CR2-binding proteins and their use in medical treatment

Abstract

The present invention provides CR2 binding proteins that bind to human CR2, pharmaceutical compositions comprising said CR2 binding proteins, and their use in the treatment or prevention of autoimmune and / or inflammatory conditions, infectious diseases and malignancies associated with Epstein-Barr virus (EBV); and their use as vaccine adjuvants / antigen carriers.

Description

[Technical field]

[0001] Field of Disclosure The present invention relates to a CR2 binding protein that binds to and neutralizes complement receptor 2 (CR2 or CD21). The present invention also relates to a method of treating a disease or disorder with said CR2 binding protein and a pharmaceutical composition comprising the CR2 binding protein. Other aspects of the present invention will become apparent from the following description. [Background technology]

[0002] Background of the disclosure The complement system is an evolutionarily conserved set of plasma proteins that are part of the innate immune response to invading pathogens. Proteolytic activation of complement occurs at activated surfaces, leading to the deposition of active complement components that have a range of effector functions, including regulation of adaptive immune responses. Complement receptor 2 (CR2), also known as CD21, is a 145 kDa membrane-bound glycoprotein expressed primarily on B lymphocytes and follicular dendritic cells (FDCs) and serves as a bridge between innate and adaptive immunity. CR2 is the receptor for the covalently linked smallest complement component 3 (C3) fragments, C3dg and C3d (together referred to as C3d / g), with weaker binding to iC3b. Interferon alpha, double-stranded DNA and Epstein-Barr virus gp350 / 220 have also been described as ligands for CR2.

[0003] CR2 plays three pivotal roles in adaptive immune responses by 1) binding opsonized antigens and transporting and retaining them in lymphoid tissues, 2) lowering the threshold for B cell activation, and 3) driving affinity maturation and germinal center formation via B cell-FDC crosstalk. The importance of these mechanisms in humans is confirmed by rare cases of CR2 deficiency, in which patients have significantly fewer memory B cells and reduced antibody diversity and serum immunoglobulin levels.

[0004] Germinal centers (GCs) and, in the context of autoimmunity, ectopic lymphoid structures (ELS) that form outside primary and secondary lymphoid organs, are central to the autoimmune response. There are several roles for CR2 in the formation, maintenance, and productivity of GCs and ELS (see Figure 1). 1) Carriage of C3d / g-opsonized antigens on noncognate B cells in the circulation and within GC and ELS; 2) a lowered threshold for B cell activation by C3d / g-opsonized antigens within the GC / ELS; 3) B cell outputs downstream of CR2 / BCR co-ligation, e.g., stimulation of antibody production; 4) retention of C3d / g-opsonized antigens within the FDC reservoir for presentation to B cells; and 5) Activation of FDC function, e.g., cytokine production.

[0005] Disruption of these CR2-dependent mechanisms is predicted to have profound effects on GC / ELS, including reduced antigen availability, disruption of FDC:B cell interactions, reduction of antigen-specific antibodies, and disruption of t follicular helper (Tfh) cell crosstalk with B cells.

[0006] Many autoimmune and / or inflammatory pathologies are characterized by high levels of autoantibodies, suggesting impaired immune tolerance and dysregulated B cell activation. For autoimmune diseases such as Sjögren's syndrome, systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), blocking complement C3d / g-CR2 interaction with CR2 blocking molecules increases the B cell activation threshold and prevents the activation and proliferation of autoantibody-producing B cells in response to self antigens.

[0007] Viral diseases, such as HIV, can be difficult to treat due to the persistence of long-lived viral reservoirs in FDCs and lymphoid tissues. Antigen binding, retention for more than a year, and presentation are highly dependent on CR2-dependent capture of C3 fragment-coated antigens. Once C3d / g-opsonized antigens are transferred from marginal zone (MZ) B cells to FDCs, the intact antigens are rapidly internalized by small non-degradable endosomal vesicles. These vesicles are then periodically recycled to the cell surface where the intact antigens and other FDC surface proteins can be recognized and acquired by cognate B cells. Similarly, C3d / g-opsonized HIV virions are captured and retained for long periods in these non-degradable endosomes, which represent long-lived viral reservoirs.

[0008] Molecules that bind to CR2 can be used as adjuvants / antigen carriers to amplify immune responses to immunization. Low doses of antigens bound to anti-CR2 antibodies have been found to induce rapid and sustained IgG immune responses in mice and cynomolgus monkeys (Whipple, EC et al., Mol. Immunol., 2007. 44(4): p. 377-388). In summary, there is a need for the treatment of autoimmune and / or inflammatory conditions, infectious diseases and other disorders or conditions that involve B cell activation or retention of C3d or C3dg opsonized antigens in FDC reservoirs. Additionally, there is a need for alternative vaccine adjuvants and antigen carriers. Summary of the Invention

[0009] Disclosure Summary The present invention provides a CR2 binding protein (hereinafter referred to as "CR2 binding protein") that binds to a novel epitope of CR2 and blocks the binding of a ligand to the CR2 receptor.

[0010] The present invention also provides a CR2 binding protein that binds to one or more residues in the linker between the two short complement repeat domains and / or near the C-terminus of human CR2. In one embodiment, the CR2 binding protein binds to one or more amino acid residues in SEQ ID NO:2 and / or SEQ ID NO:71 of human CR2. In another embodiment, the CR2 binding protein binds to one or more amino acid residues in SEQ ID NO:2 of human CR2. In another embodiment, the CR2 binding protein binds to one or more amino acid residues in SEQ ID NO:71 of human CR2. In another embodiment, the CR2 binding protein protects residues 66-70 (SEQ ID NO:2) of CR2 from deuterium exchange in HDX-MS analysis. In a further embodiment, the CR2 binding protein protects residues 66-70 (SEQ ID NO:2) and / or residues 104-127 (SEQ ID NO:71) of CR2 from deuterium exchange in HDX-MS analysis.

[0011] The present invention also provides a CR2 binding protein that is an antibody. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the monoclonal antibody is IgG1 or IgG4. In another embodiment, the monoclonal antibody is IgG1. In another embodiment, the antibody comprises in its light chain variable region a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12. In another embodiment, the antibody comprises in its heavy chain variable region a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9. In another embodiment, the antibody comprises a variable light chain amino acid sequence as shown in SEQ ID NO: 6. In another embodiment, the antibody comprises a variable heavy chain amino acid sequence as shown in SEQ ID NO: 5. In another embodiment, the antibody comprises a light chain amino acid sequence as shown in SEQ ID NO: 4. In another embodiment, the antibody comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 3. In a further embodiment, the antibody comprises a light chain amino acid sequence as set forth in SEQ ID NO:4 and a heavy chain amino acid sequence as set forth in SEQ ID NO:3.

[0012] The present invention also provides a CR2 binding protein comprising a constant region such that the CR2 binding protein has reduced ADCC and / or complement activation or effector function. In one embodiment, the CR2 binding protein constant region comprises substitutions of alanine residues at positions 235 and 237 (EU index numbering). In another embodiment, the CR2 binding protein constant region comprises substitutions of alanine residues at positions 234 and 235 (EU index numbering).

[0013] The present invention also provides CR2 binding proteins that are human, humanized or chimeric.

[0014] The present invention also provides a CR2 binding protein comprising any one or combination of the following CDRs: CDRH1, CDRH2, CDRH3 from SEQ ID NO:5 and / or CDRL1, CDRL2, CDRL3 from SEQ ID NO:6. In one embodiment, the CR2 binding protein comprises in its light chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12, or a variant of one or all of these CDRs, wherein the CDR variant has one, two or three amino acid modifications. In another embodiment, the CR2 binding protein comprises in its light chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12. In another embodiment, the CR2 binding protein comprises in its heavy chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, or a variant of one or all of these CDRs, the CDR variant having one, two or three amino acid modifications. In another embodiment, the CR2 binding protein comprises in its heavy chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9. In another embodiment, the CR2 binding protein comprises a variable light chain amino acid sequence as set forth in SEQ ID NO:6. In another embodiment, the CR2 binding protein comprises a variable heavy chain amino acid sequence as set forth in SEQ ID NO:5. In another embodiment, the CR2 binding protein comprises a variable light chain amino acid sequence as set forth in SEQ ID NO:6 and a variable heavy chain amino acid sequence as set forth in SEQ ID NO:5. In another embodiment, the CR2 binding protein comprises a light chain amino acid sequence as set forth in SEQ ID NO:4. In another embodiment, the CR2 binding protein comprises a heavy chain amino acid sequence as shown in SEQ ID NO:3.In a further embodiment, the CR2 binding protein comprises a light chain amino acid sequence as set forth in SEQ ID NO:4 and a heavy chain amino acid sequence as set forth in SEQ ID NO:3.

[0015] The present invention also provides a CR2 binding protein comprising any one or combination of the following CDRs: CDRH1, CDRH2, CDRH3 from SEQ ID NO: 17 and / or CDRL1, CDRL2, CDRL3 from SEQ ID NO: 18. In one embodiment, the CR2 binding protein comprises in its light chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, or a variant of one or all of these CDRs, wherein the CDR variant has one, two or three amino acid modifications. In another embodiment, the CR2 binding protein comprises in its light chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24. In another embodiment, the CR2 binding protein comprises in its heavy chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, or a variant of one or all of these CDRs, the CDR variant having one, two or three amino acid modifications. In another embodiment, the CR2 binding protein comprises in its heavy chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21. In another embodiment, the CR2 binding protein comprises a variable light chain amino acid sequence as set forth in SEQ ID NO: 18. In another embodiment, the CR2 binding protein comprises a variable heavy chain amino acid sequence as set forth in SEQ ID NO: 17. In another embodiment, the CR2 binding protein comprises a variable light chain amino acid sequence as set forth in SEQ ID NO: 18 and a variable heavy chain amino acid sequence as set forth in SEQ ID NO: 17. In another embodiment, the CR2 binding protein comprises a light chain amino acid sequence as set forth in SEQ ID NO: 16. In another embodiment, the CR2 binding protein comprises a heavy chain amino acid sequence as shown in SEQ ID NO:15.In a further embodiment, the CR2 binding protein comprises a light chain amino acid sequence as set forth in SEQ ID NO:16 and a heavy chain amino acid sequence as set forth in SEQ ID NO:15.

[0016] The present invention also provides a CR2 binding protein comprising any one or combination of the following CDRs: CDRH1, CDRH2, CDRH3 from SEQ ID NO:29 and / or CDRL1, CDRL2, CDRL3 from SEQ ID NO:30. In one embodiment, the CR2 binding protein comprises in its light chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:35 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:36, or a variant of one or all of these CDRs, wherein the CDR variant has one, two or three amino acid modifications. In another embodiment, the CR2 binding protein comprises in its light chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:35 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:36. In another embodiment, the CR2 binding protein comprises in its heavy chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33, or a variant of one or all of these CDRs, the CDR variant having one, two or three amino acid modifications. In another embodiment, the CR2 binding protein comprises in its heavy chain variable region a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33. In another embodiment, the CR2 binding protein comprises a variable light chain amino acid sequence as set forth in SEQ ID NO: 30. In another embodiment, the CR2 binding protein comprises a variable heavy chain amino acid sequence as set forth in SEQ ID NO: 29. In another embodiment, the CR2 binding protein comprises a variable light chain amino acid sequence as set forth in SEQ ID NO: 30 and a variable heavy chain amino acid sequence as set forth in SEQ ID NO: 29. In another embodiment, the CR2 binding protein comprises a light chain amino acid sequence as set forth in SEQ ID NO: 28. In another embodiment, the CR2 binding protein comprises a heavy chain amino acid sequence as shown in SEQ ID NO:27.In a further embodiment, the CR2 binding protein comprises a light chain amino acid sequence as set forth in SEQ ID NO:28 and a heavy chain amino acid sequence as set forth in SEQ ID NO:27.

[0017] The present invention also provides a CR2-binding protein in which the equilibrium dissociation constant (KD) of the antigen-binding protein-CR2 interaction is 0.1 to 1 nM.

[0018] The invention also provides a CR2 binding protein that competes with any one of the CR2 binding proteins for binding to CR2.

[0019] The present invention also provides a nucleic acid molecule encoding any one of the CR2 binding proteins. In one embodiment, the nucleic acid molecule sequence comprises SEQ ID NO: 13 encoding the heavy chain and / or SEQ ID NO: 14 encoding the light chain. In another embodiment, the nucleic acid molecule sequence comprises SEQ ID NO: 25 encoding the heavy chain and / or SEQ ID NO: 26 encoding the light chain. In a further embodiment, the nucleic acid molecule sequence comprises SEQ ID NO: 37 encoding the heavy chain and / or SEQ ID NO: 38 encoding the light chain.

[0020] The present invention also provides an expression vector comprising a nucleic acid molecule encoding any one of the CR2 binding proteins. In one embodiment, the expression vector comprises a nucleic acid molecule sequence comprising SEQ ID NO: 13 encoding the heavy chain and / or SEQ ID NO: 14 encoding the light chain. In another embodiment, the expression vector comprises a nucleic acid molecule sequence comprising SEQ ID NO: 25 encoding the heavy chain and / or SEQ ID NO: 26 encoding the light chain. In a further embodiment, the expression vector comprises a nucleic acid molecule sequence comprising SEQ ID NO: 37 encoding the heavy chain and / or SEQ ID NO: 38 encoding the light chain.

[0021] The present invention also provides a recombinant host cell comprising an expression vector comprising a nucleic acid molecule encoding any one of the CR2 binding proteins.

[0022] The present invention also provides a CR2 binding protein expressed by a recombinant host cell comprising an expression vector comprising a nucleic acid molecule encoding any one of the CR2 binding proteins.

[0023] The present invention also provides a method for the production of a CR2 binding protein, comprising culturing a recombinant host cell in a medium to produce the CR2 binding protein, and isolating or purifying the CR2 binding protein.

[0024] The present invention also provides pharmaceutical compositions comprising a CR2 binding protein and a pharma- ceutically acceptable carrier or excipient.

[0025] The present invention also provides CR2 binding proteins that block interactions with the C3d / g coated complex. In one embodiment, the present invention provides CR2 binding proteins that block signaling, activation and antibody production downstream of BCR:CR2 co-ligation and also prevent binding of C3d / g antigen to FDCs.

[0026] The present invention also provides CR2 binding proteins for use in therapy.

[0027] The present invention also provides a method for the treatment of an autoimmune and / or inflammatory condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In one embodiment, a method is provided for the treatment of Sjögren's syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method is provided for the treatment of rheumatoid arthritis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method is provided for the treatment of systemic lupus erythematosus in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method is provided for the treatment of vasculitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In a further embodiment, a method is provided for the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein.

[0028] The present invention also provides a method for the treatment of an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In one embodiment, a method is provided for the treatment of HIV in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein.

[0029] The present invention also provides a method for amplifying an immune response to immunization in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein.

[0030] The present invention also provides a method for the treatment of an EBV-associated malignancy in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein.

[0031] The present invention also provides for the use of a CR2 binding protein in the manufacture of a medicament for the treatment of an autoimmune and / or inflammatory condition. In one embodiment, a use of a CR2 binding protein in the manufacture of a medicament for the treatment of Sjogren's syndrome is provided. In another embodiment, a use of a CR2 binding protein in the manufacture of a medicament for the treatment of rheumatoid arthritis is provided. In another embodiment, a use of a CR2 binding protein in the manufacture of a medicament for the treatment of systemic lupus erythematosus is provided. In another embodiment, a use of a CR2 binding protein in the manufacture of a medicament for the treatment of vasculitis is provided. In a further embodiment, a use of a CR2 binding protein in the manufacture of a medicament for the treatment of antineutrophil cytoplasmic antibody (ANCA) associated vasculitis is provided.

[0032] The present invention also provides the use of a CR2 binding protein in the manufacture of a medicament for the treatment of an infectious disease, hi one embodiment, there is provided the use of a CR2 binding protein in the manufacture of a medicament for the treatment of HIV.

[0033] The invention also provides the use of a CR2 binding protein in the manufacture of a vaccine to enhance the immune response to immunization.

[0034] The invention also provides the use of a CR2 binding protein in the manufacture of a medicament for the treatment of an EBV-associated malignancy.

[0035] The present invention also provides a CR2 binding protein for use in the treatment of autoimmune and / or inflammatory conditions. In one embodiment, a CR2 binding protein is provided for use in the treatment of Sjogren's syndrome. In another embodiment, a CR2 binding protein is provided for use in the treatment of rheumatoid arthritis. In another embodiment, a CR2 binding protein is provided for use in the treatment of systemic lupus erythematosus. In another embodiment, a CR2 binding protein is provided for use in the treatment of vasculitis. In a further embodiment, a CR2 binding protein is provided for use in the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis.

[0036] The present invention also provides a CR2 binding protein for use in the treatment of infectious diseases, hi one embodiment, a CR2 binding protein for use in the treatment of HIV is provided.

[0037] The present invention also provides a CR2 binding protein for use in the treatment of EBV-associated malignancies.

[0038] The present invention also provides CR2 binding proteins for use in amplifying the immune response to immunization.

[0039] Other aspects and embodiments of the present invention will become apparent from the following detailed description. [Brief description of the drawings]

[0040] [Figure 1] Figure 1 shows the role of CR2 in GC / ELS formation and productivity. CR2-dependent processes are shown. [Diagram 2]Figure 2 shows differential deuteration from HDX-MS of CR2 peptides between samples in the absence and presence of mAb 1053. Figure 2A shows the average differential incorporation in Da per peptide; Figure 2B shows the average differential incorporation normalized per peptide residue as a percentage of the maximum differential deuteration of 1 Da per residue (assuming that peptides with n non-proline residues have n-2 amides with measurable proton exchange). Each peptide is shown with its position in the CR2 construct used and its sequence. Bars are color-coded by time of exposure to deuteration buffer (0.5 min = grey; 5 min = black). [Diagram 3] Figure 3: Synergistic activation of B cells through co-ligation of BCR and CR2 by C3dg-opsonized antigen. Antigen opsonized with the CR2 ligand C3d / g co-ligates BCR and CR2. BCR binds to its cognate antigen, and CR2 binds to C3d / g, which is covalently linked to the antigen (Ag). Co-ligation of BCR and CR2 results in enhanced signaling through both CD79 (in complex with BCR) and CD19 (part of the B cell co-receptor complex), resulting in downstream signaling and synergistic B cell activation (adapted from Carroll and Isenman, 2012, Immunity, 37 (2). 119-207). [Figure 4] Figure 4: Preincubation with mAb 1053 dose-dependently blocks binding of model C3dg-opsonized ligands to primary human non-cognate B cells. [Diagram 5] FIG. 5: Preincubation with mAb 1053 dose-dependently blocks binding of serum-opsonized PnPS14 to primary human non-cognate B cells. [Figure 6] FIG. 6: Rapid competitive dissociation of bound model C3dg opsonizing ligand from primary human non-cognate B cells. [Figure 7] FIG. 7: mAb 1053 dose-dependently inhibits CR2-mediated intracellular calcium flux in primary human B cells. [Figure 8a-8b] 8a and 8b: mAb 1053 dose-dependently inhibits CR2-mediated phosphoprotein signaling in primary human B cells. [Figure 9] FIG. 9: mAb 1053 dose-dependently inhibits CR2-mediated CD69 upregulation in primary human B cells. [Figure 10] FIG. 10: mAb 996, mAb 999 and mAb 1053 dose-dependently inhibit C3dg-dependent immunoglobulin secretion from primary human B cells. [Figure 11] FIG. 11: Targeted engagement of mAb 1053 to primary human tonsillar FDCs. [Figure 12] FIG. 12: Dose-dependent inhibition of model C3dg-opsonized ligand binding to primary human tonsillar FDCs by mAb 1053. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description of the Disclosure The term "antigen binding protein" as used herein refers to antibodies and other protein constructs, e.g., domains, that can bind to CR2. The terms "CR2 binding protein" and "antigen binding protein" and "anti-CR2 antigen binding protein" are used interchangeably herein.

[0042] The term "antibody" is used broadly herein to refer to molecules having immunoglobulin-like domains (e.g., IgG, IgM, IgA, IgD or IgE) and includes monoclonal antibodies, recombinant antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, multispecific antibodies, e.g., bispecific antibodies, and heteroconjugate antibodies; single variable domains (e.g., domain antibodies (DABs)), antigen-binding antibody fragments, Fab, F(ab')2, Fv, disulfide-linked Fv, single chain Fv, disulfide-linked scFv, diabodies, TANDABS, etc., and modified forms of any of the above (for a review of different "antibody" formats, see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126-1136).

[0043] Alternative antibody formats include another scaffold in which one or more CDRs of the antigen binding protein can be placed onto a suitable non-immunoglobulin protein scaffold or framework, such as an affibody, an SpA scaffold, an LDL receptor class A domain, an avimer (e.g., U.S. Patent Application Publication Nos. 2005 / 0053973, 2005 / 0089932, 2005 / 0164301), or an EGF domain.

[0044] The term "domain" refers to a folded polypeptide structure that retains its tertiary structure independent of the remainder of the polypeptide. Generally, a domain is responsible for a distinct functional property of the polypeptide and can often be added, removed, or transferred to other polypeptides without loss of function of the remainder of the protein and / or domain.

[0045] The term "single variable domain" refers to a folded polypeptide domain comprising sequences characteristic of an antibody variable domain. It therefore includes complete antibody variable domains such as VH, VHH and VL, as well as modified antibody variable domains, such as those in which one or more loops are replaced with sequences not characteristic of antibody variable domains, or antibody variable domains that are truncated or comprise N-terminal or C-terminal extensions, and folded fragments of variable domains that retain at least the binding activity and specificity of the full-length domain. A single variable domain may bind an antigen or epitope independently of the different variable regions or domains. A "domain antibody" or "DAB" may be considered the same as a "single variable domain". A single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent (e.g. as disclosed in WO00 / 29004A1), nurse shark and camelid VHH DAB. Camelid VHHs are immunoglobulin single variable domain polypeptides derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally lacking light chains. Such VHH domains may be humanized according to standard techniques available in the art, and such domains are considered to be "single variable domains". As used herein, VH includes camelid VHH domains.

[0046] Antigen-binding fragments can be provided by placing one or more CDRs on a non-antibody protein scaffold. "Protein scaffold" as used herein includes, but is not limited to, immunoglobulin (Ig) scaffolds, e.g., IgG scaffolds, which can be four-chain antibodies or two-chain antibodies, or can comprise only the Fc region of an antibody, or can comprise one or more constant regions from an antibody, which constant regions can be of human or primate origin, or can be artificial chimeras of human and primate constant regions.

[0047] The protein scaffold can be an Ig scaffold, for example an IgG scaffold, or an IgA scaffold. The IgG scaffold can comprise some or all of the domains of an antibody (i.e., CH1, CH2, CH3, VH, VL). The antigen-binding protein can comprise an IgG scaffold selected from IgG1, IgG2, IgG3, IgG4, or IgG4PE. For example, the scaffold can be IgG1. The scaffold can consist of or comprise, or is a part of, the Fc region of an antibody.

[0048] The protein scaffold may be a derivative of a scaffold selected from the group consisting of CTLA-4, lipocalin, protein A derived molecules such as the Z-domain of protein A (affibody, SpA), A-domain (avimer / maxibody); heat shock proteins such as GroEl and GroES; transferrin (transbody); ankyrin repeat proteins (DARPins); peptide aptamers; C-type lectin domains (tetranectin); human gamma-crystallin and human ubiquitin (affilin); PDZ domains; scorpion toxin kunitz-type domains of human protease inhibitors; and fibronectin / adnectin; which have been protein engineered to obtain binding to antigens, e.g. <antigens> other than the natural ligand.

[0049] An antigen-binding site refers to a site on an antigen-binding protein that can specifically bind to an antigen, which may be a single variable domain or a paired VH / VL domain as can be found in a standard antibody. Single chain Fv (ScFv) domains can also provide an antigen-binding site.

[0050] The term "chimeric antigen receptor" ("CAR"), as used herein, refers to an engineered receptor consisting of an extracellular antigen-binding domain (usually derived from a monoclonal antibody, or a fragment thereof, e.g., the VH and VL domains in the form of an scFv), optionally a spacer region, a transmembrane region, and one or more intracellular effector domains. CARs have also been referred to as chimeric T cell receptors or chimeric immune receptors (CIRs). CARs are transfected into hematopoietic cells, such as T cells, to redirect the specificity of the T cells to a desired cell surface antigen, resulting in CAR-T therapeutics.

[0051] The term "spacer region" as used herein refers to an oligopeptide or polypeptide that serves to link the transmembrane domain to the target binding domain. This region may also be referred to as the "hinge region" or "stalk region". The size of the spacer can be changed depending on the location of the target epitope to maintain a set distance (e.g., 14 nm) during CAR:target binding.

[0052] The term "transmembrane domain," as used herein, refers to the portion of the CAR molecule that crosses the cell membrane.

[0053] The term "intracellular effector domain" (also referred to as "signal transduction domain"), as used herein, refers to a domain in a CAR that is responsible for intracellular signal transduction after the antigen binding domain binds to a target. The intracellular effector domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be a helper activity, including cytolytic activity or secretion of cytokines.

[0054] It will be appreciated by those skilled in the art that the VH and / or VL domains disclosed herein may be incorporated into a CAR-T therapeutic, for example in the form of an scFv.

[0055] In one embodiment, the CR2 binding protein of the present disclosure exhibits cross-reactivity between human CR2 and CR2 from another species, such as cynomolgus monkey CR2. In one embodiment, the CR2 binding protein of the present invention specifically binds human CR2 and cynomolgus monkey CR2. This is particularly useful, since drug development generally requires testing lead drug candidates in animal systems before testing the drug in humans. Providing a drug that can bind to human and monkey species allows results to be tested in these systems and side-by-side comparison of data using the same drug. This eliminates the need to find a drug that acts on animal CR2 and another that acts on human CR2, and also eliminates the need to compare human and animal results using non-identical drugs. Cross-reactivity is also possible between other species used in disease models, such as dogs or mice.

[0056] Optionally, the binding affinity of the antigen binding protein to at least cynomolgus monkey CR2 and to human CR2 differs by no more than 2 or 5 fold.

[0057] Affinity, also referred to as "binding affinity", is the strength of binding at a single interaction site, i.e., of one molecule, e.g., the CR2 binding protein of the present invention, to another molecule, e.g., its target antigen, at a single binding site. The binding affinity of an antigen-binding protein to its target can be determined by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic methods (e.g., BIACORE analysis). For example, the BIACORE method described in Example 3 can be used to measure binding affinity.

[0058] Avidity, also referred to as functional affinity, is the cumulative strength of binding at multiple interaction sites, e.g., the sum of the strengths of binding of two (or more, e.g., in the case of bispecific or multispecific molecules) molecules to each other at multiple sites, taking into account, e.g., the valency of the interactions.

[0059] In one embodiment, the equilibrium dissociation constant (KD) of the antigen binding protein-CR2 interaction is 1 nM or less. In another embodiment, the equilibrium dissociation constant (KD) of the antigen binding protein-CR2 interaction is 0.5 nM or less. Alternatively, the KD can be 0.1-1 nM; or 0.2-0.5 nM. One of skill in the art will appreciate that the smaller the KD number, the stronger the binding. The reciprocal of KD (i.e., 1 / KD) is M -1 The equilibrium association constant (KA) is expressed in units of . One of skill in the art will appreciate that the higher the KA number, the stronger the binding.

[0060] The dissociation rate constant (kd) or "dissociation rate" represents the stability of the antigen-binding protein-CR2 complex, i.e., the rate at which the complex decays per second. For example, kd0.01s -1 corresponds to 1% of the complex decaying per second. In one embodiment, the dissociation rate constant (kd) is 1×10 -4 s -1 Below, 1×10 -5 s -1 or less, or 1×10 -6 s -1 kd is 1×10 -5 s -1 ~1×10 -4 s -1 ; or 1 × 10 -4 s -1 ~1×10 -3 s -1 It could be.

[0061] The association rate constant (ka) or "association rate" refers to the rate of antigen binding protein-CR2 complex formation. In one embodiment, the association rate constant (ka) is 1×10 8 M -1 s -1 In another embodiment, Ka is 1×10 7 ~1×10 8 M -1 s -1 .It could be.

[0062] The term "neutralizing" as used herein means that the biological activity of CR2 is reduced or blocked in the presence of an antigen binding protein described herein, in vitro or in vivo, as compared to the activity of CR2 in the absence of the antigen binding protein. Neutralization may be by one or more of blocking the binding of CR2 to its ligand (i.e., C3d / g), preventing activation of CR2, downregulating CR2, or affecting effector function. For example, the methods described in Examples 2, 4, and 6-11 can be used to assess the neutralizing ability of a CR2 binding protein.

[0063] The reduction or inhibition of biological activity can be partial or complete. A neutralizing antigen binding protein may neutralize the activity of CR2 by lowering the threshold for B cell activation by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% compared to CR2 activity in the absence of the antigen binding protein.

[0064] Neutralization can be determined or measured using one or more assays known to those of skill in the art or as described herein.

[0065] "CDR" is defined as the complementarity determining region amino acid sequences of an antigen binding protein. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, "CDR" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

[0066] Herein, amino acid residues in variable domain sequences and variable domain regions within a full-length antigen-binding sequence, e.g., within an antibody heavy chain sequence or an antibody light chain sequence, are numbered according to the Kabat numbering convention. Similarly, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" used in the examples follow the Kabat numbering convention. For further information, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987).

[0067] It will be obvious to those skilled in the art that there are also other numbering conventions for amino acid residues of variable domain sequences and full-length antibody sequences.There are also other numbering conventions for CDR sequences, such as those shown in Chothia et al. (1989) Nature 342: 877-883.The structure and protein folding of antigen-binding proteins may mean that other residues are also considered as part of the CDR sequence, and it will be understood by those skilled in the art.

[0068] Other numbering rules for CDR sequences available to those skilled in the art include the "AbM" (University of Bath) and "contact" (University College London) methods. The minimum overlapping region using at least two of the Kabat, Chothia, AbM and contact methods can be determined to indicate the "minimum binding unit". The minimum binding unit can be a subpart of the CDR.

[0069] Table 1 below provides one definition for each CDR or binding unit using each numbering convention. The Kabat numbering scheme is used to number the variable domain amino acid sequences in Table 1. Note that CDR definitions may vary depending on the particular publication used.

[0070] [Table 1]

[0071] Thus, there is provided an antigen binding protein comprising any one or combination of the following CDRs: CDRH1, CDRH2, CDRH3 from SEQ ID NO: 5 and / or CDRL1, CDRL2, CDRL3 from SEQ ID NO: 6. In another embodiment, there is provided an antigen binding protein comprising any one or combination of the following CDRs: CDRH1, CDRH2, CDRH3 from SEQ ID NO: 17 and / or CDRL1, CDRL2, CDRL3 from SEQ ID NO: 18. In a further embodiment, there is provided an antigen binding protein comprising any one or combination of the following CDRs: CDRH1, CDRH2, CDRH3 from SEQ ID NO: 29 and / or CDRL1, CDRL2, CDRL3 from SEQ ID NO: 30.

[0072] The CDRs or minimal binding units may be modified by at least one amino acid substitution, deletion or addition, wherein the variant antigen binding protein substantially retains the biological characteristics of the unmodified protein, such as binding to CR2.

[0073] It will be understood that each of CDRs H1, H2, H3, L1, L2, L3 may be modified alone or in combination with any other CDR in any order or combination.In one embodiment, the CDR is modified by substitution, deletion or addition of up to 3 amino acids, such as 1 or 2 amino acids, such as 1 amino acid.Generally, the modification is a substitution, in particular a conservative substitution, as shown in Table 2 below.

[0074] [Table 2]

[0075] For example, in a variant CDR, the amino acid residues of the minimal binding unit remain the same, but the adjacent residues which comprise the CDR as part of the Kabat or Chochia definition may be substituted with conservative amino acid residues.

[0076] Such antigen binding proteins comprising modified CDRs or minimal binding units as described above may be referred to herein as "functional CDR variants" or "functional binding unit variants".

[0077] The term "epitope" as used herein refers to the portion of an antigen that is in contact with a specific binding domain of an antigen-binding protein. Epitopes can be linear or conformational / discontinuous. Conformational or discontinuous epitopes comprise amino acid residues that are separated by other sequences, i.e., that are not continuous in the primary sequence of the antigen. The specific residues contained within an epitope can be determined from three-dimensional structures obtained by computer modeling programs or through methods known in the art, such as X-ray crystallography.

[0078] In one aspect of the invention, a CR2 binding protein is provided which binds to human CR2 at one or more amino acid residues of SEQ ID NO: 2. In another embodiment, a CR2 binding protein is provided which binds to human CR2 at one or more amino acid residues in the linker between the two SCR domains of human CR2. In another embodiment of the invention, a CR2 binding protein is provided which binds to human CR2 at one or more amino acid residues of SEQ ID NO: 71. In a further embodiment of the invention, a CR2 binding protein is provided which binds to human CR2 at one or more amino acid residues of SEQ ID NO: 2 and / or SEQ ID NO: 71.

[0079] In another aspect of the invention, there is provided a CR2 binding protein that protects residues 66-70 (SEQ ID NO:2) of CR2 from deuterium exchange in HDX-MS analysis, e.g., as described in Example 1. In a further embodiment of the invention, there is provided a CR2 binding protein that protects residues 66-70 (SEQ ID NO:2) and / or residues 104-127 (SEQ ID NO:71) of CR2 from deuterium exchange in HDX-MS analysis, e.g., as described in Example 1.

[0080] The competition between the antigen-binding protein of the present invention and a reference antigen-binding protein, such as a reference antibody, can be determined by competitive ELISA, FMAT or BIACORE. There are several reasons for this competition: the two proteins may bind to the same or overlapping epitopes, there may be steric inhibition of binding, or the binding of the first protein may induce a conformational change in the antigen that prevents or reduces the binding of the second protein.

[0081] The present invention is based on the identification of a CR2-binding protein that binds to a novel epitope on CR2 and blocks its activation. Blocking CR2 activation modulates the B cell activation threshold, blocks the activation and proliferation of autoantibody-producing B cells in response to self-antigens (a mechanism involved in autoimmune and / or inflammatory pathologies), and reduces the retention of C3d / g-opsonized antigens in the FDC reservoir (an important mechanism involved in viral diseases such as HIV and the generation of autoantibodies).

[0082] The antibodies and antigen-binding fragments thereof of the invention block the binding of C3d / g opsonized complexes to non-cognate B cells, thereby blocking the retention of the complexes on and trafficking to FDCs within germinal centers.

[0083] Some isotypes of human constant regions, particularly IgG4 and IgG2 isotypes, essentially lack the functions of a) classical pathway complement activation; and b) antibody-dependent cellular cytotoxicity (ADCC). Various modifications may be made to the heavy chain constant region of the antigen-binding protein to alter the effector function depending on the desired effector properties. IgG1 constant regions containing specific mutations that reduce binding to Fc receptors and reduce effector functions such as ADCC and CDC have been described (Duncan et al. Nature 1988, 332; 563-564; Lund et al. J. Immunol. 1991, 147; 2657-2662; Chappel et al. PNAS 1991, 88; 9036-9040; Burton and Woof, Adv. Immunol. 1992, 51;1-84; Morgan et al., Immunology 1995, 86; 319-324; Hezareh et al., J. Virol. 2001, 75 (24); 12161-12168).

[0084] In one embodiment of the invention, a CR2 binding protein is provided that comprises a constant region such that the effector function of the antigen binding protein is reduced, such as reduced ADCC and / or complement-dependent cytotoxicity (CDC). In one such embodiment, the heavy chain constant region may comprise an naturally occurring disordered constant region of an IgG2 or IgG4 isotype or a mutated IgG1 constant region. Examples of suitable modifications are described in EP0307434. One example comprises an alanine substitution at positions 235 and 237 (EU index numbering), i.e., L235A and G237A (commonly referred to as the "LAGA" mutation). Another example comprises an alanine substitution at positions 234 and 235 (EU index numbering), i.e., L234A and L235A (commonly referred to as the "LALA" mutation).

[0085] Further modifications and mutations to reduce effector function include (see IgG1 unless otherwise noted): glycosylation N297A or N297Q or N297G; L235E; IgG4: F234A / L235A; and chimeric IgG2 / IgG4. IgG2: H268Q / V309L / A330S / P331S, and IgG2: V234A / G237A / P238S / H268A / V309L / A330S / P331S can reduce FcγR and C1q binding (Wang et al. 2018 and U.S. Patent No. 8,961,967).

[0086] Other mutations that reduce effector function include L234F / L235E / P331S; a chimeric antibody made with CH1 and hinge regions from human IgG2 and CH2 and CH3 regions from human IgG4; IgG2m4, based on the IgG2 isotype, with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S); IgG2σ, which contains V234A / G237A / P238S / H268A / V309L / A330S / P331S substitutions to eliminate affinity for Fcγ receptors and the C1q complement protein; IgG2m4 (H268Q / V309L / A330S / P331S, changes to IgG4); IgG4 (S228P / L234A / L235A); huIgG1 L234A / L235A(AA); huIgG4 S228P / L234A / L235A; IgG1σ (L234A / L235A / G237A / P238S / H268A / A330S / P331S); IgG4σ1 (S228P / F234A / L235A / G237A / P238S); and IgG4σ2 (S228P / F234A / L235A / DG236 / G237A / P238S, where D represents a deletion) (Tam et al., Antibodies 2017, 6(3)).

[0087] The present invention also provides CR2 binding proteins that are human, humanized, or chimeric. In one embodiment, the CR2 binding protein is human.

[0088] The present invention also provides a CR2 binding protein comprising a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 9. In one embodiment, the CR2 binding protein may further comprise a heavy chain CDR2 of SEQ ID NO: 8 and a heavy chain CDR1 of SEQ ID NO: 7. In another embodiment, the CR2 binding protein may further comprise one, two or three of a light chain CDR1 of SEQ ID NO: 10, a CDR2 of SEQ ID NO: 11 and a CDR3 of SEQ ID NO: 12. In a further embodiment, the CR2 binding protein comprises a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 8, a heavy chain CDR3 of SEQ ID NO: 9, a light chain CDR1 of SEQ ID NO: 10, a light chain CDR2 of SEQ ID NO: 11 and a light chain CDR3 of SEQ ID NO: 12.

[0089] The present invention also provides a CR2 binding protein comprising a heavy chain variable region of SEQ ID NO: 5. In one embodiment, the CR2 binding protein may further comprise a light chain variable region of SEQ ID NO: 6. In another embodiment, the CR2 binding protein comprises a heavy chain variable region of SEQ ID NO: 5 and a light chain variable region of SEQ ID NO: 6.

[0090] The present invention also provides a CR2 binding protein comprising a heavy chain variable region selected from an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more or 100% identity to the amino acid sequence of SEQ ID NO: 5, and a light chain variable region selected from an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more or 100% identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment, the CR2 binding protein comprises a heavy chain variable region having 98% or more identity to the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having 98% identity to the amino acid sequence of SEQ ID NO: 6. In a further embodiment, the CR2 binding protein comprises a heavy chain of SEQ ID NO: 3 and a light chain of SEQ ID NO: 4 (mAb 1053, CHO expressed material unless otherwise stated).

[0091] The present invention also provides a CR2 binding protein comprising a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 21. In one embodiment, the CR2 binding protein may further comprise a heavy chain CDR2 of SEQ ID NO: 20 and a heavy chain CDR1 of SEQ ID NO: 19. In another embodiment, the CR2 binding protein may further comprise one, two or three of a light chain CDR1 of SEQ ID NO: 22, a CDR2 of SEQ ID NO: 23 and a CDR3 of SEQ ID NO: 24. In a further embodiment, the CR2 binding protein comprises a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 20, a heavy chain CDR3 of SEQ ID NO: 21, a light chain CDR1 of SEQ ID NO: 22, a light chain CDR2 of SEQ ID NO: 23 and a light chain CDR3 of SEQ ID NO: 24.

[0092] The present invention also provides a CR2 binding protein comprising a heavy chain variable region of SEQ ID NO: 17. In one embodiment, the CR2 binding protein may further comprise a light chain variable region of SEQ ID NO: 18. In another embodiment, the CR2 binding protein comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 18.

[0093] The present invention also provides a CR2 binding protein comprising a heavy chain variable region selected from an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more or 100% identity to the amino acid sequence of SEQ ID NO: 17, and a light chain variable region selected from an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more or 100% identity to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the CR2 binding protein comprises a heavy chain variable region having 98% or more identity to the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having 98% identity to the amino acid sequence of SEQ ID NO: 18. In a further embodiment, the CR2 binding protein comprises a heavy chain of SEQ ID NO: 15 and a light chain of SEQ ID NO: 16 (mAb 996, CHO expressed material unless otherwise noted).

[0094] The present invention also provides a CR2 binding protein comprising a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 33. In one embodiment, the CR2 binding protein may further comprise a heavy chain CDR2 of SEQ ID NO: 32 and a heavy chain CDR1 of SEQ ID NO: 31. In another embodiment, the CR2 binding protein may further comprise one, two or three of a light chain CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35 and a CDR3 of SEQ ID NO: 36. In a further embodiment, the CR2 binding protein comprises a heavy chain CDR1 of SEQ ID NO: 31, a heavy chain CDR2 of SEQ ID NO: 32, a heavy chain CDR3 of SEQ ID NO: 33, a light chain CDR1 of SEQ ID NO: 34, a light chain CDR2 of SEQ ID NO: 35 and a light chain CDR3 of SEQ ID NO: 36.

[0095] The present invention also provides a CR2 binding protein comprising a heavy chain variable region of SEQ ID NO: 29. In one embodiment, the CR2 binding protein may further comprise a light chain variable region of SEQ ID NO: 30. In another embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 30.

[0096] The present invention also provides a CR2 binding protein comprising a heavy chain variable region selected from an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more identity or 100% identity to the amino acid sequence of SEQ ID NO: 29, and a light chain variable region selected from an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more identity or 100% identity to the amino acid sequence of SEQ ID NO: 30. In one embodiment, the CR2 binding protein comprises a heavy chain variable region having 98% or more identity to the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having 98% identity to the amino acid sequence of SEQ ID NO: 30. In a further embodiment, the CR2 binding protein comprises a heavy chain of SEQ ID NO: 27 and a light chain of SEQ ID NO: 28 (mAb 999, which is CHO expressed material unless otherwise noted).

[0097] The disclosure herein contemplates CR2 binding proteins comprising any and all combinations of such heavy and light chain variable regions.

[0098] The present invention also provides a nucleic acid molecule encoding a CR2 binding protein. In one embodiment, a nucleic acid sequence of SEQ ID NO: 13 is provided. In another embodiment, a nucleic acid sequence of SEQ ID NO: 14 is provided. In another embodiment, a nucleic acid sequence of SEQ ID NO: 25 is provided. In another embodiment, a nucleic acid sequence of SEQ ID NO: 26 is provided. In another embodiment, a nucleic acid sequence of SEQ ID NO: 37 is provided. In a further embodiment, a nucleic acid sequence of SEQ ID NO: 38 is provided.

[0099] The present invention also provides expression vectors comprising the nucleic acid molecules described herein encoding the heavy and / or light chains of the CR2 binding proteins described herein. In one embodiment, the same vector comprises the nucleic acid sequences of the heavy and light chains. In another embodiment, separate vectors comprise the nucleic acid sequences of the heavy and light chains, i.e., one vector encodes the heavy chain and another vector encodes the light chain. In one embodiment, an expression vector is provided comprising the nucleic acid sequence of SEQ ID NO: 13. In another embodiment, an expression vector is provided comprising the nucleic acid sequence of SEQ ID NO: 14. In another embodiment, an expression vector is provided comprising the nucleic acid sequence of SEQ ID NO: 25. In another embodiment, an expression vector is provided comprising the nucleic acid sequence of SEQ ID NO: 26. In another embodiment, an expression vector is provided comprising the nucleic acid sequence of SEQ ID NO: 37. In a further embodiment, an expression vector is provided comprising the nucleic acid sequence of SEQ ID NO: 38.

[0100] The present invention further provides a recombinant host cell comprising such an expression vector and capable of expressing said CR2 binding protein. In one embodiment, a recombinant host cell is provided containing an expression vector comprising the nucleic acid sequence of SEQ ID NO: 13. In another embodiment, a recombinant host cell is provided comprising an expression vector comprising the nucleic acid sequence of SEQ ID NO: 14. In another embodiment, a recombinant host cell is provided comprising an expression vector comprising the nucleic acid sequence of SEQ ID NO: 25. In another embodiment, a recombinant host cell is provided comprising an expression vector comprising the nucleic acid sequence of SEQ ID NO: 26. In another embodiment, a recombinant host cell is provided comprising an expression vector comprising the nucleic acid sequence of SEQ ID NO: 37. In a further embodiment, a recombinant host cell is provided comprising an expression vector comprising the nucleic acid sequence of SEQ ID NO: 38.

[0101] The present invention also provides a CR2 binding protein expressed by a recombinant host cell as described herein.

[0102] The disclosed sequences, SEQ ID NOs: 1-74, are defined according to Kabat unless otherwise indicated.

[0103] The following examples describe a range of assays on primary human B cells that were developed to understand the impact of the antibodies of the invention and their antigen-binding fragments on human biology. Taken together, these data provide experimental evidence to support that the CR2 binding proteins of the invention effectively inhibit CR2-dependent mechanisms of autoimmunity against B cells.

[0104] In one embodiment of the invention, a CR2 binding protein is provided that inhibits the retention of opsonized autoantigens on unconjugated B cells by blocking binding of bound opsonized complexes. In another embodiment of the invention, a CR2 binding protein is provided that inhibits the retention of opsonized autoantigens on unconjugated B cells by competing with bound opsonized complexes. In another embodiment of the invention, a CR2 binding protein is provided that blocks B cell activation by opsonized autoantigens. In another embodiment of the invention, a CR2 binding protein is provided that inhibits calcium flux in human B cells. In another embodiment of the invention, a CR2 binding protein is provided that inhibits phosphoprotein signaling in human B cells. In a further embodiment, a CR2 binding protein is provided that inhibits CR2-dependent CD69 upregulation in human B cells.

[0105] Description of Use Thus, the present invention provides CR2 binding proteins for use in therapy. In one embodiment, the CR2 binding proteins can be used in the treatment of a disease or condition for which a CR2 inhibitor is indicated.

[0106] In one embodiment, a CR2 binding protein is provided for use in treating a disease involving B cell activation. In another embodiment, a use of a CR2 binding protein is provided in the manufacture of a medicament for use in treating a disease involving B cell activation. In a further embodiment, a method is provided for treating a disease involving B cell activation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein.

[0107] In one embodiment, a CR2 binding protein is provided for use in treating a disease associated with retention of C3d or C3dg opsonized antigens in the FDC reservoir. In another embodiment, a use of a CR2 binding protein in the manufacture of a medicament for use in treating a disease associated with retention of C3d or C3dg opsonized antigens in the FDC reservoir is provided. In a further embodiment, a method is provided for treating a disease associated with retention of C3d or C3dg opsonized antigens in the FDC reservoir in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein.

[0108] Autoimmune and / or inflammatory conditions Because of the role of CR2 in generating / maintaining germinal center responses, blocking this mechanism may be effective in treating autoimmune and / or inflammatory conditions such as Sjögren's syndrome, systemic lupus erythematosus, and rheumatoid arthritis. Furthermore, patients with Sjögren's syndrome, systemic lupus erythematosus, or rheumatoid arthritis have been found to contain circulating immune complexes bearing C3b / iC3b or C3d.

[0109] Given that CR2 plays a key role in lowering the threshold for B cell activation and promoting antibody production, blocking CR2 offers an excellent opportunity to reset B cell-mediated pathology.

[0110] Sjögren's syndrome is an autoimmune rheumatic disease in which lymphocytic infiltration of tissues leads to exocrine gland hyposecretion and multisystem inflammation. Disease severity is closely related to B cell activation, circulating autoantibodies, and a high frequency of germinal center foci in exocrine tissues. A subgroup of patients with Sjögren's syndrome who have high disease activity and extensive autoantibody production have low levels of complement C3, suggesting a high complement activation-induced consumption that may contribute to the pathogenesis of Sjögren's syndrome.

[0111] SLE is a chronic autoimmune disease, the etiology of which is unknown. SLE is incurable, and treatment involves preventing flares and reducing their severity and duration when they occur. Treatment includes corticosteroids, cytotoxic drugs, such as cyclophosphamide, hydroxychloroquine, and belimumab.

[0112] CR2 has been implicated in the pathogenesis of SLE, and several reports suggest that CR2 plays an important role in the recognition of foreign DNA during the host immune response, which may be a mechanism that influences the development of autoimmunity against DNA in SLE.

[0113] Activation of various parts of the complement cascade in RA has been known for many years. For example, levels of the CR2 ligand, C3d / g, are elevated in the synovial fluid of RA patients. Also, several studies have demonstrated the presence of CR2 on fibroblast-like synoviocytes (FLS) in the inflamed synovium of rheumatoid arthritis (RA) patients.

[0114] CR2 binding proteins are associated with, but are not limited to, rheumatoid arthritis (RA), Sjogren's syndrome, systemic lupus erythematosus (SLE, e.g., lupus nephritis), vasculitis (e.g., antineutrophil cytoplasmic antibody (ANCA) associated vasculitis, systemic necrotizing vasculitis, polyarteritis nodosa, allergic vasculitis and granulomatosis, polyangiitis, Wegener's granulomatosis, lymphomatoid granulomatosis, giant cell arteritis, and the like. inflammation, mucocutaneous lymph node syndrome (MLNS or Kawasaki disease), isolated CNS vasculitis, Behçet's disease, thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizing venulitis), idiopathic membranous glomerulonephritis, myasthenia gravis, pemphigus, scleroderma, systemic sclerosis, anti-glomerular basement membrane disease, bullous pemphigoid, multiple sclerosis, neuromyelitis optica, meningitis; encephalitis, hemophilia inhibitors, resistance to enzyme replacement therapy The present invention may be useful in the treatment of a variety of autoimmune and / or inflammatory conditions, including pulmonary fibrosis, IgA nephropathy, polymyositis, dermatomyositis, Graves' disease (e.g., autoimmune thyroiditis and Hashimoto's thyroiditis), antiphospholipid syndrome, type 1 diabetes mellitus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, solid organ transplantation, autoimmune pulmonary alveolar proteinosis, uveitis; osteoarthritis, autoimmune hemolytic anemia (e.g., autoimmune hemolytic anemia, immune pancytopenia, and paroxysmal nocturnal hemoglobinuria), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, and antigen-antibody complex-mediated diseases (e.g., pulmonary inflammation, pleuritis, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, and idiopathic pulmonary fibrosis (IPF)).

[0115] In one embodiment, the autoimmune and / or inflammatory condition is selected from rheumatoid arthritis (RA), Sjogren's syndrome, systemic lupus erythematosus (SLE) and vasculitis. In another embodiment, the autoimmune and / or inflammatory condition is rheumatoid arthritis (RA). In another embodiment, the autoimmune and / or inflammatory condition is Sjogren's syndrome. In another embodiment, the autoimmune and / or inflammatory condition is systemic lupus erythematosus (SLE). In another embodiment, the autoimmune and / or inflammatory condition is vasculitis. In a further embodiment, the autoimmune and / or inflammatory condition is antineutrophil cytoplasmic antibody (ANCA) associated vasculitis.

[0116] In one embodiment, a CR2 binding protein is provided for use in the treatment of autoimmune and / or inflammatory conditions. In another embodiment, a CR2 binding protein is provided for use in the treatment of rheumatoid arthritis (RA). In another embodiment, a CR2 binding protein is provided for use in the treatment of Sjogren's syndrome. In another embodiment, a CR2 binding protein is provided for use in the treatment of systemic lupus erythematosus (SLE). In another embodiment, a CR2 binding protein is provided for use in the treatment of vasculitis. In another embodiment, a CR2 binding protein is provided for use in the treatment of antineutrophil cytoplasmic antibody (ANCA) associated vasculitis.

[0117] In one embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment of an autoimmune and / or inflammatory condition. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment of rheumatoid arthritis (RA). In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment of Sjogren's syndrome. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment of systemic lupus erythematosus (SLE). In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment of vasculitis. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment of antineutrophil cytoplasmic antibody (ANCA) associated vasculitis.

[0118] Also provided is a method for the treatment of an autoimmune and / or inflammatory condition in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method for the treatment of rheumatoid arthritis (RA) in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein is provided. In another embodiment, a method for the treatment of Sjögren's syndrome in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein is provided. In another embodiment, a method for the treatment of systemic lupus erythematosus (SLE) in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein is provided. In another embodiment, a method for the treatment of vasculitis in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein is provided. In a further embodiment, a method for the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein is provided. Preferably, the subject in need thereof is a mammal, particularly a human.

[0119] infectious disease Many viral diseases, e.g., HIV, are difficult to treat due to the persistence of long-lived viral reservoirs, such as FDCs in lymphoid tissues. Antigen binding, retention for over a year, and antigen presentation are highly dependent on CR2-dependent capture of C3 fragment-coated antigens. C3d / g-opsonized HIV virions are trapped and retained for long periods in non-degradative endosomes, which represent long-lived viral reservoirs.

[0120] Current HIV treatment with combination antiretroviral therapy (cART) effectively prevents viral spread and HIV disease but falls short of eradicating the virus due to the persistence of long-lived viral reservoirs that include latently HIV-infected cells and anatomical or physiological sites that can harbor sustainable, replication-competent virus for many years.

[0121] Virus captured by FDCs represents an important mechanism for replenishing the reservoir by creating a persistent source of infectious virus in lymphoid tissues, which can lead to new infections and viral relapse after cessation of cART, preventing long-term viral remission and cure of HIV.

[0122] CR2 binding proteins may be useful in the treatment, prevention or reactivation of infectious diseases including, but not limited to, human immunodeficiency virus (HIV), hepatitis (A, B, C, D and E) and Epstein-Barr virus (EBV), bacterial infections, fungal infections, protozoan infections and parasitic infections.

[0123] In one embodiment, a CR2 binding protein is provided for use in the treatment or prevention of an infectious disease. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of a viral infection. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of HIV. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of AIDS. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of Hepatitis A. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of Hepatitis B. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of Hepatitis C. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of Hepatitis D. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of Hepatitis E. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of a bacterial infection. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of a fungal infection. In another embodiment, a CR2 binding protein is provided for use in the treatment or prevention of a protozoal infection. In a further embodiment there is provided a CR2 binding protein for use in the treatment or prevention of a parasitic infection.

[0124] In one embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of an infectious disease. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of a viral infection. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of HIV. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of AIDs. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of Hepatitis A. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of Hepatitis B. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of Hepatitis C. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of Hepatitis D. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of Hepatitis E. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of a bacterial infection. In another embodiment, there is provided a use of the CR2 binding protein in the manufacture of a medicament for the treatment or prevention of a fungal infection. In another embodiment there is provided the use of a CR2 binding protein in the manufacture of a medicament for the treatment or prevention of a protozoal infection. In a further embodiment there is provided the use of a CR2 binding protein in the manufacture of a medicament for the treatment or prevention of a parasitic infection.

[0125] Also provided is a method for the treatment or prevention of an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In one embodiment, a method for the treatment or prevention of a viral disease in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method for the treatment or prevention of HIV in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method for the treatment or prevention of AIDs in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method for the treatment or prevention of Hepatitis A in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method for the treatment or prevention of Hepatitis B in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method is provided for the treatment or prevention of hepatitis C in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method is provided for the treatment or prevention of hepatitis D in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method is provided for the treatment or prevention of hepatitis E in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method is provided for the treatment or prevention of a bacterial infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, a method is provided for the treatment or prevention of a fungal infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein.In another embodiment, a method is provided for the treatment or prevention of a protozoan infection in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein. In a further embodiment, a method is provided for the treatment or prevention of a parasitic infection in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein. Preferably, the subject in need thereof is a mammal, particularly a human.

[0126] Use as a vaccine adjuvant Low doses of antigen coupled to anti-CR2 antibodies have been shown to rapidly induce and sustain IgG immune responses in mice and cynomolgus monkeys (Whipple, EC et al., Mol. Immunol., 2007. 44(4): 377-388). In one embodiment, a CR2 binding protein is provided for use as an adjuvant / antigen carrier for amplifying an immune response to immunization. In another embodiment, a use of a CR2 binding protein in the manufacture of a medicament for amplifying an immune response to immunization is provided. In a further embodiment, a method is provided for amplifying an immune response to immunization in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein. Suitably, the subject in need thereof is a mammal, particularly a human.

[0127] Epstein-Barr virus (EBV)-associated malignancies CR2 has been identified as the major coreceptor for Epstein-Barr virus infection and may therefore play a role in the development of lymphoproliferative disorders caused by EBV (N. Neparidze et al.; Clinical Advances in Hematology & Oncology, 12(6); June 2014: 358-371).

[0128] The present invention provides a CR2 binding protein for use in the treatment or prevention of EBV-associated malignancies. The present invention also provides the use of a CR2 binding protein in the manufacture of a medicament for the treatment or prevention of EBV-associated malignancies. The present invention also provides a method for the treatment or prevention of EBV-associated malignancies in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein.

[0129] The present invention also provides a CR2 binding protein for use in the treatment or prevention of an EBV-associated malignancy selected from a B cell lymphoproliferative disorder, a T / natural killer cell lymphoproliferative disorder and an epithelial malignancy.

[0130] In one embodiment, the present invention provides a CR2 binding protein for use in the treatment or prevention of B-cell lymphoproliferative disorders. In another embodiment, the present invention provides a CR2 binding protein for use in the treatment or prevention of B-cell lymphoproliferative disorders selected from FDC sarcoma, Burkitt's lymphoma, Hodgkin's lymphoma, post-transplant lymphoproliferative disorders, HIV-associated non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, immunoblastic-plasmacytoid, centroblastic, Burkitt's lymphoma, primary central nervous system lymphoma, primary effusion lymphoma and its solid variant, oral type of plasmablastic lymphoma, EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly, lymphomatoid granulomatosis, and chronic inflammation-associated DLBCL.

[0131] In one embodiment, the present invention provides a CR2 binding protein for use in the treatment or prevention of T / natural killer cell lymphoproliferative disorders. In another embodiment, the present invention provides a CR2 binding protein for use in the treatment or prevention of T / natural killer cell lymphoproliferative disorders selected from extranodal nasal-type NK / T cell lymphoma, aggressive NK cell leukemia / lymphoma, angioimmunoblastic T cell lymphoma, enteropathy-type T cell lymphoma, EBV-associated cutaneous T cell lymphoproliferative disorder, gamma delta T cell lymphoma (hepatosplenic and non-hepatosplenic), peripheral T cell lymphoma, and chronic post-EBV infection T cell lymphoproliferative disorder.

[0132] In one embodiment, the present invention provides a CR2 binding protein for use in the treatment or prevention of an epithelial malignancy. In another embodiment, the present invention provides a CR2 binding protein for use in the treatment or prevention of an epithelial malignancy selected from nasopharyngeal carcinoma and gastric carcinoma.

[0133] The present invention also provides the use of a CR2 binding protein in the manufacture of a medicament for the treatment or prevention of an EBV-associated malignancy selected from B cell lymphoproliferative disorders, T / natural killer cell lymphoproliferative disorders and epithelial malignancies.

[0134] In one embodiment, the present invention provides the use of a CR2 binding protein in the manufacture of a medicament for the treatment or prevention of a B-cell lymphoproliferative disorder. In another embodiment, the present invention provides the use of a CR2 binding protein in the manufacture of a medicament for the treatment or prevention of a B-cell lymphoproliferative disorder selected from FDC sarcoma, Burkitt's lymphoma, Hodgkin's lymphoma, post-transplant lymphoproliferative disorder, HIV-associated non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, immunoblastic-plasmacytoid, centroblastic, Burkitt's lymphoma, primary central nervous system lymphoma, primary effusion lymphoma and its solid variant, oral type of plasmablastic lymphoma, EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly, lymphomatoid granulomatosis, and chronic inflammation-associated DLBCL.

[0135] In one embodiment, the present invention provides the use of a CR2 binding protein in the manufacture of a medicament for the treatment or prevention of a T / natural killer cell lymphoproliferative disorder. In another embodiment, the present invention provides the use of a CR2 binding protein in the manufacture of a medicament for the treatment or prevention of a T / natural killer cell lymphoproliferative disorder selected from extranodal nasal-type NK / T cell lymphoma, aggressive NK cell leukemia / lymphoma, angioimmunoblastic T cell lymphoma, enteropathy-type T cell lymphoma, EBV-associated cutaneous T cell lymphoproliferative disorder, gamma delta T cell lymphoma (hepatosplenic and non-hepatosplenic), peripheral T cell lymphoma, and T cell lymphoproliferative disorder following chronic EBV infection.

[0136] In one embodiment, the present invention provides the use of a CR2 binding protein in the manufacture of a medicament for the treatment or prevention of an epithelial malignancy. In another embodiment, the present invention provides the use of a CR2 binding protein in the manufacture of a medicament for the treatment or prevention of an epithelial malignancy selected from nasopharyngeal carcinoma and gastric carcinoma.

[0137] The present invention also provides a method for treating or preventing an EBV-associated malignancy selected from B cell lymphoproliferative disorders, T / natural killer cell lymphoproliferative disorders and epithelial malignancies in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein.

[0138] In one embodiment, the present invention provides a method for treating or preventing a B-cell lymphoproliferative disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, the present invention provides a method for treating or preventing a B-cell lymphoproliferative disorder selected from FDC sarcoma, Burkitt's lymphoma, Hodgkin's lymphoma, post-transplant lymphoproliferative disorder, HIV-associated non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, immunoblastic-plasmacytoid, centroblastic, Burkitt's lymphoma, primary central nervous system lymphoma, primary effusion lymphoma and its solid variant, oral type of plasmablastic lymphoma, EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly, lymphomatoid granulomatosis, and chronic inflammation-associated DLBCL in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein.

[0139] In one embodiment, the present invention provides a method for the treatment or prevention of a T / natural killer cell lymphoproliferative disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein. In another embodiment, the present invention provides a method for the treatment or prevention of a T / natural killer cell lymphoproliferative disorder selected from extranodal nasal-type NK / T cell lymphoma, aggressive NK cell leukemia / lymphoma, angioimmunoblastic T cell lymphoma, enteropathy-type T cell lymphoma, EBV-associated cutaneous T cell lymphoproliferative disorder, gamma delta T cell lymphoma (hepatosplenic and non-hepatosplenic), peripheral T cell lymphoma, and T cell lymphoproliferative disorder following chronic EBV infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CR2 binding protein.

[0140] In one embodiment, the present invention provides a method for the treatment or prevention of an epithelial malignancy in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein.In another embodiment, the present invention provides a method for the treatment or prevention of an epithelial malignancy selected from nasopharyngeal carcinoma and gastric carcinoma in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CR2 binding protein.

[0141] Preferably, the subject in need thereof is a mammal, particularly a human.

[0142] Drug composition / route of administration / dosage The pharmaceutical composition of the present invention can be used for therapeutic or prophylactic applications. In one embodiment of the present invention, a pharmaceutical composition is provided comprising a CR2 binding protein and a pharma- ceutically acceptable carrier or excipient thereof. In another embodiment, a pharmaceutical composition is provided comprising 1-500 mg of a CR2 binding protein. In another embodiment, a pharmaceutical composition is provided comprising 20-300 mg of a CR2 binding protein. In another embodiment, a pharmaceutical composition is provided comprising 50-200 mg of a CR2 binding protein. In a further embodiment, a pharmaceutical composition is provided comprising 50-200 mg of a CR2 binding protein, i.e., an antibody comprising a light chain amino acid sequence as shown in SEQ ID NO:4 and a heavy chain amino acid sequence as shown in SEQ ID NO:3.

[0143] In a preferred embodiment, the pharmaceutical composition comprises a composition for parenteral, transdermal, intraluminal, intraarterial, intrathecal and / or intranasal administration or by direct injection into tissue. It is envisaged that the pharmaceutical composition is administered to a patient via infusion or injection. The administration of the suitable composition can be achieved by various methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In one embodiment, a pharmaceutical composition comprising a CR2 binding protein is provided for intravenous administration. In another embodiment, a pharmaceutical composition comprising a CR2 binding protein is provided for subcutaneous administration.

[0144] These pharmaceutical compositions can be administered to subjects in suitable doses.Dosage schedules are determined by attending physicians and clinical factors.As is well known in the medical field, the dose of any patient varies according to many factors, including patient's size, body surface area, age, compound administered, sex, time, route of administration, health condition, and other drugs administered at the same time. EXAMPLES

[0145] Abbreviation ADCC antibody-dependent cytotoxicity Ag antigen Akt protein kinase B ANCA anti-neutrophil cytoplasmic antibody APC Allophycocyanin BCR B cell receptor cART combination antiretroviral therapy CDC Complement-dependent cytotoxicity CDR Complementarity Determining Region CD20 B-lymphocyte antigen CD20 CHO Chinese hamster ovary cells cIEF Capillary Isoelectric Focusing CL Clearance CR1 Complement receptor 1 CR2 Complement receptor 2 CO2 Carbon Dioxide CTL cytotoxic T lymphocytes Cyno cyno DLS Dynamic Light Scattering DMARDs Disease-modifying antirheumatic drugs DME Drug Metabolizing Enzymes DP Pharmaceuticals DNA deoxyribonucleic acid EBV Epstein-Barr virus ELS Ectopic lymphoid structures ERKs Extracellular signal-regulated kinases FDC Follicular dendritic cells FITC Fluorescein isothiocyanate FLS Fibroblast-like synoviocytes GC germinal center hCD2 humanized CR2 HEK Human Embryonic Kidney Cells HDX Hydrogen Deuterium Exchange HDMS High precision mass spectrometry HIV Human Immunodeficiency Virus HPLC High Performance Liquid Chromatography IgG Immunoglobulin G IgM Immunoglobulin M IV Intravenous KO Knockout LC-MS Liquid Chromatography Mass Spectrometry mAb Monoclonal antibody MFI Mean Fluorescence Intensity Mg / Kg milligrams per kilogram MRT average residence time MS mass spectrometry MSD Meso Scale Discovery MSX Methionine sulfoxamine MW molecular weight MZ Marginal zone NaCl Sodium chloride NaOH Sodium hydroxide NB non-bonded ng / mL nanograms per milliliter NHL Non-Hodgkin's B-cell lymphoma NHS Normal Human Serum PBS Phosphate Buffered Saline PBMCs Human peripheral mononuclear cells PBPK Physiologically Based Pharmacokinetics PLGS proteinLynx global server PMA Myristic Acid Phorbol Acetate PnPS14 Pneumococcus polysaccharide 14 RA Rheumatoid Arthritis S / C Subcutaneous SAD Single Ascending Dose SCR Short Consensus Repeat sCR2 Soluble CR2 SCS subcapsular sinus SEC-HPLC Size Exclusion Chromatography - High Performance Liquid Chromatography SEM Standard error of the mean SLE systemic lupus erythematosus SPR Surface Plasmon Resonance t 1 / 2 Half-life TA target assembly TCEP Tris(2-carboxyethyl)phosphine TCR tissue cross-reactivity TFA Trifluoroacetic acid TFF Tangential Flow Filtration Tfh T follicular helper Th T Helper TLR toll-like receptor TMDD Target-Mediated Drug Elimination Tox Toxicology UPLC Ultra High Performance Liquid Chromatography

[0146] Example 1 HDX epitope mapping - CR2 to mAb 1053 HDX-MS analysis was performed to examine the differential deuteration of CR2 (SCR1 and 2 domains) in the presence and absence of mAb 1053 (expressed in CHO).

[0147] device: Waters SYNAPT G2-Si HDMS ACQUITY M Class UPLC LEAP H / DX PAL Robot solution Quenching solution: 400 mM potassium phosphate, 6 M guanidine hydrochloride, 0.5 M TCEP pH 2.5 (after mixing 1:1 with the sample, the pH of the quenching solution was adjusted with NaOH so that it would be this pH when mixed). Dilution buffer: 50 mM sodium phosphate 100 mM sodium chloride in water, pH 7.0. protein "CR2" = human CR2 antigen (21-153), untagged; PBS 135 uM in water. "1053" = mAb; 80.7uM in PBS.

[0148] HDX In initial testing, CR2 was tested in one experiment to confirm digestion quality and signal strength. Protein was diluted to 10 uM in dilution buffer and run using the standard 1:20 dilution and quenching protocol.

[0149] A PLGS search against the database of CR2 mAb constructs used and pepsin sequences showed that the protein gave sufficient signal for excellent protein coverage. Two further runs under similar conditions were performed on the CR2 sample, yielding triplicate digests for peptide identification.

[0150] For the HDX experiment, the following two samples were prepared: Apo (control sample): 7.4ul CR2, 10ul PBS, pH 7.4 (to be compatible with mAb buffer), 82.6ul Dilution Buffer. + mAb: 7.4ul CR2, 10ul mAb 1053, 82.6ul Dilution Buffer (to give 10uM CR2, 8uM mAb).

[0151] HPLC solvent A was 0.2% formic acid + 0.03% TFA in water, solvent B was acetonitrile + 0.2% formic acid. All chromatographic steps were performed at 0°C.

[0152] Each protein was diluted 20-fold with deuterated dilution buffer stored at ambient temperature (25° C.) and used incubation times of 0, 30 and 300 seconds at 20° C. (diluted with non-deuterated buffer at time 0). After incubation, samples were quenched with one volume of quench solution at 0° C. After 1 minute, samples were injected onto an immobilized pepsin digestion column held at 15° C. and equilibrated with HPLC buffer A (Waters ENZYMATE BEH, 2.1 mm×30 mm, Part no: 186007233), and eluting peptides were captured on a C18 guard column (2.1×5 mm Waters VANGUARD BEH C18 1.8 um 130 Å guard column, Part no 186003975) maintained at 0° C. with a capture time of 4 minutes. The captured peptides were then eluted from the guard column at a flow rate of 40 ul / min onto an analytical C18 column (1.0 x 100 mm UPLC BEH C18, Part no 186002346), again at 0°C, and separated with increasing concentrations of solvent B according to the following gradient:

[0153] [Table 3]

[0154] During this phase the pepsin column was removed and washed with 2 x 100 ul injections of 2 m guanidine hydrochloride, 0.8% formic acid, 5% acetonitrile, 5% propan-2-ol, pH 2.5.

[0155] Eluted peptides were introduced into a Waters SYNAPT G2-Si mass spectrometer equipped with an electrospray ion source and a second rockspray introducing [Glul]-fibrinopeptide B in 0.2% formic acid, 50% acetonitrile for internal mass calibration. Sequential data were acquired using "resolution" mode using positive ionization over the m / z range 260-2000. For the first peptide identification samples, MS eAcquisition was used to acquire alternating scans under low- and high-energy collision cell conditions to generate full and fragmented peptide data, respectively, with deuterated samples replicated five times (at 30 and 300 seconds) or two times (at 0 deuterated sample) to increase data reliability.

[0156] MS e Initial CR2 characterization data from was used to generate peptide search lists using ProteinLynx Global Server (v3.0.2) to search a database containing the sequences of the CR2 constructs used. These peptide lists, filtered to remove low-confidence identified peptides, were used to identify peptides and their deuteration levels for samples from all time points in DynamX v3.0. Peptide and ion assignments were manually verified and refined as necessary.

[0157] Differential (Apo-mAb complex) deuteration data at the peptide level was replotted in TIBCO SPOTFIRE v.7.0 (from data provided), see Figures 2A and 2B.

[0158] result Two closely related peptides consistently showed lower deuteration levels in the presence of mAb 1053. FNKYSSCPEPIVPGGYKIRGSTPYRHGDSVT (residues 66-96 of construct SEQ ID NO:58 used) FNKYSSCPEPIVPGGYKIRGSTPYRHGDSVTF (residues 66-97 of construct SEQ ID NO:59 used)

[0159] The longer of these peptides showed larger errors in the deuteration calculations due to interference from overlapping ions, while the shorter of these peptides showed clearer data, such that the difference in deuteration between the apo and mAb 1053 samples was statistically significant. The peptide SSCPEPIVPGGYKIRGSTPYRHGDSVT (residues 70-96 of the construct used SEQ ID NO: 60), which differs only by lacking the first four residues, showed almost no differential signal. The most likely reason for this difference is that the protection induced by the mAb is mainly localized in the five amides [66-96] at the N-terminus of the peptide. Residues 66-70 (sequence FNKYS of SEQ ID NO: 2) correspond to the linker between the two SCR domains. This region is the most likely candidate for the epitope of mAb 1053.

[0160] In addition to these peptide differences, an additional region showing less protection (but a shorter peptide) was present near the C-terminus corresponding to the peptide spanning residues 104-127 (SMNGNKSVWCQANNMWGPTRLPTC, SEQ ID NO:71).

[0161] Effect of mAb 1053 on C3d / g opsonizing ligand binding to B cells. Example 2: Assay to determine the effect of mAb 1053 on C3dg ligand binding This experiment determined the ability of mAb 1053 to block binding of C3dg:streptavidin-APC complexes to non-cognate B cells. Fluorescently labeled complexes of streptavidin and biotin-C3dg (recombinant) were incubated with human PBMCs and binding of the complexes was assessed by flow cytometry.

[0162] procedure Fluorescently labeled C3dg tetramers were generated using biotinylated C3dg and streptavidin-APC by incubating biotinylated C3dg with streptavidin-APC in a 4:1 ratio.

[0163] PBMCs were isolated from human whole blood by density gradient centrifugation. PBMCs were incubated with the indicated concentrations of CHO-expressed mAb 1053, isotype control antibody, or no antibody for 30 min at 37° C. in a humidified atmosphere with 5% CO2. PBMCs were then incubated with C3dg:streptavidin-APC complex for an additional 30 min.

[0164] Cells were fixed to preserve the ligand on the surface until subsequent staining. Fixed cells were incubated with anti-CD20-PE to identify B cells within the PBMC population. After fixation of stained PBMCs, quantification of ligand binding (APC fluorescence) to CD20+ B cells was performed by flow cytometry.

[0165] result Preincubation with mAb 1053 gave an IC of 11.9 pM. 50 Dose-dependent inhibition of complex binding at 100 μg / mL (N=4 donors, 2 independent experiments, mean normalized MFI ± SEM, a single curve was fitted to the average data, a single IC 50 was determined), whereas an isotype control antibody had no effect (see FIG. 4).

[0166] These data indicate that mAb 1053 is able to block the binding of C3d / g-opsonized antigens to B cells, thereby preventing their CR2-dependent transport to FDCs.

[0167] Example 3: Affinity of anti-CR2 antibodies to human and cynomolgus monkey CR2 at 25°C procedure Binding to recombinant soluble human and cynomolgus monkey (cyno) CR2 was assessed using a BIACORE T200 (GE Healthcare) surface plasmon resonance instrument. Protein A was immobilized on a CM5 biosensor chip by primary amine coupling. Test antibodies (expressed by HEK) were diluted to 5 μg / mL and captured on the Protein A surface for 120 seconds followed by a 60 second dissociation time at 10 μL / min. The capture time to achieve 267 RU was calculated using the binding stability reporting points for each antibody for each flow cell. Human and cyno CR2 antigens were titrated over eight points in a 1:3 serial dilution from a top concentration of 22 nM and run over the capture antibody for 600 seconds at a flow rate of 3 μL / min. A 0 nM (i.e., buffer alone) injection was used to double-reference the binding curves. The Protein A surface was regenerated with two 50 mM sodium hydroxide injections for 30 seconds between each cycle of CR2 titration. Measurements were performed at 25°C using HBS-EP as the running buffer. Data were analyzed using the BIACORE analysis software native equilibrium model for setting a global R-max and for calculation of KD values. Results are shown in Table 3.

[0168] [Table 4]

[0169] Example 4: Assay to determine the effect of mAb 1053 on serum-opsonized PnPS14 binding This experiment determined the ability of mAb 1053 to block the binding of a "naturally opsonized" ligand to non-cognate B cells. The ligand used was pneumococcal polysaccharide type 14 (PnPS14; a polysaccharide antigen expressed by Streptococcus pneumoniae) opsonized with normal human serum. Opsonized PnPS14 was incubated with human PBMCs and binding was assessed by flow cytometry.

[0170] procedure Incubation of PnPS14 in 100% NHS for 1 h at 37°C resulted in the covalent deposition of opsonic C3 fragments containing C3dg.

[0171] PBMCs were isolated from human whole blood by density gradient centrifugation. PBMCs were incubated with the indicated concentrations of CHO-expressed mAb 1053, isotype control antibody, or no antibody for 30 min at 37°C, 5% CO2, in a humidified atmosphere. PBMCs were then incubated with serum-opsonized PnPS14 for an additional 30 min.

[0172] Cells were fixed to preserve the ligand on the surface until subsequent staining. Fixed cells were incubated with anti-CD20-PE to identify B cells within the PBMC population. Bound PnPS14 was detected with PnPS14 antiserum and then stained with anti-rabbit IgG-FITC. After fixing the stained PBMCs, quantification of ligand binding (FITC fluorescence) to CD20+ B cells was performed by flow cytometry.

[0173] result Preincubation with mAb 1053 inhibited the binding of serum-opsonized PnPS14 to non-cognate B cells with an IC of 37.9 pM. 50 (N=4 donors, 2 independent experiments, mean normalized MFI ± SEM, a single curve was fitted to the average data, a single IC 50 was determined), whereas an isotype control antibody did not block binding (see FIG. 5).

[0174] These data indicate that mAb 1053 is able to block the binding of C3d / g-opsonized antigens to B cells, thereby preventing their CR2-dependent transport to FDCs.

[0175] Example 5: Assay to determine the effect of mAb 1053 on bound C3dg ligand binding This experiment determined whether bound ligand could be competitively dissociated from non-cognate B cells as a model for autoantigen displacement. Fluorescent streptavidin:C3dg complexes were incubated with PBMCs and unbound ligand was washed away. mAb 1053 or an isotype control antibody was added to the cells and the amount of bound ligand was followed over time by flow cytometry.

[0176] procedure Fluorescently labeled C3dg tetramers were generated using biotinylated C3dg and streptavidin-APC by incubating biotinylated C3dg with streptavidin-APC in a 4:1 ratio.

[0177] PBMCs were isolated from human whole blood by density gradient centrifugation. PBMCs were incubated with C3dg:streptavidin-APC complexes for 30 minutes at 37°C, 5% CO2, in a humidified atmosphere. Unbound C3dg:streptavidin-APC complexes were washed away. PBMCs were then incubated with 10 nM CHO-expressed mAb 1053, an isotype control antibody, or no antibody for 0.5, 2, or 18.5 hours.

[0178] Cells were fixed to preserve the ligand on the surface until subsequent staining. Fixed cells were incubated with anti-CD20-PE to identify B cells within the PBMC population. After fixation of stained PBMCs, quantification of ligand binding (APC fluorescence) to CD20+ B cells was performed by flow cytometry.

[0179] result mAb 1053 rapidly competed off bound C3dg:streptavidin-APC complexes from non-cognate B cells, whereas the isotype control antibody had no effect. Most of this effect occurred within 2 hours of addition. The isotype control antibody did not reduce binding compared to no antibody (N=4 donors, 2 independent experiments, mean normalized MFI±SEM) (see FIG. 6).

[0180] These data indicate that mAb 1053 can rapidly and potently compete off bound C3dg-opsonized antigen from CR2-expressing cells.

[0181] Examples 6-9: Effect of mAb 1053 on B cell activation Co-ligation of CR2 and antigen-specific BCR by C3d / g-opsonized antigen lowers the threshold for B cell activation. To model this with non-cognate B cells, multimeric complexes were created to act as surrogates for C3dg-opsonized antigen. Biotinylated anti-BCR reagents (recognizing either lambda chain or IgM) were used to mimic antigen interaction with the BCR, and biotinylated recombinant C3dg was used as the CR2 ligand. These components were co-incubated with neutravidin, resulting in a complex containing both anti-BCR and C3dg; anti-BCR:C3dg complex.

[0182] Because the primary role of CR2 in this system is to lower the threshold for BCR activation, we optimized the assay by titrating anti-BCR reagents to levels that induced little or no signal. When C3dg was included in the complex, co-ligation of CR2 lowered the threshold for B cell activation, resulting in a C3dg-dependent activation window.

[0183] Example 6: Assay to determine the effect of mAb 1053 on calcium flux and phosphoprotein signaling in primary human B cells To assess the ability of mAb 1053 to block signaling events downstream of CR2-dependent BCR activation, intracellular calcium flux was monitored in B cells by flow cytometry after stimulation of PBMCs with anti-BCR:C3dg complexes.

[0184] procedure PBMCs were isolated from human whole blood by density gradient centrifugation. PBMCs were loaded with the calcium-responsive dye Fluo-4 and B cells were differentiated with anti-CD19 BV421. Baseline and post-stimulation Fluo-4 fluorescence was monitored in real time by flow cytometry. To elicit CR2-dependent release of intracellular calcium downstream of the BCR, cells were stimulated with anti-BCR:C3dg complexes. Stimulation was performed after preincubation with the indicated concentrations of CHO-expressed mAb 1053 for 30 min at room temperature. The time of peak C3dg-dependent response in the absence of mAb 1053 was extrapolated to obtain equivalent response values ​​at that time point in samples containing mAb 1053. Responses were normalized such that the maximum response for each data set=100%. N=4 individuals, mean±SD.

[0185] result A dose-dependent decrease in calcium flux signal was observed with mAb 1053, with IC 50 was 9.0 pM (see FIG. 7).

[0186] These data indicate that mAb 1053 can potently inhibit calcium signaling immediately downstream of BCR activation in primary human B cells.

[0187] Example 7: Assay to determine the effect of mAb 1053 on phosphorylated protein signaling in primary human B cells To assess the ability of mAb 1053 to block signaling events downstream of CR2-dependent BCR activation, we measured the levels of phosphorylated ERK1 / 2 (pERK) and phosphorylated AKT (pAKT) in B cells by intracellular flow cytometry after stimulation of PBMCs with anti-BCR:C3dg complexes.

[0188] procedure PBMCs were isolated from human whole blood by density gradient centrifugation and incubated for 2 h at 37°C in a humidified atmosphere with 5% CO2. PBMCs were then pre-incubated with the indicated concentrations of CHO-expressed mAb 1053 for 30 min. Cells were stimulated with anti-BCR:C3dg complexes for 10 min at 37°C, arrested by fixation, permeabilized and stored at -20°C overnight. Cells were stained with anti-CD20-PE, anti-pAKT (pS473)-ALEXA 647 and anti-pERK-ALEXA 488 to identify B cells. Fluorescence was assessed by flow cytometry and normalized %pERK+ and pAKT+ values ​​were determined in CD20+ cells. N=4 individuals, mean ± SD.

[0189] result Titering mAb 1053 levels resulted in robust inhibition of the pERK response, with IC 50 The IC value for mAb 1053 was 2.7 nM (Figure 8a). Evaluation of the phosphorylated AKT signal stimulated by the complex in the same PBMC assay revealed that the IC 50 was 3.8 nM (FIG. 8b). Taken together these data demonstrate the ability of mAb 1053 to block signaling in primary human B cells following stimulation with a model CR2 / BCR ligand complex.

[0190] mAb 1053 potently inhibited the pERK response in a dose-dependent manner, with IC 50 pAKT signaling was similarly inhibited by mAb 1053, with an IC 50 was 3.8 nM (see FIG. 8b).

[0191] These data demonstrate the ability of mAb 1053 to block phosphoprotein signaling downstream of BCR activation in primary human B cells.

[0192] Example 8: Assay to determine the effect of mAb 1053 on the activation of primary human B cells as determined by upregulation of CD69 To assess the ability of mAb 1053 to block signaling events downstream of CR2-dependent BCR activation, cell surface expression of CD69 (an activation marker) was measured by flow cytometry on primary human B cells after stimulation of PBMCs with anti-BCR:C3dg complexes.

[0193] procedure Frozen PBMCs (isolated by density gradient centrifugation from human whole blood) from combined donors were thawed and incubated with the indicated concentrations of HEK-expressed mAb 1053 or isotype control antibody for 30 min at 37°C, 5% CO2, humidified atmosphere. Cells were stimulated with anti-BCR:C3dg complexes and incubated for an additional 4 h before fixing. Cells were stained with anti-CD20-A647 to identify B cells, and anti-CD69-PE. Fluorescence was assessed by flow cytometry to determine normalized CD69 response % in CD20+ cells. N=4 independent evaluations, mean ± SD.

[0194] result mAb 1053 has an IC of 4.4 nM 50 potently inhibited CD69 upregulation, whereas an isotype control antibody did not (see FIG. 9).

[0195] These data demonstrate that mAb 1053 can block upregulation of cell surface CD69 downstream of BCR activation in primary human B cells.

[0196] Example 9: Assay to determine the effect of mAb 996, mAb 999 and mAb 1053 on antibody secretion in primary human B cells To assess the ability of mAb 1053 to block functional responses in primary human B cells, secreted immunoglobulin levels were measured in primary human B cells by MSD after stimulation of B cells with anti-BCR:C3dg complexes.

[0197] procedure B cells were isolated from single-donor, previously frozen PBMCs (isolated by density gradient centrifugation from human whole blood) by negative magnetic selection to remove non-B cells and plasma cells. Cells were pre-incubated with 5 μg / ml of HEK-expressed mAb 996, mAb 999, mAb 1053, isotype control antibody, or no mAb in culture medium at 37° C., 5% CO2, humidified atmosphere for 30 min. B cells were stimulated with anti-BCR:C3dg complexes or complexes without C3dg ("no C3dg" control). Culture medium contained suboptimal IL-21 and CD40L to maintain cell viability. After 4 days, cell supernatants were harvested and assayed for IgA using the MSD assay kit. IgA concentrations were calculated from a standard curve, and the median fold increase over the "no C3dg" control was calculated as the CR2-dependent activation window. N=11 donors, mean±SE.

[0198] result mAb 996, mAb 999, and mAb 1053, but not an isotype control antibody, potently inhibited C3dg-dependent IgA secretion from primary human B cells (see FIG. 10).

[0199] These data demonstrate that mAb 996, mAb 999, and mAb 1053 can block C3dg-dependent functional production of immunoglobulin secretion in primary human B cells.

[0200] Examples 10 and 11: Effect of mAb 1053 on primary human follicular dendritic cells (FDCs) A key role of FDCs in autoimmunity is their CR2-dependent binding to and retention of C3d / g-opsonized antigens for sustained antigen presentation to cognate B cells in GC / ELS.

[0201] Example 10: Assay to determine target engagement of mAb 1053 to primary human FDCs This experiment determined the on-cell binding affinity of mAb 1053 to primary human FDCs. Unlabeled mAb 1053 was titrated with FDC-enriched human tonsil digests followed by an excess of fluorescently labeled mAb 1053. FDCs were identified and antibody binding was assessed by flow cytometry.

[0202] procedure Human tonsils from a single donor were mechanically disrupted, FDCs were released by enzymatic digestion, and CD45 was removed from these digests by negative magnetic selection. CD45-depleted tonsil digests were preincubated for 20 min at room temperature with the indicated concentrations of unlabeled, CHO-expressed mAb 1053. Cells were then incubated for 30 min with antibodies to identify FDCs by flow cytometry and with a saturating concentration of PE-labeled mAb 1053 (mAb 1053-PE, 1 μg / ml). Lineage+ cells were identified with antibodies recognizing CD11b, CD45, CD31, and CD34. Cells were gated to identify FDCs (lineage-gp38+CD21+ using the non-competing clone Bu32). The geometric mean of uninhibited mAb 1053-PE staining was taken as 100%. Data are the mean ± SE of four independent donors.

[0203] result Target association of mAb 1053 to FDCs was concentration-dependent, with EC calculated from the binding curve on cells. 50 was 62.2 pM (see FIG. 11).

[0204] These data indicate that mAb 1053 engages the CR2 target with high affinity on primary human FDCs.

[0205] Example 11: Assay to determine the effect of mAb 1053 on the binding of C3dg ligand to primary human FDCs This experiment determined the ability of mAb 1053 to block binding of C3dg-ligand complexes to human FDCs. Fluorescently labeled streptavidin and biotin-C3dg complexes (recombinant) were incubated with human tonsillar digests and complex binding was assessed by flow cytometry.

[0206] procedure Fluorescently labeled C3dg tetramers were generated using biotinylated C3dg and streptavidin-APC by incubating biotinylated C3dg with streptavidin-APC in a 4:1 ratio.

[0207] Human tonsils from a single donor were mechanically disrupted, FDCs were released by enzymatic digestion, and CD45 was removed from these digests by negative magnetic selection. CD45-depleted tonsil digests were preincubated for 20 min at room temperature with the indicated concentrations of unlabeled, CHO-expressed mAb 1053. Cells were then incubated for 30 min with antibodies to identify FDCs by flow cytometry and 10 μg / ml C3dg:streptavidin-APC ligand complex. Lineage+ cells were identified with antibodies recognizing CD11b, CD45, CD31, and CD34. Cells were gated to identify FDCs (lineage-gp38+CD21+ using the non-competing clone Bu32). The geometric mean of C3dg(APC) staining was taken as 100%. Data are the mean ± SE of four independent donors.

[0208] result Preincubation of tonsillar digests with mAb 1053 resulted in ligand binding with an IC of 47.0 pM. 50 The effect was inhibited in a dose-dependent manner by 100 mg / kg of DMSO (see FIG. 12).

[0209] These data indicate that mAb 1053 can potently inhibit the binding of C3dg-ligand to primary human FDCs, thereby preventing the retention of complement-opsonized antigens.

[0210] Biological Data array SEQ ID NO: 1: Human CR2 sequence SEQ ID NO: 2: Human CR2 epitope FNKYS SEQ ID NO:3: Amino acid sequence of 1053 heavy chain QLQLQESGPGLVKPSETLSLTCTVSGGSISSDSYYWGWIRQPPGKGLEWIGSIHYSGSTYYNPPLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGGPRHGWSWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4: Amino acid sequence of 1053 light chain DIQMTQSPSSLSASVGDRVTITCQASQDISEYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSNLPITFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 5: Amino acid sequence of 1053 variable heavy chain QLQLQESGPGLVKPSETLSLTCTVSGGSISSDSYYWGWIRQPPGKGLEWIGSIHYSGSTYYNPPLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGGPRHGWSWGQGTLVTVSS SEQ ID NO: 6: Amino acid sequence of 1053 variable light chain DIQMTQSPSSLSASVGDRVTITCQASQDISEYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSNLPITFGGGTKVEIK SEQ ID NO: 7: 1053 Amino acid sequence of CDRH1 SDSYYWG SEQ ID NO: 8: 1053 Amino acid sequence of CDRH2 SIHYSGSTYYNPPLES SEQ ID NO: 9: 1053 Amino acid sequence of CDRH3 EGGPRHGWS SEQ ID NO: 10: 1053 Amino acid sequence of CDRL1 QASQDISEYLN SEQ ID NO:11: 1053 Amino acid sequence of CDRL2 DASNLET SEQ ID NO: 12: 1053 Amino acid sequence of CDRL3 QQDSNLPIT SEQ ID NO: 13: Nucleic acid sequence encoding 1053 heavy chain SEQ ID NO: 14: Nucleic acid sequence encoding the 1053 light chain GACATCCAGATGACTCAGTCCCCCTCTAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCAGCCAGGACATCAGCGAGTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGACGCCTCAAACCTCGAGACCGGCGTGCCTAGCAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGGACAGCAACCTGCCCATCACCTTCGGCGGAGGCACCAAGGTGGAGATTAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 15: Amino acid sequence of the 996 heavy chain QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIHNSGSTYYNPPLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGGPRHGWSWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 16: Amino acid sequence of 996 light chain DIQMTQSPSSLSASVGDRVTITCQASQDISTFLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSILPITFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 17: Amino acid sequence of 996 variable heavy chain QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIHNSGSTY YNPPLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGGPRHGWSWGQGTLVTVSS SEQ ID NO: 18: Amino acid sequence of 996 variable light chain DIQMTQSPSSLSASVGDRVTITCQASQDISTFLNWYQQKPGKAPKLLIYDASNLETGVPS RFSGSGSGTDFTFTISSLQPEDIATYYCQQDSILPITFGGGTKVEIK SEQ ID NO: 19: 996 Amino acid sequence of CDRH1 SSSYYWG SEQ ID NO:20: 996 Amino acid sequence of CDRH2 SIHNSGSTYYNPPLKS SEQ ID NO:21: 996 Amino acid sequence of CDRH3 EGGPRHGWS SEQ ID NO:22: 996 Amino acid sequence of CDRL1 QASQDISTFLN SEQ ID NO:23: 996 Amino acid sequence of CDRL2 DASNLET SEQ ID NO:24: 996 Amino acid sequence of CDRL3 QQDSILPIT SEQ ID NO: 25: Nucleic acid sequence encoding 996 heavy chain SEQ ID NO: 26: Nucleic acid sequence encoding the 996 light chain GACATCCAGATGACTCAGTCCCCCTCTAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCAGCCAGGACATCAGCACCTTCCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGACGCCTCAAACCTCGAGACCGGCGTGCCTAGCAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGGACAGCATCCTGCCCATCACCTTCGGCGGAGGCACCAAGGTGGAGATTAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 27: Amino acid sequence of the 999 heavy chain QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIHYSGSTYYNPPLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGGPRHGWSWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 28: Amino acid sequence of 999 light chain DIQMTQSPSSLSASVGDRVTITCQASHDISNFLNWYQQKPGKAPKLLIYDTSNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSILPITFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 29: Amino acid sequence of 999 variable heavy chain QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIHYSGSTY YNPPLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGGPRHGWSWGQGTLVTVSS SEQ ID NO:30: Amino acid sequence of 999 variable light chain DIQMTQSPSSLSASVGDRVTITCQASHDISNFLNWYQQKPGKAPKLLIYDTSNLETGVPS RFSGSGSGTDFTFTISSLQPEDIATYYCQQDSILPITFGGGTKVEIK SEQ ID NO:31: 999 Amino acid sequence of CDRH1 SSSYYWG SEQ ID NO:32: 999 Amino acid sequence of CDRH2 SIHYSGSTYYNPPLES SEQ ID NO:33: 999 Amino acid sequence of CDRH3 EGGPRHGWS SEQ ID NO:34: 999 Amino acid sequence of CDRL1 QASHDISNFLN SEQ ID NO:35: 999 Amino acid sequence of CDRL2 DTSNLET SEQ ID NO:36: 999 Amino acid sequence of CDRL3 QQDSILPIT SEQ ID NO: 37: Nucleic acid sequence encoding the 999 heavy chain SEQ ID NO: 38: Nucleic acid sequence encoding the 999 light chain GACATCCAGATGACTCAGTCCCCCTCTAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCAGCCACGACATCAGCAACTTCCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGACACCTCAAACCTCGAGACCGGCGTGCCTAGCAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGGACAGCATCCTGCCCATCACCTTCGGCGGAGGCACCAAGGTGGAGATTAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 39: MISCGSPPPIL SEQ ID NO: 40: MISCGSPPPILNGRISY SEQ ID NO: 41: YSTPIAVG SEQ ID NO: 42: YSTPIAVGT SEQ ID NO: 43: YSTPIAVGTVIRYSCSGT SEQ ID NO:44: TVIRYSCSGT SEQ ID NO:45: TVIRYSCSGTF SEQ ID NO:46: VIRYSCSGT SEQ ID NO:47: FRLIGEKS SEQ ID NO:48: FRLIGEKSL SEQ ID NO:49: FRLIGEKSLL SEQ ID NO:50: RLIGEKSL SEQ ID NO:51: RLIGEKSLL SEQ ID NO:52: IGEKSLL SEQ ID NO:53: GEKSLLCITKDKVDGTWDKPA SEQ ID NO:54: CITKDKVDGT SEQ ID NO:55: CITKDKVDGTWDKPAPKCEY SEQ ID NO:56: KVDGTWDKPAPKCEY SEQ ID NO:57: WDKPAPKCEY SEQ ID NO:58: FNKYSSCPEPIVPGGYKIRGSTPYRHGDSVT SEQ ID NO:59: FNKYSSCPEPIVPGGYKIRGSTPYRHGDSVTF SEQ ID NO:60: SSCPEPIVPGGYKIRGSTPYRHGDSVT SEQ ID NO:61: YKIRGSTPYRHGDSVT SEQ ID NO:62: YKIRGSTPYRHGDSVTF SEQ ID NO:63: KIRGSTPYRHGDSVT SEQ ID NO:64: KIRGSTPYRHGDSVTF SEQ ID NO:65: FACKTNF SEQ ID NO:66: SMNGNKSVW SEQ ID NO:67: SMNGNKSVWC SEQ ID NO:68: SMNGNKSVWCQA SEQ ID NO:69: SMNGNKSVWCQANNM SEQ ID NO:70: SMNGNKSVWCQANNMWGPTRL SEQ ID NO:71: SMNGNKSVWCQANNMWGPTRLPTC SEQ ID NO:72: NNMWGPTRL SEQ ID NO:73: NNMWGPTRLPTCV SEQ ID NO:74: VSVFPLE

Claims

1. A CR2 antibody, comprising, in its heavy chain variable region, a CDR1 comprising the amino acid sequence shown in SEQ ID NO:7, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:9, and, in its light chain variable region, a CDR1 comprising the amino acid sequence shown in SEQ ID NO:10, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:11, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

12.

2. 2. The CR2 antibody of claim 1, comprising a variable light chain amino acid sequence as shown in SEQ ID NO:6 and a variable heavy chain amino acid sequence as shown in SEQ ID NO:

5.

3. 3. The CR2 antibody of claim 1 or 2, comprising a light chain amino acid sequence as shown in SEQ ID NO:4 and a heavy chain amino acid sequence as shown in SEQ ID NO:

3.

4. A nucleic acid molecule encoding the CR2 antibody of claim 1.

5. 5. The nucleic acid molecule according to claim 4, wherein the sequence comprises SEQ ID NO: 13 encoding the heavy chain and / or SEQ ID NO: 14 encoding the light chain.

6. An expression vector comprising the nucleic acid molecule of claim 4.

7. A recombinant host cell comprising an expression vector as defined in claim 6.

8. A CR2 antibody expressed by a recombinant host cell as defined in claim 7.

9. 10. A method for producing a CR2 antibody, comprising culturing a recombinant host cell according to claim 7 in a medium to produce the CR2 antibody, and isolating or purifying the CR2 antibody.

10. A pharmaceutical composition comprising the CR2 antibody of claim 1 and a pharma- ceutically acceptable carrier or excipient.

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