain is a glycine/serine linker, which can be a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) or GGGGSGGGGSGGGGS (SEQ ID NO: 30). These are non-limiting examples and the linker can have varying number of GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29), or a mixture of the two. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 23) and/or GGGGA repeats (SEQ ID NO: 29) repeats. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 30 amino acids in length. In some embodiments, the linker is 35 amino acids in length. In some embodiments, the linker is from 5-50 amino acids in length.
In some embodiments, the variable heavy chain comprises the CDRs as set forth in Table 6 or Table 7. In some embodiments, the variable heavy chain comprises a HCDR1, HCDR2, and a HCDR3, wherein the HCDR1, HCDR2, and a HCDR3 are as set forth in Table 6 or Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 1 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 2 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 3 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 4 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 5 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 6 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 7 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 8 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 9 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 10 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 11 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 12 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 13 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 14 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 15 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 16 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 17 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 1 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 18 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 19 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 20 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 21 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 22 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 23 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 24 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 25 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 26 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 27 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 28 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 29 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 30 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 31 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 32 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 33 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 34 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 35 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 36 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 37 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 38 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 39 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 40 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 41 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 42 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 43 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 44 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 45 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 46 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 47 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 48 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 49 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 50 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 51 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 52 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 53 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 54 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 55 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 56 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 57 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 58 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 59 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 60 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 61 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 62 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 63 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 64 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 65 in Table 6. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 66 in Table 6.
In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 1 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 2 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 3 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 4 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 5 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 6 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 7 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 8 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 9 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 10 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 11 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 12 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 13 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 14 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 15 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 16 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 17 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 1 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 18 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 19 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 20 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 21 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 22 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 23 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 24 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 25 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 26 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 27 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 28 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 29 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 30 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 31 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 32 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 33 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 34 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 35 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 36 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 37 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 38 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 39 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 40 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 41 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 42 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 43 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 44 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 45 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 46 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 47 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 48 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 49 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 50 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 51 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 52 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 53 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 54 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 55 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 56 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 57 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 58 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 59 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 60 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 61 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 62 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 63 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 64 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 65 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 66 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 67 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 68 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 69 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 70 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 71 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 72 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 73 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 74 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 75 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 76 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 77 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 78 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 79 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 80 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 81 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 82 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 83 in Table 7. In some embodiments, the variable heavy chain has a HCDR1, HCDR2, and a HCDR3 as set forth for Clone 84 in Table 7.
In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 1 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 2 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 3 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 4 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 5 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 6 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 7 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 8 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 9 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 10 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 11 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 12 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 13 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 14 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 15 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 16 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 17 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 1 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 18 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 19 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 20 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 21 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 22 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 23 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 24 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 25 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 26 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 27 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 28 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 29 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 30 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 31 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 32 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 33 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 34 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 35 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 36 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 37 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 38 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 39 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 40 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 41 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 42 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 43 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 44 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 45 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 46 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 47 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 48 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 49 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 50 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 51 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 52 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 53 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 54 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 55 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 56 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 57 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 58 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 59 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 60 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 61 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 62 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 63 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 64 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 65 in Table 6. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 66 in Table 6.
In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 1 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 2 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 3 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 4 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 5 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 6 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 7 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 8 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 9 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 10 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 11 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 12 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 13 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 14 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 15 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 16 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 17 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 1 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 18 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 19 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 20 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 21 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 22 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 23 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 24 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 25 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 26 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 27 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 28 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 29 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 30 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 31 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 32 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 33 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 34 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 35 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 36 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 37 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 38 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 39 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 40 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 41 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 42 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 43 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 44 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 45 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 46 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 47 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 48 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 49 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 50 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 51 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 52 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 53 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 54 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 55 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 56 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 57 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 58 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 59 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 60 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 61 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 62 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 63 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 64 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 65 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 66 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 67 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 68 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 69 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 70 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 71 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 72 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 73 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 74 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 75 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 76 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 77 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 78 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 79 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 80 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 81 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 82 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 83 in Table 7. In some embodiments, the variable heavy chain has a LCDR1, LCDR2, and a LCDR3 as set forth for Clone 84 in Table 7.
In some embodiments, the CDRS are swapped for one another. For example, the HCDR1 of clone 1 can be substituted for the HCDR1 of clone 10, or vice versa. This CDR swapping can be done for any of the HCDRs of the clones provided herein (e.g., HCDR1 for HCDR1; HCDR2 for HCDR2; or HCDR3 for HCDR3) or the LCDRs (e.g., LCDR1 for LCDR1; LCDR2 for LCDR2; or LCDR3 for LCDR3). Therefore, in some embodiments, the antibody comprises a HCDR1 as set forth in any of Clones 1-66 of Table 6 or Clones 1-84 of Table 7, a HCDR2 as set forth in any of Clones 1-66 of Table 6 or Clones 1-84 of Table 7, a HCDR3 as set forth in any of Clones 1-66 of Table 6 or Clones 1-84 of Table 7, a LCDR1 as set forth in any of Clones 1-66 of Table 6 or Clones 1-84 of Table 7, a LCDR2 as set forth in any of Clones 1-66 of Table 6 or Clones 1-84 of Table 7, a LCDR3 as set forth in any of Clones 1-66 of Table 6 or Clones 1-84 of Table 7, or a variant of any of the foregoing.
In some embodiments, the MadCAM Antibody is a scFV format as shown in clones 6, 59, or 63. The linker as shown in those sequences is 20 amino acid residues in length, but could also be 5, 10, or 15 amino acid residues in length. In some embodiments, the linker the links the VH and VL(or VK) sequences of the antibody is a glycine/serine linker, which can be a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) or GGGGSGGGGSGGGGS (SEQ ID NO: 30). This is simply a non-limiting example and the linker can have varying number of GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29). In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29) repeats. Thus, the linkers shown in Table 6 are non-limiting examples and can be substituted with any other linkers, such as those provided for herein.
In some embodiments, the polypeptide comprises the formula of:







APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT


 


ELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETT


 


FMCEYADETATIVEFINRWITFSQSIISTLT-Linker1-


 


DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP


 


EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK


 


VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY


 


PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS


 


CSVMHEALHNHYTQKSLSLSPG-Linker2-Ab,






wherein Linker 1, Linker2, and Ab are as provided herein. In some embodiments, Linker 1 is GGGGSGGGGSGGGGS (SEQ ID NO: 30) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22). In some embodiments, Linker 2 is GGGGS (SEQ ID NO: 23). In some embodiments, Linker 2 is GGGGSGGGGS (SEQ ID NO: 619). In some embodiments, Linker 2 is GGGGSGGGGSGGGGS (SEQ ID NO: 30). In some embodiments, Ab is the scFV as set forth in MAdCAM Ab Table 6. In some embodiments, the Ab comprises a sequence of SEQ ID NO: 95. In some embodiments, the Ab comprises a sequence of SEQ ID NO: 364. In some embodiments, the Ab comprises a sequence of SEQ ID NO: 386. In some embodiments, the Ab comprises a VH and a VK or VL segment. In some embodiments, the VH comprises a sequence as set forth in Table 7. In some embodiments, the VK comprises a sequence as set forth in Table 7. In some embodiments, the Ab comprises a VH and a VK as set forth for the clones in Table 7. In some embodiments, the VH and VK are linked by a linker. In some embodiments, the VH and VK are linked by a peptide linker comprising a peptide of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22). In some embodiments, the VH and VK are linked by a peptide linker comprising a peptide of GGGGS. In some embodiments, the VH and VK are linked by a peptide linker comprising a peptide of GGGGSGGGG (SEQ ID NO: 619). In some embodiments, the VH and VK are linked by a peptide linker comprising a peptide of GGGGSGGGGSGGGGS (SEQ ID NO: 30). In some embodiments, the Ab comprises a VH of SEQ ID NO: 414 and a VK of SEQ ID NO: 415. In some embodiments, the Ab comprises a VH of SEQ ID NO: 591 and a VK of SEQ ID NO: 592. In some embodiments, the Ab comprises a VH of SEQ ID NO: 599 and a VK of SEQ ID NO: 600.

In some embodiments, the peptide comprises:







APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKAT


 


ELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETT


 


FMCEYADETATIVEFINRWITFSQSIISTLT-(GGGGSGGGGSGGGGS or


 


GGGGSGGGGSGGGGSGGGGS)-DKTHTCPPCPAPEAAGAPSVFLFPPKPKD


 


TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY


 


RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL


 


PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG


 


SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG-(GGGGS


 


or GGGGSGGGGS or GGGGSGGGGSGGGGS)-Ab,






wherein Ab is set forth as herein. In some embodiments, the Ab comprises a sequence of SEQ ID NO: 95. In some embodiments, the Ab comprises a sequence of SEQ ID NO: 364. In some embodiments, the Ab comprises a sequence of SEQ ID NO: 386. In some embodiments, the Ab comprises a VH and a VK or VL segment. In some embodiments, the VH comprises a sequence as set forth in Table 7. In some embodiments, the VK comprises a sequence as set forth in Table 7. In some embodiments, the Ab comprises a VH and a VK as set forth for the clones in Table 7. In some embodiments, the VH and VK are linked by a linker. In some embodiments, the VH and VK are linked by a peptide linker comprising a peptide of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22). In some embodiments, the VH and VK are linked by a peptide linker comprising a peptide of GGGGS (SEQ ID NO: 23). In some embodiments, the VH and VK are linked by a peptide linker comprising a peptide of GGGGSGGGGS (SEQ ID NO: 619). In some embodiments, the Ab comprises a VH of SEQ ID NO: 414 and a VK of SEQ ID NO: 415. In some embodiments, the Ab comprises a VH of SEQ ID NO: 591 and a VK of SEQ ID NO: 592. In some embodiments, the Ab comprises a VH of SEQ ID NO: 599 and a VK of SEQ ID NO: 600. These examples are non-limiting the combinations of VH and VK as shown in Table 7 are also provided.

In some embodiments, the therapeutic compound or polypeptide comprises a formula of a anti-PD-1 heavy and light chain, wherein the PD-1 heavy chain is linked to a MAdCAM antibody (scFV), such as those provided herein at the C-terminus of the PD-1 IgG heavy chain. The polypeptide can have the formula of A1-A2-Linker1-A4-Linker2-A5 and A6, wherein A1 is a PD-1 heavy chain, A6 is a PD-1 light chain; A2 is a IgG constant domain (e.g. IgG1 Constant domain), Linker 1 is as provided herein, such as, but not limited to, a glycine/serine linker, which can be a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) or GGGGSGGGGSGGGGS (SEQ ID NO: 30), which are simply a non-limiting example and the linker can have varying number of GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29) and in some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29) repeats; A4 is VH domain, such as those set forth in Table 7; Linker 2 is as provided herein, such as, but not limited to, a glycine/serine linker, which can be a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) or GGGGSGGGGSGGGGS (SEQ ID NO: 30), which are simply a non-limiting example and the linker can have varying number of GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29) and in some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29) repeats; and A5 is VK/VL domain, such as those set forth in Table 7. In some embodiments, Linker 2 is GGGGSGGGGSGGGGS (SEQ ID NO: 30). In some embodiments, the A4-Linker2-A5 is a scFV antibody, such as those set forth in Table 6. The linkers shown in Table 6 can be substituted with the linker of GGGGSGGGGSGGGGS (SEQ ID NO: 30). In some embodiments, the A4-Linker2-A5 comprises the HCDR sets (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) sets as set forth in Table 6 or Table 7. For the avoidance of doubt, a CDR set refers to the CDRs illustrated for each of the different antibody clones provided for in the tables. In some embodiments, A4 comprises a peptide of SEQ ID NO: 414 and A5 comprises a peptide of SEQ ID NO: 415. In some embodiments, A4 comprises a peptide of SEQ ID NO: 591 and A5 comprises a peptide of SEQ ID NO: 592. In some embodiments, A4 comprises a peptide of SEQ ID NO: 599 and A5 comprises a peptide of SEQ ID NO: 600. These examples are non-limiting the combinations of VH and VK as shown in Table 7 are also provided.
In some embodiments, A2 comprises a sequence of







(SEQ ID NO: 44)


ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH


 


TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS


 


CDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED


 


PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC


 


KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF


 


YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF


 


SCSVMHEALHNHYTQKSLSLSPG






Once expressed the heavy and light chains of the PD-1 antibody bind to one another to form the compound comprising the anti-PD-1 antibody linked to the anti-MAdCAM antibody. The anti-MAdCAM antibody can be any antibody that binds to MAdCAM, such as those provided for herein.

Polypeptides Derived from Reference, e.g., Human Polypeptides

In some embodiments, a component of a therapeutic molecule is derived from or based on a reference molecule, e.g., in the case of a therapeutic molecule for use in humans, from a naturally occurring human polypeptide. E.g., In some embodiments, all or a part of a CD39 molecule, a CD73 molecule, a cell surface molecule binder, a donor specific targeting moiety, an effector ligand binding molecule, an ICIM binding/modulating moiety, an IIC binding/modulating moiety, an inhibitory immune checkpoint molecule ligand molecule, an inhibitory molecule counter ligand molecule, a SM binding/modulating moiety, a specific targeting moiety, a target ligand binding molecule, or a tissue specific targeting moiety, can be based on or derived from a naturally occurring human polypeptide. E.g., a PD-L1 molecule can be based on or derived from a human PD-L1 sequence.
In some embodiments, a therapeutic compound component, e.g., a PD-L1 molecule:

    
    
        a) comprises all or a portion of, e.g., an active portion of, a naturally occurring form of the human polypeptide;
        b) comprises all or a portion of, e.g., an active portion of, a human polypeptide having a sequence appearing in a database, e.g., GenBank database, on Jan. 11, 2017, a naturally occurring form of the human polypeptide that is not associated with a disease state;
        c) comprises a human polypeptide having a sequence that differs by no more than 1, 2, 3, 4, 5, 10, 20, or 30 amino acid residues from a sequence of a) or b);
        d) comprises a human polypeptide having a sequence that differs at no more than by 1, 2, 3, 4, 5 10, 20, or 30% its amino acids residues from a sequence of a) or b);
        e) comprises a human polypeptide having a sequence that does not differ substantially from a sequence of a) or b); or
        f) comprises a human polypeptide having a sequence of c), d), or e) that does not differ substantially in a biological activity, e.g., ability to enhance or inhibit an immune response, from a human polypeptide having the sequence of a) or b).
    
    


In some embodiments, therapeutic compounds can comprise a plurality of effector binding/modulating moieties. For example, a therapeutic compound can comprise two or more of the following selected from:
(a) an ICIM binding/modulating moiety; (b) an IIC binding/modulating moiety; (c) an SM binding/modulating moiety, or (d) an ICSM binding/modulating moiety. In some embodiments, for example, a therapeutic compound can comprise a plurality, e.g., two, ICIM binding/modulating moieties (wherein they are the same or different); by way of example, two that activate or agonize PD-1; a plurality, e.g., two, IIC binding/modulating moieties; (wherein they are the same or different); a plurality, e.g., two, SM binding/modulating moieties (wherein they are the same or different), or a plurality, e.g., tow, ICSM binding/modulating moieties (wherein they are the same or different). In some embodiments, the therapeutic compound can comprise an ICIM binding/modulating moiety and an IIC binding/modulating moiety; an ICIM binding/modulating moiety and an SM binding/modulating moiety; an IIC binding/modulating moiety and an SM binding/modulating moiety, an ICIM binding/modulating moiety and an ICSM binding/modulating moiety; an IIC binding/modulating moiety and an ICSM binding/modulating moiety; or an ICSM binding/modulating moiety and an SM binding/modulating moiety. In some embodiments, the therapeutic compound comprises a plurality of targeting moieties. In some embodiments, the targeting moieties can be the same or different.
Pharmaceutical Compositions and Kits
In another aspect, the present embodiments provide compositions, e.g., pharmaceutically acceptable compositions, which include a therapeutic compound described herein, formulated together with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, local, ophthalmic, topical, spinal or epidermal administration (e.g. by injection or infusion). As used herein, the term “carrier” means a diluent, adjuvant, or excipient with which a compound is administered. In some embodiments, pharmaceutical carriers can also 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 carriers can also be 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. The carriers can be used in pharmaceutical compositions comprising the therapeutic compounds provided for herein.
The compositions and compounds of the embodiments provided for herein may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions. In some embodiments, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the therapeutic molecule is administered by intravenous infusion or injection. In another embodiment, the therapeutic molecule is administered by intramuscular or subcutaneous injection. In another embodiment, the therapeutic molecule is administered locally, e.g., by injection, or topical application, to a target site. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
Therapeutic compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high therapeutic molecule concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., therapeutic molecule) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
In certain embodiments, a therapeutic compound can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. Therapeutic compositions can also be administered with medical devices known in the art.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a therapeutic compound is 0.1-30 mg/kg, more preferably 1-25 mg/kg. Dosages and therapeutic regimens of the therapeutic compound can be determined by a skilled artisan. In certain embodiments, the therapeutic compound is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 40 mg/kg, e.g., 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, 1 to 10 mg/kg, 5 to 15 mg/kg, 10 to 20 mg/kg, 15 to 25 mg/kg, or about 3 mg/kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks. In one embodiment, the therapeutic compound is administered at a dose from about 10 to 20 mg/kg every other week. The therapeutic compound can be administered by intravenous infusion at a rate of more than 20 mg/min, e.g., 20-40 mg/min, and typically greater than or equal to 40 mg/min to reach a dose of about 35 to 440 mg/m2, typically about 70 to 310 mg/m2, and more typically, about 110 to 130 mg/m2. In embodiments, the infusion rate of about 110 to 130 mg/m2 achieves a level of about 3 mg/kg. In other embodiments, the therapeutic compound can be administered by intravenous infusion at a rate of less than 10 mg/min, e.g., less than or equal to 5 mg/min to reach a dose of about 1 to 100 mg/m2, e.g., about 5 to 50 mg/m2, about 7 to 25 mg/m2, or, about 10 mg/m2. In some embodiments, the therapeutic compound is infused over a period of about 30 min. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

The pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of a therapeutic molecule. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a therapeutic molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of a therapeutic molecule t is outweighed by the therapeutically beneficial effects. A “therapeutically effective dosage” preferably inhibits a measurable parameter, e.g., immune attack at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit a measurable parameter, e.g., immune attack, can be evaluated in an animal model system predictive of efficacy in transplant rejection or autoimmune disorders. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.

A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
Also within the scope of the embodiments is a kit comprising a therapeutic compound described herein. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, a therapeutic molecule to a label or other therapeutic agent, or a radioprotective composition; devices or other materials for preparing the a therapeutic molecule for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.
In some embodiments, embodiments provided herein also include, but are not limited to:
1. A therapeutic compound comprising:
i) a specific targeting moiety selected from:

    
    
        a) a donor specific targeting moiety which, e.g., preferentially binds a donor target; or
        b) a tissue specific targeting moiety which, e.g., preferentially binds target tissue of a subject; and
    
    


ii) an effector binding/modulating moiety selected from:

    
    
        (a) an immune cell inhibitory molecule binding/modulating moiety (ICIM binding/modulating moiety);
        (b) an immunosuppressive immune cell binding/modulating moiety (IIC binding/modulating moiety); or
        (c) an effector binding/modulating moiety that, as part of a therapeutic compound, promotes an immuno-suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target (SM binding/modulating moiety).

2. The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety directly binds and activates an inhibitory receptor.

3. The therapeutic compound of embodiment 2, wherein the effector binding/modulating moiety is an inhibitory immune checkpoint molecule.

4. The therapeutic compound of any of embodiments 1-3, wherein the effector binding/modulating moiety is expressed by an immune cell.

5. The therapeutic compound of embodiment 4, wherein the immune cell contributes to an unwanted immune response.

6. The therapeutic compound of embodiments 4 or 5, wherein the immune cell causes a disease pathology.

7. The therapeutic compound of embodiment 1, wherein the ability of the therapeutic molecule to agonize the molecule to which the effector binding/modulating binds is greater, e.g., 2, 5, 10, 100, 500, or 1,000 times greater, when the therapeutic compound is bound to a target through the targeting moiety than when the therapeutic compound is not bound to target through the targeting moiety.

8. The therapeutic compound of embodiments 1-7, wherein when binding as a monomer (or binding when the therapeutic compound is not multimerized), to its cognate ligand, e.g., an inhibitory immune checkpoint molecule, does not agonize or substantially agonize, the cognate ligand.

9. The therapeutic compound of embodiments 1-8, wherein at a therapeutically effective dose of the therapeutic compound, there is significant, systemic agonization of the molecule to which the effector binding/modulating moiety binds.

10. The therapeutic compound of embodiments 1-9, wherein at a therapeutically effective dose of the therapeutic compound, the agonization of the molecule to which the effector binding/modulating moiety binds occurs substantially only at a target site to which the targeting moiety binds to.

11. The therapeutic compound of embodiments 1-9, wherein binding of the therapeutic compound to its cognate ligand, e.g., an inhibitory immune checkpoint molecule, does not inhibit, or does not substantially inhibit, binding of an endogenous counter ligand to the cognate ligand, e.g., an inhibitory immune checkpoint molecule.

12. The therapeutic compound of embodiments 1-11, wherein binding of the effector binding/modulating moiety to its cognate ligand, inhibits the binding of an endogenous counter ligand to the cognate ligand of the effector binding/modulating moiety by less than 60, 50, 40, 30, 20, 10, or 5%.

13. The therapeutic compound of embodiments 1-12, wherein binding of the effector binding/modulating moiety to its cognate ligand, inhibits the binding of an endogenous counter ligand to the cognate ligand of the effector binding/modulating moiety by less than 5%.

14. The therapeutic compound of embodiments 1-11, wherein binding of the effector binding/modulating moiety to the cognate ligand, results in substantially no antagonism of the cognate ligand of the effector binding/modulating molecule.

15. The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises an ICIM binding/modulating moiety.

16. The therapeutic compound of embodiment 15, wherein the effector binding/modulating moiety comprises an ICIM binding/modulating moiety comprising an inhibitory immune checkpoint molecule ligand molecule.

17. The therapeutic compound of embodiment 16, wherein the inhibitory immune molecule counter-ligand molecule comprises a PD-L1 molecule.

18. The therapeutic compound of embodiment 15, wherein the ICIM is wherein the inhibitory immune molecule counter ligand molecule engages a cognate inhibitory immune checkpoint molecule selected from PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4.

19. The therapeutic compound of embodiment 18, wherein the ICIM is an antibody.

20. The therapeutic compound of embodiment 18, wherein the ICIM comprises an antibody that binds to PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4.

21. The therapeutic compound of embodiment 20, wherein the antibody is an antibody that binds to PD-1.

22. The therapeutic compound of embodiment 20, wherein the antibody is an antibody that binds to PD-1 and is a PD-1 agonist.

23. The therapeutic compound of embodiment 20, wherein the antibody is an antibody that binds to PD-1 and is a PD-1 agonist when tethered at a target site.

24. The therapeutic compound of embodiment 16, wherein the inhibitory immune molecule counter-ligand molecule comprises a HLA-G molecule.

25. The therapeutic compound of embodiment 15, wherein the ICIM is wherein the inhibitory immune molecule counter ligand molecule engages a cognate inhibitory immune checkpoint molecule selected from PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4.

26. The therapeutic compound of embodiment 15, wherein the inhibitory immune molecule counter ligand molecule engages a cognate inhibitory immune checkpoint molecule selected from Table 1.

27. The therapeutic compound of embodiment 15, wherein when binding as a monomer, to its cognate inhibitory immune checkpoint molecule, does not agonize or substantially agonize the inhibitory immune checkpoint molecule.

28. The therapeutic compound of embodiment 15, wherein the inhibitory immune molecule counter ligand has at least 60, 70, 80, 90, 95, 99, or 100% homology with a naturally occurring inhibitory immune checkpoint molecule ligand.

29 The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises a ICIM binding/modulating moiety which comprises a functional antibody molecule to a cell surface inhibitory molecule.

30. The therapeutic compound of embodiment 1, wherein the cell surface inhibitory molecule is an inhibitory immune checkpoint molecule.

31. The compound of embodiment 30, wherein the inhibitory immune checkpoint molecule is selected from PD-1, KIR2DL4, LILRB1, LILRB2, CTLA-4, or selected from Table 1.

32. The therapeutic compound of any of embodiments 1-31, wherein the level of systemic immune suppression at a therapeutically effective dose of the therapeutic compound, is less than that given by the standard of care with a systemic immune suppressant (if relevant), or is less than that given by an equimolar amount of free (not as a component of a therapeutic compound), effector binding/modulating molecule.

33. The therapeutic compound of embodiment 1-32, wherein the level of systemic immune activation, e.g., at a therapeutically effective dose of the therapeutic compound, is less than that given by a equimolar amount of free (not as a component of a therapeutic compound), effector binding/modulating molecule.

34. The therapeutic compound of any one of embodiments 1-33, further comprising a second effector binding/modulating moiety.

35. The therapeutic compound of embodiment 34, wherein the second effector binding/modulating moiety, binds a different target than the effector binding/modulating moiety.

36. The therapeutic compound embodiments 34 or 35, wherein the second effector binding/modulating moiety comprises a IIC binding/modulating moiety.

The therapeutic compound embodiments 34 or 35, wherein the second effector binding/modulating moiety comprises an SM binding/modulating moiety.

37. The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises an IIC binding/modulating moiety.

38. The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises an IIC binding/modulating moiety, which, increases, recruits or accumulates an immunosuppressive immune cell at the target site.

39. The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises a cell surface molecule binder which binds or specifically binds, a cell surface molecule on an immunosuppressive immune cell.

40. The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises a cell surface molecule ligand molecule that binds or specifically binds, a cell surface molecule on an immunosuppressive immune cell.

41. The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises an antibody molecule that binds a cell surface molecule on an immunosuppressive immune cell.

42. The therapeutic compound of any of embodiments 38-41, wherein the immunosuppressive immune cell comprises a T regulatory cell, such as a Foxp3+CD25+T regulatory cell.

43. The therapeutic compound of any of embodiments 1-42, wherein the effector binding/modulating moiety binds GARP, and e.g., comprises an antibody molecule that binds GARP on GARP expressing immunosuppressive cells, e.g., Tregs.

44. The therapeutic compound of embodiment 1, wherein the effector binding/modulating moiety comprises an SM binding/modulating moiety.

45. The therapeutic compound of embodiment 44, wherein SM binding/modulating moiety promotes an immuno-suppressive local microenvironment.

46. The therapeutic compound of any of embodiments 44 and 45, wherein the effector molecule binding moiety increases the availability, e.g., by increasing the local concentration or amount, of a substance which inhibits immune cell function, e.g., a substance that inhibits the activation of an immune cell or the function of an activated immune cell.

47. The therapeutic compound of any of embodiments 44-46, wherein the effector molecule binding moiety binds and accumulate a soluble substance, e.g., an endogenous or exogenous substance, having immunosuppressive function.

48. The therapeutic compound of any of embodiments 44-47, wherein the effector molecule binding moiety decreases the availability, e.g., by decreasing the local concentration or amount, or sequestering, of a substance which promotes immune cell function, e.g., a substance that promotes the activation of an immune cell or the function of an activated immune cell.

49. The therapeutic compound of any one of embodiments 44-48, wherein SM binding/modulating moiety promotes an immuno-suppressive local microenvironment, e.g., by providing in the proximity of the target, a substance that inhibits or minimizes attack by the immune system of the target.

50. The therapeutic compound of any one of embodiments 44-49, wherein the SM binding/modulating moiety comprises a molecule that inhibits or minimizes attack by the immune system of the target.

51. The therapeutic compound any one of embodiments 44-50, wherein the SM binding/modulating moiety binds and/or accumulate a soluble substance, e.g., an endogenous or exogenous substance having immunosuppressive function.

52. The therapeutic compound any one of embodiments 44-51, wherein the SM binding/modulating moiety binds and/or inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble substance, typically and endogenous soluble substance, that promotes immune attack.

53. The therapeutic compound any one of embodiments 44-52, wherein the effector molecule binding moiety decreases the availability of ATP or AMP.

54. The therapeutic compound any one of embodiments 44-53, wherein SM binding/modulating moiety binds, or comprises, a substance, e.g., CD39 or CD73, that depletes a component that promotes immune effector cell function, e.g., ATP or AMP.

55. The therapeutic compound any one of embodiments 44-54, wherein the SM binding/modulating moiety comprises a CD39 molecule.

56. The therapeutic compound any one of embodiments 44-54, wherein the SM binding/modulating moiety comprises a CD73 molecule.

57. The therapeutic compound any one of embodiments 44-54, wherein the SM binding/modulating moiety comprises an anti-CD39 molecule.

58. The therapeutic compound any one of embodiments 44-54, wherein the SM binding/modulating moiety comprises an anti-CD73 antibody molecule.

59. The therapeutic compound any one of embodiments 44-54, wherein the effector molecule binding moiety comprises an immune-suppressive substance, e.g. a fragment an immunosuppressive protein.

60. The therapeutic compound any one of embodiments 44-54, wherein SM binding/modulating moiety comprises alkaline phosphatase molecule.

61. The therapeutic compound of embodiment 1, wherein the compound has the formula from N-terminus to C-terminus:

    
    


R1---Linker Region A-R2 or R3-Linker Region B-R4,
wherein,
R1, R2, R3, and R4, each independently comprises an effector
binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC
binding/modulating moiety, or an SM binding/modulating moiety; a specific targeting moiety; or is absent; provided that an effector binding/modulating moiety and a specific targeting moiety are present.
62. The therapeutic compound of embodiment 61, wherein each of Linker Region A and Linker Region B comprises an Fc region.
63. The therapeutic compound of embodiment 61, wherein one of R1 and R2 is anti-PD-1 antibody and one of R1 and R2 is an anti-MAdCAM antibody.
64. The therapeutic compound of embodiment 61, wherein one of R1 is anti-PD-1 antibody and one R2 is an anti-MAdCAM antibody.
65. The therapeutic compound of embodiment 61, wherein one of R1 is anti-MAdCAM antibody and one R2 is an anti-PD-1 antibody.
66. The therapeutic compound of embodiment 61, wherein one of R3 and R4 is anti-PD-1 antibody and one of R3 and R4 is an anti-MAdCAM antibody.
67. The therapeutic compound of embodiment 61, wherein one of R3 is anti-PD-1 antibody and one R4 is an anti-MAdCAM antibody.
68. The therapeutic compound of embodiment 61, wherein one of R3 is anti-MAdCAM antibody and one R4 is an anti-PD-1 antibody.
69. The therapeutic compound of any of embodiments 61-68, wherein the linker is absent.
70. The therapeutic compound of any of embodiments 61-68, wherein the linker is a Fc region.
71. The therapeutic compound of any of embodiments 61-68, wherein the linker is a glycine/serine linker, such as 1, 2, 3, 4, or 5 repeats of GGGGS (SEQ ID NO: 23).
72. The therapeutic compound of any of embodiments 61-68, wherein the linker comprises a Fc region and a glycine/serine linker, such as 1, 2, 3, 4, or 5 repeats of GGGGS (SEQ ID NO: 23).
73. The therapeutic compound of any of embodiments 61-72, wherein the PD-1 antibody is a PD-1 agonist.
74. The therapeutic compound of embodiment 61, wherein:
R1 and R3 independently comprise a functional anti-PD-1 antibody molecule (an agonist of PD-1); and R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
75. The therapeutic compound of any of embodiments 73 and 74, wherein:
R1 and R3 independently comprise specific targeting moieties, e.g., an anti-tissue antigen antibody; and R2 and R4 independently comprise a functional anti-PD-1 antibody molecule (an agonist of PD-1).
76. The therapeutic compound of any of embodiments 73 and 74, wherein:
R1, R2, R3 and R4 each independently comprise: an SM binding/modulating moiety which modulates, e.g., binds and inhibits, sequesters, degrades or otherwise neutralizes a substance, e.g., a soluble molecule that modulates an immune response, e.g., ATP or AMP, e.g., a CD39 molecule or a CD73 molecule; a specific targeting moiety; or is absent;

provided that an SM binding/modulating moiety and a specific targeting moiety are present.

77. The therapeutic compound of embodiment 61, wherein:

R1 and R3 independently comprise a CD39 molecule or a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

78. The therapeutic compound of embodiment 77, wherein:

R1 and R3 each comprises a CD39 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

79. The therapeutic compound of embodiments 61 or 77, wherein:

R1 and R3 each comprises a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

80. The therapeutic compound of embodiment 61, wherein:

one of R1 and R3 comprises a CD39 molecule and the other comprises a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

81. The therapeutic compound of embodiment 61, wherein:

R1, R2, R3 and R4 each independently comprise: an HLA-G molecule; a specific targeting moiety; or is absent;

provided that an HLA-G molecule and a specific targeting moiety are present.

82. The therapeutic compound of embodiments 61 or 81, wherein:

R1 and R3 each comprise an HLG-A molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

83. The therapeutic compound of any of embodiments 81 and 82, wherein:

R1 and R3 each comprise an agonistic anti-LILRB1 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

84. The therapeutic compound of any of embodiments 81 and 82, wherein:

R1 and R3 each comprise an agonistic anti-KIR2DL4 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g., self-pairing Fc moieties or Fc moieties that do not, or do not substantially self-pair).

85. The therapeutic compound of any of embodiments 81-84, wherein:

R1 and R3 each comprise an agonistic anti-LILRB2 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

86. The therapeutic compound of any of embodiments 81-84, wherein:

R1 and R3 each comprise an agonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

87. The therapeutic compound of any of embodiments 81-84, wherein:

one of R1 and R3 comprises a first moiety chosen from, and the other comprises a different moiety chosen from: an antagonistic anti-LILRB1 antibody molecule, an agonistic anti-KR2DL4 antibody molecule, and an agonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

88. The therapeutic compound of any of embodiments 81-84, wherein:

one of R1 and R3 comprises an antagonistic anti-LILRB1 antibody molecule and the other comprises an agonistic anti-KR2DL4 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

89. The therapeutic compound of any of embodiments 81-84, wherein:

one of R1 and R3 comprises an antagonistic anti-LILRB1 antibody molecule and the other comprises an agonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.

90. The therapeutic compound of any of embodiments 81-84 wherein:

R1, R2, R3 and R4 each independently comprise: an IL-2 mutein molecule; a specific targeting moiety; or is absent; and
provided that an IL-2 mutein molecule and a specific targeting moiety are present.
91. The therapeutic compound of embodiment 90, wherein:
R1 and R3 each comprise an IL-2 mutein molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
92. The therapeutic compound of embodiments 90 or 91, wherein:
one of R1 and R3 comprises a MAdCAM binding molecule, e.g., an anti-MAdCAM antibody molecule or a GITR binding molecule, e.g., an anti-GITR antibody molecule and the other comprises an IL-2 mutein molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
93. The therapeutic compound of embodiments 90 or 91, wherein:
one of R1 and R3 comprises a GARP binding molecule, e.g., an anti-GARP antibody molecule and the other comprises an IL-2 mutein molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
94. The therapeutic compound of embodiments 90 or 91, wherein:
one of R1 and R3 comprises a GARP binding molecule, e.g., an anti-GARP antibody molecule or a GITR binding molecule, e.g., an anti-GITR antibody molecule and the other comprises an IL-2 mutein molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
95. The therapeutic compound of embodiments 90 or 91, wherein:
one of R1 and R3 comprises a GARP binding molecule, e.g., an anti-GARP antibody molecule and the other comprises an IL-2 mutein molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
96. The therapeutic compound of embodiments 90 or 91, wherein:
one of R1 and R3 comprises a GITR binding molecule, e.g., an anti-GITR antibody molecule, and the other comprises an IL-2 mutein molecule; and
R2 and R4 independently comprise specific targeting moieties, e.g., scFv molecules against a tissue antigen.
97. The therapeutic compound of embodiment 1, wherein the compound is a polypeptide or protein, wherein the polypeptide or protein comprises a targeting moiety that binds to a target cell and an effector binding/modulating moiety, wherein the effector binding/modulating moiety is a IL-2 mutant polypeptide (IL-2 mutein).

98. The therapeutic compound of embodiment 97, wherein the targeting moiety comprises an antibody that binds to a target protein on the surface of a target cell.

99. The therapeutic compound of embodiment 98, wherein the antibody is an antibody that binds to MAdCAM, OAT1 (SLC22A6), OCT2 (SLC22A2), FXYD2, TSPAN7, DPP6, HEPACAM2, TMEM27, or GPR119.

100. The therapeutic compound of embodiment 98, wherein the IL-2 mutein binds to a receptor expressed by an immune cell.

101. The therapeutic compound of embodiment 98, wherein the immune cell contributes to an unwanted immune response.

102. The therapeutic compound of any of embodiments 97-101, wherein the immune cell causes a disease pathology.

103. The therapeutic compound of any of embodiments 97-102, wherein the targeting moiety comprises an anti-MAdCAM antibody.

104. The therapeutic compound of embodiment 97, wherein the compound has the formula from N-terminus to C-terminus:

R1---Linker Region A-R2 or R3-Linker Region B-R4,
wherein,
R1, R2, R3, and R4, each independently comprises the effector
binding/modulating moiety, the targeting moiety, or is absent.
105. The therapeutic compound of embodiment 104, wherein each of Linker Region A and Linker Region B comprises an Fc region.
106. The therapeutic compound of embodiments 104 or 105 or, wherein one of R1 and R2 is the IL-mutein antibody and one of R1 and R2 is an anti-MAdCAM antibody.
107. The therapeutic compound of embodiments 104, 105, or 106, wherein R1 is the IL-mutein and R2 is an anti-MAdCAM antibody.
108. The therapeutic compound of embodiments 104, 105, or 106, wherein one of R1 is anti-MAdCAM antibody and one R2 is an anti-PD-1 antibody.
109. The therapeutic compound of embodiments 104, 105, or 106, wherein one of R3 and R4 is the IL-2 mutein and one of R3 and R4 is an anti-MAdCAM antibody.
110. The therapeutic compound of embodiments 104, 105, or 106, wherein R3 is the IL-2 mutein and R4 is an anti-MAdCAM antibody.
111. The therapeutic compound of embodiments 104, 105, or 106, wherein R3 is an anti-MAdCAM antibody and one R4 is the IL-2 mutein.
112. The therapeutic compound of any of embodiments 104-111, wherein the linker is absent.
113. The therapeutic compound of any of embodiments 104-111, wherein the linker is or comprises a Fc region.
114. The therapeutic compound of any of embodiments 104-111, wherein the linker comprises a glycine/serine linker.
115. The therapeutic compound of any of embodiments 104-111, wherein the linker comprises a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 30), GGGGSGGGGS (SEQ ID NO: 619), or GGGGS (SEQ ID NO: 23).
116. The therapeutic compound of embodiment 97, wherein the IL-2 mutein comprises a IL-2 sequence of SEQ ID NO: 6, wherein peptide comprises a mutation at a position that corresponds to position 53, 56, 80, or 118 of SEQ ID NO: 6.
117. The therapeutic compound of any of embodiments 97-116, wherein the IL-2 mutein comprises a IL-2 sequence of SEQ ID NO: 6, wherein peptide comprises a mutation at a position that corresponds to position 53, 56, 80, or 118 of SEQ ID NO: 6.
118. The therapeutic compound of embodiment 116, wherein the mutation is a L to I mutation at position 53, 56, 80, or 118.
119. The therapeutic compound of embodiment 117, wherein the mutation is a L to I mutation at position 53, 56, 80, or 118.
120. The therapeutic compound of any of embodiments 97-119, wherein the IL-2 mutein further comprises a mutation at one or more positions of 29, 31, 35, 37, 48, 69, 71, 74, 88, and 125 corresponding to those positions in SEQ ID NO: 6.
121. The therapeutic compound of any of embodiments 97-120, wherein the IL-2 mutein further comprises a mutation at one or more of positions E15, H16, Q22, D84, E95, or Q126 or 1, 2, 3, 4, 5, or each of positions E15, H16, Q22, D84, E95, or Q126 is wild-type.

122. The therapeutic compound of any of embodiments 97-121, wherein the mutation in the mutein is one or more of E15Q, H16N, Q22E, D84N, E95Q, or Q126E.

123. The therapeutic compound of any of embodiments 97-122, wherein the mutein comprises a N29S mutation in SEQ ID NO: 6.

124. The therapeutic compound of any of embodiments 97-123, wherein the mutein comprises a Y31S or a Y51H mutation.

125. The therapeutic compound of any of embodiments 97-124, wherein the mutein comprises a K35R mutation.

126. The therapeutic compound of any of embodiments 97-125, wherein the mutein comprises a T37A mutation.

127. The therapeutic compound of any of embodiments 97-126, wherein the mutein comprises a K48E mutation.

128. The therapeutic compound of any of embodiments 97-127, wherein the mutein comprises a V69A mutation.

129. The therapeutic compound of any of embodiments 97-128, wherein the mutein comprises a N71R mutation.

130. The therapeutic compound of any of embodiments 97-129, wherein the mutein comprises a Q74P mutation.

131. The therapeutic compound of any of embodiments 97-130, wherein the mutein comprises a N88D or a N88R mutation.

132. The therapeutic compound of any of embodiments 97-131, wherein the mutein comprises a C125A or C125S mutation.

133. The therapeutic compound of any of embodiments 97-132, wherein the IL-2 mutein is fused or linked to a Fc peptide.

134. The therapeutic compound of embodiment 133, wherein the Fc peptide comprises a mutation at one or more of positions of L234, L247, L235, L248, G237, and G250.

135. The therapeutic compound of embodiment 134, wherein the mutation is L to A or G to A mutation.

136. The therapeutic compound of embodiment 134, wherein the Fc peptide comprises L247A, L248A, and/or a G250A mutations (Kabat numbering).

137. The therapeutic compound of embodiment 134, wherein the Fc peptide comprises a L234A mutation, a L235A mutation, and/or a G237A mutation (EU numbering).

138. The therapeutic compound of embodiment 97, wherein the compound comprises a polypeptide comprising a first chain and a second chain that form the polypeptide, wherein

the first chain comprises:
VH-Hc-Linker-C1, wherein VH is a variable heavy domain that binds to the target cell with a VL domain of the second chain; Hc is a heavy chain of antibody comprising CH1-CH2-CH3 domain, the Linker is a glycine/serine linker, and C1 is a IL-2 mutein fused or linked to a Fc protein in either the N-terminal or C-terminal orientation; and
the second chain comprises:
VL-Lc, wherein VL is a variable light chain domain that binds to the target cell with the VH domain of the first chain, and the Lc domain is a light chain CK domain.
139. The therapeutic compound of embodiment 138, wherein the VH and VL domain are anti-MAdCAM variable domains that bind to MAdCAM expressed on a cell.
140. The therapeutic compound of embodiment 138 or 139, wherein the IL-2 mutein comprises a mutation at a position that corresponds to position 53, 56, 80, or 118 of SEQ ID NO: 6.
141. The therapeutic compound of embodiment 140, wherein the mutation is a L to I mutation at position 53, 56, 80, or 118.
142. The therapeutic compound of embodiments 140 or 141, wherein the mutein further comprises a mutation at a position that corresponds to position 69, 75, 88, and/or 125, or any combination thereof.
143. The therapeutic compound of embodiments 140 or 141, wherein the IL-2 mutein comprises a mutation selected from the group consisting of: at one of L53I, L56I, L80I, and L118I and the mutations of V69A, Q74P, N88D or N88R, and optionally C125A or C125S.
144. The therapeutic compound of embodiment 143, wherein the IL-2 mutein comprises a L53I mutation.
145. The therapeutic compound of embodiment 143, wherein the IL-2 mutein comprises a L56I mutation.
146. The therapeutic compound of embodiment 143, wherein the IL-2 mutein comprises a L80I mutation.
147. The therapeutic compound of embodiment 143, wherein the IL-2 mutein comprises a L118I mutation.
148. The therapeutic compound of embodiment 143, wherein the IL-2 mutein does not comprises any other mutations.
149. The therapeutic compound of any one of embodiments 138-148, wherein the Fc protein comprises L247A, L248A, and G250A mutations or a L234A mutation, a L235A mutation, and/or a G237A mutation according to KABAT numbering.
150. The therapeutic compound of any one of embodiments 138-149, wherein the Linker comprises a sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 30) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22).
151. The therapeutic compound of any one of embodiments 138-149, wherein the polypeptide comprises a Fc peptide comprising a sequence described herein.
152. The therapeutic compound of any of embodiments 81-84, wherein:
one of R1, R2, R3 and R4 comprises an anti-BCR antibody molecule, e.g., an antagonistic anti-BCR antibody molecule, one comprises an anti FCRL antibody molecule, and one comprises specific targeting moiety.
153. The therapeutic compound of embodiment 152, wherein:
the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRL antibody molecule, directed to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6. 154. The therapeutic compound of any of embodiments 81-84, wherein:
R1, R2, R3 and R4 each independently comprise:
i) an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, or an SM binding/modulating moiety, that minimizes or inhibits T cell activity, expansion, or function (a T cell effector binding/modulating moiety);

ii) an effector binding/modulating moiety, e.g., an ICIM binding/modulating moiety, an IIC binding/modulating moiety, or an SM binding/modulating moiety, that minimizes or inhibits B cell activity, expansion, or function (a B cell effector binding/modulating moiety);

iii) a specific targeting moiety; or

iv) is absent; provided that, a T cell effector binding/modulating moiety, a B cell effector

binding/modulating moiety, and a specific targeting moiety are present.

155. The therapeutic compound of embodiment 154, wherein:

one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody and one comprises an HLA-G molecule.

156. The therapeutic compound embodiments 154-155, wherein:

one of R1, R2, R3, and R4 comprises an SM binding/modulating moiety, e.g., a CD39 molecule or a CD73 molecule.

157. The therapeutic compound of any of embodiments 154-156, wherein:

one of R1, R2, R3, and R4 comprises an entity that binds, activates, or maintains, a regulatory immune cell, e.g., a Treg cell or a Breg cell.

158. The therapeutic compound of any of embodiments 154-157, wherein:

one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody or one comprises an HLA-G molecule.

159. The therapeutic compound of embodiment 158, wherein:

one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, one comprises an HLA-G molecule, and one comprises CD39 molecule or a CD73 molecule.

160. The therapeutic compound of any of embodiments 1-159, wherein the effector binding/modulating moiety comprises a polypeptide.

161. The therapeutic compound of any of embodiments 1-160, wherein the effector binding/modulating moiety comprises a polypeptide having at least 5, 10, 20, 30, 40, 50, 150, 200 or 250 amino acid residues.

162. The therapeutic compound of any of embodiments 1-161, wherein the effector binding/modulating moiety has a molecular weight of 5, 10, 15, 20, or 40 Kd.

163. The therapeutic compound of any of embodiments 1-162, wherein the effector binding/modulating moiety does not comprise an inhibitor of the expression of apolipoprotein CIII, protein kinase A, Src kinase, or Beta1 integrin.

164. The therapeutic compound of any of embodiments 1-162, wherein the effector binding/modulating moiety does not comprise an inhibitor of the activity of apolipoprotein CIII, protein kinase A, Src kinase, or Beta1 integrin.

165. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target a tissue selected from lung, skin, pancreas, retina, prostate, ovary, lymph node, adrenal gland, liver or gut tissue.

166. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target tubular cells, e.g., proximal tubular epithelial cells

167. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target TIE-2, APN, TEM4, TEM6, ICAM-1, nucleolin P2Z receptor, Trk-A, FLJ10849, HSPA12B, APP, or OX-45.

168. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target a luminally expressed protein.

169. The therapeutic compound of any of embodiments 1-163, wherein the donor target does not comprise a heart specific target.

170. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target lung tissue.

171. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target kidney tissue.

172. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically pancreas lung tissue.

173. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target gut tissue.

174. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target prostate tissue.

175. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target brain tissue.

176. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target CD71.

177. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target CD90.

178. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target MAdCAM.

179. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target albumin.

180. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target carbonic anhydrase IV.

181. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target ZG16-p.

182. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target dipeptidyl peptidase IV.

183. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target the luminal surface of a vascular endothelial cell membrane.

184. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target heart tissue.

185. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target a tumor, solid tumor, or the vascular of a solid tumor.

186. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target skin tissue.

187. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target epidermal tissue.

188. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target the basement membrane.

189. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target a Dsg polypeptide.

190. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target Dsg1.

191. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target Dsg3.

192. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target BP180.

193. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not specifically target desmoglein.

194. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not comprise a complement modulator, e.g., a complement inhibitor, such as, but not limited to, those described in U.S. Pat. No. 8,454,963, which is hereby incorporated by reference in its entirety.

195. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not comprise an imaging agent.

196. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not comprise an imaging agent selected from the group of: a radioactive agent, a radioisotope, a radiopharmaceutical, a contrast agent, a nanoparticle; an enzyme, a prosthetic group, a fluorescent material, a luminescent material, and a bioluminescent material, such as, but not limited to, those described in U.S. Pat. No. 8,815,235, which is hereby incorporated by reference in its entirety.

197. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not comprise a radionuclide, such as, but not limited to, those described in U.S. Pat. No. 6,232,287, which is hereby incorporated by reference in its entirety.

198. The therapeutic compound of any of embodiments 1-163, which is not internalized by a donor cell to which it binds.

199. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not enter the cell which is targeted by the specific targeting moiety.

200. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not kill the cell which is targeted by the specific targeting moiety.

201. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not enter the cell to which the effector binding/modulating moiety binds.

202. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not kill the cell to which the effector binding/modulating moiety binds.

203. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not comprise an autoantigenic peptide or polypeptide.

204. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not comprise an autoantigenic peptide or polypeptide, e.g., does not comprise a peptide or polypeptide against which the subject has autoantibodies.

205. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not comprise an antibody molecule derived from a mammal, e.g., a human, having an autoimmune disorder.

206. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not comprise an antibody molecule derived from a mammal, e.g., a human, having acute mucocutaneous PV.

207. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not comprise an antibody molecule derived from a mammal, e.g., a human, having Goodpasture's Disease

208. The therapeutic compound of any of embodiments 1-163, wherein the therapeutic compound does not comprise an antibody molecule derived from a mammal, e.g., a human, having pemphigus vulgaris.

209. The therapeutic compound of any of embodiments 1-208, comprising a donor specific targeting moiety.

210. The therapeutic compound of any of embodiments 209, that localizes preferentially to an implanted donor tissue, as opposed to tissue of a recipient.

211. The therapeutic compound of embodiments 209-210, wherein, the donor specific targeting moiety provides site-specific immune privilege for a transplant tissue, e.g., an organ, from a donor.

212. The therapeutic compound of embodiments 209-211, wherein the donor specific targeting moiety binds to a product, e.g., a polypeptide, of an allele present at a locus in the donor, which allele is not present at the locus in the recipient

213. The therapeutic compound of any of embodiments 209-212, wherein, the donor specific targeting moiety preferentially binds to an allele of a gene expressed on donor tissue, e.g., a transplant tissue, e.g., an organ, as compared with an allele of the gene expressed on subject tissue.

214. The therapeutic compound of embodiments 209-213, wherein, the donor specific targeting moiety has a binding affinity for an allele of a gene expressed on donor tissue, e.g., a transplant tissue, e.g., an organ, which is at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 fold greater than its affinity for an allele of the gene expressed on subject tissue.

215. The therapeutic compound of any of embodiments 209-214, wherein the donor specific targeting moiety binds to the product, e.g., a polypeptide, of an allele present at a locus in the donor, which allele is not present at the locus in the recipient.

216. The therapeutic compound of any one of embodiments 209-215, wherein the binding is sufficiently specific that, e.g., at a clinically effective dose of the therapeutic compound, unwanted, substantial, or clinically unacceptable, systemic immune suppression occurs.

217. The therapeutic compound of any one of embodiments 209-216, wherein the therapeutic compound accumulates at the target site, e.g., binding of the donor specific targeting moiety to results in accumulation of the therapeutic compound at the target site.

218. The therapeutic compound of any one of embodiments 209-217, wherein the donor specific targeting moiety binds a product of an allele of a locus selected from Table 2, e.g., the HLA locus, e.g., the HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ or HLA-DR locus, which allele is present in the donor but not the recipient. HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ or HLA-DR locus.

219. The therapeutic compound of any one of embodiments 209-218, wherein the donor specific targeting moiety binds an allele of HLA A, an allele of HLA-B, an allele of HLA-C, an allele of HLA-DP, an allele of HLA-, or an allele of HLA-.

220. The therapeutic compound of any one of embodiments 209-219, wherein the therapeutic compound is suitable for treating a subject that has, will have, or is in need of, a transplant.

221. The therapeutic compound of embodiment 220, wherein the transplant comprises all or part of an organ, e.g., a liver, kidney, heart, pancreas, thymus, skin or lung.

222. The therapeutic compound of any one of embodiments 209-221, wherein the donor specific targeting moiety comprises an antibody molecule.

223. The therapeutic compound of any one of embodiments 209-221, wherein the donor specific targeting moiety comprises a target specific binding polypeptide, or a target ligand binding molecule.

224. The therapeutic compound of any one of embodiments 1-223, comprising a tissue specific targeting moiety.

225. The therapeutic compound of embodiment 224, wherein the tissue specific targeting moiety is a molecule that specifically binds to MAdCAM.

226. The therapeutic compound of embodiment 224, wherein the tissue specific targeting moiety is an antibody that specifically binds to MAdCAM.

227. The therapeutic compound of any one of embodiments, 224-226, wherein the therapeutic compound is suitable for treating a subject having, or is at risk, or elevated risk, for having, an autoimmune disorder, e.g., an autoimmune disorder described herein.

228. The therapeutic compound of any of embodiments 224-227, wherein the therapeutic compound accumulates at the target site, e.g., binding of the tissue specific targeting moiety results in accumulation of the therapeutic compound at the target site.

229. The therapeutic compound of any of embodiments 224-228, wherein the therapeutic compound which localizes, preferentially to a target tissue, as opposed to other tissue of a subject.

230. The therapeutic compound of any of embodiments 224-229, wherein the therapeutic compound provides site-specific immune privilege for a subject target tissue, e.g., a target tissue undergoing, or at risk, or elevated risk, for, unwanted immune attack, e.g., in an autoimmune disorder.

231. The therapeutic compound of any of embodiments 224-229, wherein the tissue specific targeting moiety, as a component of the therapeutic compound, preferentially binds a subject target tissue undergoing unwanted immune attack, e.g., in an autoimmune disorder.

232. The therapeutic compound of any of embodiments 224-231, wherein a tissue specific targeting moiety binds to the product, e.g., a polypeptide, which is not present outside the target tissue, or is present at sufficiently low levels that, at therapeutic concentrations of therapeutic molecule, unacceptable levels of immune suppression are absent or substantially absent.

233. The therapeutic compound of any of embodiments 224-232, wherein, the tissue specific targeting moiety binds a product, or site on a product, which is more abundant in target tissue than in non-target tissue.

234. The therapeutic compound of any of embodiments 224-233, wherein, therapeutic compound binds a product, or a site on a product, that is present or expressed substantially exclusively on target tissue.

235. The therapeutic compound of any of embodiments 224-234, wherein the product, or site on a product, to which the specific targeting moiety binds, is sufficiently limited to the target tissue, that at therapeutically effective level of therapeutic compound, the subject does not suffer an unacceptable level, e.g., a clinically significant level, of systemic immune suppression.

236. The therapeutic compound of any of embodiments 224-235, wherein the therapeutic compound, preferentially binds to a target tissue or target tissue antigen, e.g., has a binding affinity for the target tissue or antigen that is greater for target antigen or tissue, e.g., at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 fold greater, than its affinity for than for non-target tissue or antigen present outside the target tissue.

237. The therapeutic compound of any of embodiments 224-236, wherein the tissue specific targeting moiety binds to a product, e.g., a polypeptide product, or site on a product, present at a preselected site, e.g., a site of unwanted immune response in an autoimmune disorder.

238. The therapeutic compound of any of embodiments 224-237, wherein therapeutic compound is suitable for the treatment of a subject having, or at risk, or elevated risk, for having, type1 diabetes.

239. The therapeutic compound of any of embodiments 224-238, wherein the target tissue comprises pancreatic tissue, e.g., pancreatic islets or pancreatic beta cells, gut tissue (e.g. gut endothelial cells), kidney tissue (e.g. kidney epithelial cells), or liver tissue (e.g. liver epithelial cells).

240. The therapeutic compound of any of embodiments 224-239, wherein the effector binding/modulating moiety or targeting moiety binds a polypeptide selected from those described herein, such as those listed in Table 3, e.g., SEZ6L2, LRP11, DISP2, SLC30A8, FXYD2, TSPAN7, or TMEM27.

241. The therapeutic compound of any of embodiments 224-236, wherein therapeutic compound is suitable for the treatment of a subject having, or at risk, or elevated risk, for having, multiple sclerosis.

242. The therapeutic compound of embodiment 241, wherein the target tissue comprises CNS tissue, myelin sheath, or myelin sheath of oligodendrocytes.

243. The therapeutic compound of any of embodiments 241-242, wherein the effector binding/modulating moiety or targeting moiety binds a polypeptide selected from those described herein and including, but not limited to, Table 3, e.g., MOG, PLP, or MBP.

244. The therapeutic compound of any of embodiments 224-236, wherein therapeutic compound is suitable for the treatment of a subject having, or at risk, or elevated risk, for having, cardiomyositis.

245. The therapeutic compound of embodiment 244, wherein the target tissue comprises cardiomyocytes, monocytes, macrophages, or myeloid cells.

246. The therapeutic compound of embodiments 244-245, wherein the effector binding/modulating moiety binds or the targeting moiety a polypeptide as described herein, including, but not limited to those selected from Table 3, e.g., SIRPA (CD172a).

247. The therapeutic compound of any of embodiments 224-236, wherein therapeutic compound is suitable for the treatment of a subject having, or at risk, or elevated risk, for having, inflammatory bowel disease, autoimmune hepatitis (AIH); Primary Sclerosing Cholangitis (PSC); Primary Biliary Sclerosis; (PBC); or transplant.

248. The therapeutic compound of any of embodiments 224-236, wherein the subject with has, is at risk or elevated risk for having Crohn's disease or ulcerative colitis.

249. The therapeutic compound of embodiments 247 or 248, wherein the target tissue comprises gut cells, such as gut epithelial cells or liver cells, such as liver epithelial cells.

250. The therapeutic compound of embodiments 247-249, wherein the effector binding/modulating moiety binds a polypeptide as described herein, including, but not limited to those selected from Table 3, e.g., PD-1.

251. The therapeutic compound of embodiments 247-249, wherein the targeting moiety binds a polypeptide as described herein, including, but not limited to MAdCAM.

252. The therapeutic compound of any of embodiments 224-236, wherein therapeutic compound is suitable for the treatment of a subject having, or at risk, or elevated risk, for having, rheumatoid arthritis.

253. The therapeutic compound of embodiment 252, wherein the target tissue comprises cardiomyocytes, monocytes, macrophages, or myeloid cells.

254. The therapeutic compound of embodiments 252 or 253, wherein the effector binding/modulating moiety or targeting moiety binds a polypeptide selected from Table 3, e.g., SIRPA (CD172a).

255. The therapeutic compound of any of embodiments 224-254, wherein the tissue specific targeting moiety comprises an antibody molecule.

256. The therapeutic compound of any of embodiments 224-254, wherein the tissue specific targeting moiety comprises a target specific binding polypeptide, or a target ligand binding molecule.

257. The therapeutic compound of any of embodiments 224-254, wherein the tissue specific targeting moiety comprises a target specific binding polypeptide binds to MAdCAM.

258. The therapeutic compound of any of embodiments 1-257, wherein the therapeutic compound binds a cell surface molecule of an immune effector cell, e.g., a T cell, B cell, NK cell, or other immune cell, which cell propagates a pro-immune response.

259. The therapeutic compound of any of embodiments 1-258, wherein the therapeutic compound reduces the ability of an immune effector cell, e.g., a T cell, B cell, NK cell, or other immune cell, to propagate a pro-immune response.

260. The therapeutic compound of any of embodiments 1-259, wherein the specific targeting moiety targets a mammalian target, e.g., a mammalian polypeptide, and the effector binding/modulating moiety binds/modulates a mammalian immune component, e.g., a human immune cell, e.g., a mammalian B cell, T cell, or macrophage.

261. The therapeutic compound of any of embodiments 1-260, wherein the specific targeting moiety targets a human target, e.g., a human polypeptide, and the effector binding/modulating moiety binds/modulates a human immune component, e.g., a human immune cell, e.g., a human B cell, T cell, or macrophage.

262. The therapeutic compound of any of embodiments 1-261, wherein the therapeutic compound is configured for use in a human.

263. The therapeutic compound of any of embodiments 1-260, wherein the therapeutic compound is configured for use in a non-human mammal.

264. The therapeutic compound of any of embodiments 1-263, wherein the therapeutic compound, e.g., the effector binding/modulating moiety, comprises a PD-1 agonist.

265. The therapeutic compound of any of the preceding embodiments, wherein the therapeutic compound comprises a IL-2 mutein of SEQ ID NO: 15, wherein the mutein comprises a mutation at position 73, 76, 100, or 138.

266. The therapeutic compound of embodiment 265, wherein the mutation is a L to I mutation at position 73, 76, 100, or 138.

267. The therapeutic compound of embodiments 265 or 266, wherein the IL-2 mutein further comprises a mutation at one or more of positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145.

268. The therapeutic compound of any of embodiments 265-267, wherein the mutein further comprises a mutation at one or more of positions E35, H36, Q42, D104, E115, or Q146 or 1, 2, 3, 4, 5, or each of E35, H36, Q42, D104, E115, or Q146 is wild-type.

269. The therapeutic compound of embodiment 268, wherein the mutation is one or more of E35Q, H36N, Q42E, D104N, E115Q, or Q146E.

270. The therapeutic compound of any one of embodiments 265-269, wherein the IL-2 mutein comprises a N49S mutation.

271. The therapeutic compound of any one of embodiments 265-270, wherein the IL-2 mutein comprises a Y51S or a Y51H mutation.

272. The therapeutic compound of any one of embodiments 265-271, wherein the IL-2 mutein comprises a K55R mutation.

273. The therapeutic compound of any one of embodiments 265-272, wherein the IL-2 mutein comprises a T57A mutation.

274. The therapeutic compound of any one of embodiments 265-272, wherein the IL-2 mutein comprises a K68E mutation, V89A (V69A) mutation, a N91R (N71R) mutation, a Q94P or Q74P mutation, a (N88D) or a N108R (N88R) mutation, a C145A(C125A) or C145S (C125S) mutation.

275. The therapeutic compound of any one of embodiments 265-274, wherein the therapeutic compound comprises a IL-2 mutein of SEQ ID NO: 6, wherein the mutein comprises a mutation at position 53, 56, 80, or 118 and one or more of the mutations recited in embodiments 265-274.

276. The therapeutic compound of any one of embodiments 265-275, wherein the IL-2 mutein is fused or linked to a Fc peptide.

277. The therapeutic compound of embodiment 276 wherein the Fc peptide comprise a mutation at one or more of positions of L234, L247, L235, L248, G237, and G250 (EU numbering).

278. A method of treating a subject with inflammatory bowel disease, the method comprising administering a therapeutic compound of any of embodiments 1-277 to the subject to treat the inflammatory bowel disease.

279. The method of embodiment 278, wherein the subject with inflammatory bowel disease has Crohn's disease.

280. The method of embodiment 278, wherein the subject with inflammatory bowel disease has ulcerative colitis.

281. A method of treating a subject with auto-immune hepatitis, the method comprising administering a therapeutic compound of any of embodiments 1-277 to the subject to treat the auto-immune hepatitis.

282. A method of treating primary sclerosing cholangitis the method comprising administering a therapeutic compound of any of embodiments 1-277 to the subject to treat the primary sclerosing cholangitis.

283. A method of treating Type 1 diabetes the method comprising administering a therapeutic compound of any of embodiments 1-277, thereby treating the subject to treat the Type 1 diabetes.

284. A method of treating a transplant subject comprising administering a therapeutically effective amount of a therapeutic compound of any of embodiments 1-277 to the subject, thereby treating a transplant (recipient) subject.

285. A method of treating GVHD in a subject having a transplanted a donor tissue comprising administering a therapeutically effective amount of a therapeutic compound of any of embodiments 1-277 to the subject.

286. The method of embodiment 285, wherein the therapeutic compound is administered to the subject: prior to receiving the transplant; prior to developing a symptom of GVHD; after or concurrent with receiving the transplant; or after or concurrent with developing a symptom of GVHD.

287. A method of treating a subject having, or at risk, or elevated risk, for having, an autoimmune disorder, comprising administering a therapeutically effective amount of a therapeutic compound of any embodiments 1-277, thereby treating the subject.

288. The method of embodiment 287, wherein the subject has received, will receive, or is in need of, allograft donor tissue.

289. The method of any of embodiments 287-288, wherein the donor tissue comprises a solid organ, e.g., a liver, kidney, heart, pancreas, thymus, or lung.

290. The method of any of embodiments 287-288, wherein the donor tissue comprises all or part of an organ, e.g., a liver, kidney, heart, pancreas, thymus, or lung.

291. The method of any of embodiments 287-288, wherein the donor tissue comprises skin.

292. The method of any of embodiments 287-288, wherein the donor tissue does not comprises skin.

293. The method of any of embodiments 287-292, wherein the donor tissue presents or expresses a product of an allele of a locus, which allele is not present or expressed in the subject.

294. The method of any of embodiments 287-292, wherein the donor tissue presents or expresses a product of an allele of a locus selected from Table 2, e.g., the HLA locus, e.g., the HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ or HLA-DR locus, which allele is not present or expressed in the subject.

295. The method of any of embodiments 287-294, comprising introducing the transplant tissue into the subject.

296. The method of any of embodiments 278-295, comprising monitoring the subject for immune cell inactivation (e.g., to monitor unwanted agonization of an immune inhibitory checkpoint molecule) at a site distant from the target site, e.g., in the peripheral circulation or the lymphatic system.

297. The method of any of embodiments 278-296, comprising monitoring the subject for immune cell activation (e.g., to monitor unwanted antagonization of an immune inhibitory checkpoint molecule) at a site distant from the target site, e.g., in the peripheral circulation or the lymphatic system.

298. The method of any of embodiments 278-297, wherein responsive to the result of monitoring, selecting a course of treatment for the subject, e.g., increasing the dose of the therapeutic compound, decreasing the dose of the therapeutic compound, continuing treatment with the therapeutic compound without a change in dose.

299. The method of any of embodiments 278-298, comprising administering the compound of embodiments 1-277, to the recipient.

300. The method of any of embodiments 278-298, wherein administering comprises systemic administration, e.g., to the peripheral circulatory system.

301. The method of any of embodiments 278-298, wherein administering comprises local administration, e.g., to the target tissue, the donor tissue or the site of at which the target tissue or the donor tissue is, or will be located.

302. The method of any of embodiments 301, comprising administering the therapeutic compound to the recipient prior to introduction of the donor tissue into the recipient.

303. The method of any of embodiments 301, comprising administering the therapeutic compound, to the recipient after introduction of the donor tissue into the recipient.

304. The method of any of embodiments 295, comprising administering the therapeutic compound to the recipient concurrent with introduction of the donor tissue into the recipient.

305. The method of embodiment 295, comprising contacting the therapeutic compound with the donor tissue prior to introduction of the donor tissue into the recipient.

306. The method of any of embodiments 295, comprising providing the therapeutic compound to the subject, wherein the transplant tissue has been contacted with therapeutic compound prior to introduction into the subject.

307. The method of any of embodiments 295, comprising contacting the therapeutic compound with the donor tissue after introduction of the donor tissue into the recipient, e.g., by local administration to the donor tissue.

308. The method of any of embodiments 278-307, comprising administering a therapeutic compound as provided for herein such that therapeutic levels are present for at least 1, 5, 10, 14, or 28 days, for example, consecutive or non-consecutive days.

309. The method of any of embodiments 278-308, wherein the subject does not receive a non-targeted immune suppressive agent.

310. The method of any of embodiments 278-308, wherein for the subject has not received a non-targeted immune suppressive agent for at least 1, 15, 30, 60, or 90 days prior to the initial administration of the therapeutic compound.

311. The method of any of embodiments 295, wherein the subject has not received a non-targeted immune suppressive agent for at least 1, 15, 30, 60, or 90 days prior to introduction of the transplant tissue.

312. The method of any of embodiments 278-311, wherein the subject does not receive a non-targeted immune suppressive agent for at least 1, 15, 30, 60, 90, or 180 days after the initial administration of the therapeutic compound.

313. The method of any of embodiments 278-311, wherein the subject does not receive a non-targeted immune suppressive agent for at least 1, 15, 30, 60, 90, or 180 days after introduction of the transplant tissue.

314. The method of any of embodiments 278-313, comprising administering a non-targeted immune suppressive agent to the subject.

315. The method of any of embodiments 278-314, wherein for the subject receives a non-targeted immune suppressive agent for at least 1, 15, 30, 60, or 90 days prior to the initial administration of the therapeutic compound.

316. The method of embodiment 295, wherein the subject receives a non-targeted immune suppressive agent for at least 1, 15, 30, 60, or 90 days prior to introduction of the transplant tissue.

317. The method of embodiment 316, wherein the subject receives a non-targeted immune suppressive agent for at least 1, 15, 30, 60, 90 or 180 days after the initial administration of the therapeutic compound.

318. The method of any of embodiments 278-317, wherein the subject receives a non-targeted immune suppressive agent for at least 1, 15, 30, 60, 90 or 180 days after introduction of the transplant tissue.

319. The method of any of embodiments 278-317, wherein for the subject receives a non-targeted immune suppressive agent prior to the initial administration of the therapeutic compound but for no more than 1, 15, 30, 60, 90 or 180 days.

320. The method of embodiment 295, wherein the subject receives a non-targeted immune suppressive agent prior to introduction of the transplant tissue but for no more than 1, 15, 30, 60, 90 or 180 days.

321. The method of any of embodiments 278-320, wherein the subject receives a non-targeted immune suppressive agent after the initial administration of the therapeutic compound but for no more than 1, 15, 30, 60, 90 or 180 days.

322. The method of embodiment 295, wherein the subject receives a non-targeted immune suppressive agent after introduction of the transplant tissue but for no more than 1, 15, 30, 60, 90 or 180 days.

323. The method of embodiment 295, wherein the subject is monitored for rejection of the transplant tissue.

324. The method of any of embodiments 278-323, a dosage of a non-targeted immune suppressive agent is selected, or wherein responsive to the monitoring, a dosage of a non-targeted immune suppressive agent is selected.

325. The method of embodiment 324, wherein the dosage is administered.

326. The method of embodiment 325, wherein the selected dosage is zero, i.e., a non-targeted immune suppressive agent is not administered.

327. The method of embodiment 325, wherein the selected dosage is non-zero, i.e., a non-targeted immune suppressive agent is administered.

328. The method of embodiment 325, wherein the dosage is less than what would be administered in the absence of administration of a therapeutic compound.

329. The method of any of embodiments 278-328, wherein the subject is a mammal, e.g., a non-human mammal.

330. The method of any of embodiments 278-328, wherein the subject is a human.

331. The method of embodiment 295, wherein the donor and subject are mismatched at an HLA locus, e.g., a major or minor locus.

332. The method of embodiment 331, wherein the subject is a mammal, e.g., a non-human mammal.

333. The method of embodiment 331, wherein the subject is a human.

334. A method of treating a subject having, or at risk, or elevated risk, for having, an autoimmune disorder, comprising administering a therapeutically effective amount of a therapeutic compound of any embodiments 1-277, thereby treating the subject.

335. The method of embodiment 334, wherein provision of the therapeutic compound is initiated prior to the onset, or prior to identification of onset, of symptoms of the autoimmune disorder.

336. The method of any of embodiments 334-335, wherein provision of the therapeutic compound is initiated after onset, or after identification of onset, of symptoms of the autoimmune disorder.

337. The method of embodiments 334-336, wherein autoimmune disorder comprises type1 diabetes.

338. The therapeutic compound of any of embodiments 334-337, wherein the target tissue comprises pancreatic islets or pancreatic beta cells, gut tissue (e.g. gut endothelial cells), kidney tissue (e.g. kidney epithelial cells), or liver tissue (e.g. liver epithelial cells).

339. The therapeutic compound of any of embodiments 334-338, wherein the effector binding/modulating moiety or targeting moiety binds a polypeptide selected from Table 3, e.g., MAdCAM, OAT1, OCT, DPP6, SEZ6L2, LRP11, DISP2, SLC30A8, FXYD2, TSPAN7, or TMEM27 polypeptide.

340. The method of any of embodiments 334-339, wherein provision of the therapeutic compound is initiated prior to the onset, or prior to identification of onset, of symptoms of type1 diabetes.

341. The method of any of embodiments 334-340, wherein provision of the therapeutic compound is initiated prior to, or prior to identification of the subject having a preselected characteristic or symptom.

342. The method of any of embodiments 334-341, wherein provision of the therapeutic compound is initiated after onset, or after identification of onset, of symptoms of type1 diabetes.

343. The method of any of embodiments 334-342, wherein provision of the therapeutic compound is initiated after, or after identification of the subject having a preselected characteristic or symptom.

344. The method of any of embodiments 334-343, wherein the therapeutic compound is a therapeutic compound of any of embodiments 1-277

345. The method of any of embodiments 334-339, wherein therapeutic compound is suitable for the treatment of a subject having, or at risk, or elevated risk, for having, multiple sclerosis.

346. The method of embodiment 345, wherein the target tissue comprises CNS tissue, myelin sheath, or myelin sheath of oligodendrocytes.

347. The method of any of embodiments 345 or 346, wherein the effector binding/modulating moiety or targeting moiety binds a polypeptide selected from Table 3, e.g., a MOG, PLP, or MBP polypeptide.

348. The method of any of embodiments 345-347, wherein provision of the therapeutic compound is initiated prior to the onset, or prior to identification of onset, of symptoms of multiple sclerosis.

349. The method of any of embodiments 345-347, wherein provision of the therapeutic compound is initiated prior to, or prior to identification of the subject a preselected characteristic or symptom.

350. The method of any of embodiments 345-347, wherein provision of the therapeutic compound is initiated after onset, or after identification of onset, of symptoms of multiple sclerosis.

351. The method of any of embodiments 345-347, wherein provision of the therapeutic compound is initiated after, or after identification of the subject having a preselected characteristic or symptom.

352. The method of any of embodiments 345-351, wherein the therapeutic compound is a therapeutic compound of any of embodiments 1-277.

353. The method of any of embodiments 334-339, wherein the therapeutic compound is suitable for the treatment of a subject having, or at risk, or elevated risk, for having, cardiomyositis.

354. The method of embodiment 353, wherein the target tissue comprises cardiomyocytes, monocytes, macrophages, or myeloid cells.

355. The method of embodiments 353 or 354, wherein the effector binding/modulating moiety or targeting moiety binds a polypeptide selected from Table 3, e.g., a SIRPA (CD172a) polypeptide.

356. The method of any of embodiments 353-355, wherein provision of the therapeutic compound is initiated prior to the onset, or prior to identification of onset, of symptoms of cardiomyositis.

357. The method of any of embodiments 353-355, wherein provision of the therapeutic compound is initiated prior to, or prior to identification of the subject having a preselected characteristic or symptom.

358. The method of any of embodiments 353-355, wherein provision of the therapeutic compound is initiated after onset, or after identification of onset, of symptoms of cardiomyositis.

359. The method of any of embodiments 353-355, wherein provision of the therapeutic compound is initiated after, or after identification of the subject having a preselected characteristic or symptom.

360. The method of any of embodiments 353-359, wherein the therapeutic compound is a therapeutic compound of any of embodiments 1-277.

361. The method of any of embodiments 334-339, wherein therapeutic compound is suitable for the treatment of a subject having, or at risk, or elevated risk, for having, rheumatoid arthritis.

362. The method of embodiment 361, wherein the target tissue comprises cardiomyocytes, monocytes, macrophages, or myeloid cells.

363. The method of embodiments 361 or 362, wherein the effector binding/modulating moiety or targeting moiety binds a polypeptide selected from Table 3, e.g., a SIRPA (CD172a) polypeptide.

364. The method of embodiments 361-363, wherein provision of the therapeutic compound is initiated prior to the onset, or prior to identification of onset, of symptoms of rheumatoid arthritis.

365. The method of embodiments 361-363, wherein provision of the therapeutic compound is initiated prior to, or prior to identification of the subject having a preselected characteristic or symptom.

366. The method of embodiments 361-363, wherein provision of the therapeutic compound is initiated after onset, or after identification of onset, of symptoms of rheumatoid arthritis.

367. The method of embodiments 361-363, wherein provision of the therapeutic compound is initiated after, or after identification of the subject having a preselected characteristic or symptom.

368. The method of embodiments 361-367, wherein the therapeutic compound is a therapeutic compound of any of embodiments 1-277.

369. The method of any of embodiments 278-368, comprising monitoring the subject for immune cell inactivation (e.g., to monitor unwanted agonization of an immune inhibitory checkpoint molecule) at a site distant from the target site, e.g., in the peripheral circulation or the lymphatic system.

370. The method of any of embodiments 278-369, comprising monitoring the subject for immune cell activation (e.g., to monitor unwanted antagonization of an immune inhibitory checkpoint molecule) at a site distant from the target site, e.g., in the peripheral circulation or the lymphatic system.

371. The method of any of embodiments 278-370, wherein responsive to the result of monitoring, selecting a course of treatment for the subject, e.g., increasing the dose of the therapeutic compound, decreasing the dose of the therapeutic compound, continuing treatment with the therapeutic compound without a change in dose.

372. The method of any of embodiments 278-371, wherein the subject monitored for autoimmune attack of the target tissue.

373. The method of embodiment 372, wherein responsive to the monitoring, a dosage of the therapeutic compound is selected.

374. The method of embodiment 373, wherein the dosage is administered.

375. The method of embodiment 372, wherein the selected dosage is zero, i.e., administration of therapeutic compound is ceased.

376. The method of embodiment 372, wherein the selected dosage is non-zero.

377. The method of embodiment 372, wherein the selected dosage is an increased dosage.

378. The method of embodiment 372, wherein the selected dosage is an reduced dosage.

379. The method of any of embodiments 278-378, wherein administering comprises systemic administration, e.g., to the peripheral circulatory system.

380. The method of any of embodiments 278-379, wherein administering comprises local administration, e.g., to the target tissue.

381. The method of any of embodiments 278-380, comprising administering a therapeutic compound provided herein such that therapeutic levels are present for at least 1, 5, 10, 14, or 28 days, e.g., consecutive or non-consecutive days.

382. The method of any of embodiments 278-381, wherein the subject is a mammal, e.g., a non-human mammal.

383. The method of any of embodiments 278-381, wherein the subject is a human.

384. A nucleic acid molecule or a plurality of nucleic acid molecules encoding a therapeutic compound of any of embodiments 1-277.

385. A vector or a plurality of vectors comprising the nucleic acid molecules of embodiment 384.

386. A cell comprising the nucleic acid molecules of embodiment 384 or the vector of embodiment 385.

387. A method of making a therapeutic compound comprising culturing a cell of embodiment 386 to make the therapeutic compound.

388. A method of making a nucleic acid sequence encoding a therapeutic compound of any of embodiments 1-277, comprising

    
    
        a) providing a vector comprising sequence encoding a targeting moiety and inserting into the vector sequence encoding an effector binding/modulating moiety to form a sequence encoding a therapeutic compound; or
        b) providing a vector comprising sequence encoding an effector binding/modulating moiety and inserting into the vector sequence encoding a targeting moiety to form a sequence encoding a therapeutic compound,
    
    


thereby making a sequence encoding a therapeutic compound.
389. The method of embodiment 388, wherein the targeting moiety is selected in response to the need of a subject.
390. The method of embodiment 388 or 389, wherein the effector binding/modulating moiety is selected in response to the need of a subject.
391. The method of any of embodiments 388 or 389, further comprising expressing the sequence encoding the therapeutic compound to produce the therapeutic compound.
392. The method of any of embodiments 388-391, further comprising transferring the sequence, or a polypeptide made from the sequence, to another entity, e.g., a health care provider who will administer the therapeutic compound to a subject.
393. A method of treating a subject comprising:
acquiring, e.g., receiving from another entity, a therapeutic compound, or a nucleic acid encoding a therapeutic compound, made by the method of any of provided herein, but not limited to embodiments 388-392;
administering the therapeutic compound, or a nucleic acid encoding a therapeutic compound to the subject, thereby treating the subject.
394. The method of embodiment 393, further comprising identifying the therapeutic compound, or nucleic acid encoding a therapeutic compound to another entity, e.g., the entity that will make the therapeutic compound, or nucleic acid encoding a therapeutic compound.

395. The method of embodiments 393 or 394, further comprising requesting the therapeutic compound, or nucleic acid encoding a therapeutic compound from another entity, e.g., the entity that made the therapeutic compound, or nucleic acid encoding a therapeutic compound.

396. The method of any of embodiments 393-395, wherein the subject has an autoimmune disorder and the therapeutic compound does not comprise an autoantigenic peptide or polypeptide characteristic of the autoimmune disorder, e.g., does not comprise a peptide or polypeptide against which the subject has autoantibodies.

397. The therapeutic compound of embodiment 97, wherein the compound has the formula from N-terminus to C-terminus:

A1---Linker A---A2---Linker B---A3 or A3---Linker A---A2---Linker B---A1,
wherein,
A1 and A3, each independently comprises the effector binding/modulating moiety, or the targeting moiety,
A2 comprises an Fc region, and
Linker A and Linker B, each are independent linkers.
398. The therapeutic compound of embodiment 397, wherein each of Linker A and Linker B comprises an independent linker.
399. The therapeutic compound of embodiment 397, wherein A1 is the anti-MAdCAM antibody and A3 is the IL-mutein antibody.
400. The therapeutic compound of embodiment 397, wherein A2 comprises an Fc region.
401. The therapeutic compound of embodiment 397-400, wherein Linker A and Linker B, each independently are glycine/serine linkers.
402. The therapeutic compound of embodiment 397-401, wherein Linker A and Linker B, each independently comprises GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 30), GGGGSGGGGS (SEQ ID NO: 619), or GGGGS (SEQ ID NO: 23).
The following examples are illustrative, but not limiting, of the compounds, compositions and methods described herein. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments.
EXAMPLES
Example 1: HLA-Targeted Pd-1 Agonizing Therapeutic Compounds
Engineering of a HLA-Targeted PD-1-Agonizing Therapeutic
Binding domains specific for HLA-A2 are obtained by cloning the variable regions of the Ig heavy and light chains from the BB7.2 hybridoma (ATCC) and converting into a single-chain Ab (scFv). Activity and specificity of the scFv can be confirmed by assessing binding of BB7.2 to HLA-A2 expressing cells in comparison to cells expressing other HLA-A alleles. The minimal PD-L1 residues required for PD-1 binding activity are identified by systematically evaluating the requirement of amino acids 3′ and 5′ of the PD-L1 IgV domain corresponding to amino acids 68-114. Expression constructs are designed and proteins synthesized and purified, with PD-1 binding activity tested by Biacore. The minimum essential amino acids required for PD-1 binding by the PD-L1 IgV domain are referred to as PD-L1-IgV. To generate a BB7.2 scFv and PD-L1-IgV bi-specific molecule, a DNA fragment is synthesized encoding the bispecific single-chain antibody BB7.2×PD-L1-IgV with the domain arrangement VLBB7.2-VHBB7.2-PD-L1-IgV-IgG4 Fc and cloned into an expression vector containing a DHFR selection cassette.
Expression vector plasmid DNA is transiently transfected into 293T cells, and BB7.2×PD-L1-IgV bispecific antibodies are purified from supernatants using a protein A/G column. BB7.2×PD-L1-IgV bispecific antibody integrity is assessed by polyacrylamide gel. Binding of the BB7.2 scFv domain to HLA-A2 and PD-L1-IgV domain to PD-1 is assessed by ELISA and cell-based FACS assay.
The in vitro function of BB7.2×PD-L1-IgV bispecific antibodies is assessed using mixed lymphocyte reaction (MLR) assay. In a 96-well plate format, 100,000 irradiated human PBMCs from an HLA-A2+ donor are aliquoted per well and used as activators. HLA-A1− responder T cells are then added together with increasing amounts of BB7.2×PD-L1-IgV bispecific antibody. The ability of responder T cells to proliferate over a period of 72 hours is assessed by BrdU incorporation, and with IFNg and IL2 cytokine production additionally evaluated in the co-culture supernatant as assessed by ELISA. BB7.2×PD-L1-IgV bispecific antibody is found to suppress MLR reaction as demonstrated by inhibiting HLA-A2− responder T cell proliferation and cytokine production.
The in vivo function of BB7.2×PD-L1-IgV bispecific antibody is assessed using a murine mouse model of skin allograft tolerance. The C57BL/6-Tg(HLA-A2.1)1Enge/J (Jackson Laboratories, Bar Harbor Me.) strain of mouse is crossed with Balb/cJ, with F1 progeny expressing the HLA-A2.1 transgene and serving as allograft donors. C57BL/6J mice are shaved and surgically engrafted with skin removed from euthanized C57BL/6-Tg(HLA-A2.1)1Enge/J×Balb/cJ F1 mice. At the same time, host mice start receiving intraperitoneal injections of the BB7.2×PD-L1-IgV bispecific antibody engineered to contain a murine IgG1 Fc or BB7.2 only or PD-L1-IgV only controls. Skin allograft rejection or acceptance is monitored over a period of 30 days, wherein hosts were euthanized and lymph node and allograft-resident lymphocyte populations quantified.
Example 2: CD39 and/or CD73 as Effector Domains Creating a Purinergic Halo Surrounding a Cell Type or Tissue of Interest
A catalytically active fragment of CD39 and/or CD73 is fused to a targeting domain. Upon binding and accumulation at the target site, CD39 phosphohydrolyzes ATP to AMP. Upon binding and accumulation at the target site, CD73 dephosphorylates extracellular AMP to adenosine. A soluble catalytically active form of CD39 suitable for use herein has been found to circulate in human and murine blood, see, e.g., Yegutkin et al. FASEB J. 2012 September; 26(9):3875-83. A soluble recombinant CD39 fragment is also described in Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39, Gayle, et al., J Clin Invest. 1998 May 1; 101(9): 1851-1859. A suitable CD73 molecule comprises a soluble form of CD73 which can be shed from the membrane of endothelial cells by proteolytic cleavage or hydrolysis of the GPI anchor by shear stress see, e.g., Reference: Yegutkin G, Bodin P, Burnstock G. Effect of shear stress on the release of soluble ecto-enzymes ATPase and 5′-nucleotidase along with endogenous ATP from vascular endothelial cells. Br J Pharmacol 2000; 129: 921-6.
The local catalysis of ATP to AMP or AMP to adenosine will deplete local energy stores required for fulminant T effector cell function. Treg function should not be impacted by ATP depletion due to their reliance on oxidative phosphorylation for energy needs (which requires less ATP), wherein T memory and other effector cells should be impacted due their reliance on glycolysis (requiring high ATP usage) for fulminant function.
Example 3: Measuring Antibody-Induced PD-1 Signaling
Jurkat cells that stably express 2 constructs, 1) a human PD-1 polypeptide fused to a b-galactosidase, which can be referred to as an “Enzyme donor” and 2) a SHP-2 polypeptide fused to a b-galactosidase, which can be referred to as an “Enzyme acceptor.” A PD-1 antibody is contacted with the cell and when the PD-1 is engaged, SHP-2 is recruited to PD-1. The enzyme acceptor and enzyme donor form a fully active b-galactosidase enzyme that can be assayed. This assay can be used to show activation of PD-1 signaling.
Example 4: Measuring PD-1 Agonism. PD-1 Agonists Inhibit T Cell Activation
Without being bound to any particular theory, PD-1 agonism inhibits anti-CD3-induced T cell activation. Human or mouse cells are preactivated with PHA (for human T cells) or ConA (for mouse T cells) so that they express PD-1. The T cells are then “reactivated” with anti-CD3 in the presence of anti-PD-1 (or PD-L1) for the PD-1 agonism assay. T cells that receive a PD-1 agonist signal in the presence of anti-CD3 will show decreased activation, relative to anti-CD3 stimulation alone. Activation can be readout by proliferation or cytokine production (IL-2, IFNg, IL-17) and possibly by other markers, such as CD69 activation marker.
Example 5. Expression and Function of Anti-MAdCAM/Mouse PD-L1 Fusion Protein is not Impacted by Molecular Configuration
A bispecific fusion molecule comprising an anti-mouse MAdCAM Ab/mouse PD-L1 molecule was expressed in two orientations. The first orientation consisted of an anti-mouse MAdCAM IgG with mouse PD-L1 fused at the c-terminus of its heavy chain. The second orientation consisted of mouse PD-L1 fused at the N-terminus of an Ig Fc domain, with a c-terminally fused anti-mouse MAdCAM scFv. Both molecules were found to be well expressed in a mammalian expression system. It was also found that the molecules can bind to their respective binding partners, MAdCAM or PD-1 in both orientations, simultaneously. These results demonstrate that a molecule consisting of an anti-MAdCAM antibody fused to PD-L1, can be expressed in configurations whereby PD-L1 is N or C-terminally fused to the Fc and retain proper functional binding activity.
Briefly, a pTT5 vector containing the single gene encoding a single polypeptide with mouse PD-L1 fused N-terminally of human IgG1 Fc domain and with c-terminal fused anti-MAdCAM scFv MECA-89 was transfected into HEK293 Expi cells. Alternatively, two plasmids were co-transfected at equimolar ratios. The first plasmid encoded the light chain of MECA-89 and the 2nd encoded the full length IgG1 heavy chain of MECA-89 with c-terminally fused mouse PD-L1. After 5-7 days, cell culture supernatants expressing the molecules were harvested, and clarified by centrifugation and filtration through a 0.22 um filtration device. The bi-specific molecules were captured on proA resin. The resin was washed with PBS pH 7.4 and the captured molecule was eluted using 100 mM Glycine pH 2.5, with neutralization using a tenth volume of 1M Tris pH 8.5. The protein was buffer exchanged into PBS pH 7.4, and analyzed by size exclusion chromatography on a Superdex 200 3.2/300. Analysis of 1 μg of purified material by reducing and non-reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted.
Both proteins, regardless of orientation were expressed at over 10 mg/L, and were over 95% monodispersed after purification as shown by size exclusion chromatography and reducing/non-reducing SDS-PAGE. Accordingly, this demonstrates the production and activity of dual function bispecific molecules with different immunomodulators and tissue targeting moieties at the N and C terminus of an Fc domain. This also shows specifically that a PD-1 agonist and binding partner can be expressed at the N or C terminus of an Ig Fc domain.
Example 6. A Bispecific Molecule Comprising a PD-1 Agonist Prototype Tethered to MAdCAM can Bind MAdCAM and PD-1 Simultaneously
Briefly, an immunosorbent plate was coated with mouse PD-1 at a concentration of 1 mg/mL in PBS pH 7.4, 75 ul/well, and incubated overnight at 4° C. Wells were washed with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer) three times, and then blocked with 200 ul/well 1% BSA in PBS pH 7.4 (block buffer) for two hours at room temperature. After three washes with wash buffer, two bispecific molecules that comprises the PD-1 Agonist prototype at either the N-terminus or C-terminus were diluted to 1 nM, 10 nM, and 100 nM in PBS containing 1% BSA and 0.05% Tween-20 (assay buffer). The diluted material was added to the mouse PD-1 coated plate at 75 ul/well for 1 hour at room temperature. After three washes with wash buffer, mouse MAdCAM was added to the plate at 75 ul/well, at a concentration of 10 nM in assay buffer for 1 hr at room temperature. After three washes with wash buffer, a goat biotinylated anti-mouse MAdCAM polyclonal antibody, diluted to 0.5 μg/mL in assay buffer, was added to the plate at 75 ul/well for 1 hr at room temperature. After three washes with wash buffer high sensitivity streptavidin HRP diluted in assay buffer at 1:5000 was added to the plate at 75 ul/well for 15 minutes at room temperature. After three washes with wash buffer and 1 wash with wash buffer (with no tween-20), the assay was developed with TMB, and stopped with 1N HCL. OD 450 nm was measured. The experiment included appropriate controls for non-specific binding to the plate/block in the absence of mouse PD-1, as well as no MAdCAM controls, and mono-specific controls, that are unable to form a bridge between mouse PD-1 and mouse MAdCAM.
The results demonstrated that at concentrations of 1 nM, 10 nM, and 100 nM, both bispecific molecules, are able to simultaneously interact with mouse MAdCAM and mouse PD-L1, whilst the monospecific controls did not create a bridging signal. Additionally, there was no binding of any compound to MAdCAM at any concentration tested, when mouse PD-1 was not present on the plate surface, indicating none of the test compounds were interacting non-specifically with the plate surface. Thus, these results demonstrate that a bispecific molecule that is targeting binding to both MAdCAM and PD-1 can successfully bind to both molecules. Although the experiments were performed with PD-L1 as a substitute for a PD-1 antibody, it is expected that the PD-1 antibody will function in a similar manner.
Example 7. A Bispecific PD-L1 Prototype Molecule Inhibits T Cells in a PD-1 Agonist Assay
A bispecific molecule that mimics a PD-1 agonist antibody was tested to demonstrate that PD-1 agonism can inhibit T cells. Briefly, 7 week old female C57LB/6 mice were sacrificed and their splenocytes were isolated. The splenocytes were exposed to ConA for 3 days and then exposed to anti-CD3 in the presence or absence of the PD-1 type molecule, which in this example was a PD-L1 bispecific molecule that was tethered to a plate using anti-human IgG. T cells were then introduced to the PD-L1 bispecific molecule. The PD-L1, which mimics a PD-1 antibody were found to be a T cell agonist and inhibit T cell activation. The same experiments were repeated using a PD-L1 bispecific molecule that was fused with an anti-MAdCAM antibody, which were tethered to a plate by interacting with a MAdCAM coated plate. The PD-1 agonist mimic, the PD-L1/anti-MAdCAM antibody were found to be effective agonists of T cell activity. These results demonstrate that a bispecific molecule that mimics a PD-1 antibody/MAdCAMAb fusion protein can exert functional inhibitory signaling into primary mouse T cell blasts when the molecule is captured via the MAdCAM antibody component at the end of the molecule.
Example 8: A Bispecific PD-1 Prototype Molecule with a Different Tissue Tether can Inhibit T Cells in a PD-1 Agonist Assay
A fusion molecule of a PD-L1 was used as a substitute for a PD-1 antibody and linked to a Class I H-2Kk antibody. The MHC Class I H-2Kk tethered PD-L1 molecule had functional binding, similar to the data described in Examples 6 and 7. Briefly, splenocytes from C57Bl/6 mice were stimulated with Concanavalin A (ConA) and IL-2 for 3 days. Plates were coated with anti-CD3 (2C11) overnight at 4 C, washed. Plates were coated with anti-human IgG for 3 hrs at 37 C and washed. Mono-specific anti-H-2Kk (16-3-22) or bi-specific anti-H-2Kk:mPD-L1 were added and incubated for 3 hr at 37 C and washed. All test articles contained a human IgG1-Fc portion. PBS (No Tx) was added to determine the assay background. ConA blasts were washed 2 times, added to the plate and incubated at 37 C. Supernatants were removed after 24 hrs. IFNg levels were determined by MSD. After 48 hrs, cell viability/metabolism was analyzed by Cell Titer-glo. When captured via the IgG Fc domain, an MHC Class I tethered PD-L1 bispecific can attenuate T cell activation in a mouse PD-1 agonism assay. Therefore, this example demonstrates that a different bispecific prototype molecule can exert functional inhibitory signaling into primary mouse T cell blasts—when the molecule is captured via a different tissue tether—in this case a mouse antibody to MHC Class I H-2Kk. Accordingly, this data demonstrates that the tethering is not specific to MAdCAM and is possible with other molecules that can act as targeting moieties as provided herein.
Example 9. PD-1 Agonists can Induce Signaling in Jurkat Cells
Jurkat cells expressing both human PD-1 fused to a beta-galactosidase enzyme donor and SHP-2 fused to a beta-galactosidase enzyme acceptor are added to test conditions in a plate and incubated for 2 hrs. Agonist PD-1 antibodies induce signaling and SHP-2 recruitment, enzyme complementation and formation of an active beta-galactosidase enzyme. Beta-galactosidase substrate was added and chemiluminescence can be measured on a standard luminescence plate reader. Agonism is measured by chemiluminescence, where the more chemiluminescence that is measured indicates the greater agonism.
Agonism of a PD-1/MAdCAM bi-specific molecule was measured in this assay. C110 (UCB) and CC-90006 (Celgene/Anaptys) were used as PD-1 agonist antibodies. Both are active and exhibit PD-1 agonism in functional assay in Ig-capture assay format. Briefly, plates were coated with anti-human IgG for overnight at 4 C and washed. Anti-tetanus toxin (TT) or benchmark agonist anti-PD-1 monoclonal antibodies, C110 or CC-90006 were added and incubated for 1 hr at 37 C and washed. All test articles contained a human IgG1-Fc. Media (No Tx) was added to determine the assay background. Plates were washed 3 times. Jurkat cells expressing both human PD-1 fused to a b-galactosidase enzyme donor and SHP-2 fused to a b-galactosidase enzyme acceptor were added and incubated for 2 hrs. Agonist PD-1 antibodies induce signaling and SHP-2 recruitment, enzyme complementation and formation of an active b-galactosidase enzyme. B-galactosidase substrate was added and chemiluminescence was measured on a standard luminescence plate reader. The two human PD-1 agonist antibodies (C110 and CC-90006) bind and induce signaling (a surrogate for agonism) in the modified Jurkat reporter assay. Thus, this assay is a functional PD-1 agonism assay. C110:MECA89 (MECA89 is a known MAdCAM antibody) is a novel bispecific molecule created by fusing MAdCAM antibody, MECA89[scFv], to C-terminus of the heavy chain of C110. This fusion protein was found to be active and exhibit PD-1 agonism in functional assay when captured via IgG Fc domain, as was C110 only protein. However, only C110:MECA89 is active in functional assay format using MAdCAM protein as capture (the monospecific components do not signal).
Briefly, plates were coated with either anti-human IgG or recombinant mMAdCAM-1 overnight at 4 C and washed. Mono-specific Anti-tetanus toxin (TT), anti-MAdCAM-1 (MECA89) or agonist anti-PD-1 (C110) or bi-specific C110:MECA89 were added and incubated for 1 hr at 37 C and washed. All test articles contained a human IgG1-Fc portion. PBS (No Tx) was added to determine the assay background. Plates were washed 2 times. Jurkat cells expressing both human PD-1 fused to a b-galactosidase enzyme donor and SHP-2 fused to a b-galactosidase enzyme acceptor were added and incubated for 2 hrs. Agonist PD-1 antibodies induce signaling and SHP-2 recruitment, enzyme complementation and formation of an active b-galactosidase enzyme. B-galactosidase substrate was added and chemiluminescence was measured on a standard luminescence plate reader. Results: Both C110, and the MAdCAM-tethered C110 bispecific molecules can induce PD-1 signaling in the Jurkat reporter assay when the plate is coated with an anti-IgG Fc capture, but only the MAdCAM-tethered bispecific can induce PD-1 signaling in the reporter assay when the plate is coated with recombinant MAdCAM protein. These results demonstrate that the molecule tethered with MAdCAM and contains a PD-1 agonist antibody are functional, which is similar to the results shown with the PD-L1 as the PD-1 agonist surrogate.
Example 10: Generation of PD-1 Agonist Antibodies
PD-1 deficient mice immunized with mouse PD-1 under conditions to generate an immune response against PD-1. 54 hybridomas were generated and identified that bind mouse PD-1. The antibodies produced by the different hybridomas were analyzed for T cell agonism according to the methods described in Examples 4 and 6. Out of the 54 hybridomas at least 6 were identified as PD-1 agonists. The antibodies were also tested for binding on PD-1 and were found to bind at the same site as the PD-L1 binding site.
Briefly, binding to the PD-L1 binding site was determined using the following assay. Immunosorbent plates were coated overnight with 75 μL of recombinant mouse PD-L1-Fc (2 μg/mL) in 1×PBS, pH 7.4. Plates were then washed 3× with 1×PBS and blocked for 2 hours at room temperature with 1×PBS supplemented with 1% BSA. Recombinant mouse PD-1-Fc (1 nM) was incubated with 100 nM of the indicated anti-mouse PD-1 antibody in 1×PBS supplemented with 1% BSA and 0.05% Tween20 (Assay Buffer) for 1 hour at room temperature, shaking. After blocking, plates were washed 3× with 1×PBS supplemented with 0.05% Tween20 PBST and the antibody-PD-1 conjugates were incubated with plate-bound mouse PD-L1. After washing away unbound PD-1 with PBST, plates were incubated with 75 μL of biotinylated, polyclonal anti-PD-1 antibody (0.5 μg/mL) in assay buffer, followed by amplification with 1:5000 streptavidin HRP also diluted in assay buffer. Plates were washed three times with PBST followed by three washes with 1×PBS before addition of 100 μL TMB followed by 100 μL 1M HCl to stop the developing. Absorbance read at 450 nm and normalized to binding of PD-1 to PD-L1 in the absence of antibody. The results showed that the active antibodies bind to the PD-L1 binding site. The inactive antibodies did not bind to the PD-L1 binding site. Therefore, this example demonstrates the ability to produce anti-PD-1 antibodies that are agonists, in addition to the previously identified PD-1 agonist antibodies described herein.
Example 11: Tethered Anti-PD-1 Antibodies Acts as PD-1 Agonists
A human antibody scFv phage library was panned against recombinant human, mouse, and cyno PD-1 proteins across iterative selection rounds to enrich for antibody clones that recognize all three aforementioned species orthologues of PD-1. The scFv clones were configured in nt-VH-Linker-VL-ct format and fused to the M13 phage surface via the pIII coat protein. After selections, clonal scFvs were screened for binding to human, mouse, and cyno PD-1 expressed on the cell surface of CHO cells. Clones that were found to be cross reactive to all three cell surface expressed PD-1 species orthologues were converted using standard molecular biology techniques, into a human IgG1 format whereby each molecule was comprised of four polypeptide chains in total (2 heavy, and 2 light chains). The two light chains were identical to each other and the two heavy chains were identical to each other as provided.
The two identical heavy chains homodimerize and the two identical light chains pair with each heavy chain to form an intact human IgG1. The Fc domain contains the L234A, L235A, and G237A mutations to ablate FcγR interactions. The converted human IgG1 anti-PD-1 antibodies were transfected and expressed in HEK293 Expi cells, and purified by protein A chromatography. The protein concentration was determined using a nanodrop spectrophotometer in conjunction with antibody specific extinction coefficients. Antibodies were formulated in PBS pH 7.4.
The anti-PD-1 antibodies were next tested in the Jurkat assay described herein for agonist activity. Briefly, tissue culture plates were coated with anti-IgG or left uncoated. For captured format, test articles or controls were added to the anti-IgG coated wells at 100 nM, 25 nM or 12.5 nM and incubated for 3 hrs at 37 C. Plates were washed and Jurkat PD-1 cells were added. For the soluble format, soluble test articles or controls were added to wells at 100 nM, 25 nM or 12.5 nM already containing Jurkat PD1 cells. Luminescence was measured in a plate reader. The results demonstrated that nine of the twelve human/mouse cross-reactive PD-1 antibodies showed dose-dependent activity in the Jurkat assay when the anti-PD-1 antibodies were captured via anti-IgG, but not in the soluble format. This data demonstrates that the anti-PD-1 antibody can act as an agonist when tethered to its target by a targeting moiety.
In conclusion, without being bound to any particular theory, the data presented herein demonstrate that a PD-1 Agonist/MAdCAM bi-specific molecule can bind to both MAdCAM and PD-1 and also agonize T cell activity. Thus, the molecules can be used to treat the various conditions provided herein and provide for localized and/or tissue specific immunomodulation and the down regulation of a T-Cell response.
Example 12: Generation of IL-2 Muteins
A pTT5 vector containing the single gene encoding the human IL-2M polypeptide fused N-terminally (SEQ ID NO: 57) or C-terminally (SEQ ID NO: 58) to human IgG1 Fc domain was transfected into HEK293 Expi cells. After 5-7 days, cell culture supernatants expressing IL-2Ms were harvested, and clarified by centrifugation and filtration through a 0.22 um filtration device. IL-2Ms were captured on proA resin. The resin was washed with PBS pH 7.4 and the captured protein was eluted using 0.25% acetic acid pH 3.5, with neutralization using a tenth volume of 1M Tris pH 8.0. The protein was buffer exchanged into 30 mM HEPES 150 mM NaCl pH 7, and analyzed by size exclusion chromatography on a Superdex 200 3.2/300 column. Analysis of 5 ug of purified material by reducing and non-reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted. The IL-2Ms were expressed at over 10 mg/L, and were over 95% monodispersed after purification as shown by size exclusion chromatography and reducing/non-reducing SDS-PAGE.
Example 13: IL-2M Molecules can Bind CD25
An immunosorbent plate was coated with CD25 at a concentration of 0.5 μg/mL in PBS pH 7.4, 75 ul/well, and incubated overnight at 4° C. Wells were washed with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer) three times, and then blocked with 200 ul/well 1% BSA in PBS pH 7.4 (block buffer) for two hours at room temperature. After three washes with wash buffer IL-2M molecules of Example 12 were diluted to eleven—two fold serial dilution in PBS containing 1% BSA and 0.05% Tween-20 (assay buffer) with 2 nM being the highest concentration. The diluted material was added to the CD25 coated plate at 75 ul/well for 1 hour at room temperature. After three washes with wash buffer, a goat biotinylated anti-IL-2 polyclonal antibody, diluted to 0.05 μg/mL in assay buffer, was added to the plate at 75 ul/well for 1 hr at room temperature. After three washes with wash buffer high sensitivity streptavidin HRP diluted in assay buffer at 1:5000 was added to the plate at 75 ul/well for 15 minutes at room temperature. After three washes with wash buffer and 1 wash with wash buffer (with no tween-20), the assay was developed with TMB, and stopped with 1N HCL. OD 450 nm was measured. The experiment included appropriate controls for non-specific binding of IL-2M molecules to the plate/block in the absence of CD25 and a negative control molecule that is unable to bind CD25.
The results indicate that at concentrations of 2 nM-1.9 pM, IL-2M molecules are able to bind CD25 with sub nanomolar EC50s. Additionally, there was no detection of any compound at any concentration tested, when CD25 was not present on the plate surface, indicating none of the test compounds were interacting non-specifically with the plate surface (data not shown).
Example 14: In Vitro P-STAT5 Assay to Determine Potency and Selectivity of IL-2M Molecules
Peripheral blood mononuclear cells (PBMCs) were prepared using FICOLL-PAQUE Premium and Sepmate tubes from freshly isolated heparinized human whole blood. PBMCs were cultured in 10% fetal bovine serum RPMI medium in the presence of wild-type IL-2 or IL-2M of Example 12 for 20 minutes and then fixed for 10 minutes with BD Cytofix. Fixed cells were sequentially permeabilized with BD Perm III and then BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10 minutes, cells were stained for 30 minutes with antibodies for phospho-STAT5 FITC, CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 and then acquired on an Attune NXT with plate reader. The IL-2M of Example 12 potently and selectively induces STAT5 phosphorylation in Tregs but not Teffs.
Example 15: Methods for Generation of Bispecific MAdCAM-Tethered IL-2M Molecules
A pTT5 vector containing the single gene encoding the single B0001 polypeptide comprising an IL-2 mutein with a N88D, V69A, and Q74P mutations fused to a Fc protein with the LALA mutations as provided for herein with a GGGGSGGGGSGGGGS (SEQ ID NO: 30) linker and scFV antibody that binds to MAdCAM or a similar molecule but with a GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) linker B0002 with human IL-2M fused N-terminally of human IgG1 Fc domain and with c-terminal fused anti-mMAdCAM scFv MECA-89 was transfected into HEK293 Expi cells. For B0003, two plasmids were co-transfected at equimolar ratios. The first plasmid encoded the light chain of MECA-89 and the 2nd encoded the full length IgG1 heavy chain of MECA-89 with c-terminally fused human IL-2M. After 5-7 days, cell culture supernatants expressing B0001, B0002, and B0003 were harvested, and clarified by centrifugation and filtration through a 0.22 um filtration device. B0001, B0002, and B0003 were captured on proA resin. The resin was washed with PBS pH 7.4 and the captured protein was eluted using 0.25% acetic acid pH 3.5, with neutralization using a tenth volume of 1M Tris pH 8.0. The protein was buffer exchanged into 30 mM HEPES 150 mM NaCl pH 7, and analyzed by size exclusion chromatography on a Superdex 200 3.2/300. Analysis of 1 μg of purified material by reducing and non-reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted.
B0001, B0002, and B0003 were expressed at over 8 mg/L, and were over 95% monodispersed after purification as shown by size exclusion chromatography and reducing/non-reducing SDS-PAGE. This experiment shows that dual function bispecific molecules with immunomodulators at either the N or C terminus can be produced and the position of the IL-2M protein (either at the N or C terminus) did not significantly alter expression and therefore, either format can be used.
Example 16: Bispecific MAdCAM-Tethered IL-2M Molecules can Bind MAdCAM and CD25 Simultaneously
An immunosorbent plate was coated with recombinant mouse MAdCAM-1 at a concentration of 1 μg/mL in PBS pH 7.4, 75 ul/well, and incubated overnight at 4° C. Wells were washed with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer) three times, and then blocked with 200 ul/well 1% BSA in PBS pH 7.4 (block buffer) for two hours at room temperature. After three washes with wash buffer, B0001, B0002, B0003 were diluted to 1 nM, 10 nM, and 100 nM in PBS containing 1% BSA and 0.05% Tween-20 (assay buffer). The diluted material was added to the mouse MAdCAM-1 coated plate at 75 ul/well for 1 hour at room temperature. After three washes with wash buffer, human CD25 was added to the plate at 75 ul/well, at a concentration of 10 nM in assay buffer for 1 hr at room temperature. After three washes with wash buffer, a goat biotinylated anti-human CD25 polyclonal antibody, diluted to 0.4 μg/mL in assay buffer, was added to the plate at 75 ul/well for 1 hr at room temperature. After three washes with wash buffer high sensitivity streptavidin HRP diluted in assay buffer at 1:5000 was added to the plate at 75 ul/well for 15 minutes at room temperature. After three washes with wash buffer and 1 wash with wash buffer (with no tween-20), the assay was developed with TMB, and stopped with 1N HCL. OD 450 nm was measured. The experiment included appropriate controls for non-specific binding of the proteins of Example 15 to the plate/block in the absence of mouse MAdCAM-1, as well as no CD25 controls, and mono-specific controls, that are unable to form a bridge between human CD25 and mouse MAdCAM.
It was found that that at concentrations of 1 nM, 10 nM, and 100 nM, the bi-specific molecules of Example 15 were able to simultaneously interact with mouse MAdCAM and human CD25, whilst the monospecific controls, did not create a bridging signal. Additionally, there was no binding of any compound to CD25 at any concentration tested, when mouse MAdCAM-1 was not present on the plate surface, indicating none of the test compounds were interacting non-specifically with the plate surface. These results demonstrate that the bispecific molecules can bind both MAdCAM and CD25 simultaneously in a functional binding assay, such as an ELISA.
Example 17: In Vitro P-STAT5 Assay Demonstrating Activity and Selectivity of Bispecific MAdCAM-Tethered IL-2M when in Solution or when Tethered
Recombinant mouse MAdCAM was coated onto wells of a 96 well high binding plate (Corning) overnight. After washing 2 times with PBS, the plate was blocked for 1 hour with 10% FBS RPMI media. A MAdCAM-tethered IL-2M bispecific of Example 15 or untethered IL-2M control (such as those prepared in Example 12) were captured for 1 hour. After washing 2 times with PBS, freshly-isolated human PBMCs were stimulated for 60 minutes with captured IL-2M or for comparison IL-2M in solution. Cells were then fixed for 10 minutes with BD Cytofix, permeabilized sequentially with BD Perm III and BioLegend FOXP3 permeabilization buffer, blocked with human serum and stained for 30 minutes with antibodies against phospho-STAT5 FITC (CST), CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 (BD) and acquired on an Attune NXT with plate loader. In solution, both molecules have comparable activity and selectivity on Treg versus Teff. Plates coated with mouse MAdCAM were able to capture the bi-specific molecule of Example 15 and the captured/immobilized bi-specific molecule was still able to selectively activate Tregs over Teffs. This example demonstrates that MAdCAM-tethered IL-2M molecules can retain biological activity and selectivity when in solution or when captured/immobilized.
Example 18: Immunogenicity of IL-2 Muteins
IL-2 Mutein sequences were analyzed using the NetMHCIIPan 3.2 software, which can be found at www “dot” cbs “dot” dtu “dot” dk/services/NetMHCIIpan/. Artificial neural networks were used to determine peptide affinity to MHC class II alleles. In that analysis, 9-residue peptides with potentially direct interaction with the MHC class II molecules were recognized as binding cores. Residues adjacent to binding cores, with potential to influence the binding indirectly, were also examined as masking residues. Peptides comprising both the binding cores and masking residues were marked as strong binders when their predicted KD to the MHC class II molecule was lower than 50 nM. Strong binders have a greater chance of introducing T cell immunogenicity.
A total of 9 MHCII alleles that are highly represented in North America and Europe were included in the in silico analysis. The panel of IL-2M (IL-2 mutein) molecules tested included the IL-2 Muteins with L53I, L56I, L80I, or L118I mutations. Only MHCII alleles DRB1_1101, DRB1_1501, DRB1_0701, and DRB1_0101 yielded hits with any of the molecules assessed. The peptide hits for DRB_1501 were identical between all constructs tested including wild-type IL-2 with the C125S mutation. The addition of L80I removes 1 T cell epitope for DRB1-0101 [ALNLAPSKNFHLRPR (SEQ ID NO: 626)] and modestly reduces the affinity of two other T cell epitopes [EEALNLAPSKNFHLR (SEQ ID NO: 627) and EALNLAPSKNFHLRP (SEQ ID NO: 628)]. For MHCII allele DRB1-0701, L80I removes 1 T cell epitope [EEALNLAPSKNFHLR (SEQ ID NO: 627)]. Therefore, the data demonstrates that a IL-2 mutein comprising the L80I mutation should be less immunogenic, which is a surprising and unexpected result from the in silico analysis.
Example 19: Generation of Additional IL-2 Muteins
A pTT5 vector containing the single gene encoding the single IL-2M (IL-2 mutein) of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 (and IL-2M control; SEQ ID NO: 50) polypeptide with human IL-2M fused N-terminally of human IgG1 Fc domain was transfected into HEK293 Expi cells. After 5-7 days, cell culture supernatants expressing SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 (and IL-2M control; SEQ ID NO: 50) were harvested, and clarified by centrifugation and filtration through a 0.22 um filtration device. SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 (and IL-2M control; SEQ ID NO: 50) were captured on proA resin. The resin was washed with PBS pH 7.4 and the captured protein was eluted using 0.25% acetic acid pH 3.5, with neutralization using a tenth volume of 1M Tris pH 8.0. The protein was buffer exchanged into 30 mM HEPES 150 mM NaCl pH 7, and analyzed by size exclusion chromatography on a Superdex 200 3.2/300 column. Analysis of 5 ug of purified material by reducing and non-reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted.
IL-2Ms SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 (and IL-2M control; SEQ ID NO: 50) expressed at over 45 mg/L, and were over 95% monodispersed after purification as shown by size exclusion chromatography and reducing/non-reducing SDS-PAGE.
Example 20: IL-2Ms of Example 19 can Bind CD25
An immunosorbent plate was coated with CD25 at a concentration of 0.5 μg/mL in PBS pH 7.4, 75 ul/well, and incubated overnight at 4° C. Wells were washed with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer) three times, and then blocked with 200 ul/well 1% BSA in PBS pH 7.4 (block buffer) for two hours at room temperature. After three washes with wash buffer IL-2Ms SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 were diluted to eleven—two fold serial dilution in PBS containing 1% BSA and 0.05% Tween-20 (assay buffer) with 2 nM being the highest concentration. The diluted material was added to the CD25 coated plate at 75 ul/well for 1 hour at room temperature. After three washes with wash buffer, a goat biotinylated anti-IL-2 polyclonal antibody, diluted to 0.05 μg/mL in assay buffer, was added to the plate at 75 ul/well for 1 hr at room temperature. After three washes with wash buffer high sensitivity streptavidin HRP diluted in assay buffer at 1:5000 was added to the plate at 75 ul/well for 15 minutes at room temperature. After three washes with wash buffer and 1 wash with wash buffer (with no tween-20), the assay was developed with TMB, and stopped with 1N HCL. OD 450 nm was measured. The experiment included appropriate controls for non-specific binding of the molecules to the plate/block in the absence of CD25. The results indicate that at concentrations of 2 nM-1.9 pM, the muteins of Example 19 were able to bind CD25 with sub nanomolar EC50s. Additionally, there was no detection of any compound at any concentration tested, when CD25 was not present on the plate surface, indicating none of the test compounds were interacting non-specifically with the plate surface. Thus, the muteins of Example 19 can bind to CD25.
Example 21: IL-2 Muteins of Example 19 are Potent and Selective
Peripheral blood mononuclear cells (PBMCs) were prepared using FICOLL-PAQUE Premium and Sepmate tubes from freshly isolated heparinized human whole blood. PBMCs were cultured in 10% fetal bovine serum RPMI medium in the presence of wild-type IL-2 or the muteins of Example 19 for 20 minutes and then fixed for 10 minutes with BD Cytofix. Fixed cells were sequentially permeabilized with BD Perm III and then BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10 minutes, cells were stained for 30 minutes with antibodies for phospho-STAT5 FITC (CST), CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 (all BD) and then acquired on an Attune NXT with plate reader. The IL-2 muteins of Example 19 were found to be potent and have selectivity against Treg versus Teff. The mutein comprising the L118I mutation was found to have increased activity and selectivity as compared to the other muteins.
Example 22: IL-2 Muteins Expand Tregs in Humanized Mice
NSG mice humanized with human CD34+ hematopoietic stem cells were purchased from Jackson Labs. On days 0 and 7, the mice were dosed subcutaneously with 1 μg IL-2 Mutein (SEQ ID NO: 50) or other IL-2 muteins SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56. On Day 7, mice were euthanized and whole blood and spleens were collected. Whole blood was aliquoted into a 96 well deep well plate and fixed for 10 minutes using BD Fix Lyse. Splenocytes were isolated using 70 um filters (BD) and red blood cells were lysed using RBC lysis buffer from BioLegend. After washing with 2% fetal bovine serum PBS, splenocytes were labeled with near infrared live dead stain (Invitrogen) for 20 minutes and then fixed for 20 minutes using BioLegend fixation buffer. Both whole blood cells and splenocytes were then permeabilized using BioLegend FOXP3 permeabilization buffer, blocked with human serum and stained for 30 minutes with antibodies against human CD8a FITC (BL), human CD25 PE (BD), human FOXP3 AF647 (BD) CD4 PerCP Cy5.5 (BD), human Siglec-8 PE Cy7 (BL), human CD3 BV421 (BL), human CD45 BV605 (BL), human CD56 BV785 (BL) and mouse CD45 (BV711) and acquired on an Attune NXT with plate loader.
Compared to vehicle control, IL-2Ms SEQ ID NO: 54 and SEQ ID NO: 56 selectively induced Tregs in mouse spleens and whole blood (p<0.0005 by ANOVA with Dunn's Multiple Comparison Test). The other IL-2Ms also increased the frequency of Tregs, though these changes compared to the vehicle group were not statistically significant. There were no significant changes in the frequencies of CD56pos NK cells, CD3pos T cells, CD8pos cytotoxic T lymphocytes, CD4pos helper T cells or CD25lo/FOXP3neg T effectors in mice dosed with SEQ ID NO: 54 and SEQ ID NO: 56. These results demonstrate that the IL-2 muteins increase the frequency of regulatory T cells.
Example 23: Generation of Bispecific mMAdCAM-Tethered IL-2M Molecule
A bispecific MAdCAM-IL-2 mutein was produced, with the antibody being the heavy and light chains of MECA89. This was produced using two plasmids encoding both heavy and light chains were co-transfected at equimolar ratios. The first plasmid encoded the light chain of MECA-89 and the second encoded the full length IgG1 heavy chain of MECA-89 with C-terminally fused to a human IL-2M comprising the L118I mutation. After 3-5 days, cell culture supernatants expressing the bispecific were harvested, and clarified by centrifugation and filtration through a 0.22 um filtration device. The bispecific was captured on proA resin. The resin was washed with PBS pH 7.4 and the captured protein was eluted using 0.25% acetic acid pH 3.5, with neutralization using a tenth volume of 1M Tris pH 8.0. The protein was buffer exchanged into 30 mM HEPES 150 mM NaCl pH 7, and analyzed by size exclusion chromatography on an AdvanceBio SEC column. Analysis of 1 μg of purified material by reducing and non-reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted.
The bispecific molecule expressed at 17 mg/L, and was over 95% monodispersed after purification as shown by size exclusion chromatography and reducing/non-reducing SDS-PAGE. These results demonstrate that it was able to produce dual function bispecific molecules with immunomodulators at the C terminus.
Example 24: Generation of MAdCAM Antibodies
A human antibody scFv phage library was panned against recombinant human, mouse, and cyno MAdCAM proteins across iterative selection rounds to enrich for antibody clones that recognize all three aforementioned species orthologues of MAdCAM. The scFv clones were configured in nt-VH-Linker-VL-ct format and fused to the M13 phage surface via the pIII coat protein. After selections, clonal scFvs were screened by ELISA for binding to human, mouse, and cyno MAdCAM expressed on the cell surface of CHO cells. Clones that were found to be cross reactive to all three cell surface expressed MAdCAM species orthologues were converted using standard molecular biology techniques or gene synthesis, into a human IgG1 format whereby each molecule was comprised of four polypeptide chains in total (2 heavy, and 2 light chains). The two light chains were identical to each other and the two heavy chains were identical to each other. The two identical heavy chains (1 and 2) homodimerize and the two identical light chains (3 and 4) pair with each heavy chain to form an intact human IgG1. The Fc domain contains the L234A, L235A, and G237A mutations to ablate FcγR interactions. The format can be illustrated as follows:

    
    
        Chain 1: nt-VH1-CH1-CH2-CH3-ct
        Chain 2: nt-VH1-CH1-CH2-CH3-ct
        Chain 3: nt-VK1-CK-ct
        Chain 4: nt-VK1-CK-ct
    
    


In addition, MAdCAM scFvs were also converted using standard molecular biology techniques (such as Gibson Cloning procedure) or gene synthesis into a bispecific format whereby an IL-2M was situated at the c-terminus of the IgG heavy chain of the MAdCAM antibody, as outlined below:

    
    
        Chain 1: nt-VH1-CH1-CH2-CH3-ct-Linker-IL-2M
        Chain 2: nt-VH1-CH1-CH2-CH3-ct-Linker-IL-2M
        Chain 3: nt-VK1-CK-ct
        Chain 4: nt-VK1-CK-ct

An ELISA was used to analyze binding of anti-MAdCAM scFvs to captured or plate bound human, cyno, and mouse MAdCAM. Biotinylated human and cyno MAdCAM were captured on a streptavidin coated plate, and mouse MAdCAM-Fc coated directly onto an immunosorbent plate. After a blocking step, the plates were washed and scFv in crude periplasmic lysate was applied to the plate surface. scFv binding was detected using an anti-V5 HRP conjugate. The assay was developed with TMB substrate and stopped with acid. The absorbance at 450 nm was measured. Appropriate wash steps were applied between each step of the ELISA. Human versus cyno and human versus mouse were evaluated. The scFv's were also analyzed using surface plasmon resonance technology. After being captured on a biosensor surface via the V5 tag, soluble monomeric human MAdCAM was titrated and both binding and dissociation measured and fit to a 1:1 binding model allowing the derivation of on and off-rates.

    
    


The results measured indicate that the majority of clones tested have human and cyno MAdCAM binding cross reactivity and a small panel have additional cross reactivity to mouse MAdCAM. Biosensor experiments demonstrated that the clones exhibited a range of binding on and off-rates against human MAdCAM with ka values ranging from 103 1/Ms through 107 1/Ms and kd values ranging 10−1 through 10−4 1/s. Certain clones have an off-rate slower than 2×10e2 1/s. Thus, MadCAM antibodies were generated and can be used in a bi-specific format.
Example 25: Generation of Bispecific Human MAdCAM-Tethered IL-2Ms of Example 19
Two plasmids each were co-transfected at equimolar ratios. The first plasmid in each case encoded the light chain of Hu.MAdCAM and the second encoded the full length IgG1 heavy chain of Hu.MAdCAM with a C-terminally fused human IL-2M comprising the L118I mutation as illustrated in the Table of MAdCAM-IL-2 Mutein Bispecific Compounds provided herein. After 3-5 days, cell culture supernatants expressing the Hu.MAdCAM-IL-2M bispecifics was harvested, and clarified by centrifugation and filtration through a 0.22 um filtration device. The Hu.MAdCAM-IL-2M bispecifics were captured on proA resin. The resin was washed with PBS pH 7.4 and the captured proteins were eluted using 0.25% acetic acid pH 3.5, with neutralization using a tenth volume of 1M Tris pH 8.0. The proteins were buffer exchanged into 30 mM HEPES 150 mM NaCl pH 7, and analyzed by size exclusion chromatography on an AdvanceBio SEC column. Analysis of 1 μg of purified material by reducing and non-reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted. The Hu.MAdCAM-IL-2M bispecifics expressed at over 10 mg/L, and was over 95% monodispersed after purification as shown by size exclusion chromatography and reducing/non-reducing SDS-PAGE. Thus, these results demonstrate that fully human dual function bispecific molecules with immunomodulators at the C terminus can be produced.
Example 26: Durability of Signaling Induced by IL-2 Muteins
Peripheral blood mononuclear cells (PBMCs) were prepared using FICOLL-PAQUE Premium and Sepmate tubes from freshly isolated heparinized human whole blood. PBMCs were cultured in 10% fetal bovine serum RPMI medium in the presence of IL-2Ms for 60 minutes. Cells were then wash 3 times and incubated for an additional 3 hours. Cells were then fixed for 10 minutes with BD Cytofix. Fixed cells were sequentially permeabilized with BD Perm III and then BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10 minutes, cells were stained for 30 minutes with antibodies for phospho-STAT5 FITC, CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 and then acquired on an Attune NXT with plate reader. All four IL-2 muteins of Example 19 induced durable signaling in Treg but not in Teff as compared to the control. An IL-2 mutein of SEQ ID NO: 56 is superior to an IL-2 mutein of SEQ ID NO: 55, SEQ ID NO: 54 or SEQ ID NO: 53. These results demonstrate that the IL-2 can induce durable and selective signaling in Treg which should lead to greater Treg expansion in vivo and permit less frequent dosing to achieve Treg expansion.
Example 27: In Vitro P-STAT5 Assay Demonstrates Activity and Selectivity of Bispecific Hu.MAdCAM-Tethered IL-2Muteins when in Solution or when Tethered
Recombinant human MAdCAM was coated onto wells of a 96 well high binding plate (Corning) overnight. After washing 2 times with PBS, the plate was blocked for 1 hour with 10% FBS RPMI media. MAdCAM-tethered IL-2M mutein bi-specifics or untethered IL-2M control were captured for 1 hour. After washing 2 times with PBS, freshly-isolated human PBMCs were stimulated for 60 minutes with captured IL-2MM or for comparison IL-2MM in solution. Cells were then fixed for 10 minutes with BD Cytofix, permeabilized sequentially with BD Perm III and BioLegend FOXP3 permeabilization buffer, blocked with human serum and stained for 30 minutes with antibodies against phospho-STAT5 FITC (CST), CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 (BD) and acquired on an Attune NXT with plate loader. Results: In solution, IL-2M bi-specifics tethered to human MAdCAM and the control have comparable activity and selectivity on Treg versus Teff. Plates coated with MAdCAM were able to capture bi-specifics, and the captured/immobilized bi-specifics were still able to selectively activate Tregs over Teffs. This example demonstrates that IL-2MM bi-specifics targeting human MAdCAM can retain biological activity and selectivity when in solution or when captured/immobilized.
Example 28. IL-2 Muteins Induce pSTAT5 in Human Tregs
Purified PBMC from heparinized whole blood from six healthy donors were treated with serial dilutions of a IL-2 mutein proteins comprising a sequence of SEQ ID NO: 59, wherein X3 is I and X1, X2, and X4 are L or a sequence of SEQ ID NO: 59, wherein X4 is I and X1, X2, and X3 are L at 37 C for 30 minutes. Cells were fixed, washed, permeabilized and washed. Cells were stained with antibodies that detect both surface markers and intracellular/nuclear markers (pSTAT5 and FOXP3). Data was collected on Attune NxT cytometer. Tregs were gated as mononuclear, singlet, CD3pos, CD4pos, CD25hi, FoxP3pos. The % of gated Tregs that express phosphorylated STAT5 was measured. Best-fit curves were fit to the dose-response of pSTAT5 and EC50 values were determined. Average EC50 values of all 6 donors were determined for IL-2 of SEQ ID NO: 59, wherein X3 is I and X1, X2, and X4 are L (37.26±7.30; n=16) and for IL-2 of SEQ ID NO: 59, wherein X4 is I and X1, X2, and X3 are L (23.11±5.35; n=15). The data demonstrate that the IL-2 muteins can induce pSTAT5 in human Tregs. The IL-2 comprising a sequence of SEQ ID NO: 59, wherein X4 is I and X1, X2, and X3 are L is more potent than the IL-2 sequence comprising SEQ ID NO: 39, but both are active across multiple populations of cells.
Example 29: IL-2 Muteins Induce pSTAT5 in Monkey PBMCs In Vitro
Purified PBMC from heparinized whole blood from three healthy monkeys were treated with serial dilutions a IL-2 mutein protein comprising a sequence of SEQ ID NO: 59, wherein X3 is I and X1, X2, and X4 are L or a sequence of SEQ ID NO: 59, wherein X4 is I and X1, X2, and X3 are L at 37 C for 60 minutes. Fluorochrome conjugated Anti-CD25 and anti-CD4 were added for the final 30 min of the IL-2 mutein treatment. Cells were fixed, washed, permeabilized and washed. Cells were stained with remaining antibodies that detect both surface markers and intracellular/nuclear markers (pSTAT5 and FOXP3). Data was collected on Attune NxT cytometer. Tregs were gated as mononuclear, singlet, CD4pos, CD25hi, FoxP3pos. The % of gated Tregs that express phosphorylated STAT5 was measured. The IL-2 muteins were found to induce pSTAT5 in monkeys.
Example 30: IL-2 Muteins Induce Expand Treg Cells and Induce Treg Proliferation In Vivo
Venous whole blood was collected in K2EDTA tubes from monkeys (cynomolgus) before dosing with IL-2 muteins of SEQ ID NO: 59, wherein X3 is I and X1, X2, and X4 are L or a sequence of SEQ ID NO: 59, wherein X4 is I and X1, X2, and X3 are L (2 timepoints/cyno, 5 cynos) and after dosing with either SEQ ID NO: 59, wherein X3 is I and X1, X2, and X4 are L (5 timepoints/cyno, 2 cynos) or SEQ ID NO: 59, wherein X4 is I and X1, X2, and X3 are L (5 timepoints/cyno, 3 cynos). Samples were divided in two and stained for two FACS panels separately. One was a “Treg panel” and one was a general immunophenotyping panel. RBCs were lysed and cells were stained for surface and intracellular markers after fixation and permeabilization. For the FACs analysis the number of total cells/ul was determined by ADVIA. The number of cells of a given subpopulation/ul was then calculated with the total number/ul and the % of total. For each monkey, the average number of a given cell type/ul of the two pre-dose bleeds was averaged and used to normalize the post-dose bleeds, such that “fold-change from pre-dose” was determined. To analyze serum cytokines and chemokines, plasma from K2EDTA whole blood was frozen until the end of the study. Chemokine and cytokine amounts were quantified by a multiplex MSD assay using serial dilutions of a standard control. The average and range of MCP-1 and IP-10 were determined in pre-dose bleeds. Both muteins were found to expand Treg and induce Treg proliferation in the monkeys. These results demonstrate that the IL-2 muteins function in an in vivo animal model that is similar to humans. It was also found that neither molecule significantly expanded Tconv cells, CD4 cells (Tnaive) or CD8 cells (Cytotoxic T), NK cells in the monkeys (non-human primate). It was also found that neither molecule significantly induced Serum chemokines. This data demonstrates that the IL-2 muteins can expand Treg cells and induce Treg cell proliferation without unwanted expansion or activation of other pathways. Thus, the IL-2 muteins are surprisingly potent, effective, and selective for Treg expansion and proliferation.
In summary, the embodiments and examples provided herein demonstrate that the IL-2 muteins that can be targeted to certain tissues can function as intended and be used to treat the diseases and conditions described herein. Furthermore, the examples provided for herein demonstrate the surprising and unexpected result that a bispecific molecule comprising a MAdCAM antibody and a IL-2 mutein can function to selectively and potently activate Tregs over Teffs, which demonstrates that the molecules can be used to treat or ameliorate the conditions described herein. The examples also demonstrate that the IL-2 mutein can function to selectively and potently activate Tregs over Teffs when used alone (or linked to a Fc protein) as provided for herein.
Example 31: Antibodies Bind to MAdCAM
Certain antibodies provided for herein were tested for their ability to bind to MAdCAM. The following table provides the binding information against the various targets and other activities. The antibodies, in either scFv or IgG format, which were tested for their ability to bind to human or mouse cells expressing MadCAM as well as binding to Cyno MadCAM protein. The results are presented either as “−” for no significant binding or as binding at different levels (e.g. “+”, “++”, and “+++”).










Activities of MADCAM Antibodies from Table 1 












Clone ID 







as in 

MAdCAM 

Cell-Based 



MAdCAM 
MAdCAM Cell 
Binding 
MAdCAM Octet 
Integrin 
ELISA Integrin 


Ab 
Binding 
ELISA 
Binding 
Blockade 
Blockade 

















Table 6 
Human 
Mouse 
Cyno 
Human 
Cyno 
Mouse 
Human 
Mouse 
Human 
Mouse




















1. 
+++
++ 
NT 
+++
+++
0.16 
−
NT 
−
+


2. 
++
+++
NT 
+
+++
+++
+
−
NT 
−


3. 
++
+++
++
NT 
NT 
NT 
+
NT 
NT 
NT 


4. 
+++
+++
NT 
+
+++
+++
+
−
+
+


5. 
+++
+++
NT 
++
+++
+++
−
NT 
−
+


6. 
+++
−
NT 
+++
+++
NB 
+
NT 
+
NT 


7. 
++
+++
NT 
+
+++
+++
+
NT 
NT 
+


8. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


9. 
+++
+++
NT 
+++
+++
+++
−
NT 
−
+


10. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


11. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


12. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


13. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


14. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


15. 
+++
−
+++
NT 
NT 
NT 
−
NT 
NT 
NT 


16. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


17. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


18. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


19. 
+++
−
NT 
+++
+++
−
−
NT 
+
NT 


20. 
+++
−
NT 
+++
+++
−
+
NT 
−
NT 


21. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


22. 
++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


23. 
+++
−
NT 
+++
+++
−
−
NT 
−
NT 


24. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


25. 
+
−
+++
NT 
NT 
NT 
NT 
NT 
NT 
NT 


26. 
++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


27. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


28 
++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


29. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


30. 
++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


31. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


32. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


33. 
++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


34. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


35. 
+++
−
+++
NT 
NT 
NT 
−
NT 
NT 
NT 


36. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


37. 
+++
−
NT 
NT 
NT 
NT 
+
NT 
NT 
NT 


38. 
++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


39. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


40. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


41. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


42. 
+++
−
++
NT 
NT 
NT 
+
NT 
NT 
NT 


43. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


44. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


45. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


46. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


47. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


48. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


49. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


50. 
+++
−
+++
NT 
NT 
NT 
−
NT 
NT 
NT 


51. 
+++
−
+++
NT 
NT 
NT 
−
NT 
NT 
NT 


52. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


53. 
+++
−
+++
NT 
NT 
NT 
−
NT 
NT 
NT 


54. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


55. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


56. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


57. 
+++
−
+++
NT 
NT 
NT 
−
NT 
NT 
NT 


58. 
+++
−
+++
NT 
NT 
NT 
−
NT 
NT 
NT 


59. 
+++
+++
NT 
+++
+++
+++
+
+
+
−


60. 
+++
++
NT 
+++
+++
NT 
−
NT 
−
+


61. 
++
+++
NT 
+++
+++
NT 
+
−
−
+


62. 
+++
+++
NT 
++
+++
NT 
−
NT 
−
+


63. 
++
+++
NT 
+
+
+++
+
+
+
+


64. 
+
+++
NT 
++
+
NT 
+
−
+
+


65. 
++
+++
NT 
+
+++
NT 
+
−
+
+


66. 
+++
+++
NT 
++
+
NT 
−
−
+
+



















Activities of MADCAM Antibodies from Table 7 












Clone ID 

MAdCAM 

Cell-Based 



as in 
MAdCAM Cell 
Binding 

Integrin 
ELISA Integrin 


MADCAM 
Binding 
ELISA 
MAdCAM Octet Binding 
Blockade 
Blockade 

















Ab Table 7 
Human 
Mouse 
Cyno 
Human 
Cyno 
Mouse 
Human 
Mouse 
Human 
Mouse




















1. 
+++
+++
NT 
NT 
NT 
NT 
−
NT 
NT 
NT 


2. 
++
+++
++
NT 
NT 
NT 
+
NT 
NT 
NT 


3. 
++
+++
++
NT 
NT 
NT 
+
NT 
NT 
NT 


4. 
+++
+++
NT 
++
++
+++
−
−
−
NT 


5. 
+++
+++
NT 
++
++
+++
−
NT 
−
NT 


6. 
+++
−
+++
+++
+++
−
+
+
+
+


7. 
++
+++
NT 
−
−
+++
NT 
NT 
−
NT 


8. 
++
+++
NT 
−
+++
+++
NT 
NT 
−
NT 


9. 
+++
−
NT 
NT 
NT 
NT 
−
NT 
−
−


10. 
+++
++
NT 
NT 
NT 
NT 
−
NT 
NT 
NT 


11. 
+++
+++
NT 
+++
+++
+++
−
NT 
+



12. 
+++
−
+++
NT 
NT 
NT 
+
NT 
+
+


13. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


14. 
+
−
NT 
−
−
−
+
NT 
−
+


15. 
+++
−
NT 
NT 
NT 
NT 
−
NT 
NT 
NT 


16. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
−
+


17. 
+++
−
NT 
+++
+++
−
−
NT 
+



18. 
+++
−
NT 
NT 
NT 
NT 
−
NT 
+
+


19. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


20. 
+++
−
NT 
+++
+++
−
−
NT 
NT 
NT 


21. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


22. 
+++
−
NT 
+++
++
−
−
NT 
+
+


23. 
+++
−
NT 
+++
+++
−
−
NT 
+
+


24. 
++
−
NT 
−
−
−
+
NT 
+
+


25. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


26. 
++
−
NT 
−
NT 
NT 
+
NT 
+
+


27. 
++
−
NT 
−
NT 
NT 
−
NT 




28. 
+
−
NT 
−
−
−
+
NT 
+
+


29. 
+
−
NT 
−
−
−
+
NT 
+
+


30. 
+++
+
NT 
+++
+++
−
−
NT 
NT 
NT 


31. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


32. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
−
+


33. 
+
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


34. 
+++
−
+++
NT 
NT 
NT 
+
NT 
−
+


35. 
+
−
NT 
−
NT 
NT 
−
NT 
−
+ 


36. 
+++
+
NT 
+++
++
NT 
−
NT 
NT 
NT 


37. 
++
−
NT 
NT 
NT 
NT 
NT 
NT 
−
+


38. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
−
+


39. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


40. 
++
−
NT 
NT 
NT 
NT 
NT 
NT 
+
−


41. 
++
−
NT 
NT 
NT 
NT 
NT 
NT 
−
−


42. 
+++
−
+++
NT 
NT 
NT 
+
NT 
+
−


43. 
+++
−
+++
NT 
NT 
NT 
+
NT 
−
−


44. 
++
−
NT 
NT 
NT 
NT 
NT 
NT 
+
−


45. 
++
−
NT 
++
+++
NT 
−
NT 
+
+


46. 
+++
−
+++
+++
inconclusive 
−
−
NT 
+
−


47. 
+++
−
+++
NT 
NT 
NT 
+
NT 
−
−


48. 
+++
−
++
NT 
NT 
NT 
+
NT 
NT 
NT 


49. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


50. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


51. 
++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


52. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


53. 
+
−
+
NT 
NT 
NT 
+
NT 
NT 
NT 


54. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


55. 
+++
−
NT 
++
+++
NT 
−
NT 
NT 
NT 


56. 
+++
−
++
NT 
NT 
NT 
+
NT 
NT 
NT 


57. 
+++
−
NT 
NT 
NT 
NT 
−
NT 
NT 
NT 


58. 
+++
−
NT 
+++
+++
NT 
−
NT 
NT 
NT 


59. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


60. 
+++
++
NT 
+++
+++
−
−
NT 
NT 
NT 


61. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


62. 
+++
−
NT 
NT 
NT 
NT 
−
NT 
NT 
NT 


63. 
+++
−
NT 
+++
+++
NT 
−
NT 
NT 
NT 


64. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


65. 
+++
−
NT 
+++
+++
−
−
NT 
NT 
NT 


66. 
+++
−
+
NT 
NT 
NT 
+
NT 
NT 
NT 


67. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


68. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


69. 
+++
−
+++
NT 
NT 
NT 
+
NT 
NT 
NT 


70. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


71. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


72. 
+++
−
+
NT 
NT 
NT 
+
NT 
NT 
NT 


73. 
+++
−
NT 
NT 
NT 
NT 
NT 
NT 
NT 
NT 


74. 
+++
−
NT 
+++
+++
−
−
NT 
NT 
NT 


75. 
+++
+++
+++
+++
+++
+++
+
+
NT 
NT 


76. 
+++
+++
NT 
+++
+++
NT 
−

NT 
NT 


77. 
+++
++
NT 
−
NT 
NT 
−
−
NT 
NT 


78. 
+++
+++
NT 
+++
+++
NT 
−

NT 
NT 


79. 
+++
+++
NT 
+
+
++
+
−
NT 
NT 


80. 
++
−
NT 
−
NT 
NT 
+
+
NT 
NT 


81. 
++
+++
NT 
−
NT 
NT 
−
−
NT 
NT 


82. 
+++
+++
NT 
++
+++
NT 
−
−
NT 
NT 


83. 
+++
++
NT 
++
+++
NT 
−
NT 
NT 
NT 


84. 
+++
++
NT 
+++
+++
NT 
−
NT 
NT 
NT









Example 32
A bispecific molecule comprising a MAdCAM antibody and a IL-2 mutein specifically Localize to High Endothelial Venules (HEV) in Gut after SC Dosing in Mice. Mice were dosed s.c. with untethered IL-2M or MAdCAM-tethered IL-2M. Intestinal tissues were harvested 4 days later, and stained for human IgG1 (to detect the test article Ig backbone of both the untethered and tethered molecules, or MECA367 (to detect MAdCAM-expressing HEV). It was found that only the MAdCAM-tethered IL-2M molecule specifically localized to the HEV whereas the untethered IL-2M did not show detectable or significant localization at the same tissues.
Example 33
Bispecific MadCAM-IL2M Do Not Block MADCAM:alpha4/beta7 interactions And Therefore Do Not Affect Cell Trafficking. A MAdCAM-tethered IL-2M molecule was tested to determine whether it blocks alpha4/beta7 integrin binding to MAdCAM. The assay demonstrated that it did not. It was also found that the bispecific did not, therefore, have an impact on cell trafficking. The binding activity was performed by ELISA or a cell interaction assay.
Example 34
IL-2M tethered to MAdCAM antibody is functional. CHO cells were transfected with human or mouse MAdCAM to generate MAdCAM-expressing CHO cells that were then grown on a plate. The test article was added, allowed to bind, then unattached test article was washed out. Human PBMC were added and 30 mins later evaluated by FACS for phosphorylation of STAT5 Tregs were pSTAT5+ revealing activation by IL-2M, Tconv cells remained inactivated, despite presumed high local concentration of the bispecific on cell surface.
Example 35
MAdCAM-Tethered-IL2M Ameliorates Weight Loss in TNBS-induced Colitis in Humanized Mice, Similar to Low-Dose IL-2. Mice were sensitized with TNBS D-7, primed with TNBS DO. Mice were dosed daily with low doses of IL-2 (+ve control) or vehicle (−ve control) from D-7 to D3. Mice dosed with the MAdCAM-Tethered-IL2M D-7 and DO. It was found that the attenuation of weight loss by the MAdCAM-Tethered-IL2M was similar to attenuation of weight loss by LD IL-2. Therefore, these results demonstrate that the tethered approach is functional even though it is specifically localizes to HEV as shown in the previous examples.
The format of the MAdCAM-Tethered-IL2M as described in Examples 22-24 was where the MAdCAM component was an IgG with IL-2 Mutein moiety fused at the c-terminus of the heavy chain. The IL-2 mutein, however, had a Fc portion at its N-terminus as described herein, such as SEQ ID NO: 56 The format of the bi-specific is a multiple chain polypeptides, which can be represented in the following format: Heavy Chain: NT-[VH_MAdCAM]-[CH1-CH2-CH3]-[Linker_B]-[IL-2_Mutein]-CT, wherein
NT=N-terminus
[VH_MAdCAM]=Any VH domain provided for herein or a VH domain comprising the CDR1, CDR2, or CDR3 as described in Table 6 or Table 7;
[CH1-CH2-CH3]=the Human IgG1 Constant Heavy 1 (CH1), Constant Heavy 2 (CH2), and Constant Heavy 3 (CH3) domains, which can have a sequence of:







(SEQ ID NO: 44)


ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH


 


TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS


 


CDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED


 


PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC


 


KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF


 


YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF


 


SCSVMHEALHNHYTQKSLSLSPG;






[Linker_B]=GGGGS (SEQ ID NO: 23), which could also be GGGGSGGGGSGGGGS (SEQ ID NO: 30);

[IL-2_Mutein]=Any IL2 Mutein provided for herein, including but not limited to SEQ ID NO: 56; and

CT=C-terminus.

The molecule can also have a light chain format of:

Light Chain: NT-[VK_MAdCAM]-[CK]-CT, wherein

NT=N-terminus;

[VK_MAdCAM]=as illustrated in Table 6 or Table 7;

[CK]=Human constant kappa domain, which can have a sequence of:








(SEQ ID NO: 45)


RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN


 


SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF


 


NRGEC;






and

CT=C-terminus.

Example 36
Various MAdCAM antibodies were tested for the ability to bind to different species of MAdCAM. The antibodies containing the CDRs of antibody 6, antibody 59, antibody 63 of Table 6. Although the antibody was tested in a scFV format as illustrated in Table 6, the MAdCAM antibody can also be as shown in Table 7 in a VH/VL format. The antibodies were found to be at able to bind to both human and cyno MAdCAM, and except for Antibody Clone 6 they could also bind to mouse MAdCAM. The antibodies can bind MAdCAM with a KD in the nanomolar to micromolar range.
Example 37: Epitope Mapping of Antibodies
Human or murine MAdCAM-avitag/polyhistidine tag was immobilized on penta-his biosensors at 0.5 ug/mL for 180s. Baseline step was established in assay buffer (1% BSA with 0.05% Tween-20 in 1×PBS) for 120 seconds. First step of association was performed in wells with 40 nM molecules comprising the antibody CDRs of antibody 6, antibody 59, antibody 63 of Table 6 and other the MAdCAM antibodies, from PF-00547659 (an integrin blocking antibody; (Pfizer; Pullen et al., Br J Pharmacol. 2009 May; 157(2):281-93), MECA89 (a non-integrin blocker), and MECA367 (an integrin blocker).
It was found that Antibody 59 of Table 6 competes with Antibody 6 for human MAdCAM binding. Antibody 63 of Table 6 did not compete with Antibody 59 or Antibody 6 of Table 6 for human MAdCAM binding. Antibody 63 competed with Pfizer benchmark antibody while the others did not compete with Pfizer benchmark antibody. The results also showed that Antibody 59 did not compete with Antibody 63 for mouse MAdCAM binding. Antibody 63 competed with MECA367 but not MECA89. Antibody 59 competed with MECA89 but not MECA367. This data illustrates that the antibodies bind to different epitopes.
Example 38
MAdCAM-IL2 mutein molecules are specific for binding to MAdCAM and IL-2 by human protein array (Retrogenix). 5528 expression vectors, encoding both ZsGreen1 and full-length human plasma membrane proteins or a cell-surface tethered human secreted protein, were arrayed in duplicate across 16 microarray slides. Human HEK293 cells were used for reverse transfection/expression. The test antibodies were added to each slide after cell fixation. Test molecules comprising Antibody 59 and IL-2 mutein comprising the L118I mutation and Antibody 63 and IL-2 mutein comprising the L118I mutation were screened as a combination. Detection of binding was performed by using the same fluorescent secondary antibody as used in the Pre-screen. Fluorescent images were analyzed and quantitated (for transfection) using ImageQuant software. A protein ‘hit’ is defined as a duplicate spot showing a raised signal compared to background levels. This is achieved by visual inspection using the images gridded on the ImageQuant software. Hits were classified as ‘strong, medium, weak or very weak’, depending on the intensity of the duplicate spots. Both molecules were found to specifically interact with their two primary targets only—IL2RA (strong intensity) and 2 isoforms of MADCAM1 (medium/strong intensity) only. No other significant interactions were detected. These results demonstrate that the bi-specific molecules are able to bind to their respective targets. The format of the molecule from N terminus to C terminus was MAdCAM antibody-IL-2 mutein, however, the inverted format has also been found to be active and able to bind to their respective targets.
Example 40. Stability
The stability of certain bi-specific molecules was tested at 4 C. Bi-specific comprising Antibody 59 or Antibody 63 and an IL-2 mutein comprising the L118I mutation were found to be stable at 4C for at least 28 days.
Example 41: MAdCAM-IL2 Mutein Bispecific Reduces TNF-α Levels in Liver Tissue from Xenogeneic Graft-Versus-Host-Disease Mice
Immunocompromised NSG mice were engrafted with human PBMCs 35 days prior to treatment. Mice were treated with MADCAM-IL-2 mutein bispecific (0.03 mg/kg) for one week and sacrificed. Colon tissues were homogenized and TNF-α concentrations were measured by ELISA in colon lysate. Values were normalized to total protein concentration to account for differences in tissue aliquot sizes. Vehicle and an untethered IL-2 mutein had TNF-alpha levels in the colon lysate of 0.982±0.271 and 0.912±0.199 (pg/mg)±S.E.M), respectively, whereas the bispecific MAdCAM Antibody 59 and IL-2 mutein comprising the L118I mutation saw levels of TNF-alpha that were less (0.538±0.167 (pg/mg)±S.E.M). Therefore, the bi-specifics and targeted to the colon expressing MAdCAM can reduce production of TNF-alpha in site specific manner. Reduced levels of this pro-inflammatory cytokines in the target tissue implies a therapeutic effect of MADCAM-IL2 Mutein and an induction immune tolerance.
Example 42. MADCAM-IL2 Mutein Bispecific Increases FOXP3 Gene Expression in Colon Tissue from Xenogeneic Graft-Versus-Host-Disease Mice
Immunocompromised NSG mice were engrafted with human PBMCs 27 days prior to treatment. Mice were treated with MADCAM-IL-2 mutein bispecific (the same molecule as in Example 41) (0.1 mg/kg) for one week and sacrificed. Liver tissues were homogenized and human FOXP3 expression was measured by qRT-PCR in liver lysate. Gene expression was normalized to human GAPDH levels. FOXP3 levels of vehicle were found to be 1.32±0.36 whereas the MAdCAM Antibody 59 and IL-2 mutein comprising the L118I mutation were found to increase FOXP3 levels to 2.21±0.36. Data represent the mean values±S.E.M. Increased expression of FOXP3 in the target tissue indicates that there has been an increase in regulatory T cells.
The Examples provided for herein demonstrate that molecules provided for herein can be used to specifically localize therapeutics, such as a IL-2M and also other therapeutic molecules, such as those described herein with specificity and with a therapeutic effect, such as a decrease in TNF-alpha as well as an increase T regulatory cells, which can be monitored, for example, through an increase in FOX3P expression.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While various embodiments have been disclosed with reference to specific aspects, it is apparent that other aspects and variations of these embodiments may be devised by others skilled in the art without departing from the true spirit and scope of the embodiments. The appended claims are intended to be construed to include all such aspects and equivalent variations.

## Claims

1. An antibody that binds to MAdCAM, comprising a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino acid sequence SEQ ID NO: 359, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 170, and the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:360; and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 361, the light chain LCDR2 has the amino acid sequence of SEQ ID NO: 362, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 363.

2. An antibody that binds to MAdCAM, or antigen binding fragment thereof, wherein the antibody, or antigen binding fragment thereof, comprises:
i) a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 90, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 91, and the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 92; and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 93, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 87, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 94;
ii) a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 135, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 381, and the heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 382; and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 383, the light chain CDR2 has the amino acid sequence of SEQ ID NO: 384, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 385; or
iii) a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino acid sequence SEQ ID NO: 359, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 170, and the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:360; and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 361, the light chain LCDR2 has the amino acid sequence of SEQ ID NO: 362, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 363.

3. The antibody of claim 2, or antigen binding fragment thereof, wherein the heavy chain variable region and the light chain variable region are in a scFv format.

4. The antibody of claim 2, or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof comprises
a VK having a sequence of SEQ ID NO: 415 and a VH having a sequence of SEQ ID NO: 414;
a VK of SEQ ID NO: 592 and a VH sequence of SEQ ID NO: 591; or
a VK of SEQ ID NO: 600 and a VH sequence of SEQ ID NO: 599.

5. The antibody of claim 4, or antigen binding fragment thereof, wherein the VK sequence comprises a sequence of SEQ ID NO: 592 and the VH sequence comprises a sequence of SEQ ID NO: 591.

6. The antibody of claim 2, or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1 sequence has the amino acid sequence SEQ ID NO: 359, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 170, and the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:360; and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain CDR1 sequence has the amino acid sequence of SEQ ID NO: 361, the light chain LCDR2 has the amino acid sequence of SEQ ID NO: 362, and the light chain CDR3 has the amino acid sequence of SEQ ID NO: 363.

7. The antibody of claim 2, or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof is linked or associated with an effector molecule.

8. A pharmaceutical composition comprising the antibody of claim 2.

9. A cell comprising a nucleic acid molecule encoding the antibody of claim 2.