WO2020089769A1

WO2020089769A1 - Antibodies specifically binding hla-dr/colii_259 complex and their uses

Info

Publication number: WO2020089769A1
Application number: PCT/IB2019/059224
Authority: WO
Inventors: Hong Zhou; Nathan FELIX
Original assignee: Janssen Biotech, Inc.
Priority date: 2018-10-29
Filing date: 2019-10-28
Publication date: 2020-05-07

Abstract

The present invention relates antibodies or antigen-binding fragments thereof specifically binding HLA-DR/ColII_259 complex, polynucleotides encoding the antibodies or fragments, and methods of making and using the foregoing.

Description

ANTIBODIES SPECIFICALLY BINDING HLA-DR/COLII_259 COMPLEX AND THEIR USES

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 18, 2019, is named JBI6012WOPCTl_SL.txt and is 173,489 bytes in size.

FIELD OF THE INVENTION

The present invention relates to antibodies and antigen-binding fragments thereof specifically binding HLA-DR/ColII_259 complexes, polynucleotides encoding the antibodies or antigen-binding fragments, and methods of making and using the foregoing.

BACKGROUND OF THE INVENTION

Pathogenic autoreactive T cells and B cells are key drivers of many autoimmune diseases, including rheumatoid arthritis (RA), type 1 diabetes (T1D), multiple sclerosis (MS), celiac disease (CD), among others. Current therapies for the treatment of autoimmune disease result in broad immunosuppression of treated individuals, increasing the risk for opportunistic infections and cancer immune evasion. Selectively targeting auto-antigen specific cells represents an important opportunity to generate novel therapeutic molecules capable of modulating pathogenic autoreactive T cells and B cells, while sparing protective immunity against infectious agents. One such approach is to target the autoantigen-MHC complex on professional antigen presenting cells (APCs; e.g. dendritic cells, monocytes, etc.) with highly specific antibodies that bind to autoantigen peptide-MHC complexes. Because autoantigen-MHC complexes will be mainly localized to the site(s) of disease, pMHC- specific antibodies can be used to selectively target and modulate autoantigen expressing APCs, while sparing APCs (and immune responses) throughout the rest of the body.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated antibody or an antigen-binding fragment thereof specifically binding a HLA-DR/ColII_259 complex. The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding a HLA-DR/ColII_259 complex having one or more of the following properties:

binds the HLA-DR ColII_259 complex with an equilibrium dissociation constant

(K_D) of less than about 5xl0 ⁸ M; or

inhibits activation of HLA-DR/ColII_259 specific T cells.

The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding a HLA-DR/ColII_259 complex comprising certain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VL and/or VL amino acid sequences as described herein.

The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding a HLA-DR ColII_259 complex conjugated to a second molecule.

The invention also provides a pharmaceutical composition comprising the isolated antibody or an antigen-binding fragment thereof specifically binding a HLA- DR/ColII_259 complex of the invention and a pharmaceutically accepted carrier.

The invention also provides a polynucleotide encoding the VH, the VL or the VH and the VL of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO:

103 or SEQ ID NO: 104; or comprising the polynucleotide sequence of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113 or SEQ ID NO: 114.

The invention also provides a vector comprising the polynucleotide of the invention.

The invention also provides a host cell comprising the vector of the invention.

The invention also provides a method of producing the antibody or the antigenbinding fragment thereof of the invention comprising culturing the host cell of the invention in conditions that the antibody is expressed, and recovering the antibody produced by the host cell.

The invention also provides a method of treating or preventing a HLA- DR/ColII_259 complex-mediated disease, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or the antigen-binding fragment thereof of the invention for a time sufficient to treat the autoimmune disease.

The invention also provides a method of suppressing an immune response towards ColII_259 presented on HLA-DR in a subject, comprising administering to a subject the antibody or the antigen-binding fragment thereof of the invention or the pharmaceutical composition of the invention for a time sufficient to suppress the immune response towards the self-antigen.

The invention also provides an anti-idiotypic antibody binding to the antibody or the antigen-binding fragment thereof of the invention.

The invention also provides a kit comprising the antibody or the antigen-binding fragment of the invention.

The invention also provides a method of detecting cells expressing on their surface a HLA-DR/ColII_259 complex in a biological sample, comprising

obtaining the biological sample;

contacting the biological sample with the antibody or an antigen-binding fragment of the invention; and

detecting the antibody bound on the cells expressing on their surface the HLA- DR/ColII_259 complex.

The invention also provides a method of isolating or detecting cells expressing on their surface a HLA-DR/ColII_259 complex in a biological sample, comprising

obtaining a biological sample from a subject;

contacting the biological sample with the antibody or the antigen-binding fragment of the invention; and

isolating or detecting the cells bound to the antibody or the antigen-binding fragment of the invention.

The invention also provides a method of detecting cells expressing on their surface a HLA-DR/ColII_259 complex in a subject, comprising

conjugating the antibody or the antigen-binding fragment of the invention to a detectable label to form a conjugate;

administering the conjugate to the subject; and

visualizing in the subject the cells expressing on their surface the HLA- DR/ColII_259 complex to which the conjugate is bound.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows the alignment of DR4B668 and DR4B 1544 VH chains DR4H771 (SEQ ID NO: 139) and DR4H887 (SEQ ID NO: 140), respectively, and their consensus VH sequence. AbM CDRs are underlined. Figure IB shows the alignment of DR4B668 and DR4B1544 VL chains DR4L334 (SEQ ID NO: 100) and DR4L436 (SEQ ID NO: 101), respectively, and their consensus VL sequence. AbM CDRs are underlined.

Figure 2A shows the alignment of DR4B683 and DR4B706 VH chains DR4H786 (SEQ ID NO: 141) and DR4H812 (SEQ ID NO: 142), respectively, and their consensus VH sequence. AbM CDRs are underlined.

Figure 2B shows the alignment of DR4B683 and DR4B706 VL chains DR4L347 (SEQ ID NO: 102) and DR4L372 (SEQ ID NO: 103), respectively, and their consensus VL sequence. AbM CDRs are underlined.

Figure 3A shows the dose response curve for binding of DR4B683, DR4B706, DR4B668, DR4B1544 and L243 to HLA-DR4/ColII_257_273 complex.

Figure 3B shows the dose response curve for binding of DR4B683, DR4B706, DR4B668, DR4B1544 and L243 to HLA-DR4/CII_1236_1249 complex (CII_1236_1249 is CII_1236 in the Figure).

Figure 3C shows the dose response curve for binding of DR4B683, DR4B706, DR4B668, DR4B1544 and L243 to HLA-DR4/CLIP complex.

Figure 3D shows the dose response curve for binding of DR4B683, DR4B706, DR4B668, DR4B1544 and L243 to HLA-DR4/HA complex.

Figure 4A shows the dose response curve for binding of DR4B683, DR4B706, DR4B668, DR4B1544 and L243 to HLA-DRl/ColII_257_273 complex.

Figure 4B shows the dose response curve for binding of DR4B683, DR4B706, DR4B668, DR4B1544 and L243 to HLA-DR1/CII_1236_1249 complex (CII_1236_1249 is CII_1236 in the Figure).

Figure 4C shows the dose response curve for binding of DR4B683, DR4B706, DR4B668, DR4B1544 and L243 to HLA-DR1/CLIP complex.

Figure 4D shows the dose response curve for binding of DR4B683, DR4B706, DR4B668, DR4B1544 and L243 to HLA-DR1/HA complex.

Figure 5 shows the dose-response curve of inhibition of IL-2 production by HLA- DR/ColII_259 specific T cells by DR4B668, DR4B683 and DR4B695. The control pan-DR antibody L243 also inhibited IL-2 production while other test antibodies (DR4B583, DR4B588, DR4B669, and isotype control PP1B40) did not inhibit IL-2 production.

Figure 6A shows that DR4B668, DR4B683 and DR4B695 did not inhibit IL-2 production by HLA-DR/HA specific T cells whereas L243 demonstrated inhibition.

Figure 6B shows that DR4B668, DR4B683 and DR4B695 did not inhibit IL-2 production by HLA-DR/CILP specific T cells whereas L243 demonstrated inhibition. Figure 6C shows that DR4B668, DR4B683 and DR4B695 did not inhibit IL-2 production by HLA-DR/fibrinogen specific T cells whereas L243 demonstrated inhibition.

Figure 7A shows that DR4B683, DR4B695 and DR4B706 bind and detect ColII_259-loaded Boleth cells (e.g. HLA-DR4/ColII_259 complexes) in antibody dose-dependent manner. Figure 7B shows that DR4B683, DR4B695 and DR4B706 demonstrated minimal binding to Boleth cells not loaded with ColII_259 (e.g. expressing endogenous peptide HLA-DR4 complexes)

Figure 8 shows antibody concentration over time in blood of DR4 transgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

As used in this specification and the appended claims, the singular forms“a,”

“an,” and“the” include plural referents unless the content clearly dictates otherwise.

Thus, for example, reference to“a cell” includes a combination of two or more cells, and the like.

“Specific binding”,“specifically binds”,“specifically binding” or“binds” refer to an antibody binding to an antigen or an epitope within the antigen with greater affinity than for other antigens. Typically, the antibody binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (K_D) of about lxlO ⁷ M or less, for example about 5xl0 ⁸ M or less, about lxlO ⁸ M or less, about lxlO ⁹ M or less, about lxlO ¹⁰ M or less, about lxlO ¹¹ M or less, or about lxlO ¹² M or less, typically with the K_D that is at least one hundred fold less than its K_D for binding to a non-specific antigen (e.g., BSA, casein). The dissociation constant may be measured using standard procedures. Antibodies that specifically bind to the antigen or the epitope within the antigen may, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus (common marmoset, marmoset). While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two distinct antigens or two distinct epitopes.

“Antibody specifically binding a HLA-DR/ColII_259 complex” refers to an antibody specifically binding HLA-DR that is in complex with collagen II peptide ColII_259. HLA-DR may be HLA-DR4 composed of for example HLA-DRA1 *01:01 a chain (SEQ ID NO: 144) or HLA-DRAl*01:02 a chain (SEQ ID NO: 15) and a HLA- DRB1*04:01 b chain (SEQ ID NO: 16). ColII_259 peptide comprises an amino acid sequence of SEQ ID NO: 123. As various HLA-DR proteins are encoded by allelic variants of the genes encoding HLA-DR a and HLA-DR b chains, the antibodies specifically binding the HLA-DR/ColII_259 complex also specifically bind complexes other than those containing HLA-DR4, such as such HLA-DR1, HLA-DR3, HLA-DR10, HLA-DR 13 and HLA-DR 16

“Antibodies” is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen-binding fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. “Full length antibody molecules” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant domain, the heavy chain constant domain comprised of subdomains CHI, hinge, CH2 and CH3. Each light chain is comprised of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and the VL may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino- to-carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

“Complementarity determining regions (CDR)” are antibody regions that bind an antigen. There are three CDRs in the VH (HCDR1, HCDR2, HCDR3) and three CDRs in the VL (LCDR1, LCDR2, LCDR3). CDRs may be defined using various delineations such as Rabat (Wu et al. (1970) J Exp Med 132: 211-50) (Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196: 901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27: 55-77) and AbM (Martin and Thornton J Bmol Biol 263 : 800-15, 1996). The correspondence between the various delineations and variable region numbering are described (see e.g. Lefranc et al. (2003) Dev Comp Immunol 27: 55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-70; International ImMunoGeneTics (IMGT) database; Web resources, http://www_imgt_org). Available programs such as abYsis by UCL Business PLC may be used to delineate CDRs. The term“CDR”,“HCDR1”,“HCDR2”,“HCDR3”,“LCDR1”,“LCDR2” and “LCDR3” as used herein includes CDRs defined by any of the methods described supra, Rabat, Chothia, IMGT or AbM, unless otherwise explicitly stated in the specification.

Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant region amino acid sequence. IgA and IgG are further sub-classified as isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (K) and lambda (l), based on the amino acid sequences of their constant domains.

“Antigen-binding fragment” refers to a portion of an immunoglobulin molecule that retains the antigen binding properties of the parental full length antibody. Exemplary antigen-binding fragments are heavy chain complementarity determining regions (HCDR) 1, 2 and/or 3, light chain complementarity determining regions (LCDR) 1, 2 and/or 3, the VH, the VL, the VH and the VL, Fab, F(ab')2, Fd and Fv fragments as well as domain antibodies (dAb) consisting of either one VH domain or one VL domain. The VH and the VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs in which the VH/VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate chains, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Pat. Publ. No. W01998/44001, Int. Pat. Publ. No. WO1988/01649; Int. Pat. Publ. No. WO1994/13804; Int. Pat. Publ. No. W01992/01047.

“Monoclonal antibody” refers to an antibody obtained from a substantially homogenous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known alterations such as removal of C-terminal lysine from the antibody heavy chain or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation. Monoclonal antibodies typically bind one antigenic epitope. A bispecific monoclonal antibody binds two distinct antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population.

Monoclonal antibody may be monospecific or multispecific such as bispecific, monovalent, bivalent or multivalent.

“Isolated” refers to a homogenous population of molecules (such as synthetic polynucleotides or a protein such as an antibody) which have been substantially separated and/or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. “Isolated antibody” refers to an antibody that is substantially free of other cellular material and/or chemicals and encompasses antibodies that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

“Humanized antibody” refers to an antibody in which at least one CDR is derived from non-human species and at least one framework is derived from human

immunoglobulin sequences. Humanized antibody may include substitutions in the frameworks so that the frameworks may not be exact copies of expressed human immunoglobulin or human immunoglobulin germline gene sequences.

“Human antibody” refers to an antibody that is optimized to have minimal immune response when administered to a human subject. Variable regions of human antibody are derived from human immunoglobulin sequences. If human antibody contains a constant region or a portion of the constant region, the constant region is also derived from human immunoglobulin sequences. Human antibody comprises heavy and light chain variable regions that are“derived from” sequences of human origin if the variable regions of the human antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci. “Human antibody” typically contains amino acid differences when compared to the immunoglobulins expressed in humans due to differences between the systems used to obtain the human antibody and human immunoglobulin loci, introduction of somatic mutations or intentional introduction of substitutions into the frameworks or CDRs, or both. Typically, “human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to an amino acid sequence encoded by human germline immunoglobulin or rearranged immunoglobulin genes. In some cases,“human antibody” may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et ak, (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al., (2010) J Mol Biol 397:385-96, and in Int. Patent Publ. No. W02009/085462.

Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of“human antibody”.

“Recombinant” refers to DNA, antibodies and other proteins that are prepared, expressed, created or isolated by recombinant means when segments from different sources are joined to produce recombinant DNA, antibodies or proteins.“Recombinant antibody” includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, or antibodies that are generated in vitro using Fab arm exchange such as bispecific antibodies.

"Epitope" refers to a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3- dimensional space through the folding of the protein molecule.

“Paratope” refers to a portion of an antibody to which an antigen specifically binds. A paratope may be linear in nature or may be discontinuous, formed by a spatial relationship between non-contiguous amino acids of an antibody rather than a linear series of amino acids. A“light chain paratope” and a“heavy chain paratope” or“light chain paratope amino acid residues” and“heavy chain paratope amino acid residues” refer to antibody light chain and heavy chain residues in contact with an antigen, respectively, or in general,“antibody paratope residues” refer to those antibody amino acids that are in contact with antigen. “Bispecific” refers to an antibody that specifically binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific antibody may have crossreactivity to other related antigens or can bind an epitope that is shared between two or more distinct antigens.

“Multispecific” refers to an antibody that specifically binds at least two distinct antigens or at least two distinct epitopes within the same antigen. Multispecific antibody may bind for example two, three, four or five distinct antigens or distinct epitopes within the same antigen.

“Polynucleotide” refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a synthetic polynucleotide.

“Polypeptide” or“protein” refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide.

“Peptide” refers to a short polypeptide up to 30 amino acids long.

“Variant” refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications, for example one or more substitutions, insertions or deletions.

“Vector” refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers that function to facilitate the duplication or maintenance of these polynucleotides in a biological system, such as a cell, vims, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The vector polynucleotide may be DNA or RNA molecules or a hybrid of these, single stranded or double stranded.

“Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

“About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result or embodiment,“about” means within one standard deviation per the practice in the art, or a range of up to 5%, whichever is larger. “Biological sample” refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary biological samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites fluids, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium, lavage fluids and the like, tissue biopsies, fine needle aspirations, surgically resected tissue, organ cultures or cell cultures.

“In combination with” means that two or more therapeutics are administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order.

"Antagonist" or“inhibitor” or“inhibits” refers to a molecule that, when bound to a cellular protein or internalized by a cell suppresses at least one reaction or activity that is induced by a natural ligand of the protein. A molecule is an antagonist when the at least one reaction or activity is suppressed by at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than the at least one reaction or activity suppressed in the absence of the antagonist (e.g., negative control), or when the suppression is statistically significant when compared to the suppression in the absence of the antagonist. An exemplary antagonist is an antibody specifically binding a HLA- DR/ColII_259 complex that by blocking interaction of antigen-presenting cell (APC) and cognate T cell thereby inhibiting activation of the cognate T cells. In some instances, the antibody specifically binding the HLA-DR/ColII_259 complex may be conjugated to an inhibitory molecule and upon internalization into APCs may reduce the ability of APCs to activate an immune response or increase the ability of the APCs to suppress an immune response.

“Subject” includes any human or nonhuman animal. “Nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms“patient” or“subject” are used interchangeably.

“Treat” or“treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder. Beneficial or desired clinical results include alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

“Treatment” can also mean prolonging survival as compared to expected survival if a subject was not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

“Therapeutically effective amount” refers to an amount effective, at doses and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic or combination of therapeutics that include, for example, improved well-being of the patient

“Human leukocyte antigen HLA-DR” or“HLA-DR” refers to a major histocompatibility complex (MHC) class II cell surface receptor. HLA-DR is a heterodimer of a and b chains with each subunit spanning the membrane once. HLA-DR a chain is encoded by HLA-DRA1 and HLA-DR b chain is encoded by HLA-DRB1 or one of its paralogues HLA-DRB3, HLA-DRB4, or HLA-DRB5. HLA-DRB1, as is well known, is hyperpolymorphic. Nomenclature, cDNA and amino acid sequences of various HLA- DR a and HLA-DR b chains are well known. For example, the international

ImMunoGeneTics information system® (IMGT®) database provides the amino acid sequences of the proteins encoded by HLA-DRA1 and HLA-DRB as well as their amino acid alignments. HLA Nomenclature provides HLA gene and protein sequences and statistics for HLA allele numbers that can be found at Http:_/_hla_alleles_org and cited in Robinson et ai, Nucleic Acids Research (2015) 43:D423-431 and arch el al, Tissue Antigens (2010) 75:291-455.

“HLA-DR4” or“DR4” refers to particular HLA antigens within serological group 4. HLA-DR4 a chain is encoded by HLA-DRA1 *01 and HLA-DR4 b chain is encoded by HLA-DRB 1 *04. HLA-DRB 1 *04 is polymorphic and encodes various variants including HLA-DRB 1*04:01, HLA-DRB 1*04:02, HLA-DRB1*04:03, HLA-DRB 1*04:04, HLA- DRB 1*04:05, etc, well known to those in the field.

“HLA-DRl” or“DR1” refers to particular HLA antigens within serological group 1. HLA-DRl a chain is encoded by HLA-DRA1 *01 and HLA-DRl b chain is encoded by the HLA-DRB 1 *01 gene. HLA-DRB 1 *01 is polymorphic and encodes various variants including HLA-DRB 1*01:01, HLA-DRB 1*01:02, HLA-DRB1*01:03, HLA- DRB 1*01:04, HLA-DRB 1*01:05, etc, well known to those in the field.

“HLA-DR3” or“DR3” refers to particular HLA antigens within serological group 3. HLA-DR3 a chain is encoded by HLA-DRA1 *01 and HLA-DR3 b chain is encoded by the HLA-DRB 1 * 03 gene. HLA-DRB 1 * 03 is polymorphic and encodes various variants including HLA-DRB 1 *03:01 , HLA-DRB 1*03:02, HLA-DRB1*03:03, HLA- DRB 1*03:04, HLA-DRB 1*03:05, etc, well known to those in the field.

“HLA-DR10” or“DR 10” refers to particular HLA antigens within serological group 10. HLA-DR10 a chain is encoded by HLA-DRA1 *01 and HLA-DR10 b chain is encoded by the HLA-DRB 1 *10 gene. HLA-DRB 1 *10 is polymorphic and encodes various variants including HLA-DRB 1*10:01, HLA-DRB1*10:02, HLA-DRB 1*10:03, HLA- DRB 1*10:04, HLA-DRB 1*10:05, etc, well known to those in the field.

“HLA-DR15” or“DR15” refers to particular HLA antigens within serological group 15. HLA-DR15 a chain is encoded by HLA-DRA1 *01 and HLA-DR15 b chain is encoded by the HLA-DRB 1 *15 gene. HLA-DRB 1 *15 is polymorphic and ecodes various HLA-DRB 1 proteins including HLA-DRB1*15:01, HLA-DRB1* 15:02, HLA- DRB1*15:03, HLA-DRB 1*15:04, HLA-DRB1*15:05, etc, well known to those in the field.

“HLA-DR” refers to human HLA-DR molecules unless explicitly noted otherwise in the specification.

“ColII_259” refers to a peptide derived from collagen II having an amino acid sequence shown in SEQ ID NO: 123.

SEQ ID NO: 123

GIAGFKGEQGPKGEP

“In complex” or“complexed” refers to the complex of HLA-DR a chain, HLA- DR b chain and one peptide residing in the well-known peptide binding groove in the HLA-DR molecule. In vivo, the peptide/ HLA-DR interaction is non-covalent. In vitro, the peptide may be covalently coupled for example to the N-terminus of the b chain. Therefore,“in complex” encompasses HLA-DR complexes with both non-covalently and covalently bound peptides. “T cell activation” refers to one or more cellular responses of a T cell, for example a CD4⁺ T cell, such as proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity and expression of activation markers.

The numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU index as described in Kabat el al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly stated.

Conventional one and three-letter amino acid codes are used herein as shown in Table 1.

Table 1.

Composition of matter - antibodies of the invention

Class II MHC is the single most significant genetic risk factor to developing rheumatoid arthritis (RA), and HLA-DRB1*04:01 (HLA-DR4) is the most prevalent risk allele for RA in people of European descent. One of the most highly studied putative autoantigens in RA is the collagen II peptide (ColII_259). This autoantigen forms strong, stable complexes with HLA-DR4, and induces a notable T cell response in RA patients during disease flares. The invention provides isolated antibodies specifically binding a HLA-DR/ColII_259 complex, polynucleotides encoding the antibodies, vectors, host cells, and methods of making and using the antibodies. The antibodies generated selectively bind the HLA-DR/ColII_259 complex and do not bind HLA-DR in complex with other autoantigenic peptides, thereby providing selective inhibition of T cell activation, restricting inhibition to T cells recognizing the HLA-DR/ColII_259 complex. The known anti-DR4 antibodies L243, Lym-1 and apolizumab are pan-DR binders recognizing HLA-DR independent of the peptide presented and hence inhibiting a broad spectrum of T cells. The antibodies of the invention described herein therefore provide a solution of selective and more safe inhibition pathways mediated by T cell recognition of HLA-DR/ColII_259.

The invention provides an isolated antibody or an antigen-binding fragment thereof specifically binding a HLA-DR/ColII_259 complex.

The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding a HLA-DR/ColII_259 complex, wherein the antibody or the antigen-binding fragment thereof has one or more of the following properties:

binds the HLA-DR/ColII_259 complex with an equilibrium dissociation constant

(K_D) of less than about 5xl0 ⁸ M; or

inhibits activation of HLA-DR/ColII_259 specific T cells.

Exemplary such antibodies are antibodies DR4B668, DR4B1544, DR4B683, DR4B706 and DR4B695. The CDR, the VH and the VL amino acid sequences of the antibodies are shown in Table 2 and Table 3.

The affinity of an antibody or the antigen-binding fragment thereof to the HLA- DR/ColII_259 complex may be determined experimentally using any suitable method.

Such methods may utilize ProteOn XPR36, Biacore 3000 or KinExA instrumentation,

ELISA or competitive binding assays known to those skilled in the art. The measured affinity of the interaction may vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other binding parameters (e.g., K_D,

K_on, K_ofi) are typically made with standardized conditions and a standardized buffer, such as the buffer described herein. The internal error for affinity measurements for example using Biacore 3000 or ProteOn (measured as standard deviation, SD) may typically be within 5-33% for measurements within the typical limits of detection. Therefore the term “about” in the context of K_D reflects the typical standard deviation in the assay. For example, the typical SD for a K_D of lxlO ⁹ M is up to +0.33xl0 ⁹M.

The HLA-DR molecules used in the experiments described herein may be expressed as soluble Fc- fusion proteins. Collagen II peptide or another antigenic peptide that is presented on HLA-DR may be covalently coupled to the N-terminus of the HLA- DR b chain to facilitate expression. Tags such as hexahistidine (SEQ ID NO: 3) or StrepII tag (SEQ ID NO: 6) may be covalently linked to the a and/or b chain or to the Fc to facilitate purification of the expressed protein. Linkers may be inserted between the presented peptide, a and/or b chain, the Fc portion and/or the various tags. Suitable linkers may be a glycine/serine linker (SEQ ID NO: 1 or 4), tobacco etch vims Nia protease cleavage site (SEQ ID NO: 2), or human rhinovirus 3C protease cleavage site (SEQ ID NO: 5). Collagen II peptide that may be presented on HLA-DR may be CII 257- 273 (SEQ ID NO: 9) or CII 259 (SEQ ID NO: 123). Other suitable peptides that may be presented on HLA-DR that may be used are hemagglutinin peptide HA 304-318 (SEQ ID NO: 7), collagen II peptides CII_1236-1249 (SEQ ID NO: 8), CLIP peptide (SEQ ID NO: 11), LCAP peptide (SEQ ID NO: 12), CILP peptide (SEQ ID NO: 127) or fibrinogen peptide (SEQ ID NO: 128). Exemplary HLA-DR molecules that may be expressed may have following configurations:

a chain: extracellular domain or the particular a chain, linker of SEQ ID NO: 1, protease cleavage site of SEQ ID NO: 2, linker of SEQ ID NO: 1, Fc domain, tag of SEQ ID NO: 3 b chain: peptide of SEQ ID NO: 9, linker of SEQ ID NO: 4, protease cleavage site of SEQ ID NO: 5, extracellular domain of the particular b chain, linker of SEQ ID NO: 4, Fc domain, tag of SEQ ID NO: 6. The a and b chains are co-expressed, and the resulting heterodimers may be purified for example using the His6 (SEQ ID NO: 3) and StrepII tags using standard methods. HLA-DP and HLA-DQ molecules may be similarly expressed.

Activation of HLA-DR-collagen II 259 specific T cells may be measured using assays described herein. For example, a B cell line Boleth homozygous for HLA- DRB1*04:01 may be incubated in the presence of collagen II 259 peptide, after which cognate T cells are added, and B-cell mediated activation of the cognate T cells measured. Readout for T cell activation may be T cell proliferation, differentiation, cytokine production, such as IL-2 production, cytotoxic effector molecule release, cytotoxic activity or expression of activation markers. T cell may be a CD4⁺ T cell. T cell activation may be inhibited by 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the isotype control, or is inhibited in a statistically significant manner when compared to inhibition in the presence of an isotype control. “Isotype control” is well known.

Table 2.

Table 3.

The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex having certain CDR, VL and VH amino acid sequences as shown in Table 2 and Table 3.

The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex, wherein the antibody or the antigen-binding fragment thereof binds HLA-DR a chain of SEQ ID NO: 15 within amino acid residues of SEQ ID NO: 119 and SEQ ID NO: 120. The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex, wherein the antibody or the antigen-binding fragment thereof binds HLA-DR a chain of SEQ ID NO: 15 within amino acid residues of SEQ ID NO: 120 and HLA-DR b chain of SEQ ID NO: 16 within amino acid residues of SEQ ID NO: 121.

The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex, wherein the antibody or the antigen-binding fragment thereof binds HLA-DR a chain of SEQ ID NO: 15 within amino acid residues of SEQ ID NO: 120.

Within” means that at least 50% of the epitope residues the antibody binds to reside within the recited amino acid stretches, and that up to 50% of the epitope residues the antibody binds to reside outside of the recited amino acid stretches.

The epitope the antibody binds to may be resolved for example using hydrogen/deuterium exchange (H/D exchange) or by analyzing a crystal structure of the antibody in complex with HLA-DR/ColII_259. The epitope residues are those which are protected by the antibody by at least 5% difference in deuteration levels through H/D exchange or those surface exposed amino acid residues determined to bind the antibody in a crystal structure of a complex of the antibody and HLA-DR/ColII_259. In the crystal structure of a complex of the antibody and HLA-DR ColII_259, the epitope residues are those HLA-DR/ColII_259 residues that reside within 4 A distance or less from any of the antibody CDR residues.

In an H/D exchange assay, HLA-DR ColII_259 protein is incubated in the presence or absence of the antibody in deuterated water for predetermined times resulting in deuterium incorporation at exchangeable hydrogen atoms which are unprotected by the antibody, followed by protease digestion of the protein and analyses of the peptide fragments using LC-MS. In an exemplary assay, 5 pL of the test antibody ( 10 pg) or 5 pL of the complex of HLA-DR ColII_259 and the test antibody (10 & 7.35 pg, respectively) is incubated with 120 pL deuterium oxide labeling buffer (50mM phosphate, lOOmM sodium chloride at pH 7.4) for 0 sec, 60 sec, 300 sec, 1800 sec, 7200 sec, and 14400 sec. Deuterium exchange is quenched by adding 63 pL of 5 M guanidine hydrochloride and final pH is 2.5. The quenched sample is subjected to on-column pepsin/protease type XIII digestion and LC-MS analysis. For pepsin/protease type XIII digestion, 5 pg of the samples in 125 pL control buffer (50mM phosphate, lOOmM sodium chloride at pH 7.4) are denatured by adding 63 pL of 5 M guanidine hydrochloride (final pH is 2.5) and incubating the mixture for 3 min. Then, the mixture is subjected to on- column pepsin/protease type XIII digestion and the resultant peptides analyzed using an UPLC-MS system comprised of a Waters Acquity UPLC coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo). Raw MS data is processed using HDX WorkBench, software for the analysis of H/D exchange MS data. The deuterium levels are calculated using the average mass difference between the deuteriated peptide and its native form (to). Peptide identification is done through searching MS/MS data against the HLA-DR ColII_259 sequence with Mascot. The mass tolerance for the precursor and product ions is 20 ppm and 0.05 Da, respectively.

For X-ray crystallography, HLA-DR/ColII_259 and the test antibody are expressed and purified using standard protocols. The HLA-DR/ColII_259/test antibody complex is incubated overnight at 4°C, concentrated, and separated from the uncomplexed species using size-exclusion chromatography. The complex is crystallized by the vapor- diffusion method from various known test solutions for example solutions containing PEG3350, ammonium citrate and 2-(N-Morpholino)ethanesulfonic acid (MES).

Antibodies binding within the specified residues on HLA-DR/ColII_259 may be generated by isolating antibodies binding HLA-DR/ColII_259 using phage display libraries, selecting those antibodies that compete with the reference antibody such as DR4B668, DR4B1544, DR4B683, DR4B706 and DR4B695, for binding to HLA- DR/ColII_259 by 100%, and confirming the epitope of the generated antibodies by solving the crystal structure of the antibody/ HLA-DR ColII_259 complex. Alternatively, mice, rats or rabbits may be immunized using the peptides encompassing the identified epitopes and the generated antibodies may be evaluated for their binding within the recited region.

DR4B668 lineage antibodies

The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding a HLA-DR ColII_259 complex comprising

a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 63 or SEQ ID NO: 73;

a HCDR2 or SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 66, SEQ ID NO: 76 or SEQ ID NO: 58;

a HCDR3 of SEQ ID NO: SEQ ID NO: 50, SEQ ID NO: 69 or SEQ ID NO: 79; a light chain complementarity determining region 1 (LCDR1) or SEQ ID NO: 82, SEQ ID NO: 90 or SEQ ID NO: 97;

a LCDR2 of SEQ ID NO: 85 or the amino acid sequence YTS; and

a LCDR3 of SEQ ID NO: 88 or SEQ ID NO: 95.

Exemplary antibodies comprising the CDRs recited are DR4B668 and DR4B1544.

In some embodiments, HLA-DR comprises a HLA-DR a chain of SEQ ID NO:

15.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof binds HLA-DR a chain of SEQ ID NO: 15 within amino acid residues of SEQ ID NO: 119 and SEQ ID NO: 120.

The epitope the antibodies of the invention bind to may be resolved for example using hydrogen/deuterium exchange (H/D exchange) or by analyzing a crystal structure of the antibody in complex with the HLA-DR/ColII complex. In an H/D exchange assay, the HLA-DR/ColII_259 complex is incubated in the presence or absence of the antibody in deuterated water for predetermined times resulting in deuterium incorporation at exchangeable hydrogen atoms which are unprotected by the antibody, followed by protease digestion of the protein and analyses of the peptide fragments using LC-MS using protocols described herein.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex comprises a heavy chain variable region (VH) of SEQ ID NO: 115 and a light chain variable region (VL) of SEQ ID NO 116.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of

SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 82, SEQ ID NO: 85 and SEQ ID NO: 88, respectively;

SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 50, SEQ ID NO: 82, SEQ ID NO: 85 and SEQ ID NO: 88, respectively;

SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 90, the amino acid sequence YTS and SEQ ID NO: 95, respectively;

SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 97, the amino acid sequence YTS and SEQ ID NO: 88, respectively; SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 82, SEQ ID NO:

85 and SEQ ID NO: 88, respectively;

SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 50, SEQ ID NO: 82, SEQ ID NO:

85 and SEQ ID NO: 88, respectively;

SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 90, the amino acid sequence YTS and SEQ ID NO: 95, respectively; or

SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 97, the amino acid sequence YTS and SEQ ID NO: 88, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the VH and the VL of SEQ ID NO: 139 and SEQ ID NO: 100, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the VH and the VL encoded by polynucleotides of SEQ ID NO: 105 and SEQ ID NO: 110, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the VH and the VL of SEQ ID NO: 140 and SEQ ID NO: 101, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the VH and the VL encoded by polynucleotides of SEQ ID NO: 106 and SEQ ID NO: 111, respectively.

In some embodiments, HLA-DR is HLA-DR4.

In some embodiments, HLA-DR is HLA-DR1.

In some embodiments, HLA-DR a chain and HLA-DR b chain comprise amino acid sequences of

SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or

SEQ ID NO: 15 and SEQ ID NO: 17, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex is a humanized antibody.

In some embodiments, the isolated antibody specifically binding the HLA- DR/ColII_259 complex comprises a heavy chain (HC) of SEQ ID NO: 129 and a light chain (LC) of SEQ ID NO: 130.

In some embodiments, the isolated antibody specifically binding the HLA- DR/ColII_259 complex comprises the HC of SEQ ID NO: 131 and the LC of SEQ ID NO: 132. DR4B683 lineage

The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding a HLA-DR/ColII_259 complex, comprising

a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 64 or SEQ ID NO: 74;

a HCDR2 or SEQ ID NO: SEQ ID NO: 48, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 77 or SEQ ID NO: 60;

a HCDR3 of SEQ ID NO: SEQ ID NO: 51, SEQ ID NO: 70 or SEQ ID NO: 80; a light chain complementarity determining region 1 (LCDR1) or SEQ ID NO: 83,

SEQ ID NO: 91 or SEQ ID NO: 98;

a LCDR2 of SEQ ID NO: SEQ ID NO: 86 or the amino acid sequence LAS; and a LCDR3 of SEQ ID NO: SEQ ID NO: 89 or SEQ ID NO: 96.

In some embodiments, HLA-DR comprises a HLA-DR a chain of SEQ ID NO:

15 and a HLA-DR b of SEQ ID NO: 16.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex binds HLA-DR a chain of SEQ ID NO: 15 within amino acid residues of SEQ ID NO: 120 and HLA-DR b chain of SEQ ID NO: 16 within amino acid residues of SEQ ID NO: 121.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises a heavy chain variable region (VH) of SEQ ID NO: 117 and a light chain variable region (VL) of SEQ ID NO 118.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of

SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO: 86 and SEQ ID NO: 89, respectively;

SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO: 86 and SEQ ID NO: 89, respectively;

SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 91, the amino acid sequence LAS and SEQ ID NO: 96, respectively;

SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 98, the amino acid sequence LAS and SEQ ID NO: 89, respectively; or SEQ ID NO: 55, SEQ ID NO: 60, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO: 86 and SEQ ID NO: 89, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the VH and the VL of SEQ ID NOs: 141 and 102, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the VH and the VL encoded by polynucleotides of SEQ ID NO: 107 and SEQ ID NO: 112, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the VH and the VL of SEQ ID NO: 142 and SEQ ID NO: 103, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the VH and the VL encoded by polynucleotides of SEQ ID NO: 108 and SEQ ID NO: 113, respectively.

In some embodiments, HLA-DR is HLA-DR4.

In some embodiments, HLA-DR is HLA-DR1.

SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or

SEQ ID NO: 15 and SEQ ID NO: 17, respectively.

In some embodiments, the isolated antibody specifically binding the HLA- DR/ColII_259 complex comprises the HC of SEQ ID NO: 133 and the LC of SEQ ID NO: 134.

In some embodiments, the isolated antibody specifically binding the HLA- DR/ColII_259 complex comprises the HC of SEQ ID NO: 135 and the LC of SEQ ID NO: 136.

DR4B695 lineage

The invention also provides an isolated antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprising

a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 65 or SEQ ID NO: 75;

a HCDR2 or SEQ ID NO: 49, SEQ ID NO: 61, SEQ ID NO: 68 or SEQ ID NO: 78; a HCDR3 of SEQ ID NO: 52, SEQ ID NO: 71 or SEQ ID NO: 81;

a light chain complementarity determining region 1 (LCDR1) or SEQ ID NO: 84, SEQ ID NO: 92 or SEQ ID NO: 99;

a LCDR2 of SEQ ID NO: SEQ ID NO: 87 or the amino acid sequence LAS; and a LCDR3 of SEQ ID NO: 89 or SEQ ID NO: 96.

In some embodiments, HLA-DR comprises a HLA-DR a chain of SEQ ID NO: 15.

In some embodiments, the isolated antibody or the antigembinding fragment thereof specifically binding the HLA-DR/ColII_259 complex binds HLA-DR a chain of SEQ ID NO: 15 within amino acid residues of SEQ ID NO: 120.

SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 84, SEQ ID NO: 87 and SEQ ID NO: 89, respechvely;

SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 52, SEQ ID NO: 84, SEQ ID NO: 87 and SEQ ID NO: 89, respechvely;

SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 92, the amino acid sequence LAS and SEQ ID NO: 96, respechvely; or

SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 99, the amino acid sequence LAS and SEQ ID NO: 89, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the VH and the VL of SEQ ID NOs 143 and SEQ ID NO: 104, respectively.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises the VH and the VL encoded by polynucleohdes of SEQ ID NO: 109 and SEQ ID NO: 114, respectively.

In some embodiments, HLA-DR is HLA-DR4.

In some embodiments, HLA-DR is HLA-DR1.

SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or

SEQ ID NO: 15 and SEQ ID NO: 17, respectively. In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex is a humanized antibody.

In some embodiments, the isolated antibody specifically binding the HLA- DR/ColII_259 complex comprises the HC of SEQ ID NO: 137 and the LC of SEQ ID NO: 138.

Homologous antibodies and antibodies with conservative mutations

Variants of the isolated antibodies or antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex provided herein are within the scope of the invention. For example, variants may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acid substitutions in the VH and/or the VL as long as the variant antibodies retain or have improved functional properties when compared to the parental antibodies. In some embodiments, the sequence identity may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the VH and/or the VL amino acid sequence of the invention. In some embodiments, the variation is in the framework regions. In some embodiments, variants are generated by conservative substitutions.

Also provided are isolated antibodies or antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex comprising the VH and the VL which are at least 80% identical to

the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 100;

the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 101;

the VH of SEQ ID NO: 141 and the VL of SEQ ID NO: 102;

the VH of SEQ ID NO: 142 and the VL of SEQ ID NO: 103; or

the VH of SEQ ID NO: 143 and the VL of SEQ ID NO: 104.

In some embodiments, the identity is 85%. In some embodiments, the identity is 90%. In some embodiments, the identity is 91%. In some embodiments, the identity is

92%. In some embodiments, the identity is 93%. In some embodiments, the identity is

94%. In some embodiments, the identity is 95%. In some embodiments, the identity is

96%. In some embodiments, the identity is 97%. In some embodiments, the identity is

98%. In some embodiments, the identity is 99%.

The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions ^c 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The percent identity between two amino acid sequences may be determined using the algorithm of E. Meyers and W. Miller (Comput Appl Biosci 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch ( JMol Biol 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http_//_www_gcg_com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

In some embodiments, variant antibodies comprise one or two conservative substitutions in any of the CDR regions, wherein the antibodies retain the desired functional properties of the parental antibodies.

“Conservative modifications” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid modifications. Conservative modifications include amino acid substitutions, additions and deletions.“Conservative substitutions” are those in which the amino acid is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amide (e.g., asparagine, glutamine), beta- branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643 :55-67; Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions to the antibodies of the invention may be made by known methods for example by PCR mutagenesis (US Pat. No.

4,683,195). Alternatively, libraries of variants may be generated for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants may be tested for their characteristics using assays described herein.

Engineered and modified antibodies

The antibodies or antigen-binding fragments thereof specifically binding the HLA- DR/ColII_259 complex provided herein may be further engineered to generate modified antibodies with similar or altered properties when compared to the parental antibodies.

The VH, the VL, the VH and the VL, the constant regions, the heavy chain framework, the light chain framework, or any or all the six CDRs may be engineered in the antibodies of the invention.

The antibodies or antigen-binding fragments thereof specifically binding the HLA- DR/ColII_259 complex may be engineered by CDR grafting. One or more CDR sequences of the antibodies of the invention may be grafted to a different framework sequence. CDR grafting may be done using known methods and methods described herein.

The framework sequences that may be used may be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA and the encoded protein sequences for human heavy and light chain variable domain genes may be found at IMGT®, the international ImMunoGeneTics information system® http_//_www-imgt_org. Framework sequences that may be used to replace the existing framework sequences of the antibodies of the invention may be those that show the highest percent (%) identity to the parental variable domains over the entire length of the VH or the VL, or over the length of FR1, FR2, FR3 and FR4. In addition, suitable frameworks may further be selected based on the VH and the VL CDR1 and CDR2 lengths or identical LCDR1, LCDR2, LCDR3, HCDR1 and HCDR2 canonical structure. Suitable frameworks may be selected using known methods, such as human framework adaptation described in U.S. Patent No. 8,748,356 or superhumanization described in U.S. Patent No. 7,709, 226.

The framework sequences of the parental and engineered antibodies may further be modified, for example by backmutations to restore and/or improve binding of the generated antibodies to the antigen as described for example in U.S. Patent No. 6,180,370. The framework sequences of the parental or engineered antibodies may further be modified by mutating one or more residues within the framework region (or alternatively within one or more CDR regions) to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as“deimmunization” and described in further detail in U.S. Patent Publ. No. US20070014796.

The CDR residues of the antibodies or the antigen-binding fragments thereof provided herein may be mutated to improve affinity of the antibodies to the HLA- DR/ColII_259 complex.

The CDR residues of the antibodies or the antigen-binding fragments thereof provided herein may be mutated to minimize risk of post-translational modifications. Amino acid residues of putative motifs for deamination (NS), acid-catalyzed hydrolysis (DP), isomerization (DS), or oxidation (W) may be substituted with any of the naturally occurring amino acids to mutagenize the motifs, and the resulting antibodies may be tested for their functionality and stability using methods described herein.

The antibodies or antigen-binding fragments thereof specifically binding the HLA- DR/ColII_259 complex provided herein which are modified to improve stability, selectivity, affinity, immunogenicity or other desirable biological or biophysical properly are within the scope of the invention. Stability of an antibody is influenced by a number of factors, including (1) core packing of individual domains that affects their intrinsic stability, (2) protein/protein interface interactions that have impact upon the HC and LC pairing, (3) burial of polar and charged residues, (4) H-bonding network for polar and charged residues; and (5) surface charge and polar residue distribution among other intra- and inter-molecular forces (Worn et al, (2001) J Mol Biol 305:989-1010). Potential structure destabilizing residues may be identified based upon the crystal structure of the antibody or by molecular modeling in certain cases, and the effect of the residues on antibody stability may be tested by generating and evaluating variants harboring mutations in the identified residues. One of the ways to increase antibody stability is to raise the thermal transition midpoint (T_m) as measured by differential scanning calorimetry (DSC). In general, the protein T_m is correlated with its stability and inversely correlated with its susceptibility to unfolding and denaturation in solution and the degradation processes that depend on the tendency of the protein to unfold (Remmele et al, (2000) Biopharm 13:36- 46). A number of studies have found correlation between the ranking of the physical stability of formulations measured as thermal stability by DSC and physical stability measured by other methods (Gupta et al, (2003) AAPS PharmSci 5E8; Zhang et al,

(2004) J Pharm Sci 93:3076-89; Maa et al, (1996) Int J P harm 140: 155-68; Bedu-Addo et al, (2004) Pharm Res 21: 1353-61; Remmele et al, (1997) Pharm Res 15:200-8).

Formulation studies suggest that a Fab T_m has implication for long-term physical stability of a corresponding mAb. Antibody isotypes, allotypes and Fc engineered antibodies

The antibodies or antigen-binding fragments thereof specifically binding the HLA- DR/ColII_259 complex provided herein may be of any known isotype or allotype with wild-type or engineered Fc.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex provided herein is an IgGl isotype.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex provided herein is an IgG2 isotype.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex provided herein is an IgG3 isotype.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex provided herein is an IgG4 isotype.

C-terminal lysine (CTL) may be removed from injected antibodies by endogenous circulating carboxypeptidases in the blood stream (Cai el al, (2011) Biotechnol Bioeng 108:404-412). During manufacturing, CTL removal may be controlled to less than the maximum level by control of concentration of extracellular Zn²⁺, EDTA or EDTA - Fe³⁺ as described in U.S. Patent Publ. No. US20140273092. CTL content in antibodies may be measured using known methods.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex provided herein has a C-terminal lysine content from about 10% to about 90%. In some embodiments, the C-terminal lysine content is from about 20% to about 80%. In some embodiments, the C-terminal lysine content is from about 40% to about 70%. In some embodiments, the C-terminal lysine content is from about 55% to about 70%. In some embodiments, the C-terminal lysine content is about 60%.

Immunogenicity of therapeutic antibodies is associated with increased risk of infusion reactions and decreased duration of therapeutic response (Baert el al, (2003) N Engl J Med 348:602-08). The extent to which therapeutic antibodies induce an immune response in the host may be determined in part by the allotype of the antibody (Stickler el al, (2011) Genes and Immunity 12:213-21). Antibody allotype is related to amino acid sequence variations at specific locations in the constant region sequences of the antibody. Table 4 shows select IgGl, IgG2 and IgG4 allotypes. In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex provided herein is an G2m(n) allotype.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex provided herein is an G2m(n-) allotype.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex provided herein is an G2m(n)/(n-) allotype.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex provided herein is an nG4m(a) allotype.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex provided herein is an Glm(17,l) allotype.

Table 4.

Fc mutations may be made to the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex provided herein to modulate antibody effector functions such as ADCC, ADCP and/or ADCP and/or pharmacokinetic properties. This may be achieved by introducing mutation(s) into the Fc that modulate binding of the mutated Fc to activating FcyRs (Fc-/RI. Fc RIIa. Fc RIII). inhibitory FcyRIIb and/or to FcRn.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex comprises at least one mutation in the antibody Fc. In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen mutations in the Fc.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex comprises at least one mutation in the Fc that modulates binding of the antibody to FcRn.

Fc positions that may be mutated to modulate antibody half-life (e.g. binding to FcRn include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434 and 435. Exemplary mutations that may be made singularly or in combination are mutations T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R. Exemplary singular or combination mutations that may be made to increase the half-life of the antibodies provided herein are mutations M428L/N434S, M252Y/S254T/T256E, T250Q/M428L, N434A and

T307A/E380A/N434A. Exemplary singular or combination mutations that may be made to reduce the half-life of the antibodies provided herein are mutations H435A,

P257I/N434H, D376V/N434H, M252Y/S254T/T256E/H433K/N434F, T308P/N434A and H435R.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises at least one mutation in the antibody Fc that reduces binding of the antibody to an activating Fey receptor (FcyR) and/or reduces Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody -dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).

Fc positions that may be mutated to reduce binding of the antibody to the activating FcyR and subsequently to reduce effector function include positions 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331 and 365. Exemplary mutations that may be made singularly or in combination are mutations K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331S in lgGl, IgG2, IgG3 or IgG4. Exemplary combination mutations that result in antibodies with reduced ADCC are mutations L234A/L235A on IgGl, V234A/G237A/ P238S/H268A/V309L/A330S/P33 IS on IgG2, F234A/L235A on IgG4, S228P/F234A/ L235A on IgG4, N297A on all Ig isotypes, V234A/G237A on IgG2, K214T/E233P/ L234V/L235A/G236- deleted/A327G/P331 A/D365E/L358M on IgGl, H268Q/V309L/A330S/P331S on IgG2, S267E/L328F on IgGl, L234F/L235E/D265A on IgGl,

L234A/L235A/G237A/P238S/H268A/A330S/P331S on IgGl,

S228P/F234A/L235A/G237A/P238S on IgG4, and S228P/F234A/L235A/G236- deleted/G237A/P238S on IgG4. Hybrid IgG2/4 Fc domains may also be used, such as Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4.

Exemplary mutation that result in antibodies with reduced CDC is a K322A mutation.

Well-known S228P mutation may be made in IgG4 antibodies to enhance IgG4 stability.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises a mutation in at least one residue position 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 322, 327, 328, 329, 330, 331 or 365.

In some embodiments, the antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises at least one mutation selected from the group consisting of K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q,

Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, K322, A330S and P331S.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises a mutation in at least one residue position 228, 234, 235, 237, 238, 268, 322, 330 or 331.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises L234A, L235A, G237A, P238S, H268A, A330S and P331S substitutions when compared to the wild-type IgGl of SEQ ID NO: 124. The amino acid sequence of a IgGl constant domain having these substitutions is shown in SEQ ID NO: 93.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises L234A and L235A substitutions when compared to the wild-type IgGl of SEQ ID NO: 124.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises V234A, G237A, P238S, H268A, V309L, A330S and P33 IS substitutions when compared to the wild-type IgG2 of SEQ ID NO: 125. The amino acid sequence of a IgG2 constant domain having these substitutions is shown in SEQ ID NO: 94.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises S228P, F234A and L235A substitutions when compared to the wild-type IgG4 of SEQ ID NO: 126.

Wild-type IgGl SEQ ID NO: 124

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC

PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Wild-type IgG2 SEQ ID NO: 125

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

LQS SGL Y SLS S WT VP S SNF GTQTYTCNVDHKP SNTKVDKT VERKCC VECPPCP AP

PVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP

REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPML

DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Wild-type IgG4 SEQ ID NO: 126

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

LQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAP

EFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNA

KTKPREEQFN STYRW S VLTVLHQD WLNGKEYKCKV SNKGLP S SIEKTISKAKGQ

PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO: 93 IgGl sigma constant domain

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC

PAPEAAGASSVFLFPPKPKDTLMISRTPEVTCWVDVSAEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 94 IgG2sigma constant domain

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQS SGL Y SLS S WT VP S SNF GTQTYTCNVDHKP SNTKVDKT VERKCC VECPPCP AP PAAASSVFLFPPKPKDTLMISRTPEVTCWVDVSAEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTFRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLD SDGSFFL Y SKLT VDKSRWQQGNVF SC S VMHEALHNHYTQKSL SL SPGK

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises a S228P mutation.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprises at least one mutation in an antibody Fc that enhances binding of the antibody to an Fey receptor (Fc-/R) and/or enhances Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody -dependent cell-mediated cytotoxicity (ADCC) and/or phagocytosis (AD CP).

Fc positions that may be mutated to increase binding of the antibody to the activating FcyR and/or enhance antibody effector functions include positions 236, 239,

243, 256,290,292, 298, 300, 305, 312, 326, 330, 332, 333, 334, 345, 360, 339, 378, 396 or 430 (residue numbering according to the EU index). Exemplary mutations that may be made singularly or in combination are a G236A mutation, a S239D mutation, a F243L mutation, a T256A mutation, a K290A mutation, a R292P mutation, a S298A mutation, an Y300L mutation, a V305L mutation, a K326A mutation, an A330K mutation, an I332E mutation, an E333 A mutation, a K334A mutation, an A339T mutation and a P396L mutation. Exemplary combination mutations that result in antibodies with increased ADCC or ADCP are a S239D/I332E mutation, a S298A/E333A/K334A mutation, a F243L/R292P/Y300L mutation, a F243L/R292P/Y300L/P396L mutation, a

F243L/R292P/Y300L/V305I/P396L mutation and a G236A/S239D/I332E mutation on IgGl.

Fc positions that may be mutated to enhance CDC of the antibody include positions 267, 268, 324, 326, 333, 345 and 430. Exemplary mutations that may be made singularly or in combination are a S267E mutation, a F1268F mutation, a S324T mutation, a K326A mutation, a K326W mutation, an E333 A mutation, an E345K mutation, an E345Q mutation, an E345R mutation, an E345Y mutation, an E430S mutation, an E430F mutation and an E430T mutation. Exemplary combination mutations that result in antibodies with increased CDC are a K326A/E333A mutation, a K326W/E333 A mutation, a H268F/S324T mutation, a S267E/H268F mutation, a S267E/S324T mutation and a S267E/H268F/S324T mutation on IgGl.

"Antibody -dependent cellular cytotoxicity", "antibody -dependent cell-mediated cytotoxicity" or“ADCC" is a mechanism for inducing cell death that depends upon the interaction of antibody -coated target cells with effector cells possessing lytic activity, such as natural killer cells (NK), monocytes, macrophages and neutrophils via Fc gamma receptors (Fc-/R) expressed on effector cells. For example, NK cells express Fc/RIIIa. whereas monocytes express Fc_/RI. Fc _/RI I and Fc/RIIIa. ADCC activity of the antibodies provided herein may be assessed using an in vitro assay using HLA/DR/ColII_259 expressing cells as target cells and NK cells as effector cells. Cytolysis may be detected by the release of label (e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells. In an exemplary assay, target cells are used with a ratio of 1 target cell to 4 effector cells. Target cells are pre-labeled with BATDA and combined with effector cells and the test antibody. The samples are incubated for 2 hours and cell lysis measured by measuring released BATDA into the supernatant. Data is normalized to maximal cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich) and minimal control determined by spontaneous release of BATDA from target cells in the absence of any antibody.

"Antibody -dependent cellular phagocytosis" ("ADCP") refers to a mechanism of elimination of antibody -coated target cells by internalization by phagocytic cells, such as macrophages or dendritic cells. ADCP may be evaluated by using monocyte-derived macrophages as effector cells and any HLA-DR/ColII_259 expressing cells as target cells also engineered to express GFP or other labeled molecule. In an exemplary assay, effectontarget cell ratio may be for example 4: 1. Effector cells may be incubated with target cells for 4 hours with or without the antibody of the invention. After incubation, cells may be detached using accutase. Macrophages may be identified with anti-CD 1 lb and anti-CD 14 antibodies coupled to a fluorescent label, and percent phagocytosis may be determined based on % GFP fluorescence in the CD11⁺CD14⁺ macrophages using standard methods. “Complement-dependent cytotoxicity”, or” CDC”, refers to a mechanism for inducing cell death in which the Fc effector domain of a target-bound antibody binds and activates complement component Clq which in turn activates the complement cascade leading to target cell death. Activation of complement may also result in deposition of complement components on the target cell surface that facilitate CDC by binding complement receptors (e.g., CR3) on leukocytes. CDC of cells may be measured for example by plating Daudi cells at 1 ^c 10⁵ cells/well (50 pL/well) in RPMI-B (RPMI supplemented with 1% BSA), adding 50 pL of test antibodies to the wells at final concentration between 0-100 gg/mL. incubating the reaction for 15 min at room temperature, adding 11 pL of pooled human serum to the wells, and incubation the reaction for 45 min at 37° C. Percentage (%) lysed cells may be detected as % propidium iodide stained cells in FACS assay using standard methods.

Binding of the antibody to FcyR or FcRn may be assessed on cells engineered to express each receptor using flow cytometry. In an exemplary binding assay, 2xl0⁵ cells per well are seeded in 96-well plate and blocked in BSA Stain Buffer (BD Biosciences, San Jose, USA) for 30 min at 4°C. Cells are incubated with a test antibody on ice for 1.5 hour at 4°C. After being washed twice with BSA stain buffer, the cells are incubated with R-PE labeled anti-human IgG secondary antibody (Jackson Immunoresearch Laboratories) for 45 min at 4°C. The cells are washed twice in stain buffer and then resuspended in 150 pL of Stain Buffer containing 1 :200 diluted DRAQ7 live/dead stain (Cell Signaling Technology, Danvers, USA). PE and DRAQ7 signals of the stained cells are detected by Miltenyi MACSQuant flow cytometer (Miltenyi Biotec, Auburn, USA) using B2 and B4 channel, respectively. Live cells are gated on DRAQ7 exclusion and the geometric mean fluorescence signals are determined for at least 10,000 live events collected. FlowJo software (Tree Star) is used for analysis. Data is plotted as the logarithm of antibody concentration versus mean fluorescence signals. Nonlinear regression analysis is performed.

“Enhance” or“enhanced” refers to enhanced effector function (e.g. ADCC, CDC and/or ADCP) or enhanced binding to an Fey receptor (FcyR) or FcRn of the antibody of the invention having at least one mutation in the Fc region when compared to the parental antibody without the mutation.“Enhanced” may be an enhancement of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or a statistically significant enhancement. “Reduce” or“reduced” refers to reduced effector function (e.g. ADCC, CDC and/or ADCP) or reduced binding to an Fey receptor (FcyR) or FcRn of the antibody of the invention having at least one mutation in the Fc region when compared to the parental antibody without the mutation.“Reduced” may be a reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or a statistically significant reduction.

“Modulate” refers to either enhanced or reduced effector function (e.g. ADCC, CDC and/or ADCP) or enhanced or reduced binding to an Fey receptor (FcyR) or FcRn of the antibody of the invention having at least one mutation in the Fc region when compared to the parental antibody without the mutation.

Anti-idiotypic antibodies

Anti-idiotypic antibodies are antibodies that specifically bind to the antibodies or antigen-binding fragments thereof specifically binding the HLA-DR/ColII_259 complex.

The invention also provides an anti-idiotypic antibody that specifically binds to the antibody comprising

the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 100;

the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 101;

the VH of SEQ ID NO: 141 and the VL of SEQ ID NO: 102;

the VH of SEQ ID NO: 142 and the VL of SEQ ID NO: 103; or

the VH of SEQ ID NO: 143 and the VL of SEQ ID NO: 104.

An anti-idiotypic (Id) antibody is an antibody which recognizes the antigenic determinants (e.g. the paratope or CDRs) of the antibody. The Id antibody may be antigen-blocking or non-blocking. The antigen-blocking Id may be used to detect the free antibody in a sample (e.g. antibodies specifically binding the HLA-DR ColII_259 complex of the invention). The non-blocking Id may be used to detect the total antibody (free, partially bond to antigen, or fully bound to antigen) in a sample. An Id antibody may be prepared by immunizing an animal with the antibody to which an anti-id is being prepared.

An anti-id antibody may also be used as an immunogen to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. An anti-anti-Id may be epitopically identical to the original mAb, which induced the anti-id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identity other clones expressing antibodies of identical specificity. Anti-Id antibodies may be varied (thereby producing anti-id antibody variants) and/or derivatized by any suitable technique, such as those described elsewhere herein with respect to the antibodies or an antigenbinding fragment thereof specifically binding the HLA-DR/ColII_259 complex.

Conjugates of the antibodies or antigen-binding fragments thereof specifically binding the HLA-DR/ColII_259 complex

The invention also provides an immunoconjugate comprising an isolated antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex conjugated to a second molecule.

In some embodiments, the second molecule is a detectable label or a cytotoxic agent.

The invention also provides an antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex conjugated to a detectable label.

The invention also provides an antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex to a cytotoxic agent.

Antibodies or antigen-binding fragments thereof specifically binding the HLA- DR/ColII_259 complex may be used to direct therapeutics to HLA-DR/ColII_259 expressing cells. Cells such as B cells or other antigen-presenting cells that present ColII_259 in the context of MHC may be targeted with antibodies or antigen-binding fragments thereof of the invention conjugated to a cytotoxic agent that kills the cell upon internalization of the antibody.

In some embodiments, the second molecule is an immunomodulatory agent.

In some embodiments, the immunomodulatory agent reduces activation of immune responses.

In some embodiments, the immunomodulatory agent enhances suppression of immune responses.

Antibodies or antigen-binding fragments thereof specifically binding the HLA- DR/ColII_259 complex conjugated to the immune modulator that reduces activation or enhances suppression of immune responses may be used to target DR-ColII_259 expressing APCs and therefore inhibit the immune responses more broadly when compared to the antibody alone.

The isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex conjugated to a detectable label may be used to identity HLA-DR_ColII_259 positive cells in vitro or in vivo.

Detectable label includes compositions that when conjugated to the antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex provided herein renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

Exemplary detectable labels include nucleic acid bar codes, radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescent quenching agents, colored molecules, radioactive isotopes, scintillates, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni²⁺, Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors, acridinium esters, and colorimetric substrates.

A detectable label may emit a signal spontaneously, such as when the detectable label is a radioactive isotope. In other cases, the detectable label emits a signal because of being stimulated by an external field.

Exemplary radioactive isotopes may be g-emitting, Auger-emitting, b-emitting, an alpha-emitting or positron-emitting radioactive isotope. Exemplary radioactive isotopes include

8⁶Y, ⁸⁹Z

and ²²⁷ Ac.

Exemplary metal atoms are metals with an atomic number greater than 20, such as calcium atoms, scandium atoms, titanium atoms, vanadium atoms, chromium atoms, manganese atoms, iron atoms, cobalt atoms, nickel atoms, copper atoms, zinc atoms, gallium atoms, germanium atoms, arsenic atoms, selenium atoms, bromine atoms, krypton atoms, rubidium atoms, strontium atoms, yttrium atoms, zirconium atoms, niobium atoms, molybdenum atoms, technetium atoms, ruthenium atoms, rhodium atoms, palladium atoms, silver atoms, cadmium atoms, indium atoms, tin atoms, antimony atoms, tellurium atoms, iodine atoms, xenon atoms, cesium atoms, barium atoms, lanthanum atoms, hafnium atoms, tantalum atoms, tungsten atoms, rhenium atoms, osmium atoms, iridium atoms, platinum atoms, gold atoms, mercury atoms, thallium atoms, lead atoms, bismuth atoms, francium atoms, radium atoms, actinium atoms, cerium atoms, praseodymium atoms, neodymium atoms, promethium atoms, samarium atoms, europium atoms, gadolinium atoms, terbium atoms, dysprosium atoms, holmium atoms, erbium atoms, thulium atoms, ytterbium atoms, lutetium atoms, thorium atoms, protactinium atoms, uranium atoms, neptunium atoms, plutonium atoms, americium atoms, curium atoms, berkelium atoms, californium atoms, einsteinium atoms, fermium atoms, mendelevium atoms, nobelium atoms, or lawrencium atoms.

In some embodiments, the metal atoms may be alkaline earth metals with an atomic number greater than twenty.

In some embodiments, the metal atoms may be lanthanides

In some embodiments, the metal atoms may be actinides

In some embodiments, the metal atoms may be transition metals

In some embodiments, the metal atoms may be poor metals.

In some embodiments, the metal atoms may be gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.

In some embodiments, the metal atoms may be metals with an atomic number of 53 (i.e. iodine) to 83 (i.e. bismuth).

In some embodiments, the metal atoms may be atoms suitable for magnetic resonance imaging.

The metal atoms may be metal ions in the form of +1, +2, or +3 oxidation states, such

Au³⁺,

and Zn²⁺. The metal atoms may comprise a metal oxide, such as iron oxide, manganese oxide, or gadolinium oxide.

Suitable dyes include any commercially available dyes such as, for example, 5(6)- carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW, ruthenium polypyridyl dyes, and the like.

Suitable fluorophores are fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.

In some embodiments, the isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex conjugated to a detectable label may be used as an imaging agent.

In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a

radioconjugate).

In some embodiments, the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, bacterial toxins such as diphtheria toxin, ricin, geldanamycin, maytansinoids or calicheamicin. The cytotoxic agent may elicit their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

In some embodiments, the cytotoxic agent is an enzymatically active toxin such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In some embodiments, the cytotoxic agent is a radionuclide, such as ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

In some embodiments, the cytotoxic agent is dolastatins or dolostatin peptidic analogs and derivatives, auristatin or monomethyl auristatin phenylalanine. Exemplary molecules are disclosed in U.S. Pat No. 5,635,483 and 5,780,588. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001) Antimicrob Agents and Chemother. 45(12):3580-3584) and have anticancer and antifungal activity. The dolastatin or auristatin drug moiety may be attached to the antibody of the invention through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety

(W002/088172), or via any cysteine engineered into the antibody.

The isolated antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex may be conjugated to a detectable label using known methods.

In some embodiments, the detectable label is complexed with a chelating agent.

In some embodiments, the detectable label is conjugated to the antibody or an antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex via a linker.

The detectable label or the cytotoxic moiety may be linked directly, or indirectly, to the antibody or an antigen-binding fragment thereof specifically binding the HLA- DR/ColII_259 complex using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenolic linkers (derivatives of N- succimidyl-benzoates; dodecaborate), chelating moieties of both macrocyclics and acyclic chelators, such as derivatives of l,4,7,10-tetraazacyclododecane-l,4,7,10,tetraacetic acid (DOTA), derivatives of diethylenetriaminepentaacetic avid (DTP A), derivatives of S-2-(4- Isothiocyanatobenzyl)-l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) and derivatives of l,4,8,l l-tetraazacyclodocedan-l,4,8,l l-tetraacetic acid (TETA), N- succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2, 4-dinitrobenzene) and other chelating moieties. Suitable peptide linkers are well known.

Kits

The invention also provides a kit comprising the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex disclosed herein.

The kit may be used for therapeutic uses and as diagnostic kits.

The kit may be used to detect the presence of HLA-DR/ColII_259 expressing cells in a sample.

In some embodiments, the kit comprises the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex provided herein and reagents for detecting the antibody. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, an antibody to a label or therapeutic agent, or a radioprotective composition; devices or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

In some embodiments, the kit comprises the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex provided herein in a container and instructions for use of the kit.

In some embodiments, the antibody in the kit is labeled.

In some embodiments, the kit comprises the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex comprising

the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 100;

the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 101;

the VH of SEQ ID NO: 141 and the VL of SEQ ID NO: 102;

the VH of SEQ ID NO: 142 and the VL of SEQ ID NO: 103; or

the VH of SEQ ID NO: 143 and the VL of SEQ ID NO: 104. Methods of detecting HLA-DR/CoII_259 presenting cells

obtaining the biological sample;

In some embodiments, HLA-DR/ColII_259 complex comprises polypeptide chains of SEQ ID NOs: 15, 16 and 9.

In some embodiments, the biological sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, synovial fluid, circulating cells, cells that are not tissue associated (/.<?.. free cells), tissues (e.g., surgically resected tissue, biopsies, including fine needle aspiration), histological preparations, and the like.

The antibodies or the antigen-binding fragments thereof of the invention bound cells expressing the HLA-DR/ColII_259 complex may be detected using known methods. Exemplary methods include direct labeling of the antibodies using fluorescent or chemiluminescent labels, or radiolabels, or attaching to the antibodies of the invention a moiety which is readily detectable, such as biotin, enzymes or epitope tags. Exemplary labels and moieties are ruthenium, ^mIn-DOTA, ^mIn- diethylenetriaminepentaacetic acid (DTP A), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes,

phenanthridine dyes, rhodamine dyes and Alexafluor® dyes.

The antibodies of the invention may be used in a variety of assays to detect the HLA-DR/ColII_259 complex in the biological sample. Exemplary assays are western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) or ELISA assay.

Methods generating antibodies

Antibodies or antigen-binding fragments thereof specifically binding the HLA- DR/ColII_259 complex may be generated using various technologies. For example, antibodies may be made in a mouse or other host animal, such as a hamster, rat or chicken by immunizing animals with recombinantly expressed and purified HLA-DR/ColII_259 complex such as the construct DR4G92 or DR4G167 as described herein followed by hybridoma generation or isolating antibodies from B cells. The hybridomas or B cells may be tested for production of antibodies with desired properties, such as specificity of binding, cross-reactivity or lack thereof, affinity for the antigen, and functionality.

Exemplary humanization techniques including selection of human acceptor frameworks include CDR grafting (U.S. Patent No. 5,225,539), SDR grafting (U.S. Patent No. 6,818,749), Resurfacing (Padlan, (1991) Mol Immunol 28:489-499), Specificity Determining Residues Resurfacing (U.S. Patent Publ. No. 2010/0261620), human framework adaptation (U.S. Patent No. 8,748,356) or superhumanization (U.S. Patent No. 7,709, 226). In these methods, CDRs or a subset of CDR residues of parental antibodies are transferred onto human frameworks that may be selected based on their overall homology to the parental frameworks, based on similarity in CDR length, or canonical structure identity, or a combination thereof.

Humanized antibodies may be further optimized to improve their selectivity or affinity to a desired antigen by incorporating altered framework support residues to preserve binding affinity (backmutations) by techniques such as those described in Int. Patent Publ. Nos. W01090/007861 and W01992/22653, or by introducing variation at any of the CDRs for example to improve affinity of the antibody.

Transgenic animals, such as mice, rat or chicken carrying human immunoglobulin (Ig) loci in their genome may be used to generate antibodies against the HLA- DR/ColII_259 complex and are described in for example U.S. Patent No. 6,150,584, Int. Patent Publ. No. WO 1999/45962, Int. Patent Publ. Nos. W02002/066630,

WO2002/43478, W02002/043478 and W01990/04036. The endogenous

immunoglobulin loci in such animal may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the genome of the animal using homologous or non-homologous recombination, using transchromosomes, or using minigenes. Companies such as Regeneron (http://_www_regeneron_com), Harbour Antibodies (http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http://_www_omtinc_net), KyMab (http://_www_kymab_com), Trianni (http://_www.trianni_com) and Ablexis (http://_www_ablexis_com) may be engaged to provide human antibodies directed against a selected antigen using technologies as described above.

Antibodies may be selected from a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions. The antibodies of the invention may be isolated for example from phage display library expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al, (2010) JMol Biol 397:385-96, and Int. Patent Publ. No. WO09/085462). The libraries may be screened for phage binding to the HLA- DR/ColII_259 complex and the obtained positive clones may be further characterized, the Fabs isolated from the clone lysates, and expressed as full length IgGs.

Preparation of immunogenic antigens and monoclonal antibody production may be performed using any suitable technique, such as recombinant protein production. The immunogenic antigens may be administered to an animal in the form of purified protein, or protein mixtures including whole cells or cell or tissue extracts, or the antigen may be formed de novo in the animal’s body from nucleic acids encoding said antigen or a portion thereof.

In some embodiments, the antibody or the antigen-binding fragment thereof specifically binding the HLA-DR/ColII_259 complex of the invention is a bispecific antibody.

In some embodiments, the antibody or the antigen-binding fragment thereof of the invention is a multispecific antibody.

The monospecific antibodies that specifically bind the HLA-DR/ColII_259 complex provided herein may be engineered into bispecific antibodies which are also encompassed within the scope of the invention.

Full length bispecific antibodies may be generated for example using Fab arm exchange (e.g., half-molecule exchange, exchanging one heavy chain-light chain pair) between two monospecific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules having distinct specificity either in vitro in cell-free environment or using co-expression. The Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of CH3 domains. The heavy chain disulfide bonds in the hinge regions of the parental monospecific antibodies are reduced. The resulting free cysteines of one of the parental monospecific antibodies form an inter heavy -chain disulfide bond with cysteine residues of a second parental monospecific antibody molecule and simultaneously CH3 domains of the parental antibodies release and reform by dissociation-association. The CH3 domains of the Fab arms may be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody having two Fab arms or half molecules which each bind a distinct epitope.

Bispecific antibodies may also be generated using designs such as the

Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-in-Hole (Genentech), CrossMAbs (Roche) and the electrostatically -induced CH3 interaction (Chugai, Amgen, NovoNordisk, Oncomed), the LUZ-Y (Genentech), the Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), the Biclonic (Merus) and as DuoBody® Products (Genmab A/S).

The Triomab quadroma technology may be used to generate full length bispecific antibodies. Triomab technology promotes Fab arm exchange between two parental chimeric antibodies, one parental mAb having IgG2a and the second parental mAb having rat IgG2b constant regions, yielding chimeric bispecific antibodies.

The“knob-in-hole” strategy may be used to generate full length bispecific antibodies. Briefly, selected amino acids forming the interface of the CH3 domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into a heavy chain of an antibody specifically binding a first antigen and an amino acid with a large side chain (knob) is introduced into a heavy chain of an antibody specifically binding a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with a“hole” with the heavy chain with a“knob”. Exemplary CH3 substitution pairs forming a knob and a hole are (expressed as modified position in the first CH3 domain of the first heavy chain/ modified position in the second CH3 domain of the second heavy chain): T366Y/F405A,

T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.

The CrossMAb technology may be used to generate full length bispecific antibodies. CrossMAbs, in addition to utilizing the“knob-in-hole” strategy to promoter Fab arm exchange, have in one of the half arms the CHI and the CL domains exchanged to ensure correct light chain pairing of the resulting bispecific antibody (see e.g. U.S. Patent No. 8,242,247).

Other cross-over strategies may be used to generate full length bispecific antibodies by exchanging variable or constant, or both domains between the heavy chain and the light chain or within the heavy chain of the bispecific antibodies, either in one or both arms. These exchanges include for example VH-CH1 with VL-CL, VH with VL, CH3 with CL and CH3 with CHI as described in Int. Patent Publ. Nos. W02009/080254, W02009/080251, W02009/018386 and W02009/080252.

Other strategies such as promoting heavy chain heterodimerization using electrostatic interactions by substituting positively charged residues at one CH3 surface and negatively charged residues at a second CH3 surface may be used, as described in US Patent Publ. No. US2010/0015133; US Patent Publ. No. US2009/0182127; US Patent Publ. No. US2010/028637 or US Patent Publ. No. US2011/0123532. In other strategies, heterodimerization may be promoted by following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain/ modified position in the second CH3 domain of the second heavy chain): L351Y F405A Y407V/T394W,

T366I K392M T394W/F405A Y407V, T366L K392M T394W/F405A Y407V, L351Y Y407A/T366A K409F, L351Y Y407A/T366V K409F, Y407A/T366A K409F, or T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in U.S. Patent Publ. No. US2012/0149876 or U.S. Patent Publ. No. US2013/0195849.

LUZ-Y technology may be utilized to generate bispecific antibodies. In this technology, a leucine zipper is added into the C terminus of the CH3 domains to drive the heterodimer assembly from parental mAbs that is removed post-purification.

SEEDbody technology may be utilized to generate bispecific antibodies.

SEEDbodies have, in their constant domains, select IgG residues substituted with IgA residues to promote heterodimerization as described in U.S. Patent No. US20070287170.

Bispecific antibodies may be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two monospecific homodimeric antibodies and forming the bispecific heterodimeric antibody from two parental monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in Int.Patent Publ. No. WO2011/131746. In the methods, the first monospecific bivalent antibody and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promoter heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange.

Substitutions that may be used are F405L in one heavy chain and K409R in the other heavy chain in IgGl antibodies. In IgG4 antibodies, one heavy chain may be a wild-type IgG4 having F at position 405 and R at position 409 and the other heavy chain may have F405L and R409K substitutions. The incubation conditions may optimally be restored to non-reducing. Exemplary reducing agents that may be used are 2- mercaptoethylamine (2- MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2- carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol. For example, incubation for at least 90 min at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used. In some embodiments, the bispecific antibodies include recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; IgG fusion molecules, wherein full length IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment; Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy -chain constant-domains, Fc-regions or parts thereof; Fab fusion molecules, wherein different Fab-fragments are fused together; ScFv- and diabody -based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy -chain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

Substitutions are typically made at the DNA level to a molecule such as the constant domain of the antibody using standard methods.

The antibodies of the invention may be engineered into various well-known antibody forms.

Polynucleotides, vectors, host cells

The invention also provides an isolated polynucleotide encoding any of the antibodies of the invention.

The invention also provides an isolated polynucleotide encoding any of the antibody heavy chain variable regions, any of the antibody light chain variable regions, or any of the antibody heavy chains and/or the antibody light chains of the invention.

The invention also provides an isolated polynucleotide encoding the VH of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NOT41, SEQ ID NO: 142 or SEQ ID NO: 143.

The invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 or SEQ ID NO: 104.

The invention also provides an isolated polynucleotide encoding the VH of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142 or SEQ ID NO: 143 and the VL of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 or SEQ ID NO: 104.

The invention also provides an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113 or SEQ ID NO: 114.

The polynucleotide sequences encoding the VH and/or the VL or antigen binding fragments thereof of the antibodies of the invention or the heavy chain and/or the light chain of the antibodies of the invention may be operably linked to one or more regulatory elements, such as a promoter or enhancer, that allow expression of the nucleotide sequence in the intended host cell. The polynucleotide may be a cDNA.

The invention also provides for a vector comprising the polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors or any other vector suitable for introduction of the polynucleotide of the invention into a given organism or genetic background by any means. For example, polynucleotides encoding light and/or heavy chain variable regions of the antibodies of the invention, optionally linked to constant regions, are inserted into expression vectors. The light and/or heavy chains may be cloned in the same or different expression vectors. The DNA segments encoding the VH, the VL, the HC and/or the LC or antigen binding fragments thereof may be operably linked to control sequences in the expression vector(s) that ensure the expression of the polypeptides. Such control sequences include signal sequences, promoters (e.g. naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are chosen to be compatible with the host cell chosen to express the antibody. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the proteins encoded by the incorporated polynucleotides.

In some embodiments, the vector comprises the polynucleotide encoding the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 100. In some embodiments, the vector comprises the polynucleotide of SEQ ID NO: 105 and 110.

In some embodiments, the vector comprises the polynucleotide encoding the VH of SEQ ID NO: 140 and the VL of SEQ ID NO: 101. In some embodiments, the vector comprises the polynucleotide of SEQ ID NO: 106 and SEQ ID NO: 111.

In some embodiments, the vector comprises the polynucleotide encoding the VH of SEQ ID NO: 141 and the VL of SEQ ID NO: 102. In some embodiments, the vector comprises the polynucleotide of SEQ ID NO: 107 and SEQ ID NO: 112.

In some embodiments, the vector comprises the polynucleotide encoding the VH of SEQ ID NO: 142 and the VL of SEQ ID NO: 103. In some embodiments, the vector comprises the polynucleotide of SEQ ID NO: 108 and SEQ ID NO: 113.

In some embodiments, the vector comprises the polynucleotide encoding the VH of SEQ ID NO: 143 and the VL of SEQ ID NO: 104. In some embodiments, the vector comprises the polynucleotide of SEQ ID NO: 109 and SEQ ID NO: 114.

Suitable expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers such as ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance to permit detection of those cells transformed with the desired DNA sequences. Glutamine synthetase system may be used to express recombinant proteins such as antibodies in cells.

Suitable promoter and enhancer elements are known in the art. For expression in a eukaryotic cell, exemplary promoters include light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex vims thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating recombinant constructs. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia), and pEE6.1 and pEE14.1 (Lonza).

The invention also provides a host cell comprising one or more vectors of the invention.“Host cell” refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but to the progeny of such a cell, and to a stable cell line generated from the particular subject cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell but are still included within the scope of the term "host cell" as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells or archeal cells. Escherichia coli, bacilli, such as Bacillus subtilis , and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species are examples of prokaryotic host cells. Other microbes, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells may be of mammalian, insect, avian or other animal origins. Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines such as SP2/0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologies, Walkersville, MD), CHO-K1 (ATCC CRL-61) or DG44.

The invention also provides a method of producing the antibody of the invention comprising culturing the host cell of the invention in conditions that the antibody is expressed, and recovering the antibody produced by the host cell. Methods of making antibodies and purifying them are known. Once synthesized (either chemically or recombinantly), the whole antibodies, their dimers, individual light and/or heavy chains, or other antibody fragments such as VH and / or VL, may be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer- Verlag, N.Y., (1982)). A subject antibody may be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99%, or more, pure, e.g., free from contaminants such as cell debris, macromolecules, etc. other than the subject antibody.

The invention also provides a method of producing an isolated antibody or an antigen-binding fragment thereof specifically binding the HLA-DR ColII_259 complex comprising:

incorporating the first polynucleotide encoding the VH of the antibody and the second polynucleotide encoding the VL of the antibody into an expression vector;

transforming a host cell with the expression vector;

culturing the host cell in culture medium under conditions wherein the VL and the VH are expressed and form the antibody; and

recovering the antibody from the host cell or culture medium.

The polynucleotides encoding certain VH or VL sequences of the invention may be incorporated into vectors using standard molecular biology methods. Host cell transformation, culture, antibody expression and purification are done using well known methods.

Pharmaceutical Compositions/Administration

The invention also provides pharmaceutical compositions comprising the antibodies or the antigen binding fragments thereof of the invention and a

pharmaceutically acceptable carrier. For therapeutic use, the antibodies of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antibodies as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the antibody of the invention is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the antibodies or the antigen-binding fragments thereof of the invention in such pharmaceutical formulation may vary, from less than about 0.5%, usually to at least about 1% to as much as 15 or 20% by weight and may be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21^st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the antibodies or the antigenbinding fragments thereof of the invention may be any suitable route that delivers the antibody to a subject, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art.

Site specific administration may be achieved by for example intratumoral, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

The antibodies or the antigen binding fragments thereof of the invention may also be administered prophylactically to reduce the risk of developing an autoimmune disease and/or delay the onset of the symptoms. The antibodies or the antigen binding fragments thereof of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional protein preparations and well known lyophilization and reconstitution techniques can be employed.

Methods and Uses

The antibodies or the antigen-binding fragments thereof of the invention have in vitro and in vivo diagnostic, as well as therapeutic and prophylactic utilities. For example, the antibodies of the invention may be administered to cells in culture, in vitro or ex vivo , or to a subject to treat, prevent, and/or diagnose a variety of disorders, such as HLA- DR/ColII_259 complex-mediated diseases such as autoimmune diseases, such as rheumatoid arthritis.

The invention also provides a method of treating or preventing a HLA- DR/ColII_259 complex-mediated disease, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or the antigen-binding fragment thereof of the invention specifically binding the HLA-DR ColII_259 complex for a time sufficient to treat the HLA-DR ColII_259 complex-mediated disease.

In some embodiments, the HLA-DR ColII_259 complex -mediated disease is an autoimmune disease.

In some embodiments, the autoimmune disease is arthritis.

In some embodiments, arthritis is juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter’s syndrome, ankylosing spondylitis, gouty arthritis or juvenile idiopathy arthritis.

The invention also provides a method of suppressing an immune response towards ColII_259 presented on HLA-DR in a subject, comprising administering to the subject the antibody or the antigen-binding fragment specifically binding HLA/DR_ColII_259 complex of the invention for a time sufficient to suppress the immune response towards ColII_259 presented on HLA-DR.

The invention also provides the antibody of the invention or the pharmaceutical composition of the invention for use in therapy.

The invention also provides the antibody of the invention or the pharmaceutical composition of the invention for use in the treatment of rheumatoid arthritis.

The invention also provides the antibody of the invention for use in the manufacture of medicament for treating rheumatoid arthritis. Combination therapies

The antibodies or antigen-binding fragments thereof specifically binding the HLA- DR/ColII_259 complex in the methods of the invention may be administered in combination with a second therapeutic agent simultaneously, sequentially or separately.

The antibodies or antigen-binding fragments thereof specifically binding the HLA- DR/ColII_259 complex of the invention may be administered in combination with any known therapy for autoimmune diseases, including any agent or combination of agents that are known to be useful, or which have been used or are currently in use, for treatment of HLA-DR/ColII_259 complex-mediated diseases. Such therapies and therapeutic agents include surgery or surgical procedures (e.g. splenectomy, lymphadenectomy, thyroidectomy, plasmapheresis, leukophoresis, cell, tissue, or organ transplantation, intestinal procedures, organ perfusion, and the like), radiation therapy, therapy such as steroid therapy and non-steroidal therapy, hormone therapy, cytokine therapy, therapy with dermatological agents (for example, topical agents used to treat skin conditions such as allergies, contact dermatitis, and psoriasis), immunosuppressive therapy, and other antiinflammatory monoclonal antibody therapy.

The second therapeutic agent may be a corticosteroid, an antimalarial drug, an immunosuppressant, a cytotoxic drug, or a B-cell modulator.

In some embodiments, the second therapeutic agent is prednisone, prednisolone, methylprednisolone, deflazcort, hydroxychloroquine, azathioprine, methotrexate, cyclophosphamide, mycophenolate mofetil (MMF), mycophenolate sodium, cyclosporine, leflunomide, tacrolimus, rituximab (Rituxan^®), or belimumab (Benlysta^®).

In some embodiments, the second therapeutic agent is corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), salicylates, sulfasalazine, cytotoxic drugs, immunosuppressive drugs, mizoribine, chlorambucil, cyclosporine, tacrolimus (FK506 ; ProGrafrM), mycophenolate mofetil, sirolimus (rapamycin), deoxyspergualin, leflunomide and its malononitriloamide analogs, clobetasol, halobetasol, hydrocortisone,

triamcinolone, betamethasone, fluocinole, fluocinonide, medications containing mesalamine (known as 5-ASA agents), celecoxib, diclofenac, etodolac, fenprofen, flurbiprofen, ibuprofen, ketoprofen, meclofamate, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, rofecoxib, salicylates, sulindac, tolmetin; phosphodiesterase-4 inhibitors, anti-TNFa antibodies infliximab (REMICADE®), golimumab (SIMPONI®) and adalimumab (HUMIRA®), thalidomide or its analogs such as lenalidomide.

Treatment effectiveness or RA may be assessed using effectiveness as measured by clinical responses defined by the American College of Rheumatology criteria, the European League of Rheumatism criteria, or any other criteria. See for example, Felson et al. (1995) Arthritis Rheum. 38: 727-35 and van Gestel et al. (1996) Arthritis Rheum. 39: 34-40. While having described the invention in general terms, the embodiments of the invention will be further disclosed in the following examples that should not be construed as limiting the scope of the claims.

Example 1. Generation of antigens and control antibodies

HLA-DR, HLA-DQ and HLA-DP heterodimeric antigens were expressed as Fc fusion proteins with covalently linked hemagglutinin, collagen, insulin, CLIP or LCAP peptides coupled to the N-terminus of the HLA b chain via cleavable linker. In some cases, CILP or a peptide derived from fibrinogen was used. The a and the b chains were expressed in format as follows:

a chain: ECD-G₄S-TEV-G₄S-Fc-His6

b chain: peptide-3xGS-HRV3C-ECD-G₄S-TEV-G₄S-Fc-StrepII

ECD: extracellular domain of the expressed HLA chain

G₄S: GGGGS (SEQ ID NO: 1)

TEV: EDLYFQ (SEQ ID NO: 2); tobacco etch vims Nia protease cleavage site His.,: HHHHHH (SEQ ID NO: 3)

3xGS: GSGSGS (SEQ ID NO: 4)

HRV3C: LEVLFQGP (SEQ ID NO: 5); human rhinovirus 3C protease cleavage site StrepII: WSHPQFEK (SEQ ID NO: 6); StrepII tag

Hemagglutinin peptide HA 304-318: ACPKYVKQNTLKLAT (SEQ ID NO: 7) Collagen II peptide CII 1236-1249: LQYMRADQAAGGLR (SEQ ID NO: 8)

Collagen II peptide CII 257-273: EPGIAGFKGEQGPKGEP (SEQ ID NO: 9)

Insulin peptide INS_1-15: FVNQLCGSHLVEAL (SEQ ID NO: 10)

CLIP peptide KMRMATPLLMQALPM (SEQ ID NO: 11)

LCAP peptide KYFAATQFEPLAARL (SEQ ID NO: 12)

CII 259 GIAGFKGEQGPKGEP (SEQ ID NO: 123)

CILP peptide (amino acids 982-996(Cit-988,991); GKLYGI{Cit}DV{Cit}STRDR, SEQ ID NO: 127)

Fibrinogen peptide (amino acids 69-81(Cit-74); GGYRA{Cit}PAKAAAT (SEQ ID NO: 128) In the pMHC constructs, a peptide with two amino acid N-terminal extension (SEQ ID NO: 9) was used instead of Collagen II peptide CII 259 (SEQ ID NO: 123) to facilitate secretion of the MHC-ColII peptide complex. Fc: modified IgG4 (SEQ ID NO: 14) (nG4m(a) allotype with S228P, F234A, L235AA) CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVD G VE VHNAKTKPREEQFN STYRW S VLT VLHQD WLNGKEYKCKVSNKGLP S SIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPP VLD SD GSFFLY SRLT VDKSRWQEGNVFS CS VMHEALHNHYTQKSL SLS L

HLA-DRA1 *01:02 (SEQ ID NO: 15)

IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEA QGALANIAVDKANLEIMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFT PPWNVTWLRNGKPVTTGVSETVFLPREDHLFRKFHYLPFLPSTED VYDCRVEH W GLDEPLLKHWEFDAPSPLPETTE

HL A-DRB 1*04:01 (SEQ ID NO: 16)

GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGR PD AEYWN SQKDLLEQKRA AVDTYCRHNY GV GESFT VQRRVYPE VT VYP AKTQP LQHHNLLVCSVNGFYPGSIEVRWFRNGQEEKTGWSTGLIQNGDWTFQTLVMLE TVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSK

HLA-DRB 1*01:01 (SEQ ID NO: 17)

GDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGR PDAEYWNSQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPL QHHNLLVCSVSGFYPGSIEVRWFRNGQEEKAGWSTGLIQNGDWTFQTLVMLET VPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSK HLA-DQA1 (SEQ ID NO: 18)

EDIVADHVASCGVNLYQFYGPSGQYTHEFDGDEQFYVDLERKETAWRWPEFSKF

GGFDPQGALRNMAVAKHNLNIMIKRYNSTAATNEVPEVTVFSKSPVTLGQPNTLI

CLVDNIFPPWNITWLSNGQSVTEGVSETSFLSKSDHSFFKISYLTFLPSADEIYDCK

VEHWGLDQPLLKHWEPEIPAPMSELTE HLA DQB 1*06:02 (SEQ ID NO: 19)

RD SPEDF VF QFKGMCYFTN GTERVRL VTRYIYNREEY ARFD SD V GVYR AVTPQG RPDAEYWNSQKEVLEGTRAELDTVCRHNYEVAFRGILQRRVEPTVTISPSRTEAL NHHNLLVCSVTDFYPGQIKVRWFRNDQEETAGVVSTPLIRNGDWTFQILVMLEM TPQRGDVYTCHVEHPSLQSPITVEWRAQSESAQSK

HLA-DPA1 (SEQ ID NO: 20)

AGAIKADHVSTYAAFVQTHRPTGEFMFEFDEDEMFYVDLDKKETVWHLEEFGQA

FSFEAQGGLANIAILNNNLNTLIQRSNHTQATNDPPEVTVFPKEPVELGQPNTLICH

IDKFFPPVLNVTWLCNGELVTEGVAESLFLPRTDYSFHKFHYLTFVPSAEDFYDCR

VEHWGLDQPLLKHWEAQEPIQMPETTE

HLA-DPB 1*04:01 (SEQ ID NO: 21)

RATPENYLFQGRQECYAFNGTQRFLERYIYNREEFARFDSDVGEFRAVTELGRPA

AEYWNSQKDILEEKRAVPDRMCRHNYELGGPMTLQRRVQPRVNVSPSKKGPLQH

HNLLVCHVTDFYPGSIQVRWFLNGQEETAGWSTNLIRNGDWTFQILVMLEMTPQ

QGDVYTCQVEHTSLDSPVTVEWKAQSDSARSK

HLA-DRA1 *01 :01 (SEQ ID NO: 144)

IK F.F.HVHQAF.FYFNPDQSGF.FMFDFDGDF.IFH VDMAKKF.TVWRFF.F.FGRFASFF.A

QGALANIAVDKANLEIMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFT

PPWNVTWLRNGKPVTTGVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHW

GLDEPLLKHWEFDAPSPLPETTENWCALGLTVGLVGIIIGTIFIIKGVRKSNAAERR

GPL

Table 5 shows the format of the expressed HLA fusion proteins. Table 6 shows the amino acid sequences of both the a and b chains. For expression and purification, HLA a and b ECD-Fc fusions were co- transfected in HEK 293 Expi cells, the soluble HLA-ECD Fc fusion proteins were purified via ProteinA/SEC. All the HLA-DR antigens were conjugated to biotin using EZ-Link™ Sulfo-NHS-LC-Biotin and Labeling Kit (Thermo, cat no 21327), the success of the biotinylation was analyzed by HABA-avidin assay (Thermo, cat no 46610) and Octet.

Table 5.

Table 6.

Antibodies Lym-1, apolizumab (1D10) and L243 were used as control antibodies after re-engineering the constant domains as IgG2sigma isotypes. The engineered IgG2sigma mAbs were renamed DR4B4 (Lym-1), DR4B5 (apolizumab) and DR4B6 (L243). IgG2sigma is an effector silent Fc and has substitutions V234A, G237A, P238S, H268A, V309L, A330S and P33 IS when compared to the wild type IgG2. IgG2sigma is described in U.S. Pat. No. 8,961,967. Lym-1 VH (SEQ ID NO: 37) QVQLKESGPGLVAPSQSLSITCTISGFSLTSYGVHWVRQPPGKGLEWLWIWSDGS

TTYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAIYYCASHYGSTLAFASWGHG

TLVTVSA

Lym-1 VL (SEQ ID NO: 38)

DIQMTQSPASLSASVGETVTIICRASVNIYSYLAWYQQKQGKSPQLLVYNAKILAE GVPSRFSGSGSGTQFSLKINSLQPEDF GSYY CQHHY GPFTF GSGTKLEIK

Apolizumab VH (SEQ ID NO: 39) was this used?

QVQLQESGPGLVKPSETLSLTCTVSGFSLTNYGVHWVRQSPGKGLEWIGVKWSG

GSTEYNAAFISRLTISKDTSKNQVSLKLNSLTAADTAVYYCARNDRYAMDYWGQ

GTLVTVSS

Apolizumab VL (SEQ ID NO: 40)

DIQMTQSP S SL S AS V GDRVTITCRASENIY S YL A WY QQKPGKAPKLL V SNAKTL AE GVPSRFSGSGSGKQFTLTISSLQPEDFATYY CQHHY GNSYPFGQGTKLEIK

L243 VH (SEQ ID NO: 41)

QIQLVQSGPELKKPGETVKISCKASGFTFTNYGMNWVKQAPGKGLKWMGWINTY TREPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTAKYFCARDITAWPTGFDY W GQGTTLTVS S

L243 VL (SEQ ID NO: 42)

DIQMTQSP ASLSVSVGETVTITCRASENIYSNLAWYRQKQGKSPQLLVFAASNL AD GVPSRFSGSGSGTQYSLKINSLQSEDFGDYYCQHFWTTPWAFGGGTNLEIK

Example 2. Generation of antibodies against HLA-DR/ColII_259 complex in mouse

As endogenous HLA-DR complex signals as a dimer with peptide sitting the grove between alpha and beta chain. Recombinant HLA-DR4 peptide complexes were expressed in multiple ways to obtain a functional recombinant peptide and HLA-DR complexes. Extracellular domains of HLA-DR4 were cloned and expressed in the following format: ECD G4S linker+TEV cleavage +G4S linker+ Fc +6xHisTag; Beta chain: Peptide +3X(GS) linker +HRV3C+ECD+G4S linker+TEV cleavage site+G4S linker + Fc+StrepII tag as is shown in Example 1. Cloning, expression and protein purification was done using standard methods. Cells expressing endogenous human HLA- DR4 (Boleth cells) were used in some assays. T cell hybridoma clones were used in some assays.

Mice were immunized with the various HLA-DR/ColII_259 complexes.

Antibody isolation and V-region cloning was performed using known methods and mAbs with human IgGl sigma were expressed from HEK cells

Example 3. Characterization of HLA-DR/ColII_259 antibodies

A plate-based assay was developed for testing the antibodies binding specificity to recombinant HLA-DR proteins covalently linked to different peptide. Briefly, recombinant HLA-DR molecules were coated on MSD plates (Meso Scale Discovery) overnight at 4°C at 5pg/ml in Dulbecco’s phosphate-buffered saline (DPBS, Thermo Fisher Scientific). The next day, the plates were washed 3 times in Tris Buffered Saline-Tween (TBS-Tween, Sigma-Aldrich) and blocked for over 30 minutes by StartingBlock blocking buffer (Thermo Fisher Scientific). Antibodies to HLA-DR were diluted to desired concentration in buffer containing DPBS and 1% Bovine Serum Albumin (BSA, Thermo Fisher Scientific) and added to the plate. After 1 hour of incubation and 3 washes with TBS- Tween, lpg/ml SulfoTag anti-Kappa or anti-Lamda secondary antibody (Meso Scale Discovery) was added to plates and incubated for another hour. The plates were once again washed three times with TBS-Tween and 150m1 of read buffer (Meso Scale Discovery) was added before it was read in plate reader (Meso Scale Discovery Sector instrument). Each condition was tested in duplicate or triplicate wells and the average was plotted and analyzed in GraphPad Prism software. An antibody is determined to be binding to the recombinant HLA-DR if the raw signal at the highest concentration is greater than 10 times the signal from background or unbound molecule.

The dose response curves for binding of DR4B683, DR4B706, DR4B668, DR4B1544 and the pan-antibody L243 (DR4B397) to DR*04:01 conjugated to ColII_257-273 (CIL259 in the Figure), CII 1236-1249 (CII: 1236 in the Figure), CLIP and HA peptides are shown in Figure 3A, Figure 3B, Figure 3C and Figure 3D, respectively.

Figure 4A, Figure 4B, Figure 4C and Figure 4D shows dose response curves for binding of DR4B683, DR4B706, DR4B668, DR4B1544 and the pan-antibody L243 to DR*01:01 conjugated to ColII_257-273 (CIL259 in the Figure), CII_1236-1249 (CII: 1236 in the Figure), CLIP and HA peptides are shown in Figure 4A, Figure 4B, Figure 4C and Figure 4D, respectively.

Summary of the results of binding of the antibodies to peptide-DR0401 and peptide-DROlOl antigens are shown in Table 7 and Table 8, respectively. DR4B668, DR4B683 and DR4B695 were specific to a complex of HLA-DR/ColII_259. No binding was demonstrated to HLA-DR/CLIP or HLA/DR/HA complexes. DR4B397 bound HLA- DR in a peptide-independent manner. Table 7.

Table 8.

Example 4. Humanization and characterization of HLA-DR/ColII_259 antibodies

DR4B668 and DR4B683 were humanized using human framework adaptation (HFA) technologies. The resulting humanized antibodies were cloned and expressed as IgGl sigma isotypes and tested for their binding to the various HLA-DR antigens as described in Example 3. Control antibodies DR4B391 (a pan-HLA-DR antibody DR4B117 cloned as IgGlsigma; DR4B117 is described in WO2017106684) and DR4B397 (L243 cloned as IgGlsigma) were also tested. The results of binding of the antibodies to peptide-DR4 complexes are shown in Table 9. The results of binding of the antibodies to peptide-DRl, DP and DQ complexes are shown in Table 10. Table 9.

Table 10.

Humanized antibodies DR4B706 and DR4B1544 demonstrated slightly reduced binding when compared to the parental antibodies and demonstrated no binding to DP and DQ. The control antibodies bound HLA-DR in peptide-independent manner.

Example 5. Generated antibodies bind human and chimeric human/mouse HLA- DR4/ColII_259 complexes with high affinity

Affinity measurements using Surface Plasmon Resonance (SPR) were performed using a ProteOn XPR36 system (BioRad). Biosensor surfaces of HLA-DR4/ColII_259 antibodies were prepared by coupling antibodies to the modified alginate polymer layer surface of a GLC chip (BioRad, Cat# 176-5011) using the manufacturer instructions for amine-coupling chemistry. Approximately, greater than 400 RU (response units) of mAbs were immobilized. The kinetic experiments were performed at 25°C in minting buffer (DPBS+0.01%P20+ lOOpg/ml BSA). To perform binding kinetic experiment for the antibodies, the antigens (DR4G90, DR4G92 and DR4G167) were injected over the immobilized mAbs at 5 concentrations (in a 3-fold serial dilution). The association phase was monitored for 6 minutes at 50 pL/min, then followed by 30 minutes of buffer flow (dissociation phase). The chip surface was regenerated with two 18 second pulses of 100 mM H3PO4 (Sigma, Cat#7961) at 100 pL/min. The collected data were processed using ProteOn Manager software. First, the data was corrected for background using inter-spots. Then, double reference subtraction of the data was performed by using the buffer injection for antigen injections. The kinetic analysis of the data was performed using either a Langmuir 1 : 1 binding or Two-states binding models. The results for each mAb was reported in the format of ka (On-rate), kd (Off-rate) and KD (equilibrium dissociation constant).

Kinetic parameters of binding of the tested antibodies to DR4G92 (human HLA- DR4/ColII_257-273) is shown in Table 11. No detectable binding was observed to DR4G90 (HLA-DR4/CII 1236). Binding of the test antibodies to DR4G167 was also tested; DR4G167 being a human-mouse chimera composed of human ColII_257-273 peptide and domain 1 of the a and b chains from human HLA-DR4 and domain 2 of both chains from corresponding mousel-E. The results are shown in Table 12. The control antibody L243 cloned as IgG2sigma bound both DR4G92 and DR4G90 with nM affinities (2.45E-10 M and 4.94E-09 M, respectively).

Table 11.

Table 12.

Example 6. Structural characterization of HLA-DR/CII 259 antibodies

The cDNA sequences and amino acid translations of the antibodies were obtained using standard techniques. After polypeptide sequence determination, some antibody cDNAs encoding the variable regions or full-length antibodies were codon optimized using standard methods for scale-up expression. Table 13 shows the CDR sequences of DR4B668. Table 14 shows the CDR sequences of DR4B1544. Table 15 shows the CDR sequences of DR4B683. Table 16 shows the CDR sequences of DR4B706. Table 17 shows the CDR sequences of DR4B695. CDR sequences are shown using the various delineations as described herein. Table 18 shows the VH and the VL amino acid sequences of the antibodies. Table 19 shows the cDNA sequences encoding the VH of the antibodies. Table 20 shows the cDNA sequences encoding the VL of the antibodies.

Table 13.

Table 14.

Table 15.

Table 16.

Table 17.

Table 18.

Table 19.

Table 20.

Table 21.

The heavy and light chains of the antibodies (cloned as IgGlsigma) are shown below. IgGlsigma is a IgGl variant with following substitutions when compared to the wild-type IgGl of SEQ ID NO: 124: L234A, L235A, G237A, P238S, H268A, A330S and P33 IS. IgGlsigma constant domain amino acid sequence is shown in SEQ ID NO: 93.

DR4B668 HC SEQ ID NO: 129

DVQLQESGPSLVRPSQTLSLTCTVTGFSINSDCYWIWIRQFPGNKLEYIGYTFYNGI TYYNPSLESRTYITRDTSKNQFSLKLRSVSTEDTATYYCARAEWYWYFDVWGA GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCWVDVSAEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPS SIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKS

LSLSPGK

DR4B668 LC SEQ ID NO: 130

DIQMTQTTSSLSASLGDRVTISCRASQDSGNYLNWYQQKPDGTVKLLIYYTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLKIKRTVAA P S VFIFPPSDEQLKS GT AS W CLLNNFYPREAK VQ WKVDNALQSGN SQES VTEQD SKD STY SL S STLTL SKAD YEKHKVY ACE VTHQGL S SP VTKSFNRGEC

DR4B1544 HC SEQ ID NO: 131 QVQLQESGPGLVKPSQTLSLTCTVSGFSINSDSYWIWIRQPPGKGLEYIGYTFYNGI

TYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARAEVVYWYFDVWGQ

GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS

GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

KTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVWDVSAEDPEVKFNW

YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPS

SIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ

PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPGK

DR4B 1544 LC SEQ ID NO: 132

DIQMTQSPSSLSASVGDRVTITCRASQDSGNYLNWYQQKPGKAVKLLIYYTSRLH SGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPWTFGQGTKLEIKRTVAA P S VFIFPPSDEQLKS GT AS W CLLNNFYPREAK VQ WKVDNALQSGN SQES VTEQD SKD STY SL S STLTL SKAD YEKHKVY ACE VTHQGL S SP VTKSFNRGEC

DR4B683 HC SEQ ID NO: 133

EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLELVAEISNSG

DNIYYPDNVKGRFTISRDNAKNTL YLQMN SLKSEDT AMYY C ARREYD AYS AWF A

YWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVE

PKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVWDVSAEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

NKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE

WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

H YTQKSL SL SPGK

DR4B683 LC SEQ ID NO: 134

DIVLTQSPASLAVSLGQRATISCRASKSVSSSGYSFMHWYQQKPGQPPKLLIYLAS NRESGVPARFTGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGVGTKLELKR TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQD SKD STY SLS STLTL SKAD YEKHKVY ACE VTHQGLS SP VTKSFNRGEC

DR4B706 HC SEQ ID NO: 135 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVSEISNSG

DNIYY AD S VKGRFTISRDNAKN SLYLQMN SLRAEDTAVYY CARRE YD AY S A WF A

YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVE

PKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVWDVSAEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

NKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE

WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

H YTQKSL SL SPGK

DR4B706 LC SEQ ID NO: 136

DIVMTQSPDSLAVSLGERATINCRASKSVSSSGYSFMHWYQQKPGQPPKLLIYLAS NRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPLTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQD SKD STYSLS STLTL SK AD YEKHKVY ACEVTHQGL S SP VTKSFNRGEC

DR4B695 HC SEQ ID NO: 137

EVQLQQSGPELVKPGASVKISCKASGYTFTDQYYMNWVKQSHGKSLEWIGYIYP

NNGDTNYNQKFKGKATLTVDKSSRTVYMELRSLTSEDSAVYYCARNGYSDYSA

WFAYWDQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS

WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVWDVSA

EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC

KVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSL SL SPGK

DR4B695 LC SEQ ID NO: 138

DIVLTQSPASLAVSLGQRATISCRASKSVSASGYSYMHWYQQKLGQPPKLLIYLAS NLESGVPVRFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELKR TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQD SKD STY SL S STLTLSK AD YEKHKVY ACEVTHQGLS SP VTKSFNRGEC

Example 7. Epitope mapping

General reagents Amine-coupling activation reagents were purchased from Pierce; activation reagents were stored at -20°C as single-use aliquots at stock concentrations of 0.4 M 1- ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride (EDC) and 0.1 M

Nhydroxy sulfosuccinimide (sulfo-NHS) in water. Amine-coupling buffers (0.1 M MES pH 4.5 or pH 5.0) were prepared using 2-(Nmorpholino)ethanesulfonic acid sodium salt (MES, catalog number M3058, Sigma). Amine -coupling blocking reagent (1 M ethanolamine HC1 pH 8.5) was purchased from GE Healthcare. All experiments were conducted at 25°C in PBS buffer supplemented with 0.01% Tween-20 (SPRi experiments) or 0.05% Tween-20 + 1 g/1 BSA (BLI experiments). Coupled mAbs were regenerated using 75 mM phosphoric acid, unless stated otherwise.

SPRi and CFM method

A CFM 2 (Wasatch Microfluidics) was used to create a microarray of 96 mAbs. It draws forty -eight 70-m1 plugs of sample from a 96-well microplate into a fluidic manifold which focuses the solutions into an array of 48 micro flow cells on the surface of the SPR substrate (a G-COOH coated prism from Ssens bv, NL) and cycles the solutions back and forth at 60 mΐ/min. A 96-well microplate was prepared with 100 mΐ of each mAb at 30 pg/rnl in MES coupling buffer pH 4.5 and loaded into bay 2 of the CFM. A second plate of freshly mixed activating reagents (150 mΐ 0.4 M EDC and 150 mΐ 0.1 M sulfo-NHS in a total of 5 ml of MES coupling buffer pH 4.5) was loaded into bay 1. The CFM was then primed with system buffer (PBS + 0.01% T20). The mAb plate contained 89 mAbs. Once docked, the activating reagents were cycled over the surface for 7 min and followed immediately by the first set of mAbs (top half of the mAb plate) and cycled for 15 min. Without undocking, the spots were rinsed with the system buffer. Since the CFM prints 48 solutions at a time, it needs to address the surface twice to create the full array of 89 mAbs. After the first print, the CFM was paused to load fresh activation reagents, and the same cycle of 7-min activation and 15 -min coupling was repeated for the second half of the mAb plate. The printed prism was then loaded into the SPRi reader (MX96, IBIS Technologies bv), which uses a single flow cell and autosampler configured to address the array with back-and-forth cycled injections of 80 mΐ per analyte. Once loaded, 1 M ethanolamine was injected across the chip for 15 min to quench the excess reactive esters. The chip was then washed with system buffer and the chip image was used to define the reaction spots (i.e., the 96-ligand array) and the interstitial reference spots (two local reference spots per reaction spot). For classical (Sandwich) binning, a co-injection was used, where both antigen and mAb analyte were transported to the flow cell in parallel lines and injected immediately after one another before continuing with regeneration. Antigen (DR4G90 or DR4G92) was injected for 3 min, followed by 20 pg/rnl mAb for a further 3 min, and then the surfaces were regenerated. All SPRi experiments were conducted in a 96X96 analyte-on-ligand format.

SPRi data analysis

Data were processed in SPRint software v. 6.15.2.1 (calibrated, locally referenced, and aligned to zero on the Y-axis prior to the binding step of interest) and analyzed in Wasatch Microfluidics’ binning software for heat map generation, sorting and node plotting. Hierarchical clustering was used to group like-behaved mAbs together in the heat map. Heat maps and node plots are alternate ways of visualizing epitope bins and their inter-bin relationships. In interpreting a node plot, it is assumed that all mAbs in the panel have been tested for pairwise competition against one another so that a chord connects two mAbs that showed a blocking relationship, and no chord represents a non-blocking relationship. MAbs that belong to the same bin are inscribed by the envelopes (the colors of the envelopes are auto-generated in the binning software and carry no meaning). The node plots for our rlsdB study are color-coded by each mAb’s Hb-blocking status, as determined by BLI, where red - blocker, green - non-blocker, and yellow - partial blocker.

89 mAbs were submitted for binning analysis with DR4G92. Competition relationships tree was generated using Combined binary dendrogram, calculated from normalized heat map. 18 Groups generated with cutoff height of 6. DR4B668 mapped to bin 7 with one other mAb. DR4B683 and DR4B695 mapped to bin 16 with six additional antibodies within the bin.

To identify the binding epitopes for 6 HLA-DR/Colff_259 mAbs, DR4B397 (pan- DR control antibody), DR4B583, DR4B590, DR4B668, DR4B683 and DR4B695, on DR4G92 protein, solution hydrogen/deuterium exchange-mass spectrometry (HDX-MS) was performed using the corresponding Fabs.

Recombinant HLA-DR4 protein (DR4G92, 4.59 mg/mL) was purified from transient 293 Expi cells. All antibodies were expressed and purified from transient 293 Expi cells.

For H/D exchange, the procedures used to analyze the Fab perturbation were similar to that described previously (Hamuro el al, J. Biomol. Techniques 14: 171-182, 2003; Horn et al, Biochemistry 45:8488-8498, 2006) with some modifications. Pepsin/protease XVIII digestion and LC-MS

15.54 pg of DR4G92 in 120 pL of control buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) was denatured by adding 120 pL of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH was 2.5) and incubating the mixture for 3 min at 25 °C. Then, the mixture was subjected to on-column pepsin/protease XIII digestion using an in-house packed pepsin/protease XIII column (2.1 x 30 mm). The resultant peptides were analyzed using an UPLC-MS system comprised of a Waters Acquity UPLC coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass

Spectrometer (Thermo). The peptides were separated on a 50 x 1 mm C8 column with a 20.5 min gradient from 2-34% solvent B (0.2% formic acid in acetonitrile). Solvent A was 0.2% formic acid in water. The injection valve and pepsin/protease XIII column and their related connecting tubings were inside a cooling box maintained at 25 °C. And the second switching valve, C8 column and their related connecting stainless steel tubings were inside another chilled circulating box maintained at 0 °C. Peptide identification was done through searching MS/MS data against the DR4G92 sequence with Mascot. The mass tolerance for the precursor and product ions was 7 ppm and 0.02 Da, respectively.

100% sequence coverage was achieved for both DR4G92-a chain and -b chain.

H/D Exchange

10 pL of native DR4G92 (15.54 pg), 10 pL of native DR4G92 & anti-HLA-DR4 mAb DR4B397 mixture (15.54 pg: 31.7 pg), or 10 pL of native DR4G92 & any other anti-HLA-DR4 mAb mixture, was incubated with 110 pL deuterium oxide labeling buffer (50mM sodium phosphate, 100 mM sodium chloride at pH 7.4) for 0 s, 60 s, 1800 s, or 7200 s at 25 °C. Hydrogen/deuterium exchange was quenched by adding 120 pL of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH was 2.5). Subsequently, the quenched samples were subjected to on-column pepsin/protease XIII digestion and LC- MS analysis as described above. The mass spectra were recorded in MS only mode.

Raw MS data was processed using HDX WorkBench, software for the analysis of H/D exchange MS data (J. Am. Soc. Mass Spectrom. 2012, 23 (9), 1512-1521). The deuterium levels were calculated using the average mass difference between the deuterated peptide and its native form (to). Table 23 shows the epitope regions identified for DR4B668, DR4B683, DR4B695 and the control antibody DR4B397 (L243 cloned as IgGlsigma). While L243 binds overlapping or nearly identical epitope when compared to DR4B668, DR4B683, DR4B695 epitopes, the distinctness of binding is evident from the fact that L243 is a pan-inhibitor while DR4B668, DR4B683 and DR4B695 are selective HLA-DR/ColII_259 binders.

Table 23.

Example 8. HLA-DR/ColII_259 antibodies selectively inhibit collagenII-DR4 specific T cells

Select antibodies were tested for their ability to selectively inhibit Collagen II- DR4 specific T cells using T cell hybridomas that specifically recognize collagen II peptide (amino acids 259-273; GIAGFKGEQGPKGEP, SEQ ID NO: 123), HA peptide (amino acids 306-318; PKYVKQNTLKLAT, (SEQ ID NO: 13), CILP peptide (amino acids 982-996(Cit-988,991); GKLYGI{Cit}DV{Cit}STRDR, SEQ ID NO: 127) or Fibrinogen peptide (amino acids 69-81(Cit-74); GGYRA{Cit}PAKAAAT, (SEQ ID NO: 128) presented by HLA-DR4. {cit}: citrulline

The Boleth B cell line (homozygous for HLA-DRBl*04:01) was obtained from the International Histocompatibility Working Group. The Boleth cells were washed and resuspended in complete medium (IMDM + 20% FBS + 1% Gentamicin + 1% Glutamax) at 1 x 10⁶ cells/ml; 50 mΐ cells were added to each well of a 96-well round bottom plate. Antibodies to be tested were added at 4X the final concentration, 50 mΐ per well, beginning at a concentration of 10 pg/ml. The plates were incubated for 1 hr at 37°C.

The T cell hybridoma lines HA5D3.9, CII259-26B6.18, CILP982-6C5.5, and Fib- b69-2C6.1 were generated at Janssen. These cells were washed with complete medium, resuspended in complete medium at a concentration of 2 x 10⁶ cells/ml, and added (50 mΐ/wcll) to the plate containing the Boleth cells. The indicated peptide was diluted in complete medium to 4X the final indicated concentration and added to the plate at 50 mΐ/well. The total volume in all wells was brought up to 200 mΐ using complete medium, if needed. The plates were incubated for 18-21 hr at 37°C. The supernatants were harvested for analysis using the mIL-2 AlphaLISA kit (Perkin Elmer) according to manufacturer’s instructions.

From the tested antibodies, DR4B668, DR4B683 and DR4B695 inhibited

ColII_259-DR4- specific T cells in a dose dependent manner as shown in Figure 5. The antibodies did not inhibit T cells specific to other antigens, such as HA (Figure 6A), CLIP (Figure 6B) and fibrinogen (Figure 6C), whereas the pan-HLA-DR antibody L243 inhibited T cells regardless of their antigen specificity.

Example 9. HLA-DR4/ColII_259 antibodies detect clonal DR4/CII 259 B cells with high specificity

Binding of select antibodies to BOLETH cells expressing HLA-DR4 loaded with ColII_259 peptide or without exogenously added peptide was assessed using FACS over a range of antibody concentrations. DR4B706 was over 31 -fold selective in binding ColII_259 loaded vs. unloaded cells and demonstrated improved selectivity over the parental mouse antibody DR4B683, which demonstrated 4-fold selectivity. Figure 7A shows the dose response curve of binding of the antibodies to ColII_259 loaded cells, and Figure 7B shows the dose response curve of binding of the antibodies to unloaded BOLETH cells. Table 24 shows the calculated MFI and fold-changes of binding to ColII_259 peptide loaded vs. unloaded cells an antibody concentration of 6.7 nM.

Table 24.

The antibodies were also tested for their binding the various peptide-HLA complexes using a kit from OneLambda, which included DRB1*01:01, DRB1*01:02, DRB1*01:03, DRB 1*03:01, DRB 1*03:03, DRB1*04:01, DRB1*04:02, DRB 1*04:03, DRB1*04:04, DRB1*04:05, DRB1*07:01, DRB1*08:01, DRB1*09:01, DRB1*10:01, DRB1*11:01, DRB 1*11:04, DRB1*12:01, DRB1*12:02, DRB1*13:01, DRB1*13:03, DRB1*14:01, DRB1*14:02, DRB1*14:54, DRB1*15:01, DRB1*15:02, DRB1*15:03, DRB1*16:01, DRB1*16:02, DRB3*01:01, DRB3*02:02, DRB3*03:01, DRB4*01:01, DRB4*01:03, DRB5*01:01, DRB5*02:02, DPAl*01:03xDPBl*01:01,

DPA1 *02:0 lxDPB 1*01:01, DPAl*01:03xDPB 1*02:01, DPA 1 *02 :02xDPB 1*05:01, DPAl*01:03xDPB 1*03:01, DPAl*01:05xDPB 1*03:01, DPAl*02:01xDPB 1*03:01, DPAl*01:03xDPB 1*04:01, DPAl*01:03xDPB 1*04:02, DPAl*02:01xDPB 1*05:01, DPA1 *02:0 lxDPB 1*06:01, DPAl*01:03xDPBl*06:01 , DPAl*02:01xDPBl*09:01, DPAl*02:02xDPB 1*10:01, DPAl*01:03xDPB 1*11:01, DPAl*01:03x DPB 1*28:01, DPA1 *02:0 lxDPB 1*13:01, DPAl*02:01x DPB 1*13:01, DPA1 *03:0 lxDPB 1*13:01, DPA1 *02:0 lx DPB 1*14:01, DPAl*02:01x DPB 1*15:01, DPA1 *02:0 lx DPB 1*17:01, DPA1 *02:0 lx DPB 1*18:01, DPAl*01:05x DPB 1*18:01, DPAl*01:04x DPB1*18:01, DPAl*01:03x DPB 1*19:01, DPAl*03:01x DPB 1*20:01, DPAl*01:03x DPB 1*23:01, DPAl*01:05xDPBl*28:01, DPAl*04:01x DPB1*28:01, DPAl*02:02x DPB1*11:01, DQAl*02:01x DQB1*02:01, DQAl*03:01x DQB1*02:01, DQAl*04:01x DQB1*02:01, DQAl*05:01x DQB1*02:01, DQAl*02:01x DQB1*02:02, DQAl*02:01x DQB1*04:01, DQAl*03:03x DQB1*04:01, DQAl*02:01x DQB1*04:02, DQAl*02:01x DQB1*04:02, DQAl*01:01x DQB1*05:01, DQAl*01:01x DQB1*05:01, DQAl*01:03x DQB1*06:01, DQAl*01:02x DQB1*06:02, DQAl*01:01xDQBl*06:02, DQAl*01:03xDQBl*06:03, DQAl*01:02x DQB1*06:04, DQAl*01:02x DQB1*06:09, DQAl*03:01x DQB1*03:01, DQAl*02:01x DQB1*03:01, DQAl*05:03x DQB1*03:01, DQAl*05:05x DQB1*03:01, DQAl*06:01x DQB1*03:01, DQAl*02:01x DQB1*03:02, DQAl*03:01x DQB1*03:02, DQAl*03:01x DQB1*03:02, DQAl*02:01x DQB1*03:03, DQAl*03:01x DQB1*03:03 and DQAl*03:02x DQB1*03:03. DR4B683 showed a small amount of binding to DRB1*01:01, *04:01, *10:01, *13:01, *DRB3*02:02, DRB3*03:01, as well as multiple DP alleles. DR4B695 showed weak background binding to DRB 1*10:01, DRB3 *02:02, and no significant DP or DQ reactivity. DR4B706 had no significant DR or DQ reactivity and only slight reactivity to DPAl*02:02x DPB1*11:01.

Example 10. HLA-DR/ColII_259 mAbs display low clearance rates

The PK of HLA-DR/ColII_259_mAbs was determined in DR4 transgenic mice. HLA-DR4 transgenic mice were purchased from Taconic. Mice were injected IP with lOmg/kg antibody and serum was collected at 0.25h, 0.5h, lh, 3h, 6h, 24h, 48h, 72h, 96h, 7d, 14d, 2 Id from groups of 4 mice per time point. The presence of HLA- DR/ColII_259_mAb in the DR4 transgenic mouse serum was measured by Human-IgG AlphaLISA according to the kit manufacturer’s instructions. Table 25 shows the Cmax and Cl/F values for the tested antibodies. DR4B397, a pan-DR antibody, had very high clearance rate of 1,386 CL/F, whereas isotype control (PP1B40) had a much lower clearance rate of 9.3 CL/F. The HLA-DR ColII_259_mAbs DR4B668 and DR4B683 had clearance rates more similar to isotype control of 77 CL/F and 82 CL/F, respectively. Figure 8 shows the antibody concentrations over time in blood of DR4 transgenic mice.

Table 25.

Claims

WHAT IS CLAIMED

1) An isolated antibody or an antigen-binding fragment thereof specifically binding a HLA-DR/ColII_259 complex.

2) The antibody or the antigen-binding fragment thereof of claim 1, wherein the antibody or the antigen-binding fragment thereof has one or more of the following properties: a) binds the HLA-DR/ColII_259 complex with an equilibrium dissociation constant (K_D) of less than about 5xl0 ⁸ M; or

b) inhibits activation of HLA-DR/ColII_259 specific T cells.

3) The antibody or the antigen-binding fragment thereof of claim 2 or 3, comprising a) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:

43, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 63 or SEQ ID NO: 73;

b) a HCDR2 or SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 66, SEQ ID NO: 76 or SEQ ID NO: 58;

c) a HCDR3 of SEQ ID NO: SEQ ID NO: 50, SEQ ID NO: 69 or SEQ ID NO: 79; d) a light chain complementarity determining region 1 (LCDR1) or SEQ ID NO: 82, SEQ ID NO: 90 or SEQ ID NO: 97;

e) a LCDR2 of SEQ ID NO: 85 or the amino acid sequence YTS; and

f) a LCDR3 of SEQ ID NO: 88 or SEQ ID NO: 95.

4) The isolated antibody or the antigen-binding fragment thereof of claim 3, wherein the antibody or the antigen-binding fragment thereof binds HLA-DR a chain of SEQ ID NO: 15 within acid residues of SEQ ID NO: 119 and SEQ ID NO: 120.

5) The antibody or the antigen-binding fragment thereof of claim 3, comprising a heavy chain variable region (VH) of SEQ ID NO: 115 and a light chain variable region (VL) of SEQ ID NO 116.

6) The antibody or the antigen-binding fragment thereof of claim 5, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 82, SEQ ID NO:

85 and SEQ ID NO: 88, respectively;

b) SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 50, SEQ ID NO: 82, SEQ ID NO:

85 and SEQ ID NO: 88, respectively;

c) SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 90, the amino acid sequence YTS and SEQ ID NO: 95, respectively; d) SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 97, the amino acid sequence YTS and SEQ ID NO: 88, respectively;

e) SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 82, SEQ ID NO:

85 and SEQ ID NO: 88, respectively;

f) SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 50, SEQ ID NO: 82, SEQ ID NO:

85 and SEQ ID NO: 88, respectively;

g) SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 90, the amino acid sequence YTS and SEQ ID NO: 95, respectively; or

h) SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 97, the amino acid sequence YTS and SEQ ID NO: 88, respectively.

7) The antibody or the antigen-binding fragment thereof of claim 6, comprising the VH and the VL of

a) SEQ ID NO: 139 and SEQ ID NO: 100, respectively; or

b) SEQ ID NO: 140 and SEQ ID NO: 101, respectively.

8) The antibody or the antigen-binding fragment thereof of claim 1 or 2, comprising a) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:

45, SEQ ID NO: 55, SEQ ID NO: 64 or SEQ ID NO: 74;

b) a HCDR2 or SEQ ID NO: SEQ ID NO: 48, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 77 or SEQ ID NO: 60;

c) a HCDR3 of SEQ ID NO: SEQ ID NO: 51, SEQ ID NO: 70 or SEQ ID NO: 80; d) a light chain complementarity determining region 1 (LCDR1) or SEQ ID NO: 83, SEQ ID NO: 91 or SEQ ID NO: 98;

e) a LCDR2 of SEQ ID NO: SEQ ID NO: 86 or the amino acid sequence LAS; and f) a LCDR3 of SEQ ID NO: SEQ ID NO: 89 or SEQ ID NO: 96.

9) The antibody or the antigen-binding fragment thereof of claim 8, wherein the antibody binds HLA-DR a chain of SEQ ID NO: 15 within amino acid residues of SEQ ID NO: 120 and HLA-DR b chain of SEQ ID NO: 16 within amino acid residues of SEQ ID NO: 121.

10) The antibody or the antigen-binding fragment thereof of claim 8, comprising a heavy chain variable region (VH) of SEQ ID NO: 117 and a light chain variable region (VL) of SEQ ID NO 118.

11) The antibody or the antigen-binding fragment thereof of claim 10, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO: 86 and SEQ ID NO: 89, respectively;

b) SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO:

86 and SEQ ID NO: 89, respectively;

c) SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 91, the amino acid sequence LAS and SEQ ID NO: 96, respectively;

d) SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 98, the amino acid sequence LAS and SEQ ID NO: 89, respectively; or

e) SEQ ID NO: 55, SEQ ID NO: 60, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO:

86 and SEQ ID NO: 89, respectively.

12) The antibody or the antigen-binding fragment thereof of claim 11, comprising the VH and the VL of

a) SEQ ID NO: 141 and SEQ ID NO: 102, respectively; or

b) SEQ ID NO: 142 and SEQ ID NO: 103, respectively.

13) The antibody or the antigen-binding fragment thereof of claim 1 or 2, comprising a) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:

46, SEQ ID NO: 56, SEQ ID NO: 65 or SEQ ID NO: 75;

b) a HCDR2 or SEQ ID NO: 49, SEQ ID NO: 61, SEQ ID NO: 68 or SEQ ID NO:

78;

c) a HCDR3 of SEQ ID NO: 52, SEQ ID NO: 71 or SEQ ID NO: 81;

d) a light chain complementarity determining region 1 (LCDR1) or SEQ ID NO: 84, SEQ ID NO: 92 or SEQ ID NO: 99;

e) a LCDR2 of SEQ ID NO: SEQ ID NO: 87 or the amino acid sequence LAS; and f) a LCDR3 of SEQ ID NO: 89 or SEQ ID NO: 96.

14) The antibody or the antigen-binding fragment thereof of claim 13, wherein the

antibody binds HLA-DR a chain of SEQ ID NO: 15 within amino acid residues of SEQ ID NO: 120.

15) The antibody or the antigen-binding fragment thereof of claim 14, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 84, SEQ ID NO:

87 and SEQ ID NO: 89, respectively;

b) SEQ ID NO: 56, SEQ ID NO: 61, SEQ ID NO: 52, SEQ ID NO: 84, SEQ ID NO:

87 and SEQ ID NO: 89, respectively;

c) SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 92, the amino acid sequence LAS and SEQ ID NO: 96, respectively; or d) SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 99, the amino acid sequence LAS and SEQ ID NO: 89, respectively.

16) The antibody or the antigen-binding fragment thereof of claim 15, comprising the VH of SEQ ID NO: 143 and the VL of SEQ ID NO: 104.

17) The antibody or the antigen-binding fragment of any one of claims 1-16, wherein HLA-DR is HLA-DR4 or HLA-DR1.

18) The antibody or the antigen-binding fragment of claim 17, wherein HLA-DR a chain and HLA-DR b chain comprise amino acid sequences of

a) SEQ ID NOs: 15 and 16, respectively; or

b) SEQ ID NOs: 15 and 17, respectively.

19) The antibody or the antigen-binding fragment of any one of claims 1-18, wherein ColII_259 comprises an amino acid sequence of SEQ ID NO: 123.

20) The antibody or the antigen-binding fragment thereof of any one of claims 1-19, wherein the antibody

a) is an IgGl isotype;

b) is an IgG2 isotype;

c) is an IgG3 isotype;

d) is an IgG4 isotype;

e) comprises at least one substitution in an Fc region that modulates binding of the antibody to FcyR or FcRn;

f) is an IgG2 isotype comprising V234A, G237A, P238S, H268A, V309L, A330S and P33 IS substitutions when compared to the wild-type IgG2 of SEQ ID NO: 125;

g) is an IgGl isotype comprising L234A, L235A, G237A, P238S, H268A, A330S and P33 IS substitutions when compared to the wild-type IgGl of SEQ ID NO: 124;

h) is an IgGl isotype comprising L234A and L235A substitutions when compared to the wild-type IgGl of SEQ ID NO: 124; or

i) is an IgG4 isotype comprising S228P, F234A and L235A substitutions when compared to the wild-type IgG4 of SEQ ID NO: 126.

21) The antibody or the antigen-binding fragment thereof of any one of claims 1-20, wherein the antibody is conjugated to a second molecule.

22) The antibody or the antigen-binding fragment thereof of claim 21, wherein the second molecule is a detectable label, a cytotoxic agent or an immunomodulatory agent. 23) The antibody or the antigen-binding fragment thereof of any one of claims 1-22, wherein the antibody is a multispecific or a bispecific antibody.

24) A pharmaceutical composition comprising the antibody or the antigen-binding

fragment of any one of claims 1-23 and a pharmaceutically accepted carrier.

25) A polynucleotide

a) encoding the VH, the VL or the VH and the VL of SEQ ID NO: 139, SEQ ID NO:

140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103 or SEQ ID NO: 104; or b) comprising the polynucleotide sequence of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113 or SEQ ID NO: 114.

26) A vector comprising the polynucleotide of claim 25.

27) A host cell comprising the vector of claim 26.

28) A method of producing the antibody or the antigen-binding fragment thereof of any one of claims 1-23, comprising culturing the host cell of claim 27 in conditions that the antibody is expressed, and recovering the antibody produced by the host cell.

29) A method of treating or preventing a HLA-DR/ColII_259 complex-mediated disease, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or the antigen-binding fragment thereof of any one of claims 1-23 or the pharmaceutical composition of claim 24 for a time sufficient to treat the autoimmune disease.

30) The method of claim 29, wherein the HLA-DR/ColII_259 complex-mediated disease is an autoimmune disease, arthritis, juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter’s syndrome, ankylosing spondylitis, gouty arthritis or systemic juvenile idiopathy arthritis.

31) The method of claim 29 or 30, wherein the antibody or the antigen-binding fragment thereof is administered in combination with a second therapeutic agent.

32) The method of claim 31, wherein the second therapeutic agent is a corticosteroid or an immunosuppressant.

33) A method of suppressing an immune response towards ColII_259 presented on HLA- DR in a subject, comprising administering to the subject the antibody or the antigenbinding fragment thereof of any one of claims 1-23 or the pharmaceutical composition of claim 24 for a time sufficient to suppress the immune response towards ColII_259 presented on HLA-DR. 34) An anti-idiotypic antibody binding to the antibody or the antigen-binding fragment thereof of any one of claims 1-23.

35) A kit comprising the antibody or the antigen-binding fragment of any one of claims 1- 23.

36) The antibody of any one of claims 1-23 or the pharmaceutical composition of claim 24 for use in therapy.

37) The antibody of any one of claims 1-23 or the pharmaceutical composition of claim 24 for use in the treatment of an autoimmune disease, arthritis, juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter’s syndrome, ankylosing spondylitis, gouty arthritis or systemic juvenile idiopathy arthritis.

38) The antibody of any one of claims 1-23 for use in the manufacture of medicament for treating an autoimmune disease, arthritis, juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter’s syndrome, ankylosing spondylitis, gouty arthritis or systemic juvenile idiopathy arthritis.

39) A method of detecting cells expressing on their surface a HLA-DR/ColII_259

complex in a biological sample, comprising

a) obtaining the biological sample;

b) contacting the biological sample with the antibody or an antigen-binding fragment of any one of claims 1-23; and

c) detecting the antibody bound on the cells expressing on their surface the HLA- DR/ColII_259 complex.

40) A method of isolating or detecting cells expressing on their surface a HLA- DR/ColII_259 complex in a biological sample, comprising

a) obtaining a biological sample;

b) contacting the biological sample with the antibody or the antigen-binding

fragment of any one of claims 1-23; and

c) isolating or detecting the cells bound to the antibody or the antigen-binding

fragment of any one of claims 1-23.

41) A method of detecting cells expressing on their surface a HLA-DR/ColII_259

complex in a subject, comprising

a) conjugating the antibody or the antigen-binding fragment of any one of claims 1- 23 to a detectable label to form a conjugate;

b) administering the conjugate to the subject; and

c) visualizing in the subject the cells expressing on their surface the HLA- DR/ColII_259 complex to which the conjugate is bound.