Glycophorin a antigen-binding protein

Abstract

To provide an antigen-binding protein such as an antibody that binds to glycophorin A.SOLUTION: In a certain aspect, antigen-binding protein of the present invention is a fully human antibody, a humanized antibody or a chimeric antibody, a binding fragment and derivative of such antibody, and polypeptide which specifically binds to glycophorin A. Some aspects relate to affinity maturation antigen-binding protein. In some aspects, an affinity maturation candidate is characterized by prediction, screening or other methods by using next generation sequencing. Furthermore, nucleic acid which encodes such an antibody, antibody fragment and derivative and polypeptide, a cell including such a polynucleotide, a method for producing such an antibody, antibody fragment and derivative and polypeptide, and a method for using such an antibody, antibody fragment and derivative and polypeptide are disclosed.SELECTED DRAWING: Figure 22

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 668,132, filed May 7, 2018, U.S. Provisional Patent Application No. 62 / 805,849, filed February 14, 2019, and U.S. Provisional Patent Application No. 62 / 829,920, filed April 5, 2019, the entire contents of each of which are incorporated herein by reference.

[0002] Reference to sequence listing The Sequence Listing submitted as an ASCII text file via EFS-Web is incorporated herein by reference in accordance with 35 U.S.C. § 1.52(e). The ASCII text file name for the Sequence Listing is ANOK036WO_ST25, the ASCII text file creation date is April 29, 2019, and the size of the ASCII text file is 197 kilobytes. [Background technology]

[0003] background Erythrocytes, also known as red blood cells (RBCs), are the most abundant cell type in mammalian blood and express a unique set of cell surface markers. Summary of the Invention

[0004] overview The compositions of the invention and related methods are described in detail below and describe specific implementations that may be performed by one of skill in the art. However, it should be understood that they may also include instructions for their implementation by another party. Thus, an implementation such as "administering an anti-human glycophorin A antigen-binding construct" includes "directing the administration of an anti-human glycophorin A antigen-binding construct."

[0005] In some embodiments, an antigen binding protein for binding to glycophorin A is provided, comprising a variable light chain domain comprising at least a first light chain complementarity determining region (LC CDR1) and a second light chain CDR (LC CDR2), and a variable heavy chain domain comprising at least a first heavy chain complementarity determining region (HC CDR1) and a second heavy chain CDR (HC CDR2), wherein the antigen binding protein is capable of binding to at least one of human glycophorin A and cynomolgus monkey glycophorin A, and wherein the antigen binding protein binds to human glycophorin A with a dissociation constant (Kd) of about 1 nM to about 100 nM.

[0006] In some embodiments, the antigen binding protein is affinity matured from a parent amino acid sequence that encodes an antigen binding protein with a Kd greater than that of the antigen binding protein. In some embodiments, the Kd is measured by flow cytometry, surface plasmon resonance, biolayer interferometry or radioimmunoassay.

[0007] In some embodiments, the antigen binding protein is not a full-length antibody. In some embodiments, the antigen binding protein does not include a constant region. In some embodiments, the antigen binding protein has fewer variable domains than a natural antibody. In some embodiments, the antigen binding protein is not an scFv. In some embodiments, the antigen binding protein comprises an antibody binding fragment (Fab), where the Fab binds to both human glycophorin A and cynomolgus monkey glycophorin A, and the Fab binds to human glycophorin A with a dissociation constant between about 10 μM and 0.1 nM.

[0008] In some embodiments, the antigen binding protein comprises or consists essentially of a Fab capable of binding to human and / or cynomolgus glycophorin A. In some embodiments, the antigen binding protein has a light chain CDR2 of the sequence LNRLH (SEQ ID NO: 298). In some embodiments, the antigen binding protein has a heavy chain CDR1 of the sequence RMTYIL (SEQ ID NO: 278). In some embodiments, the antigen binding protein has a light chain CDR1 of the sequence FRNNK (SEQ ID NO: 287).

[0009] In some embodiments, the LC CDR2 comprises an amino acid sequence selected from the group consisting of LNRLH (SEQ ID NO:298), LSRTS (SEQ ID NO:295), NTRTS (SEQ ID NO:296), NTRPS (SEQ ID NO:297), NTRLA (SEQ ID NO:299), NSRLS (SEQ ID NO:300), LSRVS (SEQ ID NO:301), LNRVS (SEQ ID NO:302), LNRLS (SEQ ID NO:303), NSRLH (SEQ ID NO:304), SSRLS (SEQ ID NO:305), SSRVS (SEQ ID NO:306), SNRLH (SEQ ID NO:307), NTRVS (SEQ ID NO:308), SNRVS (SEQ ID NO:309), HSRLS (SEQ ID NO:310), SSRLA (SEQ ID NO:311), FNRVN (SEQ ID NO:312), LNRMS (SEQ ID NO:313), LNRIS (SEQ ID NO:314) and LSHPH (SEQ ID NO:315). Some embodiments relate to a LC CDR2 that is at least about 90%, about 95% or about 98% homologous to the above sequences. In some embodiments, CDRL2 comprises a sequence selected from any one of SEQ ID NOs: 246, 243, 244, 245 and 247-263. In some embodiments, the antigen binding protein is matured from a parent sequence that comprises the sequence of VSKLD at the corresponding position in the amino acid sequence of the variable light chain domain.

[0010] In some embodiments, the HC CDR1 comprises an amino acid sequence selected from the group consisting of RMTYIL (SEQ ID NO:278), RATYIL (SEQ ID NO:269), RNIYIL (SEQ ID NO:270), KYTYIL (SEQ ID NO:271), VHTYIL (SEQ ID NO:272), RNVFIL (SEQ ID NO:273), RNIYLL (SEQ ID NO:274), RKTYIL (SEQ ID NO:275), LNVYIL (SEQ ID NO:276), KATYIL (SEQ ID NO:277), KTVYIL (SEQ ID NO:279), KHVYIL (SEQ ID NO:280), RNITMIL (SEQ ID NO:281), KDTYIL (SEQ ID NO:282), INSYIL (SEQ ID NO:283), QHTYIL (SEQ ID NO:284) and RHSYIL (SEQ ID NO:285). Some embodiments relate to HC CDR1 that is at least about 90%, about 95% or about 98% homologous to the above sequences. In some embodiments, the antigen binding protein is matured from a parent sequence that contains a sequence of KDTYML at the corresponding position in the amino acid sequence. In some embodiments, the CDR H1 comprises a sequence selected from any one of SEQ ID NOs: 227, 217-226 and 228-233.

[0011] In some embodiments, the first light chain complementarity determining region 1 (CDRL1) comprises an amino acid sequence selected from FRNNK (SEQ ID NO:287), FRNSK (SEQ ID NO:288), FKNGK (SEQ ID NO:289), FRNAK (SEQ ID NO:290), FRTGK (SEQ ID NO:291), FKNDK (SEQ ID NO:292) and YKNGK (SEQ ID NO:293). Some embodiments relate to LC CDR1 that is at least about 90%, about 95% or about 98% homologous to the above sequences. In some embodiments, CDRL1 comprises a sequence selected from any one of SEQ ID NOs:235-241. In some embodiments, the antigen binding protein is matured from a parent sequence that comprises a sequence of YSNGKT at the corresponding position in the amino acid sequence.

[0012] In some embodiments, the antigen binding protein competes with one or more of 10F7, Ter119, CLB-ery-1 (AME-1), EPR8200, YTH89.1, EPR8199, JC159, GYPA / 280, ab40844, HI264, GPHN02, JC159, SPM599, EPR8200, GYPA / 1725R, ab112201, ab114330, ab219896, BRIC256, or fragments or derivatives thereof, for binding to glycophorin A. In some embodiments, the antigen binding protein targets a different epitope than the antibodies described above, but has a higher affinity for that target.

[0013] In some embodiments, the antigen binding protein is fused to at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen or an immunogenic fragment of the at least one tolerogenic antigen is associated with multiple sclerosis.

[0014] In some embodiments, the antigen binding protein is fused to an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of at least one tolerogenic antigen comprises an immunogenic fragment of myelin basic protein (MBP), an immunogenic fragment of myelin oligodendrocyte glycoprotein (MOG), and / or an immunogenic fragment of myelin proteolipid protein (PLP). In some embodiments, a fragment of MBP is used in combination with a fragment of PLP. In some embodiments, a fragment of MOG is used in combination with a fragment of PLP. In some embodiments, a fragment of MOG is used in combination with a fragment of MBP. In some embodiments, the immunogenic fragment of the tolerogenic antigen comprises at least one of SEQ ID NOs: 169-175 and 186-202. In some embodiments, the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 169, 171-173, 175, and 188-202. Some embodiments relate to immunogenic fragments of the antigens having at least about 90%, about 95% or about 98% homology to the above sequences.Also provided is the use of antigen binding proteins fused to MS-associated antigens for use in treating multiple sclerosis.Methods of treating MS are also provided.

[0015] In some embodiments, the antigen binding protein is fused to at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen or the immunogenic fragment of the at least one tolerogenic antigen is associated with diabetes type 1. In some embodiments, the antigen binding protein is fused to an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises one or more of the following immunogenic fragments: proinsulin, insulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6 phosphatase 2, islet-specific glucose-6 phosphatase catalytic subunit associated protein. (IGRP), insulinoma-associated protein 2 (IA-2), insulinoma-associated protein 2 beta (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase and SST G-protein coupled receptor 1-5. In some embodiments, the immunogenic fragment of at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 204-214. Some embodiments relate to immunogenic fragments of antigens having at least about 90%, about 95% or about 98% homology to the above sequences. In certain embodiments, combinations of fragments from different antigens are used as well. For example, a fragment of insulin or a fragment of proinsulin may be used in combination with a fragment of IA-2, a fragment of GAD-65, and / or a fragment of GAD-67. Also provided is the use of an antigen binding protein fused to a type I diabetes associated antigen for use in treating type I diabetes. Methods of treating type I diabetes are also provided.

[0016] In some embodiments, the antigen binding protein is fused to at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen is associated with celiac disease. In some embodiments, the antigen binding protein is fused to at least one immunogenic fragment of at least one tolerogenic antigen, wherein the at least one immunogenic fragment of the tolerogenic antigen comprises one or more of the following immunogenic fragments: gluten, tissue transglutaminase, high molecular weight glutenin, low molecular weight glutenin, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, avenin and deamidated versions thereof. In some embodiments, the at least one immunogenic fragment of the tolerogenic antigen comprises at least one of SEQ ID NOs: 182-185 and 215. Some embodiments relate to immunogenic fragments of antigens that are at least about 90%, about 95% or about 98% homologous to the above sequences. In some embodiments, combinations of antigens are also used. For example, in some embodiments, fragments of gliadin are used. In further embodiments, native gliadin and deamidated gliadin fragments are used. In some embodiments, the use of antigen binding proteins fused to celiac disease associated antigens for use in treating celiac disease is also provided. Methods of treating celiac disease are also provided.

[0017] Also provided is a method for affinity maturation of antibody binding proteins. In some embodiments, the method comprises depleting a phage library of non-specific binders, where the phage library comprises a plurality of phages, each phage expressing a candidate affinity matured antibody or antibody fragment. In some embodiments, the method comprises exposing the depleted library to a target antigen, removing phages that do not bind to the target antigen (e.g., by washing or other separation methods), and amplifying phages that bind to the target antigen. In some embodiments, the exposing, removing, and amplifying steps are repeated multiple times. For example, in some embodiments, they are repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the repetition induces a selection pressure that results in the binding of the target antigen by phages expressing high affinity candidate antibodies or antibody fragments. In some embodiments, the high affinity candidate antibodies or antibody fragments are screened. In some embodiments, the screening is performed using next generation sequencing. In some embodiments, the screening is performed using ELISA. In some embodiments, optionally, the method may further include one or more of screening high affinity candidate antibodies or antibody fragments using NGS and ELISA and / or evaluating the ability of high affinity candidate antibodies or antibody fragments to be expressed in a soluble form.

[0018] In some embodiments, an antigen binding protein for binding to glycophorin A is provided, comprising a first variable domain comprising at least a first light chain complementarity determining region (LC CDR) 1 and a second light chain CDR (LC CDR2) and a second variable domain comprising at least a second heavy chain complementarity determining region (HC CDR) 1 and a second heavy chain CDR (HC CDR2), wherein the antigen binding protein can bind to both human and cynomolgus glycophorin A. In some embodiments, the antigen binding protein binds to human glycophorin A with a dissociation constant (Kd) of less than about 20 nM as measured by flow cytometry, surface plasmon resonance, biolayer interferometry or radioimmunoassay. In some embodiments, the antigen binding protein is affinity matured from a parent sequence with a Kd greater than the antigen binding protein as measured by flow cytometry, surface plasmon resonance, biolayer interferometry or radioimmunoassay.

[0019] In some embodiments, an antigen binding protein is provided that comprises a light chain variable domain that is at least about 95% identical to one or more of SEQ ID NOs: 243- 264. In some embodiments, an antigen binding protein is provided that comprises a light chain variable domain that is at least about 95% identical to one or more of SEQ ID NOs: 235-242. In some embodiments, an antigen binding protein is provided that comprises a light chain complementarity determining region (CDR) 1 comprising RASSNVX1X2MY (SEQ ID NO:49) and a light chain CDR2 comprising X3X4TSX5LAS (SEQ ID NO:50), wherein X1, X2, X3, X4 and X5 are each naturally occurring amino acids, and when X2 is Y, X3 is Y, X4 is Y, and X5 is N, X1 is not K; when X1 is K, X3 is Y, X4 is Y, and X5 is N, X2 is not Y; when X1 is K, X2 is Y, X4 is Y, and X5 is N, X3 is not Y; when X1 is K, X2 is Y, X3 is Y, and X5 is N, X4 is not Y; when X1 is K, X2 is Y, X3 is Y, and X5 is N, X4 is not Y; and when X1 is K, X2 is Y, X3 is Y, and X5 is N, X5 is not N.

[0020] In some embodiments, the light chain CDR1 comprises a sequence selected from the group consisting of RASSNVX1X2MY, where X1 is selected from F, W, and Y, and X2 is selected from F, W, Y, and Q (SEQ ID NO:51), RASSNVX1X2MY, where X1 is selected from F, W, and Y, and X2 is selected from F, W, and Y (SEQ ID NO:53), RASSNVX1X2MY, where X1 is F or Y, and X2 is selected from F, W, and Y (SEQ ID NO:55), and RASSNVX1X2MY, where X1 is F or Y, and X2 is F (SEQ ID NO:57). and the light chain CDR2 comprises a sequence selected from the group consisting of X3X4TSX5LAS, where X3 is selected from H and Y, X4 is selected from H, R and K, and X5 is a natural amino acid (SEQ ID NO:52), X3X4TSX5LAS, where X3 is H, X4 is H or R, and X5 is a natural amino acid (SEQ ID NO:54), X3X4TSX5LAS, where X3 is H, X4 is H, and X5 is a natural amino acid (SEQ ID NO:56), and X3X4TSX5LAS, where X3 is H, X4 is H, and X5 is V or D (SEQ ID NO:58).

[0021] In some embodiments, the antigen binding protein of claim 15 further comprises a light chain CDR3 comprising QQFTSSPYT (SEQ ID NO: 45).

[0022] In some embodiments, the antigen binding protein further comprises a heavy chain CDR1 comprising GYTFNSYFMH (SEQ ID NO: 46) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions, a heavy chain CDR2 comprising GMIRPNGGTTDYNEKFKN (SEQ ID NO: 47) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions, and a heavy chain CDR3 comprising WEGSYYALDY (SEQ ID NO: 48) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions. In some embodiments, the antigen binding protein further comprises a heavy chain variable domain at least about 90% identical to SEQ ID NO: 3. In some embodiments, the antigen binding protein further comprises a heavy chain variable domain at least about 95% identical to SEQ ID NO: 3. In some embodiments, the antigen binding protein further comprises a heavy chain variable domain at least about 99% identical to SEQ ID NO: 3.

[0023] In some embodiments, an antigen binding protein is provided that comprises a heavy chain variable domain that is at least about 90% identical to SEQ ID NO:3 and a light chain variable domain that is at least about 90% identical to SEQ ID NO:1. In some embodiments, an antigen binding protein is provided that comprises a heavy chain variable domain that is at least about 95% identical to SEQ ID NO:3 and a light chain variable domain that is at least about 95% identical to SEQ ID NO:1. In some embodiments, an antigen binding protein is provided that comprises a heavy chain variable domain that is at least about 99% identical to SEQ ID NO:3 and a light chain variable domain that is at least about 99% identical to SEQ ID NO:1.

[0024] In some embodiments, an antigen binding protein is provided that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-42. In some embodiments, an antigen binding protein is provided that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 11, 12, 14, 18, 19, 20, 21, 22, 24, 27, 28, 31, 38 and 42. In one embodiment, an antigen binding protein is provided that comprises the amino acid sequence of SEQ ID NO: 21. In one embodiment, an antigen binding protein is provided that comprises the amino acid sequence of SEQ ID NO: 42.

[0025] In some embodiments, a composition for inducing immune tolerance is provided comprising an antigen binding protein and further comprising at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen. In some embodiments, the composition comprises at least one immunogenic tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen comprises at least one of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin proteolipid protein (PLP), a fragment(s) of MPB, a fragment(s) of MOG, and a fragment(s) of PLP.

[0026] In some embodiments, the composition comprises at least one immunogenic tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen is selected from the group consisting of insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6 phosphatase 2, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), insulinoma-associated protein 2 (IGRP), insulinoma-associated protein 2 (IGRP), insulin-like ... and at least one of the following: insulinoma-associated protein 2 beta (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regenerative gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, and SST G-protein coupled receptor 1-5.

[0027] In some embodiments, the composition comprises at least one immunogenic, tolerogenic antigen, wherein the at least one immunogenic, tolerogenic antigen comprises at least one of tissue transglutaminase, high molecular weight glutenin, low molecular weight glutenin, gluten, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, avenin, and deamidated forms thereof.

[0028] In some embodiments, an antigen binding protein is provided. A method for treating diseases, conditions, and disorders is provided. In some embodiments, the antigen binding protein comprises or essentially consists of a light chain complementarity determining region (CDR) 1 comprising RASSNVX1X2MY (SEQ ID NO: 49) and a light chain CDR2 comprising X3X4TSX5LAS (SEQ ID NO: 50), where X1, X2, X3, X4, and X5 are each natural amino acids. In some embodiments, when X2 is Y, X3 is Y, X4 is Y, and X5 is N, X1 is not K. In some embodiments, when X1 is K, X3 is Y, X4 is Y, and X5 is N, X2 is not Y. In some embodiments, when X1 is K, X2 is Y, X4 is Y, and X5 is N, X3 is not Y. In certain embodiments, when X1 is K, X2 is Y, X3 is Y, and X5 is N, X4 is not Y. In certain embodiments, when X1 is K, X2 is Y, X3 is Y, and X4 is Y, X5 is not N. In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is selected from F, W, and Y, and X2 is selected from F, W, Y, and Q (SEQ ID NO: 51). In certain embodiments, the light chain CDR2 comprises X3X4TSX5LAS, where X3 is selected from H and Y, X4 is selected from H, R, and K, and X5 is a natural amino acid (SEQ ID NO: 52). In certain embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is selected from F, W, and Y, and X2 is selected from F, W, and Y (SEQ ID NO: 53). In one embodiment, the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H or R, and X5 is a natural amino acid (SEQ ID NO: 54). In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is F or Y, and X2 is selected from F, W, and Y (SEQ ID NO: 55). In one embodiment, the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H, and X5 is a natural amino acid (SEQ ID NO: 56).In one embodiment, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is F or Y and X2 is F (SEQ ID NO:57). In one embodiment, the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H and X5 is V or D (SEQ ID NO:58).

[0029] In one embodiment, the antigen binding protein comprises, or consists essentially of, a light chain CDR1 comprising RASSNVX1X2MY (SEQ ID NO: 49) or a variant thereof comprising one, two, three or four amino acid substitutions (e.g., conservative substitutions); and a light chain CDR2 comprising X3X4TSX5LAS (SEQ ID NO: 50) or a variant thereof comprising one, two, three or four amino acid substitutions (e.g., conservative substitutions), where X1, X2, X3, X4 and X5 are each naturally occurring amino acids. In one embodiment, the light chain CDR1 comprises RASSNVX1X2MY (wherein X1 is selected from F, W and Y, and X2 is selected from F, W, Y and Q) (SEQ ID NO: 51) or a variant thereof comprising one, two, three or four amino acid substitutions. In some embodiments, the light chain CDR2 comprises X3X4TSX5LAS, where X3 is selected from H and Y, X4 is selected from H, R and K, and X5 is a natural amino acid (SEQ ID NO: 52), or a variant thereof comprising one, two, three or four amino acid substitutions. In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is selected from F, W and Y, and X2 is selected from F, W and Y (SEQ ID NO: 53), or a variant thereof comprising one, two, three or four amino acid substitutions. In some embodiments, the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H or R, and X5 is a natural amino acid (SEQ ID NO: 54), or a variant thereof comprising one, two, three or four amino acid substitutions. In one embodiment, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is F or Y and X2 is selected from F, W and Y (SEQ ID NO:55) or a variant thereof comprising one, two, three or four amino acid substitutions. In some embodiments, the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H and X5 is a natural amino acid (SEQ ID NO:56) or a variant thereof comprising one, two, three or four amino acid substitutions. In one embodiment, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is F or Y and X2 is F (SEQ ID NO:57) or a variant thereof comprising one, two, three or four amino acid substitutions.In some embodiments, the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H, and X5 is V or D (SEQ ID NO:58), or a variant thereof containing 1, 2, 3, or 4 amino acid substitutions.

[0030] In some embodiments, the present invention also provides an antigen binding protein comprising a light chain CDR3. In some embodiments, the light chain CDR3 comprises QQFTSSPYT (SEQ ID NO: 45). In some embodiments, the light chain CDR3 comprises a variant of SEQ ID NO: 45 comprising one, two, three or four amino acid substitutions.

[0031] In some embodiments, the antigen binding protein also comprises a heavy chain CDR1, a heavy chain CDR2 and / or a heavy chain CDR3. In some embodiments, the heavy chain CDR1 comprises GYTFNSYFMH (SEQ ID NO: 46). In some embodiments, the heavy chain CDR2 comprises GMIRPNGGTTDYNEKFKN (SEQ ID NO: 47). In some embodiments, the heavy chain CDR3 comprises WEGSYYALDY (SEQ ID NO: 48). In some embodiments, the present invention also provides a variant of the heavy chain CDR1 comprising SEQ ID NO: 46, the heavy chain CDR2 comprising SEQ ID NO: 47 and / or the heavy chain CDR3 comprising SEQ ID NO: 48, wherein the variant comprises one, two, three or four amino acid substitutions.

[0032] In some embodiments, the antigen binding protein comprises a heavy chain variable domain that is at least 70% (e.g., 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, and overlapping ranges therein) identical to SEQ ID NO: 3. In some embodiments, the antigen binding protein comprises a light chain variable domain that is at least 70% (e.g., 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, and overlapping ranges therein) identical to SEQ ID NO: 1.

[0033] In some embodiments, the antigen binding protein comprises one or more amino acid sequences of SEQ ID NOs:4-42.

[0034] In some embodiments, the antigen binding protein comprises an amino acid sequence that is at least 70% identical (e.g., 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, and ranges of overlap therein) to SEQ ID NOs: 4-42. In some embodiments, the antigen binding protein comprises SEQ ID NOs: 5, 9, 11, 12, 14, 18, 19, 20, 21, 22, 24, 27, 28, 31, 38 and / or 42. In one embodiment, the antigen binding protein comprises SEQ ID NO: 21. In one embodiment, the antigen binding protein comprises SEQ ID NO: 42.

[0035] Also provided in some embodiments are humanized antigen binding proteins. In some such embodiments, the antigen binding protein comprises one or more of the following: (a) a first human light chain framework region (FR1) selected from SEQ ID NOs: 131-140; (b) a human FR2 of a light chain framework region selected from SEQ ID NOs: 141-148; (c) a human FR3 of a light chain selected from SEQ ID NOs: 149-156; (d) a human FR4 of a light chain selected from SEQ ID NOs: 157-162; (e) a first human heavy chain framework region (FR1) selected from SEQ ID NOs: 109-115; (f) a human FR2 of a heavy chain selected from SEQ ID NOs: 116-119; (g) a human FR3 of a heavy chain selected from SEQ ID NOs: 120-127; and / or (h) a human FR4 of a heavy chain selected from SEQ ID NOs: 128-130. In some embodiments, the antigen binding protein comprises a human constant region. In some embodiments, the human constant region can be IgG1, IgG2, IgG3 and / or IgG4. In one embodiment, the human constant region is IgG1.

[0036] In some embodiments, the antigen binding protein is a full-length antibody, while in other embodiments, the antigen binding protein is an antigen-binding fragment of an antibody. In some embodiments, the antigen binding protein comprises a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked Fv, VNAR domain, IgNar, intrabody, IgG-CH2, minibody, F(ab')3, tetramer, triabody, dimer, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc.

[0037] In some embodiments, the antigen binding protein specifically binds to glycophorin A (GPA). In some embodiments, the antigen binding protein specifically binds to one or more of human GPA, cynomolgus monkey GPA, porcine GPA, canine GPA, mouse GPA and / or rat GPA (e.g., the antigen binding protein is bispecific or multispecific). In one embodiment, the binding is specific to human GPA. In some embodiments, the antigen binding protein has a K of greater than 1.0 nM (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 12.0, 14.0, 20, 50 or more and overlapping ranges therein). d In some such embodiments, the binding affinity is measured by flow cytometry, surface plasmon resonance, biolayer interferometry, and / or radioimmunoassay.

[0038] In some embodiments, the antigen binding protein is affinity matured. In some embodiments, the antigen binding protein is one or more affinity matured variants of 10F7, Ter119, CLB-ery-1 (AME-1), EPR8200, YTH89.1, EPR8199, JC159, GYPA / 280, ab40844, HI264, GPHN02, JC159, SPM599, EPR8200, GYPA / 1725R, ab112201, and / or BRIC256. In one embodiment, the antigen binding protein is an affinity matured variant of 10F7. In some embodiments, the antigen binding protein competes for binding to GPA with 10F7, Ter119, CLB-ery-1 (AME-1), EPR8200, YTH89.1, EPR8199, JC159, GYPA / 280, ab40844, HI264, GPHN02, JC159, SPM599, EPR8200, GYPA / 1725R, ab112201, BRIC256, fragments thereof and / or derivatives thereof. In some embodiments, the antigen binding protein inhibits binding of 10F7, CLB-ery-1 (AME-1), EPR8200, YTH89.1, EPR8199, JC159, GYPA / 280, ab40844, HI264, GPHN02, SPM599, GYPA / 1725R, ab112201, ab114330, ab219896, BRIC256, fragments thereof and / or derivatives thereof to human GPA by at least 10% (e.g., 10-15%, 15-20%, 20-25%, 25-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, and overlapping ranges therein). In one embodiment, the inhibition of binding is at least about 50%.

[0039] In some embodiments, a humanized affinity matured antigen binding protein that specifically binds to human GPA is provided, comprising a light chain CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 59, 61, 63, 65, 67, 69, 71, 73, 76, 78, 80, 81, 83, 85, 87, 88, 90, 91, 93, 95, 96, 98, 99, 101, 102, 104, 105, 107, and 108; and / or a light chain CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77, 79, 82, 84, 86, 89, 92, 94, 97, 100, 103, and 106.

[0040] Also provided in some embodiments are antigen binding proteins that specifically bind to human GPA, comprising one, two, three, four, five, six, seven, eight, nine, or ten of the following: (a) a light chain CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 59, 61, 63, 65, 67, 69, 71, 73, 76, 78, 80, 81, 83, 85, 87, 88, 90, 91, 93, 95, 96, 98, 99, 101, 102, 104, 105, 107, and 108; (b) a light chain CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77, 79, 82, 84, 86, 89, 92, 94, 97, 100, 103 and 106; (c) a light chain CDR3 comprising SEQ ID NO: 45 or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; (d) a heavy chain CDR1 comprising SEQ ID NO: 46 or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions. (e) a heavy chain CDR2 comprising SEQ ID NO: 47 or a variant thereof comprising one, two, three or four conservative amino acid substitutions; (f) a heavy chain CDR3 comprising SEQ ID NO: 48 or a variant thereof comprising one, two, three or four conservative amino acid substitutions; (g) a first human light chain framework region (FR1) selected from SEQ ID NOs: 131-140, a human FR2 of a light chain framework region selected from SEQ ID NOs: 141-148, a human FR3 of the light chain selected from SEQ ID NOs: 149-156, and a human FR4 of the light chain selected from SEQ ID NOs: 157-162; (h) a first human heavy chain framework region (FR1) selected from SEQ ID NOs: 109-115, a human FR2 of the heavy chain selected from SEQ ID NOs: 116-119, a human FR3 of the heavy chain selected from SEQ ID NOs: 120-127, and a human FR4 of the heavy chain selected from SEQ ID NOs: 128-130; (i) a human heavy chain constant region; and / or (j) a human light chain constant region.

[0041] In some embodiments, the present invention also provides isolated polynucleotides encoding the antigen binding proteins described herein. In certain embodiments, the isolated polynucleotides encode polypeptides that are at least 70% identical (e.g., 25-50%, 50-75%, 75-100%, 100-150%, or more, and ranges of overlap therein) to one or more of SEQ ID NOs: 1-42. In some embodiments, the present invention also provides vectors comprising one or more of the polynucleotides described herein. Additionally, host cell lines (e.g., CHO, k1SV, XCeed, CHOK1SV, and GS-KO) comprising the vectors described herein are also provided in some embodiments.

[0042] In some embodiments, a method for producing an antigen binding protein as described herein is provided, comprising culturing a cell line as described herein under conditions in which the antigen binding protein is produced, and recovering the antigen binding protein. In some embodiments, the antigen binding protein comprises one or more light chains and one or more heavy chains. In some embodiments, the heavy and light chains are encoded on separate vectors, while in other embodiments, the heavy and light chains are encoded on the same vector.

[0043] Pharmaceutical compositions comprising the antigen binding proteins described herein are provided in several embodiments. In some embodiments, a pharma- ceutically acceptable carrier is also provided. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0044] In some embodiments, a composition is provided that induces immune tolerance. In some embodiments, the composition comprises or essentially consists of the antigen-protein and at least one immunogenic tolerogenic antigen or at least one immunogenic fragment of the tolerogenic antigen described herein. In some embodiments, the tolerogenic antigen can induce an undesirable immune response in a subject when the subject is exposed to the tolerogenic antigen. In one embodiment, the tolerogenic antigen can be an endogenous antigen (e.g., self-antigen(s)) or an exogenous antigen (e.g., foreign antigen(s)). In another embodiment, the tolerogenic antigen can be a foreign transplant antigen that causes a transplant recipient to develop an undesirable immune response (e.g., transplant rejection), a foreign food, animal, plant or environmental antigen that causes a patient to develop an undesirable immune response (e.g., allergic response or hypersensitivity), a therapeutic agent that causes a patient to develop an undesirable immune response (e.g., hypersensitivity and / or reduced therapeutic activity), or a self-antigen that causes a patient to develop an undesirable immune response (e.g., autoimmune disease). In some embodiments, a delivery vector (e.g., a viral vector such as an adenovirus or an adeno-associated virus) is used to deliver a therapeutic agent, but is itself capable of inducing an immune response. Thus, in some embodiments, compositions and methods are provided that induce immune tolerance to at least a portion of such vectors.

[0045] In some embodiments, the treatment methods and / or use of the compositions described herein are provided for the prevention or treatment of one or more of the following conditions: transplant rejection, type 1 diabetes, celiac disease, and / or multiple sclerosis. In the treatment methods, an effective amount of the composition is delivered to a subject (e.g., human or livestock). While undesired immune responses are disclosed in some embodiments, other embodiments are used to treat non-immune conditions (e.g., conditions that would benefit from targeting the composition to erythrocytes). In certain embodiments, diagnostic methods and diagnostic uses of the antigen binding proteins described herein are also provided. In certain embodiments, kits are provided that include one or more compounds and devices for administration (such as syringes, containers, inhalers, etc.), as well as instructions for use.

[0046] In some embodiments, the at least one immunogenic tolerance-inducing antigen comprises at least one of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin proteolipid protein (PLP), immunogenic fragments thereof, and immunogenic mimotopes. In some embodiments, the immunogenic fragment of the at least one tolerance-inducing antigen comprises at least one of SEQ ID NOs: 169-175 and 186-202. In some such embodiments, the use of the composition in the treatment of multiple sclerosis is provided.

[0047] In some embodiments, the at least one immunogenic tolerogenic antigen is selected from the group consisting of insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6 phosphatase 2, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), insulinoma-associated protein 2 (IA-2), insulinoma-associated protein 2 beta (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, SST The composition comprises at least one of G-protein coupled receptor 1-5, an immunogenic fragment thereof, and an immunogenic mimotope thereof. In some embodiments, the immunogenic fragment of at least one of the tolerogenic antigens comprises at least one of SEQ ID NOs: 204-214. In some such embodiments, the composition is used in the treatment of type 1 diabetes.

[0048] In some embodiments, the at least one immunogenic tolerance-inducing antigen comprises at least one of tissue transglutaminase, high molecular weight glutenin, low molecular weight glutenin, gluten, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, avenin, deamidated forms thereof, immunogenic fragments thereof, and immunogenic mimotopes thereof. In some embodiments, the immunogenic fragment of the at least one tolerance-inducing antigen comprises at least one of SEQ ID NOs: 182-185 and 215. In some such embodiments, the use of the composition in the treatment of celiac disease is provided.

[0049] In some embodiments, the at least one immunogenic tolerogenic antigen comprises at least one of an MHC class I protein, an MHC class II protein, a minor blood group antigen, RhCE, Kell, Kidd, Duffy, Ss, immunogenic fragments thereof, and immunogenic mimotopes thereof. In some such embodiments, the use of the composition in the treatment or prevention of transplant rejection is provided.

[0050] According to some embodiments, there is provided an antigen binding protein comprising a heavy chain complementarity determining region 1 (CDR H1) comprising amino acids selected from RATYIL, RNIYIL, KYTYIL, VHTYIL, RNVFIL, RNIYLL, RKTYIL, LNVYIL, KATYIL, RMTYIL, KTVYIL, KHVYIL, RNITMIL, KDTYIL, INSYIL, QHTYIL, RHSYIL (SEQ ID NOs: 269-285) and combinations thereof. In some embodiments, the antigen binding protein is matured from a parent sequence comprising the sequence of KDTYML (SEQ ID NO: 286) at the corresponding position in the amino acid sequence.

[0051] Further embodiments provide antigen binding proteins comprising a first light chain complementarity determining region 1 (CDRL1a) comprising an amino acid sequence selected from FRNNK, FRNSK, FKNGK, FRNAK, FRTGK, FKNDK, YKNGK (SEQ ID NOs: 287-293) and combinations thereof. In some embodiments, the antigen binding protein is matured from a parent sequence comprising the sequence of YSNGKT (SEQ ID NO: 294) at the corresponding position in the amino acid sequence.

[0052] Yet further embodiments provide antigen binding proteins comprising a light chain complementarity determining region (CDRL2) comprising an amino acid sequence selected from LSRTS, NTRTS, NTRPS, LNRLH, NTRLA, NSRLS, LSRVS, LNRVS, LNRLS, NSRLH, SSRLS, SSRVS, SNRLH, NTRVS, SNRVS, HSRLS, SSRLA, FNRVN, LNRMS, LNRIS, LSHPH (SEQ ID NO:295-315) and combinations thereof. In some embodiments, CDLR2 is matured from a parent sequence comprising the sequence of VSKLD (SEQ ID NO:316) at the corresponding position in the amino acid sequence.

[0053] In some embodiments, an antigen binding protein comprising CDRH1, CDRL1a and / or CDRL2 binds to a human target antigen and a cynomolgus monkey target antigen. In some embodiments, the human target antigen and the cynomolgus monkey target antigen is glycophorin A. In some embodiments, an antigen binding protein comprising CDRH1, CDRL1a and / or CDRL2 is bound to an antigen to which tolerance is desired.

[0054] In some embodiments, compositions for inducing immune tolerance are provided that include an antigen binding protein that has been affinity matured using NGS fusion (e.g., recombinantly linked, chemically linked, etc.) or other linkage to at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen. In some embodiments, the at least one immunogenic tolerogenic antigen includes at least one of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin proteolipid protein (PLP), a fragment(s) of MPB, a fragment(s) of MOG, and a fragment(s) of PLP. In some embodiments, the immunogenic fragment of the at least one tolerogenic antigen includes at least one of SEQ ID NOs: 169-175 and 186-202. In some embodiments, the immunogenic fragment of the at least one tolerogenic antigen includes at least one of SEQ ID NOs: 169, 171-173, 175, and 188-202. In some embodiments, such embodiments are for use in the treatment of multiple sclerosis.

[0055] In a further embodiment, the composition comprises an antigen binding protein that has been affinity matured with NGS and at least one immunogenic, tolerogenic antigen, wherein the at least one immunogenic, tolerogenic antigen is selected from the group consisting of insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6 phosphatase 2, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), insulin, In some embodiments, the composition comprises at least one of the following immunogenic fragments: insulinoma-associated protein 2 (IA-2), insulinoma-associated protein 2 alpha (IA-2 alpha), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreatic / pancreatic associated protein, S100 alpha, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, and SST G-protein coupled receptor 1-5. In some embodiments, the composition comprises an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises SEQ ID NO: 204-214. In some embodiments, such a composition is for use in treating type 1 diabetes.

[0056] In further embodiments, the composition comprises an antigen binding protein affinity matured with NGS and at least one immunogenic tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen comprises at least one of tissue transglutaminase, high molecular weight glutenin, low molecular weight glutenin, gluten, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, avenin, and deamidated forms thereof. In certain embodiments, the composition comprises an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises SEQ ID NOs: 182-185 and 215. In certain embodiments, such a composition is for use in treating celiac disease.

[0057] Provided herein are methods of treating multiple sclerosis, type 1 diabetes or celiac disease, comprising administering to a subject an antigen binding protein that has been affinity matured using NGS and an antigen or immunogenic fragment associated with MS, T1D or celiac disease.

[0058] Also provided herein is a method for affinity maturing an antibody or antibody fragment to increase its affinity for a target, comprising depleting a phage library of non-specific binders, wherein the phage library comprises a plurality of phages, each phage expressing a candidate affinity matured antibody or antibody fragment, exposing the depleted library to a target antigen, removing phages that do not bind to the target antigen by washing, amplifying phages that bind to the target antigen, repeating the exposing, removing and amplifying steps multiple times to induce selection pressure that results in binding of the target antigen by phage expressing high affinity candidate antibodies or antibody fragments, and screening for high affinity candidate antibodies or antibody fragments using next generation sequencing.

[0059] In some embodiments, the method may further comprise one or more of screening the high affinity candidate antibodies or antibody fragments using ELISA and evaluating the ability of the high affinity candidate antibodies or antibody fragments to be expressed in a soluble form. [Brief description of the drawings]

[0060] [Figure 1]Figures 1A-1C show the sequences of the humanized 10F7 antibody used in the affinity maturation campaign. Figure 1A shows the sequences of the constant and variable light and heavy chains of the humanized antibody clone ("m10") selected as the non-limiting antibody sequence for follow-on affinity maturation. The randomized antigen-binding residues of the complementarity determining regions in the affinity maturation campaign are shown in bold and underlined. Figure 1B shows the positions of the variable light and heavy chain CDRs and the paratome antigen-binding regions (ABRs) according to the Kabat and Clothia scheme. Figure 1C shows five separate phage display libraries generated by TRIM (trinucleotide mutagenesis). [Diagram 2] Figure 2 shows an overview of the affinity maturation selection process used according to some embodiments. The phage display library was selected three times against recombinant biotinylated human glycophorin A (GPA) and then further screened by ELISA. Positive clones were sequenced, recombinantly expressed, purified, and their affinities were characterized by ELISA, biolayer interferometry, surface plasmon resonance, and flow cytometry assays. [Diagram 3] Figure 3 shows the sequence of the light chain variable domain of the affinity matured fAb. The randomized antigen-binding residues in CDR1 and CDR2 are shown in bold and underlined. [Figure 4] FIG. 4 shows sequence analysis of the 3-103 antibody variable light (VL) and variable heavy (VH) chains. [Diagram 5] FIG. 5 shows a non-limiting schematic of affinity maturation of antibodies (including fragments) described herein. [Figure 6] FIG. 6 shows a non-limiting schematic diagram of the method flow used to affinity mature antibodies. [Figure 7] FIG. 7 shows a non-limiting embodiment of a selection scheme as described herein. [Figure 8]Figures 8A-8C show data related to the maturation campaign of the CDR-L1b library. Figure 8A shows the convergence sequence of the hits from the maturation campaign. Figure 8B shows a schematic ribbon diagram of 3-103 where the various CDR regions were identified. Figure 8C shows the extent of binding to human erythrocytes of the candidate matured antibody fragments compared to the parent antibody fragment (non-mature). [Figure 9] 9A-9I show data relating fold sequence enrichment to enhanced affinity of antibodies screened from the CDRL1b library. Wild type 3-103 is indicated by an arrow for reference. [Figure 10] Figures 10A-10D show data for CDRL1a clones isolated from culture supernatants. Figure 10A shows the extent of binding of candidate mature antibody fragments to human erythrocytes compared to the parent antibody fragment (immature). Figure 10B shows the convergent sequences of hits from the maturation campaign. Figure 10C shows a comparison of the number and affinity of sequences identified by ELISA and NGS. Figure 10D shows a percentage breakdown of sequences identified by ELISA or NGS methods and that could be expressed as soluble Fabs, sorted by affinity for human erythrocytes (Note - this graph shows the breakdown of those Fabs tested by affinity, where the sum of Fab types across all affinity ranges equals 100%). [Figure 11] 11A-11F show data relating sequence enrichment to enhanced affinity of resulting antibodies screened from the CDRL1a library. Wild type 3-103 is indicated by an arrow for reference. [Figure 12]Figures 12A-12D show data related to CDRL2 clones isolated from culture supernatants. Figure 12A shows the extent of binding of candidate mature antibody fragments to human erythrocytes compared to the parent antibody fragment (immature). Figure 12B shows the convergent sequences of hits from the maturation campaign. Figure 12C shows a comparison of the number and affinity of sequences identified by ELISA and NGS. Figure 12D shows a percentage breakdown of sequences identified by ELISA or NGS methods that could be expressed as soluble Fabs, sorted by affinity to human erythrocytes (Note - this graph shows the breakdown of those Fabs tested by affinity, where the sum of Fab types across all affinity ranges equals 100%). [Figure 13] 13A-13F show data relating to the enrichment of sequences from the CDRL2 library from screening output from rounds 1 through 3. Wild type 3-103 is indicated by an arrow for reference. [Figure 14] Figures 14A-14D show data on CDRH1 clones isolated from culture supernatants. Figure 14A shows the extent of binding of candidate mature antibody fragments to human erythrocytes compared to the parent antibody fragment (immature). Figure 14B shows the convergent sequences of hits from the maturation campaign. Figure 14C shows a comparison of the number and affinity of sequences identified by ELISA and NGS. Figure 14D shows a percentage breakdown of sequences identified by ELISA or NGS methods and that can be expressed as soluble Fabs, sorted by their affinity for human erythrocytes (Note - this graph shows the breakdown of those Fabs tested by affinity, where the sum of Fab types across all affinity ranges equals 100%). [Figure 15] Figures 15A-15F show the relative enrichment of sequences from the CDRH1 library from screening outputs from rounds 1 through 3. Wild type 3-103 is indicated by an arrow for reference. [Figure 16]Figures 16A-16D show data related to CDRH2 clones isolated from culture supernatants. Figure 16A shows the extent of binding of candidate mature antibody fragments to human erythrocytes compared to the parent antibody fragment (immature). Figure 16B shows the convergent sequences of hits from the maturation campaign. Figure 16C shows a comparison of the number and affinity of sequences identified by ELISA, manual mutagenesis and NGS. Figure 16D shows a percentage breakdown of sequences identified by ELISA, manual mutagenesis or NGS methods that can be expressed as soluble Fabs, sorted by affinity for human erythrocytes (Note - this graph shows the breakdown of those Fabs tested by affinity, where the sum of Fab types across all affinity ranges equals 100%). [Figure 17] Figures 17A-17I show the relative enrichment of sequences from the CDRH2 library from screening results from rounds 1 through 3. Wild type 3-103 is indicated by an arrow for reference. [Figure 18] Figures 18A-18B show data relating to binding of clones selected from the CDRL1a, L2 and H1 libraries to human (18A) or cynomolgus monkey (18B) erythrocytes. [Figure 19] FIG. 19 shows an analysis of the efficiency of enhanced affinity from the indicated CDR libraries. [Figure 20] FIG. 20 shows the sequences of selected clones and data relating to human and cynomolgus affinity. [Figure 21] Figures 21A-21F provide a summary of the data presented in the preceding figures relative to a comparison of antibody affinity enhancement by either ELISA-based or next-generation sequencing (NGS)-based technologies. [Figure 22] FIG. 22 shows data correlating increased affinity for targets on red blood cells with improved disease severity. [Figure 23]Figures 23A-23C show data related to the assessment of enrichment of selected sequences as a function of affinity improvement. Figure 23A shows a scatter plot of sequence enrichment versus affinity from various libraries. Figure 23B shows affinity data from library 1, enriched for wild-type and wild-type-like sequences. Figure 23C shows affinity data from library 4, enriched for non-parental sequences and showing enhanced affinity. [Figure 24] FIG. 24 shows affinity data for cynomolgus monkey erythrocytes with selected clones that showed enhanced affinity for human red blood cells. [Diagram 25] Figures 25A-25B show kinetic analysis data for clones (B) that exhibit enhanced affinity relative to wild type (A). [Figure 26] FIG. 26 shows additional summary graphs comparing the ELISA and NGS screening methods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] Detailed Description Antigen-binding proteins that bind to glycophorin A Erythrocytes, also known as red blood cells (RBCs), are the most abundant cell type in mammalian blood. They are small, discoid, anucleated, biconcave cells whose primary function is to transport oxygen and carbon dioxide between tissues. Red blood cells express a characteristic set of cell surface markers, including human blood group antigens, glycophorin, band 3, and Lewis antigen.

[0062] Human glycophorin A (GPA) is a protein encoded by the GYPA gene (CD235a) and refers to a single transmembrane domain protein with a highly glycosylated extracellular domain. Glycophorin A is expressed exclusively in erythroid cells and in blood by RBCs.

[0063] Some embodiments relate to antigen binding proteins such as antibodies that bind to glycophorin A. In certain embodiments, the antigen binding proteins of the invention are full-length human, humanized or chimeric antibodies, binding fragments and derivatives of such antibodies, and polypeptides that specifically bind to glycophorin A. Some embodiments relate to affinity matured antigen binding proteins. Additionally described are nucleic acids encoding such antibodies, antibody fragments and derivatives, and polypeptides, cells comprising such polynucleotides, methods of making such antibodies, antibody fragments and derivatives, and polypeptides, and methods of using such antibodies, antibody fragments and derivatives, and polypeptides.

[0064] Highly homologous GPA and glycophorin B (GPB) are encoded by two genes, one derived from the other after a gene duplication event. The sequence of the 26 N-terminal amino acids of GPB is identical to one of the two alleles of the N-terminus of GPA. GPA and GPB are single transmembrane domain proteins with highly glycosylated extracellular domains. GPA and GPB have been found to bind within the erythrocyte membrane. In some embodiments, the antigen binding protein binds to GPA in a monomeric form. In some embodiments, the antigen binding protein binds to GPB in a dimeric form. In some embodiments, the antigen binding protein binds to GPB in a monomeric form. In some embodiments, the antigen binding protein binds to GPB in a dimeric form. In some embodiments, the antigen binding protein binds to a heterodimer of GPA and GPB.

[0065] Glycophorin carries several blood group antigens, such as the M and N blood group antigens on GPA, and the N, S and s antigens on GPB. In addition, glycophorin carries antigens that are not dependent on blood type, and there are several mouse monoclonal antibodies that target such epitopes that are always present on blood type-independent molecules. In some embodiments, the antigen binding protein recognizes GPA on red blood cells independent of the blood group antigen carried by the molecule. In some embodiments, the reactivity of the antigen binding protein is the same regardless of whether the blood group phenotype is M+N+, M-N+, or M+N-. In some embodiments, the antigen binding protein recognizes an epitope of GPA that is independent of the blood group determinant carried by this protein.

[0066] In some embodiments, the antigen binding protein binds to a single species of GPA. In some embodiments, the antigen binding protein binds to one or more species of GPA, including but not limited to human, cynomolgus monkey, pig, dog, mouse and rat. In some embodiments, the antigen binding protein binds to human GPA. In some embodiments, the antigen binding protein binds to cynomolgus monkey GPA. In some embodiments, the antigen binding protein binds to human GPA and cynomolgus monkey GPA.

[0067] Antigen-binding proteins In some embodiments, an antigen binding protein is provided. The term "antigen binding protein" as used herein has its normal meaning and refers to an antigen binding fragment that binds to an antigen, and optionally a protein that includes a scaffold or framework portion that allows the antigen binding fragment to adopt a conformation that facilitates binding of the antigen binding protein to the antigen. In some embodiments, the antigen is GPA or a fragment thereof. In some embodiments, the antigen binding fragment includes at least one CDR from an antibody that binds to the antigen. In some embodiments, the antigen binding fragment includes all three CDRs from the heavy chain of the antibody that binds to the antigen or the light chain of the antibody that binds to the antigen. Further, in some embodiments, the antigen binding fragment includes all six CDRs, the three CDRs from the heavy chain and the three CDRs from the light chain of the antibody that binds to the antigen. In some embodiments, the antigen binding fragment includes one, two, three, four, five or six CDRs from the antibody that binds to the antigen, and in some embodiments, the CDRs can be any combination of heavy and / or light chain CDRs. In some embodiments, the antigen binding fragment is an antibody fragment.

[0068] Non-limiting examples of antigen-binding proteins include antibodies, antibody fragments (e.g., antigen-binding fragments of antibodies), antibody derivatives, and antibody analogs. Further specific examples include, but are not limited to, single chain variable fragments (scFv), nanobodies (e.g., VH domains of camelid heavy chain antibodies; VHH fragments), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity determining region (CDR) fragments. These molecules can be from any mammal, such as human, mouse, rat, rabbit or pig, dog, or camel. Antibody fragments can compete with intact antibodies for binding to target antigens, and can be produced by modification of intact antibodies (e.g., enzymatic or chemical cleavage) or synthesized de novo using recombinant DNA technology or peptide synthesis. Antigen-binding proteins can include, for example, alternative protein scaffolds or artificial scaffolds with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds that contain, for example, mutations introduced to stabilize the three-dimensional structure of the antigen binding protein, as well as fully synthetic scaffolds that contain, for example, biocompatible polymers. Additionally, scaffolds based on peptide antibody mimetics ("PAMs"), as well as antibody mimetics that utilize fibronectin components as the scaffold, can be used.

[0069] In some embodiments, the antigen binding protein comprises one or more antibody fragments incorporated into a single polypeptide chain or multiple polypeptide chains. For example, antigen binding proteins include, but are not limited to, bispecific antibodies; intrabodies; domain antibodies (a single VL or VH domain or two or more VH domains linked by a peptide linker); maxibodies (two scFvs fused to an Fc region); trimers; tetramers; minibodies (scFvs fused to a CH3 domain); peptibodies (one or more peptides bound to an Fc region); linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions with complementary light chain polypeptides); small modular immunopharmaceuticals; and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0070] In some embodiments, the antigen binding protein has the structure of an immunoglobulin. As used herein, the term "immunoglobulin" has its ordinary meaning and can also refer to a tetrameric molecule, where each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0071] Within light and heavy chains, the variable (V) and constant (C) regions are joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 more amino acids. The variable regions of each light / heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.

[0072] Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From the N-terminus to the C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0073] Human light chains are classified as kappa and lambda light chains. As used herein, the term "light chain" has its usual meaning and can also refer to a polypeptide that includes, from amino to carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α) and epsilon (ε), which define the antibody isotypes as IgM, IgD, IgG, IgA and IgE, respectively. As used herein, the term "heavy chain" has its usual meaning and can also refer to a polypeptide that includes, from amino to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3) and optionally immunoglobulin heavy chain constant domain 4 (CH4). The IgG class is further divided into subclasses, namely IgG1, IgG2, IgG3 and IgG4. The IgA class is further divided into subclasses, namely IgA1 and IgA2. IgM includes, but is not limited to, subclasses IgM1 and IgM2. The heavy chains of IgG, IgA and IgD antibodies have three domains (CH1, CH2 and CH3), whereas the heavy chains of IgM and IgE antibodies have four domains (CH1, CH2, CH3 and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. The antibody chains are linked together via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the antibody heavy chains.

[0074] In some embodiments, the antigen-binding protein is an antibody. The term "antibody" as used herein has its usual meaning and also refers to an intact immunoglobulin of any isotype, including, for example, chimeric antibodies, humanized antibodies, human antibodies, and bispecific antibodies. An intact antibody generally comprises at least two full-length heavy chains and two full-length light chains. The antibody sequence may be derived from only a single species or may be "chimeric", i.e., different portions of the antibody may be derived from two different species, as further described below. Unless otherwise specified, the term "antibody" also includes antibodies comprising two substantially full-length heavy chains and two substantially full-length light chains, provided that the antibody retains the same or similar binding and / or function as an antibody consisting of two full-length light chains and a heavy chain. For example, antibodies with substitutions, insertions or deletions of 1, 2, 3, 4 or 5 amino acid residues at the N-terminus and / or C-terminus of the heavy and / or light chains are included in the definition if the antibody retains the same or similar binding and / or function as an antibody comprising two full-length heavy chains and two full-length light chains. Furthermore, unless expressly excluded, antibodies include, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies and synthetic antibodies. In certain embodiments, monoclonal and polyclonal antibodies are provided. As used herein, the term "polyclonal antibody" has its ordinary meaning and may also refer to a population of antibodies that generally differ widely in composition and binding specificity. As used herein, the term "monoclonal antibody" ("mAb") has its ordinary meaning and may also refer to one or more of a population of antibodies with identical sequence. A monoclonal antibody binds to an antigen at a specific epitope of the antigen.

[0075] In some embodiments, the antigen binding protein is a "fragment" or "antigen-binding fragment" of an antibody. The term "antibody fragment" as used herein has its ordinary meaning and can also refer to Fab, Fab', F(ab')2 and Fv fragments that contain at least one CDR of an immunoglobulin sufficient to provide specific antigen binding to GPA. Antibody fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

[0076] In some embodiments, Fab fragments are provided. Fab fragments are monovalent fragments with VL, VH, CL and CH1 domains; F(ab')2 fragments are bivalent fragments with two Fab fragments linked by disulfide bridges at the hinge region; Fd fragments have VH and CH1 domains; Fv fragments have VL and VH domains of a single arm of an antibody; and dAb fragments have one VH domain, one VL domain or antigen-binding fragments of VH or VL domains. In some embodiments, these antibody fragments can be incorporated into single domain antibodies, single chain antibodies, maxibodies, minibodies, intrabodies, dimers, trimers, tetramers, V-NARs and bis-scFvs. In some embodiments, the antibody comprises at least one CDR as shown in Table 2 herein.

[0077] The present invention also provides, in some embodiments, single chain variable fragments. The term "single chain variable fragment" ("scFv") as used herein has its usual meaning and also refers to a fusion protein in which the VL and VH regions are linked via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, where the linker is long enough to allow the protein chain to fold back on itself to form a monovalent antigen binding site. For clarity, unless otherwise specified, a "single chain variable fragment" is not an antibody or antibody fragment as defined herein. A dimer is a bivalent antibody comprising two polypeptide chains, where each polypeptide chain comprises a VH and a VL domain connected by a linker that is too short to allow pairing between the two domains of the same chain or is in a configuration that does not allow pairing, thus allowing each domain to pair with a complementary domain of another polypeptide chain. According to some embodiments, if the two polypeptide chains of a dimer are identical, the dimer resulting from the pairing may have two identical antigen binding sites. Polypeptide chains with different sequences can be used to create dimers with two different antigen binding sites. Similarly, trimers and tetramers are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen binding sites, respectively, which may be the same or different.

[0078] In some embodiments, an antigen binding protein comprises one or more CDRs. As used herein, the term "CDR" has its usual meaning and can also refer to the complementarity determining regions (also called "minimal recognition units" or "hypervariable regions") within an antibody variable sequence. CDRs allow an antigen binding protein to specifically bind to a particular antigen of interest. There are three heavy chain variable region CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable region CDRs (CDRL1, CDRL2 and CDRL3). The CDRs of each of the two chains are usually aligned by framework regions to form a structure that specifically binds to a particular epitope or domain on the target protein. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions usually contain these elements in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of the amino acids that make up each of these domains is made according to a numbering system. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity determining regions (CDRs) and framework regions (FRs) of a particular antibody can be identified using this system. Other numbering systems for amino acids in immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001). One or more CDRs can be incorporated into a molecule, either covalently or non-covalently, to make it an antigen binding protein.

[0079] In some embodiments, the antigen binding proteins of the present invention comprise one or more CDRs as part of a larger polypeptide chain. In some embodiments, the antigen binding proteins covalently link one or more CDRs to another polypeptide chain. In some embodiments, the antigen binding proteins can incorporate one or more CDRs non-covalently. In some embodiments, the antigen binding proteins can comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In some embodiments, the biocompatible framework structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support, or framework, or scaffold, capable of displaying one or more sequences of amino acids that bind to an antigen (e.g., CDRs, variable regions, etc.) in a localized surface region. Such structures can be native polypeptides or polypeptide "folds" (structural motifs) or can have one or more modifications, such as additions, deletions and / or substitutions of amino acids, compared to native polypeptides or folds. Depending on the embodiment, these scaffolds can be derived from various species (or species) of polypeptides, such as humans, non-human primates or other mammals, other vertebrates, invertebrates, plants, bacteria or viruses.

[0080] Depending on the embodiment, the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains. In some such embodiments, the framework structures are based on fibronectin, ankyrin, lipocalin, neocarzinostatin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain and / or tendamistat domain.

[0081] In some embodiments, antigen-binding proteins having two or more binding sites are also provided. In some embodiments, the binding sites are identical to each other, while in some embodiments, the binding sites are different from each other. For example, an antibody usually has two identical binding sites, but a "bispecific" or "bifunctional" antibody has two different binding sites. The two binding sites of a bispecific antigen-binding protein or antibody can bind to two different epitopes that may be present on the same or different protein targets.

[0082] The term "chimeric antibody" as used herein has its ordinary meaning and also refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In certain embodiments, one or more of the CDRs are from an anti-GPA antibody. In some embodiments, all of the CDRs are from an anti-GPA antibody. In some embodiments, CDRs from two or more anti-GPA antibodies are mixed and matched to form a chimeric antibody. For example, a chimeric antibody may contain CDR1 from a light chain of a first anti-GPA antibody, CDR2 and CDR3 from a light chain of a second anti-GPA antibody, and CDRs from a heavy chain from a third anti-GPA antibody. Additionally, the framework regions of the antigen binding proteins described herein may be derived from one of the same anti-GPA antibodies, one or more different antibodies, such as a human antibody, or a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and / or light chain is identical to, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain(s) is identical to, homologous to, or derived from an antibody from another species or belonging to another antibody class or subclass. Also provided herein are fragments of such antibodies that exhibit the desired biological activity.

[0083] In certain embodiments, an antigen binding protein is provided comprising a heavy chain variable domain having at least 90% identity to any of the VH domain amino acid sequences set forth in Table 1. In certain embodiments, an antigen binding protein comprises a heavy chain variable domain having at least 95% identity to any of the VH domain amino acid sequences set forth in Table 1. In certain embodiments, an antigen binding protein comprises a heavy chain variable domain having at least 99% identity to any of the VH domain amino acid sequences set forth in Table 1. In some embodiments, the heavy chain variable domain may have one or more additional mutations in any of the VH domain amino acid sequences set forth in Table 1, but retains specific binding to GPA. In some embodiments, the heavy chain variable domain may have one or more additional mutations in any of the VH domain amino acid sequences set forth in Table 1, but has improved specific binding to GPA.

[0084] In certain embodiments, the antigen binding protein comprises a light chain variable domain having at least 90% identity to any of the VL domain amino acid sequences set forth in Table 1. In certain embodiments, the antigen binding protein comprises a light chain variable domain having at least 95% identity to any of the VL domain amino acid sequences set forth in Table 1. In certain embodiments, the antigen binding protein comprises a light chain variable domain having at least 99% identity to any of the VL domain amino acid sequences set forth in Table 1. In some embodiments, the light chain variable domain may have one or more additional mutations in any of the VL domain amino acid sequences set forth in Table 1, but retains specific binding to GPA. In some embodiments, the light chain variable domain may have one or more additional mutations in any of the VL domain amino acid sequences set forth in Table 1, but has improved specific binding to GPA.

[0085] In certain embodiments, the antigen binding protein comprises a heavy chain variable domain having at least 90% identity to any of the VH domain amino acid sequences listed in Table 1, and a light chain variable domain having at least 90% identity to any of the VL domain amino acid sequences listed in Table 1. In certain embodiments, the antigen binding protein comprises a heavy chain variable domain having at least 95% identity to any of the VH domain amino acid sequences listed in Table 1, and a light chain variable domain having at least 95% identity to any of the VL domain amino acid sequences listed in Table 1. In certain embodiments, the antigen binding protein comprises a heavy chain variable domain having at least 99% identity to any of the VH domain amino acid sequences listed in Table 1, and a light chain variable domain having at least 99% identity to any of the VL domain amino acid sequences listed in Table 1.

[0086] In certain embodiments, the antigen binding protein comprises a heavy chain variable domain having any of the VH domain amino acid sequences set forth in Table 1, and a light chain variable domain having any of the VL domain amino acid sequences set forth in Table 1. In certain embodiments, the light chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence of a light chain variable domain set forth in Table 1. In certain embodiments, the heavy chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence of a heavy chain variable domain set forth in Table 1.

[0087] In certain embodiments, the light chain variable domain comprises an amino acid sequence encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a polynucleotide sequence set forth in Table 1. In certain embodiments, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding a light chain variable domain selected from the sequences set forth in Table 1. In certain embodiments, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide encoding a light chain variable domain selected from the group consisting of the sequences set forth in Table 1.

[0088] In certain embodiments, the heavy chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence of a heavy chain variable domain selected from the sequences set forth in Table 1. In certain embodiments, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding a heavy chain variable domain selected from the sequence set forth in Table 1. In certain embodiments, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide encoding a heavy chain variable domain selected from the group consisting of the sequences set forth in Table 1.

[0089] [Table 1] [Table 2]

Table 3

Table 4

Table 5

[0090] In one embodiment, an antigen binding protein is provided which comprises one or more VH CDR1, VH CDR2 and / or VH CDR3 domains having an amino acid sequence identical to or including one, two or three amino acid residue substitutions, deletions and / or insertions in each CDR compared to the VH CDR1, VH CDR2 or VH CDR3 sequences set forth in Table 2. In one embodiment, the antigen binding protein comprises one or more VL CDR1, VL CDR2 and / or VL CDR3 having an amino acid sequence identical to or including one, two or three amino acid residue substitutions, deletions and / or insertions in each CDR compared to the VL CDR1, VL CDR2 or VL CDR3 sequences set forth in Table 2. In certain embodiments, the antigen binding protein comprises one or more VH CDR1, VH CDR2 and / or VH CDR3 having an amino acid sequence identical to or including a substitution, deletion or insertion of one, two or three amino acid residues in each CDR compared to the VH CDR1, VH CDR2 or VH CDR3 sequence set forth in Table 2, and one or more VL CDR1, VL CDR2 and / or VL CDR3 having an amino acid sequence identical to or including a substitution, deletion and / or insertion of one, two or three amino acid residues in each CDR compared to the VL CDR1, VL CDR2 or VL CDR3 sequence set forth in Table 2. In one embodiment, the antigen binding protein comprises one VH CDR1, VH CDR2 and / or VH CDR3 having an amino acid sequence identical to or including substitutions, deletions and / or insertions of one, two or three amino acid residues in each CDR compared to the VH CDR1, VH CDR2 or VH CDR3 sequence set forth in Table 2, and one VL CDR1, VL CDR2 and / or VL CDR3 having an amino acid sequence identical to or including substitutions, deletions and / or insertions of one, two or three amino acid residues in each CDR compared to the VL CDR1, VL CDR2 or VL CDR3 sequence set forth in Table 2.In one embodiment, the antigen binding protein comprises two VH CDR1s, VH CDR2s and / or VH CDR3s that have amino acid sequences identical to or including substitutions, deletions and / or insertions of one, two or three amino acid residues in each CDR compared to the VH CDR1, VH CDR2 and VH CDR3 sequences set forth in Table 2, and two VL CDR1s, VL CDR2s and / or VL CDR3s that have amino acid sequences identical to or including substitutions, deletions and / or insertions of one, two or three amino acid residues in each CDR compared to the VL CDR1, VL CDR2 or VL CDR3 sequences set forth in Table 2. In some embodiments, the antigen binding protein comprises a VH CDR1, VH CDR2 and VH CDR3 having an amino acid sequence identical to or including about one, two or three amino acid residue substitutions, deletions and / or insertions in the CDRs compared to the VH CDR1, VH CDR2 and VH CDR3 sequences set forth in Table 2, and a VL CDR1, VL CDR2 and VL CDR3 having an amino acid sequence identical to or including one, two or three amino acid residue substitutions, deletions and / or insertions in each CDR compared to the VL CDR1, VL CDR2 or VL CDR3 sequences set forth in Table 2. In some embodiments, the antigen binding protein comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 having an amino acid sequence identical to any of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences set forth in Table 2. In certain embodiments, the VL CDR1, VL CDR2 and / or VL CDR3 may have one or more additional mutations in any of the VL CDR1, VL CDR2 and / or VL CDR3 sequences set forth in Table 2, but retain specific binding to GPA (e.g., human GPA and / or mouse and / or cynomolgus monkey, depending on the embodiment).In some embodiments, VLCDR1, VLCDR2 and / or VLCDR3 may have one or more additional mutations in any of the VLCDR1, VLCDR2 and / or VLCDR3 amino acid sequences set forth in Table 2, but have improved specific binding to GPA (e.g., human GPA and / or mouse and / or cynomolgus monkey, depending on the embodiment). In some embodiments, VH CDR1, VH CDR2 and / or VH CDR3 may have one or more additional mutations in any of the VH CDR1, VH CDR2 and / or VH CDR3 sequences set forth in Table 2, but retain specific binding to GPA (e.g., human GPA and / or mouse and / or cynomolgus monkey, depending on the embodiment). In some embodiments, the VH CDR1, VH CDR2 and / or VH CDR3 may have one or more additional mutations in any of the VH CDR1, VH CDR2 and / or VH CDR3 amino acid sequences set forth in Table 2, but have improved specific binding to GPA (e.g., human GPA and / or mouse and / or cynomolgus monkey, depending on the embodiment).

[0091] [Table 6] [Table 7] [Table 8]

[0092] Humanization of antigen-binding proteins In some embodiments, humanized antigen binding proteins are provided. As used herein, the term "humanized antigen binding protein" has its ordinary meaning and also refers to an antigen binding protein having a sequence that differs from that of an antigen binding protein derived from a non-human species by one or more amino acid substitutions, deletions and / or additions such that the humanized antigen binding protein is less likely to elicit an immune response and / or elicits a less severe immune response when administered to a human subject compared to the non-human species antigen binding protein. In some embodiments, specific amino acids within the framework and / or constant domains of the heavy and / or light chains of the non-human species antigen binding protein are mutated to generate a humanized antigen binding protein. In another embodiment, the constant domain(s) from a human antigen binding protein are fused to the variable domain(s) of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of the non-human antigen binding protein are modified to reduce the potential immunogenicity of the non-human antigen binding protein when administered to a human subject, wherein the modified amino acid residues are not residues primarily involved in immunospecific binding of the antigen binding protein to its antigen, or the modifications made to the amino acid sequence are conservative modifications such that binding of the humanized antigen binding protein to the antigen is not significantly altered compared to binding of the non-human antigen binding protein to the antigen.

[0093] According to some embodiments, humanization of antigen binding proteins involves CDR grafting, which involves recombining the CDRs of a non-human antigen binding protein, and in some embodiments, only these CDRs on human variable region framework regions and human constant regions. In some embodiments, this change substantially reduces or eliminates immunogenicity. In some embodiments, overall immunogenicity is reduced, but allotypic or idiotypic differences may still exist. Depending on the embodiment, some framework residues of the original antigen binding protein may need to be preserved. Such framework residues may be identified via computer modeling, or potentially by comparing known antigen binding site structures. Residues that may affect antigen binding are classified into several groups. The first group includes residues that are adjacent to the antigen site surface and therefore may directly contact the antigen. These residues include, in some embodiments, amino-terminal residues and residues adjacent to the CDRs. The second group includes residues that, in some embodiments, may change the structure or relative arrangement of the CDRs by contacting the CDRs or another peptide chain in the antigen binding protein. According to some embodiments, the third group comprises amino acids with buried side chains that may affect the structural integrity of the variable domain.

[0094] In certain embodiments, the antigen binding protein further comprises one or more of the non-limiting embodiments of the FR (framework region) exemplified in Tables 3-4. In certain embodiments, the antigen binding protein comprises one or more of the heavy chain FR1 sequence, the heavy chain FR2 sequence, the heavy chain FR3 sequence, and the heavy chain FR4 sequence exemplified in Table 3. In certain embodiments, the antigen binding protein comprises one or more of the light chain FR1 sequence, the light chain FR2 sequence, the light chain FR3 sequence, and the light chain FR4 sequence shown in Table 4. In certain embodiments, any one or more of the FR regions in Tables 3 and 4 can be combined with any one or more of the CDR sequences provided herein. Thus, in certain embodiments, an antigen binding protein is provided that comprises six CDRs (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) and eight FRs (e.g., HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4).

[0095] [Table 9]

[0096] [Table 10]

[0097] Affinity maturation of antigen-binding proteins In some embodiments, antigen binding proteins with increased affinity for a target antigen are provided. In some embodiments, the target antigen is glycophorin A. In some embodiments, affinity maturation of one or more of the CDRs listed in Table 1 is obtained by a number of affinity maturation protocols, including, but not limited to, maintaining the CDRs, chain shuffling, using mutant strains of E. coli, DNA shuffling, phage display and additional PCR techniques.

[0098] Affinity can be determined using a variety of techniques, including but not limited to equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); or radioimmunoassay (RIA)), or surface plasmon resonance assays or other mechanism-based kinetic assays (e.g., BIACORE® analysis or Octet® analysis (forteBIO)), and other methods such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize one or more labels of the component being tested and / or may use a variety of detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. One example of a competitive binding assay is a radioimmunoassay, which involves incubation of a labeled antigen with an antigen-binding protein in the presence of increasing amounts of unlabeled antigen, and detection of the antigen-binding protein bound to the labeled antigen. The affinity and binding off-rate of an antigen-binding protein for a particular antigen can be determined from the data by Scatchard plot analysis. Competition with a second antigen-binding protein can also be determined using a radioimmunoassay. In this case, the antigen is incubated in the presence of an antigen binding protein bound to a labeled compound and increasing amounts of a second, unlabeled antigen binding protein.

[0099] 10 -7 The equilibrium dissociation constant (K D or the corresponding K D An antigen binding protein "specifically binds" to an antigen, such as GPA, if it binds to the antigen with a strong binding affinity, as determined by a β-Affinity (Affinity) value. An antigen binding protein that specifically binds to human GPA may be able to bind to GPA of other species as well, with the same or different affinity.

[0100] In one embodiment, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 Less than M, 10 -11Less than M, 10 -12 Less than M, 10 -13 Less than M or 5x10 -13 Equilibrium dissociation constant or K less than M (smaller values ​​indicate higher binding affinity) D (k off / k on In one embodiment, the present invention provides an antigen binding protein (e.g., an antibody or fragment) that specifically binds to GPA at about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M, about 10 -13 Less than M or about 5x10 -13 Equilibrium dissociation constant or K less than M D (k off / k on ) provides an antigen-binding protein that specifically binds to GPA.

[0101] In one embodiment, about 10 -7 About 10 meters from -8 M, about 10 -8 About 10 meters from -9 M, about 10 -9 About 10 meters from -10 M, about 10 -10 About 10 meters from -11 M, about 10 -11 About 10 meters from -12 M, about 10 -12 About 10 meters from -13 Equilibrium dissociation constant of M or K D (k off / k on In one embodiment, the present invention provides an antigen binding protein (e.g., an antibody fragment) that specifically binds to GPA at 10 -7 M to 10 -8 M, 10 -8 M to 10 -9 M, 10 -9 M to 10 -10 M, 10 -10 M to 10 -11 M, 10 -11 M to 10 -12 M, 10-12 M to 10 -13 Equilibrium dissociation constant of M or K D (k off / k on ), and an antigen binding protein that specifically binds to GPA, having a dissociation constant between the enumeration, including an end point.

[0102] Antigen binding proteins with identical or overlapping epitopes can often compete for binding to an antigen (e.g., human GPA). Thus, in some embodiments, antigen binding proteins (e.g., antibodies or antibody fragments thereof) are provided that compete with antigen binding proteins 10F7, Ter119 (in embodiments where the antigen binding protein cross-reacts with mouse GPA), CLB-ery-1 (AME-1), EPR8200, YTH89.1, EPR8199, JC159, GYPA / 280, ab40844, HI264, GPHN02, SPM599, GYPA / 1725R, ab112201, BRIC256, or fragments or derivatives thereof. As used herein, "compete" or "competition" has its ordinary meaning and can refer to antigen binding proteins that compete for the same epitope or binding site on a target. Such competition can be determined by an assay in which a control antigen-binding protein (e.g., an antibody or antibody fragment thereof) prevents or inhibits specific binding of the test antigen-binding protein. A number of competitive binding assays can be used to determine whether a test molecule competes with a control molecule for binding. Examples of assays that can be used include solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays, solid-phase direct biotin-avidin EIA, solid-phase direct labeling assays, solid-phase direct labeling sandwich assays, Luminex, and surface plasmon resonance. Competitive inhibition can be measured by determining the amount of labeled ligand bound to a solid surface or cell in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antigen-binding proteins or antibodies identified by competitive assays (competing antigen-binding proteins or antibodies) include antigen-binding proteins that bind to the same epitope as the control antigen-binding protein and antigen-binding proteins that bind to adjacent epitopes sufficiently close to the epitope bound by the control antigen-binding protein to create steric hindrance.Typically, when a competing antigen binding protein is present in excess, it can inhibit (e.g., reduce) specific binding of a control antigen binding protein to a target antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more than 75% (or any percentage of inhibition between the listed values). In some embodiments, binding is inhibited by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or more than 97% (including up to 100% inhibition).

[0103] Nucleic Acids Encoding Antigen-Binding Proteins The antigen binding proteins of the present invention can be produced by any method known in the art for the synthesis of proteins (eg, antibodies), particularly by chemical synthesis or recombinant expression techniques.

[0104] In some embodiments, nucleic acids are provided that encode the antigen binding proteins of the present invention. The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably herein and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleotide analogs), and hybrids thereof. Nucleic acids include, for example, polynucleotides encoding all or a portion of an antigen binding protein, such as a single or both strands of an antibody, or a fragment, derivative, mutein or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, antisense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the above. Nucleic acids can be of any length appropriate for a desired use or function, can include one or more additional sequences, such as regulatory sequences, and / or can be part of a larger nucleic acid, such as a vector. One of skill in the art will appreciate that due to the degeneracy of the genetic code, each of the polypeptide sequences described herein is encoded by numerous other nucleic acid sequences. The present invention provides each modified nucleotide sequence encoding each antigen binding protein described herein.

[0105] Mutant antigen-binding proteins Nucleotide sequences encoding the antigen binding proteins described herein can be modified, for example, by random mutagenesis and / or site-specific mutagenesis (e.g., oligonucleotide-directed site-specific mutagenesis) to generate modified polynucleotides containing one or more nucleotide substitutions, deletions, and / or insertions compared to the non-mutated polynucleotide. These and other methods can be used to generate derivatives of antigen binding proteins that have, for example, desired properties, such as increased affinity, avidity, or specificity for a desired target, increased activity or stability in vivo or in vitro, or reduced side effects in vivo, compared to the non-derivatized antibody. In other embodiments, mutations introduced into the nucleic acids of the invention do not significantly alter the biological activity of the polypeptide they encode.

[0106] In some embodiments, the antigen binding proteins (e.g., antibodies or fragments thereof) of the present invention may have at least one amino acid substitution, provided that the antigen binding protein retains the same or better (e.g., higher affinity, lower Kd) desired binding specificity. Thus, in some embodiments, modifications to the structure of the antigen binding protein are provided. In some embodiments, the antigen binding proteins (e.g., but not limited to, antibodies) comprise sequences that differ from the CDR sequences set forth in Table 2 herein by the addition, substitution, and / or deletion of 5, 4, 3, 2, 1, or 0 single amino acids, each independently. These may include amino acid substitutions that may be conservative or non-conservative that do not destroy the desired binding ability of the antibody. Conservative amino acid substitutions may include non-natural amino acid residues, which are generally incorporated by chemical peptide synthesis rather than synthesis in a biological system. These include peptidomimetics and other reversed or inverted forms of amino acid sites. Conservative amino acid substitutions may also include the replacement of a natural amino acid residue with a normative residue, such that it has little or no effect on the polarity or charge of the amino acid residue at that position.

[0107] Non-conservative substitutions may involve the exchange of a member of one class for an amino acid or amino acid mimetic of a member of another class that has different physical properties (e.g., size, polarity, hydrophobicity, charge, replacement of an L-isoform with a D-isoform, etc.) In certain embodiments, such substituted residues may be introduced into regions of the antigen binding protein that are homologous to non-human antibodies, or into the non-homologous regions of the fragment.

[0108] Non-limiting examples of desirable amino acid substitutions are: (1) those that reduce susceptibility to proteolysis, (2) those that reduce susceptibility to oxidation, (3) those that change the binding affinity for forming protein complexes, (4) those that change the binding affinity, and / or (5) those that impart or modify other physicochemical or functional properties to such polypeptides. For example, in some embodiments, the amino acid substitutions can reduce (or increase) the propensity for post-translational modification such as glycosylation. In such embodiments, the degree of post-translational modification can be adjusted to a desired level for a given application. In some embodiments, single or multiple amino acid substitutions (in some embodiments, conservative amino acid substitutions) can be made in the native sequence (in some embodiments, the portion of the polypeptide outside the domain(s) that form intermolecular contacts). In some embodiments, conservative amino acid substitutions generally may not substantially alter the structural characteristics of the parent sequence (e.g., the replacement amino acid does not tend to disrupt helices occurring in the parent sequence or other types of secondary structures that characterize the parent sequence).

[0109] Vectors encoding antigen-binding proteins In some embodiments, a vector comprising one or more of the nucleic acids described herein is further provided. As used herein, the term "vector" has its ordinary meaning and refers to a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), in which additional DNA segments can be introduced into the viral genome. In some embodiments, vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors that contain a bacterial origin of replication). In other embodiments, vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing or location of expression) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can exert its effect, for example, directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals).

[0110] The recombinant expression vector of the present invention comprises one or more regulatory sequences, which are selected based on the host cell used for expression and are operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter and cytomegalovirus promoter), those that direct expression of a nucleotide sequence only in a particular host cell (e.g., tissue-specific regulatory sequences, see Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science 236:1237, the entire contents of which are incorporated herein by reference), and those that direct inducible expression of a nucleotide sequence in response to a particular treatment or disease state (e.g., metallothionine promoter in mammalian cells and tetracycline-responsive (tet-responsive) and / or streptomycin-responsive promoters in both prokaryotic and eukaryotic systems (see, ibid.)). Depending on the embodiment, the design of the expression vector can depend on factors such as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by the nucleic acids described herein.

[0111] Host cells expressing antigen-binding proteins In some embodiments, a host cell is provided into which a recombinant expression vector described herein has been introduced. As used herein, the term "host cell" has its ordinary meaning and also refers to a cell that can be used to express a nucleic acid, e.g., a nucleic acid encoding an antigen binding protein described herein. A host cell can be any prokaryotic or eukaryotic cell (and / or lysates or transcription and / or translation active components, e.g., cell-free systems derived from such cells). Prokaryotic host cells, in some embodiments, include gram-negative or gram-positive organisms, e.g., E. coli or Bacillus. Eukaryotic cells, in some embodiments, include unicellular eukaryotes (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells or insect cells), or hybridomas. Examples of suitable mammalian host cell lines include, but are not limited to, Chinese Hamster Ovary (CHO) cells or derivatives thereof, such as Veggie CHO and related cell lines that grow in serum-free medium, or the CHO line DXB-11, which is deficient in DHFR. Additional CHO cell lines of the present invention include CHO-K1 (ATCC#CCL-61), EM9 (ATCC#CRL-1861) and UV20 (ATCC#CRL-1862). Additional host cells include, but are not limited to, the COS-7 line of monkey kidney cells (ATCC CRL 1651), L cells, C127 cells, 3T3 cells (ATCC CCL 163), AM-1 / D cells, HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1 / EBNA cell line (ATCC CCL 70) derived from the African green monkey kidney cell line CV1, human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epithelial A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissues, primary explants, HL-60, U937, HaK or Jurkat cells.

[0112] In some embodiments, a host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, thereby allowing it to be expressed in the host cell. The term "recombinant host cell" as used herein has its usual meaning and also refers to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express the nucleic acid at a desired level unless a regulatory sequence is introduced into the host cell so that the nucleic acid is operably linked to the nucleic acid. The term "host cell" as used herein is meant to refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Some modifications may occur in subsequent generations, for example, due to mutations or environmental influences, and such progeny may not, in fact, be identical to the parent cell, but are still within the scope of the term as used herein. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. In the case of stable transfection of mammalian cells, only a small proportion of cells can integrate the foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants, in some embodiments, a gene encoding a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the gene of interest. Further examples of selectable markers include those that confer drug resistance (e.g., G418, hygromycin, and methotrexate). Cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have integrated the selectable marker gene survive and other cells die), among other methods.

[0113] In some embodiments, the host cell is co-transfected with two expression vectors, such as a first vector encoding an antibody heavy chain derived polypeptide and a second vector encoding an antibody light chain derived polypeptide. In some such embodiments, the two vectors can contain the same selectable marker that allows for equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used that can encode and express both antibody heavy and light chain polypeptides, for example. In some such embodiments, the light chain is placed before the heavy chain to avoid excess non-toxic heavy chain. The coding sequences of the heavy and light chains can comprise cDNA or genomic DNA.

[0114] When an antigen binding protein is produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified, for example, by chromatography (e.g., ion exchange, affinity, particularly for a particular antigen following Protein A, and size exclusion chromatography), centrifugation, differential solubility, or any other standard technique for purification of proteins. Additionally, the antigen binding protein may be fused to a heterologous polypeptide sequence as described herein or otherwise known to facilitate purification. In certain embodiments, antigen binding proteins are provided that are fused or chemically conjugated (including both covalent and non-covalent) to a polypeptide. The fused or conjugated antigen binding proteins of the present invention may be used to enhance purification. Additionally, the antigen binding proteins of the present invention may be fused to a marker sequence, such as a peptide, to facilitate purification. In certain embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector to facilitate purification of the fusion protein. Non-limiting examples of other peptide tags useful for purification include, but are not limited to, the "HA" tag and the "flag" tag, which correspond to an epitope derived from the influenza hemagglutinin protein.

[0115] Conjugates In some embodiments, a conjugate of an antigen binding protein of the present invention and another molecule is provided. In some embodiments, the antigen binding protein is conjugated to one or more non-proteinaceous compounds. Non-limiting examples of non-proteinaceous compounds include, but are not limited to, chemotherapeutic agents such as cytotoxic agents, cytostatic agents, toxins and / or radioactive substances. Other derivatives of the antigen binding protein of the present invention include covalent or aggregate conjugates of the antigen binding protein of the present invention with other proteins or polypeptides. In some embodiments, the antigen binding protein is conjugated to one or more therapeutic agents. Non-limiting examples of therapeutic agents suitable for conjugation include, but are not limited to, single chain variable fragments (scFv), antibody fragments, small molecule drugs, bioactive peptides, bioactive proteins and / or bioactive biomolecules. In some embodiments, a conjugate of an antigen binding protein provided herein and an antigenic component is provided, including, but not limited to, a full-length antigen, an antigenic and / or immunogenic fragment thereof, or an antigenic and / or immunogenic mimotope thereof.

[0116] The term "conjugate" or "molecular fusion" as used herein has its usual meaning and refers to direct or indirect attachment by chemical bonds, including but not limited to covalent bonds, electrostatic ions, charges. In some embodiments, conjugation produces a unit that is held together by chemical bonds. The term "direct conjugation" as used herein has its usual meaning and refers to chemical attachment to a molecule, with or without an intermediate linker or chemical group. Indirect conjugation as used herein has its usual meaning and refers to chemical attachment to a carrier. In some embodiments, a molecular fusion is formed between an antigen-binding protein described herein and a second polypeptide or protein. In some embodiments, the fusion comprises an antigen-binding protein and another component directly attached to each other. In other embodiments, the fusion comprises an antigen-binding protein and another component indirectly (e.g., via a linker) conjugated to each other. Non-limiting examples of linkers include, but are not limited to, peptides, polymers, aptamers, nucleic acids, and / or particles. In some embodiments, the particle is a microparticle, nanoparticle, polymersome, liposome, or micelle. In some embodiments, the polymer is natural or synthetic. In some embodiments, the polymer is linear or branched. A fusion protein comprising an antigen-binding protein and a second polypeptide is an example of a molecular fusion of polypeptides, and the fusion protein comprises polypeptides in which the polypeptides are directly linked to each other or have a linker sequence and / or additional sequence intervening at one or both ends. Conjugation to the linker can be via a covalent bond or an ionic bond.

[0117] In some embodiments, conjugation is achieved by covalently binding the antigen-binding protein to another molecule, with or without the use of a linker.The formation of such a conjugate is within the skill of the art, and various techniques for achieving the conjugate are known, and the choice of a particular technique is guided by the material to be conjugated.As described further below, the addition of amino acids to a polypeptide (C-terminus or N-terminus) that contain ionizable side chains, i.e., aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine or tyrosine, and that are not included in the active portion of the polypeptide sequence, function in their unprotonated state as strong nucleophiles that participate in various bioconjugation reactions with reactive groups attached to the polymer, i.e., homo- or hetero-bifunctional PEG.

[0118] In some embodiments, covalent modifications of antigen-binding proteins are provided. Depending on the embodiment, the covalent modifications are made in some embodiments by chemical synthesis or by enzymatic or chemical cleavage of the antigen-binding protein. In other embodiments, the covalent modifications of the antigen-binding protein are introduced into the molecule by reacting targeted amino acid residues of the antibody with organic derivatizing agents that can react with selected side chains or N- or C-terminal residues.

[0119] In some embodiments, cysteinyl residues of antigen binding proteins are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. In some embodiments, iodine reagents are used. In further embodiments, cysteinyl residues are derivatized by reaction with bromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoic acid, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0120] In certain embodiments, histidyl residues of the antigen binding protein are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0, as this agent is relatively specific for histidyl side chains. In certain embodiments, parabromophenacyl bromide is used for derivatization; in such embodiments, the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

[0121] In some embodiments, lysyl- and amino-terminal residues of antigen-binding proteins are reacted with succinic acid or other carboxylic acid anhydrides. In some embodiments, derivatization with these reactants has the effect of reversing the charge of lysinyl residues. Non-limiting examples of other suitable reagents for derivatizing α-amino- and / or e-amino-containing residues include, but are not limited to, transaminase-catalyzed reactions with imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, 0-methylisourea, 2,4-pentanedione, and glyoxylate.

[0122] In some embodiments, the arginyl residues of the antigen-binding protein are modified by reaction with one or more conventional reagents, such as, for example, phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and / or ninhydrin. In some embodiments, derivatization of arginyl residues is generally performed under alkaline conditions due to the high pKa of the guanidine functional group. In such embodiments, these reagents can react with the ε-amino group of lysine as well as the ε-amino group of arginine.

[0123] In some embodiments, specific modification of tyrosyl residues of antigen binding proteins is performed.In some such embodiments, spectral labels are introduced into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.In some embodiments, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Tyrosyl residues are iodized using I125 or I131 to prepare labeled proteins for use in radioimmunoassay.

[0124] In certain embodiments, carboxyl side groups (aspartyl or glutamyl) of antigen binding proteins are selectively modified by reaction with carbodiimides (RN=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Moreover, in certain embodiments, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0125] In some embodiments, the glutaminyl and / or asparaginyl residues of the antigen binding protein are deamidated to the corresponding glutamyl and aspartyl residues, respectively. Depending on the embodiment, these residues are deamidated under neutral or basic conditions. In some embodiments, the antigen binding protein contains one or more deamidated forms of these residues.

[0126] Other exemplary modifications of the antigen binding proteins of the invention include, but are not limited to, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino group of lysine, arginine and histidine side chains, acetylation of the N-terminal amine and / or amidation of any C-terminal carboxyl group.

[0127] In some embodiments, the covalent modification of the antigen binding protein involves chemically or enzymatically coupling glycosides to the antibody. In some embodiments, these procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine, (d) free hydroxyl groups such as serine, threonine or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine.

[0128] Binding to the erythrocyte cell surface In some embodiments, the antigen binding protein binds to an erythrocyte. In some embodiments, the antigen binding protein binds to a single erythrocyte. In some embodiments, the antigen binding protein binds erythrocytes of one or more species, including but not limited to human, cynomolgus monkey, pig, dog, mouse and / or rat. In some embodiments, the antigen binding protein binds human erythrocytes. In some embodiments, the antigen binding protein binds to cynomolgus monkey erythrocytes. In some embodiments, the antigen binding protein binds to human erythrocytes and cynomolgus monkey erythrocytes. In some embodiments, the antigen binding protein specifically binds to human erythrocytes and cynomolgus monkey erythrocytes with similar specificity.

[0129] In some embodiments, antigen binding proteins are provided that bind to red blood cell surfaces. In some embodiments, the antigen binding proteins bind to red blood cell surfaces without altering cell morphology. In some embodiments, the antigen binding proteins bind to red blood cell surfaces without cytoplasmic translocation. In some embodiments, the antigen binding proteins bind to red blood cell surfaces without causing apoptosis of the bound red blood cells. In some embodiments, the antigen binding proteins specifically bind to red blood cells in vivo. In some embodiments, the antigen binding proteins specifically bind to red blood cells ex vivo. In some embodiments, the antigen binding proteins have a dissociation constant between about 10 μM and 0.1 nM as determined by equilibrium binding measurements between the antigen binding protein and red blood cells.

[0130] In some embodiments, the antigen binding protein binds non-covalently to red blood cells. In some embodiments, the antigen binding protein binds non-covalently and specifically to red blood cells and does not specifically bind to other blood components. In some such embodiments, the other blood components are one or more of blood proteins, albumin, fibronectin, platelets, and / or white blood cells. In some such embodiments, the other blood components are substantially all of the components found in a blood sample taken from a typical human. With respect to a blood sample, the term "substantially all" refers to components that are normally present, but exclude concomitant components at very low concentrations, that would not otherwise effectively reduce the titer of bioavailable ligand.

[0131] Diagnostic Uses of Antigen Binding Proteins In some embodiments, the antigen binding proteins of the present invention are useful for detecting the presence of GPA in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In some embodiments, the biological sample comprises a cell or tissue. In some embodiments, a method for detecting the presence of GPA in a biological sample is provided. In some such embodiments, the method comprises contacting the biological sample with the antigen binding protein under conditions permissive for binding of the antigen binding protein to GPA, and detecting whether a complex is formed between the antigen binding protein and GPA. Such methods include, but are not limited to, antigen binding assays well known in the art, such as Western blots, radioimmunoassays, ELISA (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC).

[0132] In some embodiments, the antigen binding protein is labeled. Labels include, but are not limited to, directly detectable labels or moieties (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, radioactive labels, etc.), as well as indirectly detectable moieties, such as enzymes or ligands, e.g., moieties that are detected via an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes 32P, 14C, 125I, 3H and 131I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g., firefly luciferase and bacterial luciferase, luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated to enzymes that use hydrogen peroxide to oxidize dye precursors, such as HRP, lactoperoxidase or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0133] In some embodiments, the antigen-binding protein is immobilized on an insoluble matrix. Immobilization involves separating the antigen-binding protein described herein from GPA, which remains free in solution. This is conventionally accomplished by either insolubilizing the antigen-binding protein prior to the assay procedure, by adsorption to a water-insoluble matrix or surface, or by covalent binding (e.g., using glutaraldehyde cross-linking), or by insolubilizing the antigen-binding protein after complex formation between the antigen-binding protein and GPA, e.g., by immunoprecipitation.

[0134] Tolerogenic Compositions In some embodiments, a tolerogenic composition is provided that includes one or more antigen binding proteins as described herein and one or more tolerogenic antigens. In some embodiments, the tolerogenic composition specifically binds to erythrocytes via one or more antigen binding proteins as described herein, and the tolerogenic antigen is presented to the immune system, thereby inducing antigen-specific tolerance. As used herein, a "tolerogenic antigen" is any substance that serves as a target for a receptor of an immune response (e.g., an adaptive immune response), such as a T cell receptor, major histocompatibility complex class I and II, a B cell receptor, or an antibody. In some embodiments, the tolerogenic antigen can be derived from within the body (e.g., "self", "auto", or "endogenous"). In further embodiments, the tolerogenic antigen can be derived from outside the body ("non-self", "foreign", or "exogenous"), entering the body, for example, by inhalation, ingestion, injection, or implantation, transdermal administration, etc. In some embodiments, the exogenous antigen can be biochemically modified in the body. Foreign antigens include, but are not limited to, food antigens, animal antigens, plant antigens, environmental antigens, therapeutic agents, as well as antigens present in allografts.

[0135] In some embodiments, the tolerance-inducing compositions described herein are used for other applications, such as, for example, treatment of transplant rejection, immune responses to therapeutic agents, autoimmune diseases, and food allergies.

[0136] In some embodiments, the tolerogenic compositions described herein are used to modulate, and in particular down-regulate, unwanted antigen-specific immune responses.

[0137] In some embodiments, the tolerogenic compositions described herein are used to specifically bind and clear from circulation undesirable proteins, including antibodies (e.g., not exogenous antibodies administered to the patient), peptides, etc., produced endogenously within the patient that cause autoimmune and related pathologies, allergies, inflammatory immune responses, and anaphylaxis.

[0138] According to some embodiments, the present invention provides tolerance-inducing compositions and methods for treating unwanted immune responses to self- and foreign antigens, including but not limited to: foreign transplant antigens to which transplant recipients generate unwanted immune responses (e.g., transplant rejection), foreign antigens to which patients develop unwanted immune responses (e.g., allergies or hypersensitivity), therapeutic agents to which patients develop unwanted immune responses (e.g., hypersensitivity and / or reduced therapeutic activity), and self-antigens to which patients develop unwanted immune responses (e.g., autoimmune disease). In some embodiments, the therapeutic agents are delivered, for example, by use of gene therapy vectors. In some such embodiments, an immune response may be generated against a portion of such vectors and / or their cargo (e.g., therapeutic agents). Thus, in some embodiments, antigens to which tolerance is desired include gene therapy vectors, including but not limited to adenoviruses and adeno-associated viruses (and corresponding mutant forms -1, -2, -5, -6, -8, -9 and / or other parvoviruses), lentiviruses, and retroviruses.

[0139] Autoimmune disease conditions that may be treated using the methods and tolerance-inducing compositions of the present invention include, but are not limited to, acute disseminated encephalomyelitis (ADEM); acute interstitial allergic nephritis (drug allergy); acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; alopecia areata; alopecia universalis; ankylosing spondylitis; juvenile arthritis; psoriatic arthritis; rheumatoid arthritis; atopic dermatitis; autoimmune aplastic anemia; autoimmune gastritis; autoimmune hepatitis; autoimmune hypophysitis; autoimmune folliculitis; autoimmune orchitis; autoimmune polyendocrine syndrome type 1; autoimmune polyendocrine syndrome type 2; autoimmune thyroiditis; Behcet's disease; bronchiolitis obliterans; pemphigoid; celiac disease; Churg-Strauss syndrome;Chronic inflammatory demyelinating polyneuropathy;Cicatricial pemphigoid;Crohn's disease;Coxsackie myocarditis;Dermatitis herpetiformis of Juhring;Diabetes mellitus (type 1 diabetes);Erythema nodosum;Epidermolysis bullosa acquisita, giant cell arteritis (temporal arteritis);Giant cell myocarditis;Goodpasture's syndrome;Graves' disease;Guillain-Barré syndrome;Hashimoto's encephalitis;Hashimoto's thyroiditis;IgG4-related sclerosing disease;Lambert-Eaton syndrome;Mixed connective tissue disease;Much-Habermann disease;Multiple sclerosis;Myasthenia gravis;Optic neuritis;Neuromyelitis optica;Parkinson's disease;Pemphigus vulgaris and variants;Pernicious anemia;Pituitary autoimmune diseases;Polymyositis;Postpericardiotomy syndrome syndrome); premature ovarian failure; primary biliary cirrhosis; primary sclerosing cholangitis; psoriasis; rheumatic heart disease; Sjogren's syndrome; systemic lupus erythematosus; systemic sclerosis; ulcerative colitis; undifferentiated connective tissue disease (UCTD); uveitis; leukoplakia; and Wegener's granulomatosis.

[0140] Particular groups of autoimmune disease conditions that may be treated using the methods and tolerance-inducing compositions of the present invention include, but are not limited to, acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; psoriatic arthritis; rheumatoid arthritis; autoimmune aplastic anemia; autoimmune hypophysitis; autoimmune gastritis; autoimmune polyendocrine syndrome type 1; pemphigoid; celiac disease; Coxsackie myocarditis; Juhring's dermatitis herpetiformis; diabetes mellitus (type 1 diabetes); epidermolysis bullosa acquisita; giant cell myocarditis; Goodpasture's syndrome; Graves' disease; Hashimoto's thyroiditis; mixed connective tissue disease; multiple sclerosis; myasthenia gravis; neuromyelitis optica; Parkinson's disease; pernicious anemia; pemphigus vulgaris and variants; pituitary autoimmune diseases; premature ovarian failure; rheumatic heart disease; systemic sclerosis; Sjogren's syndrome; systemic lupus erythematosus; and vitiligo.

[0141] In embodiments using tolerogenic antigens to which an unwanted immune response occurs, such as food antigens, treatment can be provided for reactions to, for example, peanuts, apples, milk, egg whites, egg yolks, mustard, celery, shrimp, wheat (and other grains), strawberries, and bananas.

[0142] According to some embodiments, a patient can be tested to identify the foreign antigen to which an unwanted immune response was generated, and a tolerogenic composition (comprising an antigen binding protein and one or more tolerogenic antigens described herein) can be developed based on that tolerogenic antigen.

[0143] test The effect on immune regulation can be tested, for example, by measuring the proliferation of OT-I CD8+ cells (transplanted into a host mouse) in response to administration of the tolerogenic composition described herein, compared to administration of the tolerogenic antigen alone and / or vehicle. According to some embodiments, the tolerogenic composition described herein, when tested in this manner, shows increased OT-I cell proliferation and increased CD8+ T cell cross-priming, which is indicative of induction of immune tolerance, compared to the tolerogenic antigen alone or vehicle. To distinguish T cells that have been expanded to a functional effector phenotype from those that have been expanded and deleted, the proliferating OT-I CD8+ T cells can be phenotyped for molecular hallmarks of exhaustion [programmed cell death-1 (PD-1), FasL, etc.], and Annexin-V binding as a hallmark of apoptosis and therefore deletion. OT-I CD8+ T cells can also be evaluated for responsiveness to tolerogenic antigen exposure with adjuvants to demonstrate functional non-responsiveness to the tolerogenic antigen, and therefore immune tolerance. To this end, cells are analyzed for inflammatory indicators after administration of the tolerogenic composition to the host mouse followed by antigen challenge. According to some embodiments, the tolerogenic compositions described herein, when tested in this manner, exhibit significantly lower levels (e.g., background) of inflammatory OT-I CD8+ T cell responses to OVA compared to control groups, thus indicating immune tolerance.

[0144] According to some embodiments, humoral immune responses can be tested by administering a tolerogenic antigen composition as described herein, comprising one or more tolerogenic antigens as described herein, and measuring the levels of antibodies obtained, compared to administering the tolerogenic antigen alone or vehicle alone. According to some embodiments, when tested in this manner, the tolerogenic compositions of the invention show significantly lower (e.g., background) levels of antibody formation in response to their administration and to vehicle administration, and show significantly higher levels of antibody formation in response to administering the tolerogenic antigen alone. Reduced antibody formation is indicative of induction of immune tolerance.

[0145] The effectiveness of tolerization to a tolerogenic antigen can be tested as described above with reference to humoral immune responses, where a group of subjects is challenged with the tolerogenic antigen alone several weeks after treatment(s) with a tolerogenic composition as described herein, and the level of antibodies to the tolerogenic antigen is then measured. According to some embodiments, the tolerogenic compositions of the invention when tested in this manner show lower levels of antibody formation in response to challenge with the tolerogenic antigen in groups pretreated with such tolerogenic compositions compared to groups that have not been pretreated.

[0146] Disease-focused experimental models include, but are not limited to, the NOD (or non-obese diabetic) mouse model of autoimmunity and tolerance of type 1 diabetes, and the EAE (experimental autoimmune encephalomyelitis) model of multiple sclerosis, a human inflammatory demyelinating disease. According to some embodiments, such models can demonstrate effective induction of tolerance using the tolerance-inducing compositions described herein.

[0147] Administration According to some embodiments, the tolerance-inducing compositions of the present invention are administered in a therapeutically effective dose, e.g., a dose sufficient to provide treatment for the disease conditions described above. Administration of the tolerance-inducing compositions of the present invention, or pharma- ceutically acceptable salts thereof, can be via any of the accepted modes of administration of agents that provide similar utilities.

[0148] Although human dosage levels have yet to be optimized for the tolerogenic compounds of the present invention, they may be administered initially at a dose of about 10 μg to about 100 μg in mice. In general, individual human doses are about 0.01 to 20.0 mg / kg, preferably about 0.1 to 10 mg / kg, and most preferably about 0.3 to 2.0 mg / kg of body weight. Treatments may be administered for one or several days and may be repeated at intervals of several days, one to several weeks, or one to several months. Administration may be as a single dose (e.g., as a bolus), or an initial bolus followed by continuous infusion of the remainder of the full dose over time, e.g., for one to seven days. Dosages of active compounds may vary depending on any or all of the following: the subject and condition to be treated, the severity of the affliction, the method and schedule of administration, and the judgment of the prescribing physician. It may be understood that dosages may also vary depending on the molecular weight of the tolerogenic antigen, antibody, antibody fragment (or other antigen-binding fragments described herein) as well as the size of the linker.

[0149] The tolerance-inducing compositions of the present invention may be administered alone or in combination with other pharma- ceutically acceptable excipients. Common routes of administration used in accordance with the embodiment include, but are not limited to, oral, topical, transdermal, injection (intramuscular, intravenous or intraarterial), etc. Depending on the embodiment, the formulation may contain, if necessary, a conventional pharmaceutical carrier or excipient and the tolerance-inducing composition of the present invention or a pharma-ceutically acceptable salt thereof. In addition, these tolerance-inducing compositions may contain other drugs, pharmaceutical agents, carriers, etc., including, but not limited to, therapeutic proteins, peptides, antibodies or antibody-like molecules corresponding to the antigens used in the tolerance-inducing compositions of the present invention, as well as other active agents that may act as immunomodulators, more specifically, folate antagonists, immunosuppressants, cryosuppressants, mitotic inhibitors, and antimetabolites, or combinations thereof, and may have an inhibitory effect on B cells.

[0150] In general, depending on the intended method of administration, a pharma- ceutically acceptable composition will contain from about 0.1% to about 95% by weight of the tolerance-inducing composition of the present invention, with the remainder being suitable pharmaceutical excipients, carriers, etc. For example, in some embodiments, a pharma- ceutically acceptable composition will contain the tolerance-inducing composition described herein in an amount of from about 0.1% to about 1% by weight, from about 1% to about 5% by weight, from about 5% to about 15% by weight, from about 15% to about 25% by weight, from about 25% to about 50% by weight, from about 50% to about 75% by weight, from about 75% to about 95% by weight (including any amount between the ranges recited, including the end points). Dosage forms or compositions containing active ingredient in the range of 0.005% to about 95% can be prepared, with the balance being made up of a non-toxic carrier. For example, in some embodiments, the dosage form may contain from about 0.005% to about 0.01%, from about 0.01% to about 0.05%, from about 0.05% to about 0.1%, from about 0.1% to about 1%, from about 1% to about 5%, from about 5% to about 15%, from about 15% to about 25%, from about 25% to about 50%, from about 50% to about 75%, or from about 75% to about 95% (including any amount between the recited ranges, inclusive of the endpoints) of active ingredient.

[0151] Liquid pharma- ceutically administrable tolerance-inducing compositions can be prepared, for example, by dissolving, dispersing, etc., the active tolerance-inducing composition of the invention (e.g., lyophilized powder) and, optionally, pharmaceutical adjuvants in a carrier, such as water (water for injection), saline, aqueous dextrose, glycerol, glycol, ethanol, and the like (except galactose), thereby forming a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting agents, emulsifiers, stabilizers, solubilizers, pH buffers, and the like, osmolytes, such as sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, and triethanolamine oleate, amino acids, sugars and carbohydrates, proteins and polymers, salts, surfactants, chelating agents and antioxidants, preservatives, and specific ligands, and the like.

[0152] Tolerogenic antigens The tolerogenic antigens used in the tolerogenic compositions comprising the antigen binding proteins described herein may be proteins or peptides, for example, the tolerogenic antigen may be a full or partial therapeutic agent, a full length transplantation protein or peptide thereof, a full length autoantigen or peptide thereof, a full length allergen or peptide and / or nucleic acid, or a mimetic of said tolerogenic antigens. The listing of a particular tolerogenic antigen in association with one category or a particular disease or response does not exclude that tolerogenic antigen from being considered part of another category or from being associated with another disease or response.

[0153] The tolerogenic antigens used in the practice of the present invention may be one or more of the following: Therapeutic agents that are proteins, peptides, antibodies and antibody-like molecules, including antibody fragments and fusion proteins of antibodies and antibody fragments, and vectors for gene therapy. These include human, non-human (such as mouse) and non-natural (i.e., man-made) proteins, antibodies, chimeric antibodies, humanized antibodies, viruses and virus-like particles, and non-antibody binding scaffolds such as fibronectin, DARPins, knottins, etc. · Human allograft antigens to which transplant recipients may generate undesirable immune responses. Autoantigens that trigger unwanted autoimmune responses. Autoantigens are of endogenous origin in a patient (e.g., an autoimmune patient), although the polypeptides used in the tolerogenic compositions of the invention may, in some embodiments, be exogenously synthesized (as opposed to purified and concentrated from a source). Foreign antigens, such as food antigens, animal antigens, plant antigens and environmental antigens, to which the patient experiences an undesirable immune response. Therapeutic proteins may also be considered foreign antigens due to their exogenous origin, but for purposes of clarity of description of the present invention, such therapeutics are described as separate groups. Similarly, plant or animal antigens may be eaten and considered food antigens, and environmental antigens may be derived from plants. However, these are considered foreign antigens. For simplicity, no attempt is made to separately describe and define all of such potentially overlapping groups, since the skilled artisan will be able to understand the tolerogenic antigens that may be used in the tolerogenic compositions of the present invention, especially in light of the detailed description and examples.

[0154] The tolerogenic antigen may be a complete protein, a portion of a complete protein, a peptide, etc., may be derivatized (as described above) for attachment to a linker and / or antigen binding site, may contain mutations and / or conservative substitutions, in particular may maintain sequence identity, and / or may be desialylated.

[0155] In embodiments in which the tolerogenic antigen is a therapeutic protein, peptide, antibody, or antibody-like molecule, specific tolerogenic antigens include, but are not limited to, abatacept, abciximab, adalimumab, adenosine deaminase, adotrastuzumab emtansine, agalsidase alfa, agalsidase beta, aldeslukin, alglucerase, alglucosidase alfa, alpha-1-proteinase inhibitors, anakinra, anistreplase (Abatacept), ... abatacept, abatacept, abatacept, abatacept Nisoylated plasminogen streptokinase activator complex, antithrombin III, antithymocyte globulin, Ateplase, Bevacizumab, Bivalirudin, Botulinum toxin type A, Botulinum toxin type B, C1-esterase inhibitors, Canakinumab, Carboxypeptidase G2 (Glucarpidase, Voraxaze), Certolizumab pegol, Cetuximab, Collagenase, Crotalidae immune Fab Fab), Darbepoetin-α, Denosumab, Digoxin Immune Fab, Dornase alfa, Eculizumab, Etanercept, Factor VIIa, Factor VIII, Factor IX, Factor XI, Factor XIII, Fibrinogen, Filgrastim, Galsulfase, Golimumab, Histrelin acetate, Hyaluronidase, Idursulphase, Imiglucerase, Infliximab, Insulin [including recombinant human insulin ("rHu insulin") and bovine insulin], interferon-alpha 2a, interferon-alpha 2b, interferon-beta 1a, interferon-beta 1b, interferon-gamma 1b, ipilimumab, L-arginase, L-asparaginase, L-methionase, lactase, laronidase, lepirudin / hirudin, mecasermin, mecasermin rinfabaterinfabate), methoxynatalizumab, octreotide, ofatumumab, oprelvekin, pancreatic amylase, pancreatic lipase, papain, PEG-asparaginase, PEG-doxorubicin HCl, PEG-epoetin-β, pegfilgrastim, PEG-interferon-α2a, PEG-interferon-α2b, pegloticase, pegvisomant, phenylalanine ammonia-lyase (PAL), protein C, rasburicase (uricase), sacrosidase, salmon calcitonin, sargramostim, streptokinase, tenecteplase, teriparatide, tocilizumab (atlizumab), trastuzumab, type 1 alpha-interferon, ustekinumab, vW factor. Therapeutic proteins can be obtained from natural sources (e.g., enriched and purified) or synthetic, e.g., recombinantly obtained, and include antibody therapeutics, which are generally IgG monoclonals or fragments or fusions.

[0156] Particular therapeutic proteins, peptides, antibodies or antibody-like molecules include, but are not limited to, abciximab, adalimumab, agalsidase alfa, agalsidase beta, aldeslukin, alglucosidase alfa, factor VIII, factor IX, infliximab, insulin (including rHu insulin), L-asparaginase, laronidase, natalizumab, octreotide, phenylalanine ammonia lyase (PAL) or rasburicase (uricase), and typically IgG monoclonal antibodies thereof in their various forms.

[0157] Some embodiments utilize hemostatic agents (eg, Factors VIII and IX), insulin (including rHu insulin), and the non-human therapeutic agents uricase, PAL, and asparaginase.

[0158] In some embodiments, therapeutic agents are delivered, for example, by using gene therapy vectors. In some such embodiments, immune responses can be generated against parts of such vectors and / or their cargo (e.g., therapeutic agents). Thus, in some embodiments, antigens for which tolerance is desired include gene therapy vectors, including, but not limited to, adenoviruses and adeno-associated viruses (and corresponding mutants-1, -2, -5, -6, -8, -9 and / or other parvoviruses), lentiviruses and retroviruses.

[0159] Undesirable immune responses in hematology and transplantation include autoimmune aplastic anemia, transplant rejection (generally) and graft-versus-host disease (bone marrow transplant rejection). In embodiments where the tolerance-inducing antigen is a human allograft transplantation antigen, the specific sequence can be selected from the following: subunits of various MHC class I and MHC class II haplotype proteins (e.g., donor / recipient differences identified in tissue cross-matching), and single amino acid polymorphisms on minor blood group antigens, including RhCE, Kell, Kidd, Duffy and Ss. Such tolerance-inducing compositions can be individually prepared for a given donor / recipient pair.

[0160] In embodiments in which the tolerogenic antigen is an autoantigen, the specific tolerogenic antigens (and the autoimmune diseases with which they are associated) may be selected.

[0161] In type 1 diabetes, antigens include, but are not limited to, insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6 phosphatase 2 (IGRP or islet-specific glucose-6 phosphatase catalytic subunit-related protein), insulinoma-associated protein 2 (IA-2) and insulinoma-associated protein 2β (IA-2β). Other antigens include ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, islet-specific glucose-6-phosphatase catalytic subunit-associated protein, and SST G protein-coupled receptor 1-5, or immunogenic fragments or portions of any of such antigens. It is noted that insulin is an example of an antigen that can be characterized as both an autoantigen and a therapeutic protein antigen. For example, rHu insulin and bovine insulin are therapeutic protein antigens (subjects of unwanted immune attack), whereas endogenous human insulin is an autoantigen (subject of unwanted immune attack). Since endogenous human insulin cannot be used in pharmaceutical compositions, in certain embodiments of the tolerance-inducing compositions of the present invention, recombinant forms are used.

[0162] In some embodiments, human insulin, including exogenously derived forms, useful in the tolerance-inducing compositions of the present invention has the following sequence (UNIPROT P01308): MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 163).

[0163] In some embodiments, GAD-65, including exogenously derived forms, useful in the tolerance-inducing compositions of the invention has the following sequence (UNIPROT Q05329):MASPGSGFWSFGSEDGSGDSENPGTARAWCQVAQKFTGGIGNKLCALLYGDAEKPAESGGSQPPRAAARKAACACDQKPCSCSKVDVNYAFLHATDLLPACDGERPTLAFLQDVMNILLQYVVKSFDRSTKVIDFHYPNELLQEYNWELADQPQNLEEILMHCQTTLKYAIKTGHPRYFNQLSTGLDMVGLAADWLTSTANTNMFTYEIAPVFVLLEYVTLKKMREIIGWPGGSGDGIFSPGGAISNMYAMMIARFKMFPEVKEKGMAALPRLIAFTSEHSHFSLKKG AAALGIGTDSVILIKCDERGKMIPSDLERRILEAKQKGFVPFLVSATAGTTVYGAFDPLLAVADICKKYKIWMHVDAAWGGGLLMSRKHKWKLSGVERANSVTWNPHKMMGVPLQCSALLVREEGLMQNCNQMHASYLFQQDKHYDLSYDTGDKALQCGRHVDVFKLWLMWRAKGTTGFEAHVDKCLELAEYLYNIIKNREGYEMVFDGKPQHTNVCFWYIPPSLRTLEDNEERMSRLSKVAPVIKARMMEYGTTMVSYQPLGDKVNFFRMVISNPAATHQDIDFLIEEIERLGQDL (SEQ ID NO: 164).

[0164] In some embodiments, an IGRP, including an exogenously derived form, useful in the tolerance-inducing compositions of the invention has the following sequence (UNIPROT QN9QR9):MDFLHRNGVLIIQHLQKDYRAYYTFLNFMSNVGDPRNIFFIYFPLCFQFNQTVGTKMIWVAVIGDWLNLIFKWILFGHRPYWWVQETQIYPNHSSPCLEQFPTTCETGPGSPSGHAMGASCVWYVMVTAALSHTVCGMDKFSITLHRLTWSFLWSVFWLIQISVCISRVFIATHFPHQVILGVIGGMLVAEAFEHTPGIQTASLGTYLKTNLFLFLFAVGFYLLLRVLNIDLLWSVPIAKKWCANPDWIHIDTTPFAGLVRNLGVLFGLGFAINSEMFLLSCRGGNNYTLSFRLLCALTSLTILQLYHFLQIPTHEEHLFYVLSFCKSASIPLTVVAFIPYSVHMLMKQSGKKSQ (SEQ ID NO: 165).

[0165] In some embodiments, human proinsulin, including exogenously derived forms, useful in the tolerance-inducing compositions of the present invention has the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 203).

[0166] Depending on the embodiment, peptides / epitopes useful in the tolerogenic compositions of the invention for treating type 1 diabetes include some or all of the following sequences, either individually in the tolerogenic composition or together in a cocktail of tolerogenic compositions: Human proinsulin 1-70: FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQ (SEQ ID NO: 204); Human proinsulin 9-70: SHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQ (SEQ ID NO: 205); Human proinsulin 9-38: SHLVEALYLVCGERGFFYTPKTRREAEDLQ (SEQ ID NO: 206); Human proinsulin 1-38: FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQ (SEQ ID NO: 207); Human proinsulin 9-23: SHLVEALYLVCGERG (SEQ ID NO: 208); Human proinsulin 45-71 (C13-A6): GGGPGAGSLQPLALEGSLQKRGIVEQC (SEQ ID NO: 209); Human proinsulin C24-A1: LALEGSLQKRG (SEQ ID NO: 210); Human proinsulin C19-A3: GSLQPLALEGSLQKRGIV (SEQ ID NO: 211); Human proinsulin C13-32: GGGPGAGSLQPLALEGSLQK (SEQ ID NO: 212); Human proinsulin B9-C4: SHLVEALYLVCGERGFFYTPKTRREAED (SEQ ID NO: 213); Human proinsulin C22-A5: QPLALEGSLQKRGIVEQ (SEQ ID NO: 214).

[0167] In autoimmune diseases of the thyroid gland, including Hashimoto's thyroiditis and Graves' disease, the major antigens include, but are not limited to, thyroglobulin (TG), thyroid peroxidase (TPO), and thyroid-stimulating hormone receptor (TSHR). Other antigens include sodium iodide symporter (NIS) and megalin. In thyroid-associated ophthalmopathy and dermatosis, in addition to thyroid autoantigens including TSHR, the antigen is insulin-like growth factor 1 receptor. In hypoparathyroidism, the major antigen is the calcium-sensing receptor.

[0168] In Addison's disease, the major antigens include, but are not limited to, 21-hydroxylase, 17α-hydroxylase, and P450 side-chain cleavage enzyme (P450scc). Other antigens include ACTH receptor, P450c21, and P450c17.

[0169] In premature ovarian failure, the major antigens include, but are not limited to, the FSH receptor and alpha-enolase.

[0170] In autoimmune hypophysitis or autoimmune diseases of the pituitary gland, major antigens include, but are not limited to, pituitary-specific protein factors (PGSF) 1a and 2. Another antigen is type 2 iodothyronine deiodinase.

[0171] In multiple sclerosis, major antigens include, but are not limited to, myelin basic protein ("MBP"), myelin oligodendrocyte glycoprotein ("MOG"), and myelin proteolipid protein ("PLP").

[0172] MBP, including exogenously derived forms, useful in the tolerance-inducing compositions of the present invention has the following sequence (UNIPROT P02686):MGNHAGKRELNAEKASTNSETNRGESEKKRNLGELRTTSEDNEVFGEADANQNNGTSSQDTAVTDSKRTADPKNAWQDAHPADPGSRPHLIRLFSRDAPGREDNTFKDRPSESDELQTIQEDSAATSESLDVMASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKDSHHPARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLGGRDSRSGSPMARR (SEQ ID NO: 166).

[0173] MOG, including exogenously derived forms, useful in the tolerance-inducing compositions of the present invention has the following sequence (UNIPROT Q16653):MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPGVLVLLAVLPVLLLQITVGLIFLCLQYRLRGKLRAEIENLHRTFDPHFLRVPCWKITLFVIVPVLGPLVALIICYNWLHRRLAGQFLEELRNPF (SEQ ID NO: 167).

[0174] PLP, including exogenously derived forms, useful in the tolerance-inducing compositions of the present invention has the following sequence (UNIPROT P60201):MGLLECCARCLVGAPFASLVATGLCFFGVALFCGCGHEALTGTEKLIETYFSKNYQDYEYLINVIHAFQYVIYGTASFFFLYGALLLAEGFYTTGAVRQIFGDYKTTICGKGLSATVTGGQKGRGSRGQHQAHSLERVCHCLGKWLGHPDKFVGITYALTVVWLLVFACSAVPVYIYFNTWTTCQSIAFPSKTSASIGSLCADARMYGVLPWNAFPGKVCGSNLLSICKTAEFQMTFHLFIAAFVGAAATLVSLLTFMIAATYNFAVLKLMGRGTKF (SEQ ID NO: 168).

[0175] Peptides / epitopes useful in the tolerogenic compositions of the invention for treating multiple sclerosis include some or all of the following sequences, either individually in the tolerogenic composition or together in combinations (e.g., cocktails) of the tolerogenic composition: · MBP13-32: KYLATASTMDHARHGFLPRH (SEQ ID NO: 169); · MBP83-99: ENPWHFFKNIVTPRTP (SEQ ID NO: 170); · MBP111-129: LSRFSWGAEGQRPGFGYGG (SEQ ID NO: 171); · MBP146-170: AQGTLSKIFKLGGRDSRSGSPMARR (SEQ ID NO: 172); · MOG1-20: GQFRVIGPRHPIRALVGDEV (SEQ ID NO: 173); · MOG35-55:MEVGWYRPPFSRWHLYRNGK (SEQ ID NO:174); · PLP139-154:HCLGKWLGHPDKFVGI (SEQ ID NO: 175); · MOG35-55:MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 186); · MOG30-60: KNATGMEVGWYRSPFSRVVHLYRNGKDQDAE (SEQ ID NO: 187); · MBP83-99: ENPVVHFFKNIVTPRTP (SEQ ID NO: 188); · MOG35-55:MEVGWYRPPFSRVVHLYRNGK (SEQ ID NO: 189); MBP82-98: DENPVVHFFKNIVTPRT (SEQ ID NO: 190); · MBP82-99: DENPVVHFFKNIVTPRTP (SEQ ID NO: 191); MBP82-106: DENPVVHFFKNIVTPRTPPPSQGKG (SEQ ID NO: 192); MBP87-106: VHFFKNIVTPRTPPPSQGKG (SEQ ID NO: 193); · MBP131-155: ASDYKSAHKGLKGVDAQGTLSKIFK (SEQ ID NO: 194); · PLP41-58: GTEKLIETYFSKNYQDYE (SEQ ID NO: 195); · PLP89-106: GFYTTGAVRQIFGDYKTT (SEQ ID NO: 196); · PLP95-116: AVRQIFGDYKTTICGKGLSATV (SEQ ID NO: 197); · PLP178-197: NTWTTCQSIAFPSKTSASIG (SEQ ID NO: 198); ·PLP190-209:SKTSASIGSLCADARMYGVL (sequence number 199); · MOG11-30:PIRALVGDEVELPCRISPGK (sequence number 200); · MOG21-40:ELPCRISPGKNATGMEVGWY (SEQ ID NO:201); MOG64-86:EYRGRTELLKDAIGEGKVTLRIR (sequence number 202).

[0176] In rheumatoid arthritis, major antigens include, but are not limited to, collagen II, immunoglobulin binding proteins, fragment crystallizable regions of immunoglobulin G, double-stranded DNA, and native and citrullinated forms of proteins involved in the pathology of rheumatoid arthritis, including fibrin / fibrinogen, vimentin, collagen I and II, and alpha-enolase.

[0177] In autoimmune gastritis, the major antigen is H + , K + -ATPase.

[0178] In pernicious anemia, the major antigen is intrinsic factor.

[0179] In celiac disease, the main antigens include, but are not limited to, tissue transglutaminase and native and deamidated forms of gluten or gluten-like proteins, such as alpha-, gamma- and omega-gliadins, glutenins, hordeins, secalins and avenins.Those skilled in the art can understand that, for example, the main antigen in celiac disease is alpha-gliadins, but alpha-gliadins are modified in the body to become more immunogenic through deamidation by tissue transglutaminase, which converts the glutamine of alpha-gliadins to glutamic acid.Thus, alpha-gliadins are originally foreign food antigens, but when modified in the body to become more immunogenic, they can be characterized as self-antigens in some aspects.

[0180] In vitiligo, the major antigens are tyrosinase, and tyrosinase-related proteins 1 and 2.

[0181] MART1, a melanoma antigen recognized by T cell 1, Melan-A, including exogenously derived forms useful in the tolerance-inducing compositions of the present invention, has the following sequence (UNIPROT Q16655): MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPPYSP (SEQ ID NO: 176).

[0182] Tyrosinase, including exogenously derived forms, useful in the tolerance-inducing compositions of the present invention has the following sequence (UNIPROT P14679): MLLAVLYCLLWSFQTSAGHFPRACVSSKNLMEKECCPPWSGDRSPCGQLSGRGSCQNILLSNAPLGPQFPFTGVDDRESWPSVFYNRTCQCSGNFMGFNCGNCKFGFWGPNCTERRLLVRRNIFDLSAPEKDKFFAYLTLAKHTISSDYVIPIGTYGQMKNGSTPMFNDINIYDLFVWMHYYVSMDALLGGSEIWRDIDFAHEAPAFLPWHRLFLLRWEQEIQKLTGDENFTIPYWDWRDAEKCDICTDEYMGGQHPTNP NLLSPASFFSSWQIVCSRLEEYNSHQSLCNGTPEGPLRRNPGNHDKSRTPRLPSSADVEFCLSLTQYESGSMDKAANFSFRNTLEGFASPLTGIADASQSSMHNALHIYMNGTMSQVQGSANDPIFLLHHAFVDSIFEQWLRRHRPLQEVYPEANAPIGHNRESYMVPFIPLYRNGDFFISSKDLGYDYSYLQDSDPDSFQDYIKSYLEQASRIWSWLLGAAMVGAVLTALLAGLVSLLCRHKRKQLPEEKQPLLMEKEDYHSLYQSHL (SEQ ID NO: 177).

[0183] The melanocyte protein PMEL, gp100, including exogenously derived forms useful in the tolerance-inducing compositions of the present invention, has the following sequence (UNIPROT P40967):MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQ VPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRLMKQDFSVPQLPHSSSHWLRLPRIFCSCPIGENSPLLSGQQV (SEQ ID NO: 178).

[0184] In myasthenia gravis, the primary antigen is the acetylcholine receptor.

[0185] In pemphigus vulgaris and variants, major antigens include, but are not limited to, desmogleins 3, 1 and 4. Other antigens include penfaxin, desmocollin, plakoglobin, purplakin, desmoplakin and the acetylcholine receptor.

[0186] In pemphigoid, major antigens include, but are not limited to, BP 180 and BP 230. Other antigens include plectin and laminin 5.

[0187] In Duhring's dermatitis herpetiformis, major antigens include, but are not limited to, endomysial and tissue transglutaminase.

[0188] In epidermolysis bullosa acquisita, the major antigen is collagen VII.

[0189] In systemic sclerosis, major antigens include, but are not limited to, matrix metalloproteinases 1 and 3, collagen-specific molecular chaperone heat shock protein 47, fibrillin-1, and PDGF receptor. Other antigens include Scl-70, U1 RNP, Th / To, Ku, Jo1, NAG-2, centromere proteins, topoisomerase I, nucleolar proteins, RNA polymerase I, II, and III, PM-Slc, fibrillarin, and B23.

[0190] In mixed connective tissue disease, the major antigen is U1snRNP.

[0191] In Sjogren's syndrome, major antigens include, but are not limited to, nuclear antigens SS-A and SS-B. Other antigens include fodrin, poly(ADP-ribose) polymerase and topoisomerase, muscarinic receptors and Fc-gamma receptor IIIb.

[0192] In systemic lupus erythematosus, major antigens include, but are not limited to, nuclear proteins including the "Smith antigen", SS-A, high mobility group box 1 (HMGB1), nucleosomes, histone proteins and double-stranded DNA (against which autoantibodies are made during the disease process).

[0193] In Goodpasture's syndrome, major antigens include, but are not limited to, glomerular basement membrane proteins, including collagen IV.

[0194] In rheumatic heart disease, the major antigen is cardiac myosin.

[0195] Autoimmune polyendocrine syndrome type 1 antigens include, but are not limited to, aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine ​​sulfinic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450 cytochromes P4501A2 and 2A6, SOX-9, SOX-10, calcium-sensing receptor protein, and type 1 interferons, interferon alpha, beta, and gamma.

[0196] In neuromyelitis optica, the major antigen is AQP4.

[0197] Aquaporin-4, including exogenously derived forms, useful in the tolerance-inducing compositions of the present invention has the following sequence (UNIPROT P55087): MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV (SEQ ID NO: 179).

[0198] In uveitis, major antigens include, but are not limited to, retinal S-antigen or "S-arrestin" and interphotoreceptor retinoid binding protein (IRBP) or retinol binding protein 3.

[0199] S-arrestin, including exogenously derived forms, useful in the tolerance-inducing compositions of the present invention has the following sequence (UNIPROT P10523):MAASGKTSKSEPNHVIFKKISRDKSVTIYLGNRDYIDHVSQVQPVDGVVLVDPDLVKGKKVYVTLTCAFRYGQEDIDVIGLTFRRDLYFSRVQVY PPVGAASTPTKLQESLLKKLGSNTYPFLLTFPDYLPCSVMLQPAPQDSGKSCGVDFEVKAFATDSTDAEEDKIPKKSSVRLLIRKVQHAPLEMGPQPRAEAAW QFFMSDKPLHLAVSLNKEIYFHGEPIPVTVTVTNNTEKTVKKIKAFVEQVANVVLYSSDYYVKPVAMEEAQEKVPPNSTLTKTLTLLPLLANNRERRGIALDG KIKHEDTNLASSTIIKEGIDRTVLGILVSYQIKVKLTVSGFLGELTSSEVATEVPFRLMHPQPEDPAKESYQDANLVFEEFARHNLKDAGEAEEGKRDKNDVDE (Sequence number 180).

[0200]

[0201] In embodiments where the tolerogenic antigen is a foreign antigen against which an unwanted immune response may be generated, e.g., a food antigen, specific antigens include, but are not limited to, the following: · From peanut: conaratin (Ara h 1), allergen II (Ara h 2), peanut agglutinin, conglutinin (Ara h 6); Conarachin, for example, has the sequence identified as UNIPROT Q6PSU6. · From apple: Major allergen / disease resistance protein homologue of 31 kda (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1); · From milk: alpha-lactalbumin (ALA), lactotransferrin; from kiwi: actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), kiwellin (Act d 5); · From egg white: ovomucoid, ovalbumin, ovotransferrin and lysozyme; From egg yolk: livetin, apovitilin and bosvetin; · From mustard: 2S albumin (Sin a 1), 11S globulin (Sin a 2), lipid transfer protein (Sin a 3), profilin (Sin a 4); · From celery: profilin (Api g 4), a high molecular weight glycoprotein (Api g 5); From shrimp: Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen m 2), fast tropomyosin isoform; From wheat and / or other cereals: high molecular weight glutenins, low molecular weight glutenins, alpha-, gamma- and omega-gliadins, hordeins, secalins and / or avenins; Peptides / epitopes useful in the tolerogenic composition of the invention for treating celiac disease include some or all of the following 2-5, 5-10, 10-15, 15-20 (and overlapping ranges therein) or more sequences, either individually in the tolerogenic composition or together in a combination (e.g. cocktail) of a tolerogenic composition: - DQ-2 related, alpha-gliadin “33-mer” native form: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 182) - DQ-2 related, alpha-gliadin “33-mer” deamidation: LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO: 183) - DQ-8 related, alpha-gliadin: QQYPSGQGSFQPSQQNPQ (SEQ ID NO:184) - DQ-8 related, omega-gliadin (wheat, U5UA46): QPFPQPEQPFPW (SEQ ID NO:185) -alpha-gliadin “15mer” fragment: ELQPFPQPELPYPQP (SEQ ID NO:215) Strawberry-derived: Major strawberry allergen Fra a 1-E (Fra a 1); and · From banana: Profilin (Mus xp 1).

[0202] In Parkinson's disease, the major antigen is alpha synuclein. Alpha synuclein, including exogenously derived forms, useful in the tolerance-inducing compositions of the present invention has the following sequence (UNIPROT P37840): MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 216).

[0203] In embodiments where the tolerogenic antigen is a foreign antigen against which an unwanted immune response is generated, such as animal antigens, plant antigens and environmental antigens, the specific antigen may be, for example: cat, mouse, dog, horse, bee, dust, tree and goldenrod, including proteins or peptides derived from: · Weeds (including ragweed allergens amb a 1, 2, 3, 5, 6, and Amb t 5; ragweed Che a 2, 5; and other weed allergens Par j 1, 2, 3, and Par o 1). Grass (contains the major allergens Cyn d 1, 7 and 12; Dac g 1, 2 and 5; Hol I 1.01203; Lol p 1, 2, 3, 5 and 11; Mer a 1; Pha a 1; Poa p 1 and 5). Pollen from ragweed and other weeds (including curly dock, lambs quarters, pigweed, plantain, sheep sorrel and sagebrush), grasses (including Bermuda, Johnson, Kentucky, orchard, sweet vernal and timothy grass), trees (including catalpa, elm, hickory, olive, pecan, sycamore and walnut); The major allergens from the Dermatophagoides pteronyssinus species, such as dust (Der p 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 18, 20, 21 and 23); the major allergens from the Dermatophagoides farina species, such as Der f 1, 2, 3, 6, 7, 10, 11, 13, 14, 15, 16, 18, 22 and 24; the major allergens from the Blomia tropicalis species, such as Blo t 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 19 and 21; and the allergens from Euroglyphus maynei, such as Tyrophagus putrescentiae), and allergens Bla g 1, 2 and 4; Per a 1, 3 and 7 from the cockroach); · pets (including cats, dogs, rodents and livestock; major cat allergens include Fel d 1-8, feline IgA, BLa g 2 and feline albumin; major dog allergens include Can f 1-6 and dog albumin); Bee stings, which contain the main allergens API m 1-12, and Fungi (including allergens from Aspergillus and Penicillium species, as well as Alternaria alternata, Davidiella tassiana and Trichophyton rubrum species).

[0204] As will be appreciated by those skilled in the art, patients can be tested to identify the tolerogenic antigen against which an unwanted immune response occurred, and proteins, peptides, etc. can be developed based on that tolerogenic antigen and incorporated into the tolerogenic compositions of the present invention.

[0205] The methods and materials used in the non-limiting examples described below should not be read as limiting the embodiments described herein. EXAMPLES

[0206] Example 1: Humanization of the murine antibody 10F7 An anti-human glycophorin A (GPA) antibody (10F7) isolated from a mouse (Bigbee et al., 1983) was selected as a candidate for erythrocyte targeting. 10F7 binds to 100% of the human population, regardless of blood type. Before affinity maturing 10F7 to improve binding properties, the antibody was first converted to an antibody fragment form and humanized. The heavy and light chains of the mouse 10F7 antibody were humanized by grafting mouse complementarity determining regions (CDRs) and human framework regions (FRs), generating 10 candidate antibody sequences as Fab antibody fragments ("fAbs"). Back mutations were introduced to maintain structure and affinity. These fAb antibodies were expressed, purified, and evaluated for binding to human GPA by kinetic binding assays (ELISA, surface plasmon resonance) and for binding to human erythrocytes by flow cytometry. Figure 1A shows the constant and variable light and heavy chains of a selected antibody sequence (called "clone m10") that was selected for subsequent affinity maturation. The candidate GPA binding residues that were mutated in the affinity maturation campaign of Example 2 are shown in bold and underlined.

[0207] Example 2: 10F7 affinity maturation campaign Library Design and Construction Computational structure-based modeling using the Paratome algorithm was used to identify putative antigen-binding regions (ABRs) of 10F7, which are amino acids in the CDRs believed to make physical contact with human GPA. Figure 1B shows the locations of the light and heavy chain complementarity determining regions of the variable region, as well as the Paratome ABRs, according to the scheme of Kabat and Clothia. These ABRs were aligned with the CDRs, and the identified ABR residues in the light and heavy chains were randomized in five separate libraries to affinity mature 10F7 (Figure 1C). Phage display libraries for affinity maturation screening were created by GeneArt / ThermoFisher Scientific using TRIM (trinucleotide mutagenesis) - an assembly of preassembled trinucleotide blocks for customizable randomization of libraries.

[0208] Selection Process The resulting fAb library was displayed on M13 bacteriophage and subjected to three rounds of selection against recombinant biotinylated human GPA previously bound to streptavidin-coated magnetic beads. To facilitate the selection of fAb antibody clones with increased affinity for GPA, the intensity of the selection process was increased in each successive round of selection by decreasing the GPA concentration and the library bead binding time. After the third round of selection, the selected library output was screened by ELISA against human GPA to identify individual fAb clones and characterize their GPA-binding properties. Clones that bound to GPA were sequenced, focusing primarily on the light chain variable region of the fAb. A schematic of the selection process is shown in Figure 2.

[0209] A total of 14 campaigns were completed and the total number of hits identified from each library is shown in Table 5. An initial review of the libraries confirmed that the L1+L2 CDR library (containing mutations in the light chain CDR1 and CDR2 ABRs) exhibited high affinity for human GPA. Figure 3 shows the sequence of the light chain variable domain of the affinity matured fAb (all other domains are the same as wild type 10F7-M10) with the randomized ABR residues in CDR1 and CDR2 in bold and underlined.

[0210] [Table 11]

[0211] Characterization and Ranking Sequence-verified clones were recombinantly expressed in soluble form (not fused to phage) in HEK cells and purified. All clones were assessed by analytical SEC to ensure that the Fabs were >97% monomeric prior to characterization. The affinity of the top hits for human GPA was characterized by ELISA, biolayer interferometry, surface plasmon resonance and flow cytometry assays on human erythrocytes. The starting (non-affinity matured 10F7-M10) was also characterized as a control. The results of this analysis are shown in Table 6, and in particular clones L1+2-17 and L1+2-39 showed unexpectedly high affinity for human GPA.

[0212] [Table 12] [Table 13]

[0213] Example 3: Next generation sequencing-based affinity maturation overview This example relates to methods and resulting antibodies, the discovery of candidate antibodies or fragments thereof (e.g., Fabs) that bind to human erythrocytes regardless of blood group antigens. Another goal was to achieve inter-species cross-reactivity, in particular such that successful candidates would also be cross-reactive to cynomolgus monkey (cyno) homologue proteins. An affinity maturation campaign was developed to identify high affinity clones that bind to both human and cyno target protein scaffolds independent of post-translational modifications. Phage-displayed combinatorial scfv libraries were designed to randomize each of the CDRs of the parent Fabs individually. Each CDR library was then panned against several variants of the human and cyno target proteins to maintain cross-reactivity and ensure that differences in post-translational modifications do not affect binding. A next generation sequencing (NGS)-based primary screen was developed to identify affinity matured hits. Data from the NGS screen was compared to data from the scfv-phage ELISA screen, showing that although both screens identified the top hits from each campaign, the NGS analysis provided broader insight into the success of the selection. By profiling the output of each round of phage panning via NGS, incremental enrichment of sequence motifs could be more easily detected and affinity improvements could be better predicted. The data also show that the NGS approach could identify hits more efficiently than the traditional ELISA approach, reducing labor and costs. By implementing this streamlined method, we were able to affinity mature Fabs 6-fold and 16-fold against human and cynomolgus monkey (cyno) erythrocytes, respectively, and achieve high affinity binding to all blood types tested.

[0214] Discovery and Humanization During a hybridoma campaign in which mice were immunized with cynoRBCs and boosted with cynoglycophorin A, an IgG antibody called 3-103 was discovered. It is noted that the methods described herein are applicable to a variety of antibody types, including subtypes of IgA, IgD, IgE, IgM, or any such antibody class. In some embodiments, the methods of the present invention are particularly suited to IgG2 antibodies, such as, by way of example only, IgG2a. The heavy and light chains of the 3-103 antibody were humanized by grafting mouse CDRs into human framework regions to generate candidate constructs. Back mutations were introduced to maintain the structure, affinity, and improved E. coli expression of the antibody as a soluble Fab. These constructs were expressed as soluble Fabs, purified, and evaluated by stability and kinetic assays with human and cyno GPA and erythrocytes. Clone M1 ("3-103M1") was selected for further affinity maturation.

[0215] Library Design and Construction Computer modeling was used to identify predicted CDR residues. Libraries were rationally designed to target and randomize the predicted light chain CDR1 and CDR2 and heavy chain CDR1 and CDR2, and five libraries were generated for affinity maturation screening. Genes were synthesized at GeneArt and cloned into the pBs-3-103M1-CMVd1 vector for the generation of scfv-phage combinatorial libraries.

[0216] Selection Process The libraries were phage displayed and three rounds of selection were performed against biotinylated recombinant human GPA, recombinant cynomolgus monkey GPA and human GPA extracted from erythrocytes (extract). Scfv-phage bound to biotinylated antigen was captured with streptavidin-coated magnetic beads. Nine different selection conditions were performed for each library to ensure that each was selected on the three forms of GPA and that each library could find a combination of both antigen species. After each selection round, the antigen type or concentration was changed and the binding incubation time of the library and antigen was decreased. After the third round of selection, the library output was sent for AmpliconEZ NGS analysis by GeneWiz and the phage output was screened in parallel by scfv-phage spot ELISA. Positive hits in the ELISA were confirmed by Sanger sequencing of the appropriate variable regions.

[0217] NGS analysis NGS-based amplicon sequencing was performed using the GeneWiz AmpliconEZ service. Amino acid-specific sequence frequency analysis was performed. The enrichment fold of each sequence was calculated by dividing the frequency of each sequence by the frequency observed in the previous round of phage display or selection. A total enrichment fold score was also calculated considering the enrichment of a sequence across all selection rounds. The total enrichment was calculated by adding the enrichment folds of at least two selection rounds. Clones were typically ranked by the sum of the 3rd round output vs 2nd round output + 3rd round output vs 1st round output + 3rd round enrichment fold. Sequences were selected as hits for further characterization if they had a total enrichment fold score that exceeded the median score of all clones. For example, if the median total enrichment fold of the list of top hits from one comparison condition was 32, clones with a total enrichment fold score above 32 were typically selected for recombinant expression. The sequences were further evaluated for potential sequence liability (glycosylation sites, Asn deamidation and Asp isomerization) and sequences with potential liability were eliminated.

[0218] Characterization and Ranking The top sequences identified by NGS and ELISA were expressed in HEK cells as soluble Fabs in a high-throughput manner. The Fab concentrations in the supernatants were quantified by Biolayer Interferometry (BLI / Octet® analysis), and clones in the supernatants were tested for affinity to human erythrocytes by flow cytometry. The top ranked clones were purified with CH1 resin and characterized for binding to human and cynomolgus erythrocytes by flow cytometry.

[0219] Library Panning Figure 7 shows a non-limiting schematic of the library panning protocol. A given CDR library is subjected to three (or more) selection rounds according to some embodiments. Round 1 is performed with three concentrations of target antigen. Any target of the candidate antibody can be used. As a non-limiting example, selection was performed with human glycophorin A or cynomolgus glycophorin A as the target, although other targets or species can be used according to some embodiments. The output titer of a given round is used to select the starting antigen concentration for the subsequent round. In this study, 1x10 6 ~1×10 8 The condition with the lowest antigen concentration that produced the first round output titer in CFU / mL was selected. The antigen concentration was reduced by a factor of 10 in each subsequent round (unless the antigen was changed). In further embodiments, different concentration reductions can be used, such as 2x, 5x, 8x, 12x, 15x, 20x, 50x, etc. When alternating antigens (e.g., from human to cynomolgus monkey or vice versa), the antigen concentration was kept constant between rounds. The binding incubation of the library to the biotin-antigen complex was reduced with each successive round of selection, while the capture time and washing of the streptavidin beads were kept constant.

[0220] Results and Discussion As described above, an affinity maturation campaign was conducted based on the various CDRs of the identified antibodies that bind to both human and cynomolgus glycophorin A. Figure 4 is a schematic diagram of a candidate antibody capable of binding to both species of glycophorin A, called 3-103. Fabs were generated, humanized, and screened, and hF-3-103M1 was identified as a highly cross-reactive clone. Figure 4 shows the sequence alignment of the VL and VH chains of 3-103M1 and identifies several CDR regions that were targeted for affinity maturation.

[0221] FIG. 5 shows a schematic of the affinity maturation protocol. The phage library of Fabs is first depleted by incubating the library with streptavidin (SA) beads to remove non-specific binders from the library. The depleted library is then incubated with biotin-human glycophorin A coated SA beads. Selection pressure is applied to identify antibody fragments with higher affinity to glycophorin A through successive reductions in antigen concentration applied to the phage library with simultaneous reductions in incubation time. This allows for the identification of Fabs with higher affinity to the target. Phages bound to glycophorin A beads are amplified for subsequent screening rounds. In this schematic, three rounds of screening are performed, although in some embodiments, more screening rounds may be performed. After three rounds, the output candidate Fabs are evaluated by both ELISA-based and NGS-based methods. Finally, the hits are characterized as soluble Fabs. FIG. 6 shows a schematic of the workflow pathway used according to some embodiments to affinity mature antibodies or antibody fragments.

[0222] As described above in Materials and Methods, Figure 7 shows a non-limiting example of a selection scheme used to identify high affinity Fabs. Shown here is the selection process for the CDRL1B library. As described above, round 1 was performed with three concentrations of human glycophorin A target antigen. The output titer of a particular round is used to select the starting antigen concentration for the subsequent round. With this approach, the output of each round should selectively remove antibodies or antibody fragments with low affinity to the target from the candidate population. Candidates showing high affinity can then be characterized.

[0223] Figures 8A-8C show data related to the output of the selection process applied to the CDRL1B affinity maturation campaign. Figure 8A shows a schematic of the weighted consensus sequence. The selection process showed that the library converged to a sequence similar to the wild-type (immature) sequence (TYLH). It is also evident that amino acid residues 2 and 4 are essentially fixed as -YH based on this selection campaign. Figure 8B shows a schematic ribbon structure of the antibody with the position of CDR1B identified by a box. Subsequent characterization of the candidates showed that more than half of the hits were not expressed as soluble fAbs. Furthermore, as shown by the binding assay results in Figure 8C, those that were expressed did not have significantly improved affinity for either human or cynomolgus RBCs over the parental fAb (generally identified by an arrow). There were many curves that yielded reduced MFI compared to the parental fAb, indicating a lower affinity for human erythrocytes (which express the target glycophorin A). Additional data are presented in Figures 9A-9I, which plot sequence fold enrichment vs. affinity for human erythrocytes for each of the output parameters of the selection scheme depicted in Figure 7. A sum fold enrichment score was also calculated considering the enrichment of the sequence across all rounds of selection. The sum enrichment was calculated by adding the enrichment folds obtained in at least two rounds of selection. Clones were typically ranked by the sum of enrichments in round 3 output vs round 2 output + round 3 output vs round 1 output + round 3 output. Sequences were selected as hits for further characterization if their sum enrichment fold score was equal to or greater than the median score of all clones. As an example, Figure 9A shows data related to affinity obtained from round 3 output 1 of Figure 7 (top right box - hGPA extract, 20 fmol) compared to affinity in round 1 output A (hGPA extract, 200 pmol). Similarly, Figure 9B shows data for round 3 output 2 of Figure 7 compared to round 1 output A.Similar to the binding data shown in Figure 8C, these scatter plots show that the majority of sequences enriched during this campaign show similar affinity for human erythrocytes compared to the wild-type parental Fab (indicated by the arrow). These data further strongly indicate that the parental Fab sequences were also enriched during selection. This reinforces the initial hypothesis that this domain of CDRL1 may need to be conserved to maintain its potential role in structural or other properties of the Fab supporting binding to antigen.

[0224] Figures 10A-10D show data summarizing the characteristics of clones run through the selection process of Figure 7, but utilizing the CDRL1a library. Figure 10A shows that a number of CDRL1a clones generated antibodies with enhanced affinity for human erythrocytes compared to the parent Fab (generally indicated by an arrow). Figure 10B shows a schematic of the convergent sequences of hits selected from the library. Figure 10C shows data comparing the affinity of antibody fragments identified by either the ELISA-based method (black circles) or the NGS-based method described herein (and used in this example). These data show that many more high affinity antibodies have been identified using NGS. The higher numbers are due, at least in part, to the greater throughput that the NGS-based method offers. Nearly 40 sequences were screened, compared to 6-8 using the ELISA method. Furthermore, many of these antibodies were in the low nM affinity range, which was a higher affinity than many of the Fabs identified by ELISA. Figure 10D presents the data from Figure 10C as the percentage of clones identified from the NGS or ELISA primary screen grouped by affinity range for human erythrocytes (Note - the sum of Fab types in all affinity ranges equals 100%). As shown, approximately 10% of the sequences identified using NGS have an affinity of <6 nM for human erythrocytes, compared to approximately 38% using ELISA. However, at affinities between 6-10 nM or >10 nM, the percentage of sequences identified using the NGS method was greater than the percentage of sequences expressed using the ELISA method. Although the ELISA screen identified 5-fold fewer clones than the NGS screen, a higher percentage of clones identified in this library had an EC of <6 nM for human erythrocytes. 50Fabs with enhanced affinity for the target were high affinity binders. It is important to note that these same high affinity clones were also identified by the NGS screen. Figures 11A-11F show data related to fold enrichment of CDRL1a candidates. As noted above, each panel relates to a specific output comparison from the selection scheme to determine enrichment fold. Figure 11A shows data related to a comparison of output 1 from round 3 with output 1 from round 2. In contrast to the data in Figure 8, the data in Figures 11A-11F show that as a result of the selection / maturation process, the affinity for the target was enhanced and the parental sequence was outperformed by the affinity matured clones. Notably, many of the data points are shifted to affinity values ​​in the nM range (e.g., higher affinity for the target) compared to the parental Fab (indicated by the arrow). Thus, the maturation campaign and selection process using NGS methods identified Fabs with enhanced affinity for the target. Given the more rapid nature of NGS-based methods and the Fabs with higher affinity identified, in some embodiments, the use of NGS-based methods may provide a more robust screening of candidate antibodies at a lower cost.

[0225] Figures 12A-12D show data summarizing the characteristics of clones run through the selection process of Figure 7, but utilizing the CDRL2 library. Figure 12A shows that a number of CDRL2 clones generated antibodies with enhanced affinity for human erythrocytes compared to the parent Fab (generally indicated by an arrow). Figure 12B shows a schematic of the convergent sequences of selected hits from the library. Figure 12C shows data on the affinity of antibody fragments identified by the ELISA-based method (filled circles) compared to the NGS-based method described herein (and used in this example). As above, these data show that more high affinity antibodies were identified using NGS, with the NGS method having a higher throughput, resulting in 45 or more candidate clones. Figure 12D shows the data from Figure 12C as a percentage of clones identified from the NGS and ELISA primary screens sorted by affinity for human erythrocytes. As shown, nearly 40% of the sequences identified using NGS had affinities <6 nM, compared to approximately 25% of the sequences identified using ELISA. Those with affinities between 6-10 nM and those with affinities above 10 nM were similar between ELISA and NGS. Figures 13A-13F show data on fold enrichment of CDRL2 candidates. Again, many candidates show significant enrichment, indicating higher affinity compared to wild type (arrow).

[0226] Figures 14A-14D show data summarizing the characteristics of clones run through the selection process of Figure 7, but utilizing the CDRH1 library. Figure 14A shows a broad distribution of affinities for human erythrocytes from CDRH1 clones compared to the parental Fab. Figure 14B shows a schematic of the convergent sequences of selected hits from the library. Figure 14C shows data comparing the affinities of antibody fragments identified by the ELISA-based method (black circles) and the NGS-based method (triangles) described herein (and used in this example). As above, these data show that many more high affinity antibodies were identified using NGS, and that the NGS method has a higher throughput, resulting in 30 or more candidate clones. Figure 14D shows the data from Figure 14C as a percentage of clones identified from the NGS and ELISA primary screens sorted by affinity for human erythrocytes. As shown, the number of sequences with <6 nM identified using NGS was approximately twice the number of sequences identified using ELISA. Those with affinities between 6 and 10 nM and those with affinities above 10 nM were similarly expressed between the two methods. Figures 15A-15F show data on fold enrichment of CDRH1 candidates. Again, many candidates show higher affinity compared to wild type (arrow). These data support the notion that, in some embodiments, NGS-based methods are highly efficient in predicting successful identification of antibodies or antibody fragments that show enhanced affinity for targets.

[0227] Figures 16A-16D show data summarizing the characteristics of clones run through the selection process of Figure 7, but utilizing the CDRH2 library. Figure 16A shows a broad distribution of affinities for human erythrocytes from CDRH2 clones compared to the parental Fab. Figure 16B shows a schematic of the convergent sequences of selected hits from the library. Figure 16C shows data comparing the affinity of antibody fragments identified by the ELISA-based method (filled circles) and the NGS-based method described herein (and used in this example) (triangles) to sequences designed with manual mutations (squares). As above, these data show that more high affinity antibodies were identified using NGS, and that the NGS method has a greater throughput compared to the ELISA or manual methods. Figure 16D shows the data from Figure 16C as a percentage of clones identified from the NGS and ELISA primary screens sorted by affinity for human erythrocytes. As shown, the number of sequences with affinities <6 nM identified using NGS was greater than sequences identified using ELISA, and more than two-fold greater than manual mutations. Those with affinities between 6 and 10 nM and those with affinities >10 nM had similar expression between NGS and ELISA. EC<6 nM for human erythrocytes 50Although there was some variability in the percentage of high affinity clones with , there was no substantial difference in clonal attenuation between the two screening formats, and this was true for all library outputs analyzed in this experiment. At the lowest affinity range, manual means outperformed both ELISA and NGS methods. However, as seen with other CDR libraries, the higher throughput and higher rate of identification of high affinity clones indicates that NGS is a very efficient method. Figures 17A-17I show data related to fold enrichment of CDRH2 candidates. As with the H1 library, many of the candidates show enhanced affinity compared to wild-type Fab (arrow). These data support the notion that, in some embodiments, NGS-based methods are highly efficient in predicting successful identification of antibodies or antibody fragments that show enhanced affinity for a target.

[0228] Figures 18A-18B show data regarding the ability of selected clones with enhanced target affinity to bind to both human (18A) and cynomolgus monkey (18B) erythrocytes. As can be seen, a large number of clones show binding to erythrocytes of both species, with the majority of clones showing enhanced affinity for the target compared to the wild-type parent Fab.

[0229] Figure 19 shows a comparison of the efficiency of each library tested in this example. Overall, a total of 305 hits were characterized, with a total of 136 hits showing enhanced potency for erythrocyte binding, and in the L2 library, 52 hits showed improved affinity, with a frequency of 0.38. Thus, L2 was the most successful library in producing the best affinity matured hits for human and cynomolgus monkey erythrocytes.

[0230] Figure 20 summarizes data related to affinity measured by FACS for both cynomolgus monkey and human erythrocytes. The goal of the affinity maturation campaign was to have single-digit nM affinity (1-9 nM) for human erythrocytes. As shown in the right-most column, single-digit nM affinity (2.5-4.4 nM) was achieved for at least 10 identified clones. The second column from the right also shows enhanced affinity for cynomolgus monkey erythrocytes.

[0231] Figures 21A-21F show summary data related to the affinity (and quantity) of sequences assessed by ELISA vs. NGS. Taking the above data together, Figures 21A, 21C and 21E show that one advantage of screening by NGS is the ability to screen a wider panel of clones covering a wide range of affinities. Advantageously, this can reduce the cost and time spent on the maturation campaign. Also, the clones showing the highest affinity have been screened by both ELISA and NGS, allowing for quality control by two methods. Furthermore, although there is some variability in the percentage of high affinity clones identified (e.g., affinity for human erythrocytes <6 nM), there is no significant difference in candidate attrition between the two screening formats. Thus, this example shows the particular advantages of using NGS-based methods to screen affinity maturation candidates.

[0232] Example 3A: Further data on next generation sequencing-based affinity maturation Further improving on Example 3 above, a study was performed to identify fragment antibodies (fAbs) specific to glycophorin A, a red blood cell membrane protein. The Fab constructs were affinity matured 6-fold and 16-fold to human and cynomolgus monkey (cyno) erythrocytes (referred to as hRBC or cRBC, respectively). Cross-reactive high affinity binding to erythrocytes was achieved regardless of blood group antigens. To identify affinity matured hits, a next generation sequencing (NGS) based primary screen was developed. It was demonstrated that the NGS method can not only identify hits more efficiently than the traditional ELISA method, but also with less labor and cost. The ELISA and NGS methods are summarized in Table 6 below.

[0233] [Table 14]

[0234] method As described above in Example 3, a combinatorial scFv-phage library was designed to randomize each CDR individually. Selection of candidate antibodies was performed using both human and cynomolgus monkey target antigens to maintain cross-reactivity. In addition, multiple antigen formats were used to ensure selection of clones that bind regardless of post-translational modifications. Primary screening of both NGS and ELISA was used to identify binding candidates. The resulting clones were first ranked by affinity to human erythrocytes, then by affinity to cynomolgus monkey erythrocytes. Finally, binding kinetics to target proteins from both species was measured by Octet Biolayer Interferometry. The selection method is summarized in Figure 5.

[0235] result FIG. 22 shows data relating to the correlation between affinity and tolerance response. As described above, antigens, fragments thereof or mimotopes thereof can be attached to binding sites as described herein. The antigens were fused to one of two different Fabs, each with a different affinity for glycophorin A. Fab 2.0 has approximately 200-fold higher affinity for the target compared to that of Fab 1.0. Disease severity was scored in animals following administration of antigen-Fab fusions and used as an index of antigen-specific immune response. As shown in the figure, Fab-2.0, which has 200-fold higher affinity for erythrocytes, induced a significant and sustained reduction in disease severity. In contrast, the lower affinity Fab (Fab 1.0) only slightly reduced disease severity. Thus, according to some embodiments, the method of screening and identifying high affinity candidate binders correlates with those binders that result in improved antigen-specific immune tolerance.

[0236] Figures 23A-23C relate to the types of sequences that can be enriched and correlate with improved affinity. Figure 23A shows plots of the affinity of the clones tested against sequence enrichment fold from library 1 and library 4. Library 1 was enriched primarily for parental (WT or wild type) and wild type-like sequences. Figure 23B shows the binding of clones from library 1 to human erythrocytes, illustrated as the fluorescence intensity of the Fab binding signal measured by flow cytometry. As shown, selection of this population of clones did not lead to improved affinity for human erythrocytes (plot of binding affinity of the clones is right shifted compared to the wild type curve). In contrast, selection with library 4 was successful, as shown in Figure 23C, where the flow cytometry affinity curves of the clones are shifted to the left compared to the wild type curve, indicating a higher affinity for human erythrocytes. This library produced clones with approximately 6-fold higher affinity for human erythrocytes compared to the wild type parental sequences.

[0237] As described above, screening was performed using human and cynomolgus antigens to select clones with cross-reactivity to both human and cynomolgus species. Figure 24 shows data showing that clones that showed improved affinity to hRBC also showed improved affinity to cyno (cRBC). Flow cytometry was used to assess binding, and the top clones showed approximately 16-fold higher affinity to cRBC compared to wild type. Advantageously, in some embodiments, such clones can be used in preclinical (e.g., primate studies) and human clinical trial work.

[0238] To further elucidate the mechanism underlying the improved affinity, binding kinetics was evaluated using Octet Biolayer Interferometry. Sensograms of Fab binding kinetics are shown in Figures 25A-25B. Figure 25A shows the kinetic data of the wild type, and Figure 25B shows the data of a selected higher affinity clone. As indicated by the arrows, the off-rate differs between the two clones tested, with the off-rate of the higher affinity clone in 25B being about 4-fold slower than the wild type. In some embodiments, the enhanced affinity may also be related to another kinetic property, such as the binding constant (e.g., "on-rate" which indicates how quickly a binder binds to its target).

[0239] As described above in Example 3, the NGS primary screen advantageously provides a higher sampling rate of clones and a broader range of affinities. The data from Example 3 above is shown in FIG. 26 with a breakdown of the top performing clones based on the screening method used into those that met the target affinity goal and those that did not. As shown in FIG. 26 and described above in Example 3, both ELISA and NGS screens were able to identify high affinity binders. Furthermore, in some embodiments, all or substantially all of the top hits found by ELISA may also be identified by NGS. Advantageously, the NGS approach can reduce the cost and time spent on a maturation campaign. Also, because the clones exhibiting the highest affinities were identified by both ELISA and NGS screening, quality control by two methods can be performed. Thus, Examples 3 and 3A are non-limiting examples that show certain advantages of screening affinity maturation candidates using NGS-based methods.

[0240] Further non-limiting embodiments are described below. In some embodiments, an antigen binding protein is provided, comprising: a light chain complementarity determining region (CDR) 1 comprising RASSNVX1X2MY (SEQ ID NO: 49) and a light chain CDR2 comprising X3X4TSX5LAS (SEQ ID NO: 50), wherein X1, X2, X3, X4 and X5 are each a naturally occurring amino acid; when X2 is Y, X3 is Y, X4 is Y and X5 is N, X1 is K but not; when X1 is K, X3 is Y, X4 is Y, and X5 is N, then X2 is not Y; when X1 is K, X2 is Y, X4 is Y, and X5 is N, then X3 is not Y; when X1 is K, X2 is Y, X3 is Y, and X5 is N, then X4 is not Y; when X1 is K, X2 is Y, X3 is Y, and X4 is Y, then X5 is not N).

[0241] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is selected from F, W and Y, and X2 is selected from F, W, Y and Q (SEQ ID NO: 51); the light chain CDR2 comprises X3X4TSX5LAS, where X3 is selected from H and Y, X4 is selected from H, R and K, and X5 is a natural amino acid (SEQ ID NO: 52).

[0242] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is selected from F, W and Y, and X2 is selected from F, W and Y (SEQ ID NO:53); the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H or R, and X5 is a natural amino acid (SEQ ID NO:54).

[0243] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is F or Y and X2 is selected from F, W and Y (SEQ ID NO:55); the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H, and X5 is a natural amino acid (SEQ ID NO:56).

[0244] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is F or Y and X2 is F (SEQ ID NO:57); the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H, and X5 is a natural amino acid (SEQ ID NO:56).

[0245] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is F or Y and X2 is F (SEQ ID NO:57); the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H, and X5 is V or D (SEQ ID NO:58).

[0246] In some embodiments, an antigen binding protein is provided that comprises a light chain CDR1 comprising RASSNVX1X2MY (SEQ ID NO: 49) and a light chain CDR2 comprising X3X4TSX5LAS (SEQ ID NO: 50), where X1, X2, X3, X4 and X5 are each a naturally occurring amino acid.

[0247] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is selected from F, W and Y, and X2 is selected from F, W, Y and Q (SEQ ID NO: 51); the light chain CDR2 comprises X3X4TSX5LAS, where X3 is selected from H and Y, X4 is selected from H, R and K, and X5 is a natural amino acid (SEQ ID NO: 52).

[0248] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is selected from F, W and Y, and X2 is selected from F, W and Y (SEQ ID NO: 53); the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H or R, and X5 is a natural amino acid (SEQ ID NO: 54).

[0249] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is F or Y and X2 is selected from F, W and Y (SEQ ID NO:55); the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H, and X5 is a natural amino acid (SEQ ID NO:56).

[0250] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is F or Y and X2 is F (SEQ ID NO:57); the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H, and X5 is a natural amino acid (SEQ ID NO:56).

[0251] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY, where X1 is F or Y and X2 is F (SEQ ID NO:57); the light chain CDR2 comprises X3X4TSX5LAS, where X3 is H, X4 is H, and X5 is V or D (SEQ ID NO:58).

[0252] In some embodiments, the antigen binding protein further comprises a light chain CDR3 comprising QQFTSSPYT (SEQ ID NO:45).

[0253] In some embodiments, an antigen binding protein is provided that comprises a light chain CDR1 comprising RASSNVX1X2MY (SEQ ID NO:49), or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; and a light chain CDR2 comprising X3X4TSX5LAS (SEQ ID NO:50), or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions, where X1, X2, X3, X4 and X5 are each naturally occurring amino acids.

[0254] In some embodiments, the antigen binding protein light chain CDR1 comprises RASSNVX1X2MY (SEQ ID NO:51) or a variant thereof comprising one, two, three or four conservative amino acid substitutions, where X1 is selected from F, W and Y, and X2 is selected from F, W, Y and Q; and the light chain CDR2 comprises X3X4TSX5LAS (SEQ ID NO:52) or a variant thereof comprising one, two, three or four conservative amino acid substitutions, where X3 is selected from H and Y, X4 is selected from H, R and K, and X5 is a naturally occurring amino acid.

[0255] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY (SEQ ID NO:53) or a variant thereof comprising one, two, three or four conservative amino acid substitutions, where X1 is selected from F, W and Y, and X2 is selected from F, W and Y; the light chain CDR2 comprises X3X4TSX5LAS (SEQ ID NO:54) or a variant thereof comprising one, two, three or four conservative amino acid substitutions, where X3 is H, X4 is H or R, and X5 is a naturally occurring amino acid.

[0256] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY (SEQ ID NO:55) or a variant thereof comprising one, two, three or four conservative amino acid substitutions, where X1 is F or Y and X2 is selected from F, W and Y; the light chain CDR2 comprises X3X4TSX5LAS (SEQ ID NO:56) or a variant thereof comprising one, two, three or four conservative amino acid substitutions, where X3 is H, X4 is H and X5 is a natural amino acid.

[0257] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY (SEQ ID NO:57) or a variant thereof comprising one, two, three or four conservative amino acid substitutions, where X1 is F or Y and X2 is F; the light chain CDR2 comprises X3X4TSX5LAS (SEQ ID NO:56) or a variant thereof comprising one, two, three or four conservative amino acid substitutions, where X3 is H, X4 is H and X5 is a natural amino acid.

[0258] In some embodiments, the light chain CDR1 comprises RASSNVX1X2MY (SEQ ID NO:57) or a variant thereof comprising one, two, three or four conservative amino acid substitutions (wherein X1 is F or Y and X2 is F); the light chain CDR2 comprises X3X4TSX5LAS (SEQ ID NO:58) or a variant thereof comprising one, two, three or four conservative amino acid substitutions (wherein X3 is H, X4 is H, and X5 is V or D).

[0259] In some embodiments, the antigen binding protein also comprises a light chain CDR3 comprising QQFTSSPYT (SEQ ID NO:45), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

[0260] In some embodiments, the antigen binding protein also comprises a heavy chain CDR1 comprising GYTFNSYFMH (SEQ ID NO: 46); a heavy chain CDR2 comprising GMIRPNGGTTDYNEKFKN (SEQ ID NO: 47); and a heavy chain CDR3 comprising WEGSYYALDY (SEQ ID NO: 48).

[0261] In some embodiments, the antigen binding protein also comprises a heavy chain CDR1 comprising GYTFNSYFMH (SEQ ID NO: 46) or a variant thereof comprising one, two, three or four conservative amino acid substitutions; a heavy chain CDR2 comprising GMIRPNGGTTDYNEKFKN (SEQ ID NO: 47) or a variant thereof comprising one, two, three or four conservative amino acid substitutions; and a heavy chain CDR3 comprising WEGSYYALDY (SEQ ID NO: 48) or a variant thereof comprising one, two, three or four conservative amino acid substitutions.

[0262] In some embodiments, the antigen binding protein also comprises a heavy chain variable domain that is at least about 90% identical to SEQ ID NO: 3. In some embodiments, the antigen binding protein also comprises a heavy chain variable domain that is at least about 95% identical to SEQ ID NO: 3. In some embodiments, the antigen binding protein also comprises a heavy chain variable domain that is at least about 99% identical to SEQ ID NO:3.

[0263] In some embodiments, an antigen binding protein is provided that comprises a heavy chain variable domain that is at least about 90% identical to SEQ ID NO:3 and a light chain variable domain that is at least about 90% identical to SEQ ID NO:1.

[0264] In some embodiments, an antigen binding protein is provided that comprises a heavy chain variable domain that is at least about 95% identical to SEQ ID NO:3 and a light chain variable domain that is at least about 95% identical to SEQ ID NO:1.

[0265] In some embodiments, an antigen binding protein is provided that comprises a heavy chain variable domain that is at least about 99% identical to SEQ ID NO:3 and a light chain variable domain that is at least about 99% identical to SEQ ID NO:1.

[0266] In some embodiments, an antigen binding protein is provided that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-42.

[0267] In some embodiments, an antigen binding protein is provided that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 11, 12, 14, 18, 19, 20, 21, 22, 24, 27, 28, 31, 38 and 42.

[0268] In some embodiments, an antigen binding protein is provided that comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, an antigen binding protein is provided that comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antigen binding protein is humanized.

[0269] In some embodiments, the antigen binding protein also comprises (a) a first human light chain framework region (FR1) selected from SEQ ID NOs: 131-140, a human FR2 of the light chain framework region selected from SEQ ID NOs: 141-148, a human FR3 of the light chain selected from SEQ ID NOs: 149-156, and a human FR4 of the light chain selected from SEQ ID NOs: 157-162; and (b) a first human heavy chain framework region (FR1) selected from SEQ ID NOs: 109-115, a human FR2 of the heavy chain selected from SEQ ID NOs: 116-119, a human FR3 of the heavy chain selected from SEQ ID NOs: 120-127, and a human FR4 of the heavy chain selected from SEQ ID NOs: 128-130.

[0270] In some embodiments, the antigen binding protein comprises a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3 and / or IgG4. In some embodiments, the constant region is IgG1.

[0271] In some embodiments, the antigen binding protein is a full-length antibody. In some embodiments, the antigen binding protein is an antigen-binding fragment of an antibody.

[0272] In some embodiments, the antibody or antigen-binding fragment comprises a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked Fv, a VNAR domain, an IgNar, an intrabody, an IgG-CH2, a minibody, F(ab')3, a tetramer, a trimer, a bispecific antibody, a single domain antibody, a DVD-Ig, an Fcab, a mAb2, an (scFv)2, or a scFv-Fc.

[0273] In some embodiments, the antigen binding protein specifically binds to glycophorin A (GPA). In some embodiments, the antigen binding protein specifically binds to one or more of human GPA, cynomolgus monkey GPA, porcine GPA, bovine GPA, mouse GPA, or rat GPA. In some embodiments, the antigen binding protein specifically binds to human GPA.

[0274] In some embodiments, the antigen binding protein has a K d In some embodiments, K binds to human GPA. d is about 1.0 nM or more (e.g., less than or equal to 1.0 nM). In some embodiments, the binding affinity is measured by flow cytometry, surface plasmon resonance, biolayer interferometry, or radioimmunoassay.

[0275] In some embodiments, the antigen binding protein is affinity matured. In some embodiments, the antigen binding protein is an affinity matured variant of 10F7. In some embodiments, the antigen binding protein competes with GPA for binding to 10F7, Ter119, CLB-ery-1 (AME-1), EPR8200, YTH89.1, EPR8199, JC159, GYPA / 280, ab40844, HI264, GPHN02, JC159, SPM599, EPR8200, GYPA / 1725R, ab112201, ab114330, ab219896, BRIC256, or fragments or derivatives thereof. In some embodiments, the antigen binding protein inhibits binding of 10F7, Ter119, CLB-ery-1 (AME-1), EPR8200, YTH89.1, EPR8199, JC159, GYPA / 280, ab40844, HI264, GPHN02, SPM599, GYPA / 1725R, ab112201, BRIC256, or a fragment or derivative thereof, to human GPA by at least about 50%.

[0276] In some embodiments, an antigen binding protein is provided that specifically binds human glycophorin A (GPA) and comprises a light chain CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 59, 61, 63, 65, 67, 69, 71, 73, 76, 78, 80, 81, 83, 85, 87, 88, 90, 91, 93, 95, 96, 98, 99, 101, 102, 104, 105, 107, and 108; a light chain CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77, 79, 82, 84, 86, 89, 92, 94, 97, 100, 103, and 106, wherein the antigen binding protein is affinity matured and humanized.

[0277] In some embodiments, the light chain CDR1 specifically binds to human glycophorin A (GPA) and comprises: (a) the amino acid sequence of any one of SEQ ID NOs: 59, 61, 63, 65, 67, 69, 71, 73, 76, 78, 80, 81, 83, 85, 87, 88, 90, 91, 93, 95, 96, 98, 99, 101, 102, 104, 105, 107, and 108; (b) SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77, 79, 82, 84 , 86, 89, 92, 94, 97, 100, 103 and 106; (c) a light chain CDR3 comprising QQFTSSPYT (SEQ ID NO: 45) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; (d) a heavy chain CDR1 comprising GYTFNSYFMH (SEQ ID NO: 46) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; (e) GMIRPNGGTTDYNEKFKN (SEQ ID NO: 47). (f) a heavy chain CDR3 comprising WEGSYYALDY (SEQ ID NO:48) or a variant thereof comprising one, two, three or four conservative amino acid substitutions; (g) a first human light chain framework region (FR1) selected from SEQ ID NOs: 131-140, a human FR2 of a light chain framework region selected from SEQ ID NOs: 141-148, a human FR3 of the light chain selected from SEQ ID NOs: 149-156, and a human FR4 of the light chain selected from SEQ ID NOs: 157-162; (h) a first human heavy chain framework region (FR1) selected from SEQ ID NOs: 109-115, a human FR2 of the heavy chain selected from SEQ ID NOs: 116-119, a human FR3 of the heavy chain selected from SEQ ID NOs: 120-127, and a human FR4 of the heavy chain selected from SEQ ID NOs: 128-130; (i) a human heavy chain constant region; and (j) a human light chain constant region.

[0278] In some embodiments, an isolated polynucleotide encoding an antigen binding protein of the present invention is provided.

[0279] In some embodiments, an isolated polynucleotide is provided that encodes a polypeptide at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, an isolated polynucleotide is provided that encodes a polypeptide at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, an isolated polynucleotide is provided that encodes a polypeptide at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, a vector is provided that comprises such a polynucleotide. In some embodiments, a host cell is provided that comprises the vector. In some embodiments, the cell line is selected from the group consisting of CHO, k1SV, XCeed, CHOK1SV and GS-KO.

[0280] In some embodiments, a method for producing an antigen binding protein that specifically binds to GPA is provided, comprising culturing a cell line under conditions in which the antigen binding protein is produced; and recovering the antigen binding protein. In some embodiments, the antigen binding protein comprises one or more light chains and one or more heavy chains, wherein the heavy and light chains are encoded on separate vectors. In some embodiments, the antigen binding protein comprises one or more light chains and one or more heavy chains, wherein the heavy and light chains are encoded on the same vector. In some embodiments, a pharmaceutical composition is provided comprising the antigen binding protein and a pharma- ceutical acceptable carrier.

[0281] In some embodiments, compositions are provided that induce immune tolerance and include an antigen binding protein and at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen.

[0282] In some embodiments, the composition comprises at least one immunogenic tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen comprises at least one of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin proteolipid protein (PLP), a fragment(s) of MPB, a fragment(s) of MOG, and a fragment(s) of PLP. In some embodiments, the composition comprises an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 169-175 and 186-202. In some embodiments, the composition comprises an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 169, 171-173, 175, and 188-202. In some embodiments, the composition is for use in treating multiple sclerosis. In some embodiments, a method of treating multiple sclerosis is provided comprising administering such a composition to a subject.

[0283] In some embodiments, the composition comprises at least one immunogenic tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen is selected from the group consisting of insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6 phosphatase 2, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), insulinoma-associated protein 2 (IGRP), insulinoma-associated protein 2 (IGRP), insulin-like ... IA-2), insulinoma-associated protein 2 beta (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, and at least one of SST G-protein coupled receptors 1-5. In some embodiments, the composition comprises an immunogenic fragment of at least one tolerance-inducing antigen, wherein the immunogenic fragment of the at least one tolerance-inducing antigen comprises SEQ ID NO: 204-214. In some embodiments, the composition is for use in treating type 1 diabetes. In some embodiments, a method of treating type 1 diabetes is provided that comprises administering such a composition to a subject.

[0284] In some embodiments, the composition comprises at least one immunogenic tolerance-inducing antigen, wherein the at least one immunogenic tolerance-inducing antigen comprises at least one of tissue transglutaminase, high molecular weight glutenin, low molecular weight glutenin, gluten, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, avenin, and deamidated forms thereof. In some embodiments, the composition comprises an immunogenic fragment of at least one tolerance-inducing antigen, wherein the immunogenic fragment of the at least one tolerance-inducing antigen comprises SEQ ID NOs: 182-185 and 215. In some embodiments, the composition is for use in treating celiac disease. In some embodiments, a method of treating celiac disease is provided, comprising administering such a composition to a subject.

[0285] In some embodiments, the composition comprises at least one immunogenic tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen comprises at least one of MHC class I protein, MHC class II protein, minor blood group antigen, RhCE, Kell, Kidd, Duffy and Ss.In some embodiments, the composition is for use in preventing or treating transplant rejection.In some embodiments, a method for treating transplant rejection is provided, comprising administering such a composition to a subject.

[0286] In some embodiments, the use of the compositions of the invention is for use in inducing immune tolerance to an antigen of interest bound to the antigen binding protein.

[0287] In some embodiments, an antigen binding protein is provided that comprises a heavy chain complementarity determining region 1 (CDR H1) comprising an amino acid sequence selected from RATYIL, RNIYIL, KYTYIL, VHTYIL, RNVFIL, RNIYLL, RKTYIL, LNVYIL, KATYIL, RMTYIL, KTVYIL, KHVYIL, RNITMIL, KDTYIL, INSYIL, QHTYIL and RHSYIL. In some embodiments, the protein is matured from a parent sequence that comprises a sequence of KDTYML at the corresponding position of the amino acid sequence. In some embodiments, the CDR H1 comprises a sequence selected from SEQ ID NO: 217-233 or 269-285.

[0288] In some embodiments, an antigen binding protein is provided that comprises a first light chain complementarity determining region 1 (CDRL1a) comprising an amino acid sequence selected from FRNNK, FRNSK, FKNGK, FRNAK, FRTGK, FKNDK and YKNGK. In some embodiments, the protein is matured from a parent sequence that comprises a sequence of YSNGKT at the corresponding position of the amino acid sequence. In some embodiments, CDRL1a comprises a sequence selected from SEQ ID NOs: 235-241 or 287-293.

[0289] In some embodiments, an antigen binding protein is provided that comprises a light chain complementarity determining region (CDRL2) comprising an amino acid sequence selected from LSRTS, NTRTS, NTRPS, LNRLH, NTRLA, NSRLS, LSRVS, LNRVS, LNRLS, NSRLH, SSRLS, SSRVS, SNRLH, NTRVS, SNRVS, HSRLS, SSRLA, FNRVN, LNRMS, LNRIS and LSHPH. In some embodiments, the protein is matured from a parent sequence that comprises the sequence of VSKLD at the corresponding position of the amino acid sequence. In some embodiments, CDRL2 comprises one sequence selected from SEQ ID NO: 243-263 or 295-315.

[0290] In some embodiments, the antigen binding protein binds to a human target antigen and a cynomolgus monkey target antigen. In some embodiments, the human target antigen and the cynomolgus monkey target antigen are glycophorin A. In some embodiments, the antigen binding protein is fused to an antigen against which tolerance is desired.

[0291] In some embodiments, compositions are provided which induce immune tolerance and comprise an antigen binding protein of the invention and further comprise at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen.

[0292] In some embodiments, the composition comprises at least one immunogenic tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen comprises at least one of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin proteolipid protein (PLP), a fragment(s) of MPB, a fragment(s) of MOG, and a fragment(s) of PLP. In some embodiments, the composition comprises an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 169-175 and 186-202. In some embodiments, the composition comprises an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 169, 171-173, 175, and 188-202. In some embodiments, the composition is for use in the treatment of multiple sclerosis.

[0293] In some embodiments, the composition comprises at least one immunogenic tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen is selected from the group consisting of insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6 phosphatase 2, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), insulinoma-associated protein 2 (IGRP), insulinoma-associated protein 2 (IGRP), insulin-like ... In some embodiments, the composition comprises at least one of the following immunogenic fragments: insulinoma-associated protein 2β (IA-2β), insulinoma-associated protein 2β (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular carcinoma-intestinal-pancreatic / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, and SST G-protein coupled receptor 1-5. In some embodiments, the composition comprises an immunogenic fragment of at least one tolerance-inducing antigen, wherein the immunogenic fragment of the at least one tolerance-inducing antigen comprises SEQ ID NOs: 204-214. In some embodiments, the composition is for use in treating type 1 diabetes.

[0294] In some embodiments, the composition comprises at least one immunogenic tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen comprises at least one of tissue transglutaminase, high molecular weight glutenin, low molecular weight glutenin, gluten, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, avenin, and deamidated forms thereof. In some embodiments, the composition comprises an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises SEQ ID NOs: 182-185 and 215. In some embodiments, the composition is for use in treating celiac disease.

[0295] In some embodiments, compositions of the invention are provided that induce immune tolerance to an antigen of interest, wherein the antigen of interest is fused to an antigen binding protein.

[0296] In some embodiments, methods are provided for affinity maturing antibodies or antibody fragments to have enhanced affinity for a target, the methods comprising: depleting a phage library of non-specific binders, the phage library comprising a plurality of phages, each phage expressing a candidate affinity matured antibody or antibody fragment; exposing the depleted library to the target antigen; removing phages that do not bind to the target antigen by washing; amplifying phages that bind to the target antigen; repeating the exposing, removing and amplifying steps multiple times to induce selection pressure that results in binding of the target antigen by phage expressing high affinity candidate antibodies or antibody fragments; and screening for high affinity candidate antibodies or antibody fragments using next generation sequencing.

[0297] In some embodiments, the method may further include one or more of screening the high affinity candidate antibodies or antibody fragments using ELISA and evaluating the ability of the high affinity candidate antibodies or antibody fragments to be expressed in a soluble form.

[0298] Although the above has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be understood by those skilled in the art that modifications may be made without departing from the spirit of the invention. It will therefore be clearly understood that the forms described herein are illustrative only and are not intended to limit the scope of the invention, but rather to cover all modifications and alternatives which come within the true scope and spirit of the present invention.

[0299] It is contemplated that various combinations or subcombinations of the specific features and aspects of the above embodiments may be made and fall within the scope of one or more of the present inventions. Furthermore, the description herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, etc., associated with one embodiment may be used in all other embodiments described herein. Thus, it should be understood that the various features and aspects of the described embodiments may be combined with or substituted for one another to form various forms of the described invention. Thus, it is intended that the scope of the invention described herein should not be limited by the specific described embodiments above. Moreover, the present invention is susceptible to various modifications and alternative forms, specific examples of which have been shown in the drawings and described in detail herein. However, the present invention should not be limited to the specific forms or methods described, but rather, it should be understood that the present invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the various described embodiments and the appended claims. Any method described herein need not be performed in the order described. The methods described herein include specific operations performed by those skilled in the art, but may also include, explicitly or implicitly, any third party instructions regarding those operations. For example, an act such as "administering an antigen binding protein" includes "instructing the administration of the antigen binding protein." Furthermore, when features or aspects herein are described in terms of Markush groups, one of skill in the art will recognize that the description is also described in terms of any individual members or subgroup members of the Markush group thereby.

[0300] Ranges described herein also encompass any and all overlaps, subranges, and combinations thereof. The terms "up to," "at least," "greater than," "less than," "between," and the like, include the recited numerical values. Numbers preceded by the terms "about" or "approximately," and the like, include the recited numerical values. For example, "about 90%" includes "90%." In certain embodiments, at least 95% homology includes 96%, 97%, 98%, 99%, and 100% homology to the reference sequence. Furthermore, when a sequence is described as "comprising" a nucleotide or amino acid sequence, such reference also includes that the sequence "comprises," "consists of," or "consists essentially of" the recited sequence, unless otherwise specified.

[0301] Terms and phrases used in this application, and variations thereof, particularly in the claims, should be construed as open-ended and not limiting, unless expressly stated. As an example above, the term "including" should be construed to mean "including, without limitation," "including, but not limited to," etc.

[0302] The indefinite article "a" or "an" does not exclude a plurality. For example, the term "about" used herein to define molecular weight values ​​and ranges means that the indicated value and / or range limits may vary, for example, within ±20%, for example, within ±10%. The use of "about" before a numerical value includes the numerical value itself. For example, "about 5" explicitly supports "5". Numeric values ​​provided in ranges include overlapping ranges and integers therebetween; for example, the ranges 1-4 and 5-7 include, for example, 1-7, 1-6, 1-5, 2-5, 2-7, 4-7, 1, 2, 3, 4, 5, 6, and 7.

Claims

1. An antigen binding protein for binding to glycophorin A, comprising: a variable light chain domain comprising at least a first light chain complementarity determining region (LC CDR1) and a second light chain CDR (LC CDR2); and a variable heavy chain domain comprising at least a first heavy chain complementarity determining region (HC CDR1) and a second heavy chain CDR (HC CDR2); wherein the antigen binding protein is a single chain fragment variable (scFv) and is capable of binding to at least one of human glycophorin A and cynomolgus glycophorin A; the antigen binding protein binds to human glycophorin A with a dissociation constant (Kd) of about 1 nM to about 100 nM as measured by flow cytometry, surface plasmon resonance, biolayer interferometry, or radioimmunoassay; and the antigen binding protein has been affinity matured from a parent amino acid sequence encoding an antigen binding protein having a greater Kd than the antigen binding protein as measured by flow cytometry, surface plasmon resonance, biolayer interferometry or radioimmunoassay; Antigen-binding proteins.

2. The antigen binding protein of claim 1, wherein the antigen binding protein comprises a fragment of an antibody (Fab), wherein the Fab binds to both human glycophorin A and cynomolgus monkey glycophorin A, and the Fab binds to human glycophorin A with a dissociation constant of between about 10 μM and 0.1 nM.

3. LC CDR2 is LNRLH (SEQ ID NO:298), LSRTS (SEQ ID NO:295), NTRTS (SEQ ID NO:296), NTRPS (SEQ ID NO:297), NTRLA (SEQ ID NO:299), NSRLS (SEQ ID NO:300), LSRVS (SEQ ID NO:301), LNRVS (SEQ ID NO:302), LNRLS (SEQ ID NO:303), NSRLH (SEQ ID NO:304), SSRLS (SEQ ID NO:305), SSRVS (SEQ ID NO:306), 2. The antigen binding protein of claim 1, comprising an amino acid sequence selected from the group consisting of: NTRVS (SEQ ID NO:306), SNRLH (SEQ ID NO:307), NTRVS (SEQ ID NO:308), SNRVS (SEQ ID NO:309), HSRLS (SEQ ID NO:310), SSRLA (SEQ ID NO:311), FNRVN (SEQ ID NO:312), LNRMS (SEQ ID NO:313), LNRIS (SEQ ID NO:314) and LSHPH (SEQ ID NO:315).

4. 4. The antigen binding protein of claim 3, wherein CDRL2 comprises a sequence selected from any one of SEQ ID NOs: 246, 243, 244, 245 and 247-263.

5. 4. The antigen binding protein of claim 3, wherein the protein is matured from a parent sequence that contains the sequence of VSKLD at the corresponding position in the amino acid sequence of the variable light domain.

6. 2. The antigen binding protein of claim 1, wherein HC CDR1 comprises an amino acid sequence selected from the group consisting of RMTYIL (SEQ ID NO:278), RATYIL (SEQ ID NO:269), RNIYIL (SEQ ID NO:270), KYTYIL (SEQ ID NO:271), VHTYIL (SEQ ID NO:272), RNVFIL (SEQ ID NO:273), RNIYLL (SEQ ID NO:274), RKTYIL (SEQ ID NO:275), LNVYIL (SEQ ID NO:276), KATYIL (SEQ ID NO:277), KTVYIL (SEQ ID NO:279), KHVYIL (SEQ ID NO:280), RNITMIL (SEQ ID NO:281), KDTYIL (SEQ ID NO:282), INSYIL (SEQ ID NO:283), QHTYIL (SEQ ID NO:284) and RHSYIL (SEQ ID NO:285).

7. 7. The antigen binding protein of claim 6, wherein the protein is matured from a parent sequence that contains the sequence KDTYML at the corresponding position in the amino acid sequence.

8. 7. The antigen binding protein of claim 6, wherein CDR H1 comprises a sequence selected from any one of SEQ ID NOs: 227, 217-226 and 228-233.

9. 2. The antigen binding protein of claim 1, wherein the first light chain complementarity determining region 1 (CDRL1) comprises an amino acid sequence selected from FRNNK (SEQ ID NO:287), FRNSK (SEQ ID NO:288), FKNGK (SEQ ID NO:289), FRNAK (SEQ ID NO:290), FRTGK (SEQ ID NO:291), FKNDK (SEQ ID NO:292) and YKNGK (SEQ ID NO:293).

10. 10. The antigen binding protein of claim 9, wherein CDRL1 comprises a sequence selected from any one of SEQ ID NOs: 235-241.

11. 10. The antigen binding protein of claim 9, wherein the protein is matured from a parent sequence that contains the sequence YSNGKT at the corresponding position in the amino acid sequence.

12. 2. The antigen binding protein of claim 1, wherein the antigen binding protein competes for binding to glycophorin A with one or more of 10F7, Ter119, CLB-ery-1 (AME-1), EPR8200, YTH89.1, EPR8199, JC159, GYPA / 280, ab40844, HI264, GPHN02, JC159, SPM599, EPR8200, GYPA / 1725R, ab112201, ab114330, ab219896, BRIC256 or fragments or derivatives thereof.

13. 13. The antigen binding protein of any one of claims 1 to 12, wherein the antigen binding protein is fused to at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen or an immunogenic fragment of the at least one tolerogenic antigen is associated with multiple sclerosis.

14. 14. The antigen protein of claim 13, wherein the antigen binding protein is fused to an immunogenic fragment of at least one tolerogenic antigen, the immunogenic fragment of the at least one tolerogenic antigen comprising an immunogenic fragment of myelin basic protein (MBP), an immunogenic fragment of myelin oligodendrocyte glycoprotein (MOG) and / or an immunogenic fragment of myelin proteolipid protein (PLP).

15. The antigen protein according to claim 14, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 169-175 and 186-202.

16. 15. The antigen binding protein of claim 14, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 169, 171-173, 175 and 188-202.

17. 17. Use of an antigen binding protein according to any one of claims 13 to 16 for use in the treatment of multiple sclerosis.

18. 13. The antigen binding protein of any one of claims 1 to 12, wherein the antigen binding protein is fused to at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen or an immunogenic fragment of the at least one tolerogenic antigen is associated with type 1 diabetes.

19. The antigen binding protein is fused to an immunogenic fragment of at least one tolerogenic antigen, the immunogenic fragment of the at least one tolerogenic antigen being selected from the group consisting of proinsulin, insulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6 phosphatase 2, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), insulinoma-associated protein, and the like.

18. The antigen binding protein of claim 17, comprising one or more immunogenic fragments of insulinoma-associated protein 2 (IA-2), insulinoma-associated protein 2 beta (IA-2β), ICA69, ICA12 (SOX-13), carboxypeptidase H, immunogen 38, GLIMA 38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular carcinoma-intestine-pancreas / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, and SST G-protein coupled receptor 1-5.

20. 20. The antigen binding protein of claim 18, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 204-214.

21. 21. Use of an antigen binding protein according to any one of claims 18 to 20 for use in the treatment of type 1 diabetes.

22. 13. The antigen binding protein of any one of claims 1 to 12, wherein the antigen binding protein is fused to at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen, wherein the at least one immunogenic tolerogenic antigen or an immunogenic fragment of the at least one tolerogenic antigen is associated with celiac disease.

23. 23. The antigen binding protein of claim 22, wherein the antigen binding protein is fused to an immunogenic fragment of at least one tolerogenic antigen, the immunogenic fragment of at least one tolerogenic antigen comprising one or more immunogenic fragments of gluten, tissue transglutaminase, high molecular weight glutenin, low molecular weight glutenin, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, avenin and deamidated forms thereof.

24. 24. The antigen binding protein of claim 23, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 182-185 and 215.

25. 25. An antigen binding protein according to any one of claims 22 to 24 for use in the treatment of coeliac disease.

26. 17. A method of treating multiple sclerosis comprising administering to a subject a composition comprising the antigen binding protein of any one of claims 13 to 16.

27. 21. A method of treating type 1 diabetes comprising administering to a subject a composition comprising the antigen binding protein of any one of claims 18 to 20.

28. 25. A method of treating celiac disease comprising administering to a subject a composition comprising the antigen binding protein of any one of claims 22 to 24.

29. 2. A method for affinity maturation of an antibody binding protein according to claim 1, comprising: depleting a phage library of non-specific binders, wherein the phage library comprises a plurality of phage, each phage expressing a candidate affinity matured antibody or antibody fragment; exposing the depleted library to the target antigen; removing phages that do not bind to the target antigen by washing; amplifying phage that bind to the target antigen; repeating the revealing, removing and amplifying steps multiple times to induce selection pressure that results in binding of the target antigen by phage expressing high affinity candidate antibodies or antibody fragments; and Screening high affinity candidate antibodies or antibody fragments using next generation sequencing A method comprising:

30. 30. The method of claim 28, further comprising one or more of screening the high affinity candidate antibodies or antibody fragments using ELISA and evaluating the ability of the high affinity candidate antibodies or antibody fragments to be expressed in a soluble form.

31. An antigen binding protein for binding to glycophorin A, comprising: a first variable domain comprising at least a first light chain complementarity determining region (LC CDR) 1 and a second light chain CDR (LC CDR2); and a second variable domain comprising at least a second heavy chain complementarity determining region (HC CDR) 1 and a second heavy chain CDR (HC CDR2); wherein the antigen binding protein is a single chain fragment variable (scFv) and is capable of binding to both human glycophorin A and cynomolgus glycophorin A; the antibody binding fragment (Fab) binds to human glycophorin A with a dissociation constant (Kd) of less than about 20 nM as measured by flow cytometry, surface plasmon resonance, biolayer interferometry or radioimmunoassay; and the antibody binding fragment (Fab) has been affinity matured from a parent sequence with a greater Kd than the antibody binding fragment (Fab) as measured by flow cytometry, surface plasmon resonance, biolayer interferometry or radioimmunoassay; Antigen-binding proteins.

32. An antigen binding protein comprising a light chain variable domain that is at least about 95% identical to one or more of SEQ ID NOs:243-264.

33. An antigen binding protein comprising a light chain variable domain that is at least about 95% identical to one or more of SEQ ID NOs:235-242.

34. RASSNVX １ X ２ A light chain complementarity determining region (CDR) 1 comprising MY (SEQ ID NO:49); and X ３ X ４ TSX ５ a light chain CDR2 comprising LAS (SEQ ID NO:50); Here, X １ , X ２ , X ３ , X ４ and X ５ are each naturally occurring amino acids; X ２ is Y and X ３ is Y and X ４ is Y and X ５ When is N, X １ is not K; X １ is K and X ３ is Y and X ４ is Y and X ５ When is N, X ２ is not Y; X １ is K and X ２ is Y and X ４ is Y and X ５ When is N, X ３ is not Y; X １ is K and X ２ is Y and X ３ is Y and X ５ When is N, X ４ is not Y; and X １ is K and X ２ is Y and X ３ is Y and X ４ When Y, X ５ is not N, Antigen-binding proteins.

35. Light chain CDR1 is RASSNVX １ X ２ MY (in the formula, X １ is selected from F, W and Y; X ２ is selected from F, W, Y and Q (SEQ ID NO:51); RASSNVX １ X ２ MY (in the formula, X １ is selected from F, W and Y; X ２ is selected from F, W and Y (SEQ ID NO:53); RASSNVX １ X ２ MY (in the formula, X １ is F or Y, and X ２ is selected from F, W and Y (SEQ ID NO:55), and RASSNVX １ X ２ MY (in the formula, X １ is F or Y, and X ２ is F (SEQ ID NO:57), Light chain CDR2 is X ３ X ４ TSX ５ LAS (where X ３ is selected from H and Y; X ４ is selected from H, R and K, and X ５ is a natural amino acid) (SEQ ID NO:52); X ３ X ４ TSX ５ LAS (where X ３ is H, and X ４ is H or R, and X ５ is a natural amino acid) (SEQ ID NO:54); X ３ X ４ TSX ５ LAS (where X ３ is H, and X ４ is H and X ５ is a natural amino acid) (SEQ ID NO:56), and X ３ X ４ TSX ５ LAS (where X ３ is H, and X ４ is H and X ５ is V or D (SEQ ID NO:58), 35. The antigen-binding protein of claim 34.

36. 16. The antigen binding protein of claim 15, further comprising a light chain CDR3 comprising QQFTSSPYT (SEQ ID NO:45).

37. a heavy chain CDR1 comprising GYTFNSYFMH (SEQ ID NO:46) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; A heavy chain CDR2 comprising GMIRPNGGTTDYNEKFKN (SEQ ID NO:47), or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; and A heavy chain CDR3 comprising WEGSYYALDY (SEQ ID NO:48) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions.

37. The antigen binding protein of any one of claims 34 to 36, further comprising:

38. 38. The antigen binding protein of any one of claims 34 to 37, further comprising a heavy chain variable domain that is at least about 90% identical to SEQ ID NO:

3.

39. 38. The antigen binding protein of any one of claims 34 to 37, further comprising a heavy chain variable domain that is at least about 95% identical to SEQ ID NO:

3.

40. 38. The antigen binding protein of any one of claims 34 to 37, further comprising a heavy chain variable domain that is at least about 99% identical to SEQ ID NO:

3.

41. a heavy chain variable domain having at least about 90% identity to SEQ ID NO:3, and A light chain variable domain having at least about 90% identity to SEQ ID NO:

1.

1. An antigen-binding protein comprising:

42. a heavy chain variable domain having at least about 95% identity to SEQ ID NO:3, and A light chain variable domain having at least about 95% identity to SEQ ID NO:

1.

1. An antigen-binding protein comprising:

43. a heavy chain variable domain having at least about 99% identity to SEQ ID NO:3, and A light chain variable domain having at least about 99% identity to SEQ ID NO:

1.

1. An antigen-binding protein comprising:

44. An antigen binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:4-42.

45. An antigen binding protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 11, 12, 14, 18, 19, 20, 21, 22, 24, 27, 28, 31, 38 and 42.

46. An antigen-binding protein comprising the amino acid sequence of SEQ ID NO:

21.

47. An antigen-binding protein comprising the amino acid sequence of SEQ ID NO:

42.

48. 48. A composition for inducing immune tolerance comprising an antigen-binding protein according to any one of claims 34 to 47, further comprising at least one immunogenic tolerogenic antigen or an immunogenic fragment of at least one tolerogenic antigen.

49. 49. The composition of claim 48, comprising at least one immunogenic, tolerance-inducing antigen, wherein the at least one immunogenic, tolerance-inducing antigen comprises at least one of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin proteolipid protein (PLP), one or more fragments of MPB, one or more fragments of MOG, and one or more fragments of PLP.

50. 49. The composition of claim 48, comprising an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 169-175 and 186-202.

51. 49. The composition of claim 48, comprising an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 169, 171-173, 175 and 188-202.

52. 52. A composition according to any one of claims 48 to 51 for use in the treatment of multiple sclerosis.

53. at least one immunogenic, tolerogenic antigen, wherein the at least one immunogenic, tolerogenic antigen is selected from the group consisting of insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65 or glutamic acid decarboxylase 2), GAD-67, glucose-6 phosphatase 2, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), insulinoma-associated protein 2 (IA-2), insulinoma-associated protein 2 beta (IA-2β), ICA69, ICA12, 49. The composition of claim 48, comprising at least one of the following proteins: (SOX-13), carboxypeptidase H, immunogen 38, GLIMA 38, chromogranin A, HSP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocellular carcinoma-intestine-pancreas / pancreatic associated protein, S100β, glial fibrillary acidic protein, regeneration gene II, pancreatic duodenal homeobox 1, myotonic dystrophy kinase, and SST G-protein coupled receptors 1-5.

54. 49. The composition of claim 48, comprising an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 204-214.

55. 55. A composition according to claim 53 or 54 for use in the treatment of type 1 diabetes.

56. 49. The composition of claim 48, comprising at least one immunogenic, tolerogenic antigen, wherein the at least one immunogenic, tolerogenic antigen comprises at least one of tissue transglutaminase, high molecular weight glutenin, low molecular weight glutenin, gluten, alpha-gliadin, gamma-gliadin, omega-gliadin, hordein, secalin, avenin, and deamidated forms thereof.

57. 49. The composition of claim 48, comprising an immunogenic fragment of at least one tolerogenic antigen, wherein the immunogenic fragment of the at least one tolerogenic antigen comprises at least one of SEQ ID NOs: 182-185 and 215.

58. 58. A composition according to any one of claims 56 and 57 for use in the treatment of celiac disease.

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