RU2798422C2 - Antibodies against cxcr5, their compositions and use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an isolated antibody or its antigen-binding fragment that specifically binds to the C-X-C-chemokine 5 receptor (CXCR5), to a composition containing it, as well as to a method for producing it. Also an isolated nucleic acid encoding the above antibody or a fragment thereof, as well as a vector and a cell containing it are disclosed. The invention also relates to a method for detecting CXCR5 in a sample, tissue or cell, using the above antibody or a fragment thereof.

EFFECT: invention is effective for the treatment or prevention of an immunological disease, disorder or condition mediated by CXCR5.

47 cl, 9 dwg, 16 tbl, 17 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Patent Application No. 62/593830, filed December 1, 2017, and US Patent Application No. 62/732,985, filed September 18, 2018, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LIST

[0002] The present disclosure further includes by reference the sequence listing filed with it on November 28, 2018. Under 37 C.F.R. § 1.52(e)(5), the sequence listing text file named PC72320A_Seq_Listing_ST25.txt is 112891 bytes in size and was created on November 15, 2018. The sequence listing filed electronically does not depart from the scope of the present disclosure and thus does not contain new features of the invention.

PARTIES TO A JOINT RESEARCH AGREEMENT

[0003] The invention described in this application was created by or on behalf of the following parties to a joint research agreement. The joint research agreement was in effect on or before the date of creation of the invention described in the application, and the invention described in the application was created as a result of actions taken within the scope of the joint research agreement. The parties to the joint research agreement are the Board of Regents of the University of California, on behalf of its San Diego campus, and PFIZER INC.

FIELD OF TECHNOLOGY

[0004] The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to the C-X-C chemokine receptor type 5 (CXCR5), and their compositions, methods and uses. The antibodies are fucosylated and/or afucosylated and may show altered effector function.

BACKGROUND OF THE INVENTION

[0005] C-X-C chemokine receptor type 5 (CXCR5), also known as CD185 or Burkitt's lymphoma receptor 1 (BLR1), is a naturally occurring G-protein coupled receptor expressed by B cells, true follicular T helper cells (Tfh) and circulating Tfh-like cells (referred to interchangeably herein as "cTfh" and "Tfh-like" cells). CXCR5 plays an important role in the immune response and is a potential target for the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE).

[0006] Germinal centers (GCs), a critical component of the humoral immune response, are sites where antigen-activated B cells proliferate, differentiate, and undergo somatic hypermutation and class switching of the immunoglobulin (Ig) antibodies they produce. True Tfh cells provide instructive signals to facilitate this process, culminating in the production of affinity matured antibodies. The humoral "memory" of the GC response is maintained by long-lived plasma cells that maintain antibody levels and by memory B cells, which, upon restimulation with antigen, trigger secondary GC responses with Tfh memory cells, resulting in the production of more high-affinity plasma cells (McHeyzer-Williams et al., 2011, Nature Rev. Immunol.12(1):24-34). It is believed that circulating Tfh-like cells (hereinafter referred to as "Tfh-like" or "cTfh" cells) also differentiate into true Tfh cells after antigen re-stimulation to support these anamnestic responses (Crotty et al., 2011 , Annu Rev. Immmunol 29:621-663).

[0007] Importantly, the formation of autoreactive B cells can also result from GC reactions with undesirable consequences. In fact, numerous chronic systemic autoimmune diseases such as SLE, RA, myositis, Sjögren's syndrome, ANCA-associated vasculitis, and scleroderma show signs of autoreactive humoral responses. For example, many of the autoantibodies that are hallmarks of these diseases are high-affinity, somatically mutated Ig isotype-switched antibodies suggesting they are the result of autoreactive GC reactions (Vinuesa et al., 2009, Nature Rev. Immunol. 9(12 ):845-857). In addition, increased numbers of circulating Tfh-like cells are found in the peripheral blood of patients with many of these autoimmune diseases, levels of which often correlate with autoantibody titers and/or disease severity (Tangye et al., 2013, Nature Rev. Immunol. 13(6 ):412-426). Taken together, these data suggest that B cells, true Tfh cells, and circulating Tfh-like cells are potential therapeutic targets for many systemic autoimmune diseases.

[0008] CXCR5 is expressed by B cells, true Tfh cells and circulating Tfh-like cells and mediates their migration to GC and participation in GC reactions along with the CXCR5 ligand gradient, CXCL13 (Vinuesa and Cyster, 2011, Immunity 35(5): 671-680, Ansel et al., 1999, J. Exp. Med., 190(8):1123-1134, Ansel et al., 2000, Nature 406(6793):309-314, Cyster et al., 1999, Curr Top Microbiol Immunol 246:87-92 Hardtke et al 2005 Blood 106(6):1924-1931 Haynes et al 2007 J Immunol 179(8):5099-5108 ). In this regard, targeting CXCR5 may be of therapeutic benefit in the treatment of autoimmune diseases.

[0009] In addition, targeting CXCR5 may also be of therapeutic benefit in cancers characterized by the proliferation of cells expressing CXCR5, such as pancreatic cancer, colon cancer, bladder cancer, T-cell leukemia, and B-cell leukemia.

SUMMARY OF THE INVENTION

[00010] This application describes isolated antibodies and their antigen-binding fragments that specifically bind to CXCR5 (C-X-C chemokine receptor type 5). In some aspects, the antibodies and antigen-binding fragments thereof bind to CXCR5 and reduce the binding of CXCR5 to CXCL13. In other aspects, the antibodies may be fucosylated, but more preferably they are afucosylated. In some aspects, antibodies and antigen-binding fragments exhibit an altered effector function. In some aspects, antibodies and antigen-binding fragments thereof are afucosylated and exhibit increased antibody-dependent cellular cytotoxicity (ADCC) compared to otherwise identical, but fucosylated, antibodies and antigen-binding fragments thereof.

[00011] In some aspects, the present invention provides isolated antibodies and antigen-binding fragments thereof specifically binding to CXCR5, wherein the antibody and antigen-binding fragment thereof bind to an epitope containing leucine (Leu; L) at amino acid residue position 11 (L11) according to numbering SEQ ID NO:32.

[00012] In another aspect, the present invention provides isolated antibodies, or antigen-binding fragments thereof, specifically binding to CXCR5, wherein the antibody and antigen-binding fragment thereof bind to an epitope containing aspartate (Asp; D) at amino acid residue position 22, according to SEQ ID numbering. NO:32.

[00013] Those of skill in the art will recognize many equivalents to the specific embodiments presented herein, or may ascertain them using no more than routine experimentation. Such equivalents are intended to be included in the following embodiments (E).

E1. An isolated antibody or antigen-binding fragment thereof that specifically binds to the C-X-C chemokine receptor 5 (CXCR5), wherein the antibody or antigen-binding fragment thereof binds to an epitope in the N-domain of human CXCR5 (hCXCR5) or cynomolgus monkey CXCR5 (cynoCXCR5).

E2. An antibody or antigen-binding fragment thereof according to claim E1, wherein the antibody or antigen-binding fragment thereof binds to an epitope at amino acid residues 1-50 of hCXCR5 according to the amino acid sequence numbering of SEQ ID NO:32 or binds to an epitope at amino acid residues 1-50 of cynoCXCR5 according to with the numbering of SEQ ID NO:33.

E3. An antibody or antigen-binding fragment thereof according to any one of claims E1-E2, wherein the antibody or antigen-binding fragment thereof binds to an epitope containing leucine at amino acid residue position 11 according to the amino acid sequence numbering of SEQ ID NO:32.

E4. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E3, wherein the antibody or antigen-binding fragment thereof binds to an epitope containing aspartate at amino acid residue position 22 according to the amino acid sequence numbering of SEQ ID NO:32.

E5. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E4, wherein the antibody or antigen-binding fragment thereof binds to an epitope containing leucine at amino acid residue position 11 and aspartate at amino acid residue position 22 in accordance with the amino acid sequence numbering of SEQ ID NO:32 .

E6. An antibody or antigen-binding fragment thereof according to any one of claims E1-E5, wherein the antibody or antigen-binding fragment thereof binds to an epitope containing leucine at amino acid residue position 11 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to hCXCR5 if said residue is not leucine.

E7. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E6, wherein the antibody or antigen-binding fragment thereof binds to an epitope containing leucine at amino acid residue position 11 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to hCXCR5 if said residue is threonine.

E8. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E7, wherein the antibody or antigen-binding fragment thereof binds to an epitope containing aspartate at amino acid residue position 22 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to hCXCR5, if the indicated residue is not an aspartate.

E9. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E8, where the antibody or antigen-binding fragment thereof binds to an epitope containing aspartate at amino acid residue position 22, in accordance with the amino acid sequence numbering of SEQ ID NO:32, but does not bind to hCXCR5, if said residue is alanine.

E10. An antibody or antigen-binding fragment thereof according to any one of claims E1-E9, wherein the antibody or antigen-binding fragment thereof binds to an epitope containing leucine at amino acid residue position 11 and aspartate at amino acid residue position 22 in accordance with the amino acid sequence numbering of SEQ ID NO:32, but does not bind to said epitope if leucine is substituted with threonine and/or aspartate is substituted with alanine.

E11. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E10, wherein the antibody or antigen-binding fragment thereof binds to hCXCR5 expressed on human B cells with an apparent affinity EC ₅₀ of approximately 6.60 pM with a standard deviation of approximately ±2.33 pM .

E12. An antibody or antigen-binding fragment thereof according to any one of claims E1-E11, wherein the antibody or antigen-binding fragment thereof binds to hCXCR5 expressed on circulating human follicular T-helper-like (Tfh-like) cells with an apparent affinity EC ₅₀ of approximately 5.89 pM with a standard deviation of approximately ±1.40 pM.

E13. An antibody or antigen-binding fragment thereof according to any one of claims E1-E12, wherein the antibody or antigen-binding fragment thereof binds to hCXCR5 expressed on human follicular T helper (Tfh) cells with an apparent EC ₅₀ affinity of approximately 10.6 pM.

E14. An antibody or antigen-binding fragment thereof according to any one of claims E1-E13, wherein the antibody or antigen-binding fragment thereof binds to cynoCXCR5 expressed on cynomolgus monkey B cells with an apparent affinity of EC ₅₀ of approximately 1.32 pM.

E15. An antibody or antigen-binding fragment thereof according to any one of claims E1-E14, wherein the antibody or antigen-binding fragment thereof binds to cynoCXCR5 expressed on Tfh-like cynomolgus monkey cells with an apparent EC ₅₀ affinity of approximately 10.5 pM.

E16. An antibody or antigen-binding fragment thereof according to any one of claims E1-E15, wherein the antibody or antigen-binding fragment thereof antagonizes CXCR5-CXCL13 signaling in a cAMP reporter assay with an EC ₅₀ of approximately 961 pM.

E17. An antibody or antigen-binding fragment thereof according to any one of items E1-E16, wherein the antibody or antigen-binding fragment thereof exhibits ADCC activity against human B cells expressing hCXCR5 with an EC _{50 of} approximately 2.01 pM with a standard deviation of approximately ±2.28 pM.

E18. An antibody or antigen-binding fragment thereof according to any one of claims E1-E17, wherein the antibody or antigen-binding fragment thereof exhibits ADCC activity against human Tfh-like cells expressing hCXCR5 with an EC ₅₀ of approximately 4.28 pM with a standard deviation of approximately ±2.88 pM .

E19. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E18, wherein the antibody or antigen-binding fragment thereof exhibits ADCC activity against human Tfh cells expressing hCXCR5 with an EC ₅₀ of approximately 0.11 pM.

E20. An antibody or antigen-binding fragment thereof according to any one of claims E1-E19, wherein the antibody or antigen-binding fragment thereof exhibits ADCC activity against cynoCXCR5 expressing cynoCXCR5-expressing cynoCXCR5-expressing EC ₅₀ of approximately 15.3 pM with a standard deviation of approximately ±11.7 pM .

E21. An antibody or antigen-binding fragment thereof according to any one of claims E1-E20, wherein the antibody or antigen-binding fragment thereof binds to hCXCR5 but does not detectably bind to human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 and XCR1.

E22. An antibody or antigen-binding fragment thereof according to any one of E1-E21, wherein the antibody or antigen-binding fragment thereof depletes B cells in the peripheral blood.

E23. An antibody or antigen-binding fragment thereof according to item E22, wherein the depletion of B cells in the peripheral blood is reversible.

E24. An antibody or antigen-binding fragment thereof according to any one of claims E1-E23, wherein the antibody or antigen-binding fragment thereof depletes Tfh-like cells in peripheral blood.

E25. An antibody or antigen-binding fragment thereof according to any one of E1-E24, wherein the antibody or antigen-binding fragment thereof depletes Tfh cells in the spleen.

E26. An antibody or antigen-binding fragment thereof according to any one of claims E1-E24, wherein the antibody or antigen-binding fragment thereof interferes with the humoral anamnestic immune response.

E27. An antibody or antigen-binding fragment thereof according to item E26, wherein the impaired humoral anamnestic immune response is a response to tetanus toxoid in cynomolgus monkeys.

E28. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E27, wherein the antibody or antigen-binding fragment thereof inhibits the binding of CXCR5 to CXCL13.

E29. An antibody or antigen-binding fragment thereof according to any one of claims E1-E28, wherein the antibody, or antigen-binding fragment thereof, inhibits CXCL13 inhibition of cAMP production in a cell otherwise triggered by forskolin, resulting in an increase in cAMP levels relative to cAMP levels in the absence of antibody, or its antigen-binding fragment.

E30. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E29, wherein the antibody or antigen-binding fragment thereof inhibits CXCL13 inhibition of cAMP production with an EC ₅₀ of approximately 961 pM.

E31. An antibody or antigen-binding fragment thereof according to claim E30, wherein the maximum inhibition of CXCL13 inhibition is at least about 60%, 70%, or 80%.

E32. An antibody or antigen-binding fragment thereof according to any one of claims E1-E31, wherein the antibody or antigen-binding fragment thereof binds to CXCR5-expressing human B cells (e.g., CXCR5 ⁺ human B cells) with an apparent affinity EC ₅₀ of less than about 26 pM, but does not bind to cells expressing mouse, rat, or rabbit CXCR5 orthologues.

E33. An antibody or antigen-binding fragment thereof according to any one of claims E1-E32, wherein the antibody or antigen-binding fragment thereof triggers ADCC of CXCR5-expressing cells in peripheral blood mononuclear cells (PBMC) of a human donor and cynomolgus monkey and tonsillar mononuclear cells (TMC) of a human donor .

E34. An isolated antibody that specifically binds to hCXCR5 and competes with an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E33.

E35. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E34, wherein the antibody or antigen-binding fragment thereof binds to human CXCR5 ⁺ B cells with an apparent EC ₅₀ affinity of less than about 26 pM, but does not substantially bind to cells expressing orthologues CXCR5. In some embodiments, the antibody, or antigen-binding fragment thereof, exhibits undetectable binding to cells expressing CXCR5 orthologues, for example, based on any of the assays provided herein; or binds to cells expressing CXCR5 orthologues with an apparent EC ₅₀ affinity at least 10,000 times higher than when bound to hCXCR5.

E36. The antibody or antigennegative fragment according to any one of paragraphs E1-E35, where the antibody or antigennegative fragment contains a CDR-L1 containing the amino acid sequence of SEQ ID NO:2; CDR-L2 containing the amino acid sequence of SEQ ID NO:3; CDR-L3 containing the amino acid sequence of SEQ ID NO:4; CDR-H1 containing the amino acid sequence of SEQ ID NO:7; CDR-H2 containing the amino acid sequence of SEQ ID NO:8; and CDR-H3 containing the amino acid sequence of SEQ ID NO:9.

E37. The antibody or antigennegative fragment according to any one of paragraphs E1-E36, where the antibody or antigennegative fragment contains a CDR-L1 containing the amino acid sequence of SEQ ID NO:2; CDR-L2 containing the amino acid sequence of SEQ ID NO:3; CDR-L3 containing the amino acid sequence of SEQ ID NO:4; CDR-H1 containing the amino acid sequence of SEQ ID NO:7; CDR-H2 containing the amino acid sequence of SEQ ID NO:8; and CDR-H3 containing the amino acid sequence of SEQ ID NO:11.

E38. The antibody or antigennegative fragment according to any one of paragraphs E1-E37, where the antibody or antigennegative fragment contains a CDR-L1 containing the amino acid sequence of SEQ ID NO:14; CDR-L2 containing the amino acid sequence of SEQ ID NO:15; CDR-L3 containing the amino acid sequence of SEQ ID NO:16; CDR-H1 containing the amino acid sequence of SEQ ID NO:19; CDR-H2 containing the amino acid sequence of SEQ ID NO:20; and CDR-H3 containing the amino acid sequence of SEQ ID NO:21.

E39. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E38, wherein the antibody or antigen-binding fragment thereof comprises a VL containing an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1, 5, 13, 35, 37, 48 -51, 39 and 58-62, and a VH containing an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 6, 10, 12, 17, 18, 36, 40, 53-57 and 63.

E40. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E39, wherein the antibody or antigen-binding fragment thereof comprises a VL containing an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1, 5, 13, 35, 37, 48 -51, 39 and 58-62, and a VH containing an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 6, 10, 12, 17, 18, 36, 40, 53-57 and 63.

E41. An antibody or antigen-binding fragment thereof according to any one of claims E1-E40, wherein the antibody or antigen-binding fragment thereof comprises a VL containing an amino acid sequence encoded by an insert of a plasmid deposited with ATCC and having accession number PTA-124324, and a VH containing an amino acid sequence encoded by by inserting a plasmid deposited with ATCC and having accession number PTA-124323.

E42. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35 and E39-E40, wherein the antibody or antigen-binding fragment thereof comprises a VL containing the amino acid sequence of SEQ ID NO:13 and a VH containing the amino acid sequence of SEQ ID NO:17.

E43. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35 and E38-E40, wherein the antibody or antigen-binding fragment thereof comprises a VL containing the amino acid sequence of SEQ ID NO:13 and a VH containing the amino acid sequence of SEQ ID NO:18.

E44. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35 and E38-E40, wherein the antibody or antigen-binding fragment thereof comprises a VL containing the amino acid sequence of SEQ ID NO:37 and a VH containing the amino acid sequence of SEQ ID NO:38.

E45. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E36 and E39-E41, wherein the antibody or antigen-binding fragment thereof comprises a VL containing the amino acid sequence of SEQ ID NO:35 and a VH containing the amino acid sequence of SEQ ID NO:36.

E46. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E36 and E39-E41, wherein the antibody or antigen-binding fragment thereof comprises a VL containing the amino acid sequence of SEQ ID NO:47 and a VH containing the amino acid sequence of SEQ ID NO:52.

E47. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E36 and E39-E41, wherein the antibody or antigen-binding fragment thereof comprises a VL containing the amino acid sequence of SEQ ID NO:5 and a VH containing the amino acid sequence of SEQ ID NO:6.

E48. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E36 and E39-E41, wherein the antibody or antigen-binding fragment thereof comprises a VL containing the amino acid sequence of SEQ ID NO:5 and a VH containing the amino acid sequence of SEQ ID NO:10.

E49. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E36 and E39-E41, wherein the antibody or antigen-binding fragment thereof comprises a VL containing the amino acid sequence of SEQ ID NO:1 and a VH containing the amino acid sequence of SEQ ID NO:6.

E50. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35, E37, and E39-E41, where the antibody or antigen-binding fragment contains a VL containing the amino acid sequence of SEQ ID NO:1, and a VH containing the amino acid sequence of SEQ ID NO:12 .

E51. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35, E37, and E39-E41, wherein the antibody or antigen-binding fragment thereof comprises a VL containing the amino acid sequence of SEQ ID NO:39 and a VH containing the amino acid sequence of SEQ ID NO:40 .

E52. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35, E37, and E39-E41, where the antibody or antigen-binding fragment contains an LC containing the amino acid sequence of SEQ ID NO:22, and a HC containing the amino acid sequence of SEQ ID NO:23 .

E53. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35 and E38-E40, wherein the antibody or antigen-binding fragment thereof comprises an LC containing the amino acid sequence of SEQ ID NO:24 and an HC containing the amino acid sequence of SEQ ID NO:25.

E54. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35, E37, and E39-E41, wherein the antibody or antigen-binding fragment thereof comprises an LC containing the amino acid sequence of SEQ ID NO:26 and an HC containing the amino acid sequence of SEQ ID NO:27 .

E55. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35, E37, and E39-E41, wherein the antibody or antigen-binding fragment thereof comprises an LC containing the amino acid sequence of SEQ ID NO:28 and an HC containing the amino acid sequence of SEQ ID NO:29 .

E56. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35, E37, and E39-E41, wherein the antibody or antigen-binding fragment thereof comprises a VL encoded by the nucleic acid sequence of SEQ ID NO:95 and a VH encoded by the nucleic acid sequence of SEQ ID NO :96.

E57. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E35, E37, and E39-E41, wherein the antibody or antigen-binding fragment thereof comprises an LC encoded by the nucleic acid sequence of SEQ ID NO:97 and an HC encoded by the nucleic acid sequence of SEQ ID NO :98.

E58. An antibody or antigen-binding fragment thereof according to any one of claims E1-E57, wherein the antibody or antigen-binding fragment thereof comprises a VL framework sequence and a VH framework sequence, and wherein one or both of the VL framework sequence or the VH framework sequence are at least 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the human germline sequence from which they are derived, and where the human VL germline sequence from which the framework sequence is derived VL, selected from the group consisting of DPK9, DPK12, DPK18, DPK24, HK102_V1, DPK1, DPK8, DPK3, DPK21, Vg_38K, DPK22, DPK15, DPL16, DPL8, V1-22, Vλ consensus, Vλ1 consensus, Vλ consensus 3, consensus Vκ, Vκ1 consensus, Vκ2 consensus, and Vκ3, and wherein the human germline VH sequence from which the VH framework sequence is derived is selected from the group consisting of DP54, DP47, DP50, DP31, DP46, DP71, DP75, DP10, DP7, DP49 , DP51, DP38, DP79, DP78, DP73, VH3, VH5, VH1 and VH4.

E59. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E58, wherein the antibody or antigen-binding fragment thereof is afucosylated.

E60. An antibody or antigen-binding fragment thereof according to item E59, wherein the antibody or antigen-binding fragment thereof exhibits increased ADCC.

E61. An isolated antibody or antigen-binding fragment thereof, containing the sequence of CDR-H1, CDR-H2 and CDR-H3 VH, containing an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:6, 7, 8, 25, 17, 18, 23, 27, 52, 53, 54, 55, 56, 57 and 63.

E62. An isolated antibody or antigen-binding fragment thereof, containing the sequence of CDR-L1, CDR-L2 and CDR-L3 VL, containing an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1, 2, 13, 22, 24, 26, 47, 48, 49, 50, 51, 58, 59, 60, 61 and 62.

E63. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E62, containing the sequences CDR-H1, CDR-H2 and CDR-H3 shown in the amino acid sequence of SEQ ID NO:6.

E64. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E62, containing the sequences CDR-L1, CDR-L2 and CDR-L3 from the amino acid sequence of SEQ ID NO:1.

E65. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E64, containing one or more of the following amino acid substitutions:

1, 2, 3, 4, 5 or 6 substitutions in CDR-L1 in the corresponding residue of the human germline VL sequence,

1, 2, 3, 4, or 5 substitutions in CDR-L2 at the corresponding residue of the human germline VL sequence,

1, 2, 3, 4, 5, or 6 substitutions in CDR L3 in the corresponding residue of the human germline VL sequence,

1, 2, 3, 4, 5, or 6 substitutions in CDR-H1 at the corresponding residue of the human germline VH sequence,

1, 2, 3, 4, 5, 6, 7, or 8 substitutions in CDR-H2 at the corresponding residue of the human germline VH sequence,

where the human germline VL sequence is selected from the group consisting of DPK9, DPK12, DPK18, DPK24, HK102_V1, DPK1, DPK8, DPK3, DPK21, Vg_38K, DPK22, DPK15, DPL16, DPL8, V1-22, consensus Vκ1, consensus V κ2 and consensus Vκ3, and the human germline VH sequence is selected, consisting of DP54, DP47, DP50, DP31, DP46, DP71, DP75, DP10, DP7, DP49, DP51, DP38, DP79, DP78, DP73, VH3, VH5, VH1, and VH4 .

E66. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E65, comprising a VH framework sequence derived from a human germline VH sequence selected from the group consisting of DP54, DP47, DP50, DP31, DP46, DP71, DP75, DP10, DP7 , DP49, DP51, DP38, DP79, DP78, DP73, VH3, VH5, VH1 and VH4.

E67. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E66, containing a VH framework sequence derived from a human germline VH3 sequence.

E68. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E67, comprising a VH framework sequence derived from a human germline VH sequence selected from the group consisting of DP54, DP47, DP50, DP31, DP46, DP49 and DP51.

E69. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E68, containing a VH framework sequence derived from a human germline VH sequence selected from the group consisting of DP54, DP47, DP50 and DP31.

E70. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E69, containing a VH framework sequence derived from a human germline DP54 sequence.

E71. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E70, comprising a VL framework sequence derived from a human germline VL sequence selected from the group consisting of DPK9, DPK12, DPK18, DPK24, HK102_V1, DPK1, DPK8, DPK3, DPK21 , Vg_38K, DPK22, DPK15, DPL16, DPL8, V1-22, Vκ consensus, Vκ1 consensus, Vκ2 consensus, and Vκ3 consensus.

E72. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E71, comprising a VL framework sequence derived from a human germline VL sequence selected from the group consisting of DPK9, DPK12, DPK18, DPK24, HK102_V1, DPK1, DPK8, DPK3, DPK21 , Vg_38K, DPK22, DPK15, consensus Vκ, consensus Vκ1, consensus Vκ2 and Vκ3.

E73. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E72, containing a VL framework sequence derived from a human germline Vκ1 sequence.

E74. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E73, containing a VL framework sequence derived from a human germline VL sequence selected from the group consisting of DPK9, HK102_V1, DPK1 and DPK8.

E75. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E74, containing a VL framework sequence derived from a human germline DPK9 sequence.

E76. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E75, containing a VL framework sequence and a VH framework sequence, and where one or both of the VL framework sequence or the VH framework sequence are at least 90% identical to the human germline sequence, from which they were received.

E77. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E76, containing a VL framework sequence and a VH framework sequence, and where one or both of the VL framework sequence or the VH framework sequence are at least 66%, 76%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identical to the human germline from which they are derived.

E78. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E77, comprising a VL framework sequence and a VH framework sequence, and wherein one or both of the VL framework sequence or the VH framework sequence are identical to the human germline sequence from which they are derived.

E79. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E78, containing a VH containing an amino acid sequence at least 90% identical to SEQ ID NO:6.

E80. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E79, containing a VH containing an amino acid sequence at least 92% identical to SEQ ID NO:6.

E81. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E80, containing a VH containing the amino acid sequence of SEQ ID NO:6.

E82. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E81, containing a VL containing an amino acid sequence at least 66% identical to SEQ ID NO:1.

E83. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E82, containing a VL containing an amino acid sequence of at least 66%, 76%, 80%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% identical to SEQ ID NO:1.

E84. An antibody or antigen-binding fragment thereof according to any one of paragraphs E61-E83, containing a VL containing the amino acid sequence of SEQ ID NO:1.

E85. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E84 containing an Fc domain.

E86. An antibody or antigen-binding fragment thereof according to item E85, where the Fc domain is the Fc domain of IgA (for example, IgA1 or IgA2), IgD, IgE, IgM or IgG (for example, IgG1, IgG2, IgG3 or IgG4).

E87. An antibody or antigen-binding fragment thereof according to item E86, wherein the Fc domain is an IgG Fc domain.

E88. An antibody or antigen-binding fragment thereof according to item E87, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3, or IgG4.

E89. The antibody or antigen-binding fragment of item E88, where the IgG is IgG1.

E90. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E89, containing a heavy chain containing an amino acid sequence at least 90% identical to SEQ ID NO:29.

E91. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E89, containing a heavy chain containing an amino acid sequence of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29.

E92. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E89, containing a heavy chain containing the amino acid sequence of SEQ ID NO:29.

E93. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E93, containing an LC containing an amino acid sequence at least 90% identical to SEQ ID NO:28.

E94. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E93, containing an LC containing an amino acid sequence of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % identical to SEQ ID NO:28.

E95. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E94, containing an LC containing the amino acid sequence of SEQ ID NO:28.

E96. An isolated antibody or antigen-binding fragment thereof containing a VH sequence encoded by an insert of a plasmid deposited with ATCC and bearing ATCC accession number PTA-124323.

E97. An isolated antibody or antigen-binding fragment thereof, containing the VL sequence encoded by an insert of a plasmid deposited with ATCC and having ATCC registration number PTA-124324.

E98. An antibody or antigen-binding fragment thereof that competes for binding to human CXCR5 with one or more of mouse 11G2, chimeric 11G2, VH h11G2 (XC152)/VL (XC151), VH h11G2 (XC152)/VL (XC153), VH h11G2 (XC152) /VL (XC154), VH h11G2 (XC152) / VL (XC346), VH h11G2 (XC152) / VL (XC347), VH h11G2 (XC152) / VL (XC348), VH h11G2 (XC152) / VL (XC349), VH h11G2 (XC155)/VL (XC151), VH h11G2 (XC155)/VL (XC153), VH h11G2 (XC155)/VL (XC154), VH h11G2 (XC155)/VL (XC346), VH h11G2 (XC155)/ VL (XC347) h11G2 (XC156)/VL (XC154), VH h11G2 (XC156)/VL (XC346), VH h11G2 (XC156)/VL (XC347), VH h11G2 (XC156)/VL (XC348), VH h11G2 (XC156)/VL (XC349) (XC157)/VL (XC347), VH h11G2 (XC157)/VL (XC348), VH h11G2 (XC157)/VL (XC349), VH h11G2 (XC350)/VL (XC151), VH h11G2 (XC350)/VL ( XC153), VH h11G2 (XC350)/VL (XC154), VH h11G2 (XC350)/VL (XC346), VH h11G2 (XC350)/VL (XC347), VH h11G2 (XC350)/VL (XC348), VH h11G2 ( XC350)/VL (XC349), VH h11G2 (XC351)/VL (XC151), VH h11G2 (XC351)/VL (XC153), VH h11G2 (XC351)/VL (XC154), VH h11G2 (XC351)/VL (XC346) ), VH h11G2 (XC351)/VL (XC347), VH h11G2 (XC351)/VL (XC348) and VH h11G2 (XC351)/VL (XC349), VH h11G2 (XC352)/VL (XC151), VH h11G2 (XC352) )/VL (XC153), VH h11G2 (XC352)/VL (XC154), VH h11G2 (XC352)/VL (XC346), VH h11G2 (XC352)/VL (XC347), VH h11G2 (XC352)/VL (XC348) , VH h11G2 (XC352)/VL (XC349), VH h11G2 (XC353)/VL (XC151), VH h11G2 (XC353)/VL (XC153), VH h11G2 (XC353)/VL (XC154), VH h11G2 (XC353) /VL (XC346), VH h11G2 (XC353) / VL (XC347), VH h11G2 (XC353) / VL (XC348), VH h11G2 (XC353) / VL (XC349), VH h11G2 (XC354) / VL (XC151), VH h11G2 (XC354)/VL (XC153), VH h11G2 (XC354)/VL (XC154), VH h11G2 (XC354)/VL (XC346), VH h11G2 (XC354)/VL (XC347), VH h11G2 (XC354)/ VL (XC348), VH h11G2 (XC354)/VL (XC349), 41A10 mouse, chimeric 41A10, VH h41A10 (XC147)/VL (XC142), VH h41A10 (XC147)/VL (XC143), VH h41A10 (XC147)/ VL (XC144) h41A10 (XC148)/VL (XC143), VH h41A10 (XC148)/VL (XC144), VH h41A10 (XC148)/VL (XC145), VH h41A10 (XC148)/VL (XC146), VH h41A10 (XC148)/VL (XC149) (XC150)/VL (XC146), VH h41A10 (XC150)/VL (XC149), mouse 5H7 and chimeric 5H7.

E99. An antibody or antigen-binding fragment thereof that competes for binding to human CXCR5 with CXCL13 and an antibody according to any one of paragraphs E1-E98.

E100. An antibody or antigen-binding fragment thereof according to any one of claims E1-E99, wherein the antibody or antigen-binding fragment is an Fc fusion protein, a monobody, a maxibody, a bifunctional antibody, a scFab, a scFv, a peptibody.

E101. An antibody or antigen-binding fragment thereof according to E1-E100, wherein the antibody or antigen-binding fragment thereof binds to human CXCR5 with a KD approximately equal to or less than a value selected from the group consisting of approximately 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM and 1 pM.

E102. An antibody or antigen-binding fragment thereof according to items E1-E101, wherein the antibody, or antigen-binding fragment thereof, binds to cynomolgus monkey CXCR5 with a KD approximately equal to or less than a value selected from the group consisting of approximately 10 nM, 5 nM, 2 nM , 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 13 pM, 10 pM, 5 pM and 1 pM.

E103. An antibody or antigen-binding fragment thereof according to paragraphs E1-E102, where the KD of binding of the antibody or antigen-binding fragment to cynomolgus monkey CXCR5 is within 1 order of the KD of binding of the antibody or antigen-binding fragment to human CXCR5.

E104. An antibody or antigen-binding fragment thereof according to paragraphs E1-E103, where the ratio of KD binding of the antibody or antigen-binding fragment to human CXCR5 and binding to cynomolgus monkey CXCR5 is from 5:1 to 1:5.

E105. An antibody or antigen-binding fragment thereof according to paragraphs E1-E104, where the ratio of KD binding of the antibody or antigen-binding fragment to human CXCR5 and binding to cynomolgus monkey CXCR5 is from 2:1 to 1:2.

E106. An antibody or antigen-binding fragment thereof according to paragraphs E1-E105, where the ratio of KD binding of the antibody or antigen-binding fragment to cynomolgus monkey CXCR5 and binding to human CXCR5 is in the range, the lower limit of which is selected from the group consisting of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1, 9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4, 4, 4.5 and 6.2, and the upper limit of which is selected from the group consisting of 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2, 1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 3.1, 3.2, 3.3, 3 .4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 6.2 , 9, 9.2 and 10.

E107. An antibody or antigen-binding fragment thereof according to paragraphs E1-E106, where the ratio of KD binding of the antibody or antigen-binding fragment to cynomolgus monkey CXCR5 SEQ ID NO:33 and binding to human CXCR5 SEQ ID NO:32 is from about 1.0 to about 10.0 .

E108. An antibody or antigen-binding fragment thereof according to any one of E1-107, wherein the predicted human half-life is in the range of from about one (1) day to twenty-one (21) days.

E109. An antibody or antigen-binding fragment thereof according to any one of E1-108, wherein the predicted human half-life is approximately seventeen (17) days.

E110. An antibody or antigen-binding fragment thereof, comprising the CDR of an antibody or an antigen-binding fragment thereof, selected from the group consisting of mouse VH 11G2, mouse VL 11G2, chimeric VH 11G2, chimeric VL 11G2, humanized VH 11G2 (XC152), VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353), VH h11G2 (XC354), VL h11G2 (XC151), VL h11G2 ( XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348), VL h11G2 (XC349), VH 41A10 mouse, VL 41A10 mouse, chimeric VH 41A10, chimeric VL 41A10, humanized noah VH 41A10 (XC147) VH h41A10 (XC148) VH h41A10 (XC150) VL h41A10 (XC142) VL h41A10 (XC143) VL h41A10 (XC144) VL h41A10 (XC145) h41A10 (XC149), mouse VH 5H7, mouse VL 5H7, chimeric VH 5H7 and chimeric VL 5H7.

E111. An antibody or an antigen-binding fragment thereof, comprising the VL and VH of an antibody or an antigen-binding fragment thereof, selected from the group consisting of mouse VH 11G2, mouse VL 11G2, VH 11G2 chimeric, VL 11G2 chimeric, VH h11G2 (XC152), VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353), VH h11G2 (XC354), VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348), VL h11G2 (XC349), VH 41A10 mouse, VL 41A10 mouse, chimeric VH 41A10, chimeric VL 41A10, humanized VH 41A10 (XC147), VH h41A10 (XC148), VH h41A10 (XC150), VL h41A10 (XC142), VL h41A10 (XC143), VL h41A10 (XC144), VL h41A10 (XC145), VL h41A10 (XC146) ), VL h41A10 (XC149), mouse VH 5H7, mouse VL 5H7, chimeric VH 5H7 and chimeric VL 5H7.

E112. An antibody, or antigen-binding fragment thereof, selected from the group consisting of

a. an antibody containing mouse 11G2 VH and mouse 11G2 VL;

b. an antibody containing chimeric VH 11G2 and chimeric VL 11G2;

c. an antibody comprising a humanized VH 11G2 (XC152) and a VL selected from the group consisting of mouse VL 11G2, chimeric VL 11G2, VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348) and VL h11G2 (XC349);

d. an antibody comprising a humanized VH 11G2 (XC155) and a VL selected from the group consisting of mouse VL 11G2, chimeric VL 11G2, VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348) and VL h11G2 (XC349);

e. an antibody comprising a humanized VH 11G2 (XC156) and a VL selected from the group consisting of mouse VL 11G2, chimeric VL 11G2, VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348) and VL h11G2 (XC349);

f. an antibody comprising a humanized VH 11G2 (XC157) and a VL selected from the group consisting of mouse VL 11G2, chimeric VL 11G2, VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348) and VL h11G2 (XC349);

g. an antibody comprising a humanized VH 11G2 (XC350) and a VL selected from the group consisting of mouse VL 11G2, chimeric VL 11G2, VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348) and VL h11G2 (XC349);

h. antibody containing VH h11G2 (XC351) and VL selected from the group consisting of VL 11G2 mouse, chimeric VL 11G2, VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348) and VL h11G2 (XC349);

i. an antibody containing VH h11G2 (XC352) and a VL selected from the group consisting of mouse VL 11G2, chimeric VL 11G2, VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348) and VL h11G2 (XC349);

j. antibody containing VH h11G2 (XC353) and VL selected from the group consisting of VL 11G2 mouse, chimeric VL 11G2, VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348) and VL h11G2 (XC349);

k. antibody containing VH h11G2 (XC354) and VL selected from the group consisting of VL 11G2 mouse, chimeric VL 11G2, VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348) and VL h11G2 (XC349);

l. an antibody comprising VL h11G2 (XC151) and a VH selected from the group consisting of mouse VH 11G2, chimeric VH 11G2, VH h11G2 (XC152), VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353) and VH h11G2 (XC354);

m. an antibody comprising VL h11G2 (XC153) and a VH selected from the group consisting of mouse VH 11G2, chimeric VH 11G2, VH h11G2 (XC152), VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353) and VH h11G2 (XC354);

n. an antibody comprising VL h11G2 (XC154) and a VH selected from the group consisting of mouse VH 11G2, chimeric VH 11G2, VH h11G2 (XC152), VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353) and VH h11G2 (XC354);

o. an antibody comprising VL h11G2 (XC346) and a VH selected from the group consisting of mouse VH 11G2, chimeric VH 11G2, VH h11G2 (XC152), VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353) and VH h11G2 (XC354);

p. an antibody comprising VL h11G2 (XC347) and a VH selected from the group consisting of mouse VH 11G2, chimeric VH 11G2, VH h11G2 (XC152), VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353) and VH h11G2 (XC354);

q. an antibody comprising VL h11G2 (XC348) and a VH selected from the group consisting of mouse VH 11G2, chimeric VH 11G2, VH h11G2 (XC152), VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353) and VH h11G2 (XC354);

r. an antibody comprising VL h11G2 (XC349) and a VH selected from the group consisting of mouse VH 11G2, chimeric VH 11G2, VH h11G2 (XC152), VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353) and VH h11G2 (XC354);

s. an antibody comprising a mouse VH 41A10 and a VL selected from the group consisting of mouse VL 41A10, chimeric VL 41A10, VL h41A10 (XC142), VL h41A10 (XC143), VL h41A10 (XC144), VL h41A10 (XC145), VL h41A10 ( XC146) and VL h41A10 (XC149);

t. an antibody comprising a chimeric VH 41A10 and a VL selected from the group consisting of mouse VL 41A10, chimeric VL 41A10, VL h41A10 (XC142), VL h41A10 (XC143), VL h41A10 (XC144), VL h41A10 (XC145), VL h41A10 ( XC146) and VL h41A10 (XC149);

u. an antibody comprising a humanized VH 41A10 (XC147) and a VL selected from the group consisting of mouse VL 41A10, chimeric VL 41A10, VL h41A10 (XC142), VL h41A10 (XC143), VL h41A10 (XC144), VL h41A10 (XC145), VL h41A10 (XC146) and VL h41A10 (XC149);

v. an antibody containing VH h41A10 (XC148) and a VL selected from the group consisting of mouse VL 41A10, chimeric VL 41A10, VL h41A10 (XC142), VL h41A10 (XC143), VL h41A10 (XC144), VL h41A10 (XC145), VL h41A10 (XC146) and VL h41A10 (XC149);

w. VH h41A10 (XC150) and VL selected from the group consisting of mouse VL 41A10, chimeric VL 41A10, VL h41A10 (XC142), VL h41A10 (XC143), VL h41A10 (XC144), VL h41A10 (XC145), VL h41A10 (XC14 6 ), VL h41A10 (XC149) and VL h41A10 (XC142);

x. an antibody comprising mouse VL 41A10 and a VH selected from the group consisting of mouse VH 41A10, chimeric VH 41A10, VH h41A10 (XC147), VL h41A10 (XC148), and VH h41A10 (XC150);

y. an antibody comprising a chimeric VL 41A10 and a VH selected from the group consisting of mouse VH 41A10, chimeric VH 41A10, VH h41A10 (XC147), VL h41A10 (XC148), and VH h41A10 (XC150);

z. an antibody comprising VL h41A10 (XC143) and a VH selected from the group consisting of mouse VH 41A10, chimeric VH 41A10, VH h41A10 (XC147), VL h41A10 (XC148) and VH h41A10 (XC150);

a.a. an antibody comprising VL h41A10 (XC144) and a VH selected from the group consisting of mouse VH 41A10, chimeric VH 41A10, VH h41A10 (XC147), VL h41A10 (XC148) and VH h41A10 (XC150);

b.b. an antibody comprising VL h41A10 (XC145) and a VH selected from the group consisting of mouse VH 41A10, chimeric VH 41A10, VH h41A10 (XC147), VL h41A10 (XC148) and VH h41A10 (XC150);

cc. an antibody comprising VL h41A10 (XC146) and a VH selected from the group consisting of mouse VH 41A10, chimeric VH 41A10, VH h41A10 (XC147), VL h41A10 (XC148) and VH h41A10 (XC150);

dd. an antibody comprising VL h41A10 (XC149) and a VH selected from the group consisting of mouse VH 41A10, chimeric VH 41A10, VH h41A10 (XC147), VL h41A10 (XC148) and VH h41A10 (XC150);

ee. an antibody comprising a mouse 5H7 VH and a VL selected from the group consisting of a mouse 5H7 and a 5H7 chimeric VL;

ff. a mouse 5H7 VL and a VH selected from the group consisting of a mouse 5H7 VH and a chimeric 5H7 VH;

gg. an antibody containing VH h11G2 (XC152) and VL h11G2 (XC151);

h.h. an antibody containing VH h11G2 (XC155) and VL h11G2 (XC153);

ii. an antibody containing VH h11G2 (XC155) and VL h11G2 (XC154);

jj. an antibody containing VH h11G2 (XC156) and VL h11G2 (XC153);

k.k. antibodies containing VH h11G2 (XC157) and h11G2 (XC154);

ll. an antibody containing mouse 11G2 VH and mouse 11G2 VL;

mm. antibodies containing chimeric HC 11G2 and chimeric LC 11G2;

nn. an antibody containing VH h11G2 (XC152) and VL h11G2 (XC151);

oo. an antibody containing VH h11G2 (XC155) and VL h11G2 (XC154);

pp. an antibody containing VH h11G2 (XC156) and VL h11G2 (XC153);

qq. an antibody containing VH h11G2 (XC157) and VL h11G2 (XC154);

rr. an antibody containing mouse VH 41A10 and mouse VL 41A10;

ss. antibodies containing chimeric HC 41A10 and chimeric LC 41A10;

tt. antibodies containing VH h41A10 (XC147) and VL h41A10 (XC142);

uu. antibodies containing VH h41A10 (XC147) and VL h41A10 (XC143);

vv. antibodies containing VH h41A10 (XC147) and VL h41A10 (XC144);

ww. antibodies containing VH h41A10 (XC147) and VL h41A10 (XC145);

xx. antibodies containing VH h41A10 (XC148) and VL h41A10 (XC142);

yy. antibodies containing VH h41A10 (XC148) and VL h41A10 (XC143);

zz. antibodies containing VH h41A10 (XC148) and h41A10VL (XC144);

aaa. an antibody containing mouse VH 5H7 and mouse VL 5H7; And

bbb. an antibody containing chimeric HC 5H7 and chimeric LC 5H7.

E113. An antibody, or antigen-binding fragment thereof, selected from the group consisting of:

(a) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:36 and a VL containing the amino acid sequence of SEQ ID NO:35;

(b) an antibody containing a CDR-H1 containing the sequence of SEQ ID NO:7, CDR-H2 containing the amino acid sequence of SEQ ID NO:8, CDR-H3 containing the amino acid sequence of SEQ ID NO:9, CDR-L1 containing the amino acid the sequence of SEQ ID NO:2, CDR-L2 containing the amino acid sequence of SEQ ID NO:3, and CDR-L3 containing the sequence of SEQ ID NO:4;

(c) an antibody containing a CDR-H1 containing the sequence of SEQ ID:7, CDR-H2 containing the amino acid sequence of SEQ ID NO:8, CDR-H3 containing the amino acid sequence of SEQ ID NO:11, CDR-L1 containing the amino acid sequence SEQ ID NO:2, CDR-L2 containing the amino acid sequence of SEQ ID NO:3, and CDR-L3 containing the sequence of SEQ ID NO:4;

(d) an antibody containing CDR-H1 containing the sequence of SEQ ID:19, CDR-H2 containing the amino acid sequence of SEQ ID NO:20, CDR-H3 containing the amino acid sequence of SEQ ID NO:21, CDR-L1 containing the amino acid sequence SEQ ID NO:14, CDR-L2 containing the amino acid sequence of SEQ ID NO:15, and CDR-L3 containing the sequence of SEQ ID NO:16;

(e) an antibody comprising CDR-H1, CDR-H2, CDR-H3 as shown in the amino acid sequence of SEQ ID NO:6 and CDR-L1, CDR-L2 and CDR-L3 as shown in the amino acid sequence of SEQ ID NO:1 ;

(f) an antibody comprising CDR-H1, CDR-H2, CDR-H3 encoded by the plasmid insert deposited with the ATCC with accession number PTA-124323 and CDR-L1, CDR-L2 and CDR-L3 encoded by the plasmid insert, deposited with the ATCC and bearing registration number PTA-124324;

(g) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:52 (XC152) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:1, 5, 35, 47, 48, 49, 50 and 51;

(h) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:6 (XC155) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:1, 5, 35, 47, 48, 49, 50 and 51;

(i) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:10 (XC156) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:1, 5, 35, 47, 48, 49, 50 and 51;

(j) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:12 (XC157) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:1, 5, 35, 47, 48, 49, 50 and 51;

(k) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:53 (XC350) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:1, 5, 35, 47, 48, 49, 50 and 51;

(l) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:54 (XC351) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:1, 5, 35, 47, 48, 49, 50 and 51;

(m) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:55 (XC352) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:1, 5, 35, 47, 48, 49, 50 and 51;

(n) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:56 (XC353) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:1, 5, 35, 47, 48, 49, 50 and 51;

(o) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:55 (XC354) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:1, 5, 35, 47, 48, 49, 50 and 51;

(p) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:47 (XC151) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, 10, 12, 36, 52, 53, 54 , 55, 56 and 57;

(q) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:5 (XC153) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, 10, 12, 36, 52, 53, 54 , 55, 56 and 57;

(r) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:1 (XC154) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, 10, 12, 36, 52, 53, 54 , 55, 56 and 57;

(s) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:48 (XC346) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, 10, 12, 36, 52, 53, 54 , 55, 56 and 57;

(t) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:49 (XC347) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, 10, 12, 36, 52, 53, 54 , 55, 56 and 57;

(u) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:50 (XC348) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, 10, 12, 36, 52, 53, 54 , 55, 56 and 57;

(v) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:51 (XC349) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, 10, 12, 36, 52, 53, 54 , 55, 56 and 57;

(w) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:38 (VH m41A10) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:13, 37, 58, 59, 60, 61 and 62;

(x) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:17 (XC148), and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:13, 37, 58, 59, 60, 61 and 62 ;

(y) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:18 (XC147) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:13, 37, 58, 59, 60, 61 and 62 ;

(z) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:63 (XC150) and a VL containing an amino acid sequence selected from the group consisting of SEQ ID NO:13, 37, 58, 59, 60, 61 and 62 ;

(aa) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:37 (mouse VL h41A10) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 38, and 63;

(bb) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:13 (VL h41A10 XC142) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 38, and 63;

(cc) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:58 (VL h41A10 XC143) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 38, and 63;

(dd) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:59 (VL h41A10 XC144) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 38, and 63;

(ee) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:60 (VL h41A10 XC145) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 38, and 63;

(ff) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:61 (VL h41A10 XC1446) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 38 and 63;

(gg) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:62 (VL h41A10 XC149) and a VH containing an amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 38 and 63;

(hh) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:40 (VH 5H7 mouse) and a VL containing the amino acid sequence of SEQ ID NO:39;

(ii) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:52 and a VL containing the amino acid sequence of SEQ ID NO:47;

(jj) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:6 and a VL containing the amino acid sequence of SEQ ID NO:5;

(kk) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:6 and a VL containing the amino acid sequence of SEQ ID NO:1;

(ll) (ii) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:10 and a VL containing the amino acid sequence of SEQ ID NO:5;

(mm) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:10 and a VL containing the amino acid sequence of SEQ ID NO:5; And

(nn) an antibody comprising a VH containing the amino acid sequence of SEQ ID NO:12 and a VL containing the amino acid sequence of SEQ ID NO:1.

E114. An antibody or antigen-binding fragment thereof that competes for binding to human CXCR5 with an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E113.

E115. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114.

E116. An isolated nucleic acid molecule comprising at least one nucleic acid sequence encoding an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E109.

E117. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of the sequences shown as SEQ ID NOS: 95, 96, 97 and 98.

E118. An isolated nucleic acid molecule containing the nucleic acid sequence given as SEQ ID NO:95.

E119. An isolated nucleic acid molecule containing the nucleic acid sequence shown as SEQ ID NO:96.

E120. An isolated nucleic acid molecule comprising the nucleic acid sequence given as SEQ ID NO:95 and the nucleic acid sequence given as SEQ ID NO:96.

E121. An isolated nucleic acid molecule containing the nucleic acid sequence shown as SEQ ID NO:97.

E122. An isolated nucleic acid molecule containing the nucleic acid sequence shown as SEQ ID NO:98.

E123. An isolated nucleic acid molecule comprising the nucleic acid sequence given as SEQ ID NO:97 and the nucleic acid sequence given as SEQ ID NO:98.

E124. An isolated nucleic acid molecule containing the nucleic acid sequence of the plasmid insert deposited with the ATCC and assigned accession number PTA-124323.

E125. An isolated nucleic acid molecule containing the nucleic acid sequence of the plasmid insert deposited with the ATCC and assigned accession number PTA-124324.

E126. An isolated nucleic acid encoding a VH, VL, or both of an antibody or antigen-binding fragment thereof that specifically binds to CXCR5, wherein said nucleic acid comprises the nucleic acid sequence of SEQ ID NO:95, the nucleic acid sequence of SEQ ID NO:107, or both , and another.

E127. An isolated nucleic acid encoding a heavy chain, a light chain, or both of an antibody or antigen-binding fragment thereof that specifically binds to CXCR5, wherein said nucleic acid comprises the nucleic acid sequence of SEQ ID NO:97, the nucleic acid sequence of SEQ ID NO:98, or both one and the other.

E128. An isolated nucleic acid encoding the VH of an antibody or antigen-binding fragment thereof that specifically binds to CXCR5, wherein said nucleic acid contains the nucleic acid sequence of an insert of a plasmid deposited with ATCC and has accession number PTA-124323.

E129. An isolated nucleic acid encoding the VL of an antibody or antigen-binding fragment thereof that specifically binds to CXCR5, wherein said nucleic acid contains the nucleic acid sequence of an insert of a plasmid deposited with ATCC and has accession number PTA-124324.

E130. An isolated nucleic acid encoding the VL and VH of an antibody or antigen-binding fragment thereof that specifically binds to CXCR5, wherein said nucleic acid contains the nucleic acid sequence of the plasmid insert deposited with ATCC and has accession number PTA-124324, and the nucleic acid sequence of the plasmid insert deposited with ATCC and has registration number PTA-124323.

E131. A vector containing a nucleic acid molecule according to any one of paragraphs E115-E130.

E132. A host cell containing a nucleic acid molecule according to any one of paragraphs E115-E130 or a vector according to paragraph E131.

E133. The host cell of claim E132, wherein said cell is a mammalian cell.

E134. The host cell of claim E133, wherein said host cell is a CHO cell, a COS cell, a HEK-293 cell, an NS0 cell, a PER.C6® cell, or an Sp2.0 cell.

E135. A host cell according to any one of claims E132-134, wherein said host cell lacks a functional alpha-1,6-fucosyltransferase (FUT8).

E136. A host cell according to any one of claims E132-135, wherein said cell does not express a functional alpha-1,6-fucosyltransferase enzyme.

E137. A host cell according to any one of claims E132-136, wherein said cell lacks the FUT8 gene encoding a functional enzyme.

E138. A host cell according to any one of claims E132-137, wherein said cell lacks a gene encoding a functional FUT8 gene.

E139. A host cell according to any one of claims E132-139, wherein said cell is a Potelligent® CHOK1SV cell or a Lec13 CHO cell.

E140. The host cell of claim E139, wherein said cell is a Potelligent® CHOK1SV cell.

E141. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing a host cell according to any one of paragraphs E1-E114 under conditions in which said antibody or antigen-binding fragment is expressed by said host cell.

E142. The method of claim E141, further comprising isolating said antibody or antigen-binding fragment thereof.

E143. A method for producing an afucosylated anti-CXCR5 antibody or antigen-binding fragment thereof, comprising culturing a host cell containing a nucleic acid molecule according to any one of paragraphs E115-E130 or a vector according to paragraph E131, where functional FUT8 is absent in said host cell.

E144. The method of claim E143, wherein the host cell is a Potelligent® CHOK1SV cell.

E145. An isolated antibody, or antigen-binding fragment thereof, obtainable by the method of any one of paragraphs E141-E114.

E146. The isolated antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114, wherein said antibody or antigen-binding fragment thereof is afucosylated.

E147. An afucosylated antibody or antigen-binding fragment thereof according to E146, wherein said afucosylated antibody or antigen-binding fragment thereof exhibits increased antibody-dependent cellular cytotoxicity (ADCC) compared to an otherwise identical antibody or antigen-binding fragment thereof that is fucosylated.

E148. An afucosylated antibody or antigen-binding fragment thereof according to E146, wherein said afucosylated antibody or antigen-binding fragment thereof exhibits approximately 2-fold, approximately 5-fold, approximately 7-fold, approximately 10-fold, approximately 20-fold, approximately 30-fold, approximately 40-fold, approximately 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 110 times, about 120 times, about 130 times, about 140 times, and about 143 times higher ADCC compared to the rest an identical antibody or antigen-binding fragment that is fucosylated.

E149. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 and a pharmaceutically acceptable carrier or excipient.

E150. The pharmaceutical composition of claim E149, wherein said antibody or antigen-binding fragment thereof is afucosylated.

E151. A method for reducing CXCR5 activity, comprising administering to an individual in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150.

E152. A method for treating an inflammatory disease, comprising administering to an individual in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149-E150.

E153. A method of treating an individual in need of immunosuppression, comprising administering to the individual in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150.

E154. A method for treating an autoimmune disease, disorder or condition, comprising administering to an individual in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150.

E155. A method for reducing the number of cells expressing CXCR5 in a patient, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150.

E156. The method of claim E155, wherein the cells express CXCR5 on their surface.

E157. The method of claim E156, wherein the cells are B cells and Tfh-like cells.

E158. The method according to any one of claims E151-E157, wherein said individual is a human.

E159. The method according to any one of paragraphs E151-E158, including intravenous administration of the specified antibody or its antigen-binding fragment or pharmaceutical composition.

E160. The method according to any one of paragraphs E151-E158, including subcutaneous administration of the specified antibody or its antigen-binding fragment or pharmaceutical composition.

E161. The method according to any one of claims E151-E160, wherein said antibody or antigen-binding fragment or pharmaceutical composition is administered approximately twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months , once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, one once every eleven months or once every twelve months.

E162. A method for reducing the number of CXCR5 ⁺ cells in a sample, comprising contacting said cell with an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150.

E163. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150 for use as a medicine.

E164. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150 for use in reducing the activity of CXCR5 in an individual.

E165. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150 for use in the treatment of an individual in need of immunosuppression.

E166. An antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150 for use in the treatment of an autoimmune disease, disorder or condition in an individual.

E167. The use of an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 in the manufacture of a medicament for the treatment of an immunological disease, disorder or condition.

E168. The use of a pharmaceutical composition according to any one of claims E149 and E150 in the manufacture of a medicament for the treatment of an immunological disease, disorder or condition.

E169. A method for treating a medical condition, comprising administering to an individual in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150.

E170. The method according to item E169, where the condition is selected from the group consisting of inflammatory responses, such as inflammatory diseases of the skin, including psoriasis and dermatitis (eg, atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; gastritis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions including T cell infiltration and chronic inflammatory responses; atherosclerosis; lack of adhesion of leukocytes; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes mellitus (eg type I diabetes mellitus or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and immune responses associated with acute and delayed hypersensitivity reactions mediated by cytokines and T-lymphocytes, usually found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; Wegener's granulomatosis; pernicious anemia (Addison's disease); diseases, including diapedesis of leukocytes; inflammatory disorders of the central nervous system (CNS); multiple organ failure syndrome; hemolytic anemia (including, but not limited to, cryoglobulinemia or autoimmune hemolytic anemia with incomplete heat agglutinins); myasthenia gravis; diseases mediated by antigen-antibody complexes; anti-BMC antibody disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; myasthenic Lambert-Eaton syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; vitiligo; Reiter's disease; muscle stiffness syndrome; Behçet's disease; giant cell arteritis; immunocomplex nephritis; IgA nephropathy; IgM polyneuropathies; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic diseases; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveoretinitis; Guillain-Barré syndrome; Goodpasture's syndrome; mixed connective tissue disease; autoimmune-associated infertility; nodous polyarteritis; alopecia areata; idiopathic myxedema; graft-versus-host reactions; muscular dystrophy (Duchenne's dystrophy, Becker's dystrophy, myotonic dystrophy, limb girdle dystrophy, shoulder-blade-facial dystrophy, congenital dystrophy, oculopharyngeal dystrophy, distal dystrophy, Emery-Dreyfus dystrophy) and control of proliferation of malignant cells expressing CXCR5, such as cancer pancreatic, colon cancer, bladder cancer, T-cell leukemia and B-cell leukemia.

E171. A method for detecting CXCR5 in a sample, tissue, or cell using an antibody or antigen-binding fragment thereof according to any one of paragraphs E1-E114 and E145-E148 or a pharmaceutical composition according to any one of paragraphs E149 and E150, which includes bringing the sample, tissue or cell into contact with antibody and detection antibody.

E172. An isolated antibody or antigen-binding fragment thereof that specifically binds to CXCR5, wherein the antibody is at least one antibody selected from the group consisting of:

(a) an antibody containing a CDR-L1 containing the amino acid sequence of SEQ ID NO:2; CDR-L2 containing the amino acid sequence of SEQ ID NO:3; CDR-L3 containing the amino acid sequence of SEQ ID NO:4; CDR-H1 containing the amino acid sequence of SEQ ID NO:7; CDR-H2 containing the amino acid sequence of SEQ ID NO:8; and CDR-H3 containing the amino acid sequence of SEQ ID NO:9;

(b) an antibody containing a CDR-L1 containing the amino acid sequence of SEQ ID NO:2; CDR-L2 containing the amino acid sequence of SEQ ID NO:3; CDR-L3 containing the amino acid sequence of SEQ ID NO:4; CDR-H1 containing the amino acid sequence of SEQ ID NO:7; CDR-H2 containing the amino acid sequence of SEQ ID NO:8; and CDR-H3 containing the amino acid sequence of SEQ ID NO:11;

(c) an antibody containing a CDR-L1 containing the amino acid sequence of SEQ ID NO:14; CDR-L2 containing the amino acid sequence of SEQ ID NO:15; CDR-L3 containing the amino acid sequence of SEQ ID NO:16; CDR-H1 containing the amino acid sequence of SEQ ID NO:19; CDR-H2 containing the amino acid sequence of SEQ ID NO:20; and CDR-H3 containing the amino acid sequence of SEQ ID NO:21;

(d) an antibody comprising a VL containing the amino acid sequence encoded by the plasmid insert deposited with the ATCC, accession number PTA-124324, and a VH containing the amino acid sequence encoded by the plasmid insert deposited with the ATCC, accession number PTA-124323;

(e) an antibody containing a VL containing the amino acid sequence of SEQ ID NO:1 and a VH containing the amino acid sequence of SEQ ID NO:6;

(f) an antibody containing a VL containing the amino acid sequence of SEQ ID NO:13 and a VH containing the amino acid sequence of SEQ ID NO:18;

(g) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:47 and a VH containing the amino acid sequence of SEQ ID NO:52;

(h) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:5 and a VH containing the amino acid sequence of SEQ ID NO:6;

(i) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:5 and a VH containing the amino acid sequence of SEQ ID NO:10;

(j) an antibody containing a VL containing the amino acid sequence of SEQ ID NO:13 and a VH containing the amino acid sequence of SEQ ID NO:17;

(k) an antibody comprising a VL containing the amino acid sequence of SEQ ID NO:1 and a VH containing the amino acid sequence of SEQ ID NO:12;

(l) an antibody containing an LC containing the amino acid sequence of SEQ ID NO:22 and an HC containing the amino acid sequence of SEQ ID NO:23;

(m) an antibody comprising an LC containing the amino acid sequence of SEQ ID NO:24 and an HC containing the amino acid sequence of SEQ ID NO:25;

(n) an antibody comprising an LC containing the amino acid sequence of SEQ ID NO:26 and an HC containing the amino acid sequence of SEQ ID NO:27;

(o) an antibody comprising an LC containing the amino acid sequence of SEQ ID NO:28 and an HC containing the amino acid sequence of SEQ ID NO:29;

(p) an antibody comprising a VL encoded by the nucleic acid sequence of SEQ ID NO:95 and a VH encoded by the nucleic acid sequence of SEQ ID NO:96; And

(q) an antibody containing an LC encoded by the nucleic acid sequence of SEQ ID NO:97 and an HC encoded by the nucleic acid sequence of SEQ ID NO:98.

E173. An isolated antibody or antigen-binding fragment thereof that specifically binds to the C-X-C chemokine receptor 5 (CXCR5), wherein the antibody or antigen-binding fragment thereof is at least one antibody selected from the group consisting of:

(a) an antibody or antigen-binding fragment thereof that binds to an hCXCR5 epitope containing leucine at amino acid residue position 11 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to said epitope if said residue is not leucine;

(b) an antibody or antigen-binding fragment thereof that binds to an hCXCR5 epitope containing leucine at amino acid residue position 11 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to said epitope if said residue is threonine;

(c) an antibody or antigen-binding fragment thereof that binds to an hCXCR5 epitope containing aspartate at amino acid residue position 22 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to said epitope if said residue is not aspartate;

(d) an antibody or antigen-binding fragment thereof that binds to an hCXCR5 epitope containing aspartate at amino acid residue position 22 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to said epitope if said residue is an alanine;

(e) an antibody or antigen-binding fragment thereof that binds to an hCXCR5 epitope containing leucine at amino acid residue position 11 and aspartate at amino acid residue position 22 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to said epitope if said leucine substituted with threonine and/or said aspartate is substituted with alanine;

(f) an antibody or antigen-binding fragment thereof that binds to hCXCR5 or a fragment thereof containing leucine at amino acid residue position 11 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to hCXCR5 or a fragment thereof, if said residue is not leucine ;

(g) an antibody or antigen-binding fragment thereof that binds to hCXCR5 or a fragment thereof containing leucine at amino acid residue position 11 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to hCXCR5 or a fragment thereof if said residue is threonine;

(h) an antibody or antigen-binding fragment thereof that binds to hCXCR5 or a fragment thereof containing aspartate at amino acid residue position 22 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to hCXCR5 or a fragment thereof, if said residue is not an aspartate ;

(i) an antibody or antigen-binding fragment thereof that binds to hCXCR5 or a fragment thereof containing aspartate at amino acid residue position 22 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to hCXCR5 or a fragment thereof, if said residue is an alanine; And

(j) an antibody or antigen-binding fragment thereof that binds to hCXCR5 or a fragment thereof containing leucine at amino acid residue position 11 and aspartate at amino acid residue position 22 according to the amino acid sequence numbering of SEQ ID NO:32, but does not bind to hCXCR5 or a fragment thereof when said leucine is substituted with threonine and/or said aspartate is substituted with alanine.

E174. An isolated antibody or antigen-binding fragment thereof that specifically binds to the C-X-C chemokine receptor 5 (CXCR5), wherein the antibody or antigen-binding fragment thereof is at least one antibody selected from the group consisting of:

(a) an antibody or antigen-binding fragment thereof that binds to hCXCR5 expressed on human B cells with an apparent affinity EC _{50 of} approximately 6.60 pM with a standard deviation of approximately ±2.33 pM;

(b) an antibody or antigen-binding fragment thereof that binds to hCXCR5 expressed on circulating human follicular T-helper-like cells with an apparent affinity of EC _{50 of} approximately 5.89 pM with a standard deviation of approximately ±1.40 pM;

(c) an antibody or antigen-binding fragment thereof that binds to hCXCR5 expressed on human follicular T helper (Tfh) cells with an apparent affinity of EC ₅₀ of approximately 10.6 pM;

(d) an antibody or antigen-binding fragment thereof that binds to cynoCXCR5 expressed on cynomolgus monkey B cells with an apparent affinity of EC ₅₀ of approximately 1.32 pM;

(e) an antibody or antigen-binding fragment thereof that binds to cynoCXCR5 expressed on Tfh-like cynomolgus monkey cells with an apparent affinity of EC ₅₀ of approximately 10.5 pM;

(f) an antibody or antigen-binding fragment thereof that antagonizes CXCR5-CXCL13 signaling in a cAMP reporter assay with an EC ₅₀ of approximately 961 pM;

(g) an antibody or antigen-binding fragment thereof exhibiting ADCC activity against human B cells expressing hCXCR5 with an EC ₅₀ of approximately 2.01 pM with a standard deviation of approximately ±2.28 pM;

(h) an antibody or antigen-binding fragment thereof exhibiting ADCC activity against human Tfh-like cells expressing hCXCR5 with an EC _{50 of} approximately 4.28 pM with a standard deviation of approximately ±2.88 pM;

(i) an antibody or antigen-binding fragment thereof exhibiting ADCC activity against human Tfh cells expressing hCXCR5 with an EC ₅₀ of approximately 0.11 pM;

(j) an antibody, or antigen-binding fragment thereof, exhibiting ADCC activity against cynoCXCR5 expressing cynoCXCR5-expressing cynomolgus monkey B cells with an EC _{50 of} approximately 15.3 pM with a standard deviation of approximately ±11.7 pM;

(k) an antibody or antigen-binding fragment thereof that binds to hCXCR5 but does not bind detectably to the human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3 , CXCR4, CXCR6, CXCR7 and XCR1;

(l) an antibody or antigen-binding fragment thereof that inhibits the binding of CXCR5 to CXCL13.

(m) an antibody or antigen-binding fragment thereof that binds to human CXCR5 ⁺ B cells with an apparent EC ₅₀ affinity of less than about 26 pM, but does not bind to cells expressing mouse, rat, or rabbit CXCR5 orthologues;

(n) an antibody or antigen-binding fragment thereof that antagonizes CXCL13 inhibition of cAMP release triggered by forskolin;

(o) an antibody or antigen-binding fragment thereof that triggers ADCC of CXCR5-expressing cells in human donor and cynomolgus monkey PBMC and human donor TMC;

(p) an antibody or antigen-binding fragment thereof that binds to human CXCR5 but does not bind to the human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3 , CXCR4, CXCR6, CXCR7 or XCR1;

(q) an antibody or antigen-binding fragment thereof that depletes B cells in peripheral blood;

(r) an antibody or antigen-binding fragment thereof that depletes Tfh-like cells in peripheral blood;

(s) an antibody or antigen-binding fragment thereof that depletes true Tfh cells in the spleen; And

(t) an antibody or antigen-binding fragment thereof that interferes with the humoral anamnestic immune response.

E175. An antibody according to any one of claims E172-E174, wherein said antibody, or antigen-binding fragment thereof, exhibits at least one of the following biological activities:

(a) binds to human CXCR5 ⁺ B cells with an apparent EC ₅₀ affinity of less than about 26 pM, but does not bind to cells expressing mouse, rat, or rabbit CXCR5 orthologues;

(b) antagonistically acts on CXCL13 inhibition of cAMP release triggered by forskolin;

(c) triggers ADCC of CXCR5-expressing cells in human-donor and cynomolgus monkey PBMCs and human-donor TMCs;

(d) binds to human CXCR5 but does not bind to human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 or XCR1;

(e) depletes B cells in peripheral blood;

(f) depletes Tfh-like cells in peripheral blood;

(g) depletes true Tfh cells in the spleen; or

(h) impairs the humoral anamnestic immune response.

E176. An antibody according to any one of claims E172-E175, wherein said antibody or antigen-binding fragment thereof is afucosylated.

E177. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of paragraphs E172-E176.

E178. An isolated nucleic acid encoding the VH, VL, or both of an antibody or an antigen-binding fragment thereof that specifically binds to CXCR5, wherein said nucleic acid comprises the nucleic acid sequence of SEQ ID NO:95, the nucleic acid sequence of SEQ ID NO:96, or both , and another.

E179. An isolated nucleic acid encoding a heavy chain, a light chain, or both of an antibody or an antigen-binding fragment thereof that specifically binds to CXCR5, wherein said nucleic acid comprises the nucleic acid sequence of SEQ ID NO:97, the nucleic acid sequence of SEQ ID NO:98, or both one and the other.

E180. An isolated nucleic acid encoding a VH, VL, or both of an antibody or an antigen-binding fragment thereof that specifically binds to CXCR5, wherein said nucleic acid contains the nucleic acid sequence of an insert of a plasmid deposited with ATCC and has accession number PTA-124323, a nucleic acid sequence inserting a plasmid deposited with ATCC and having accession number PTA-124324, or both.

E181. A vector containing a nucleic acid according to any one of paragraphs E177-E180.

E182. A host cell containing the vector according to item E181.

E183. The host cell of claim E182, wherein said host cell is a mammalian cell selected from the group consisting of a CHO cell, COS cell, HEK-293 cell, NS0 cell, PER.C6® cell, or Sp2.0 cell.

E184. A host cell according to item E183, wherein said cell lacks a functional alpha-1,6-fucosyltransferase (FUT8).

E185. The host cell of claim E184, wherein said cell is a Potelligent® CHOK1SV cell or a Lec13 CHO cell.

E186. A method for producing an antibody or antigen-binding fragment thereof, comprising using a Potelligent® CHOK1SV cell according to item E185 under conditions in which said antibody or antigen-binding fragment thereof is expressed by said host cell and is afucosylated.

E187. The method of claim E186, further comprising isolating said antibody or antigen-binding fragment thereof.

E188. An afucosylated antibody or antigen-binding fragment thereof according to E186, wherein said antibody exhibits increased ADCC activity compared to an otherwise identical antibody or antigen-binding fragment thereof that is fucosylated.

E189. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of paragraphs E172-E176 and E188 and a pharmaceutically acceptable carrier or excipient.

E190. An antibody or antigen-binding fragment thereof according to any one of paragraphs E172-E176, E188 and a pharmaceutical composition according to paragraph E189 for use in the treatment of an immunological disease, disorder or condition.

E191. The use of an antibody or antigen-binding fragment thereof according to any one of paragraphs E172-E176, E188 or a pharmaceutical composition according to paragraph E189 for the treatment of an immunological disease, disorder or condition.

E192. A method of treating or preventing an immunological disease, disorder or condition mediated by CXCR5 in a patient, comprising administering to the individual an effective amount of the pharmaceutical composition according to item E189, where the specified disease, disorder or condition is selected from the group consisting of inflammatory responses, such as inflammatory diseases of the skin including psoriasis and dermatitis (eg, atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; gastritis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions including T cell infiltration and chronic inflammatory responses; atherosclerosis; lack of adhesion of leukocytes; rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); diabetes mellitus (eg type I diabetes mellitus or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and immune responses associated with acute and delayed hypersensitivity reactions mediated by cytokines and T-lymphocytes, usually found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; Wegener's granulomatosis; pernicious anemia (Addison's disease); diseases, including diapedesis of leukocytes; inflammatory disorders of the central nervous system (CNS); multiple organ failure syndrome; hemolytic anemia (including, but not limited to, cryoglobulinemia or autoimmune hemolytic anemia with incomplete heat agglutinins); myasthenia gravis; diseases mediated by antigen-antibody complexes; anti-BMC antibody disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; myasthenic Lambert-Eaton syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; vitiligo; Reiter's disease; muscle stiffness syndrome; Behçet's disease; giant cell arteritis; immunocomplex nephritis; IgA nephropathy; IgM polyneuropathies; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic diseases; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveoretinitis; Guillain-Barré syndrome; Goodpasture's syndrome; mixed connective tissue disease; autoimmune-associated infertility; nodous polyarteritis; alopecia areata; idiopathic myxedema; graft-versus-host reactions; muscular dystrophy (Duchenne's dystrophy, Becker's dystrophy, myotonic dystrophy, limb girdle dystrophy, shoulder-blade-facial dystrophy, congenital dystrophy, oculopharyngeal dystrophy, distal dystrophy, Emery-Dreyfus dystrophy) and control of proliferation of malignant cells expressing CXCR5, such as cancer pancreatic, colon cancer, bladder cancer, T-cell leukemia and B-cell leukemia.

E193. The method of claim E192, wherein said disease is SLE or rheumatoid arthritis.

E194. The use of an antibody or antigen-binding fragment thereof according to any one of claims E172-E176 or E188 in the manufacture of a medicament for the treatment of an immunological disease, disorder or condition.

E195. A method for detecting CXCR5 in a sample, tissue, or cell using an antibody or antigen-binding fragment thereof according to any one of paragraphs E172-E176, comprising contacting the sample, tissue, or cell with an antibody and a detection antibody.

E196. A method for reducing the biological activity of CXCR5 in a patient, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of paragraphs E172-E176 or E188 or a pharmaceutical composition according to paragraph E189.

E197. The method of claim E196, wherein the antibody mediates depletion of at least one CXCR5 expressing cell selected from the group consisting of a Tfh cell in the spleen, a B cell in the peripheral blood, and a Tfh-like cell in the peripheral blood.

E198. A method for inhibiting a humoral immune response in a patient, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of paragraphs E172-E176 or E188 or a pharmaceutical composition according to paragraph E189.

E199. The method of claim E198, wherein the antibody mediates depletion of at least one CXCR5 expressing cell selected from the group consisting of a Tfh cell in the spleen, a B cell in the peripheral blood, and a Tfh-like cell in the peripheral blood.

BRIEF DESCRIPTION OF THE DRAWINGS

[00014] The summary of the invention described above, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. For illustrative purposes, embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the specific embodiments and devices presented.

[00015] FIG. 1 is a diagram showing the canonical bianthenary glycoform typically present in immunoglobulin heavy chain constant regions. The diagram shows an asparagine-linked (N-linked) glycoform on the IgG constant domain at amino acid residue position 297 (asparagine 297; Asn297 ) according to Eu numbering according to Edelman et al., 1969, Proc. Natl. Acad. sci. USA 63(1):78-85 as described in Kabat et al., 1991.

[00016] FIG. 2A-2G are diagrams showing examples of bianthenary glycoforms typically present in immunoglobulin heavy chain constant regions. The designation "G0" refers to a bianthenary structure where there are no terminal sialic acids (NeuAc) or Gal, the designation "G1" refers to a bianthenary structure containing one Gal and no NeuAc, and the designation "G2" refers to a bianthenary structure with two terminal Gal and without NeuAc. In each glycoform, fucose is typically present in antibodies produced in mammalian cells such as CHO cells. In FIG. 2A shows G2S2, FIG. 2B shows G2S1, FIG. 2C shows G2, FIG. 2D shows G1, FIG. 2E shows G0, FIG. 2F shows G -1 and FIG. 2G shows G-2.

[00017] FIG. 3 is a bar graph showing the binding of various monoclonal antibodies (indicated on the x-axis) to HEK 293 cells expressing human CXCR5 (hCXCR5 293) (lighter bars) and/or HEK 293 cells expressing cynomolgus monkey CXCR5 (cynoCXCR5 293). ) (darker bars). The monoclonal antibodies shown in Fig. 3 are, from left to right, 11B9, 18C7, 21A3, 23F1, 30C3, 31F3, 39H4, 47D11, 51D11, 52E7, 56A9, 56G2, 57H5, 58C12, 59F3, 4F1, 6E9, 12E11, 16 D10, 18G4, 19D9, 19F8, 20G4, 26E2, 41A10, 48A10, 13F4, 17D5, 18H9, 23C6, 1A11, 1D10, 1C11, 3A1, 5H3, 6D6, 9G11, 10G2, 12C1, 12C3, 15A11, 1 5C10, 15D5, 18A11, 19C11, 20D10, 21B2, 21G3, 25D4, 25E1, 5H7, 11G2, 18B11, 23D5, 23F7, 31E5, 19G2 and 31G7.

[00018] FIG. 4a is a diagram showing the absence of detectable binding of various anti-CXCR5 antibodies to cells expressing CXCR1.

[00019] FIG. 4B is a graph showing the lack of binding of various anti-CXCR5 antibodies to cells expressing CXCR2.

[00020] FIG. 4C is a graph showing the lack of binding of various anti-CXCR5 antibodies to cells expressing CXCR3.

[00021] FIG. 4D is a diagram showing the binding of various anti-CXCR5 antibodies to Jurkat cells naturally expressing CXCR4.

[00022] FIG. 5 is a graph showing the binding affinity for CXCR5-expressing antibody cells: afucosylated h11G2 XC154/XC155 (open circles), fucosylated h11G2 XC154/XC155 (solid circles), 2C9 (solid triangles), 16D7 (solid squares), and 11A7 ( shaded ovals).

[00023] FIG. 6 shows the amino acid sequence alignment of human and mouse CXCR5. The figure also shows the various extracellular domains of CXCR5 labeled "N", "L1", "L2" and "L3" which are underlined.

[00024] FIG. 7 is a bar graph showing that certain amino acid residues were critical for antibody binding to hCXCR5. Those. altering D10G or inserting SI into the human sequence abolished the binding of the 2C9 antibody, but did not affect the binding of the other three antibodies. More importantly, L11T or D22A substitution abolished 11G2 binding but did not affect 2C9, 16D7 or 11A7 binding. The substitution of W for K at amino acid residue 19 (W19K) abolished the binding of antibodies 16D7 and 11A7, but did not affect the binding of 11G2 and 2C9. These data indicate that these four antibodies do not share a common epitope on hCXCR5.

[00025] FIG. 8A is a graph showing the depletion and recovery of peripheral blood B cells in male cynomolgus monkeys. Peripheral blood B cell counts per µl blood in males up to day 352 of the exploratory toxicity study are shown. B cells were defined as CD3 ^- CD20 ⁺ . Data for individual animals are presented. The archival range of B cells in male monkeys (272-2503 cells per µl) is indicated by dotted lines.

[00026] FIG. 8B is a graph showing the depletion and recovery of peripheral blood B cells in female cynomolgus monkeys. Peripheral blood B cell counts per µl of blood in females up to day 352 of the exploratory toxicity study are shown. B cells were defined as CD3 ^- CD20 ⁺ . Data for individual animals are presented. Archival ranges of B cells in female monkeys (233-1700 cells per µl) are indicated by dotted lines.

[00027] FIG. 8C is a graph showing depletion and recovery of Tfh-like (also referred to as "cTfh" or "circulating Tfh") cells in male cynomolgus monkeys. Numbers of Tfh-like cells in peripheral blood per µl of male blood up to day 352 of the exploratory toxicity study are shown. Tfh-like cells were defined as the sum of CD3 ⁺ CD4 ⁺ CD95 ⁺ CXCR5 ⁺ cells and CD3 ⁺ CD4 ⁺ CD95 ⁺ hIgG ⁺ cells, since the test drug interferes with the anti-CXCR5 antibody used for immunophenotyping. Data for individual animals are presented.

[00028] FIG. 8D is a graph showing the depletion and recovery of Tfh-like (also referred to as "cTfh" or "circulating Tfh") cells in female cynomolgus monkeys. The numbers of Tfh-like cells in peripheral blood per µl of blood in females up to day 352 of the exploratory toxicity study are shown. Tfh-like cells were defined as the sum of CD3 ⁺ CD4 ⁺ CD95 ⁺ CXCR5 ⁺ cells and CD3 ⁺ CD4 ⁺ CD95 ⁺ hIgG ⁺ cells, since the test drug interferes with the anti-CXCR5 antibody used for immunophenotyping. Data for individual animals are presented.

[00029] FIG. 9A is a graph showing the depletion and recovery of peripheral blood B cells in a cynomolgus monkey. Peripheral blood B cell counts per µl of monkey blood up to day 393 of the GLP pivotal toxicity study are shown. B cells were defined as CD3 ^- CD20 ⁺ HLA-DR ⁺ cells. Group means (males and females combined) ± standard deviation are shown.

[00030] FIG. 9B is a graph showing the depletion and recovery of CXCR5 ⁺ peripheral blood B cells in cynomolgus monkeys. Numbers of CXCR5 ⁺ peripheral blood B cells per µl blood in monkeys up to day 393 of the GLP pivotal toxicity study are shown. B cells were defined as CD3 ^- CD20 ⁺ HLA-DR ⁺ cells. Group means (males and females combined) ± standard deviation are shown.

[00031] FIG. 9C is a graph showing the depletion and recovery of circulating peripheral blood follicular T helper cells (cTfh; also referred to elsewhere herein as "Tfh-like cells") in cynomolgus monkeys. The numbers of peripheral blood cTfh cells per µl of monkey blood up to day 393 of the GLP pivotal toxicity study are shown. cTfh cells were defined as CD3 ⁺ CD4 ⁺ CD95 ⁺ cells. Group means (males and females combined) ± standard deviation are shown.

[00032] FIG. 9D is a graph showing the depletion and recovery of peripheral blood CXCR5 ⁺ cTfh cells (also referred to elsewhere herein as "Tfh-like cells") with detectable surface CXCR5 in cynomolgus monkeys. Numbers of CXCR5 ⁺ cTfh peripheral blood cells per µl blood in monkeys up to day 393 of the GLP-compliant pivotal toxicity study are shown. cTfh cells were defined as CD3 ⁺ CD4 ⁺ CD95 ⁺ cells. Group means (males and females combined) ± standard deviation are shown.

DETAILED DESCRIPTION

[00033] The present invention relates to antibodies that specifically bind to CXCR5, and, in addition, antibodies that antagonize the activity of CXCR5 or its interaction with CXCL13. The present invention relates to methods for producing antibodies against CXCR5, compositions containing these antibodies, and methods of using these antibodies.

[00034] The present invention relates to fucosylated and afucosylated antibodies that bind to CXCR5. In some embodiments, the present invention also provides afucosylated antibody heavy chains and light chains capable of generating antibodies that bind to CXCR5. In some embodiments, the present invention relates to afucosylated antibodies, heavy chains and light chains, containing one or more specific complementarity determining regions (CDRs). In some embodiments, afucosylated anti-CXCR5 antibodies have an altered effector function. In some embodiments, antibodies of the invention have increased ADCC activity relative to otherwise identical fucosylated anti-CXCR5 antibodies of the invention.

[00035] The present invention relates to polynucleotides encoding antibodies that bind to CXCR5, or antigen-binding fragments thereof. The present invention also relates to polynucleotides encoding heavy chains or light chains of an antibody. The present invention relates to host cells expressing fucosylated and/or afucosylated anti-CXCR5 antibodies. The present invention relates to methods of treatment using afucosylated and fucosylated anti-CXCR5 antibodies. Such methods include, but are not limited to, methods for treating a disease associated with or mediated by CXCR5 expression and/or binding to CXCL13, including, but not limited to, inflammatory and immunological diseases.

[00036] The section headings used in this specification are for organizational purposes only and should not be construed as limiting the present invention.

[00037] All references cited herein, including patent applications, patent publications, and Genbank registration numbers, are incorporated herein by reference as if each individual reference were specifically and separately indicated as being incorporated by reference in its entirety.

[00038] The methods described or referred to herein are generally well understood and commonly used by those skilled in the art using conventional methodology, such as the commonly used methods described in Sambrook et al, Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): OCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); Current Protocols in Immunology (J. E. Coligan et al, eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J. B. Lippincott Company, 1993); and their updated versions.

[00039] Anti-CXCR5 antibodies, whether fucosylated or afucosylated, can be used in the prevention, treatment, and/or amelioration of diseases, disorders, or conditions caused by and/or associated with CXCR5 activity. Such diseases, disorders, or conditions include, but are not limited to, inflammatory responses such as systemic lupus erythematosus (SLE); chronic inflammatory responses; atherosclerosis; lack of adhesion of leukocytes; rheumatoid arthritis; diabetes mellitus (eg, type I diabetes mellitus or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and immune responses associated with acute and delayed hypersensitivity reactions, mediated by cytokines and T-lymphocytes, usually found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; Wegener's granulomatosis; pernicious anemia (Addison's disease); diseases, including diapedesis of leukocytes; inflammatory disorder of the central nervous system (CNS); multiple organ failure syndrome; hemolytic anemia (including, but not limited to, cryoglobulinemia or autoimmune hemolytic anemia with incomplete heat agglutinins); myasthenia gravis; diseases mediated by antigen-antibody complexes; anti-GBM antibody disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; myasthenic Lambert-Eaton syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; vitiligo; Reiter's disease; muscle stiffness syndrome; Behcet's disease; giant cell arteritis; immunocomplex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic diseases; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveoretinitis; Guillain-Barré syndrome; Goodpasture's syndrome; mixed connective tissue disease; autoimmune-associated infertility; nodous polyarteritis; alopecia areata; idiopathic myxedema; graft-versus-host reaction; muscular dystrophy (Duchenne's dystrophy, Becker's dystrophy, myotonic dystrophy, limb-girdle dystrophy, shoulder-blade-facial dystrophy, congenital dystrophy, oculopharyngeal dystrophy, distal dystrophy, Emery-Dreyfus dystrophy) and control of proliferation of malignant cells expressing CXCR5, such as cancer pancreatic cancer, colon cancer, bladder cancer, T-cell leukemia and B-cell leukemia, as will be understood by a person skilled in the art in view of the ideas presented in the present description.

I. DEFINITIONS

[00040] The present invention will be better understood with reference to the following detailed description of examples of embodiments of the invention and the examples included therein.

[00041] Unless otherwise indicated, all technical and scientific terms used in the present description have the same meaning, which is generally understood by a person skilled in the field to which the present invention belongs. In the event of a conflict, the present description, including the definition, will take precedence.

[00042] In addition, unless the context otherwise requires or otherwise specified, singular terms should include the plural, and plural terms should include the singular.

[00043] It is to be understood that aspects and embodiments of the invention presented herein include "consisting of" and/or "essentially consisting of" aspects and embodiments. Within the scope of the invention, unless otherwise indicated, terms in the singular include reference to the plural.

[00044] In this application, the use of the terms "or" means "and/or", unless otherwise specifically indicated or understood by a person skilled in the art. With respect to claims dependent on other dependent claims, the use of the term "or" includes reference to several preceding independent or dependent claims.

[00045] The term "approximately" when used with respect to measurable numeric variables refers to the specified value of the variable and all values of the variable that are within observational error of the specified value (e.g., within the 95% confidence interval for the mean) or within 10 percent from the specified value, whichever is greater. Numeric ranges are inclusive of the numbers that define the range.

[00046] Although the numerical ranges and parameters reflecting the broad scope of the invention are approximate, the numerical values given in the specific examples are as accurate as possible. However, any numerical value, by definition, contains some errors, inevitably resulting from the standard deviation found in the corresponding test measurements. In addition, all ranges provided in the present description, should be understood as including any and all subranges included in them. For example, a specified range of "1 to 10" should be considered to include any and all sub-ranges from (and including) a minimum value of 1 to a maximum value of 10; those. all subranges starting with a minimum value of 1 or more, such as 1 to 6.1, and ending with a maximum value of 10 or less, such as 5.5 to 10.

[00047] Throughout the present description and claims, the term "comprise" or variations thereof such as "comprises" or "comprising" should be understood to mean the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or groups of integers. Unless the context otherwise requires, singular terms must include the plural, and plural terms must include the singular. Any examples following the term "for example" are not meant to be exhaustive or limiting.

[00048] It should be understood that in any case where embodiments are presented herein with the term "comprising", they also include otherwise similar embodiments described in terms of "consisting of" and/or "consisting essentially of ".

[00049] If aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention applies not only to the entire specified group as a whole, but to each member of the group separately and all possible subgroups of the main group, as well as to the main group , which lacks one or more members of the group. The present invention also provides for the express exclusion of one or more of any members of the group from the invention described in the application.

[00050] It should be understood that the terminology used in the present description is intended solely to describe specific embodiments and is not limiting. In the present description and claims, reference is made to a number of terms that should have the following meaning.

[00051] The term "isolated molecule" (if the molecule is, for example, a polypeptide, polynucleotide or antibody or fragment thereof) is a molecule that, due to its origin or source of production (1), is not associated with naturally associated components that accompany it in the native state , (2) essentially does not contain other molecules of the same species, (3) is expressed by a cell of a different species, or (4) does not occur naturally. Thus, a molecule that is chemically synthesized or expressed in a cellular system other than the cell from which it naturally originates will be "isolated" from its naturally associated components. The molecule can also be made substantially free of naturally associated components by isolation using purification methods well known in the art. The purity or homogeneity of a molecule can be analyzed in a number of ways well known in the art. For example, the purity of the analysis of the polypeptide can be analyzed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using methods well known in this field. For some purposes, high resolution can be achieved using HPLC or other purification methods well known in the art.

[00052] In the context of the invention, the term "substantially pure" means that the target species is the predominant species present (i.e., on a mole basis, it is more numerous than any other species in the composition), and preferably, significantly a highly purified fraction is a composition where the target species (eg, glycoprotein, including antibody or receptor) is at least about 50 percent (on a mole basis) of all macromolecular species present. Typically, a substantially pure composition will contain greater than about 80 percent of all macromolecular species present in the composition, more preferably greater than about 85%, 90%, 95%, and 99%. Most preferably, the target species is purified to significant homogeneity (contaminants cannot be detected in the composition by conventional detection methods), where the composition consists essentially of one kind of macromolecules. In some embodiments, the substantially pure material is at least 50% pure (i.e. free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, still more preferably at least 98% pure, and most preferably at least 99% pure.

[00053] As is known in the art, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence comparison. In this area, the term "identity" also means the degree of relationship between the sequences of polypeptide molecules or nucleic acid molecules, respectively, as determined by the match between the chains of nucleotide or amino acid sequences. "Identity" measures the percentage of identical matches between two or more sequences using a gap-to-gap alignment performed by a specific mathematical model in computer programs (ie, "algorithms").

[00054] The term "similarity" is a related concept, but unlike the term "identity", it refers to a measure of similarity that includes identical matches and conservative substitutions. Because conservative substitutions refer to polypeptides, not nucleic acid molecules, the term "similarity" refers only to the comparison of polypeptide sequences. If two polypeptide sequences have, for example, 10 out of 20 identical amino acids, and all the others are non-conservative substitutions, then each percent identity and similarity will be 50%. If in the same example there are 5 more positions that have conservative substitutions, then the percentage of identity remains 50%, but the percentage of similarity will be 75% (15 out of 20). Thus, in cases where there are conservative substitutions, the degree of similarity between two polypeptide sequences will be greater than the percent identity between the two sequences.

[00055] "Fragments" or "parts" of a polypeptide or antibody of the invention can be obtained by shortening, for example, by removing one or more amino acids from the N- and/or C-terminus of the polypeptide. Thus, up to 10, up to 20, up to 30, up to 40 or more amino acids can be removed from the N- and/or C-terminus. Fragments or parts can also be obtained through one or more internal deletions.

[00056] An antibody variant may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and/or deletions and/or insertions in the specific sequences and fragments described above. "Deletion" variants may include deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid regions such as deletion of specific amino acid domains or other features. "Insert" variants may include the insertion of single amino acids, the insertion of small groups of amino acids, such as 2, 3, 4, or 5 amino acids, or the insertion of larger amino acid regions, such as the insertion of specific amino acid domains or other features. "Substitution" options preferably include replacing one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid can be replaced with an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some properties of the 20 main amino acids that can be used to select suitable substituents are described below.

[00057] Substitution variants contain at least one amino acid residue removed from the antibody molecule and another residue inserted in its place. The regions of greatest interest for substitutional mutagenesis include hypervariable regions, but changes in the framework are also contemplated. Conservative substitutions are listed in Table 1 under the heading "conservative substitutions". If such substitutions result in a change in biological activity, then more significant substitutions, referred to as "examples of substitutions" below or described below with respect to amino acid classes, can be included and the products screened.

TABLE 1

Amino acids and substitutions

original balance Conservative substitutions Substitution examples Alanine Ala (A) Val Val; Leu; ile Arginine Arg(R) Lys Lys; gln; Asn Asparagine Asn (N) Gln gln; His; Asp, Lys; Arg Aspartic Acid Asp(D) Glu Glu; Asn Cysteine Cys(C) Ser Ser; Ala Glutamine Gln (Q) Asn asn; Glu Glutamic Acid Glu(E) asp asp; Gln Glycine Gly(G) Ala Ala Histidine His(H) Arg asn; gln; Lys; Arg Isoleucine Ile(I) Leu Leu; Val; met; Ala; Ph; norleucine Leucine Leu (L) ile Norleucine; Ile; Val; met; Ala; Phe Lysine Lys(K) Arg Arg; gln; Asn Methionine Met (M) Leu Leu; Ph; ile Phenylalanine Phe(F) Tyr Leu; Val; Ile; Ala; Tyr Proline Pro (P) Ala Ala Serine Ser(S) Thr Thr Threonine Thr(T) Ser Ser Tryptophan Trp (W) Tyr Tyr; Phe Tyrosine Tyr(Y) Phe trp; Ph; Thr; Ser Valine Val(V) Leu Ile; Leu; met; Ph; Ala; norleucine

[00058] Significant modifications to the biological properties of an antibody are made by selecting substitutions that differ significantly in their effect in maintaining (a) the structure of the polypeptide backbone in the region of the substitution, for example, in the form of a beta sheet or helical conformation, (b) the charge or hydrophobicity of the molecule in target site, or (c) side chain volume. Natural residues are divided into groups based on the general properties of the side chain:

i. Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile;

ii. Polar uncharged: Cys, Ser, Thr, Asn, Gln;

iii. Acidic (negatively charged): Asp, Glu;

iv. Basic (positively charged): Lys, Arg;

v. Residues affecting chain orientation: Gly, Pro; And

vi. Aromatic: Trp, Tyr, Phe, His.

[00059] Non-conservative substitutions are made by replacing a member of one of these classes with a member of another class.

[00060] One type of substitution, for example, that can be made is the replacement of one or more cysteines in an antibody, which may be chemically reactive, with another residue such as, but not limited to, alanine or serine. For example, a non-canonical cysteine substitution can be made. The substitution can be made in the CDR or framework region of a variable domain or in the constant region of an antibody. In some embodiments, the cysteine is canonical. Any cysteine residue not involved in maintaining the correct conformation of the antibody can also be replaced, typically with serine, to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine bonds can be added to an antibody to improve its stability, particularly if the antibody is an antibody fragment, such as an Fv fragment.

[00061] An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. For the purposes of the invention, the term includes not only intact polyclonal or monoclonal antibodies, but also, unless otherwise indicated, any antigen-binding fragment thereof that competes with an intact antibody for specific binding, fusion proteins containing the antigen-binding fragment, and any other modified configuration of an immunoglobulin molecule containing antigen recognition site. Antigen binding fragments include, for example, Fab, Fab', F(ab') ₂ , Fd, Fv, domain antibodies (dAb, e.g. shark and camel antibodies), fragments including complementarity determining regions (CDRs), single chain antibodies from a variable fragment (scFv), maxibody, minibody, intrabody, diabody, triabody, tetrabody, v-NAR and bis-scFv, and polypeptides containing at least an immunoglobulin fragment sufficient to confer specific antigen binding to the polypeptide. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or a subclass thereof), and the antibody may not belong to any particular class. Depending on the amino acid sequence of the antibody constant region in their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant regions corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[00062] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion"), as used interchangeably herein, refer to one or more antibody fragments that retain the ability to specifically bind to an antigen (e.g., CXCR5). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the term "antigen binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab') ₂ fragment, bivalent fragment containing two Fab-fragment connected by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of one antibody arm, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) isolated complementarity determining region (CDR), disulfide-linked Fvs (dsFv), and anti-idiotypic antibodies (anti-Id) and intrabodies. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected recombinantly using a synthetic linker, allowing them to form a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single chain Fv (scFv)); see, for example, Bird et al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. sci. USA 85:5879-5883 (1988)). Such single chain antibodies are also intended to be included in the term "antigen binding fragment" of an antibody. Also included are other forms of single chain antibodies such as diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on the same polypeptide chain, but they use a linker that is too short to allow pairing between two domains on the same chain, thus causing the domains to pair with the complementary domains of the other. chain and give two antigen-binding sites (see, for example, Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure 2:1121-1123).

[00063] Antibodies can be obtained from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g., chickens) , fish (eg sharks) and camelids (eg llamas).

[00064] The term "variable region" of an antibody refers to an antibody light chain variable region (VL) or an antibody heavy chain variable region (VH), alone or in combination. As is known in the art, the heavy and light chain variable regions each consist of four framework regions (FRs) connected by three "complementarity determining regions (CDRs)", also known as hypervariable regions (HVRs), and participate in the formation of the antigen-binding region of an antibody. . If variants of the target variable region are desired, in particular with a substitution at amino acid residues outside the CDR region (i.e. in the framework region), a suitable amino acid substitution, preferably a conservative amino acid substitution, can be identified by comparing the target variable region with variable regions of other antibodies containing CDR1 and CDR2 sequences in the same canonical class as the target variable region (Chothia and Lesk, J. Mol. Biol. 196(4): 901-917, 1987).

[00065] In some embodiments, precise delimitation of CDRs and identification of residues containing an antibody binding site is performed by antibody structure resolution and/or antibody-ligand complex structure resolution. In some embodiments, which can be obtained by any of a variety of methods known to those skilled in the art, such as x-ray crystallography. In some embodiments, various analysis methods can be used to identify or approximate CDR regions. In some embodiments, various analysis methods can be used to identify or approximate CDR regions. Non-limiting examples of such methods include Kabat determination, Chothia determination, AbM determination, Contact system determination, and conformational determination.

[00066] The Kabat definition is the standard for numbering residues in an antibody and is generally used to identify CDR regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the positions of specific areas of structural loops. See, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The definition of AbM uses a built-in software package produced by the Oxford Molecular Group, with which the structure of an antibody is modeled. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular Ltd. AbM detection models the tertiary structure of an antibody from a primary sequence using a combination of information databases and ab initio methods such as those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198.

[00067] The determination by the Contact system is based on the analysis of the available crystal structures of the complexes. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as "conformational definition" of CDRs, CDR positions can be identified as residues making an enthalpy contribution to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. While other definitions of CDR boundaries may not follow exactly one of the above approaches, they will nonetheless overlap with at least a portion of the Kabat CDR, although they may be shortened or lengthened to account for prediction or experimental evidence that particular residues or groups of residues do not significantly affect antigen binding. Within the scope of the invention, the term "CDR" may refer to CDRs determined by any approach known in the art, including combinations of approaches. In the methods used in the present description, you can use the CDR, determined in accordance with any of these approaches. In the case of any specified embodiment containing multiple CDRs, the CDR may be determined according to any one of the Kabat definition, the Chothia definition, the extended definition, the AbM definition, the Contact definition, and/or the conformational definition.

[00068] As used herein, the term "contact residue" in relation to an antibody or antigen specifically bound by it refers to an amino acid residue present on an antibody/antigen containing at least one heavy atom (i.e., not a hydrogen) ranging from 4 Å or less from the heavy atom of the amino acid residue present on the cognate antibody/antigen.

[00069] "Framework" (FR) residues are antibody variable domain residues other than CDR residues. The VH or VL domain framework contains four framework subregions, FR1, FR2, FR3 and FR4, interspersed with CDRs in the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[00070] As described above in the present description, the residues in the variable domain, as a rule, are numbered according to Kabat, which is the numbering system used for heavy chain variable domains or light chain variable domains of a population of antibodies. See, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. Using this numbering system, the exact linear amino acid sequence may contain fewer amino acids or additional amino acids corresponding to a shortening or insertion in the FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insertion (Kabat residue 52a) after H2 residue 52 and inserted residues (eg, Kabat residues 82a, 82b, and 82c) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for the indicated antibody by aligning regions of antibody sequence homology with a "standard" Kabat numbered sequence. Various algorithms are available for assigning Kabat numbering. The algorithm implemented in version 2.3.3 of the Abysis release (www.abysis.org) can be used to assign Kabat numbering to the variable regions CDR-L1, CDR-L2, CDR-L3, CDR-H2, and CDR-H3, and then you can use the AbM definition for CDR-H1.

[00071] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, ie. the individual antibodies that make up the population are identical except for possible natural mutations, which may be present in small amounts. Monoclonal antibodies are highly specific, directed against one site of the antigen. In addition, unlike polyclonal antibody preparations, which typically include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a separate determinant on the antigen. The term "monoclonal" refers to the nature of an antibody as being derived from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be obtained by the hybridoma method first described in Kohler and Milstein, 1975, Nature 256:495, or can be obtained by recombinant DNA methods, such as those described in US patent No. 4816567. Monoclonal antibodies can also be isolated from phage libraries obtained by methods described, for example, in McCafferty et al., 1990, Nature 348:552-554. As used herein, the term "humanized" antibody refers to non-human (e.g., murine) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ , or other antigen-binding subsequences of antibodies ) containing the minimum sequence derived from a non-human immunoglobulin. Preferably, the humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the CDR of the recipient antibody are replaced with residues from the CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. The humanized antibody may contain residues not found either in the recipient antibody or in the grafted CDR or framework sequences, but included to further refine and optimize the properties of the antibody.

[00072] An antibody or antigen-binding fragment thereof of the invention may be affinity matured. For example, an affinity matured antibody can be prepared by methods known in the art (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813 ; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7) :3310-9; Hawkins et al., 1992, J. Mol. Biol., 226:889-896 and WO2004/058184).

[00073] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or obtained by any of the methods for producing human antibodies described herein. This definition of a human antibody specifically excludes a humanized antibody containing non-human antigen-binding residues.

[00074] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are regions are derived from a human antibody, or vice versa. The term also includes an antibody containing the V region of one individual of one species (eg, a first mouse) and a constant region of another individual of the same species (eg, a second mouse).

[00075] The term "antigen (Ag)" refers to a molecule used to immunize an immunocompetent vertebrate to produce an Ag-recognizing antibody (Ab) or to screen an expression library (e.g., but not limited to a phage, yeast, or ribosome display library). As used herein, the term "Ag" is defined in a broader sense, and is generally intended to include target molecules specifically recognized by an Ab, thus including fragments or mimetics of the molecule used in immunization to produce an Ab or in library screening for an Ab. Ab selection. Thus, in the case of antibodies of the invention that bind to CXCR5, full-length mammalian (e.g., human, monkey, mouse, and rat) CXCR5, including their monomers and multimers such as dimers, trimers, etc., as well as truncated and other variants of CXCR5 are referred to as an antigen.

[00076] Typically, the term "epitope" refers to the region of an antigen (eg, protein, nucleic acid, carbohydrate, or lipid, etc.) to which an antibody specifically binds, i. area in physical contact with the antibody. Thus, the term "epitope" refers to a portion of a molecule that can be recognized and bound by an antibody at one or more of the antibody's antigen-binding regions. Typically, an epitope is defined in the context of a molecular interaction between an "antibody or antigen-binding fragment thereof" (Ab) and its corresponding antigen. Epitopes often consist of surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. In some embodiments, the epitope may be a protein epitope. Protein epitopes may be linear or conformational. In a linear epitope, all points of interaction between a protein and an interacting molecule (such as an antibody) are located linearly along the protein's primary amino acid sequence. A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides (or amino acids) in an antigenic protein to which an antibody specific for the epitope binds. Within the scope of the invention, the term "antigenic epitope" is defined as the portion of an antigen to which an antibody can specifically bind, as determined by any method well known in the art, eg, by conventional immunoassays. Alternatively, information about the desired epitopes can be ascertained at the time of discovery during antibody production and characterization. With this information, antibodies can be competitively screened for binding to the same epitope. This can be achieved using competition and cross-competition assays to detect antibodies competing or cross-competing with each other for binding to CXCR5, eg, antibodies competing for binding to an antigen.

[00077] An antibody "preferentially binding" or "specifically binding" (terms used interchangeably herein) to an epitope is a term well known in the art, and methods for determining such specific or preferential binding are also well known in the art. . A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, for longer duration, and/or with higher affinity for a particular cell or substance than for alternative cells or substances. An antibody "specifically binds" or "predominantly binds" to a target if it binds with higher affinity, avidity, more easily, and/or longer duration than other substances. In addition, an antibody "specifically binds" or "predominantly binds" to a target if it binds with higher affinity, avidity, more easily and/or longer duration to that target in the sample than to other substances present in the sample. For example, an antibody that specifically or preferentially binds to an epitope of CXCR5 is an antibody that binds to that epitope with higher affinity, avidity, more readily, and/or longer duration than other CXCR5 epitopes or non-CXCR5 epitopes. It should also be understood from this definition, for example, that an antibody (or substance or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. In this regard, "specific binding" or "preferential binding" may not require exclusive binding (although it may include it). Typically, but not necessarily, a binding reference means a preferential binding. "Specific binding" or "preferential binding" includes a compound, such as a protein, nucleic acid, antibody, or the like, that recognizes and binds to a specific molecule, but does not necessarily recognize or bind other molecules in the sample. For example, an antibody or peptide receptor that recognizes and binds to a cognate ligand or binding partner (e.g., an anti-CXCR5 antibody that binds to CXCR5) in a sample, but does not substantially recognize or bind to other molecules in the sample, binds specifically to this cognate ligand or binding partner. Thus, under the conditions of the assay described, said binding agent (e.g., an antibody or antigen-binding fragment thereof, or a receptor or ligand-binding fragment thereof) binds preferentially to a particular target molecule and does not bind to a significant extent to other components present in the test sample.

[00078] A variety of assay formats can be used to select an antibody or peptide that specifically binds to a molecule of interest. For example, ELISA immunoassay, immunoprecipitation, Biacore™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescence-activated cell sorting (FACS), Octet™ (FortéBio, Inc., Menlo Park, CA), and Western blot analysis are some of assays that can be used to identify an antibody that specifically reacts with an antigen or receptor, or a ligand-binding fragment thereof that specifically binds to a cognate ligand or binding partner. Typically, a specific or selective response will be at least twice the background signal or noise, more typically more than 10 times, even more typically more than 50 times, more typically more than 100 times, even more typically, more than 500 times, even more typically, more than 1,000 times, and even more typically, more than 10,000 times. In addition, an antibody is said to "specifically bind" to an antigen if the equilibrium dissociation constant (K _D ) is ≤1 μM, preferably ≤100 nM, more preferably ≤10 nM, even more preferably ≤100 pM, even more preferably ≦10 pM, and even more preferably ≦1 pM. In some embodiments, an antibody is said to "specifically bind" to an antigen if the equilibrium dissociation constant (K _D ) is ≦7 nM.

[00079] As used herein, the term "binding affinity" is used as a measure of the strength of a non-covalent interaction between two molecules, eg, an antibody or fragment thereof and an antigen. The term "binding affinity" is used to describe monovalent interactions (characteristic activity).

[00080] In addition, to determine the binding affinity of anti-CXCR5 antibodies to CXCR5 expressing cells, cell binding experiments can be performed to determine apparent affinity. The apparent binding affinity of an antibody to cells expressing the target can be calculated as EC ₅₀ equilibrium binding titration curves in which the geometric mean fluorescence intensity (gMFI) of the antigen-binding population is quantified by flow cytometry.

[00081] The binding affinity between two molecules, eg, an antibody or fragment thereof and an antigen, via a monovalent interaction can be quantified by determining the dissociation constant (K _D ). In turn, K _D can be determined by measuring the kinetics of formation and dissociation of complexes using, for example, the method of surface plasmon resonance (SPR) (Biacore). The rate constants corresponding to the association and dissociation of the monovalent complex are referred to as the association rate constant k _a (or k _on ) and the dissociation rate constant k _d (or k _off ), respectively. K _D is related to k _a and k _d through the equation K _D = k _d / k _a . The value of the dissociation constant can be determined directly by well-known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K _D can be determined using a double nitrocellulose filter binding assay, such as the assay described in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays for evaluating the binding capacity of ligands such as antibodies against target antigens are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry, and other assays, examples of which are given elsewhere in this specification. The binding kinetics and binding affinity of an antibody can also be analyzed using standard assays known in the art such as surface plasmon resonance (SPR), for example using the Biacore™ or KinExA system.

[00082] A competitive binding assay can be performed in which the binding of an antibody to an antigen is compared to the binding of a target to another ligand on that target, such as another antibody or soluble receptor, that otherwise binds to the target. The concentration at which 50% inhibition occurs is known as K _i . Under ideal conditions, K _i is equivalent to K _D . The value of K _i is never less than K _D , so the measurement of K _i can be substituted to obtain an upper limit on K _D .

[00083] Following the definition above, the binding affinities associated with different molecular interactions, for example, the binding affinity of different antibodies for a given antigen, can be compared by comparing the K _D values of individual antibody/antigen complexes. The K _D values of antibodies or other binding partners can be determined by methods well known in the art. One way to determine K _D is to use surface plasmon resonance, typically with a biosensor system such as the Biacore® system.

[00084] Similarly, the specificity of an interaction can be analyzed by determining and comparing the K _D value of an interaction of interest, e.g., a specific interaction between an antibody and an antigen, with the K _D value of an interaction of no interest, e.g., a control antibody known to be does not bind to CXCR5.

[00085] An antibody that specifically binds to a target can bind to a target with high affinity, ie. has a low K _D as above and can bind to other non-target molecules with lower affinity. For example, an antibody can bind to non-target molecules with a K _D of 1×10 ^-6 M or more, more preferably 1×10 ^-5 M or more, more preferably 1×10 ^-4 M or more, more preferably 1×10 ^-3 M or more, even more preferably 1×10 ^-2 M or more. An antibody of the invention can preferably bind to a target with an affinity of at least two times, 10 times, 50 times, 100 times, 200 times, 500 times, 1000 times, or 10,000 times or more its binding affinity to another molecule, non-CXCR5.

[00086] As used herein, the term "competes" with respect to an antibody means that the first antibody, or antigen-binding fragment thereof, binds to an epitope in a manner substantially similar to binding of a second antibody, or antigen-binding fragment thereof, such that the result of binding of the first antibody to its cognate the epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Optionally, there may be an alternative, where the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody. Those. the first antibody can inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to the corresponding epitope. However, if each antibody detectably inhibits the binding of another antibody to its cognate epitope or ligand, to the same, greater or lesser extent, indicate that the antibodies "cross-compete" with each other for binding to the respective epitopes. Competing and cross-competing antibodies are within the scope of the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or a portion thereof), those skilled in the art, in view of the teachings presented herein, will recognize that such competing and/or cross-competing antibodies, and can be used in the methods presented in the present description.

[00087] Standard competition assays can be used to determine if two antibodies compete with each other. One suitable antibody competition assay involves the use of Biacore technology, which can measure the extent of interactions using surface plasmon resonance (SPR), typically with a biosensor system (such as the BIACORE® system). For example, the SPR can be used in an in vitro competitive binding inhibition assay to determine the ability of one antibody to inhibit the binding of a second antibody. Another assay uses an ELISA-based approach to measure antibody competition.

[00088] In addition, international patent application No. WO2003/48731 describes a high throughput method for "binning" antibodies depending on their competition. Competition occurs if one antibody (or fragment) reduces the binding of another antibody (or fragment) to CXCR5. For example, a sequential binding competition assay can be used using different antibodies added sequentially. The first antibody can be added to achieve near saturation binding. Then a second antibody is added. If the binding of the second antibody to CXCR5 is undetectable or significantly reduced (e.g., a decrease of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least by about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%) compared to the parallel assay in the absence of the first antibody (the value in which can be taken as 100%), the two antibodies are considered to compete with each other.

[00089] In addition, an example of an antibody epitope binning analysis using domain shuffling between human and mouse CXCR5 proteins to evaluate potential epitopes among multiple antibodies is provided in Example 8. Those skilled in the art will appreciate, given the ideas presented herein, that there is a wide range of assays known in the art that can be used to determine target binding of at least two antibodies relative to each other, and such assays are included herein.

[00090] Anti-CXCR5 antibodies can be characterized by methods well known in the art. For example, one way is to identify the epitope to which it binds, or "epitope mapping". There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including crystal structure resolution of the antibody-antigen complex, competition assays, gene fragment expression assays, and assays using synthetic peptides, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In a further example, epitope mapping can be used to determine the sequence to which an anti-CXCR5 antibody binds. Epitope mapping is commercially available from various sources such as Pepscan Systems (15 Edelhertweg 8219 PH Lelystad, The Netherlands). The epitope may be a linear epitope, ie. contained in a single amino acid fragment, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not be contained in a single fragment. Peptides of various lengths (eg, at least 4-6 amino acids) can be isolated or synthesized (eg, recombinantly) and used for binding assays using an anti-CXCR5 antibody.

[00091] In addition, the epitope to which an anti-CXCR5 antibody binds can be determined by system screening using overlapping peptides derived from the CXCR5 sequence and determining antibody binding. According to gene fragment expression assays, the open reading frame encoding CXCR5 can be fragmented randomly or with specific genetic constructs and the reactivity of the expressed CXCR5 fragments determined with the antibody to be tested. Gene fragments can be obtained, for example, using PCR, and then transcribed and translated into protein in vitro in the presence of radioactively labeled amino acids. The binding of the antibody to radiolabeled CXCR5 fragments is then determined by immunoprecipitation and gel electrophoresis.

[00092] Some epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) or yeast (yeast display). Alternatively, a specific library of overlapping peptide fragments can be tested for binding to a test antibody in simple binding assays. In a further example, antigen mutagenesis, domain shuffling experiments, and alanine scan mutagenesis can be performed to identify residues necessary, sufficient, and/or required for epitope binding. For example, alanine scan mutagenesis experiments can be performed using mutant CXCR5 in which various residues of the CXCR5 polypeptide are replaced with alanine. By analyzing the binding of an antibody to a mutant CXCR5, the importance of specific CXCR5 residues for antibody binding can be assessed.

[00093] Another method that can be used to characterize a CXCR5 antibody is to use competition assays with other antibodies known to bind to the same antigen, ie. different fragments on CXCR5 to determine if the anti-CXCR5 antibody binds to the same epitope as other antibodies. Competitive assays are well known to those skilled in the art.

[00094] In addition, an epitope for a given antibody/antigen binding pair can be determined and characterized at various levels using various experimental and computational epitope mapping techniques. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen-deuterium exchange mass spectrometry (H/D-MS), and various competitive binding methods well known in the art. Because each method is based on a unique principle, the description of an epitope is directly related to the way in which it is determined. Thus, the epitope for said antibody/antigen pair will be determined differently depending on the epitope mapping method used.

[00095] At the most detailed level, an epitope for an interaction between Ag and Ab can be determined from the spatial coordinates defining the atomic contacts that take place during the Ag-Ab interaction, as well as information about their relative contribution to binding thermodynamics. At a less detailed level, an epitope can be characterized by the spatial coordinates defining the atomic contacts between Ag and Ab. At an even less detailed level, an epitope can be characterized by the amino acid residues it contains, as determined by specific criteria, such as the distance between atoms (eg, heavy, ie, non-hydrogen atoms) in Ab and Ag. At an even less detailed level, an epitope can be characterized by function, for example by competitive binding to other Abs. An epitope can also be defined more generally as containing amino acid residues in which case substitution with a different amino acid will change the characteristics of the interaction between Ab and Ag (eg using alanine scanning).

[00096] From the fact that the description and definitions of epitopes, depending on the method of epitope mapping used, are obtained at different levels of detail, it follows that comparison of epitopes for different Abs on the same Ag can similarly be carried out at different levels of detail.

[00097] Epitopes described at the amino acid level, such as those defined by x-ray structure, are said to be identical if they contain the same set of amino acid residues. Epitopes are said to overlap if at least one amino acid is common to the epitopes. Epitopes are said to be separate (unique) if the epitopes do not share common amino acid residues.

[00098] Epitopes characterized by competitive binding are said to overlap if the binding of the respective antibodies is mutually exclusive, ie. binding of one antibody precludes simultaneous or sequential binding of another antibody. Epitopes are said to be separate (unique) if the antigen can accommodate both corresponding antibodies simultaneously binding.

[00099] The definition of the term "paratope" follows from the above definition of "epitope" in the opposite sense. Thus, the term "paratope" refers to the region on an antibody that specifically binds to an antigen, ie. amino acid residues on the antibody in contact with the antigen (CXCR5) as defined by "contact" elsewhere in this specification.

[000100] The epitope and paratope for said antibody/antigen pair can be identified by conventional methods. For example, the general location of an epitope can be determined by assessing the ability of an antibody to bind to different fragments or variants of CXCR5 polypeptides. Specific amino acids in CXCR5 contacting an antibody (epitope) and specific amino acids in an antibody contacting CXCR5 (paratope) can also be determined by conventional methods, such as those described in the examples. For example, an antibody and a target molecule can be combined and the antibody/antigen complex crystallized. The crystal structure of the complex can be determined and used to identify specific interaction sites between an antibody and its target.

[000101] An antibody of the invention may bind to the same CXCR5 epitope or domain as the antibodies of the invention as specifically described herein. Assays that can be used for this identification include assays that evaluate competition for CXCR5 binding between the antibody of interest and the CXCR5 receptor in the biological activity assays as described in Examples 1-10 and antibody crystal structure assays.

[000102] An antibody of the invention may be able to compete or cross-compete with another antibody of the invention for binding to CXCR5 as described herein. For example, an antibody of the invention may compete or cross-compete with the antibodies provided herein for binding to CXCR5 or a suitable fragment or variant of CXCR5 bound by the antibodies provided herein.

[000103] That is, if the first antibody competes with the second antibody for binding to CXCR5 but does not compete, if the second antibody binds to CXCR5 first, it is said to "compete" with the second antibody (also referred to as unidirectional competition). If an antibody competes with another antibody, regardless of which antibody binds to CXCR5 first, then the antibody "cross-competes" for binding to CXCR5 with the other antibody. Such competing or cross-competing antibodies can be identified based on their ability to compete/cross-compete with a known antibody of the invention in standard binding assays. For example, SPR can be used to demonstrate competition/cross-competition, eg using the Biacore™ system, ELISA assays, or flow cytometry. Such competition/cross-competition may suggest that two antibodies bind to identical, overlapping, or similar epitopes.

[000104] Thus, an antibody of the invention can be identified by a method including a binding assay that assesses whether a test antibody can compete/cross-compete with a reference antibody for a binding site on a target molecule. Methods for performing competitive binding assays are provided herein and/or are well known in the art. For example, they may include binding a reference antibody of the invention to a target molecule using conditions under which the antibody can bind to the target molecule. The antibody/target complex can then be exposed to the test/second antibody and assess the extent to which the test antibody can displace the reference antibody of the invention from the antibody/target complexes. An alternative method may involve bringing a test antibody into contact with a target molecule under conditions that allow binding of the antibody, and then adding a reference antibody of the invention capable of binding to the target molecule and assessing the extent to which the reference antibody of the invention can displace the test antibody from antibody/target complexes or simultaneously bind to the target (i.e. non-competitive antibody).

[000105] The ability of a test antibody to inhibit the binding of a reference antibody of the invention to a target indicates that the test antibody can compete with the reference antibody of the invention for binding to the target, and thus that the test antibody binds to the same or substantially the same epitope or region on the CXCR5 protein as the reference antibody of the invention. A test antibody identified as competing with a reference antibody of the invention in such a method is also an antibody of the present invention. That the test antibody can bind to CXCR5 in the same region as the reference antibody of the invention and can compete with the reference antibody of the invention suggests that the test antibody may act as a ligand at the same binding site as the antibody. of the invention, and that the test antibody can thus mimic the action of a reference antibody and is thus an antibody of the invention. This can be confirmed by comparing the activity of CXCR5 in the presence of a test antibody with the activity of CXCR5 in the presence of a reference antibody under otherwise identical conditions using an assay that is presented in more detail elsewhere in this specification.

[000106] The reference antibody of the invention may be an antibody as described herein, such as 11G2, 41A10, 5H7, and any variant or portion thereof as described herein that retains the ability to bind to CXCR5. An antibody of the invention may bind to the same epitope as the reference antibodies provided herein, or any variant or portion thereof as provided herein that retains the ability to bind to CXCR5.

[000107] As noted above elsewhere in this specification, specific binding can be analyzed in terms of the binding of an antibody to a non-target molecule. This comparison can be made by comparing the ability of an antibody to bind to a target and another molecule. This comparison can be made as described above for estimating K _D or K _i . The other molecule used in such a comparison may be any molecule that is not the target molecule. Preferably, the other molecule is not identical to the target molecule. Preferably, the target molecule is not a fragment of the target molecule.

[000108] Another molecule used to determine specific binding may be unrelated to the target in structure or function. For example, the other molecule may be an unrelated material or a material associated with the environment.

[000109] The other molecule used to determine specific binding may be another molecule in the same in vivo pathway as the target molecule, ie. CXCR5. By ensuring that an antibody of the invention has specificity for CXCR5 relative to another such molecule, undesirable in vivo cross-reactivity can be avoided.

[000110] An antibody of the invention may retain the ability to bind to certain molecules that are related to the target molecule.

[000111] Alternatively, an antibody of the invention may have specificity for a particular target molecule. For example, it may bind to a target molecule as described herein, but may not bind, or may bind with significantly reduced affinity, to another target molecule as described herein. For example, full-length mature human CXCR5 can be used as a target, but an antibody that binds to that target may be unable to bind, or may bind with less affinity, for example, to other CXCR5 proteins from other species, such as other mammalian CXCR5. In some embodiments, the antibody binds to human and mouse CXCR5.

[000112] An "Fc fusion" protein is a protein where one or more polypeptides are operably linked to an Fc polypeptide. In the Fc fusion protein, the Fc region of an immunoglobulin is combined with a fusion partner.

[000113] A "native Fc region sequence" contains an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. An "Fc region variant" contains an amino acid sequence that differs from the native Fc region sequence by at least one amino acid modification, but retains at least one effector function of the native Fc region sequence. Preferably, the Fc region variant contains at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions. in the native sequence of the Fc region or in the Fc region of the parent polypeptide. A variant Fc region as used herein will preferably have at least about 80% sequence identity to the native sequence Fc region and/or Fc region of the parent polypeptide, and most preferably at least about 90% sequence identity to relative to it, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity relative to it.

[000114] As is known in the art, the term "constant region" of an antibody refers to an antibody light chain constant region or an antibody heavy chain constant region, alone or in combination.

[000115] The terms "IgG Fc region", "Fc region", "Fc domain", and "Fc", as used interchangeably herein, refer to the portion of the IgG molecule associated with the crystallizable fragment obtained by papain cleavage of the molecule. IgG. Within the scope of the invention, the terms refer to the constant region of an antibody, excluding the first domain of an immunoglobulin constant region, and additionally refer to portions of this region. Thus, the term "Fc" refers to the last two IgA constant region domains, IgD and IgG, the last three IgE and IgM constant region domains, and the flexible hinge region N-terminal to these domains or portions thereof. In the case of IgA and IgM, the Fc may include a J chain. In the case of IgG, Fc contains the Cγ2 and Cγ3 immunoglobulin domains (C gamma 2 and C gamma 3) and a hinge region between Cγ1 (C gamma 1) and Cγ2 (C gamma 2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as containing C226 or P230 residues at the carboxyl terminus, where the numbering corresponds to the EU index according to Edelman et al., 1969, Proc. Natl. Acad. sci. USA 63(1):78-85 as described in Kabat et al., 1991. Typically, the Fc domain contains amino acid residues from about 236 to about 447 of the human IgG1 constant domain. An example of the amino acid sequence of the Fc domain of human IgG1 wild type is shown in SEQ ID NO:31. The term "Fc polypeptide" may refer to this region when isolated or this region in the context of an antibody or antigen-binding fragment or Fc fusion protein.

[000116] The heavy chain constant domain comprises an Fc region and further comprises a CH1 domain and a hinge region, as well as CH2 and CH3 domains (and optionally CH4 from IgA and IgE) of an IgG heavy chain.

[000117] A "functional Fc region" has at least one effector function of a native sequence Fc region. Examples of "effector functions" include C1q binding; complement-mediated cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity; phagocytosis; downregulation of cell surface receptors (eg B-cell receptor), etc. Such effector functions typically require the combination of an Fc region with a binding domain (e.g., an antibody variable domain or an antigen-binding fragment thereof) and can be assessed using various assays known in the art for evaluating such antibody effector functions.

[000118] A "native Fc region sequence" contains an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. The native sequence of the human Fc region includes the native sequence of the human IgG1 Fc region (non-A and A allotypes), the native sequence of the human IgG2 Fc region, the native sequence of the human IgG3 Fc region and the native sequence of the human IgG4 Fc region, as well as their natural options.

[000119] A "variant Fc region" contains an amino acid sequence that differs from the native sequence of the Fc region as a result of at least one amino acid modification.

[000120] The term "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcyR is a native human FcR. In some embodiments, the FcR is an IgG (gamma receptor) antibody-binding FcR and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA ("activating receptor") and FcyRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The FcyRIIA activating receptor contains an immunoreceptor tyrosine activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). For reviews of FcR see, for example, Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et ah, Immunomethods 4:25-34 (1994); and de Haas et ah, J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including FcRs that will be identified in the future, are included in the term "FcR" in the present description.

[000121] The term "Fc receptor" or "FcR" also includes the neonatal FcRn receptor responsible for the transport of maternal IgG to the fetus (Guyer et ah, J. Immunol. 117:587 (1976) and Kim et ah, J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997 ); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.)).

[000122] The term "effector functions" refers to the types of biological activity attributed to the Fc region of an antibody and varying depending on the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg B-cell receptor) and activation of B-cells.

[000123] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcyRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK), monocytes, macrophages, cytotoxic T cells, and neutrophils. Effector cells can be isolated from a native source such as blood.

[000124] The term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Fc receptor-bound (FcR) Ig present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) allow these cytotoxic effector cells specifically bind to an antigen-bearing target cell and then destroy the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, only express FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is shown in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as described in US Pat. Nos. 5,500,362 or 5,821,337 or US Pat. Effector cells that can be used for such assays include PBMCs and NK cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assayed in vivo , for example in an animal model such as that described in Clynes et al. Proc. Natl. Acad. sci. (USA) 95:652-656 (1998). Additional antibodies with altered Fc region amino acid sequences and increased or decreased ADCC activity are described, for example, in US Pat. No. 7,923,538 and US Pat. No. 7,994,290.

[000125] The term antibody having "enhanced ADCC activity" refers to an antibody that is more effective at mediating ADCC in vitro or in vivo compared to the parent antibody, where the antibody and the parent antibody differ in at least one structural aspect, and if the amounts such an antibody and the parent antibody used in the assay are essentially the same. In some embodiments, the antibody and parent antibody have the same amino acid sequence, but the antibody is afucosylated while the parent antibody is fucosylated. In some embodiments, ADCC activity will be determined using an in vitro ADCC assay as described herein, but other assays or methods for determining ADCC activity, such as in an animal model, etc., are contemplated. In some embodiments, an antibody with enhanced ADCC activity has increased affinity for Fc gamma RIIIA. In some embodiments, an antibody with increased ADCC activity has increased affinity for Fc gamma RIIIA (V158). In some embodiments, an antibody with enhanced ADCC activity has increased affinity for Fc gamma RIIIA (F158).

[000126] An antibody with "altered" FcR binding affinity or ADCC activity is an antibody having increased or decreased FcR binding activity and/or ADCC activity compared to the parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect. An antibody "showing increased binding" to an FcR binds to at least one FcR with better affinity than the parent antibody. An antibody "showing reduced binding" to an FcR binds to at least one FcR with lower affinity than the parent antibody. Such antibodies showing reduced FcR binding may have little or no FcR binding, such as 0-20 percent FcR binding compared to native IgG Fc region sequence.

[000127] The term "increased affinity for Fc gamma RIIIA" refers to an antibody having a higher affinity for Fc gamma RIIIA (also referred to in some cases as CD16a) than the parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect. . In some embodiments, the antibody and parent antibody have the same amino acid sequence, but the antibody is afucosylated while the parent antibody is fucosylated. You can use any suitable method for determining the affinity for Fc gamma RIIIA. In some embodiments, the implementation of the affinity for Fc gamma RIIIA is determined by the method presented in the present description. In some embodiments, an antibody with increased affinity for Fc gamma RIIIA has increased ADCC activity. In some embodiments, an antibody with increased affinity for Fc gamma RIIIA has increased affinity for Fc gamma RIIIA(V158). In some embodiments, an antibody with increased affinity for Fc gamma RIIIA has increased affinity for Fc gamma RIIIA(F158).

[000128] L-fucose, also referred to as 6-deoxy-L-glactose, is a monosaccharide that is a component of several N- and O-linked glycans and glycolipids in animals. See Becker and Lowe, Glycobiology 13:41R-51R (2003). Fucose is typically added as a terminal modification to glycans, including glycans attached to blood group antigens, selectins, and antibodies. Fucose can attach to glycans through α(1,2)-, α(1,3)-, α(1,4)- and α(1,6)-bonds using specific fucosyltransferases. α(1,2)-fucose bonds are usually associated with the H-group of blood antigens. α(1,3)- and α(1,4)-fucose bonds are associated with the modification of Lewis X antigens. α(1,6)-fucose bonds are associated with N-linked GlcNAc molecules, such as molecules on antibodies.

[000129] The carbohydrate molecules of the present invention will be described with reference to common nomenclature for describing oligosaccharides. A review of carbohydrate chemistry using this nomenclature can be found in Hubbard and Ivatt (1981) Ann. Rev. Biochem. 50:555-583. This nomenclature includes, for example, Man, which corresponds to mannose; GIcNAc, which corresponds to 2-N-acetylglucosamine; Gal, which corresponds to galactose; Fuc, which corresponds to fucose; and Glc corresponding to glucose. Sialic acids are described by the abbreviation NeuNAc for 5-N-acetylneuraminic acid and NeuNGc for 5-glycolylneuraminic acid (IUB-IUPAC Joint Commission on Biochemical Nomenclature, 1982, J. Biol. Chem. 257: 3347-3351; (1982) J Biol Chem 257:3352).

[000130] The carbohydrate structures of the present invention are on a protein expressed as N-linked oligosaccharides. The term "N-linked glycosylation" refers to the attachment of a carbohydrate molecule via GlcNAc to an asparagine residue in a polypeptide chain. All N-linked carbohydrates share the core structure Man 1-6(Man1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-R. Thus, in the core structure described, R corresponds to the asparagine residue of the glycoprotein produced. The sequence of the protein produced will contain asparagine-X-serine, asparagine-X-threonine and asparagine-X-cysteine, where X is any amino acid except proline (Asn-Xaa-Ser/Thr). In contrast, "O-linked" carbohydrates share a common core structure, which is a GalNAc attached to a threonine or serine hydroxyl group, but a consensus sequence is not necessary. Of the N-linked carbohydrates, the most important are "complex" N-linked carbohydrates such as the "bianthenary" structures described herein.

[000131] Those skilled in the art will recognize that immunoglobulin G (IgG) glycoprotein is associated with three types of complex bianthenary structures containing zero, one, or two galactose residues (Wormland et al., 1997, Biochemistry 36:1370-1380), commonly known as G0, G1 and G2, respectively. With respect to human IgG antibody molecules, each contains an N-linked oligosaccharide attached to the amide side chain of the Asn 297 β-4-bend of the inner surface of the CH2 domain of the Fc region (Beale and Feinstein, 1976, Q. Rev. Biophys. 9 :253-259; Jefferis et al., 1995, Immunol Letts 44:111-117). The oligosaccharide residue attached to Asn 297 of the IgG CH2 domain is of a complex bianthenary type having an identified hexasaccharide core structure and variable external sugar residues (see Jefferis et al., 1997, supra; Wyss and Wagner, 1996, Current Opinions in Biotech. 7:409-416). The core structure (GIcNAc2Man3GIcNAc) is typical of bianthenary oligosaccharides and is shown schematically in FIG. 1.

[000132] Because each core structure can contain bisected N-acetylglucosamine, core fucose, and external saccharides galactose or sialic acid, for a total of 36 structurally unique oligosaccharides that can occupy the Asn 297 site (Jefferis and Lund, supra). It should also be understood that, in a particular CH2 domain, Asn 297 glycosylation may be asymmetric due to different oligosaccharide chains attached to the Asn 297 residue in the double-stranded Fc domain. For example, although the heavy chain synthesized in a single antibody-secreting cell may be homogeneous in its amino acid sequence, it is typically differentially glycosylated, resulting in a large number of structurally unique Ig glycoforms.

[000133] The main types of complex oligosaccharide structures, also referred to as "glycoforms", found in the CH2 domain of IgG are described in International Patent Publication No. WO 99/22764 on page 7.

[000134] Within the scope of the invention, the term "G0" refers to a bianthenary structure in which there are no terminal sialic acids (NeuAc) or Gal, the term "G1" refers to a bianthenary structure containing one Gal and no NeuAc, and the term "G2" refers to bianthenary structure with two terminal Gal and no NeuAc. See, for example, FIG. 2A-2G showing examples of G0, G1, G-1, and G2 structures.

[000135] The terms "afucosylated" antibody or antibody "lacking fucose" refers to an IgG1 or IgG3 isotype antibody that lacks fucose in the glycosylation of the constant region. Glycosylation of human IgG1 or IgG3 occurs at Asn297, since glycosylation of the core fucosylated bianthenary complex oligosaccharide ends with up to 2 Gal residues. In some embodiments, the afucosylated antibody lacks fucose in Asn297. These structures are designated G0, G1 (1.6 or 1.3) or G2 glycan residues depending on the number of terminal Gal residues. See, for example, Raju, T. S., BioProcess Int. 1:44-53 (2003). Fc glycosylation of a CHO type antibody is described, for example, in Routier, F. FL, Glycoconjugate J. 14: 201-207 (1997). Various antibody glycoforms are shown in FIG. 2A-2G.

[000136] In some embodiments, the terms "fucosyl" or "afucosylated" antibody, as used interchangeably herein, refer to an antibody glycoengineered to not contain crustal fucose. Antibodies with reduced fucose content in glycan residues have increased affinity for FcγRIIIa (CD16) and as a result have increased antibody-dependent cellular cytotoxicity (ADCC) activity. Afucosylated antibodies can be generated using the Potelligent® CHOK1SV cell line (Lonza Biologics) which lacks both alleles of the gene responsible for the addition of fucose (α1,6-fucosyltransferase). Afucosylated or fucose-reduced antibodies can also be obtained by modifying the activity of oligosaccharide biosynthesis in various ways. For example, overexpression of N-acetylglucosamine transferase III (GnTIII) in the Golgi apparatus of a producing cell line leads to the formation of bisect oligosaccharide structures associated with the Fc constant region of the antibody and suppresses fucosylation. In such expression systems, the level of GnTIII expression correlates with the formation of afucosylated IgG1 glycoforms and leads to increased ADCC activity. Fucosylation can also be reduced in cell culture using non-limiting examples of sugar analogs such as fucose analogs as described in WO 2012/019165. Thus, afucosylated or fucose-reduced antibodies can be generated using a wide variety of methods well known in the art.

[000137] In some embodiments, the afucosylated antibody has increased affinity for Fc gamma RIIIA. In some embodiments, the afucosylated antibody has increased affinity for Fc gamma RIIIA (V158). In some embodiments, the afucosylated antibody has increased affinity for Fc gamma RIIIA (F158).

[000138] The term "glycoform" refers to a complex oligosaccharide structure containing bonds of various carbohydrate units. Such structures are described, for example, in Essentials of Glycobiology Varki et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999), which also provides an overview of standard glycobiology nomenclature. Such glycoforms include, as non-limiting examples, G2, G1, G0, G-1 and G-2 (see, for example, International Patent Publication No. WO 99/22764).

[000139] The term "glycosylation profile" is defined as the profile of carbohydrate units covalently attached to a protein (e.g., glycoform) as well as the sites where the glycoforms are covalently attached to the peptide backbone of the protein, more specifically, to the immunoglobulin protein.

[000140] In some embodiments, at least 85 percent of a batch of antibodies recombinantly expressed in non-glycomodified CHO host cells are fucosylated at Asn297. For a composition containing multiple antibodies, antibodies are considered afucosylated if less than about 5 percent of the antibodies in the composition contain fucose in at least one Asn297. Even more preferably, the level of afucosylation is about 100%, i. e. do not detect fucose on any Asn297 heavy chain glycoform using standard methods for measuring antibody fucosylation. Methods for measuring fucose include any methods known in this field, including the methods presented in the present description. In some embodiments, fucose is undetectable in a composition containing a plurality of afucosylated antibodies. In some embodiments, the afucosylated antibody has increased ADCC activity.

[000141] The term "complement-mediated cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) bound to their cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Antibodies with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described, for example, in US Pat. No. 6,194,551 B1, US Pat. No. 7,923,538, US Pat. See also, for example, Idusogie et al, J.

[000142] As used herein, the terms "wild-type amino acid", "wild-type IgG", "wild-type antibody", or "wild-type mAb" refer to a sequence of amino acids or nucleic acids naturally occurring in a population (e.g., humans, mice , rats, cages, etc.).

[000143] The terms "C-X-C chemokine receptor type 5" or "CXCR5" used interchangeably herein, also referred to in this field as "CD185" and "Burkitt's lymphoma receptor 1 (BLR1)", mean G-protein coupled receptor expressed in some cells. The term "CXCR5" includes homologs and orthologs of CXCR5, including but not limited to human, cynomolgus, rat, rabbit, and mouse homologs and orthologs. As used herein, the term "CXCR5" refers to mammalian CXCR5, such as human, rat, or mouse CXCR5, as well as non-human primate, bovine, ovine, or porcine CXCR5. Non-limiting examples of CXCR5 include human CXCR5 (see, e.g., Genbank accession number P60568, SEQ ID NO:32), cynomolgus monkey (see, e.g., Genbank accession number Q29615, SEQ ID NO:33), and mouse (SEQ ID NO: 34). The term "CXCR5" also includes fragments, variants, isoforms, and other homologues of such CXCR5 molecules. Variant CXCR5 molecules will generally differ in having the same type of activity as native CXCR5, such as the ability to bind to the CXCR5 receptor, the ability to induce mediated receptor activity, and the ability to bind or not bind to an antibody or antigen-binding fragment of the invention.

[000144] CXCR5 may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, or fifteen or more available surface residues of CXCR5. In FIG. 6 shows the amino acid sequence of wild-type mouse and human CXCR5, with examples of available surface residues underlined. If CXCR5 contains a homomultimeric form of CXCR5, the target may contain one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more or fifteen or more accessible surface residues of the first CXCR5 subunit, and one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more , ten or more, twelve or more, or fifteen or more accessible surface residues of the second CXCR5 subunit. The target molecule may contain a known epitope from CXCR5.

[000145] As noted above, the aligned amino acid sequences of human CXCR5 (hCXCR5) and murine CXCR5 (mCXCR5) wild-type, indicating (underlined) various examples of extracellular regions (labeled "N", "L1", "L2" and "3" ) proteins are shown in FIG. 5.

[000146] As indicated elsewhere in this description, you can change some positions of the antibody molecule. In the context of the invention, the term "position" means a place in the sequence of a protein. Positions can be numbered sequentially or according to a predetermined format, for example, the EU suffix and the Kabat suffix can be used to number the amino acid residues of an antibody. For example, position 297 is a position in a human IgG1 antibody. Appropriate positions are determined as above, typically by alignment with other parent sequences.

[000147] As used herein, the term "residue" means a position in a protein and the type of amino acid associated with it. For example, asparagine 297 (also referred to as Asn297, also referred to as N297) is a residue in a human IgG1 antibody.

[000148] The terms "Tfh cell" or "Tfh" or "true Tfh" or "germinal center Tfh cell" or "GC Tfh cell" as used interchangeably herein refer to follicular helper T -cells found in the germinal center (GC), which is a structure consisting of Tfh GC cells, B GC cells, follicular dendritic cells (FDC), macrophages and stroma. See Crotty, 2014, Immunity 41(4):529-542. Tfh cells are identified by constitutive expression of the B cell follicle homing receptor CXCR5. Functionally, Tfh cells provide instructional signals to B cells, guiding isotype switches, somatic hypermutations, and rapid cell division to populate germinal centers.

[000149] The terms "Tfh-like cells", "circulating Tfh cells" and "cTfh", used interchangeably herein, refer to Tfh cells that have left the germinal center. Upon exiting the GC, the cells acquire a less activated, less polarized phenotype and are referred to as "circulating follicular helper T cells" (cTfh) or "Tfh-like cells". See Crotty, 2014, Immunity 41(4):529-542. These cells express CXCR5. Compared to germ center Tfh cells (i.e. "true Tfh cells" or "GC Tfh cells" or "Tfh"), cTfh cells (i.e. Tfh-like cells) express reduced levels of ICOS, Bcl-6, and cellular activation markers such as CD69 and HLA-DR, but retain the ability to stimulate Ab production and Ig class switching in B cells in vitro after reactivation with cognate antigens.

[000150] As is known in the art, the terms "polynucleotide" or "nucleic acid", as used interchangeably herein, refer to chains of nucleotides of any length and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be inserted into the chain by DNA or RNA polymerases. The polynucleotide may contain modified nucleotides such as methylated nucleotides and their analogs. If a modification is present in the structure of the nucleotide, it can be included before or after chain assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example by conjugation with a labeled moiety. Other types of modifications include, for example, capping, replacement of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, modifications with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged bonds (e.g. phosphorothioates, phosphorodithioates, etc.), modifications containing side residues, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications with intercalators (for example, acridine, psoralen, etc.), modifications containing chelators (for example, metals, radioactive metals, boron, oxidizing metals, etc.), modifications containing alkylators, modifications with modified bonds (for example , alpha-anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides. In addition, any of the hydroxyl groups typically present in sugars can be replaced with, for example, phosphonate groups, phosphate groups, protected with standard protecting groups, or activated to make additional bonds to additional nucleotides, or can be conjugated to solid supports. The 5' and 3' OH can be phosphorylated or substituted with amines or organic caps of 1-20 carbons. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose sugars generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose , carbocyclic sugar analogs, alpha or beta anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and nucleoside analogs without a nitrogen base such as methyl riboside. One or more phosphodiester bonds can be replaced by alternative linker groups. These alternative linker groups include, but are not limited to, embodiments where phosphate is replaced by P(O)S ("thioatom"), P(S)S ("dithioatom"), (O)NR ₂ ("amidate"), P(O)R, P(O)OR', CO or CH ₂ ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C), optionally containing an ether bond (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or apaldyl. Not all bonds in a polynucleotide need to be identical. The previous description applies to all polynucleotides mentioned in the present description, including RNA and DNA.

[000151] As used herein, the term "vector" means a construct that can deliver and preferably express one or more genes or sequences of interest in a host cell. Non-limiting examples of vectors include viral vectors, deproteinized DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors coupled to cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and some eukaryotic cells such as producer cells.

[000152] The term "host cell" includes a single cell or cell culture that can be or are a recipient for vectors for inserting polynucleotide inserts. Host cells include the progeny of a single host cell, and the progeny may not be completely identical (in morphology or genomic DNA) to the original parent cell due to natural, accidental or intentional mutation. A host cell includes cells transfected and/or transformed in vivo using a polynucleotide of the invention.

[000153] Host cells can be prokaryotic cells or eukaryotic cells. Examples of eukaryotic cells include mammalian cells such as primate cells or non-primate animal cells; fungal cells such as yeast; plant cells and insect cells.

[000154] Any host cell receptive to cell culture and protein or polypeptide expression can be used in accordance with the present invention. In some embodiments, the implementation of the host cell belongs to a mammal. Mammalian cell lines available as hosts for expression are well known in the art and include many of the immortalized cell lines available from the American Type Culture Collection (ATCC). Non-limiting examples of mammalian cells include NS0 cells, HEK 293 cells, and Chinese Hamster Ovary (CHO) cells and their derivatives such as 293-6E cells and CHO DG44, CHO DXB11, and Potelligent® CHOK1SV cells (BioWa/Lonza, Allendale, NJ). Mammalian host cells also include, as non-limiting examples, human cervical carcinoma cells (HeLa, ATCC CCL 2), newborn hamster kidney cells (BHK, ATCC CCL 10), monkey kidney (COS) cells, and human hepatocellular carcinoma cells (e.g. , Hep G2). Other non-limiting examples of mammalian cells that can be used in accordance with the present invention include human retinoblasts (PER.C6®; CruCell, Leiden, The Netherlands); monkey liver cell line CV1 transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line 293 (HEK 293) or 293 cells subcloned for growth in suspension culture (Graham et al., 1977, J. Gen Virol. 36:59); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); dog kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; human hepatoma cell line (Hep G2); and numerous myeloma cell lines, including, but not limited to, the BALB/c mouse myeloma cell line (NS0/1, ECACC No. 85110503), NS0 cells, and Sp2/0 cells.

[000155] In addition, any number of commercially and non-commercially available cell lines expressing polypeptides or proteins can be used in the present invention. One of skill in the art will appreciate that different cell lines may have different nutrient requirements and/or may require different culture conditions for optimal growth and expression of a polypeptide or protein, and one of skill in the art will be able to modify the conditions as needed.

[000156] The invention includes any eukaryotic expression system known in the art or provided herein for the production of proteins of interest, such as an insect cell expression system, a yeast expression system, or a mammalian cell system, as non-limiting examples, such as CHO cells.

[000157] As used herein, the term "expression control sequence" means a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence may be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[000158] In the context of the invention, "treatment" is an approach to obtain favorable or desired clinical results. For purposes of the present invention, beneficial or desirable results include, but are not limited to, one or more of the following: improved survival rate (reduced mortality), reduced inflammatory response to disease, reduced tissue fibrosis, improved appearance of disease foci, reduced pathological foci to focal foci, reducing the extent of disease damage, reducing the duration of the disease, and/or reducing the number, extent, or duration of symptoms associated with the disease. The term includes the administration of the compounds or agents of the present invention to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease, alleviate symptoms, or stop or inhibit the further development of a disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease or to prevent the onset of its clinical or subclinical symptoms) or therapeutic suppression or relief of symptoms after the onset of the disease.

[000159] The term "improvement" means a reduction or improvement in one or more symptoms compared to no administration of the CXCR5 antibody. The term "improvement" also includes a reduction in the duration of a symptom.

[000160] As used herein, an "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results. In more specific aspects, the effective amount is capable of preventing, alleviating or ameliorating the symptoms of a disease or infection, and/or prolonging the survival of the individual being treated. In the case of prophylactic use, beneficial or desirable results include the elimination or reduction of risk, reduction in severity or delay in the onset of the disease, including the biochemical, histological and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during the development of the disease. In the case of therapeutic use, beneficial or desirable results include clinical results such as reduction of one or more symptoms of a CXCR5-mediated disease, disorder or condition, reduction in the dose of other drugs needed to treat the disease, enhancement of the effect of another drug, and/or delay in the progression of the disease. in patients. An effective dose may be administered in one or more administrations. For the purposes of the present invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, a prophylactic or therapeutic treatment. As should be understood in a clinical context, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, the term "effective dose" may be considered in the context of the administration of one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if, when combined with one or more other agents, the desired result can be achieved or is achieved.

[000161] "Individual" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals (eg, cows, pigs, horses, chickens, etc.), sport animals, pets, primates, horses, dogs, cats, mice, and rats. In some embodiments, the individual is considered to be at risk of developing a disease, disorder, or condition mediated or associated with CXCR5 binding to its receptor and signaling mediated by it. In some embodiments, the individual has an autoimmune disease, disorder, or condition, such as type 1 diabetes. In some embodiments, the individual is in need of immunosuppressive therapy.

[000162] As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the individual's immune system. Non-limiting examples include any of the standard pharmaceutical carriers such as phosphate saline, water, emulsions such as an oil-in-water emulsion, and various types of humectants. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well known, conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

[000163] Examples of methods and materials are provided herein, although methods and materials similar or equivalent to the methods and materials presented herein may also be used in the practice or testing of the present invention. The materials, methods and examples are illustrative only and are not intended to limit the invention.

II. ANTIBODIES AGAINST CXCR5

[000164] The present invention relates to antibodies that bind to CXCR5. Preferably, the antibodies specifically bind to CXCR5, ie. they bind to CXCR5, but they do not bind detectably or bind with lower affinity to other molecules. The invention further relates to anti-CXCR5 antibodies showing altered effector function. In some embodiments, the altered effector function is increased ADCC. In some embodiments, the antibodies lack fucose or contain detectably reduced levels of fucose (ie, they are afucosylated). The invention also relates to compositions containing such antibodies, as well as to the use of such antibodies, including therapeutic and pharmaceutical uses.

[000165] In one embodiment, the present invention provides any of the following antibodies or compositions (including pharmaceutical compositions) comprising an antibody comprising a light chain sequence or fragment thereof and a heavy chain sequence or fragment thereof derived from any of the following antibodies: 11G2 , 41A10 and 5H7.

[000166] Antibodies that can be used in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (eg, Fab, Fab', F(ab') ₂ , Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugated antibodies, single-chain antibodies (ScFv), their mutants, fusion proteins containing antibody fragments (for example, a domain antibody), humanized antibodies and any other modified configuration of an immunoglobulin molecule containing an antigen recognition site of the required specificity, including variants of antibody glycosylation, amino acid sequence variants of antibodies; and covalently modified antibodies. The antibodies may be mouse, rat, human, or any other source (including chimeric or humanized antibodies). In some embodiments, the anti-CXCR5 antibody is a monoclonal antibody. In some embodiments, the antibody is a human antibody or a humanized antibody.

[000167] Anti-CXCR5 antibodies of the invention can be made by any method known in the art. General methods for producing human and mouse antibodies are known in the art and/or provided herein.

[000168] After initial identification, the activity of a candidate anti-CXCR5 antibody can be further confirmed and refined by known biological assays to test for targeted biological activities. In some embodiments, an in vitro cellular assay is used to further characterize a candidate anti-CXCR5 antibody. For example, biological assays can be used to directly screen candidates. Some of the methods for identifying and characterizing an anti-CXCR5 antibody are detailed in the examples.

[000169] In some aspects, the antibody or antigen-binding fragment thereof comprises at least one amino acid sequence selected from the group consisting of the sequence of VH 11G2 mouse, VL 11G2 mouse, VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157 ), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353), VH h11G2 (XC354), VL h11G2 (XC151), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348), VL h11G2 (XC349), VH 41A10 mice, VL 41A10 mice, VH h41A10 (XC147), VH h41A10 (XC148), VH h41A10 (XC150), VL h41A10 (XC142), VL h41A10 (XC143), VL h41A10 (XC144), VL h41A10 (XC145), VL h41A10 (XC146), VL h41A10 (XC149), VH 5H7 mouse and VL 5H7 mouse.

[000170] In some aspects, the antibody or antigen-binding fragment competes with any of the above antibodies for binding to CXCR5.

[000171] In some aspects, an antibody or antigen-binding fragment thereof comprises a VH amino acid sequence and a VL amino acid sequence, including the following combinations: an antibody or antigen-binding fragment thereof that competes for binding to human CXCR5 with one or more of mouse 11G2, chimeric 11G2, VH h11G2 ( XC152)/VL (XC151), VH h11G2 (XC152)/VL (XC153), VH h11G2 (XC152)/VL (XC154), VH h11G2 (XC152)/VL (XC346), VH h11G2 (XC152)/VL (XC347) ), VH h11G2 (XC152)/VL (XC348), VH h11G2 (XC152)/VL (XC349), VH h11G2 (XC155)/VL (XC151), VH h11G2 (XC155)/VL (XC153), VH h11G2 (XC155 )/VL (XC154), VH h11G2 (XC155)/VL (XC346), VH h11G2 (XC155)/VL (XC347), VH h11G2 (XC155)/VL (XC3484), VH h11G2 (XC155)/VL (XC349) , VH h11G2 (XC156)/VL (XC151), VH h11G2 (XC156)/VL (XC153), VH h11G2 (XC156)/VL (XC154), VH h11G2 (XC156)/VL (XC346), VH h11G2 (XC156) /VL (XC347), VH h11G2 (XC156) / VL (XC348), VH h11G2 (XC156) / VL (XC349), VH h11G2 (XC157) / VL (XC151), VH h11G2 (XC157) / VL (XC153), VH h11G2 (XC157)/VL (XC154), VH h11G2 (XC157)/VL (XC346), VH h11G2 (XC157)/VL (XC347), VH h11G2 (XC157)/VL (XC348), VH h11G2 (XC157)/ VL (XC349) h11G2 (XC350)/VL (XC347), VH h11G2 (XC350)/VL (XC348), VH h11G2 (XC350)/VL (XC349), VH h11G2 (XC351)/VL (XC151), VH h11G2 (XC351)/VL VH h11G2 (XC351)/VL (XC154), VH h11G2 (XC351)/VL (XC346), VH h11G2 (XC351)/VL (XC347), VH h11G2 (XC351)/VL (XC348) (XC351)/VL (XC349), VH h11G2 (XC352)/VL (XC151), VH h11G2 (XC352)/VL (XC153), VH h11G2 (XC352)/VL (XC154), VH h11G2 (XC352)/VL ( XC346), VH h11G2 (XC352)/VL (XC347), VH h11G2 (XC352)/VL (XC348), VH h11G2 (XC352)/VL (XC349), VH h11G2 (XC353)/VL (XC151), VH h11G2 ( XC353)/VL (XC153), VH h11G2 (XC353)/VL (XC154), VH h11G2 (XC353)/VL (XC346), VH h11G2 (XC353)/VL (XC347), VH h11G2 (XC353)/VL (XC348) ), VH h11G2 (XC353)/VL (XC349), VH h11G2 (XC354)/VL (XC151), VH h11G2 (XC354)/VL (XC153), VH h11G2 (XC354)/VL (XC154), VH h11G2 (XC354) )/VL (XC346), VH h11G2 (XC354)/VL (XC347), VH h11G2 (XC354)/VL (XC348), VH h11G2 (XC354)/VL (XC349), mouse 41A10, chimeric 41A10, VH h41A10 (XC147 )/VL (XC142), VH h41A10 (XC147)/VL (XC143), VH h41A10 (XC147)/VL (XC144), VH h41A10 (XC147)/VL (XC145), VH h41A10 (XC147)/VL (XC146) , VH h41A10 (XC147)/VL (XC149), VH h41A10 (XC148)/VL (XC142), VH h41A10 (XC148)/VL (XC143), VH h41A10 (XC148)/VL (XC144), VH h41A10 (XC148) /VL (XC145), VH h41A10 (XC148) / VL (XC146), VH h41A10 (XC148) / VL (XC149), VH h41A10 (XC150) / VL (XC142), VH h41A10 (XC150) / VL (XC143), VH h41A10 (XC150)/VL (XC144), VH h41A10 (XC150)/VL (XC145), VH h41A10 (XC150)/VL (XC146), VH h41A10 (XC150)/VL (XC149), mouse 5H7 and chimeric 5H7.

[000172] In some aspects, the antibody or antigen-binding fragment competes with any of the above antibodies for binding to CXCR5.

[000173] In some aspects, the antibody or antigennegative fragment thereof contains CDR-L1, CDR-L2 and CDR-L3 shown in the amino acid sequence of at least one of SEQ ID NOs: 1, 5, 35, 47, 48, 49, 50 and 51.

[000174] In some aspects, the antibody or antigen-binding fragment further comprises CDR-H1, CDR-H2, and CDR-H3 shown in the amino acid sequence of at least one of SEQ ID NOs: 6, 10, 12, 36, 52, 53, 54 , 55, 56, 57.

[000175] In some aspects, the antibody or antigennegative fragment thereof contains CDR-L1, CDR-L2, CDR-L3, shown in the amino acid sequence of SEQ ID NO: 1, and CDR-H1, CDR-H2 and CDR-H3, given in the amino acid the sequence of SEQ ID NO:6.

[000176] In some aspects, the antibody or antigennegative fragment thereof contains CDR-L1, CDR-L2, CDR-L3, shown in the amino acid sequence of SEQ ID NO: 1, and CDR-H1, CDR-H2 and CDR-H3, given in the amino acid the sequence of SEQ ID NO:10.

[000177] In some aspects, an antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO:1 and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence the sequence of SEQ ID NO:12.

[000178] In some aspects, an antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, CDR-L3 as shown in the amino acid sequence of SEQ ID NO:1 and CDR-H1, CDR-H2 and CDR-H3 as shown in the amino acid sequence the sequence of SEQ ID NO:52.

[000179] In some aspects, the antibody or antigennegative fragment thereof further comprises the CDR-H1, CDR-H2 and CDR-H3 shown in the amino acid sequence of SEQ ID NO:6 and the CDR-L1, CDR-L2 and CDR-L3 shown in the amino acid sequence of at least one of SEQ ID NOs: 1, 5, 35, 47, 48, 49, 50 and 51.

[000180] In some aspects, the antibody, or antigen-binding fragment thereof, further comprises CDR-H1, CDR-H2, and CDR-H3 as shown in the amino acid sequence of SEQ ID NO:10, and CDR-L1, CDR-L2, and CDR-L3 as shown in in the amino acid sequence of at least one of SEQ ID NOs: 1, 5, 35, 47, 48, 49, 50 and 51.

[000181] In some aspects, the antibody, or antigen-binding fragment thereof, further comprises CDR-H1, CDR-H2, and CDR-H3 as shown in the amino acid sequence of SEQ ID NO:12, and CDR-L1, CDR-L2, and CDR-L3 as shown in in the amino acid sequence of at least one of SEQ ID NOs: 1, 5, 35, 47, 48, 49, 50 and 51.

[000182] In some aspects, the antibody, or antigen-binding fragment thereof, further comprises CDR-H1, CDR-H2, and CDR-H3 as shown in the amino acid sequence of SEQ ID NO:52, and CDR-L1, CDR-L2, and CDR-L3 as shown in in the amino acid sequence of at least one of SEQ ID NOs: 1, 5, 35, 47, 48, 49, 50 and 51.

[000183] In some aspects, the antibody or antigennegative fragment thereof further comprises CDR-H1, CDR-H2, and CDR-H3 as set forth in the amino acid sequence of SEQ ID NO:27, and CDR-L1, CDR-L2, and CDR-L3 as set forth in amino acid sequence of SEQ ID NO:26.

[000184] In some aspects, the antibody or antigen-binding fragment thereof contains CDR-L1, CDR-L2 and CDR-L3, given in the amino acid sequence encoded by the insert of the plasmid deposited with ATCC and having the registration number PTA-124324.

[000185] In some aspects, an antibody or antigen-binding fragment thereof comprises a CDR-H1, a CDR-H2, and a CDR-H3 specified in the amino acid sequence encoded by an insert of a plasmid deposited with ATCC, accession number PTA-124323.

[000186] In some aspects, an antibody, or an antigen-binding fragment thereof, comprises the amino acid sequence of CDR-L1, CDR-L2, and CDR-L3 encoded by an insert of a plasmid deposited with ATCC and has accession number PTA-124324, and the amino acid sequence of CDR-H1, CDR- H2 and CDR-H3 encoded by a plasmid insert deposited with the ATCC with accession number PTA-124323.

[000187] In some aspects, the antibody or antigen-binding fragment competes with any of the above antibodies for binding to CXCR5.

[000188] In some aspects, an antibody or antigen-binding fragment thereof comprises a CDR-L1 containing the amino acid sequence of SEQ ID NO:2, a CDR-L2 containing the amino acid sequence of SEQ ID NO:3, a CDR-L3 containing the amino acid sequence of SEQ ID NO:4 , CDR-H1 containing the amino acid sequence of SEQ ID NO:7, CDR-H2 containing the amino acid sequence of SEQ ID NO:8, and CDR-H3 containing the amino acid sequence of SEQ ID NO:9.

[000189] In some aspects, an antibody or antigen-binding fragment thereof comprises a CDR-L1 containing the amino acid sequence of SEQ ID NO:2, a CDR-L2 containing the amino acid sequence of SEQ ID NO:3, a CDR-L3 containing the amino acid sequence of SEQ ID NO:4 , CDR-H1 containing the amino acid sequence of SEQ ID NO:7, CDR-H2 containing the amino acid sequence of SEQ ID NO:8, and CDR-H3 containing the amino acid sequence of SEQ ID NO:11.

[000190] In some aspects, an antibody or antigen-binding fragment thereof comprises a CDR-L1 containing the amino acid sequence of SEQ ID NO:2, a CDR-L2 containing the amino acid sequence of SEQ ID NO:3, a CDR-L3 containing the amino acid sequence of SEQ ID NO:4 , CDR-H1 containing the amino acid sequence of SEQ ID NO:19, CDR-H2 containing the amino acid sequence of SEQ ID NO:20, and CDR-H3 containing the amino acid sequence of SEQ ID NO:21.

[000191] In some aspects, an antibody or antigen-binding fragment thereof comprises a CDR-L1 containing the amino acid sequence of SEQ ID NO:14, a CDR-L2 containing the amino acid sequence of SEQ ID NO:15, a CDR-L3 containing the amino acid sequence of SEQ ID NO:16 , CDR-H1 containing the amino acid sequence of SEQ ID NO:19, CDR-H2 containing the amino acid sequence of SEQ ID NO:20, and CDR-H3 containing the amino acid sequence of SEQ ID NO:21.

[000192] In some aspects, an antibody or antigen-binding fragment thereof comprises a CDR-L1 containing the amino acid sequence of SEQ ID NO:14, a CDR-L2 containing the amino acid sequence of SEQ ID NO:15, a CDR-L3 containing the amino acid sequence of SEQ ID NO:16 , CDR-H1 containing the amino acid sequence of SEQ ID NO:7, CDR-H2 containing the amino acid sequence of SEQ ID NO:8, and CDR-H3 containing the amino acid sequence of SEQ ID NO:9.

[000193] In some aspects, an antibody or antigen-binding fragment thereof comprises a CDR-L1 containing the amino acid sequence of SEQ ID NO:14, a CDR-L2 containing the amino acid sequence of SEQ ID NO:15, a CDR-L3 containing the amino acid sequence of SEQ ID NO:16 , CDR-H1 containing the amino acid sequence of SEQ ID NO:8, CDR-H2 containing the amino acid sequence of SEQ ID NO:9, and CDR-H3 containing the amino acid sequence of SEQ ID NO:10.

[000194] In some aspects, an antibody or antigen-binding fragment thereof comprises the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 shown in at least one of SEQ ID NOs: 6, 10, 12, 36, 52, 53, 54, 55, 56 and 57.

[000195] In some aspects, an antibody or antigen-binding fragment thereof comprises the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 shown in at least one of SEQ ID NOs: 1, 5, 35, 47, 48, 49, 50 and 51.

[000196] In some aspects, the antibody or antigennegative fragment contains the amino acid sequences CDR-L1, CDR-L2 and CDR-L3 shown in SEQ ID NO:5.

[000197] An antibody or antigen-binding fragment thereof may comprise a VH containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:6. The VH may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:6. VH may contain the amino acid sequence of SEQ ID NO:6.

[000198] An antibody or antigen-binding fragment thereof may comprise a VH containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:10. The VH may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:10. VH may contain the amino acid sequence of SEQ ID NO:10.

[000199] An antibody, or antigen-binding fragment thereof, may comprise a VH containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:12. The VH may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:12. VH may contain the amino acid sequence of SEQ ID NO:12.

[000200] The antibody or antigen-binding fragment may comprise a VL containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1. VL may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:1. VL may contain the amino acid sequence of SEQ ID NO:1.

[000201] The antibody or antigen-binding fragment may comprise a VL containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:5. VL may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:5. VL may contain the amino acid sequence of SEQ ID NO:5.

[000202] An antibody or antigen-binding fragment thereof may comprise a VH containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:17. The VH may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:17. VH may contain the amino acid sequence of SEQ ID NO:17.

[000203] An antibody, or antigen-binding fragment thereof, may comprise a VH containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:18. The VH may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:18. VH may contain the amino acid sequence of SEQ ID NO:18.

[000204] The antibody or antigen binding fragment may comprise a VL containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:13. VL may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:13. VL may contain the amino acid sequence of SEQ ID NO:13.

[000205] The antibody or antigen-binding fragment may comprise a VL containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:58. VL may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:58. VL may contain the amino acid sequence of SEQ ID NO:58.

[000206] In one aspect, the afucosylated antibody or antigen-binding fragment thereof may comprise a heavy chain containing a VH selected from a VH containing the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:36, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57 and additionally containing an IgG1 constant domain (SEQ ID NO:31). In one aspect, said antibody variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 additions and/or deletions in the full length heavy chain. In a further aspect, said variant has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least at least 98% or at least 99% sequence identity to the full length heavy chain, and wherein said antibody or antigen-binding fragment specifically binds to CXCR5.

[000207] An afucosylated antibody of the invention may comprise a VL containing light chain comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:35, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51, wherein the antibody further comprises a light chain constant domain. As detailed elsewhere herein, the light chain constant domain of an afucosylated antibody can be selected from a Cκ or Cλ constant region, eg, the Cκ constant region of SEQ ID NO:30. In one aspect, said antibody variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 additions and/or deletions in the full length light chain. In a further aspect, said variant has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least at least 98% or at least 99% sequence identity to the full length light chain, and wherein said antibody or antigen-binding fragment specifically binds to CXCR5.

[000208] An antibody or antigen binding fragment may comprise a HC containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid the sequence of SEQ ID NO:29. HC may contain the amino acid sequence of SEQ ID NO:29. Preferably, the antibody is afucosylated.

[000209] An antibody or antigen binding fragment may comprise an LC containing an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:28. The LC may contain the amino acid sequence of SEQ ID NO:28. Preferably, the antibody is afucosylated.

[000210] Preferably, the antibody or antigen-binding fragment of the invention is afucosylated. Even more preferably, the antibody or antigen-binding fragment thereof is afucosylated and exhibits enhanced ADCC effector function compared to an otherwise identical antibody that is fucosylated.

Germline substitutions

[000211] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following heavy chain CDR sequences: (i) a CDR-H1 containing SEQ ID NO:7, a CDR-H2 containing SEQ ID NO:8, and a CDR-H3 containing SEQ ID NO: 9 or 11; and/or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO:2, CDR-L2 containing SEQ ID NO:3, and CDR-L3 containing SEQ ID NO:4. They are mouse CDRs and are preferably grafted or otherwise added to the context of the human VH and VL domains. A wide variety of human germline acceptor sequences are available, and methods for "humanizing" antibodies of non-human species for use in humans are well known in the art and are also provided elsewhere herein. Thus, those skilled in the art will appreciate that the above mouse CDR sequences can be placed within the context of human V domain amino acid sequences. However, in order to maintain antibody binding and other desirable characteristics of the original parental (ie, donor) antibody, as a rule, changes can be made in the acceptor sequences of the human germline. Both CDRs and frame regions (FWs) can be constructed as follows.

[000212] In some embodiments, no more than 11, or no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than more than 2 or at most 1 substitution relative to the amino acid sequence of SEQ ID NO:2. In some embodiments, no more than 6, no more than 5, no more than 4, no more than 3, no more than 3, no more than 2, or no more than one substitution is made in CDR-L2 relative to the amino acid sequence of SEQ ID NO:3. In some embodiments, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 3, no more than 2, or no more than one substitution is made in CDR-L3 relative to the amino acid sequence of SEQ ID NO:4. In some embodiments, CDR-H1 makes no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution relative to amino acid sequence of SEQ ID NO:7. In some embodiments, no more than no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11 or no more than 10, no more than 9, no more than 8 , no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 substitution relative to the amino acid sequence of SEQ ID NO:8. In some embodiments, no more than 12, no more than 11, or no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, are made in CDR-H3, no more than 2 or no more than 1 substitution relative to the amino acid sequence of SEQ ID NO:9 or relative to the amino acid sequence of SEQ ID NO:11. In some embodiments, the substitutions do not change the binding affinity (K _D ) value by more than 1000 times, more than 100 times, or 10 times. In some embodiments, the substitution is a conservative substitution according to Table 1.

[000213] In some embodiments, the replacement is a human germline replacement in which a (donor) CDR residue is replaced with a corresponding (acceptor) human germline residue to increase human amino acid content and potentially reduce antibody immunogenicity, as described, for example, in a patent application publication US No. 2017/0073395 and Townsend et al., 2015, Proc. Nat. Acad. sci. USA 112(50):15354-15359). For example, if a human germline DPK9 framework is used and an example VL 11G2 (XC154) mouse antibody or humanized VL 11G2 (XC154) is compared, then the CDR-L1 alignment of VL 11G2 antibody (XC154 mouse and humanized XC154) (SEQ ID NO:2) and The human germline DPK9 is as follows:

Position 27 28 29 thirty 31 32 33 34 35 DPK9 human germline Q S I S S Y L N W VL 11G2 (SEQ ID NO:2) E S V E Y H G T S

[000214] In the case of amino acid position 28 ( in italics ), the human germline residue (acceptor) and the corresponding residues VL 11G2 (XC154) (donor) are the same, and germline replacement is not possible. In the case of positions 27, 29, 30, 31, 32, 33, 34 and 35 (in bold and underlined), the human germline residue (acceptor) and the corresponding VL 11G2 (XC154) residue (donor) are different. VL 11G2 (XC154) residues at these positions can be replaced with the corresponding human germline DPK9 residue to further increase human residue levels. The same can be used for each heavy and light chain CDR to increase the content of human amino acid residues while maintaining binding characteristics, e.g., epitope binding, affinity, etc., and minimizing the content of mouse residues, thus reducing potential immunogenicity, e.g., immune a human anti-mouse antibody (HAMA) response to an antibody in a human.

[000215] Methods and libraries for inserting human germline residues into antibody CDRs are detailed in US Patent Application Publication No. 2017/0073395, and Townsend et al., 2015, Proc. Natl. Acad. sci. USA. 112(50):15354-15359, included in the present description by reference in its entirety.

[000216] The antibody, or antigen-binding fragment thereof, may comprise a VH framework containing a human germline VH framework sequence. The VH framework sequence can be derived from the VH3 germline, VH1 germline, VH5 germline, or VH4 human germline. Preferred human germline heavy chain frameworks are those derived from the VH1, VH3 or VH5 germline. For example, VH scaffolds from the following germlines can be used: IGHV3-23, IGHV3-7, or IGHV1-69 (germline names are based on the IMGT germline definition). Preferred human germline light chain frameworks are those derived from the Vκ or Vλ germline. For example, VL scaffolds from the following germlines can be used: IGKV1-39 or IGKV3-20 (germline names are based on the IMGT germline definition). Alternatively or additionally, the framework sequence may be a human germline consensus framework sequence, such as the human Vλ1 consensus framework, Vκ1 consensus sequence, Vκ2 consensus sequence, Vκ3 consensus sequence, VH3 germline consensus sequence, VH1 germline consensus sequence, germline consensus sequence left line VH5 or the VH4 germline consensus sequence. Human germline scaffold sequences are available from various public databases such as V-base, IMGT, NCBI or Abysis.

[000217] The antibody, or antigen-binding fragment thereof, may comprise a VL framework containing a human germline VL framework sequence. The VL framework may contain one or more amino acid substitutions, additions or deletions while maintaining functional and structural similarity to the germline from which it is derived. In some aspects, the VL scaffold is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human germline VL framework sequence. In some aspects, the antibody or antigen-binding fragment thereof comprises a VL framework containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions, or deletions relative to the human germline VL framework sequence. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, or deletions are the only ones in the framework regions. In some aspects, % identity is based on similarity to VL, except for fragments defined herein as CDRs.

[000218] The human germline VL scaffold may be a DPK9 scaffold (IMGT name: IGKV1-39). The human germline VL scaffold may be a DPK12 scaffold (IMGT name: IGKV2D-29). The human germline VL scaffold may be a DPK18 scaffold (IMGT name: IGKV2-30). The human germline VL scaffold may be a DPK24 scaffold (IMGT name: IGKV4-1). The human germline VL scaffold may be the HK102_V1 scaffold (IMGT name: IGKV1-5). The human germline VL scaffold may be a DPK1 scaffold (IMGT name: IGKV1-33). The human germline VL scaffold may be a DPK8 scaffold (IMGT name: IGKV1-9). The human germline VL scaffold may be a DPK3 scaffold (IMGT name: IGKV1-6). The human germline VL scaffold may be a DPK21 scaffold (IMGT name: IGKV3-15). The human germline VL scaffold may be the Vg_38K scaffold (IMGT name: IGKV3-11). The human germline VL scaffold may be a DPK22 scaffold (IMGT name: IGKV3-20). The human germline VL scaffold may be a DPK15 scaffold (IMGT name: IGKV2-28). The human germline VL scaffold may be the DPL16 scaffold (IMGT name: IGLV3-19). The human germline VL scaffold may be a DPL8 scaffold (IMGT name: IGLV1-40). The human germline VL scaffold may be the V1-22 scaffold (IMGT name: IGLV6-57). The human germline VL framework may be a human Vλ consensus sequence framework. The human germline VL framework may be a human Vλ1 consensus sequence framework. The human germline VL framework may be the human Vλ3 consensus sequence framework. The human germline VL backbone may be a human Vκ consensus sequence backbone. The human germline VL backbone may be the human Vκ1 consensus sequence backbone. The human germline VL framework may be a human Vκ2 consensus sequence framework. The human germline VL framework may be the human Vκ3 consensus sequence framework.

[000219] In some aspects, the VL framework is DPK9. Other similar framework regions are predicted to produce preferred antibodies of the invention comprising CDRs of SEQ ID NOs: 2, 3, 4, 7, 8, 9, 11, 14, 15, 16; and CDRs defined by the following VL amino acid sequences: 1, 5, 13, 28, 35, 37, 39, 47, 48, 48, 50, 51, 58, 59, 60, 61, 62, 97, 98, including DPK5, DPK4, DPK1, IGKV1-5*01, DPK24, DPK21, DPK15, IGKV1-13*02, IGKV1-17*01, DPK8, IGKV3-11*01 and DPK22 having 99, 97, 97, 96, 80, 76 , 66, 97, 97, 96, 76, and 74% identity, respectively, to the DPK-9 FW region and one or fewer amino acid differences in common structural features (Kabat numbering): (A) residues immediately below the CDR (zone Vernier), L2, L4, L35, L36, L46, L47, L48, L49, L64, L66, L68, L69, L71, (B) VH/VL chain packing residues: L36, L38, L44, L46, L87 and (C) canonical CDR structural support residues L2, L48, L64, L71 (see Lo, "Antibody Humanization by CDR Grafting", (2004) Antibody Engineering, Vol. 248, Methods in Molecular biology pp 135-159 and O'Brien and Jones, "Humanization of Monoclonal antibodies by CDR Grafting", (2003) Recombinant Antibodies for Cancer Therapy, Vol. 207, Methods in Molecular biology pp 81-100). Particularly preferred are DPK5, DPK4, DPK1, IGKV1-5*01, DPK24, DPK21 and DPK15 framework regions having 99%, 97%, 97%, 96%, 80%, 76%, 66% identity to DPK9, respectively, and no difference amino acids in these common structural features. In some aspects, % identity is based on similarity to VL, except for fragments defined herein as CDRs.

[000220] The antibody, or antigen-binding fragment thereof, may comprise a VH framework containing a human germline VH framework sequence. The VH framework may contain one or more amino acid substitutions, additions or deletions while maintaining functional and structural similarity to the germline from which it is derived. In some aspects, the VH framework is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human germline VH framework sequence. In some aspects, the antibody or antigen-binding fragment thereof comprises a VH framework containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions, or deletions relative to the human germline VH framework sequence. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, or deletions are the only ones in the framework regions. In some aspects, % identity is based on similarity to VH, except for fragments defined herein as CDRs.

[000221] The human germline VH framework may be a DP54 or IGHV3-7 framework. The human germline VH scaffold can be a DP47 or IGHV3-23 scaffold. The human germline VH scaffold can be a DP71 or IGHV4-59 scaffold. The human germline VH scaffold can be a DP75 or IGHV1-2_02 scaffold. The human germline VH scaffold can be a DP10 or IGHV1-69 scaffold. The human germline VH scaffold can be a DP7 or IGHV1-46 scaffold. The human germline VH scaffold can be a DP49 or IGHV3-30 scaffold. The human germline VH scaffold can be a DP51 or IGHV3-48 scaffold. The human germline VH scaffold can be a DP38 or IGHV3-15 scaffold. The human germline VH scaffold can be a DP79 or IGHV4-39 scaffold. The human germline VH scaffold may be a DP78 or IGHV4-30-4 scaffold. The human germline VH scaffold can be a DP73 or IGHV5-51 scaffold. The human germline VH scaffold can be a DP50 or IGHV3-33 scaffold. The human germline VH scaffold can be a DP46 or IGHV3-30-3 scaffold. The human germline VH scaffold can be a DP31 or IGHV3-9 scaffold. The human germline VH framework may be a human germline VH consensus sequence framework. The human germline VH framework may be a human VH3 germline consensus sequence framework. The human germline VH framework may be the human germline VH5 consensus sequence framework. The human germline VH framework may be a human VH1 germline consensus sequence framework. The human germline VH framework may be the human germline VH4 consensus sequence framework.

[000222] In some aspects, the VH framework is DP-54. Other similar framework regions are predicted to produce preferred antibodies of the invention containing CDRs of SEQ ID NOs: 7, 8, 9, 11,19, 20, 21 and CDRs defined by the following VH amino acid sequences: 6, 10, 12, 17, 18, 36, 38, 40, 52, 53, 54, 55, 56, 57, 63, 96, including DP-50, IGHV3-30*09, IGHV3-30*15, IGHV3-48*01, DP -77, DP-51, IGHV3-66*01, DP-53, DP-48, IGHV3-53*01, IGHV3-30*02 and DP-49 having 93, 92, 92, 99, 97, 97, 96, 96, 94, 94, 93, 92% identity, respectively, to the DP-54 FW region and one or more amino acid differences in common structural features (Kabat numbering): (A) residues immediately below the CDR (Vernier zone) , H2, H47, H48, and H49, H67, H69, H71, H73, H93, H94, (B) VH/VL chain packing residues: H37, H39, H45, H47, H91, H93 and (C) canonical structural supporting CDR residues H24, H71, H94 (see Lo 2004, and O'Brien and Jones 2003). Particularly preferred are DP-50, IGHV3-30*09, IGHV3-30*15 framework regions having 93%, 92% and 92% identity to DP-54, respectively, and no amino acid differences in these common structural features. In some aspects, % identity is based on similarity to VH, except for fragments defined herein as CDRs.

[000223] In some embodiments, an antibody or antigen-binding fragment provided herein comprises (i) a VH containing an amino acid sequence of at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8, and/or (ii) a VL containing an amino acid sequence of at least 50%, at least 60%, at least 66%, at least 70%, at least 75%, at least 76%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. The invention also includes any combination of these VL and VH sequences.

[000224] In some embodiments, an antibody or antigen-binding fragment provided herein comprises (i) an HC containing an amino acid sequence of at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29; and/or (ii) an LC containing an amino acid sequence of at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:28. The invention also includes any combination of these HC and LC sequences.

[000225] In some embodiments, the implementation of the antibody or its antigennegative fragment presented in the present description, contain an Fc domain. The Fc domain can be derived from IgA (eg IgA ₁ or IgA ₂ ), IgG, IgE or IgG (eg IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ ).

[000226] The present invention also provides an antibody or antigen-binding fragment thereof that competes for binding to human CXCR5 with any of the antibodies or antigen-binding fragments thereof provided herein, such as any of the antibodies provided herein (or an antigen-binding fragment thereof). For example, if binding of an antibody or antigen-binding fragment thereof to human CXCR5 prevents subsequent binding of CXCL13 to human CXCR5, the antibody or antigen-binding fragment thereof competes with CXCL13 for binding to human CXCR5.

[000227] Antibodies and antigen-binding fragments of the invention include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab') ₂ , Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugated antibodies, single-chain antibodies (ScFv), their mutants, fusion proteins containing part of an antibody, domain antibodies (dAb), humanized antibodies and any other modified configuration of an immunoglobulin molecule containing an antigen recognition site of the required specificity, including variants of antibody glycosylation, variants of the amino acid sequence of antibodies and covalently modified antibodies. The antibodies and antigen-binding fragments may be mouse, rat, human, or any other source (including chimeric or humanized antibodies). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody.

Epitope mapping

[000228] The present invention also provides an antibody or antigen-binding fragment thereof that binds to the same human CXCR5 epitope as the antibody or antigen-binding fragment thereof described herein. For example, competitive antibody assays (and analysis of overlapping epitopes) can be performed by SPR, flow cytometry, and any other assay known in the art.

[000229] In one aspect, the present invention provides an antibody, or antigen-binding fragment thereof, that binds to human CXCR5 and cynomolgus monkey CXCR5 but does not bind to mouse CXCR5.

[000230] The present invention also includes an antibody, or antigen-binding fragment thereof, that binds to the N-terminal ("N") region of human CXCR5. The N-terminus of CXCR5 contains amino acid residues 1-51 according to the amino acid sequence numbering of SEQ ID NO:31.

[000231] The present invention includes an antibody, or antigen-binding fragment thereof, that specifically binds to CXCR5 at an epitope containing leucine (L) at amino acid residue position 11 according to the amino acid sequence numbering of SEQ ID NO:32.

[000232] In one aspect, the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to CXCR5 at an epitope containing aspartate (D) at amino acid residue position 22, according to the amino acid sequence numbering of SEQ ID NO:32.

[000233] In one aspect, the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to CXCR5 at an epitope containing leucine at amino acid residue position 11 and aspartate at amino acid residue position 22, according to the amino acid sequence numbering of SEQ ID NO:32.

[000234] The present invention includes an antibody or antigen-binding fragment thereof that specifically binds to CXCR5 when the amino acid residue at position 11 is leucine, according to the amino acid sequence numbering of SEQ ID NO:32.

[000235] The present invention includes an antibody or antigen-binding fragment thereof that specifically binds to CXCR5 if the amino acid residue at position 11 is leucine, but does not bind to CXCR5 if the amino acid residue at position 11 is threonine, according to the amino acid sequence numbering of SEQ ID NO: 32.

[000236] In one aspect, the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to wild-type human CXCR5 but does not bind if the amino acid residue at position 11 according to the amino acid sequence numbering of SEQ ID NO:32 is not leucine.

[000237] The present invention includes an antibody or antigen-binding fragment thereof that specifically binds to CXCR5 when the amino acid residue at position 22 is aspartate, according to the amino acid sequence numbering of SEQ ID NO:32.

[000238] In one aspect, the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to wild-type human CXCR5, but does not bind if the amino acid residue at position 22 is not an aspartate according to the amino acid sequence numbering of SEQ ID NO:32.

[000239] In one aspect, the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to wild-type human CXCR5 but does not bind if the amino acid residue at position 22 is alanine according to the amino acid sequence numbering of SEQ ID NO:32.

[000240] The present invention includes an antibody or antigen-binding fragment thereof that specifically binds to CXCR5 when the amino acid residue at position 11 is leucine and the amino acid residue at position 22 is aspartate, according to the amino acid sequence numbering of SEQ ID NO:32.

[000241] In one aspect, the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to human CXCR5 but does not bind to CXCR5 if the amino acid residue at position 11 is not leucine and the amino acid residue at position 22 is not aspartate, according to by numbering the amino acid sequence of SEQ ID NO:32.

[000242] In one aspect, the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to human CXCR5 but does not bind if the amino acid residue at position 11 is threonine and the amino acid residue at position 22 is alanine, according to SEQ amino acid sequence numbering. ID NO:32.

[000243] Those of skill in the art, in view of the teachings presented herein, will appreciate that the amino acid residue at position 11 and/or position 22, according to the amino acid sequence numbering of SEQ ID NO:32, is critical for binding an antibody of the invention. More specifically, these amino acid residues are critical for binding to the CXCR5 11G2 antibody or antigen-binding fragment thereof. Thus, those skilled in the art will appreciate that the present invention includes an antibody or antigen-binding fragment thereof that specifically binds to CXCR5 when the amino acid residue at position 11 is leucine and the amino acid residue at position 22 is aspartate. Substitution or deletion of these amino acid residues can result in loss of binding to CXCR5. Some substitutions of the amino acid residue at position 11 may result in retention of binding, but not the substitution of this amino acid residue (leucine) with threonine. Likewise, some substitutions or deletions of the amino acid residue at position 22 may result in retention of binding, but not the replacement of the aspartate at that position of the amino acid residue with alanine. Thus, it is a feature of an 11G2 antibody, or an antigen-binding fragment thereof, or an antibody competing with them for binding, that the antibody binds to human CXCR5 if leucine 11 and aspartate 22 are present, according to the amino acid sequence numbering of SEQ ID NO:32, but the antibody does not bind if leucine 11 is replaced with threonine or another amino acid residue, and where the antibody does not bind if aspartate 22 is replaced with alanine or another amino acid residue. Testing for loss of binding following an amino acid change at amino acid residue position 11 and/or 22 can be performed using a wide range of methods well known in the art, including binding assays using point mutation polypeptides, as in the case of the methods exemplified herein. description.

[000244] In view of the ideas presented herein, one of skill in the art will appreciate that an antibody of the invention can compete with, for example, 11G2 and still lack an epitope containing leucine at amino acid residue 11 and/or aspartate at position amino acid residue 22 according to the amino acid sequence numbering of SEQ ID NO:32. Those. an antibody can compete with an antibody of the invention for binding to CXCR5, but binding of a competing antibody is not affected if the leucine at amino acid residue position 11 or the aspartate at amino acid residue position 22 is replaced with another amino acid (e.g., threonine for leucine and/or alanine for aspartate ), such as 2C9, 11A7 and 16D7. Thus, an antibody of the invention competes for binding to CXCR5 and also does not bind to CXCR5 if the amino acid residue at position 11 is not leucine, and more specifically it is threonine, and/or if the amino acid residue at position 22 is not aspartate, and more specifically, it is an alanine. As noted above elsewhere in this specification, the production of mutant CXCR5 proteins and assays to evaluate competitive antibody binding are well known in the art, including the methods described herein. Thus, in view of the teachings presented herein, those skilled in the art can easily identify antibodies that bind to CXCR5, compete for binding to an antibody of the invention, and lose their ability to bind to CXCR5 muteins if certain amino acids are substituted, for example, leucine 11, aspartate 22 or both, according to the amino acid sequence numbering of SEQ ID NO:32.

[000245] In another aspect, the present invention includes an antibody or antigen-binding fragment thereof that competes for binding with an 11G2 antibody or antigen-binding fragment thereof, wherein the antibody does not bind to CXCR5 if the amino acid residue at position 11 is not leucine according to the SEQ ID amino acid sequence numbering. NO:32.

[000246] In another aspect, the present invention includes an antibody or antigen-binding fragment thereof that competes for binding with an 11G2 antibody or antigen-binding fragment thereof, wherein the antibody does not bind to CXCR5 if the amino acid residue at position 22 is not an aspartate according to the amino acid sequence numbering of SEQ ID NO:32.

[000247] In another aspect, the present invention includes an antibody or antigen-binding fragment thereof that competes for binding with an 11G2 antibody or antigen-binding fragment thereof, wherein the antibody does not bind to CXCR5 if the amino acid residue at position 11 is not leucine and the amino acid residue at position 22 is not is an aspartate according to the amino acid sequence numbering of SEQ ID NO:32.

[000248] In another aspect, the present invention includes an antibody or antigen-binding fragment thereof that competes for binding with an 11G2 antibody or antigen-binding fragment thereof, wherein the antibody does not bind to CXCR5 if the amino acid residue at position 11 is threonine according to the amino acid sequence numbering of SEQ ID NO :32.

[000249] In another aspect, the present invention includes an antibody or antigen-binding fragment thereof that competes for binding with an 11G2 antibody or antigen-binding fragment thereof, wherein the antibody does not bind to CXCR5 if the amino acid residue at position 22 is alanine according to the amino acid sequence numbering of SEQ ID NO :32.

[000250] In another aspect, the present invention includes an antibody or antigen-binding fragment thereof that competes for binding with an 11G2 antibody or antigen-binding fragment thereof, wherein the antibody does not bind to CXCR5 if the amino acid residue at position 11 is threonine and the amino acid residue at position 22 is alanine in accordance with the numbering of the amino acid sequence of SEQ ID NO:32.

Biological activity of antibodies against CXCR5

[000251] In addition to binding an epitope to CXCR5, an antibody or antigen-binding fragment thereof of the invention may mediate biological activity. Those. the present invention includes an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to CXCR5 and mediates at least one detectable activity selected from the following: (a) binds to CXCR5 ⁺ cells with high apparent affinity, but does not bind to cells expressing murine CXCR5 orthologues , rat or rabbit; (b) antagonistically acts on CXCL13 inhibition of cAMP release triggered by forskolin; (c) triggers ADCC of CXCR5-expressing cells in human-donor and cynomolgus monkey PBMCs and human-donor TMCs; (d) binds to human CXCR5 but does not bind to human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 or XCR1; (e) depletes B cells in peripheral blood and/or secondary lymphoid organs (ie, lymph nodes, spleen, Peyer's patches, and mucosal lymphoid tissue); (f) depletes Tfh-like cells in peripheral blood; (g) depletes true Tfh cells in secondary lymphoid organs; and (h) disrupts the humoral anamnestic immune response.

[000252] In one aspect, the present invention includes an antibody that binds to CXCR5 ⁺ cells with high apparent affinity, but does not bind to cells expressing murine, rat, or rabbit CXCR5 orthologues. Apparent binding affinity can be assessed using flow cytometry to detect antibody binding to cells expressing the target protein (eg, CXCR5). Cells can be transiently or stably transfected using a nucleic acid encoding CXCR5. Alternatively, the cells may be cells that naturally express CXCR5 on their surface. Regardless of the sources of CXCR5 ⁺ cells, the binding of antibodies to cells can be easily analyzed using various methods known in this field. The antibody, or antigen-binding fragment thereof, binds to human CXCR5 or cynomolgus monkey CXCR5, but does not bind detectably, or binds to a much lesser extent, to mouse, rat, or rabbit CXCR5.

[000253] The present invention includes an antibody or antigen-binding fragment thereof that specifically binds to CXCR5 and antagonizes activity mediated by CXCL13 binding to CXCR5. There are many assays known in the art for determining inhibition of activity mediated by CXCR5-CXCL13 signaling. One such assay is the cAMP reporter assay. In this assay, forskolin induces cAMP production, which is inhibited by CXCR5-CXCL13 signaling in a cell stably expressing CXCR5. The ability of an anti-CXCR5 antibody to bind to CXCR5 and antagonize the effect of CXCR5-CXCL13 signaling is thus assessed by measuring the level of cAMP produced in the presence or absence of the antibody. Preferably, the antibody can mediate a dose-dependent increase in cAMP levels with an EC ₅₀ of about 50 pM, about 100 pM, about 200 pM, about 400 pM, about 600 pM, about 700 pM, about 750 pM, about 790 pM, about 800 pM, about 850 pM, approximately 900 pM, approximately 950 pM, approximately 960 pM, approximately 970 pM, approximately 980 pM, approximately 990 pM, or approximately 1000 pM. More preferably, the antibody, or antigen-binding fragment thereof, inhibits CXCL13 inhibition of cAMP release triggered by forskolin with an EC ₅₀ of approximately 961 pM.

[000254] The present invention includes an antibody or antigen-binding fragment thereof that specifically binds to CXCR5 and triggers ADCC of CXCR5-expressing cells in human-donor and cynomolgus monkey PBMCs and human-donor tonsillar mononuclear cells (TMCs). Many ADCC assays can be used to evaluate ADCC activity of an antibody. One example of such assays is provided herein in Examples 6 and 7, but the present invention is not limited to this particular ADCC assay. The present invention includes an antibody or antigen-binding fragment thereof showing ADCC activity against human B cells, human Tfh-like cells, human Tfh cells, and cynomolgus B cells with an EC ₅₀ of approximately 0.11 pM, 0.2 pM, 0 .5 pM, 1 pM. 1.5 pM, 2.0 pM, 2.5 pM, 3.0 pM, 4.5 pM, 4.8 pM, 5.0 pM, 6.0 pM, 7.0 pM, 8.0 pM, 9.0 pM, 10 pM, 11 pM, 12 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM or 40 pM. More preferably, the antibody, or antigen-binding fragment thereof, exhibits ADCC activity with an EC ₅₀ of about 2.01±2.28 pM against human B cells, about 4.82±2.88 pM against human Tfh-like cells, about 0.11 pM against human Thf cells and approximately 15.3±11.7 pM against cynomolgus B cells. Even more preferably, the antibody, or antigen-binding fragment thereof, is afucosylated.

[000255] The present invention relates to an antibody or antigen-binding fragment thereof that binds to human CXCR5 but does not bind detectably to the proteins CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, Human CXCR2, CXCR3, CXCR4, CXCR6, CXCR7, or XCR1.

[000256] The present invention relates to an antibody, or antigen-binding fragment thereof, that specifically binds to CXCR5 and exhibits a dose-dependent depletion of B cells in peripheral blood. The depletion may be permanent or, more preferably, the depletion is transient and/or reversible. In one aspect, the dose of antibody sufficient to mediate B cell depletion may be in the range of 0.001 to about 0.2 mg, where 0.0001 mg/kg may mediate B cell depletion and Tfh-like cells in the peripheral blood of a Javanese. macaque by about 50% compared to the percentage of B-cells or Tfh-like cells in peripheral blood if no antibody is administered. In addition, the antibody can mediate the maximum depletion of B-cells and Tfh-like cells in the peripheral blood of cynomolgus monkeys when administered at a dose of less than 5 mg/kg intravenously (IV). Preferably, after administration of the antibody, partial or complete recovery of B cells, Tfh-like cells and Tfh cells occurs.

[000257] The present invention includes an antibody, or antigen-binding fragment thereof, that specifically binds to CXCR5 and impairs the humoral history of the immune response. In one aspect, the humoral anamnestic response is assessed using a secondary vaccine response assay. Those. the antibody is administered to an individual who has previously been vaccinated with an antigen, such as tetanus toxoid (TT). The subject is subjected to a second challenge with the antigen and the immune response, eg, IgM and IgG titers, is compared to that of an otherwise identical individual receiving the antibody. Those of skill in the art, given the teachings presented herein, will appreciate that many humoral history assays can be used to evaluate the ability of an antibody or antigen-binding fragment thereof to interfere with a humoral history response. In addition, those skilled in the art will recognize that the ability to interfere with the humoral history response is a desirable characteristic for an antibody or antigen-binding fragment thereof to be used as a therapeutic agent for the treatment or prevention of an immunological disease in a patient.

[000258] The present invention relates to an antibody or antigen-binding fragment showing at least one, preferably two, even more preferably three, even more preferably four, even more preferably five, even more preferably six, more preferably seven, and even more preferably all of the above biological activities.

III. AFUCOSYLATED ANTIBODIES AGAINST CXCR5

[000259] In one embodiment, the present invention relates to antibodies having a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region (ie, afucosylated antibodies). For example, the amount of fucose in a composition containing a plurality of such antibodies can be from 0 percent to about 30 percent, from 0 percent to about 20 percent, from 0 percent to about 15 percent, from 0 percent to about 10 percent, and more preferably - from 0 percent to about 5 percent. In some embodiments, a composition containing a plurality of such antibodies contains at least 80 percent, more preferably at least 85 percent, even more preferably at least 90 percent, even more preferably at least 95 percent afucosylated antibodies, still more preferably at least 99 percent, and most preferably at least 99.5 percent afucosylated antibodies. In some embodiments, the antibodies are 100 percent afucosylated; those. fucose is not detected for Asn297 using a method known in the art for detecting fucose in an antibody. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain in Asn297 relative to the sum of all glycostructures attached to Asn 297 (eg complex, hybrid and high mannose structures).

[000260] In some embodiments, the level of fucosylation is no more than 0.5%, based on the limit of quantification (LOQ) of the testing method. Thus, in some embodiments, the level of afucosylation is 99.5% or more. The N-linked oligosaccharide profiling method can be used to determine the level of fucosylation, sialylation, mannosylation, and terminal glycosylation in a sample. The N-linked oligosaccharide profiling method can be used to evaluate N-linked glycans. Briefly, N-linked glycans are enzymatically released from the protein by peptide-N-glycosidase F. The glycans are then derivatized with a fluorescent agent and analyzed using hydrophilic interaction liquid chromatography and fluorescence detection. The chromatographic profile is then compared with that of the reference material. This method and many other methods are known in the art for assaying antibody fucosylation and can be used to determine the level of fucosylation of an antibody of the present invention.

[000261] Non-limiting examples of methods for detecting fucose in an antibody include MALDI-TOF mass spectrometry (see, for example, WO 2008/077546), HPLC measurement of released fluorescently labeled oligosaccharides (see, for example, Schneider et al, "N-Glycan analysis of monoclonal antibodies and other glycoproteins using UHPLC with fluorescence detection," Agilent Technologies, Inc. (2012); Lines, J. Pharm. Biomed. Analysis, 14: 601-608 (1996); Takahasi, J. Chrom., 720: 217-225 (1996)), capillary electrophoresis of released fluorescently labeled oligosaccharides (see, e.g., Ma et al., Anal. Chem., 71: 5185-5192 (1999)) and HPLC with pulsed amperometric detection for measuring the composition of monosaccharides ( see, for example, Hardy, et al, Analytical Biochem., 170: 54-62 (1988)). The designation "Asn297" refers to an asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 can also be approximately ±3 amino acids upstream or downstream of position 297, ie. between positions 294 and 300, as a result of small changes in the sequence of antibodies. In the anti-CXCR5 antibody provided herein, Asn297 is found in the QYNST sequence and is bold and underlined in Table 16 (eg, SEQ ID NO:31 of wild-type human IgG1 Fc domain).

[000262] Fucosylation variants may have improved ADCC function. See, for example, US Patent Publication Nos. 2003/0157108 (Presta, L.); 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "afucosylated" or "devoid of fucose" antibodies include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO2003/085119; WO2003/084570; WO2005/035586; W02005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines that can produce afucosylated antibodies include CHO Lec 13 cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. 2003/0157108 A1, Presta, L; and WO 2004/056312, Adams et al., especially in example 11), and knockout cell lines, such as cell lines lacking a functional alpha-1,6-fucosyltransferase gene, FUT8, such as CHO knockout cells ( see, for example, Yamane-Ohnuki et al Biotech Bioeng 87:614 (2004), Kanda, Y et al, Biotechnol Bioeng 94(4):680-688 (2006), and WO2003/085107 ).

[000263] The present invention also relates to antibodies with bisected oligosaccharides, for example, in which the bisected oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibodies may have reduced fucosylation and/or improved ADCC function. Examples of such antibodies are described, for example, in WO 2003/011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umaña et al.) and US 2005/0123546 (Umaña et al.). The present invention relates to antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibodies may have improved CDC function. Such antibodies are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.).

[000264] In some embodiments, the afucosylated antibody mediates ADCC in the presence of human effector cells more efficiently than the fucose-containing parental antibody. In general, ADCC activity can be determined using an in vitro ADCC assay as described herein, but other assays or methods for determining ADCC activity, such as in an animal model, etc., are contemplated.

[000265] In some embodiments, afucosylated anti-CXCR5 antibodies have increased ADCC activity in vitro and/or in vivo . In some embodiments, afucosylated anti-CXCR5 antibodies have increased ADCC activity in vitro . In some embodiments , in vitro ADCC activity is determined by the method described herein. Briefly, serial dilutions of anti-CXCR5 antibodies or isotype control are incubated with peripheral blood mononuclear cells (PBMC) from healthy human donors or cynomolgus monkeys. In this assay, PBMCs are the source of natural killer (NK) effector cells and target CXCR5 ⁺ B cells and Tfh-like cells. Flow cytometry was used to analyze the number of B cells and Tfh-like cells remaining after approximately 20 hours. Cytotoxicity titration curves were generated by plotting percent cytotoxicity of the antigen-binding population against the logarithm of PF-06835375 antibody concentration. EC ₅₀ values were determined using GraphPad Prism® (version 6.0, GraphPad Software, Inc, San Diego, CA) by fitting a non-linear regression curve and log sigmoid in an agonist dose-response model according to the following equation:

Log (agonist) vs. answer - variable slope (four parameters)

Y=Min+(Max-Min/(1+10^((LogEC ₅₀ -X)*Hill slope))

Where Y is the percent cytotoxicity, X is the antibody concentration, Maximum is the maximum Y value corresponding to the upper plateau of the sigmoid curve, Minimum is the minimum Y value corresponding to the lower plateau of the sigmoid curve (limited by 0), and LogEC ₅₀ is the logarithm of the antibody concentration at the inflection point of the curve .

[000266] EC ₅₀ values are summarized across experiments using mean and standard deviation (STDEV). In some embodiments, the ability of humanized mAbs to induce ADCC of true Tfh cells from human tonsils was analyzed in a similar manner using CD4 ⁺ T cells isolated from tonsillar mononuclear cells supplemented with NK cells isolated from PBMC. In some embodiments, Ba/F3 cells expressing CXCR5 are used as target cells. In some embodiments, cytotoxicity is determined by assaying LDH using a non-radioactive CytoTox cytotoxicity assay (Promega, Madison, WI).

[000267] In some embodiments, maximum lysis is determined using 5% Triton X-100 and spontaneous release is determined in the absence of antibody. In some embodiments, the implementation of the percentage of specific lysis can be determined using the formula: (experimental - spontaneous release) / (maximum - spontaneous release) × 100 = percent specific lysis. In some embodiments, an afucosylated anti-CXCR5 antibody having increased ADCC activity results in a specific lysis that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points more higher than specific lysis using the same amount of fucosylated antibody in at least one test antibody concentration. In some embodiments, an afucosylated anti-CXCR5 antibody having increased ADCC activity results in a specific lysis that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points more higher than specific lysis using a fucosylated antibody, where each antibody is at a concentration of 0.01 to 1 μg/ml and the target cells are Ba/F3 cells expressing CXCR5. In some embodiments, antibodies are tested at concentrations ranging from 0.000005 µg/mL to 5 µg/mL.

[000268] In some embodiments, afucosylated anti-CXCR5 antibodies have increased affinity for Fc gamma RIIIA. In some embodiments, afucosylated anti-CXCR5 antibodies have increased affinity for Fc gamma RIIIA(V158). In some embodiments, afucosylated anti-CXCR5 antibodies have increased affinity for Fc gamma RIIIA(F158). In some embodiments, the affinity of an antibody for Fc gamma RIIIA is determined using surface plasmon resonance and/or as described for Fc gamma RIIIA(V158), but which is also suitable for determining affinity for Fc gamma RIIIA(F158). Briefly, in some embodiments, a fucosylated or afucosylated anti-CXCR5 antibody is fixed on a protein A coated dextran chip. Fc gamma RIIIA (V158) (available, for example, from R&D Systems) is injected at various concentrations. The association constant, dissociation constant, and affinity for Fc gamma RIIIA (V158) of a fucosylated and afucosylated anti-CXCR5 antibody can be determined, for example, using software supplied with a surface plasmon resonance system (e.g., 1:1 binding models in Biacore T200 software evaluation software). In some embodiments, an afucosylated anti-CXCR5 antibody may have increased affinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) or Fc gamma RIIIA(F158)) and can bind to Fc gamma RIIIA with at least 2-fold, at least at least 3 times, at least 4 times, at least 5 times, at least 7 times, at least 10 times, at least 12 times, at least 15 times, at least 17 times, 20 times, 30 times, 50 times, 100 times, 500 times or at least 1000 times higher affinity than fucosylated anti-CXCR5 antibody.

IV. EXPRESSION AND PRODUCTION OF ANTIBODY AGAINST CXCR5

NUCLEIC ACIDS ENCODING ANTIBODIES AGAINST CXCR5

[000269] The invention also relates to polynucleotides encoding any of the antibodies, including fragments of antibodies and modified antibodies presented in the present description. The invention also relates to a method for obtaining any of the polynucleotides presented in the present description. Polynucleotides can be generated and expressed by methods known in the art.

[000270] The sequence of a desired antibody, a particular antibody fragment or antigen-binding fragment thereof, and a nucleic acid encoding such antibody or fragment thereof can be determined by standard sequencing methods. A nucleic acid sequence encoding a desired antibody, a particular antibody fragment, or an antigen-binding fragment thereof can be inserted into various vectors (such as cloning and expression vectors) for recombinant production and characterization. A nucleic acid encoding a heavy chain, a specific antibody fragment, or a heavy chain antigen-binding fragment, and a nucleic acid encoding a light chain, a specific antibody fragment, or a light chain antigen-binding fragment can be cloned into the same vector or different vectors.

[000271] In one aspect, the invention provides polynucleotides encoding the amino acid sequences of any of the following anti-CXCR5 antibodies and antigen-binding fragments thereof: mouse VH 11G2, mouse VL 11G2, chimeric VH 11G2, chimeric VL 11G2, VH h11G2 (XC152), VH h11G2 (XC155), VH h11G2 (XC156), VH h11G2 (XC157), VH h11G2 (XC350), VH h11G2 (XC351), VH h11G2 (XC352), VH h11G2 (XC353), VH h11G2 (XC354) ), VL h11G2 (XC153), VL h11G2 (XC154), VL h11G2 (XC346), VL h11G2 (XC347), VL h11G2 (XC348), VL h11G2 (XC349), VH 41A10 mouse, VL 41A10 mouse, chimeric VH 41A10, chimeric VL 41A10, humanized VH 41A10 (XC147), VH h41A10 (XC148), VH h41A10 (XC150), VL h41A10 (XC142), VL h41A10 (XC143), VL h41A10 (XC144), VL h41A10 (XC145), VL h41A10 ( XC146), VL h41A10 (XC149), VH 5H7 mouse, VL 5H7 mouse, chimeric VH 5H7 and chimeric VL 5H7. The polynucleotide encoding the above amino acid sequences encodes an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical and preferably more identical to the amino acid sequence antibodies or its antigennegative fragment of the present invention, presented in the present description.

[000272] The invention relates to polynucleotides encoding the amino acid sequences of an antibody or antigen-binding fragment thereof that binds to essentially the same epitope as an antibody selected from the group consisting of: chimeric 41A10, humanized 41A10 (142/147), humanized 41A10 (142/148), Chimeric 11G2, Afucosylated Chimeric 11G2, Humanized 11G2 (151/152), Humanized 11G2 (153/155), Humanized 11G2 (153/156), Humanized 11G2 (154/155), Humanized 11G2 (154 /157) and afucosylated humanized 11G2 (154/155).

[000273] The invention relates to polynucleotides encoding the amino acid sequences of an antibody or an antigen-binding fragment thereof that competes for binding to CXCR5 with an antibody selected from the group consisting of: chimeric 41A10, humanized 41A10 (142/147), humanized 41A10 (142/148) , chimeric 11G2, afucosylated chimeric 11G2, humanized 11G2 (151/152), humanized 11G2 (153/155), humanized 11G2 (153/156), humanized 11G2 (154/155), humanized 11G2 (154/157), and afucosylated humanized 11G2 (154/155).

[000274] The invention relates to polynucleotides encoding one or more proteins containing an amino acid sequence selected from the group consisting of: SEQ ID NO:1-29, SEQ ID NO:35-40, and SEQ ID NO:47-63.

[000275] The invention relates to polynucleotides containing the nucleic acid sequence shown as one or more of SEQ ID NO:106, 107, 108 and 109. The invention relates to a polynucleotide containing the nucleic acid sequence shown as SEQ ID NO:95. The invention relates to a polynucleotide containing the nucleic acid sequence given as SEQ ID NO:96. The invention relates to a polynucleotide containing the nucleic acid sequence given as SEQ ID NO:97. The invention relates to a polynucleotide containing the nucleic acid sequence given as SEQ ID NO:98.

[000276] The invention relates to a polynucleotide containing one or both of the nucleic acid sequences of a plasmid DNA insert deposited with ATCC and having accession number PTA-124323 and accession number PTA-124324.

[000277] The invention relates to a polynucleotide containing the nucleic acid sequence of an insert in a plasmid deposited with the ATCC and having the accession number PTA-124323.

[000278] The invention relates to a polynucleotide containing the nucleic acid sequence of an insert in a plasmid deposited with the ATCC and having the accession number PTA-124324.

[000279] The invention relates to a polynucleotide containing the nucleic acid sequence of an insert of a plasmid deposited with ATCC and having accession number PTA-124323 and accession number PTA-124324.

[000280] The invention relates to a polynucleotide containing the nucleic acid sequence of a plasmid insert deposited with the ATCC and having the accession number PTA-124323.

[000281] The invention relates to a polynucleotide containing the nucleic acid sequence of a plasmid insert deposited with the ATCC and having the accession number PTA-124324.

[000282] The invention relates to cells containing one or more nucleic acid molecules listed in one or more of SEQ ID NOs: 106, 107, 108 and 109. The invention relates to cells containing one or more nucleic acid molecules listed in SEQ ID NOs: 106 and 107. The invention relates to cells containing one or more nucleic acid molecules shown in SEQ ID NOs: 108 and 109.

[000283] In another aspect, the invention provides polynucleotides and variants thereof encoding an anti-CXCR5 antibody, wherein such polynucleotide variants have at least 70%, at least 75%, at least 80%, at least 85%, at least at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% sequence identity with respect to any of the specific nucleic acid sequences provided herein. These amounts are not meant to be limiting, and increments between the percentages listed are specifically part of the present invention.

[000284] The invention relates to polypeptides encoded by the nucleic acid molecules presented in the present description.

[000285] In one embodiment, the VH and VL domains, or antigen-binding fragments thereof, or full-length HC or LC, are encoded by separate polynucleotides. Alternatively, both VH and VL, or their antigen-binding fragments, or HC and LC, are encoded by the same polynucleotide.

[000286] Polynucleotides complementary to any such sequences are also included in the present invention. Polynucleotides may be single stranded (coding or antisense) or double stranded and may be DNA (genomic DNA, cDNA or synthetic DNA) or RNA molecules. RNA molecules include HnRNA molecules containing introns and corresponding to the DNA molecule one to one, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may optionally be present in the polynucleotide of the present invention, and the polynucleotide may optionally be coupled to other molecules and/or support materials.

[000287] Polynucleotides may contain a native sequence (ie, an endogenous sequence encoding an antibody or portion thereof) or may contain a variant of such a sequence. Variant polynucleotides contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the native immunoreactive molecule. The effect of the immunoreactivity of the encoded polypeptide, as a rule, can be analyzed, as presented in the present description. In some embodiments, the variants have at least about 70% identity, in some embodiments at least about 80% identity, in some embodiments at least about 90% identity, and in some embodiments at least about 95% identity with respect to the polynucleotide sequence encoding the native antibody or part thereof. These amounts are not meant to be limiting, and increments between the percentages listed are specifically part of the present invention.

[000288] Indicate that two polynucleotide or polypeptide sequences are "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned with the maximum match, as described below. Comparison of two sequences is typically performed by comparing sequences in a comparison window to identify and compare local areas of sequence similarity. As used herein, the term "comparison window" refers to a segment of at least about 20 contiguous positions, typically 30 to about 75 or 40 to about 50, in which the sequence can be compared to a reference sequence of the same number of contiguous positions after optimal alignment of two sequences.

[000289] Optimal alignment of sequences for comparison can be performed using the MegAlign® program in the Lasergene® bioinformatics software package (DNASTAR®, Inc., Madison, WI) using default parameters. This program implements several alignment schemes described in the following sources: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. at Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5:151-153; Myers, E.W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E.D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Acad. sci. USA 80:726-730.

[000290] In some embodiments, "percent sequence identity" is determined by comparing two optimally aligned sequences in a comparison window of at least 20 positions, where a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) 20 percent or less, typically 5 to 15 percent or 10 to 12 percent compared to reference sequences (containing no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions by dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the comparison window size) and multiplying result in 100 to get percent sequence identity.

[000291] Additionally or alternatively, the variants may be substantially homologous to the native gene or part or complementary strand. Such polynucleotide variants can hybridize under conditions of moderate stringency to the native DNA sequence encoding the native antibody (or a complementary sequence).

[000292] Suitable "moderate stringency conditions" include prewash in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization at 50°C-65°C in 5x SSC overnight followed by washing twice at 65° C. for 20 minutes each with 2x, 0.5x, and 0.2x SSC containing 0.1% SDS.

[000293] In the context of the invention, "high stringency conditions" or "high stringency conditions" are conditions in which: (1) low ionic strength and high temperature are used for washing, e.g., 0.015 M sodium chloride/0.0015 M sodium citrate /0.1% sodium dodecyl sulfate at 50°C; (2) a denaturant such as formamide is used during hybridization, e.g. 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% ficoll/0.1% polyvinylpyrrolidone/50 mM buffer sodium phosphate at pH 6.5 with 750 mm sodium chloride, 75 mm sodium citrate at 42°C; or (3) use 50% formamide, 5x SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon milt DNA (50 µg/ml), 0.1% SDS and 10% dextran sulfate at 42°C with washes at 42°C in 0.2x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C followed by a high stringency wash consisting of 0.1x SSC containing EDTA at 55°C. Those skilled in the art will understand how to adjust temperature, ionic strength, and so on. as needed to adapt to factors such as probe length, etc.

[000294] Specialists in this field should be understood that as a result of the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptide presented in the present description. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Despite this, polynucleotides that may vary due to differences in codon usage are specifically included in the present invention. In addition, alleles of genes containing the polynucleotide sequences presented in the present description are included in the scope of the present invention. Alleles are endogenous genes altered by one or more mutations such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may optionally have an altered structure or function. Alleles can be identified by standard methods (such as hybridization, amplification and/or comparison with sequence databases).

[000295] The polynucleotides of the invention can be prepared using chemical synthesis, recombinant methods, or PCR. Methods for the chemical synthesis of polynucleotides are well known in the art and need not be described in detail herein. To obtain the desired DNA sequence, a person skilled in the art can use the sequences presented in the present description, and a commercial DNA synthesizer.

[000296] To obtain polynucleotides by recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and the vector, in turn, can be inserted into a suitable host cell for replication and amplification, as described hereinafter. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by inserting an exogenous polynucleotide via direct capture, endocytosis, transfection, F-pairing, or electroporation. Once inserted, the exogenous polynucleotide can be maintained in the cell as a non-integrated vector (such as a plasmid) or inserted into the genome of the host cell. The thus amplified polynucleotide can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al. , 1989.

[000297] Alternatively, PCR allows the reproduction of DNA sequences. PCR technology is well known in the art and is described in US Pat. Nos. 4,683,195; 4,800,159 ; eds., Birkauswer Press, Boston, 1994.

[000298] RNA can be generated using isolated DNA in an appropriate vector and inserted into a suitable host cell. If the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated by methods well known to those skilled in the art, as described, for example, in Sambrook et al. , 1989.

[000299] In some embodiments, the implementation of the first vector contains a polynucleotide encoding a heavy chain, and the second vector contains a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are used to transfect host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, host cells are transfected with a first vector and a second vector in a 5:1 to 1:5 molar or mass ratio. In some embodiments, a 1:1 to 1:5 weight ratio of a heavy chain encoding vector and a light chain encoding vector is used. In some embodiments, a 1:2 weight ratio of the heavy chain encoding vector to the light chain encoding vector is used.

Vectors

[000300] In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO cells or cells derived from CHO or NSO cells. Examples of such vectors are described, for example, in Running Deer et al, Biotechnol. Prog. 20:880-889 (2004).

[000301] Suitable cloning and expression vectors may include various components such as a promoter, enhancer, and other transcriptional regulatory sequences. It is also possible to design a vector to allow subsequent cloning of the antibody variable domain into other vectors. Suitable cloning vectors can be constructed by standard methods or can be selected from a wide variety of cloning vectors available in the art. Although the chosen cloning vector may vary depending on the host cell used, cloning vectors that can be used will generally be self-replicating, can have a single target for a particular restriction endonuclease, and/or can carry marker genes that can be used in selection of clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g. pUC18, pUC19, Bluescript (e.g. pBS SK+) and their derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors such as pSA3 and pAT28 . These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen. The present invention also relates to expression vectors. Expression vectors are typically replicable polynucleotide constructs containing a polynucleotide of the invention. The expression vector is expected to be replicable in host cells as episomes or an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vectors as described in PCT Publication No. WO 87/04462. The components of the vectors, as a rule, may include, as non-limiting examples, one or more of the following: a signal sequence; site of origin of replication; one or more marker genes; suitable transcriptional control elements (such as promoters, enhancers and terminator). Expression (ie, translation) typically also requires one or more transcriptional control elements, such as ribosome binding sites, translation initiation sites, and stop codons.

[000302] The vectors containing the polynucleotides of interest and/or the polynucleotides themselves can be inserted into the host cell by any of a number of suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microparticle bombardment; lipofection and infection (for example, if the vector is an infectious agent such as vaccinia virus). The choice of insert vectors or polynucleotides will often depend on the characteristics of the host cell.

Host cells

[000303] An antibody, or antigen-binding fragment thereof, can be produced recombinantly using a suitable host cell. The nucleic acid encoding the antibody or antigen-binding fragment thereof can be cloned into an expression vector, which can then be inserted into a host cell such as an E. coli cell, a yeast cell, an insect cell, a COS monkey cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell, where the cell does not otherwise produce the immunoglobulin protein to achieve antibody synthesis in the recombinant host cell. Preferred host cells include a CHO cell, a human embryonic kidney HEK-293 cell, or an Sp2.0 cell, in addition to many cells well known in the art. An antibody fragment can be produced by proteolytic or other degradation of a full-length antibody, by recombinant methods, or by chemical synthesis. An antibody polypeptide fragment, in particular shorter polypeptides up to about 50 amino acids in length, can be obtained by chemical synthesis. Methods for the chemical synthesis of proteins and peptides are known in the art and are commercially available.

[000304] In various embodiments, anti-CXCR5 antibody heavy chains and/or anti-CXCR5 antibody light chains can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, by methods known in the art. Non-limiting examples of eukaryotic cells that can be used to express polypeptides include COS cells, including COS 7 cells; cells 293, including cells 293-6E; CHO cells, including CHO-S cells, CHO DG44, CHO Lecl3 cells and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, anti-CXCR5 antibody heavy chains and/or anti-CXCR5 antibody light chains can be expressed in yeast. See, for example, US Patent Publication No. 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to effect the desired post-translational modifications in the anti-CXCR5 antibody heavy chains and/or anti-CXCR5 antibody light chains. For example, in some embodiments, CHO cells produce polypeptides having a higher level of sialylation than the same polypeptide produced in 293 cells.

[000305] Incorporation of one or more nucleic acids into a desired host cell can be accomplished by any method including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, and etc. Non-limiting examples of methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into desired host cells by any suitable method.

[000306] In some embodiments, afucosylated anti-CXCR5 antibodies are generated in cells capable of producing afucosylated antibodies, such as Lec3 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); patent US application No. 2003/0157108; and WO 2004/056312, especially in example 11), and knockout cell lines, such as cell lines that lack a functional FUT8 alpha-1,6-fucosyltransferase gene, for example, CHO knockout cells (see ., for example, Yamane-Ohnuki et al Biotech Bioeng 87:614 (2004); Kanda et al, Biotechnol Bioeng 94(4):680-688 (2006); and WO2003/085107). In some embodiments, afucosylated anti-CXCR5 antibodies are generated in CHO cells that lack a functional FUT8 gene. In some embodiments, afucosylated anti-CXCR5 antibodies are generated in Potelligent® CHOK1SV cells (BioWa/Lonza, Allendale, NJ).

[000307] Anti-CXCR5 antibodies can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the CXCR5 ECD and ligands that bind to antibody constant regions. For example, a protein A, protein G, protein A/G, or antibody affinity column can be used to bind the constant region and purify an anti-CXCR5 antibody. Hydrophobic interaction chromatography, for example using a butyl or phenyl column, may also be suitable for the purification of some polypeptides. Many methods are known in the art for the purification of polypeptides.

[000308] In some embodiments, the implementation of the antibody against CXCR5 receive in a cell-free system. Non-limiting examples of cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al, Biotechnol. Adv. 21:695-713 (2003).

V. USE AND MEDICAL THERAPY

[000309] In some aspects, the present invention relates to therapeutic methods for removing, inhibiting or reducing the activity or signaling of CXCR5 using an anti-CXCR5 antibody or antigen-binding fragment, where the therapeutic methods include administering a therapeutically effective amount of a pharmaceutical composition containing an anti-CXCR5 antibody or an antigen-binding fragment thereof. fragment. A treatable disorder is any disease or condition that is ameliorated, inhibited, or prevented by eliminating, inhibiting, or reducing CXCR5 activity or signaling.

[000310] In some aspects, the present invention provides an anti-CXCR5 antibody, or antigen-binding fragment thereof, for use in eliminating, inhibiting, or reducing CXCR5 activity or signaling. In some embodiments, the use may include administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-CXCR5 antibody or antigen-binding fragment thereof. In some embodiments, inhibition or reduction of CXCR5 activity or signaling can be used to treat any disease or condition that is ameliorated, inhibited, or prevented by eliminating, inhibiting, or reducing CXCR5 activity or signaling.

[000311] Such diseases, disorders, or conditions include, but are not limited to, inflammatory responses such as systemic lupus erythematosus (SLE); chronic inflammatory responses; atherosclerosis; lack of adhesion of leukocytes; rheumatoid arthritis; diabetes mellitus (eg, type I diabetes mellitus or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and immune responses associated with acute and delayed hypersensitivity reactions, mediated by cytokines and T-lymphocytes, usually found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; Wegener's granulomatosis; pernicious anemia (Addison's disease); diseases, including diapedesis of leukocytes; inflammatory disorder of the central nervous system (CNS); multiple organ failure syndrome; hemolytic anemia (including, but not limited to, cryoglobulinemia or autoimmune hemolytic anemia with incomplete heat agglutinins); myasthenia gravis; diseases mediated by antigen-antibody complexes; anti-GBM antibody disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; myasthenic Lambert-Eaton syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; vitiligo; Reiter's disease; muscle stiffness syndrome; Behcet's disease; giant cell arteritis; immunocomplex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic diseases; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveoretinitis; Guillain-Barré syndrome; Goodpasture's syndrome; mixed connective tissue disease; autoimmune-associated infertility; nodous polyarteritis; alopecia areata; idiopathic myxedema; graft-versus-host reaction; muscular dystrophy (Duchenne's dystrophy, Becker's dystrophy, myotonic dystrophy, limb-girdle dystrophy, shoulder-blade-facial dystrophy, congenital dystrophy, oculopharyngeal dystrophy, distal dystrophy, Emery-Dreyfus dystrophy) and control of proliferation of malignant cells expressing CXCR5, such as cancer pancreatic cancer, colon cancer, bladder cancer, T-cell leukemia and B-cell leukemia, as will be understood by a person skilled in the art in view of the ideas presented in the present description.

[000312] Anti-CXCR5 antibodies or antigen-binding fragments thereof of the present invention can also be used to detect and/or measure CXCR5 or CXCR5-expressing cells in a sample, for example, for diagnostic purposes. For example, an anti-CXCR5 antibody or fragment thereof can be used to diagnose a condition or disease characterized by abnormal expression (eg, overexpression, underexpression, lack of expression, etc.) of CXCR5. Examples of CXCR5 diagnostic assays may include, for example, contacting a sample obtained from a patient with an anti-CXCR5 antibody of the invention, wherein the anti-CXCR5 antibody is labeled with a detectable label or reporter molecule.

[000313] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the individual's immune system. Non-limiting examples include any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as an oil-in-water emulsion, and various types of humectants. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well known, conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).

VI. COMPOSITIONS

[000314] The present invention also provides pharmaceutical compositions comprising an effective amount of an anti-CXCR5 antibody or antigen-binding fragment thereof as described herein. Examples of such compositions, as well as formulation methods, are also provided herein. In some embodiments, the implementation of the composition contains one or more antibodies against CXCR5. In other embodiments, the anti-CXCR5 antibody recognizes CXCR5. In other embodiments, the anti-CXCR5 antibody is a human antibody. In other embodiments, the anti-CXCR5 antibody is a humanized antibody. In some embodiments, the anti-CXCR5 antibody contains a constant region capable of triggering the desired immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the anti-CXCR5 antibody contains a constant region that is afucosylated and provides increased ADCC compared to an otherwise identical antibody that is fucosylated. In other embodiments, the anti-CXCR5 antibody comprises one or more CDRs of the antibody (eg, one, two, three, four, five, or, in some embodiments, all six CDRs).

[000315] It should be understood that the compositions may contain multiple anti-CXCR5 antibodies or antigen-binding fragments thereof (eg, a mixture of anti-CXCR5 antibodies recognizing different epitopes of CXCR5). Other exemplary compositions contain multiple anti-CXCR5 antibodies recognizing the same epitopes or different types of anti-CXCR5 antibodies binding to different epitopes of CXCR5. In some embodiments, the compositions comprise a mixture of anti-CXCR5 antibodies that recognize different variants of CXCR5.

[000316] the Composition used in the present invention may additionally contain pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and practice of Pharmacy 20th Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at doses and concentrations and may contain buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; complex compounds with metals (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONIC™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are additionally presented in the present description.

[000317] The anti-CXCR5 antibody, antigen binding fragment thereof, and compositions thereof can also be used in combination with other agents to enhance and/or supplement the efficacy of the agents.

[000318] In some embodiments, the anti-CXCR5 antibody, or antigen-binding fragment thereof, is complexed with CXCR5 prior to administration. In some embodiments, the anti-CXCR5 antibody is not complexed with CXCR5 prior to administration.

[000319] The present invention also relates to compositions comprising pharmaceutical compositions containing any of the polynucleotides of the invention. In some embodiments, the implementation of the composition contains an expression vector containing a polynucleotide encoding an antibody presented in the present description. In other embodiments, the composition comprises an expression vector containing a polynucleotide encoding any of the antibodies provided herein. In other embodiments, the composition comprises any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:1 and SEQ ID NO:6, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:1 and SEQ ID NO: 10, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:1 and SEQ ID NO:12, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:5 and SEQ ID NO:6, either or both of the polynucleotides encoding the sequence shown in SEQ ID NO:5 and SEQ ID NO:10, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:5 and SEQ ID NO:12, any or both of the polynucleotides, encoding the sequence shown in SEQ ID NO:13 and SEQ ID NO:17, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:13 and SEQ ID NO:18, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:13 and SEQ ID NO:63, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:1 and SEQ ID NO:52, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO :1 and SEQ ID NO:53, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:1 and SEQ ID NO:54, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:1 and SEQ ID NO:55, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:1 and SEQ ID NO:56, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:1 and SEQ ID NO:57 , any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:6 and SEQ ID NO:48, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:6 and SEQ ID NO:49, either or both of the polynucleotides encoding the sequence shown in SEQ ID NO:6 and SEQ ID NO:50, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:6 and SEQ ID NO:51, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:17 and SEQ ID NO:58, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:17 and SEQ ID NO:59, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:17 and SEQ ID NO:60, any or both of the polynucleotides containing the sequence shown in SEQ ID NO:17 and SEQ ID NO:61, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO: 17 and SEQ ID NO:62, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:52 and SEQ ID NO:1, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:52 and SEQ ID NO:5, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:52 and SEQ ID NO:47, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:52 and SEQ ID NO:48, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:52 and SEQ ID NO:49, any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:52 and SEQ ID NO:50, any or both of polynucleotides encoding the sequence shown in SEQ ID NO:52 and SEQ ID NO:51, or any or both of the polynucleotides encoding the sequence shown in SEQ ID NO:35 and SEQ ID NO:36.

[000320] In another aspect, a polynucleotide can encode for VH, VL and/or both VH and VL of an antibody of the invention. Those. the composition contains one polynucleotide or multiple polynucleotides encoding the antibody or antigen-binding fragment of the invention.

[000321] The pharmaceutical compositions of the invention can also be administered as a combination treatment, such as in combination with other agents. For example, a combination treatment may include an anti-CXCR5 antibody or antigen-binding fragment thereof of the invention in combination with at least one other treatment, where the treatment may be surgery, immunotherapy, or drug therapy.

[000322] The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and basic addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Basic addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., as well as from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[000323] The pharmaceutical composition of the invention may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[000324] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil , and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and using surfactants.

[000325] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the presence of microorganisms can be achieved through sterilization and the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of an injectable pharmaceutical form can be achieved by the inclusion of absorption delaying agents such as aluminum monostearate and gelatin.

[000326] Pharmaceutical compositions generally must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. In many cases it may be appropriate to include isotonic agents, for example sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, in the compositions. Prolonged absorption of injectable compositions can be achieved by including in the composition an absorption delaying agent, for example, monostearate salts and gelatin.

[000327] Sterile injectable solutions can be obtained by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the ingredients listed above, if necessary, followed by microfiltration sterilization.

[000328] Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, vacuum drying and lyophilization are preferred methods of preparation, whereby a powder of the active ingredient and any additional desired ingredient is obtained from their previously sterile filtered solution.

[000329] The pharmaceutical composition of the invention can be prepared, packaged or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops comprising, for example, a 0.1%-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or liquid oily vehicle. Such drops may further contain buffers, salts, or one or more of the other additional ingredients described herein. Other ophthalmic formulations that may be used include formulations containing the active ingredient in microcrystalline or liposomal formulation.

[000330] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating agents and disintegrating agents; binding agents; lubricants; sweeteners; flavors; dyes; preservatives; physiologically degradable compositions such as gelatin; aqueous carriers and solvents; oil carriers and solvents; suspending means; dispersants or wetting agents; emulsifiers, emollients; buffers; salt; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in pharmaceutical compositions of the invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Genaro, ed., Mack Publishing Co., Easton, PA (1985), incorporated herein in as a link.

[000331] In one embodiment, the anti-CXCR5 antibody, or antigen binding fragment thereof, is administered in an intravenous formulation as a sterile aqueous solution containing 5 mg/mL, or in some embodiments, about 10 mg/mL, or in some embodiments, about 15 mg /ml, or in some embodiments, about 20 mg/ml of antibody, or in some embodiments, about 25 mg/ml, or in some embodiments, about 50 mg/ml with sodium acetate, polysorbate 80, and sodium chloride at a pH in the range of about 5 to 6. In some embodiments, the intravenous formulation is a sterile aqueous solution containing 5 or 10 mg/ml antibody with 20 mM sodium acetate, 0.2 mg/ml polysorbate 80, and 140 mM sodium chloride at pH 5.5. In addition, a solution containing an antibody or antigen-binding fragment thereof, among many other compounds, may contain histidine, mannitol, sucrose, trehalose, glycine, polyethylene glycol, EDTA, methionine, and any combination thereof, and many other compounds known in this field.

[000332] In one embodiment, the pharmaceutical composition of the invention contains the following components: 50 mg/ml of an anti-CXCR5 antibody or antigen-binding fragment of the invention, 20 mM histidine, 8.5% sucrose, and 0.02% polysorbate 80, 0.005% EDTA with pH 5.8; in another embodiment, the pharmaceutical composition of the present invention contains the following components: 100 mg/ml of an anti-CXCR5 antibody or antigen-binding fragment of the invention, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80 at pH 5.8. This composition can be obtained in the form of a liquid formulation or lyophilized powder. When the powder is reconstituted in its entirety, the composition retains the same composition. Alternatively, the powder can be reconstituted at half volume, in which case the composition contains 100 mg of an anti-CXCR5 antibody or antigen-binding fragment of the invention, 20 mM histidine, 10% sucrose, and 0.02% polysorbate 80 at pH 5.8.

[000333] In one embodiment, a portion of the dose is administered by intravenous bolus and the remainder by infusion of the antibody formulation. For example, a 0.01 mg/kg intravenous injection of an anti-CXCR5 antibody or antigen-binding fragment thereof may be administered as a bolus and the remainder of the antibody dose may be administered by intravenous injection. A predetermined dose of the anti-CXCR5 antibody or antigen-binding fragment thereof may be administered over an hour and a half hour to two hours or five hours, for example.

[000334] With regard to a therapeutic agent, if the agent is, for example, a small molecular weight compound, it can be in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, for example, in combination with a physiologically acceptable cation or anion, which is well known in this field.

[000335] the Pharmaceutical compositions presented in the present description can be obtained by any method known in the field of pharmacology, or by a method that will be developed in the future. In general, such preparations include the step of bringing the active ingredient into contact with the carrier or one or more other auxiliary ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single-dose or multi-dose container.

[000336] In one embodiment, the compositions of the invention are pyrogen-free formulations substantially free of endotoxins and/or related pyrogens. Endotoxins include toxins that reside within a microorganism and are released when the microorganism is destroyed or killed. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both substances can cause fever, hypotension, and shock when administered to humans. Due to potential adverse effects, it is preferable to remove even small amounts of endotoxins from intravenously administered pharmaceutical drug solutions. The Food and Drug Administration ("FDA") has set an upper limit of 5 units of endotoxin (EU) per dose per kilogram of body weight in one one-hour period for intravenous drug administration (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). If the therapeutic proteins are administered in amounts of several hundreds or thousands of milligrams per kilogram of body weight, it is preferable to remove even small amounts of endotoxin. In one embodiment, the levels of endotoxin and pyrogens in the composition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg, or less than 0.001 EU/mg. In another embodiment, the levels of endotoxin and pyrogens in the composition are less than about 10 EU/mg, or less than about 5 EU/mg, or less than about 1 EU/mg, or less than about 0.1 EU/mg, or less than about 0.01 EU/mg, or less than about 0.001 EU/mg.

[000337] In one embodiment, the present invention includes administering a composition, wherein said administration is oral, parenteral, intramuscular, intranasal, vaginal, rectal, lingual, sublingual, buccal, intrabuccal, intravenous, dermal, subcutaneous, or transdermal.

[000338] In another embodiment, the present invention further includes administering the composition in combination with other treatments such as surgery, chemotherapy, hormonal therapy, biological therapy, immunotherapy, or radiation therapy.

VII. DOSING / ADMINISTRATION

[000339] To prepare pharmaceutical or sterile compositions comprising an anti-CXCR5 antibody or antigen-binding fragment of the invention, the antibody is mixed with a pharmaceutically acceptable carrier or excipient. Therapeutic and diagnostic formulations can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, suspensions, aqueous solutions, or lotions (see, for example, Hardman, et al. (2001) Goodman and Gilman's Pharmaceutical Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY Lieberman, et al (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY Lieberman, et al (eds.) (1990) Pharmaceutical Dosage Forms : Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

[000340] The choice of administration schedule for a therapeutic agent depends on several factors, including the rate of circulation of the substance in serum or tissue, the level of symptoms, the immunogenicity of the substance, and the availability of target cells in the biological matrix. In some embodiments, the administration schedule maximizes the amount of therapeutic agent delivered to the patient in accordance with an acceptable level of side effects. Thus, the amount of biological agent delivered depends in part on the particular substance and the severity of the condition being treated. Guidance is available on the selection of appropriate doses of antibodies, cytokines and small molecule compounds (see e.g. Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.), 1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N. Y.; Baert, et al., 2003, New Engl. J. Med. 348: 601-608; Milgrom, et al., 1999, New Engl. J. Med. 341:1966-1973; Slamon, et al., 2001, New Engl. J. Med. 344:783-792; Beniaminovitz, et al. ., 2000, New Engl. J. Med. 342:613-619; Ghosh, et al., 2003, New Engl. J. Med. 348:24-32; Lipsky, et al., 2000, New Engl. J Med. 343:1594-1602).

[000341] Determination of an appropriate dose is left to the clinician, for example, using parameters or factors known or expected in the art to influence treatment or are expected to influence treatment. Typically, the dose is started at slightly less than the optimal dose and then increased in small increments until the desired or optimal effect is achieved with respect to any negative side effects. Important diagnostic measures include measuring symptoms, such as inflammation or the level of inflammatory cytokines produced.

[000342] The exact dosage levels of the active ingredients in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and route of administration, without toxicity to the patient. The selected dose level will depend on various pharmacokinetic factors, including the potency of the particular compositions of the invention or their ester, salt or amide used, route of administration, time of administration, rate of excretion of the particular compound used, duration of treatment, other drugs, compounds and/or materials. used in combination with the specific compositions used, age, sex, body weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field.

[000343] Compositions containing antibodies against CXCR5 or antigennegative fragments of the invention can be administered by continuous infusion or at dose intervals, for example, one day, one week, or 1-7 times a week. Doses can be administered intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebral, or by inhalation. A specific administration protocol is a protocol including the maximum dose or frequency of administration to avoid significant undesirable side effects. The total weekly dose may be at least 0.05 µg/kg of body weight, at least 0.2 µg/kg, at least 0.5 µg/kg, at least 1 µg/kg, at least 10 µg /kg, at least 100 μg/kg, at least 0.2 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, at least 10 mg/kg, according to at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, or at least 50 mg/kg (see, for example, Yang, et al., 2003, New Engl. J. Med 349:427-434; Herold, et al., 2002, New Engl. J. Med. 346:1692-1698; Liu, et al., 1999, J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al., 2003, Cancer Immunol Immunother 52: 133-144). The dose may be at least 15 µg, at least 20 µg, at least 25 µg, at least 30 µg, at least 35 µg, at least 40 µg, at least 45 µg, at least 50 µg , at least 55 micrograms, at least 60 micrograms, at least 65 micrograms, at least 70 micrograms, at least 75 micrograms, at least 80 micrograms, at least 85 micrograms, at least 90 micrograms, at least 95 µg or at least 100 µg. The number of doses administered to an individual may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more.

[000344] In the case of antibodies against CXCR5 or antigennegative fragments thereof according to the invention, the dose administered to the patient may be from 0.0001 mg/kg to 100 mg/kg of the patient's body weight. The dose can be from 0.0001 mg/kg to 20 mg/kg, from 0.0001 mg/kg to 10 mg/kg, from 0.0001 mg/kg to 5 mg/kg, from 0.0001 to 2 mg/kg. kg, 0.0001 to 1 mg/kg, 0.0001 mg/kg to 0.75 mg/kg, 0.0001 mg/kg to 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg /kg or from 0.01 to 0.10 mg/kg of the patient's body weight.

[000345] The dose of an anti-CXCR5 antibody or antigen-binding fragment thereof can be calculated using the patient's body weight in kilograms (kg) multiplied by the administered dose in mg/kg. The dose of antibodies of the invention may be 150 μg/kg or less, 125 μg/kg or less, 100 μg/kg or less, 95 μg/kg or less, 90 μg/kg or less, 85 μg/kg or less, 80 μg /kg or less, 75 mcg/kg or less, 70 mcg/kg or less, 65 mcg/kg or less, 60 mcg/kg or less, 55 mcg/kg or less, 50 mcg/kg or less, 45 mcg/ kg or less, 40 mcg/kg or less, 35 mcg/kg or less, 30 mcg/kg or less, 25 mcg/kg or less, 20 mcg/kg or less, 15 mcg/kg or less, 10 mcg/kg or less, 5 mcg/kg or less, 2.5 mcg/kg or less, 2 mcg/kg or less, 1.5 mcg/kg or less, 1 mcg/kg or less, 0.5 mcg/kg or less or 0.1 µg/kg patient body weight or less.

[000346] A single dose of anti-CXCR5 antibodies or antigen-binding fragments thereof according to the invention may be from 0.1 mg to 200 mg, from 0.1 mg to 175 mg, from 0.1 mg to 150 mg, from 0.1 mg to 125 mg, 0.1 mg to 100 mg, 0.1 mg to 75 mg, 0.1 mg to 50 mg, 0.1 mg to 30 mg, 0.1 mg to 20 mg, 0, 1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg , 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[000347] Using a dose of antibodies against CXCR5 or their antigen-binding fragments according to the invention, it is possible to achieve a serum titer of at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 µg/ml, at least 5 µg/ml, at least 6 µg/ml, at least 10 µg/ml, at least 15 µg/ml, at least 20 µg/ml, at least 25 µg/ml, at least 50 µg/ml, at least 100 µg/ml, at least 125 µg/ml, at least 150 µg/ml, at least 175 µg/ml, at least 200 µg/ml ml, at least 225 µg/ml, at least 250 µg/ml, at least 275 µg/ml, at least 300 µg/ml, at least 325 µg/ml, at least 350 µg/ml, at least 375 μg/ml or at least 400 μg/ml in an individual. Alternatively, a serum titer of at least 0.1 µg/mL, at least 0.5 µg/mL, at least 1 µg/mL, at least 2 µg/mL, at least at least 5 µg/ml, at least 6 µg/ml, at least 10 µg/ml, at least 15 µg/ml, at least 20 µg/ml, at least 25 µg/ml, at least 50 µg/ml, at least 100 µg/ml, at least 125 µg/ml, at least 150 µg/ml, at least 175 µg/ml, at least 200 µg/ml, at least 225 µg/ml ml, at least 250 µg/ml, at least 275 µg/ml, at least 300 µg/ml, at least 325 µg/ml, at least 350 µg/ml, at least 375 µg/ml, or at least 400 μg/ml in an individual.

[000348] Doses of anti-CXCR5 antibodies or antigen-binding fragments thereof of the invention may be repeated and administrations may be separated by intervals of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or at least 6 months.

[000349] The effective amount in the case of a particular patient may vary depending on such factors as the condition being treated, the general health of the patient, the route, route of administration and dose and severity of side effects (see, for example, Maynard, et al., 1996, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent, 2001, Good Laboratory and Good Clinical Practice, Urch Publ, London, UK).

[000350] The route of administration may be, for example, topical or dermal application, injection or infusion via intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intraspinal, intralesional routes, or via sustained release systems or implant (see, for example, Sidman et al., 1983, Biopolymers 22:547-556; Langer, et al., 1981, J. Biomed. Mater. Res. 15: 167-277; Langer, 1982, Chem. Tech. 12:98-105; Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030-4034; U.S. Patent Nos. 6350466 and 6316024). If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. In addition, pulmonary administration, for example by means of an inhaler or nebulizer, and an aerosol formulation may also be used. See, for example, US Pat. , WO 98/31346 and WO 99/66903, each of which is incorporated herein by reference in its entirety. In one embodiment, an anti-CXCR5 antibody, or antigen-binding fragment thereof, or composition of the invention is administered using Alkermes AIR™ pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).

[000351] The composition of the invention may also be administered by one or more routes of administration by one or more of the various routes known in the art. As will be understood by those skilled in the art, the route and/or route of administration will vary depending on the desired results. Selected routes of administration of the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. Parenteral administration may be administration routes other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal , subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the composition of the invention may be administered by a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, eg, intranasal, oral, vaginal, rectal, sublingual, or topical administration.

[000352] If the anti-CXCR5 antibodies or antigen-binding fragments thereof of the invention are administered in a controlled release or sustained release system, a pump can be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:501; Saudek et al., 1989, N. Engl. J. Med. 321:514).

[000353] Polymeric materials can be used to achieve controlled or sustained release of the therapeutic agents of the invention (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. ScL Rev. Macromol. Chem. 23:61; also see Levy et al, 1985, Science 11 225:190, During et al, 19Z9, Ann Neurol 25:351, Howard et al, 1989, J Neurosurg 71:105, U.S. Patent No. 5,679,377, U.S. Patent No. 5,916,597 ; US Patent No. 5912015; US Patent No. 5989463; US Patent No. 5128326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). Non-limiting examples of polymers used in sustained release formulations include poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA) and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable. The controlled or sustained release system can be placed near the prophylactic or therapeutic target and thus only a fraction of the systemic dose is needed (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[000354] Controlled release systems are reviewed by Langer, 1990, Science 249:1527-1533. Any method known to the person skilled in the art can be used to prepare sustained release formulations containing one or more antibodies of the invention or conjugates thereof. See, for example, U.S. Patent No. 4,526,938, International Patent Publication Nos. WO 91/05548, WO 96/20698, Ning et al., 1996, "Intratumoral Radioimmunotheraphy of Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy and Oncology 59:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science and Technology 50:372-397, Cleek et ah, 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application, Pro. ml. Symp. control. Rel. Bioact. mater. 24:853-854, and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. ml. Symp. Control Rel. Bioact. mater. 24:759-160, each of which is incorporated herein by reference in its entirety.

[000355] When an anti-CXCR5 antibody or antigen-binding fragment thereof of the invention is administered topically, it can be formulated as an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well known to those skilled in the art. areas. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). In the case of non-sprayable topical dosage forms, as a rule, viscous and semi-solid or solid forms are used, containing a carrier or one or more excipients compatible with topical use and having a dynamic viscosity, in some cases greater than that of water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, and the like, which, if desired, are sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, humectants, buffers, or salts) to influence various properties such as, for example, osmotic pressure. Other suitable topical dosage forms include spray-on aerosol formulations where the active ingredient, in some cases in combination with a solid or liquid inert carrier, is packaged in admixture with a pressurized volatile substance (e.g. a gaseous propellant such as Freon) or in a soft bottle. If desired, humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.

[000356] When compositions containing anti-CXCR5 antibodies or antigen-binding fragments thereof are administered intranasally, they can be formulated in aerosol, spray, or drop form. In particular, prophylactic or therapeutic agents for use according to the invention may be administered in the form of a pressurized aerosol spray or nebulizer using a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by using a valve to deliver a metered amount. Capsules and cartridges (composed of, for example, gelatin) for use in an inhaler or insufflator can be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

[000357] Methods of co-administration or treatment using a second therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation therapy, are well known in the art (see, e.g., Hardman, et al. (eds.) (2001 ) Goodman and Gilman's Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, N.Y. Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams and Wilkins, Phila. , Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams and Wilkins, Phila., Pa.). With an effective amount of a therapeutic agent, symptoms can be reduced by at least 10 percent, at least 20 percent; at least about 30 percent; at least 40 percent or at least 50 percent.

[000358] Additional agents (e.g., prophylactic or therapeutic agents) that can be administered in combination with CXCR5 antibodies or antigen-binding fragments of the invention may be administered at intervals of less than 5 minutes, less than 30 minutes, 1 hour, about 1 hour, from about 1 to about 2 hours, from about 2 hours to about 3 hours, from about 3 hours to about 4 hours, from about 4 hours to about 5 hours, from about 5 hours to about 6 hours, from about 6 hours to about 7 hours , from about 7 hours to about 8 hours, from about 8 hours to about 9 hours, from about 9 hours to about 10 hours, from about 10 hours to about 11 hours, from about 11 hours to about 12 hours, from about 12 hours to 18 hours, from 18 hours to 24 hours, from 24 hours to 36 hours, from 36 hours to 48 hours, from 48 hours to 52 hours, from 52 hours to 60 hours, from 60 hours to 72 hours, from 72 hours to 84 hours, 84 hours to 96 hours, or 96 hours to 120 hours relative to the antibodies of the invention. Two or more agents may be administered within a single visit by a patient to a healthcare facility.

[000359] The CXCR5 antibodies or antigen-binding fragments thereof of the invention and other agents can be administered cyclically. Cycling therapy includes administering a first agent (e.g., a first prophylactic or therapeutic agent) over a period of time followed by administering a second agent (e.g., a second prophylactic or therapeutic agent) over a period of time, optionally followed by administration of a third agent (e.g., a prophylactic or therapeutic agent) over a period of time, etc., and repeating this sequential administration, i.e. cycle, to reduce the development of resistance to one of the agents, to avoid or reduce the side effects of one of the agents and / or to improve the effectiveness of the agents.

[000360] In one embodiment, the anti-CXCR5 antibodies of the invention may be co-administered with compositions for the treatment of autoimmune diseases and disorders, including, but not limited to, adriamycin, azathioprine, busulfan, cyclophosphamide, cyclosporin A, cytoxan, fludarabine, 5-fluorouracil , methotrexate, mycophenolate mofetil, 6-mercaptopurine, corticosteroid, non-steroidal anti-inflammatory drugs, sirolimus (rapamycin), and tacrolimus (FK-506). In alternative embodiments, the immunomodulatory or immunosuppressive agent is an antibody selected from the group consisting of muromonab-CD3, alemtuzumab (Campath®), basiliximab, daclizumab, muromonab (OKT3®), rituximab, antithymocyte globulin, and IVIg, and others known to those skilled in the art. this area.

[000361] In one embodiment, the anti-CXCR5 antibodies of the invention may be co-administered with compositions for the treatment of diabetes, including, but not limited to, biguanides (e.g., buformin, metformin, and phenformin), hormones, and their analogs (amylin, insulin, insulin aspart, insulin detemir, insulin glargine, insulin glulisine, insulin lispro, liraglutide and pramlintide), sulfonylcarbamide derivatives (acetohexamide, carbutamide, chlorpropamide, glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glizoxepide, glyburide, glibutiazole, glibusol, glihexamide , glymidine, tolazamide, tolbutamide, and tolcyclamide), thiazolidinediones (pioglitazone, rosiglitazone, and troglitazone), acarbose, exenatide, miglitol, mitiglinide, muraglitazar, nateglinide, repaglinide, sitagliptin, tezaglitazar, vildagliptin, and voglibose.

[000362] In some embodiments, the anti-CXCR5 antibodies or antigen-binding fragments thereof of the invention can be formulated to ensure proper distribution in vivo . For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To allow penetration of the therapeutic compounds of the invention through the BBB (if desired), they can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, US Pat. Ranade, 1989, J. Clin. Pharmacol. 29:685). Examples of targeting agents include folate or biotin (see, for example, US Pat. No. 5,416,016); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun. 153: 1038); antibodies (PG Bloeman et al., 1995, FEBS Lett. 357: 140; M. Owais et al., 1995, Antimicrob. Agents Chemother. 39: 180); surfactant A protein receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; ML Laukkanen, 1994, FEBS Lett. 346:123; killion; Fidler, 1994; Immunomethods 4:273.

[000363] The present invention relates to methods of administering to an individual in need thereof a pharmaceutical composition comprising the anti-CXCR5 antibodies or antigen-binding fragments thereof of the invention, alone or in combination with other agents. Means (eg, prophylactic or therapeutic agents) within the combined treatment according to the invention can be administered to the individual simultaneously or sequentially. The agent (eg prophylactic or therapeutic agents) within the combination treatment according to the invention can also be administered cyclically. Cyclic therapy includes administering a first agent (eg, a first prophylactic or therapeutic agent) over a period of time followed by administering a second agent (eg, a second prophylactic or therapeutic agent) over a period of time and repeating this sequential administration, i. cycle, to reduce the development of resistance to one of the agents in order to avoid or reduce the side effects of one of the agents and / or to improve the effectiveness of the agents.

[000364] Agents (eg, prophylactic or therapeutic agents) within the combination treatment of the invention can be administered to an individual simultaneously. The term "simultaneously" is not limited to administering the agent (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather means that the pharmaceutical composition comprising the anti-CXCR5 antibodies or antigen-binding fragments thereof of the invention is administered to the individual in that sequence and within such a time interval that the antibodies of the invention, or conjugates thereof, can act in conjunction with other agents to provide a beneficial effect enhanced relative to their use otherwise. For example, each agent can be administered to an individual simultaneously or sequentially in any order at different points in time; however, if they are not administered simultaneously, they should be administered close enough in time so as to provide the desired therapeutic or prophylactic response. Each agent can be administered to the individual separately, in any suitable form and by any suitable route. In various embodiments, the agents (e.g., prophylactic or therapeutic agents) are administered to the individual at intervals of less than 15 minutes, less than 30 minutes, less than 1 hour, about 1 hour, from about 1 hour to about 2 hours, from about 2 hours to about 3 hours , from about 3 hours to about 4 hours, from about 4 hours to about 5 hours, from about 5 hours to about 6 hours, from about 6 hours to about 7 hours, from about 7 hours to about 8 hours, from about 8 hours up to about 9 hours, from about 9 hours to about 10 hours, from about 10 hours to about 11 hours, from about 11 hours to about 12 hours, 24 hours, 48 hours, 72 hours, or 1 week. In other embodiments, two or more agents (eg, prophylactic or therapeutic agents) are administered within a single visit by a patient to a healthcare facility.

[000365] Prophylactic or therapeutic agents in combination treatment can be administered to an individual in a single pharmaceutical composition. Alternatively, prophylactic or therapeutic agents within the combined treatment can be administered to the individual simultaneously in separate pharmaceutical compositions. Prophylactic or therapeutic agents can be administered to the individual by the same or different routes of administration.

VIII. SETS

[000366] The present invention also relates to kits containing any or all of the antibodies presented in the present description. Kits of the invention include one or more containers containing an anti-CXCR5 antibody provided herein and instructions for use in accordance with any of the methods of the invention provided herein. As a rule, these instructions contain a description of the introduction of antibodies for the therapeutic treatment described above. In some embodiments, the kits are designed to provide a single dose dosage unit. In some embodiments, the kit may comprise a first container containing dried protein and a second container containing an aqueous formulation. In some embodiments, kits are provided that contain an applicator, such as single-chamber and multi-chamber pre-filled syringes (eg, liquid syringes and lyophilisate syringes).

[000367] Instructions for use of an anti-CXCR5 antibody typically include information on dosage, administration schedule, and route of administration for the intended treatment. Containers may include unit doses, multi-dose packs, or dosage subunits. The instructions supplied in the kits of the invention are typically written instructions on a label or package insert (for example, a paper sheet included in the kit), but machine-readable instructions (for example, instructions found on a magnetic or optical disk) are also acceptable.

[000368] The kits of the invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Packages are also provided for use in combination with a specific device such as an inhaler, a nasal device (eg an aerosol inhaler), or an infusion device such as a minipump. The kit may contain a sterile entry port (for example, the container may be an intravenous solution bag or a vial with a hypodermic stopper). The container may also contain a sterile entry port (for example, the container may be an intravenous solution bag or a vial with a hypodermic stopper). At least one active agent in the composition is an anti-CXCR5 antibody of the invention. The container may further contain a second pharmaceutically active agent.

[000369] Sets may optionally include additional components such as buffers and interpreted information. Typically, the kit contains a container and a label or package insert on or with the container.

[000370] The present invention also relates to diagnostic kits containing any or all of the antibodies presented in the present description. Diagnostic kits can be used, for example, to detect the presence of CXCR5 in a sample. In some embodiments, a diagnostic kit can be used to identify an individual with a latent disease, disorder, or condition that may be at risk for developing a CXCR5-mediated disease, disorder, or condition, or a CXCR5 deficient disease, disorder, or condition. In some embodiments, a diagnostic kit can be used to detect the presence and/or level of CXCR5 in an individual believed to have a CXCR5-mediated disease or a CXCR5-deficient disease, disorder, or condition.

[000371] Diagnostic kits of the invention include one or more containers containing an anti-CXCR5 antibody provided herein and instructions for use in accordance with any of the methods of the invention provided herein. Typically, these instructions include a description of the use of an anti-CXCR5 antibody to detect the presence of CXCR5 in individuals at risk of developing or suspected to have a CXCR5-mediated or CXCR5-deficient disease, disorder or condition. In some embodiments, an exemplary diagnostic kit may be configured to contain reagents such as, for example, an anti-CXCR5 antibody, a negative control sample, a positive control sample, and instructions for using the kit.

IX. EQUIVALENTS

[000372] The above description and the following examples describe in detail some specific embodiments of the invention and the best mode of implementation provided by the authors of the present invention. However, it should be understood that no matter how detailed it is described in the text, the present invention can be practiced in a variety of ways, and the present invention should be construed in accordance with the claims and any equivalents thereof.

[000373] Although the ideas have been described with reference to various uses, methods, kits, and compositions, it should be understood that various changes and modifications can be made without deviating from the ideas set forth in the present description and the claims below. The following examples are presented to better illustrate the ideas described and are not intended to limit the scope of the present invention. While the present invention has been described in terms of these exemplary embodiments, those skilled in the art will appreciate that numerous changes and modifications to these exemplary embodiments are possible without undue experimentation. All such changes and modifications are within the scope of the present invention.

[000374] All references cited herein, including patents, patent applications, articles, manuals, and the like, and references cited therein, are hereby incorporated by reference in their entirety. In the event that one or more of the included literature and related materials differ or conflict with this application, including, but not limited to, certain terms, use of terms, methods described, or the like, this application takes precedence.

X. GENERAL METHODS

[000375] It should be understood that the present invention is not limited to specific synthetic methods of obtaining, which, of course, can be varied. Unless otherwise indicated in the present description, scientific and technical terms used in relation to the present invention should have a meaning generally understood by specialists in this field. In addition, unless the context otherwise requires, singular terms must include the plural, and plural terms must include the singular. As a rule, the nomenclature and methods of cell and tissue culture, molecular biology, immunology, microbiology, genetics and chemistry of proteins and nucleic acids and hybridization presented in the present description are well known and commonly used in this field.

[000376] Unless otherwise indicated, conventional methods of molecular biology (including recombinant methods), microbiology, cell biology, biochemistry and immunology known to those skilled in the art are used in the practice of the present invention. Such methods are fully described in the literature, for example, in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

[000377] Enzymatic reactions and purification methods are carried out according to the manufacturer's instructions, as is customary in this field or presented in the present description. The nomenclature used and the laboratory methods of analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medicinal and pharmaceutical chemistry presented herein are well known and commonly used in the art. For chemical synthesis, chemical analyses, preparation, formulation and administration of pharmaceutical preparations and treatment of patients, standard methods are used.

XI. BIOLOGICAL DEPOSIT

[000378] The basic materials of the present invention are deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, July 26, 2017. The h11G2-VH (XC155) vector, ATCC registration number PTA-124323, contains a DNA insert encoding the h11G2 antibody heavy chain variable region (XC155), and the h11G2-VL (XC154) vector, ATCC registration number PTA-124324, contains an insert DNA encoding the light chain variable region of the h11G2 antibody (XC154). The deposits were made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and regulations (Budapest Treaty). This ensures that live cultures of the deposit are maintained for 30 years from the date of deposit. The deposit will be made available to ATCC under the Budapest Agreement and will be subject to an agreement between Pfizer Inc. and ATCC, which provides permanent and unrestricted access to the progeny of the deposit culture to the public after the grant of a pertinent U.S. patent or after the publication of any U.S. or foreign patent application, whichever occurs first, and ensures that the progeny is available to a person authorized by the Patent and Trademark Office U.S. marks pursuant to 35 U.S.C. section 122 and relevant Office rules (including 37 C.F.R. section 1.14 citing 886 OG 638).

[000379] The owner of the present application agrees that if a culture of deposit materials dies or is lost or destroyed when cultivated under suitable conditions, the materials will be immediately replaced upon notification with another of the same material. The availability of the deposited material should not be construed as a license to practice the present invention in violation of the rights granted by permission of any government under patent law.

EXAMPLES

[000380] The present invention is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and should not be construed as limiting unless otherwise noted. Thus, the present invention should in no way be construed as being limited to the following examples, but rather as including any and all variations that become apparent from the teachings presented herein.

Example 1: Hybridomas binding to human CXCR5 and cynomolgus monkey CXCR5

[000381] Female BALB/c mice were immunized three times with a mixture containing 1×10 ⁶ BaF3 cells overexpressing human CXCR5 (SEQ ID NO:32) or 300.19 cells expressing human CXCR5 (SEQ ID NO:32) with 5 μg adjuvant CPG (ODN1826) (Invivogen) in series at intervals of three weeks. At the final booster, mice were immunized with 20 μg of virus-like particles (VLPs) derived from HEK-293 cells overexpressing human CXCR5 (SEQ ID NO:32) using the MembranePro™ functional protein expression system (Thermo Fisher). Five rounds of fusion were carried out and eighty-eight clones were obtained that bind HEK-293 cells expressing human CXCR5 (hCXCR5-293). These monoclonal antibodies were purified with protein A and tested for binding to HEK-293 cells expressing hCXCR5 and HEK-293 cells expressing cynomolgus monkey CXCR5 (SEQ ID NO:33), ie. cynoCXCR5-293. hCXCR5-293 or cynoCXCR5-293 cells were stained with monoclonal antibodies at 10 μg/ml before staining with PE-conjugated anti-mouse IgG (Southern Biotech). Cells were analyzed by flow cytometry using a FACSVerse Analyzer (BD Biosciences). When screened for binding to hCXCR5 HEK-293 and cynoCXCR5 HEK-293 cells, thirty-four antibodies were identified that bind to human and cynomolgus monkey CXCR5 (FIG. 3).

Example 2: Anti-CXCR5 Antibodies Binding to Raji Cells and Antagonistic Activity in a Calcium Transfer Assay

[000382] Antibodies binding to hCXCR5-293 and cynoCXCR5-293 cells were further analyzed for binding to Raji cells by flow cytometry using a FACSVerse Analyzer (BD Biosciences, Franklin Lakes, NJ). The apparent binding affinity of the antibody to Raji cells was calculated as EC ₅₀ equilibrium binding titration curves in which the geometric mean fluorescence intensity (gMFI) of the antigen-binding population was quantified by flow cytometry and the results are presented in Table 2.

[000383] Evidence suggests that some antibodies bind to Raji cells to a greater/less degree than other antibodies. These data suggest that antibodies recognize endogenous CXCR5 on Raji cells derived from a patient with Burkitt's lymphoma, suggesting that antibodies can bind to B cells in vivo . In addition, the affinity of CXCR5 antibody 11G2 on Raji cells is higher than that of the reference anti-CXCR5 antibody 16D7 (WO 2009/032661), suggesting that 11G2 may be more active than 16D7.

[000384] Antibodies binding to both hCXCR5-293 and cynoCXCR5-293 were also tested in the calcium transfer assay for antagonist activity. In addition, reference antibodies against CXCR5 16D7 (WO 2009/032661) were included as controls. Briefly, hCXCR5-HEK-293 cells were seeded in a 96-well plate in high glucose DMEM media. After overnight incubation, the media was removed and 100 μl of 1x Fluo-4 NW (Thermo Fisher Scientific, Waltham, MA) was added to the cells. The cells were then incubated at 37°C for one hour. Serial dilutions of monoclonal antibodies were added to the cells and incubated at room temperature for one hour. Calcium transfer was measured with a Flexstation (Molecular Devices, Sunnyvale, CA) at 485 nM excitation wavelength and 525 nM emission wavelength by adding 111 nM recombinant hCXCL13 (BPS Bioscience, San Diego, CA). Data was recorded at intervals of 1.52 seconds. Calcium transfer was determined using the device by subtracting the smallest reading from the largest reading. Ligand-induced calcium transport inhibition _IC50 values were determined using GraphPad Prism® software (version 6.0, GraphPad Software, Inc, San Diego, CA) by fitting a non-linear regression curve and log sigmoid in an agonist dose-response model (Table 2) . Inhibition of calcium transfer is evidence of functional antagonism. Thus, the 11G2 antibody is not only a potent ADCC debilitating agent, but also an antagonist, which is the second mechanism of action. The antagonism of the 11G2 antibody is comparable to comparator antibodies, eg 2C9 (reference antibody, see eg WO 2012/010582) and 16D7 (reference antibody, see eg WO 2009/032661). Data reporter analysis cAMP also indicate functional antagonism (see below).

TABLE 2

Antibody (hybridoma) EU ₅₀ Raji cell binding (nM) IC ₅₀ Ca transfer ²⁺ (nM) 5H3 4.767 13.60 5H7 1.014 5.555 11G2 0.9679 5.929 31E5 0.7292 5,050 18G4 0.7454 13.86 19D9 0.9022 9.395 20G4 0.5178 9.853 21A3 1.226 9.043 23F1 0.4062 6.982 39H4 0.3154 Not tested 47D11 0.8493 9.448 56G2 0.3871 14.36 4F1 0.4202 9.739 15D5 0.7949 11.21 20D10 0.3259 11.03 6E9 0.5879 14.28 12E11 0.6493 13.40 16D10 0.7862 14.47 26E2 1.424 18.40 1A11 0.4936 35.06 1D10 0.2164 33.66 10G2 0.4571 14.74 9G11 0.4976 38.61 12C3 0.4868 35.01 25E1 0.7682 14.13 18G11 0.3161 5.034 23D5 0.1483 5,886 31G7 0.2848 9.084 31F3 0.2705 18.23 51D11 0.2525 7.429 41A10 0.8046 14.67 48A10 0.7083 21.07 13F4 0.6225 Not tested 3A1 0.2572 20.22 19F8 2.378 30.03 2C9 0.21 3.486 16D7 0.65 11.43

Example 3: Cloning of heavy and light chain variable regions of anti-CXCR5 antibody

[000385] The antibody producing hybridomas assayed in Example 2 were lysed using the RNeasy mini kit (Qiagen, Hilden, Germany) and then first strand cDNA was synthesized using the Superscript III First Strand Synthesis System (Thermo Fisher Scientific, Waltham, MA ). The light and heavy chain variable regions (VL and VH) were amplified by PCR using degenerate mouse light and heavy chain primers containing the nucleic acid sequences of SEQ ID NO:64-88 shown in Table 16. After PCR, VL and VH were cloned into vectors Zero Blunt TA (Thermo Fisher Scientific, Waltham, MA) and then sequenced. The mouse VL and VH amino acid sequences from the 11G2, 5H7 and 41A10 antibodies are shown in Table 16 as SEQ ID NOS:35-40. CDRs are underlined.

Example 4: Obtaining afucosylated and fucosylated chimeric antibodies

[000386] To evaluate the effect of afucosylation and fucosylation on antibody activity, fucosylated and afucosylated versions of the antibody of Example 3 were prepared in mammalian cells.

[000387] The VL and VH of the antibodies of interest were further cloned into vectors containing human kappa chain constant regions and human IgG1 constant regions, respectively. The heavy chain variable regions were cloned into the pSMED2 mammalian expression vector containing the human IgG1 constant region (SEQ ID NO:89) to give chimeric full-length mouse-human heavy chains. The light chain variable regions were cloned into the pSMEN3 mammalian expression vector containing human kappa chain (Cκ) constant regions (SEQ ID NO:90) to generate chimeric full-length mouse-human light chains.

[000388] HEK-293F cells (Thermo Fisher Scientific, Waltham, MA) were transiently transfected with vectors containing the chimeric antibody genes to produce a fucosylated chimeric antibody. The fucosylated chimeric antibodies were then purified using protein A columns. The apparent affinity (EC ₅₀ cell binding) of binding of the chimeric antibody to Raji cells was determined by flow cytometry using a FACSVerse Analyzer (BD Bioscience, Franklin Lakes, NJ) and is shown in Table 3.

TABLE 3

Antibody (chimeric) EU ₅₀ Raji cell binding (nM) (fucosylated) EU ₅₀ CynoCXCR5-300.19 cells (nM) (fucosylated) 5H7 0.04215 0.06027 11G2 0.01746 0.02419 31E5 Not tested 0.05151 23F1 0.1687 66.03 47D11 0.4292 13.85 56G2 0.07613 6.376 4F1 0.3209 14.08 15D5 0.2695 1.387 20D10 0.1624 0.1478 6E9 Not tested 18.26 12E11 0.2883 Not tested 16D10 0.2909 Not tested 1A11 0.08464 0.2575 1D10 0.1530 Not tested 10G2 1.008 Not tested 12C3 0.1035 0.1767 25E1 0.4487 11.73 23D5 0.03410 4.168 31G7 Not tested 12.83 31F3 0.07858 4.072 51D11 0.08732 49.63 41A10 0.3899 0.8308 13F4 0.5557 4.381 3A1 0.09156 0.3059 2C9 0.09989 0.1305

[000389] Afucosylated chimeric antibodies were generated using the Potelligent® CHOK1SV cell line (BioWa/Lonza, Allendale, NJ) that lacks both alleles of the fucose addition gene (α-1,6-fucosyltransferase, FUT8 ). The chimeric light chain containing the human kappa constant region was cloned into the Lonza pEE12.4 GS vector (Lonza Biologics, Basel, Switzerland) and the chimeric heavy chain containing the human IgG1 constant region was cloned into the Lonza pEE6.4 GS vector (Lonza Biologics , Basel, Switzerland). The heavy chain expression cassette from pEE6.4 was purified by digestion with NotI and PvuI and then cloned at the NotI and PvuI regions in pEE12.4 containing the chimeric light chain. The final DGV vector containing the heavy and light chain expression cassettes was linearized with PvuI and then electroporated into Potelligent® CHOK1SV cells. 24 hours after transfection, Potelligent® cells were seeded in 96-well plates at 3-5K cells/well in CDCHO media (Thermo Fisher Scientific, Waltham, MA) supplemented with 1x HT (Thermo Fisher Scientific, Waltham, MA), 1 mM uridine (Sigma, St. Louis, MO) and 50 μM MSX (EMD Millipore, Billerica, MA). After 3-4 weeks of incubation, clone supernatants were analyzed for antibody titer using Octet (Pall Fortebio, Fremont, CA). High-expressing clones were further grown for antibody production. The afucosylated chimeric antibodies were then purified on protein A columns.

[000390] To determine the specificity of antibodies to CXCR5, the binding of antibodies to cells expressing other cytokines was analyzed. Briefly, FlowCellect chemokine receptor cell lines expressing CXCR1, CXCR2, or CXCR3 (EMD Millipore, Billerica, MA) and Jurkat cells (ATCC, Manassas, VA) expressing CXCR4 were stained with 5 µg/mL or 10 µg /ml of each chimeric antibody against CXCR5. Cells were then incubated with PE-conjugated goat anti-human antibody (Southern Biotech, Birmingham, AL) before analysis by flow cytometry using a FACSVerse Analyzer (BD Biosciences, Franklin Lakes, NJ). As shown in FIG. 4A-4D, all chimeric antibodies showed very low binding to cells expressing CXCR1 (Fig. 4A), CXCR2 (Fig. 4B), CXCR3 (Fig. 4C), or CXCR4/Jurkat cells (Fig. 4D) compared to positive controls. .

Example 5: Humanization of mouse monoclonal antibodies

[000391] To avoid any potential immunogenicity with the formation of HAMA (human anti-mouse antibodies), chimeric antibodies 41A10 (C-41A10) and 11G2 (C-11G2) were humanized using human germline framework sequences from IGKV1-39 (light chain variable domain DPK9, GenBank accession number X93627.1, SEQ ID NO:93) and a human germline framework sequence from IGHV3 (heavy chain variable domain DP54, GenBank accession number AB019440, SEQ ID NO:91).

[000392] Humanized versions of chimeric 11G2 antibodies (C-11G2) and chimeric 41A10 antibodies (C-41A10) were generated by grafting complementarity determining regions (CDRs) (referred to as "CDR grafted antibodies"). Those. Heavy chain CDRs were grafted onto a human DP-54 framework (VH3 subgroup; SEQ ID NO:91) with a JH4 segment (SEQ ID NO:92), while light chain CDRs were grafted onto a human DPK9 framework (VKI subgroup; SEQ ID NO:93) with a JK4 segment (SEQ ID NO:94).

[000393] Humanized VH regions were fused to a human IgG1 constant region (SEQ ID NO:89) and then subcloned with a self expression vector to produce CDR grafted heavy chains including, but not limited to, SEQ ID NO:96 (VH 11G2 with grafted CDRs). Humanized VL regions were fused to a human kappa chain constant region (SEQ ID NO:90) and then subcloned into a native expression vector to produce CDR grafted light chains including, but not limited to, SEQ ID NO:97 (VL 11G2 with grafted CDRs).

Example 6: Binding affinity of afucosylated or fucosylated anti-CXCR5 antibodies

[000394] The apparent affinity of chimeric and humanized fucosylated and afucosylated anti-CXCR5 antibodies to CXCR5 on the cell surface was analyzed. More specifically, to determine the apparent affinity of chimeric and humanized anti-CXCR5 antibodies to CXCR5 expressing cells, cell binding experiments were performed using human and cynomolgus monkey peripheral blood mononuclear cells (PBMC) and human tonsillar mononuclear cells (TMC). The apparent binding affinity of the anti-CXCR5 mAb to CXCR5 ⁺ cells (B cells, true Tfh cells, and circulating Tfh-like cells) was calculated as the EC ₅₀ equilibrium binding titration curves, where the geometric mean of the fluorescence intensity (gMFI) of the antigen-binding population was quantified by flow cytometry. .

[000395] Healthy human donor cells prepared with the Trima® Apheresis System and enriched with PBMC were obtained from Blood Centers of the Pacific (San Francisco, CA). PBMCs were isolated by density gradient centrifugation using SepMate ^™ tubes and Lymphoprep ^™ media according to the manufacturer's instructions (STEMCELL Technologies, Vancouver, BC, Canada).

[000396] Tonsillar mononuclear cells (TMC) were isolated from human tonsils obtained from BioOptions (Brea, CA). Briefly, tonsils were dissected into small fragments (3-4 mm) in cold RPMI 1640 medium using a sterile scalpel. The tonsil tissue was then digested for 30 minutes at 37° C. in digestion medium (3 ml 10x collagenase, 300 μl 100x deoxyribonuclease (DNase), 6.7 ml RPMI 1640). RPMI 1640 supplemented with 10% fetal calf serum (FBS) was added to neutralize enzymatic activity, and then the tissue was filtered successively through cell sieves with nylon filters (70 µm and 40 µm nylon). After centrifugation, erythrocyte pellets were lysed using ammonium chloride lysis buffer. Platelets were removed using low speed centrifugation (200×g) for 10 minutes in phosphate buffered saline (PBS)/2% FBS/2 mM ethylenediaminetetraacetic acid (EDTA). CD4 ⁺ T cells were purified from the TMC mixture using the EasySep ^™ human CD4 ⁺ T cell enrichment kit according to the manufacturer's instructions (STEMCELL Technologies, Vancouver, BC, Canada).

[000397] After isolation, PBMC were seeded at a density of 1.0×10 ⁵ cells per well and CD4 ⁺ TMC were seeded at a density of 2.0×10 ⁵ cells per well in isolation buffer (PBS containing 1% FBS and 1 mM EDTA) in 96-well U-bottom plates. The plates were then centrifuged at 1500 rpm. for 5 minutes at room temperature. PBMC and CD4 ⁺ TMC were resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS containing 1% FBS) containing a human Fc crystallizable fragment blocker (2 μl per well; Biolegend) and 4-fold antibody serial dilutions (dilution series with 11 points starting at 5000 ng/ml for PBMC and 312.5 ng/ml for CD4 ⁺ TMC) and incubated on ice for 2 hours. PBMC and CD4 ⁺ TMC were then washed 3 times with FACS buffer and resuspended in 50-100 µl of FACS buffer containing fluorescently labeled antibodies to stain lymphocyte subpopulations and fluorescently labeled secondary anti-human Ig antibody to detect monoclonal antibodies (mAb) against CXCR5. After incubation for 30 minutes at 4°C, PBMC and CD4 ⁺ TMC were washed 2 times with FACS buffer and resuspended in 125 μl of 0.5% paraformaldehyde (PFA) in PBS. The plates were stored at 4° C. until analyzed by flow cytometry (BD LSRFortessa™ Cell Analyzer, BD Biosciences, Franklin Lakes, NJ).

[000398] Cell binding titration curves were obtained by plotting the gMFI antigen-binding population against the logarithm of anti-CXCR5 antibody concentration. EC ₅₀ values were determined using GraphPad Prism® software (version 6.0, GraphPad Software, Inc, San Diego, CA) by fitting a non-linear regression curve and log sigmoid in an agonist dose-response model according to the following equation:

Log (agonist) vs. answer - variable slope (four parameters)

Y=Min+(Max-Min/(1+10^((LogEC ₅₀ -X)*Hill slope))

[000399] Where Y is gMFI, X is the antibody concentration, Maximum is the maximum Y value corresponding to the upper plateau of the sigmoid curve, Minimum is the minimum Y value corresponding to the lower plateau of the sigmoid curve, and LogEC ₅₀ is the logarithm of the antibody concentration at the inflection point of the curve.

[000400] EC ₅₀ values are summarized across experiments using mean and standard deviation (STDEV) with multiple repetitions. The results (i.e., mean apparent affinity of chimeric and humanized anti-CXCR5 mAbs that are fucosylated or afucosylated for CXCR5 expressing cells) are shown in Table 4. The results also include binding affinity data obtained using control anti-CXCR5 antibodies 2C9 (WO 2012/010582), 16D7 (WO 2009/032661) and 11A7 (WO 2016/028573) which were included for comparison purposes. It should be noted that fucosylated and afucosylated chimeric and humanized 11G2 antibodies containing various combinations of VL and VH (i.e. h11G2 XC51/XC152, h11G2 XC153/XC155, h11Gh11G2 XC153/XC156, h11G2 XC154/XC155 and h11G2 XC 154/XC157), have an apparent affinity for human B cells that is approximately equal. Those. the data suggest that afucosylation does not appear to affect the affinity of the antibody for CXCR5-expressing cells. In addition, the data indicate that the h11G2 XC154/XC155 antibodies (fucosylated and afucosylated) showed approximately 10-fold higher affinity than the control anti-CXCR5 antibody 2C9 and approximately 100-fold higher affinity than 11A7. The control anti-CXC5 antibody 16D7 did not show saturable binding. These data indicate that afucosylation did not affect the affinity of 11G2 antibodies when bound to CXCR5-expressing cells. Because affinity is a measure of the strength of the interaction between an epitope and the antigen-binding site of an antibody (i.e., paratope), these data suggest that even though antibodies bind to a similar epitope, they do not bind to the epitope with the same strength as 11G2.

TABLE 4

CXCR5 mAb Apparent affinity
Average EC ₅₀ ± STDEV (pM) human B cells Tfh-like human cells Human Tfh cells NHP B cells Tfh-like NHP cells Chimeric 41A10 161.28±33.41 ND ND 548.55 ND Humanized 41A10 142/147 153.38±57.21 ND ND 2049.50 ND Humanized 41A10 142/148 152.29±75.51 ND ND 1961.00 ND Chimeric 11G2 17.94±8.48 ND 8.46 6.95 ND Afucosylated chimeric 11G2 8.09±2.93 ND 45.59 ND ND Humanized 11G2 151/152 13.05±10.9 ND 3.33 10.06 ND Humanized 11G2 153/155 7.79±6.89 ND 4.82 7.03 ND Humanized 11G2 153/156 8.88±6.39 ND 3.23 7.06 ND Humanized 11G2 154/155 7.08±4.48 5.92±1.55 8.25 4.13 5.65 Humanized 11G2 154/157 19.12±16.83 ND 7.24 8.65 ND Afucosylated humanized 11G2 154/155 6.60±2.33 5.89±1.40 10.59 1.32 10.47 2C9 (reference antibody) 65.59±37.95 ND ND ND ND 16D7 (reference antibody) Non-saturable binding ND ND ND ND 11A7 (reference antibody) 563.63±249.39 ND ND ND ND

Example 7: Concentration-dependent binding of afucosylated or fucosylated humanized 11G2 antibodies versus fucosylated reference antibodies

[000401] Concentration dependent binding of fucosylated humanized 11G2 (VL h11G2 XC154/VH XC155, also referred to as h11G2 154/155 or h11G2 XC154/XC155) and afucosylated humanized 11G2 CXCR5 (afucosylated h11G2 154/15 5) with B-cells from human PBMC compared with the binding of comparator mAbs 2C9, 16D7 and 11A7. Cell binding titration curves were obtained by plotting the gMFI antigen-binding population against the logarithm of anti-CXCR5 antibody concentration and are shown in FIG. 5.

[000402] Evidence suggests that fucosylated and afucosylated h11G2 154/155 have an identical curve, suggesting that the presence or absence of fucose on the N-linked glycoform at position Asn297 on the constant regions of at least one, but preferably both chains The antibody does not affect the affinity of the antibody for CXCR5 present on the cells.

[000403] In addition, the data further suggest that the EC ₅₀ of fucosylated (solid circles, 9.630×10 ^-12 M) and afucosylated (open circles, 1.188×10 ^-11 M) h11G2 154/155 antibodies was much lower than EC ₅₀ comparator antibodies: 2C9 (solid triangles, 6.24×10 ^-11 ), 11A7 (solid diamonds, 9.265×10 ^-10 ) and 16D7 (solid squares, approximately 0.004945 and binding was not saturable). These data indicate that h11G2 154/155 antibodies have different binding characteristics than comparator antibodies. Thus, these data indicate that antibodies 11G2 and 2C9, 11A7 and 16D7 do not bind to the same epitope on CXCR5.

Example 8: ADCC activity of chimeric and humanized antibodies against CXCR5

[000404] To determine the potency and efficacy with which a cohort of chimeric and humanized anti-CXCR5 antibodies stimulates antibody-dependent cellular cytotoxicity (ADCC), serial dilutions of anti-CXCR5 antibodies or isotype control were incubated with peripheral blood mononuclear cells (PBMC) from healthy human donors or cynomolgus monkeys . In this assay, PBMCs are the source of natural killer (NK) effector cells and target B cells and Tfh-like cells. Flow cytometry was used to analyze the number of B cells and Tfh-like cells remaining after approximately 20 hours. Similarly, the ability of humanized antibodies to induce ADCC of true Tfh cells from human tonsils was assessed using CD4 ⁺ T cells isolated from tonsillar mononuclear cells, with the addition of NK cells isolated from PBMC.

[000405] As described in Example 6, healthy human donor cells prepared with the Trima® Apheresis System and enriched for PBMC were obtained from Blood Centers of the Pacific (San Francisco, CA). PBMCs were isolated by density gradient centrifugation using SepMate ^™ tubes and Lymphoprep ^™ media according to the manufacturer's instructions (STEMCELL Technologies, Vancouver, BC, Canada). After isolation, PBMC were seeded in complete RPMI medium at a density of 2.0×10 ⁵ cells per well in 96-well U-bottomed plates.

[000406] In this assay, PBMCs are the source of natural killer (NK) effector cells and target B cells and Tfh-like cells. Serial dilutions of anti-CXCR5 mAb or isotype controls were added to wells and cells were incubated at 37° C., 5% CO ₂ for approximately 20 hours. The plates were then centrifuged at 1800 rpm. for 5 minutes at room temperature (RT). The PBMCs were then washed with ice-cold FACS buffer and resuspended in 50 μl ice-cold FACS buffer containing fluorescently labeled antibodies to stain lymphocyte subpopulations. After incubation for 15-30 minutes at 4° C., PBMCs were washed 2 times with ice-cold FACS buffer and resuspended in 100 μl of 0.5% paraformaldehyde (PFA) in PBS. CountBright ^™ Absolute Counting Beads (Thermo Fisher Scientific) (15 µl per well) were added to each well. The plates were stored at 4° C. until analyzed by flow cytometry (BD LSRFortessa ^™ cell analyzer).

[000407] Tonsillar mononuclear cells (TMC) were also isolated as described in Example 6 above.

[000408] Cytotoxicity titration curves were generated using the methods described in Example 6. EC ₅₀ values are summarized across experiments using mean and standard deviation (STDEV) with multiple repetitions. The results are shown in Table 5. Data generated using reference antibodies against CXCR5 2C9, 16D7 and 11A7 are included for comparison purposes.

TABLE 5

Average cytotoxicity of chimeric and humanized anti-CXCR5 mAbs against CXCR5-expressing cells

mAbs against CXCR5 ADCC activity
Mean EC ₅₀ ±STDEV (pM) human B cells Tfh-like human cells Human Tfh cells NHP B cells Chimeric 41A10 11732.28±24781.14 ND ND ND Afucosylated chimeric 41A10 54.47±89.71 ND ND ND Humanized 41A10 142/147 1245±881.03 ND ND ND Humanized 41A10 142/148 1109.25±1081.15 ND ND ND Chimeric 11G2 564.01±1643.77 ND 1.55 115.26 Afucosylated chimeric 11G2 4.39±3.08 ND 0.07 ND Humanized 11G2 151/152 376.74±606.13 ND ND 43.61 Humanized 11G2 153/155 463.17±670.85 ND ND 20.72 Humanized 11G2 153/156 567.68±847.34 ND ND 28.06 Humanized 11G2 154/155 253.72±672.14 7.978 0.77 39.14 Humanized 11G2 154/157 517.33±1062.82 ND ND 54.02 Afucosylated humanized 11G2 154/155 2.01±2.28 4.82±2.88 0.11 15.26±11.65 2C9 380.31±757.04 ND ND ND 16D7 2590.61±6343.88 ND ND ND 11A7 4.35±4.03 ND ND ND

[000409] The data presented herein indicate that even when fucosylated, the h11G2 154/155 antibody has higher ADCC activity (253.72±672.14) than the 2C9 comparator antibodies (380.31±757.04 ) and 16D7 (2590.61±6343.88). The fucosylated 11A7 antibody showed higher ADCC activity than any fucosylated antibody tested. However, data suggest that afucosylated h11G2 154/155 had higher ADCC activity than any antibody tested, including fucosylated 11A7. The characterization studies provided herein showed that the 41A10 and 11G2 chimeric anti-CXCR5 mAbs triggered human B cell ADCC with EC _50s of 11.73 nM and 0.56 nM, respectively. Afucosylated versions of the 41A10 and 11G2 chimeric anti-CXCR5 mAbs potentiated ADCC activity by at least about 100-fold. The humanized variants triggered human B cell ADCC with powers comparable or superior to those of the corresponding chimeric antibodies. As with the chimeric anti-CXCR5 mAbs, the afucosylated humanized variant increased the power of ADCC over the normal corresponding fucosylated antibody by at least about 100-fold. Afucosylated h11G2 was more potent for ADCC than the fucosylated versions of 2C9 and 16D7, and afucosylated h11G2 was at least comparable to fucosylated 11A7.

Example 9: 11G2 antibodies antagonize CXCL13 signaling

[000410] Cyclic adenosine monophosphate reporter assay was used to determine if anti-CXCR5 antibodies could functionally antagonize CXCL13 signaling. (cAMP) in an engineered cell line stably expressing human CXCR5. In these cells, CXCL13 inhibits forskolin-driven cAMP production in a concentration dependent manner.

[000411] To determine whether anti-CXCR5 antibodies can functionally antagonize CXCL13 signaling, a Hit Hunter® cAMP assay (DiscoveRx Corporation, Fremont, CA) was performed in CHO-K1 cells stably expressing human CXCR5. In this assay, active β-galactosidase (β-gal) is generated by complementation of enzyme fragments when cAMP is formed. β-gal can then convert the chemiluminescent substrate, generating an output signal detectable with a standard microplate reader spectrophotometer. The CXCR5 ligand, CXCL13, inhibits cAMP production triggered by forskolin (20 μM) in a concentration dependent manner. To test the potency and efficacy of anti-CXCR5 antibodies in inhibiting CXCL13-mediated forskolin-induced cAMP production, serial dilutions of each anti-CXCR5 antibody were added to CXCR5-expressing CHO-K1 cells in the presence of forskolin (20 μM) and CXCL13 (used at its IC _{80 of} 600 pM ).

[000412] Functional antagonism titration curves were prepared as described in Example 6, including determination of the EC ₅₀ value. The results are presented in table 6.

TABLE 6

Stimulation of CXCL13-inhibited cAMP production in vitro in CXCR5 ⁺ CHO-K1 cells

mAbs against CXCR5 EU ₅₀ (pM) % CXCL13 inhibition Humanized 11G2 154/155 752.9 120.6 2C9 793.1 113.5 16D7 1104 102.4 11A7 1401 105.9

[000413] In a separate study, afucosylated humanized 11G2 154/155 was tested for functional antagonism and acted similarly to humanized fucosylated 11G2 154/155 (i.e., EC ₅₀ =961.4 pM).

[000414] Data suggest that h11G2 154/155 inhibited CXCL13 signaling at least as well as reference antibodies 2C9, 16D7, 11A7. In fact, h11G2 inhibited CXCL13 signaling (120.6%) to a significantly greater extent than 16D7 (102.4%) and 11A7 (105.9%). These data suggest that 11G2 antibodies may be a useful new therapeutic agent for the treatment of a disease, disorder or condition mediated or associated with CXCR5-mediated CXCL13 signaling.

Example 10 : Mapping of 11G2 antibody binding sites to hCXCR5

[000415] The binding site of afucosylated humanized 11G2 154/155 on CXCR5 was identified by grafting amino acids from the extracellular N-terminus of mouse CXCR5 onto human CXCR5.

[000416] More specifically, the mapping strategy took advantage of the fact that 11G2 binds to human CXCR5 (SEQ ID NO:32) but does not bind to mouse CXCR5 (SEQ ID NO:34). The amino acid sequence alignment of human CXCR5 (hCXCR5) and mouse CXCR5 (mCXCR5) is shown in FIG. 6. Amino acid residues of the extracellular domains of CXCR5 are underlined. The extracellular domains (ECDs) of the murine and human CXCR5 proteins are shown in bold and labeled "N", "L1", "L2", and "L3" below the sequence of each region in FIG. 6. These ECDs were swapped between mouse and human proteins to identify regions responsible for 11G2 specificity for human CXCR5 and no binding to mCXCR5. Each of these regions from mouse CXCR5 was replaced with the same region in human CXCR5 to generate chimeric proteins designated as XC251, XC252, XC253, XC254, XC255 and XC256, where the human CXCR5 protein contains the corresponding defined mouse CXCR5 domain. Table 7 shows which regions of the mouse (m) protein are rearranged in human CXCR5.

TABLE 7

(chimeric mouse/human ECD swapped CXCR5 proteins)

XC251 hCXCR5-mL3 XC252 hCXCR5-mL2 XC253 hCXCR5-mN XC254 hCXCR5-mL2-mL3 XC255 hCXCR5-mN-mL3 XC256 hCXCR5-mN-mL2

[000417] More specifically, the mouse L3 domain was swapped with the corresponding human L3 domain to generate a chimeric XC251 protein containing the mouse L3 domain in the context of the human CXCR5 protein (hCXCR5-mL3). Similarly, mouse L2 was swapped with the corresponding human domain to generate the XC252 chimeric protein (hCXCR5-mL2); The mouse N-domain was swapped with the corresponding human N-domain to generate the XC253 chimeric protein (hCXCR5-mN); the mouse L2 and L3 domains were swapped with the corresponding human domains to generate the XC254 chimeric protein (hCXCR5-mL2-mL3); the mouse mN and L3 domains were swapped with the corresponding human domains to generate a chimeric XC255 protein (hCXCR5-mN-mL3); and the mouse mN and L2 domains were swapped with the corresponding human domains to generate a chimeric XC255 protein (hCXCR5-mN-mL2).

[000418] Binding of 11G2 to mouse CXCR5, human CXCR5, and various ECD-swapped mouse-human CXCR5 chimeric proteins (XC251-XC256) was analyzed and compared to the binding of various antibodies, ie. rat anti-mouse CXCR5 antibody (rat antibody) (cat. no. MAB6198, R&D systems, Minneapolis, MN), rabbit anti-human CXCR5 antibody (Rb) (cat. no. ab46218, Abcam, Cambridge, MA), 2C9, 16D7. The results are shown in Table 8. Numbers in bold indicate antibodies that bind to the protein.

TABLE 8

(FACS analysis of antibody binding to mouse-human CXCR5 chimeric proteins with rearranged ECDs)

Rb (hCXCR5)
(GMFI) Rat antibody (mCXCR5)
(GMFI) 2C9
(GMFI) 16D7
(GMFI) 11G2
(GMFI) hCXCR5 18575 2457 117099 149936 138584 XC251 hCXCR5-mL3 6594 3932 13287 19927 14559 XC252 hCXCR5-mL2 12496 3842 22383 26963 21650 XC254 hCXCR5-mL2-mL3 4681 3826 11446 16877 11471 mCXCR5 1037 12537 1183 958 901 XC253 hCXCR5-mN 1549 37152 1779 1264 1215 XC255 hCXCR5-mN-mL3 1607 39099 1954 1341 1318 XC256 hCXCR5-mN-mL2 1525 31061 1966 1376 1262

[000419] The data suggest that if an antibody is specific for mouse CXCR5, it binds to ECD-swapped mouse-human chimeric CXCR5, provided that the chimeric protein contains the mouse N-domain. Similarly, in the case of antibodies that bind to human CXCR5 (11G2, 2C9, 16D7 and Rb), the antibodies bind to chimeric CXCR5 if the human N-domain was present in the chimeric protein. Thus, the species specificity of antibodies seems to be regulated by the binding site located in the N-domain. More importantly, these data indicate that the N-domain of human CXCR5 was required for antibody binding 11G2, 2C9 and 16D7.

Example 11 : Identification of amino acid residues unique to hCXCR5 binding antibody 11G2

[000420] In order to more accurately determine the specific amino acid residues key to antibody binding to hCXCR5 and to further differentiate anti-human CXCR5 antibodies, more detailed studies of the N-domain of CXCR5 were performed. More specifically, point mutations were performed in a fragment of the N-domain of human CXCR5 (amino acids 1-58 according to SEQ ID NO:32) to produce point mutated proteins, each containing the corresponding mouse amino acid residue from CXCR5. Received stable transfectants expressing each mutein, and the cells were analyzed for antibody binding using flow cytometry. Those. obtained nineteen (19) N-domain mutant human CXCR5 proteins, referred to as XC276-XC294, each containing a single amino acid substitution, where the human residue from CXCR5 was replaced with the corresponding mouse residue, as shown in Table 9. Human CXCR5 muteins additionally contained an epitope FLAG (DYKDDDDK), shown in Table 9 using lowercase letters, at the N-terminus to allow normalization based on expression level; the FLAG tag does not affect antibody binding to muteins. Generally, substitutions were not made where substitution of a human amino acid residue for a mouse amino acid residue would be a conservative substitution, and these residues are indicated in italics.

[000421] Table 9 shows the amino acid sequence of the hCXCR5 N region (first 58 amino acids shown) compared to the amino acid sequence of the mouse CXCR5 N region. Amino acids that differ between the two sequences are underlined. The figure also shows the various constructs produced (XC276-XC294) where the mouse amino acid residue was replaced with the corresponding human amino acid in hCXC5. For example, XC276 is human CXCR5 with a single D (human) to G (mouse) amino acid substitution. The rest of the amino acid sequence of hCXCR5 (SEQ ID NO:32) is not shown because she was not changed. Muteins were generated to determine which amino acid residues are critical for antibody binding to hCXCR5, as the antibodies tested did not bind to mouse CXCR5.

TABLE 9 Protein Mutation relative to SEQ ID NO:32 Protein sequence showing change relative to human CXCR5 hCXCR5 wt MdykddddkNYPLTLEMD--LENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAY mCXCR5 M--------NYPLTLD M GSI TY N MD DL Y K EL AFYS N ST E IP L Q D SNF C STV EGPL L T SFKA VFMPVAY XC276 D10G G XC277 SI (insertion) SI XC278 L11T T XC279 E12Y Y XC280 W19K K XC281 D22A A XC282 R23F F XC283 L24Y Y XC284 D25S S XC285 Y27S S XC286 N28T T XC287 T30I I XC288 S31P P XC289 V33Q Q XC290 N35S S XC291 H36N N XC292 L37F F XC293 P39S S XC294 A40T T

[000422] HEK-293T cells were transiently transfected with the nucleic acids encoding each mutant peptide. 2 days after transfection, cells were harvested and stained with 1 μg/ml PE-conjugated anti-FLAG antibody (Biolegend) or 1 μg/ml or 3 μg/ml afucosylated humanized 11G2 154/155; 2C9; S16D7 or 11A7 followed by a PE-conjugated anti-human IgG antibody (Southern Biotech, Birmingham, AL). Cells were analyzed by flow cytometry using Accuri (BD Biosciences Franklin Lakes, NJ). The geometric mean fluorescence intensity (gMFI) was determined and the ratio of gMFI of each of the anti-CXCR5 antibody and anti-FLAG antibody was calculated.

[000423] As shown in FIG. 7, all four antibodies bound to wild-type human CXCR5 (XC275; WT). The antibodies then showed different binding profiles to peptides with different point mutations. Those. 2C9 did not bind to XC276 (containing the D10G0 mutation) or XC277 (containing the insertion of amino acid residues S and I in the N-domain of human CXCR5), while 11G2, 16D7, and 11A7 bound to these proteins. These data suggest that the insertion of these two amino acids into the hCXCR5 sequence abolished 2C9 binding but did not affect the binding of the other three antibodies, suggesting that the epitope for 2C9 is different from that for 11G2, 16D7 and 11A7. More importantly, the mutation of leucine (L) to threonine (T) at amino acid residue position 11 (based on SEQ ID NO:32) completely abolished the binding of only 11G2 to XC278, but did not affect the binding of antibodies 2C9, 16D7 or 11A7 to this protein. Thus, the leucine residue at position 11 (relative to the amino acid sequence shown in SEQ ID NO:32) appears to be critical for 11G2 binding to human CXCR5, but not essential for 2C9, 16D7, or 11A7 binding to hCXCR5. These data indicate that the 11G2 epitope is not the same as the epitope for those antibodies. In addition, in FIG. 7 shows that all four antibodies bound to XC279 such that an E to Y substitution at amino acid residue position 12 (relative to SEQ ID NO:32) was not critical for any of these antibodies to bind to hCXCR5.

[000424] Binding of these four antibodies to XC280 containing a W substitution with lysine (K) at amino acid residue position 19 (according to SEQ ID NO:32) resulted in loss of binding to XC280 of antibodies 16D7 and 11A7, but did not affect binding of antibodies 11G2 and 2C9 to mutein (Fig. 7). These results further suggest that these four antibodies do not bind to the same epitope on hCXCR5.

[000425] FIG. 7 also shows that substitution of D at position 22 (according to SEQ ID NO:32) with alanine (A) completely eliminates 11G2 binding to the XC281 mutein, but does not affect the binding of antibodies 2C9, 16D7, and 11A7. This again emphasizes that the epitope for 11G2 is not the same as the epitopes for antibodies 2C9, 16D7 and 11A7.

[000426] All four antibodies bound to XC285-294 equally, and their binding was not affected by any amino acid substitutions in these muteins. These data suggest that amino acid residues at these positions (27, 28, 30, 31, 33, 35, 36, 37, 39 and 40; see Table 9) are not involved in antibody binding to CXCR5 in the case of antibodies 11G2, 2C9 , 11A7 and 16D7.

[000427] Thus, these data indicate that the leucine (L) residue at amino acid residue position 11 and the aspartate residue (D) at amino acid residue position 22 (relative to the amino acid sequence numbering of SEQ ID NO:32) are critical for antibody binding 11G2 with hCXCR5 because replacement of either (or both) of these residues with the corresponding mouse amino acid residue at these positions abolishes antibody binding to hCXCR5. These data also indicate that the 11G2 antibody does not share the same epitope as the 2C9, 16D7, or 11A7 antibody, as substitution of L11 and/or W22 does not affect the binding of these antibodies to hCXCR5.

[000428] FIG. 7 also shows that all four antibodies bind to the CXCR5 XC282-XC284 mutant proteins. All four antibodies also bound to the XC285-XC294 proteins. These data suggest that the epitope for antibodies 11G2, 2C9, 16D7 and 11A7 is not located in the N-domain region of hCXCR5, shown as XC282-XC294 muteins.

Example 12: 11G2 is selective for CXCR5 and does not bind to other members of the chemokine GPCR family

[000429] The selectivity of afucosylated humanized 11G2 154/155 was analyzed against twenty (20) members of the chemokine GPCR family using a β-arrestin binding assay (DiscoveRx). This assay uses whole cells stably transfected with each receptor and directly measures GPCR activity by detecting the interaction of β-arrestin with activated GPCR. Because arrestin recruitment is independent of G protein signaling, these assays provide a versatile screening and profiling platform that can be used for virtually any Gi-, Gs-, or Gq-coupled receptor, providing a standardized and efficient comparison across receptors or families of receptors (regardless of G-protein coupling). The assay was performed in agonistic (no ligand) and antagonistic (in the presence of ligand at EC90) modes. The selectivity panel included the following chemokine receptors: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1.

[000430] No significant activity was observed for afucosylated humanized 11G2 154/155 (tested at 200 nM) against any other chemokine receptor when tested in agonistic mode, and it was only significantly active as a CXCR5 antagonist (92% inhibition of ligand-induced β binding -arrestin) when compared in antagonistic mode with all other evaluated chemokine receptors, i.e. no significant inhibition was observed for any other chemokine receptor tested.

Example 13 : h11G2 does not show polyreactivity against DNA, insulin, or LPS

[000431] A polyreactivity assay is used to determine if antibodies can potentially react with various unrelated antigens in vivo . The lack of polyreactivity suggests that 11G2 is a CXCR5-specific antibody and does not bind to non-target targets in vivo .

[000432] Black DELFIA plates (Thermoscientific) were coated with 10 μg/ml double-stranded DNA (dsDNA; Millipore), 5 μg/ml lipopolysaccharide (LPS; Sigma), 10 μg/ml insulin (Sigma, St. Louis, MO) or phosphate- saline buffer (PBS) and incubated at 4°C overnight. The plates were then washed with water using a Biotek ELx405 microplate washer (Biotek, Winooski, VT) and then the plate was blocked with 200 µl of assay buffer containing 1x PBS, 0.05% Tween 20 and 1 mM EDTA at room temperature in within one hour. After an additional washing step, the plates were incubated at room temperature for one hour with serial dilutions of anti-CXCR5 antibodies (all antibodies were fucosylated). For detection, 100 μl of DELFIA-Eu-N1 anti-human IgG antibodies (50 μg/ml; Perkin Elmer, Waltham, MA) were added to the plates and incubated at room temperature for one hour. Finally, 100 µl of DELFIA enhancement solution was added to each well and the plates were shaken at room temperature for 15 minutes before being read with a VictorX4 multichannel plate reader (PerkinElmer, Waltham, MA) under europium fluorescence. The data are presented in table 10.

TABLE 10

Fluorescence (readings) in case of anti-CXCR5 mAb polyreactivity

Fluorescence (counts) mAbs against CXCR5 Dose (nM) Insulin coverage DNA coating LPS coating PBS coating Humanized 11G2 154/155 1.2 161 1099 190 1788 eleven 305 1780 829 1145 100 6090 2696 4988 2314 2C9 1.2 267 919 328 523 eleven 1690 1483 4144 1408 100 21100 13970 21447 7758 16D7 1.2 175 701 222 1874 eleven 1752 1974 1889 1733 100 4899 14356 3850 2286 11A7 1.2 614 29862 334 1669 eleven 3252 75133 1521 1608 100 31720 108952 6510 10188

[000433] In a separate study, afucosylated humanized 11G2 154/155 was tested for polyreactivity and no binding to insulin, DNA, LPS, or PBS was observed, consistent with the results for fucosylated humanized 11G2 154/155 shown in Table 10 above.

[000434] These data indicate that 11G2 did not react with insulin, DNA or LPS and is less reactive than comparator antibodies 2C9, 16D7 and 11A7. These data suggest that 11G2 is a potential useful new therapeutic agent. Polyreactive antibodies, by definition, bind in a non-specific manner to a range of biological molecules. The fact that 11G2 is not reactive in the global polyreactivity assay further confirms that 11G2 is specific for CXCR5. PK, TK and toxicity data also confirm that 11G2 has specificity for CXCR5.

Example 14 : Summary of differences between 11G2 and comparator antibodies

[000435] Table 11 below shows the characteristics of 11G2 and various comparator antibodies, presented above elsewhere in this description. Mean NDs were not determined.

[000436] These data indicate that the 11G2 antibody, both fucosylated and afucosylated, is very different from the comparator antibodies 2C9, 16D7 and 11A7. Those. 11G2 binding is abolished by substitution of mouse amino acid residues at positions 11 and 22, while comparator antibody binding is not affected by these residues (numbered according to the amino acid sequence of SEQ ID NO:32). In addition, amino acid residues W19, D22, and R23 are required for 2C9 to bind to hCXCR5, but no antibody requires all three of them, even though 11A7 binding to hCXCR5 is abolished by changing alanine to W at position 19 (in according to the amino acid sequence numbering of SEQ ID NO:32). Unlike all other antibodies, binding of 16D7 to hCXCR5 is abolished by the insertion of two mouse residues between D10 and L11 of human CXCR5 (according to the amino acid sequence numbering of SEQ ID NO:32) (see FIG. 7). Thus, the data presented herein suggests that 11G2 does not have the same epitope as the 2C9, 16D7 and 11A7 antibodies and that these antibodies also have different epitopes relative to each other.

[000437] In addition, the data presented herein indicate that 11G2 has an affinity for hCXCR5 (expressed as EC ₅₀ ) that is at least 10 times higher (7.08 pM) than 2C9 (65 .59 pM), and at least 100 times higher than that of 11A7 (563.63 pM), and very different from 16D7, which cannot be compared, since in this case, the binding was not saturable.

TABLE 11

mAbs against CXCR5 Epitope (critical residues for binding) Polyreactivity Apparent affinity (EC ₅₀ , pM) ADCC (EC ₅₀ , pM) Functional antagonism
(EC ₅₀ , pM) Humanized 11G2 154/155 L11, D22 No 7.08 253.72 752.9 Afucosylated humanized 11G2 154/155 ND No 6.60 2.38 961.4 2C9 W19, D22, R23 No 65.59 380.31 793.1 16D7 D10, SI insert No Non-saturable binding 2590.61 1104 11A7 W19 DNA reactivity 563.63 4.35 1401

[000438] These data suggest that 11G2 is significantly different from the 2C9, 16D7, and 11A7 comparator antibodies and is a potential new useful therapeutic agent for the treatment or prevention of a disease, disorder, or condition mediated or associated with CXCR5 biological activity, including, but not limited to examples, CXCR5-mediated CXCL13 signaling. This is especially true in the case of the afucosylated version of 11G2, which exhibits significantly increased ADCC activity, thus increasing its potential utility as a therapeutic agent for the treatment of, for example, immunological diseases mediated or associated with CXCR5 expression in cells.

Example 15 : Pharmacodynamics of afucosylated h11G2 XC154/XC155 in vivo

[000439] In vivo doses ranging from 0.001 to 400 mg/kg/dose IV or SC have been studied in numerous studies of afucosylated humanized 11G2 154/155 in cynomolgus monkeys. Within this experiment, formulations IV and SC contained the following components: 50 mg/ml anti-CXCR5 antibody or antigen binding fragment of the invention, 20 mM histidine, 8.5% sucrose and 0.02% polysorbate 80, 0.005% EDTA at pH 5.8 . Table 12 below shows the design of an in vivo afucosylated 11G2 XC154/XC155 study in cynomolgus monkeys.

TABLE 12

Study Description Dose (route of administration) Scheme of introduction Single dose pharmacodynamic (PD) study in cynomolgus monkeys 0.001, 0.002, 0.005, 0.1 and 0.2 mg/kg (IV) single dose Exploratory repeat toxicity toxicity study in cynomolgus monkeys (with 48-week recovery phase) 40 and 400 mg/kg/dose (IV), 260 (SC) Three weekly doses on days 1, 8 and 15 GLP compliant repeat dose toxicity study in cynomolgus monkeys (with 11-month recovery phase) 5 and 200 mg/kg/dose (IV), 20 (SC) Five biweekly doses on days 1, 15, 29, 43, and 57 Study of PK, PD, immunocompetence (secondary vaccine response) of afucosylated VL h11G2 XC154/VH XC155 in cynomolgus monkeys 2 and 10 mg/kg/dose (IV) Two weekly doses on days 1 and 8

[000440] In cynomolgus monkeys, a dose-dependent depletion of B-cells and Tfh-like cells in peripheral blood was observed with single doses of afucosylated VL h11G2 XC154/VH XC155 (afucosylated h11G2 XC154/XC155) ranging from 0.001 to 0.2 mg/kg; the lowest dose tested (0.001 mg/kg) resulted in approximately 50% depletion of B-cells and Tfh-like (ie cTfh) cells in the peripheral blood.

[000441] In exploratory and baseline GLP toxicity studies in cynomolgus monkeys, doses of afucosylated h11G2 XC154/XC155 ≥5 mg/kg IV resulted in significant depletion of B-cells and Tfh-like cells in peripheral blood and reduced cellularity of lymphoid follicles in spleen and other lymphoid tissues (axillary and mesenteric lymph nodes and intestinal lymphoid tissue); these effects correlated with the pharmacologically mediated reduced cellularity of B-cell regions observed immunohistochemically. Partial or complete recovery of B-cells, Tfh-like cells and Tfh-cells was observed in exploratory and basic studies. The kinetics of depletion and subsequent recovery is related to levels of exposure to afucosylated h11G2 XC154/XC155.

[000442] To determine whether afucosylated h11G2 XC154/XC155 impairs humoral anamnestic responses, a secondary vaccine response study was performed in cynomolgus monkeys. Briefly, afucosylated h11G2 XC154/XC155 was administered by IV bolus injection on days 1 and 8 at levels of 2 and 10 mg/kg/dose to cynomolgus monkeys (N=6 per group) vaccinated against TT (tetanus toxoid) prior to the study. Administration of afucosylated h11G2 XC154/XC155 was well tolerated and produced the expected pharmacological results (ie, reduction in circulating B cells and Tfh-like cells). Increased anamnestic responses with the formation of immunoglobulin M (IgM) and IgG compared with day 15 were observed in all animals in all dose groups on day 22, 7 days after the booster immunization with TT. These responses were reduced in animals treated with afucosylated H11G2 XC154/XC155 (10 mg/kg/dose), with a peak mean IgG titer against TT 42667 compared to 72,000 in the control group and a peak mean IgM titer against TT 1583 compared to 3500 in the control group. Rituxan® (rituximab), a B-cell-depleting antibody used as a comparator, did not affect the anamnestic responses to generate anti-TT IgG. In conclusion, these data suggest that afucosylated h11G2 XC154/XC155 impairs humoral anamnestic responses in vivo .

Safety pharmacology study

[000443] The design of the baseline toxicity study included electrocardiography and heart rate measurements. No deviations in electrocardiographic results attributed to the administration of afucosylated h11G2 XC154/XC155 were observed. All electrocardiograms were qualitatively and quantitatively within the normal range, and no arrhythmia was observed.

Example 16 : Pharmacokinetics and metabolism of afucosylated h11G2 XC154/XC155 in vivo

[000444] To detect the presence of ADA in cynomolgus monkey serum, electrochemiluminescent analysis (ECL) was performed using the Meso Scale Discovery® (MSD) analytical platform. To monitor the performance of the assay, each plate included a positive control, an anti-idiotypic antibody against CXCR5 added to cynomolgus monkey serum, and a negative control consisting of pooled normal cynomolgus monkey serum. Biotin labeled afucosylated h11G2 XC154/XC155 and ruthenium labeled afucosylated h11G2 XC154/XC155 were co-incubated with test samples, positive control and negative control. Anti-afucosylated h11G2 XC154/XC155 antibodies present in the samples should bind to biotin and ruthenium labeled versions of afucosylated h11G2 XC154/XC155 detected in this assay. The complexes were fixed through biotinylated afucosylated h11G2 XC154/XC155 on streptavidin-coated multichannel MSD plates read with an MSD Sector Imager. Final detection was performed using ruthenium labeled afucosylated h11G2 XC154/XC155 and tripropylamine to generate an ECL signal (RLU) in an MSD Sector Imager.

[000445] Test samples were tested for ADA using a multilevel strategy. Initially, samples are tested in the screening assay at a dilution factor of 75. Samples generating RLUs below the assay cutoff were scored negative (<1.88). Samples generating RLUs at or above the assay cutoff were reanalyzed in a full dilution series to determine antibody titer. Antibody titer was defined as the dilution factor of a sample that would generate an RLU equal to the RLU threshold. The logarithm (base 10) of this titer was recorded.

[000446] Conclusions regarding ADA induction were made based on a comparison of pre-dose samples collected on study day 1 and post-dose samples. If a pre-dose sample was negative for ADA and the corresponding post-dose sample was positive, the animal was considered positive for ADA induction. If both pre-dose and post-dose samples were positive for ADA, the animal was considered positive for ADA induction only if the post-dose sample titer was at least 0.48 (log 3, serial dilution factor) or exceeded the titer by sample before dosing.

Single dose pharmacokinetics

[000447] The PK of afucosylated h11G2 XC154/XC155 was characterized after a single IV dose of 0.001, 0.002, 0.005, 0.1, or 0.2 mg/kg of afucosylated h11G2 XC154/XC155 as part of a study to assess depletion and recovery of B-cells and follicular T-helper (Tfh)-like (Tfh-like) peripheral blood cells in male and female cynomolgus monkeys (n=1/sex/dose group). For this experiment, formulations IV and SC contained the following components: 50 mg/mL anti-CXCR5 antibody or antigen binding fragment of the invention, 20 mM histidine, 8.5% sucrose and 0.02% polysorbate 80, 0.005% EDTA at pH 5.8 .

[000448] Following a single IV dose, systemic exposure increased with dose and the PK of afucosylated h11G2 XC154/XC155 was characterized by low CL and low V _ss with mean values ranging depending on dose levels from about 0.2 to 2.6 ml /h/kg and 0.03 to 0.1 l/kg, respectively. Mean _t½ values, depending on dose levels, ranged from about 1 to 4 days. At low doses (0.001-0.005 mg/kg), evidence of a higher CL was observed, probably as a result of antibody-dependent cellular cytotoxicity (ADCC)-mediated cell depletion, which may also lead to clearance of the afucosylated h11G2 XC154/XC155/CXCR5 complex (Kryzanski et al. , 2016, J. Pharmacokinet Pharmacodyn 43(5):513-527; Wang et al., 2010, AAPS J. 12(4):729-740). A similar clearance/nonlinearity mechanism can be observed in humans at low doses. As described elsewhere herein above, a dose-dependent depletion of B cells and Tfh-like cells in peripheral blood was observed, and the depletion of circulating B cells was more robust than the depletion of Tfh-like cells.

Toxicokinetics (TK) upon repeated administration in the cynomolgus monkey

[000449] TK and ADA were assessed after administration of IV or SC afucosylated h11G2 XC154/XC155 at 5 (IV), 20 (SC) or 200 (IV) mg/kg every 2 weeks (5 doses total) to male and female cynomolgus monkeys (n=3 or 4/gender/dose group) as part of a repeat-dose toxicity study complying with GLP requirements.

[000450] No measurable concentrations of afucosylated h11G2 XC154/XC155 were observed in the samples collected and analyzed in the vehicle control group. No obvious sex differences in systemic exposure were observed in dose groups; thus group-average TK parameters are presented using combined data for male and female cynomolgus monkeys (Table 13).

[000451] Following biweekly administration at 5(IV), 20(IV) mg/kg, or 200(IV) mg/kg, systemic exposure was higher after repeated administration with accumulation factors (study day 43/study day 1) in the range of approximately from 1.4 to 1.7. In addition, systemic exposure increased in proportion to dose following IV administration. The last measurable concentrations of afucosylated h11G2 XC154/XC155 in animals from the recovery group from the 5 mg/kg dose group (IV) were observed on study days 148-281 of the 11-month recovery period. Following SC administration, the bioavailability of afucosylated h11G2 XC154/XC155 was estimated to be at least about 50%.

[000452] The incidence of ADA against afucosylated h11G2 XC154/XC155 was 19% (3/16 animals), 17% (1/6 animals) and 0% (0/6 animals) after administration of afucosylated h11G2 XC154/XC155 at a dose of 5 ( IV), 20 (SC) or 200 (IV) mg/kg, respectively, and serum concentrations on study day 43 were generally lower in ADA-positive animals compared to ADA-negative animals. It should be noted that concentrations of circulating afucosylated h11G2 XC154/XC155 may interfere with ADA detection.

TABLE 13

Dose (mg/kg/week)/Route Research Day _Cmax
(µg/ml) _Tmax
(watch) AUC ₁₆₈
(μg h/ml) 5(IV) 1 112 0.61 8340 43 142 5.5 11300 20 (SC) 1 131 100 18800 43 219 40 31600 200(IV) 1 4610 0.25 316000 43 5220 1.2 438000 AUC ₁₆₈ = Area under the drug concentration-time curve from zero to 168 hours after administration; C _max =maximum observed concentration; IV=intravenous administration; NA=not applicable; SC=subcutaneous administration; T _max = time to first occurrence of C _max

Distribution

[000453] Protein binding and tissue distribution studies have not been performed for afucosylated h11G2 XC154/XC155 in non-clinical species. The V _{ss of} afucosylated h11G2 XC154/XC155 in cynomolgus monkeys ranged from approximately 0.03 to 0.1 L/kg after a single IV administration, which corresponds to the limited distribution expected for IgG (Lin et al., 1999, J. Pharmacol. Exp. Ther. 288(1):371-378; Mascelli et al., 2007, J. Clin. Pharmacol. 47(5):553-565).

Metabolism

[000454] Metabolism studies were not performed for afucosylated h11G2 XC154/XC155. Like other therapeutic proteins with molecular weights above the glomerular filtration threshold, afucosylated h11G2 XC154/XC155 is expected to be metabolized primarily via catabolic degradation (Lobo et al., 2004, J. Pharm. Sci. 93(11):2645- 2668; Mascelli et al., 2007, supra; Vugmeyster et al., 2012, World J. Biol. Chem. 3(4):73-95).

Pharmacokinetic drug interactions

[000455] In vitro or in vivo pharmacokinetic drug interaction studies have not been conducted. Afucosylated h11G2 XC154/XC155 is a humanized monoclonal antibody (mAb) against CXCR5 and has been shown to modulate cytokine release in vitro but not in vivo . Cytokines have been shown to modulate the expression of cytochrome P450 (CYP) enzymes and transporters (Lee et al., 2010, Clin. Pharmacokinet. 49(5):295-310; Mahmood & Green, 2007, J. Clin. Pharmacol. 47(12 ):1540-1554). Thus, treatment with afucosylated h11G2 XC154/XC155 has the potential to affect CYP enzyme and transporter levels and thus modulate the clearance of concomitant drugs that are substrates for these enzymes or transporters. However, cytokine-mediated drug interactions observed clinically for other drugs were modest, resulting in less than 2-fold changes in exposure to co-administered small molecule drugs (Huang et al., 2010, Clin. Pharmacol. Ther. 87(4):497-503; Evers et al., 2013, Drug Metab. Dispos. 41(9):1598-1609). Thus, without wishing to be bound by any particular theory, it is believed that if a concomitant drug alters target expression, it could potentially affect the PK of afucosylated h11G2 XC154/XC155.

Prediction of pharmacokinetics in humans

[000456] The PK profiles of afucosylated h11G2 XC154/XC155 in the cynomolgus monkey were typical of the human IgG1 mAb in the monkey. Thus, predicted human 2-compartment PK parameters are expected to be the same as a typical therapeutic IgG1 mAb and are expected to be linear over the dose range tested. The PK parameter values used were similar to those previously described (Singh et al., 2015, In: Developability of Biotherapeutics: Computational Approaches, S. Kumar, Singh S. Kumar, Eds., CRC Press). These parameters were as follows: 3.2 L in case of central volume (V _c ), 2.2 L in case of peripheral volume (V _p ), 0.25 L/day in case of central clearance (CL _c ), 0.45 L /day in the case of distributive clearance (Q), 0.26 l/day in the case of a rate constant of absorption (k _a ) with the introduction of SC and 60% in the case of bioavailability with the introduction of SC. Given the data presented herein, the predicted human serum half-life of afucosylated h11G2 XC154/XC155 is approximately 17 days.

Prediction of effective dose in humans

[000457] A model-based approach was adapted to characterize the relationship between dose, concentrations of afucosylated h11G2 XC154/XC155, and modulation of B-cells and Tfh-like cells in cynomolgus monkey serum. Published pharmacokinetic/pharmacodynamic (PK/PD) data of SBI-087, a humanized small-sized modular protein (SMIP) immunopharmaceutical, which is similar to rituximab, binds to and depletes CD20 on B cells, in monkey and human (Cohen et al., 2016, Clin. Ther. 38(6):1417-1434; Dunussi-Joannopoulos et al., 2008, Ann. Rheum Dis. 64(Suppl II):190 (Abstr THU0171) In contrast to B cells, data on depletion of Tfh-like cells in humans are not available, thus the transformation of the depletion of Tfh-like cells is considered to be similar to that in the case of B Cell Depletion Using baseline numbers of B cells and Tfh-like cells in SLE patients (Belouski et al., 2010, Cytometry B Clin. Cytom. 78(1):49-58) and predicted parameters of human cell depletion, we simulated the kinetics of depletion of B-cells and Tfh-like cells after administration of afucosylated h11G2 XC154/XC155 to humans.

[000458] The predicted effective dose of afucosylated h11G2 XC154/XC155 in humans is approximately 10 to 30 mg (IV) and is based on the assumption that blood B cells need to be depleted to ≤1 cell/ml for clinical efficacy for approximately 8 weeks. Serum concentrations of afucosylated h11G2 XC154/XC155 after the second dose of 10 to 30 mg IV (doses on day 1 and day 29) were analyzed as above and the predicted _Cmax was approximately 5 to 15 µg/mL, predicted area under the concentration curve - time per dosing interval tau (AUC _tau ) was approximately 38 to 114 μg/day/ml, and the predicted mean concentration (C _av ; calculated as AUC _tau /28) was 1.36 to 4.06 μg/ml.

Example 17 : Toxicological studies of afucosylated h11G2 XC154/XC155

[000459] Afucosylated h11G2 XC154/XC155 was analyzed in a series of non-clinical toxicity studies in a review of toxicity testing studies, as shown in Table 14. Intravenous (IV) or subcutaneous (SC) routes of administration were chosen because they are the intended routes for clinical administration. For this experiment, formulations IV and SC contained the following components: 50 mg/mL anti-CXCR5 antibody or antigen binding fragment of the invention, 20 mM histidine, 8.5% sucrose and 0.02% polysorbate 80, 0.005% EDTA at pH 5.8 . As presented elsewhere herein, studies using human or cynomolgus PBMCs have shown that afucosylated h11G2 XC154/XC155 binds to CXCR5-expressing cells with comparable affinity and triggers antibody-dependent cellular cytotoxicity (ADCC) in a similar manner to human and monkey. As presented elsewhere in this specification, afucosylated h11G2 XC154/XC155 does not bind to mouse, rat, or rabbit CXCR5 orthologues. Thus, non-clinical toxicity studies have only been performed on cynomolgus monkeys.

TABLE 14

Study ^b concentration or dose GLP Status Repeated dose toxicity Exploratory research A 17-day exploratory toxicity study of intravenous and subcutaneous administration of afucosylated h11G2 XC154/XC155 in cynomolgus monkeys with a recovery period 0 (IV, SC), 40 (IV), 260 (SC), 400 IV (once a week) No Base Study A 2-month toxicity study of intravenous and subcutaneous administration of afucosylated h11G2 XC154/XC155 in cynomolgus monkeys with a recovery period 0 (IV, SC), 5 (IV), 20 (SC), 200 (IV) (every 2 weeks) Yes Other toxicity studies Preliminary study to develop a method for immunohistochemical staining for afucosylated h11G2 XC154/XC155 0.5-25 µg/ml No Study of tissue cross-reactivity of biotinylated afucosylated h11G2 XC154/XC155 in human and cynomolgus monkey normal tissues 1.5mcg/ml Yes In vitro human cytokine release assay by afucosylated h11G2 XC154/XC155 1-1000 µg/ml or 1-100 µg/well ^d No

[000460] The abbreviations in Table 14 are as follows: GLP=Good Laboratory Practice; IV=intravenous administration; OECD=Organization for Economic Cooperation and Development; PBMC=peripheral blood mononuclear cells; SC = subcutaneous injection.

[000461] All GLP-compliant studies were conducted in a Member State of the OECD Mutual Recognition Agreement. All in vivo studies were performed on male and female animals. Unless otherwise indicated, all doses are expressed as mg protein per kg body weight per dose. Human whole blood was analyzed at 1, 10, 100, or 1000 μg/ml (soluble phase) or human PBMC was analyzed at 1, 10, or 100 μg/well (solid phase).

[000462] Afucosylated h11G2 XC154/XC155 was administered to monkeys in the IV and SC studies up to 400 mg/kg/dose (IV) weekly for 17 days (3 doses total) or every 2 weeks up to 200 mg/kg/dose (IV ) for 2 months (5 doses in total). The 17 day monkey exploratory study included a recovery phase up to day 352. The baseline 2 month monkey study (GLP) included an 11 month recovery phase (day 401). Afucosylated h11G2 XC154/XC155 induced cytokine release in an in vitro assay. The target organ identified in these studies was the hematolymphopoietic system. The No Apparent Adverse Event Level (NOAEL) in the 2-month monkey study was the highest dose of 200 mg/kg/dose (IV) with a C _{max of} 5220 µg/mL and AUC _{of 168,438,000} µg h/mL on day 43.

Repeated dose toxicity

[000463] Exploratory and baseline repeated dose toxicity studies were performed using afucosylated h11G2 XC154/XC155 in cynomolgus monkeys.

Exploratory toxicity study

[000464] In a non-GLP exploratory study, afucosylated h11G2 XC154/XC155 was administered by IV or SC injection to monkeys (1-2/sex/dose) once a week at 0 (IV, vehicle SC), 40 (IV) , 260 (SC) or 400 (IV) mg/kg/dose (3 doses total) followed by a recovery phase in vehicle groups, 40 (IV) and 400 (IV) (1/sex/group) until day 352. All animals survived to scheduled necropsy on day 17 or day 352. No test drug-related clinical signs and effects on body weight, food intake, or serum cytokines were observed.

[000465] In the administration phase, a decrease in the total number of B cells, CXCR5 ⁺ B cells, and circulating Tfh-like cells in peripheral blood relative to baseline was observed (0.00-0.02-fold, 0.00-0.02-fold and 0.03-0.09 times, respectively) starting on days 2 or 3, with the largest decrease from baseline observed on day 17. The amount of depletion was similar for all doses. Significantly lower numbers of B cells, CXCR5 ⁺ B cells, and true Tfh cells (0.018-0.144-fold, 0.003-0.033-fold, and 0.037-0.245-fold, respectively) were observed in the spleen for all doses compared to controls. In peripheral blood, a decrease (0.33-0.67-fold relative to baseline) in absolute lymphocyte count was observed in all but one animal at ≥40 mg/kg/dose by day 2 and an increase (by 1.11-1. 81-fold from baseline) immunoglobulin (Ig) G in animals at ≥40 mg/kg/dose per day 17. A transient decrease in natural killer (NK) counts was observed on day 2 (0.05-0.14-fold from baseline ) with partial or complete recovery by day 6 in most animals. Observed moderately or significantly reduced cellularity in the lymphoid follicles of the spleen; minimally reduced cellularity in the lymphoid follicles of the submandibular, axillary and inguinal lymph nodes, and at ≥40 mg/kg/dose and 260 mg/kg/dose (SC) in the tonsils, a mixed cellular infiltration was observed by microscopy of the injection sites. Immunohistochemistry showed a dose-independent decrease in CD20-positive cells and CXCR5-positive cells in the spleen and a decrease in CD20-positive cells in the submandibular and inguinal lymph nodes. Effects on lymphoid tissues correlated with a decrease in B-cells and Tfh-like cells, as well as total peripheral blood lymphocytes, and this was consistent with pharmacological data for afucosylated h11G2 XC154/XC155, which is a depleting antibody that targets CXCR5-expressing cells.

[000466] In the recovery phase, reduced numbers of B cells, CXCR5 ⁺ B cells, and Tfh-like cells, peripheral blood increased (slowly) by day 199, and by the end of the recovery phase on day 352, all animals had partial or complete recovery of these cell types . During the recovery phase, NK cells and IgG were restored to the baseline range (FIGS. 8A-8D). No test drug-related microscopy results were observed (including immunohistochemical evaluation of CD20 and CXCR5 in the spleen and submandibular and inguinal lymph nodes), and in the spleen, the numbers of B cells, CXCR5 ⁺ B cells, and true Tfh cells were similar to or greater than those in animals in carrier control group, suggesting complete recovery.

[000467] No effects were observed on serum cytokines or T cells, T helper cells or cytotoxic T cells in peripheral blood or spleen at any dose. This study did not detect the apparent presence of anti-drug antibodies (ADA).

[000468] FIG. 8A-8D are graphs showing the depletion and recovery of B-cells and Tfh-like peripheral blood cells in cynomolgus monkeys. Levels of B-cells (Fig. 8A and Fig. 8B) and Tfh-like cells (Fig. 8C and Fig. 8D) of peripheral blood per µl of blood in males (Fig. 8A and Fig. 8C) and females (Fig. 8B and Fig. 8D) are given up to day 352 of the exploratory toxicity study. B cells were defined as CD3 ^- CD20 ⁺ cells. Tfh-like cells were defined as the sum of CD3 ⁺ CD4 ⁺ CD95 ⁺ CXCR5 ⁺ cells and CD3 ⁺ CD4 ⁺ CD95 ⁺ hIgG ⁺ cells, since the test drug antagonizes the anti-CXCR5 antibody used for immunophenotyping. Historical ranges of B cells in male monkeys (FIG. 8A, 272-2503 cells/µl) and female monkeys (FIG. 8B, 233-1700 cells/µL) are indicated by dotted lines.

Basic Toxicity Study (GLP)

[000469] In the baseline study, afucosylated h11G2 XC154/XC155 was administered by IV or SC injection to monkeys (3-8/gender/group) every 2 weeks at 0 (IV, SC), 5 (IV), 20 (SC), or 200 (IV) mg/kg/dose (5 doses total) followed by an 11 week recovery phase (day 401). To assess the primary and secondary T-cell dependent humoral response (TDAR), monkeys on day 22 (introduction phase) and day 253 (recovery phase) were injected with saucer limpet hemocyanin (KLH) and tetanus toxoid (TT), respectively. Animals were immunized with TT but were KLH-naive at the start of the study. Blood samples were collected pre-study (to assess baseline) on days 22, 25, 29, 36, 43, 58, 253 (prior to recovery phase immunization), 256, 260, 267, 274, and 281 and assessed for IgM production against KLH, anti-KLH IgG, anti-TT IgM, and anti-TT IgG.

[000470] All administration phase animals survived to scheduled necropsy on day 58. No test drug related clinical signs and effects were observed with respect to body weight, food intake, plasma cytokines, coagulation, clinical biochemistry, TDAR-KLH parameters, or electrocardiogram parameters. All afucosylated h11G2 XC154/XC155 outcomes in this study included non-adverse and reversible changes in hematological parameters, peripheral blood immunophenotyping parameters, dose-independent reduction in IgG TDAR-TT values, and altered microscopy and immunophenotyping in the spleen and/or lymph nodes in males and females at 5 and 200 mg/kg/dose IV and 20 mg/kg/dose SC. During the recovery phase, one control female was euthanized on day 330 due to complications with blood sampling. All other animals in the recovery group survived to necropsy at day 401.

[000471] Hematologic changes associated with afucosylated h11G2 XC154/XC155 included a generally dose-independent, mild to moderate decrease in lymphocytes (0.34-0.60 times baseline) at all doses with the greatest frequency per day 2 and lower frequency at subsequent time points, contributing to the overall decrease in the number of leukocytes (0.25-0.59 times relative to baseline) in the case of individual animals at all doses. Lymphocytes approached baseline and/or control values during the first 1-2 months of the recovery phase. A slight decrease in basophils (0.17-0.50-fold) was also observed at 200 mg/kg/dose IV on day 2 and in selected animals at 5 mg/kg/dose IV and 20 mg/kg/dose SC thereafter. time points followed by recovery during the first 1–3 months of the recovery phase and a transient minimal decrease (0.79–0.82 fold) in RBC mass (hemoglobin, RBC count, and hematocrit) in females at 200 mg/kg/dose IV a day 6.

[000472] A significant decrease in the total number of B-cells, CXCR5 ⁺ B-cells and Tfh-like cells (0.00-0.56-fold) in peripheral blood relative to baseline was observed with afucosylated h11G2 XC154/XC155 in all animals and in all dose groups as early as day 2, and it persisted until the end of the administration phase (day 58) with a return to baseline or had absolute values for each subpopulation of animals that were administered 5 mg/kg/dose IV in the recovery phase by day 393, in the range of values observed in animals in the vehicle control group (FIGS. 9A-9D). We also observed a significant decrease in the number of NK cells in the peripheral blood on day 2 in all animals relative to the initial level (by 0.04-0.46 times); however, most animals showed partial or complete recovery by day 6, although NK cells remained below baseline levels in some animals at one or more subsequent time points, returning to baseline by the end of the recovery phase. Also, in some animals, a transient decrease in the total number of T cells, T helper cells and cytotoxic T cells was observed on day 2 (0.45-0.66 times); however, these cell populations returned to baseline levels by day 6 in most animals and by day 36 in all animals. No afucosylated h11G2 XC154/XC155 related changes in interleukin (IL)-2, IL-6, IL-10, IL-13, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were observed at any dose.

[000473] Afucosylated h11G2 XC154/XC155, dose-independent decrease in cellularity of splenic and axillary and mesenteric lymph nodes and intestinal lymphoid tissue (GALT) lymphoid follicles was observed at ≥5 mg/kg/dose IV and 20 mg/kg/dose SC. This was associated with a decrease in the cellularity of lymphoid follicles in relation to CD20 ⁺ and CXCR5 ⁺ cells in the spleen, assessed by immunohistochemistry, with the exception of 1 male at 20 mg/kg/dose SC; however, exposure to afucosylated h11G2 XC154/XC155 in this animal may have been affected by proliferation of ADA starting at day 15. In addition, lower numbers of B cells, CXCR5 ⁺ B cells, and Tfh cells were detected at day 58 necropsy. in the spleen compared to vehicle-treated controls. In some animals, lower numbers of NK cells were determined in the spleen compared to controls. By the end of recovery on day 401, the absolute values for each study population in all animals treated with afucosylated h11G2 XC154/XC155 were in the range of values for animals in the vehicle control group, indicating recovery from afucosylated h11G2 XC154/XC155 the decrease in cell numbers observed during the administration phase of this study.

[000474] Decreased lymphoid cellularity in the spleen and lymphoid follicles in the lymph nodes, together with lower numbers of B cells, CXCR5 ⁺ B cells and Tfh cells in the spleen, correlated with a decrease in the total number of lymphocytes and B cells, CXCR5 ⁺ B cells and Tfh-like cells in peripheral blood at ≥5 mg/kg/dose IV and 20 mg/kg/dose SC. By the end of the recovery phase (Day 401), no afucosylated h11G2 XC154/XC155 associated microscopy results were observed in the 5 mg/kg IV dose group, indicating complete recovery. These results are consistent with pharmacological data on afucosylated h11G2 XC154/XC155, which is expected to deplete CXCR5-expressing cells. Lower numbers of NK cells in the peripheral blood and/or spleen may result from the death of effector NK cells as described for NK cells after antibody-dependent cellular cytotoxicity (Warren et al., 2011, J. Immunol. Meth. 370:86- 92), and/or tissue redistribution; however, other mechanisms cannot be excluded.

[000475] No effects associated with afucosylated h11G2 XC154/XC155 on primary T-cell dependent humoral response (TDAR) to sea saucer hemocyanin (KLH) were observed. Responses with the formation of immunoglobulins (Ig) M and IgG against KLH were observed in all groups. All monkeys were vaccinated against tetanus toxoid (TT) prior to enrollment in the study. Increased anamnestic responses with the formation of IgM and IgG were observed in all animals in all dose groups. Afucosylated h11G2 XC154/XC155-associated, dose-independent reduction in secondary TDAR at TT (IgG midpoint titer values) was observed in male and female animals in the 5 IV, 20 SC and/or 200 IV mg/kg/dose groups at 7, 14 , 21 and 36 days after immunization. A statistically significant reduction in group mean anti-TT IgG antibodies was observed in the 5 and 200 mg/kg/dose IV groups 7 days post-immunization.

[000476] Following recovery phase immunization, a history of anti-KLH IgM and IgG responses was observed in all animals in the 5 mg/kg dose group of afucosylated h11G2 XC154/XC155 at 1 or more time points within the range of the vehicle control group . During reconstitution, afucosylated h11G2 XC154/XC155-associated statistically significant decrease in central point titer (CPT) anti-TT IgG values in the group was observed in animals in the 5 mg/kg dose group 7 days post-immunization in the recovery phase. The CPT values of all animals in the recovery phase were within the range of the control group, with the exception of 2 of 8 animals in the 5 mg/kg dose group, which had CPT values below the control range at all time points in the recovery phase up to 28 days after immunization with TT in the recovery phase . No change in anti-TT IgM values attributable to administration of afucosylated h11G2 XC154/XC155 was observed in the administration or recovery phase.

[000477] Observed primary responses with the formation of IgM and IgG against KLH in all groups; some animals in the 5 and 200 mg/kg/dose groups had a CPT above the control range at 1 or more time points after primary immunization. No statistically significant difference was observed between group mean values of anti-KLH IgM or IgG antibodies in any of the groups at any time point. Because changes in individual animals were sporadic, independent of dose, and CPT values in the group were in the range of control animals, they were not considered associated with afucosylated h11G2 XC154/XC155. During the recovery phase, all animals were within the control range, with the exception of 1 of 8 animals in the 5 mg/kg dose group, which had CPT values slightly below the control range at 14, 21, and 28 days after KLH immunization. These data (introduction or recovery phase) indicate that all animals treated with afucosylated h11G2 XC154/XC155 (male and female) could generate primary IgM and IgG responses to KLH immunization similar to control animals.

[000478] Despite a significant decrease in the total number of B cells, CXCR5 ⁺ B cells, Tfh-like cells and NK cells in peripheral blood and a significantly lower number of B cells, CXCR5 ⁺ B cells and Tfh cells in the spleen , as determined by immunophenotyping, decreased lymphocyte counts, and reduced lymphoid cellularity in the spleen, lymph nodes, and GALT, were all non-favorable due to the absence of associated clinical signs and no effect on primary TDAR. Other haematological findings, including a decrease in total leukocyte, basophil, and erythrocyte parameters, were not unfavorable due to limited severity and lack of correlation with microscopy findings. No afucosylated h11G2 XC154/XC155 associated microscopy and immunohistochemistry at day 401 was noted, indicating complete recovery.

[000479] The NOAEL for afucosylated h11G2 XC154/XC155 in monkeys after SC or IV administration every other week for 2 months was 200 mg/kg/dose. Systemic exposure (C _max and AUC ₁₆₈ ) at NOAEL was 5220 µg/ml and 438000 µg h/ml, respectively. Antibodies against afucosylated h11G2 XC154/XC155 were determined in this study and data are provided elsewhere in this specification. Elsewhere in this specification, an assessment of the risk of target organ toxicity in a repeat dose toxicity study is provided. Elsewhere in this description, threshold concentrations of afucosylated h11G2 XC154/XC155 associated with key responses in monkeys can be found.

Reproductive and embryonic toxicity

[000480] No test drug-related effects were observed on male or female reproductive tissues as assessed in a repeat-dose toxicity study in cynomolgus monkeys.

Local tolerance

[000481] No independent local tolerability studies have been performed using afucosylated h11G2 XC154/XC155, although injection sites have been examined macroscopically and microscopically in in vivo toxicity studies.

[000482] In an exploratory study in monkeys, moderate perivascular mixed leukocyte cell infiltration (mononuclear leukocytes, neutrophils and fewer eosinophils) was observed at the SC injection site at 260 mg/kg/dose compared to minimal and mild infiltrates at the SC injection sites in control animals . In a male treated with 260 mg/kg/dose of SC, the infiltrate spread multifocally into the muscularis propria of small vessels. In a female treated with 260 mg/kg/dose of SC, several multinucleated giant cells infiltrated the subcutaneous collagen, and intracytoplasmic basophilic or eosinophilic fragments of fibrillar material were detected in these cells. In the pivotal 2-month study in monkeys, microscopy results at IV and SC injection sites were not considered related to the test drug.

antigenicity

[000483] Immunogenicity was assessed by measuring ADA levels in repeated dose toxicity studies in cynomolgus monkeys; these data are presented above in the present description.

Immunotoxicity

[000484] In vitro and in vivo studies characterized the effect of afucosylated h11G2 XC154/XC155 on the immune system. The ability of afucosylated h11G2 XC154/XC155 to induce cytokine release (TNFα, IL-6 and IFNγ) was determined in human cytokine release assays using 2 different in vitro formats: a homogeneous assay using whole blood and a solid phase assay using immobilized afucosylated h11G2 XC154/XC155 and peripheral blood mononuclear cells (PBMC). Samples from 8 healthy human donors were evaluated in each of these formats. In both cytokine release assay formats, afucosylated h11G2 XC154/XC155 induced release of cytokines (TNFα, IL-6 and IFNγ) was observed. In vivo exploratory and baseline repeat toxicity studies in monkeys characterized the in vivo cytokine release profile and the effects of afucosylated h11G2 XC154/XC155 on cells and tissues of the immune system. Afucosylated h11G2 XC154/XC155 did not induce cytokines in vivo . Depletion of lymphocytes, B cells, CXR5 ⁺ B cells and Tfh/Tfh-like cells was observed in the peripheral blood and spleen of monkeys administered afucosylated h11G2 XC154/XC155, as presented elsewhere in this description, the effects consistent with the expected mechanism of action of CXCR5- depleting antibodies. After the recovery phase in the exploratory and baseline toxicity studies, lymphocyte parameters in the spleen and lymph nodes were comparable to vehicle controls.

[000485] Primary and secondary TDAR responses to KLH and TT, respectively, were assessed in a baseline 2-month toxicity study, as presented elsewhere herein. No effects associated with afucosylated h11G2 XC154/XC155 on primary TDAR on KLH were observed. All animals developed a secondary TDAR response to TT, but an afucosylated h11G2 XC154/XC155-associated decrease in midpoint titers was observed in groups 5 and 200 mg/kg/dose IV 7 days post-immunization during the administration and recovery phases of the study.

Tissue Cross Reactivity (TCR)

[000486] In a preliminary study of a staining method using afucosylated h11G2 XC154/XC155, extensive preliminary method development work was carried out, including the use of numerous assays and methods. Under none of the conditions of this assay, stable staining was observed in the expected cells and tissues. A GLP compliant tissue cross-reactivity study assessed the binding of biotinylated afucosylated h11G2 XC154/XC155 (afucosylated h11G2 XC154/XC155-Bio; 1 and 5 µg/mL) to cryosections of cynomolgus and human tissues. Staining profiles overlapped between cynomolgus and human tissues. Staining common to humans and monkeys was observed in mononuclear cells, reticuloendothelial cells, various types of epithelium, glial cells and/or pituicites, consistent with the expected reactivity of afucosylated h11G2 XC154/XC155 (Breitfeld et al., 2000, J Exp Med 192(11 ):1545-1552; Carlsen et al., 2002, Gut 51(3):364-371; Flynn et al., 2003, J. Neuroimmunol.136(1-2):84-93; Schaerli et al., 2000, J. Exp. Med. 192(11):1553-1562; Schmutz et al., 2005, Arthritis Res. Ther. 7(2):R217-R229), as well as thyroid colloid. Positive staining with afucosylated h11G2 XC154/XC155 was observed in other monkey or human tissues. In the case of human tissue, staining has been observed in optic and spinal nerve root neurons, prostate smooth muscle cells, testicular interstitial cells, and lens fibers in the eye.

[000487] Tissues stained only in the cynomolgus monkey were hematopoietic progenitor cells in the bone marrow, mast cells in the skin and cervix, neuropil in the brain and spinal cord, cardiomyocytes, spindle cells in the placenta, and optic nerve sheath. Although this may represent an unexpected cross-reactivity of the test drug, the staining was cytoplasmic in nature, lacking histological specificity and clearly staining the plasma membrane. Such cytoplasmic staining cannot sufficiently differentiate between CXCR5-expressing and non-expressing cells and tissues. Cytoplasmic staining is not expected to be available for the drug under test in vivo and is generally considered to have little or no toxicological significance (Hall et al., 2008, In: Preclinical Safety Evaluation of Biopharmaceuticals: A Science-Based Approach to Facilitating Clinical Trials, pp. 208-240, Cavagnaro, JA, ed., Wiley-Interscience; Leach et al., 2010, Toxicol Pathol 38(7):1138-1166). The lack of clear membrane staining suggests that a reliable method cannot be obtained to achieve a complete score. The absence of adverse results in the 2-month repeated-dose toxicity study confirms that the staining observed in the ex vivo tissue cross-reactivity study does not translate into in vivo effects. Although many attempts have been made to obtain a reliable method of TCR, the observed binding did not lead to safety problems in excess of what would be expected, given the expected pharmacological properties.

Relationship between results and pharmacokinetics

[000488] Serum concentrations of afucosylated h11G2 XC154/XC155 were determined after administration of SC or IV to male and female monkeys at doses of 5 (IV), 20 (SC), or 200 (IV) mg/kg/dose every 2 weeks for 2 months ( only 5 doses). No obvious sex differences in systemic exposure were observed between dose groups; thus group mean TK parameters were presented using combined data for male and female cynomolgus monkeys. Systemic exposure was higher after repeated administration with accumulation factors (study day 43/study day 1) ranging from about 1.4 to 1.7. In addition, systemic exposure increased in proportion to dose following IV administration. Following SC administration, the bioavailability of afucosylated h11G2 XC154/XC155 was estimated to be >50%.

[000489] Thresholds for afucosylated h11G2 XC154/XC155 serum concentrations associated with key responses and exposure limits calculated relative to these key responses are presented in Table 15.

TABLE 15

Key Answers Dose (mg/kg/dose) C _av (µg/ml) C _max (μg h/ml) Exposure limits Repeated exposure toxicity studies Monkey 17-Day Toxicity Study (1-2/sex/dose) - Administration once a week (all results were not unwanted) Peripheral blood: ↓ total B cells, ↓ CXCR5 ⁺ B cells, ↓ Tfh-like cells, ↓ NK cells; spleen: ↓ total B cells, ↓ total CXCR5 ⁺ B cells, ↓ true Tfh cells; ↓ lymphocytes; ↑ IgG ; spleen: ↓ cellularity (follicular CD20-positive cells and CXCR5-positive cells); lymph nodes and tonsils: ↓ cellularity (in lymph nodes: follicular CD20-positive cells) 40(IV) 844 1020 208 Same as above, also at the injection site: perivascular leukocyte cell infiltration 260 (SC) 2810 3340 692 Same as 40 mg/kg/dose 400(IV) 7500 9610 1850 2-month toxicity study in monkeys (3-8/sex/dose) - Administration every 2 weeks (all results were not unwanted) Peripheral blood: ↓ total B cells, ↓ CXCR5 ⁺ B cells, ↓ Tfh-like cells, ↓ NK cells, ↓ T cells, ↓ T helper cells, ↓ cytotoxic T cells, ↓ WBCs, ↓ lymphocytes , ↓ basophils, spleen: ↓ B cells, ↓ total number of CXCR5 ⁺ B cells, ↓ true Tfh cells, ↓ NK cells; spleen: ↓ cellularity (follicular CD20 ⁺ cells and CXCR5 ⁺ cells); lymph nodes and GALT: ↓ lymphoid cellularity; TDAR: secondary (anamnestic) anti-TT IgG antibody response 5(IV) 67.3 142 17 Same as above 20 (SC) 188 219 46 Same as above plus ↓ RBC mass 200(IV)
NOAEL 2610 5220 643

[000490] The abbreviations used in Table 15 were as follows: AUC=area under the concentration-time curve; C _av =mean concentration; C _max = maximum (mean) plasma concentration; Tfh-like cells (alternatively, cTfh); CXCR5=chemokine receptor type 5; GALT=intestinal lymphoid tissue; IgG=immunoglobulin G; IV=intravenous administration; NK=natural killers; NOAEL=level with no visible adverse events; SC=subcutaneous administration; TDAR=T-cell dependent humoral response; Tfh=follicular T helper cells; TT=tetanus toxoid; WBC=leukocytes.

[000491] In repeat administration studies, C _max values correspond to mean plasma concentrations. C _av values are calculated by dividing the AUC by the sampling interval (48 or 168 hours). The reported values were obtained towards the end of the administration phase.

[000492] Exposure limits were calculated by dividing C _av values in animal toxicity studies by a predicted human C _av of 4.06 μg/mL at a predicted effective human dose of 30 mg, IV.

Target organ toxicity

[000493] Based on non-clinical studies, the hematolymphopoietic system (spleen, lymph nodes, GALT, tonsils, parameters of circulating lymphocytes, leukocytes, erythrocytes, in vitro release of cytokines) was identified as target organs/tissues. In a non-baseline 17-day study in monkeys using afucosylated h11G2 XC154/XC155, test drug-related changes in these tissues were consistent with those observed in the baseline 2-month toxicity study. All afucosylated h11G2 XC154/XC155 results fully or partially reverted to baseline or values in the vehicle control group during the recovery phase in the non-baseline and baseline toxicity studies. None of the test drug-related effects were considered undesirable due to the absence of any clinical findings or opportunistic infections and due to the minimal effect on immune function as measured by the TDAR assay. Thus, NOAEL 200 mg/kg/dose IV in monkeys was determined for afucosylated h11G2 XC154/XC155 related effects.

Hematolymphopoietic system

[000494] In repeat-dose toxicity studies in cynomolgus monkeys, afucosylated h11G2 XC154/XC155-associated reduction in peripheral blood lymphocytes was observed at doses ≥5 mg/kg/dose (IV) and 20 mg/kg/dose (SC) starting at study day 2. This decrease correlated with the observed decrease in total B cells, CXCR5 ⁺ B cells, and Tfh-like cells, persisting until day 57. A complete or partial return of these reduced lymphocyte subpopulations to baseline values was observed by the end of the recovery phase in the exploratory toxicity study. Afucosylated h11G2 XC154/XC155 related reductions in NK cells, total T cells, T helper cells, and cytotoxic T cells were also observed at ≥5 mg/kg/dose from day 2, but achieved a partial or complete return to baseline values by the end of the administration phase. In addition to a decrease in peripheral blood lymphocyte populations, a transient minimal decrease in erythrocyte mass was observed in female monkeys administered 200 mg/kg/dose on day 6.

[000495] Decreased peripheral blood lymphocyte populations correlated with afucosylated h11G2 XC154/XC155 associated lower lymphoid cellularity in the spleen and lymph nodes at ≥5 mg/kg/dose. Lower lymphoid cellularity was reflected in lower total B cells, lower numbers of CXCR5 ⁺ B cells, true Tfh cells, and NK cells, and lower numbers of follicular CD20 ⁺ and CXCR5 ⁺ cells in the spleen. Lower follicular lymphoid cellularity was observed in lymph nodes and GALT at ≥5 mg/kg/dose and in tonsils at ≥40 mg/kg/dose.

[000496] After the recovery phase in the exploratory and baseline toxicity studies, lymphocyte parameters in the spleen and lymph nodes were comparable to vehicle-treated controls.

[000497] The decrease in the total number of B cells, CXCR5 ⁺ B cells and Tfh/Tfh-like cells is consistent with the expected mechanism of action of afucosylated h11G2 XC154/XC155, which is expected to deplete CXCR5-expressing cells. The lower numbers of NK cells in peripheral blood and spleen are likely the result of effector cell death, which is known to occur in NK cells after ADCC.

[000498] Although a decrease in lymphocyte parameters was observed in the peripheral blood and spleen of monkeys treated with afucosylated h11G2 XC154/XC155, no effect associated with afucosylated h11G2 XC154/XC155 on primary TDAR on KLH was observed. All animals developed secondary TDAR but TT, but a statistically significant reduction was observed at ≥5 mg/kg/dose.

[000499] All results associated with afucosylated h11G2 XC154/XC155 were not considered undesirable due to the absence of relevant clinical signs or opportunistic infections. The effects of afucosylated h11G2 XC154/XC155 on peripheral blood lymphocytes, total B cells, CXCR5 ⁺ B cells, Tfh-like cells and NK cells, and primary and secondary TDAR responses can be monitored in humans in a clinical setting.

[000500] Afucosylated h11G2 XC154/XC155 induced release of cytokines (TNFα, IL-6 and IFNγ) was observed in homogeneous and solid phase in vitro cytokine release assay formats. In vitro cytokine release induced by afucosylated h11G2 XC154/XC155 was expected due to the expected pharmacological properties of this molecule. Afucosylated h11G2 XC154/XC155 did not induce cytokine release in exploratory or pivotal in vivo monkey studies. Serum cytokines and clinical indicators of systemic cytokine release can be easily monitored in a clinical setting.

TABLE 16

(SEQUENCES)

(CDR amino acid residues are underlined)

Name Sequence identification number Subsequence Humanized (h) VL 11G2 (XC154) SEQID NO:1 DIQMTQSPSSLSASVGDRVTITCRAS ESVEYHGTS LMHWYQQKPGKAPKLLIY AASNVESG VPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSRKVPWT FGQGTKVEIK CDR-L1 VL h11G2 (XC154) SEQ ID NO:2 ESVEYHGTS CDR-L2 VL h11G2 (XC154) SEQ ID NO:3 AASNVESG CDR-L3 VL h11G2 (XC154) SEQ ID NO:4 QQSRKVPWT VL h11G2 (XC153) SEQ ID NO:5 DIQMTQSPSSLSASVGDRVTITCRAS ESVEYHGTS LMHWYQQKPGKAPKLLIY AASNVESG VPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQSRKVPWT FGQGTKVEIK VH h11G2 (XC155) SEQ ID NO:6 EVQLVESGGGLVQPGGSLRLSCAAS GFTFTDFY MSWVRQAPGKGLEWVGF IRNKANGYT TEYSASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC A RVYGSTLHY WGQGTLVTVSS CDR-H1VH h11G2 (XC155) (also XC156) SEQ ID NO:7 GFFTDFY CDR-H2 VH h11G2 (XC155) (also XC156) SEQ ID NO:8 IRNKANGYT CDR-H3 VH h11G2 (XC155) SEQ ID NO:9 A RVYGSTLHY VH h11G2 (XC156) SEQ ID NO:10 EVQLVESGGGLVQPGGSLRLSCATS GFTFTDFY MSWVRQAPGKGLEWVGF IRNKANGYT TEYSASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC VRVYGSTLHY WGQGTLVTVSS CDR-H3 VH h11G2 (XC156) SEQ ID NO:11 VRVYGSTLHY VH h11G2 (XC157) SEQ ID NO:12 EVQLVESGGGLVQPGGSLRLSCATS GFTFTDFY MSWVRQAPGKGLEWVGF IRNKANGYT TEYSASVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYC VRVYGSTLHY WGQGTLVTVSS VL h41A10 (XC142) SEQ ID NO:13 DIQMTQSPSSLSASVGDRVTITCSAS SSVNY IHWYQQKPGKAPKRLIY ETSRLASG VPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPLT FGQGTKVEIK CDR-L1 VL h41A10 (XC142) SEQ ID NO:14 SSVNY CDR-L2 VL h41A10 (XC142) SEQ ID NO:15 ETSRLASG CDR-L3 VL h41A10 (XC142) SEQ ID NO:16 QQWSSNPLT VH h41A10 (XC148) SEQ ID NO:17 EVQLVESGGGLVQPGGSLRLSCAAS GFTFRSYG MSWVRQAPGKGLEWVAT ISSGGTY TFYPDILKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARRGEDYRGALEH WGQGTLVTVSSA VH h41A10 (XC147) SEQ ID NO:18 EVQLVESGGGLVQPGGSLRLSCAAS GFTFRSYG MSWVRQAPGKGLEWVAT ISSGGTY TFYPDILKGRITISRDNAKNSLYLQMNSLRAEDTAVYYC ARRGEDYRGALEH WGQGTLVTVSSA CDR-H1 VH h41A10 (XC147) (same as XC148) SEQ ID NO:19 GFTFRSYG CDR-H2 h41A10 (XC147) (same as XC148) SEQ ID NO:20 ISSGGTY CDR-H3 h41A10 (XC147) (same as XC148) SEQ ID NO:21 ARRGEDYRGALEH Chimeric c11G2 full-length light chain (cLC) (Mouse VL is indicated in uppercase letters and human kappa chain constant region is indicated in lowercase letters)
* stop codon indicated SEQ ID NO:22 DVVVTQSPDSLAVSLGQRATISCRAS ESVEYHGTS LMHWYQQKPGQPPKLLIY AASNVESG VPARFSGSGSGTDFSLNIHPVEEGDIAMYFC QQSRKVPWT FGGGTKLEIKrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstys lsstltlskadyekhkvyacevthqglsspvtksfnrgec* Chimeric full-length c11G2 heavy chain (cHC) (mouse VH is indicated in uppercase letters and human IgG1 constant region is indicated in lowercase letters)
* stop codon indicated SEQ ID NO:23 VRVYGSTLHY WGQGTILTVSSastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvv tvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknq vsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg * Chimeric light chain c41A10
* stop codon indicated SEQ ID NO:24 QIVLTQSPAVMSASPGEKVTMTCSAS SSVNY IHWYQQKSGTSPKRWIY ETSRLAS GVPVRFSGSGSGTSYSLTISTMEAEDAATYYC QQWSSNPLT FGAGTKLELKrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekh kvyacevthqglsspvtksfnrgec* Chimeric heavy chain c41A10
(Mouse VH is indicated in capital letters, and human IgG1 constant region is indicated in lowercase letters)
* stop codon indicated SEQ ID NO:25 EVQLVESGGDLVKPGGSLKLSCAAS GFTFRSYG MSWVRQTPDKRLEWVAT ISSGGTYTF YPDILKGRITISRDNAKNTLYLQMSNLKSEDTAMYYC ARRGEDYRGALEH WGQGTSVTVSSastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglys lssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreem tknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg* Chimeric full-length light chain c5H7 (cLC)
* stop codon indicated SEQ ID NO:26 DVVVTQSPDSLAVSLGQRATISCRAS ESVEYHGTS LMHWYQQKPGQPPKLLIY AASNVES GVPARFSGSGSGTDFSLNIHPVEEGDVSMFFC QQSRKVPWT FGGGTKLEIKrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdsty slsstltlskadyekhkvyacevthqglsspvtksfnrgec* Chimeric full-length c5H7 heavy chain (cHC) (mouse VH is indicated in uppercase letters and human IgG1 constant region is indicated in lowercase letters)
* stop codon indicated SEQ ID NO:27 VRVYGSTLHY WGQGTILTVSSastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvv tvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknq vsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg * Full length light chain h11G2 (XC154) (VL is in upper case and Cκ is in lower case) SEQ ID NO:28 QQSRKVPWT FGQGTKVEIKrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstysls stltlskadyekhkvyacevthqglsspvtksfnrgec Full-length h11G2 heavy chain (XC155) (VL is in uppercase letters and IgG1 Fc is in lowercase letters) SEQ ID NO:29 EVQLVESGGGLVQPGGSLRLSCAAS GFTFTDFY MSWVRQAPGKGLEWVGF IRNKANGYT TEYSASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC A RVYGSTLHY WGQGTLVTVSSastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglysls svvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemt knqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg Human kappa light chain constant domain (Cκ) SEQ ID NO:30 rtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec Wild type human IgG1 Fc (includes part of C _H 1 and hinge domain, C _H 2 and C _H 3)
Wild-type effector function sequence LLGG is indicated in italics
N-linked glycosylation site Asn297 NST SEQ ID NO:31 astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpape llgg psvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhn aktkpreeqy NST yrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg Human CXCR5 (epitope L11 and D22 amino acid residues critical for h11G2 binding are in bold and italics) SEQ ID NO:32 MNYPLTLEMD L ENLEDLFWEL D RLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEILFAKVSQGHHNNSLPRCTFSQ ENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPSWRRSSLSESENATSLTTF CXCR5 cynomolgus macaque SEQ ID NO:33 MNYPLMLEMDLENLEDLFLEFDKFDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLFALPEILFAKVSQAHPNNSLPTFSQENQ AETHAWFTSRFLYHVAGFLLPMLVMGWCYVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLVRLKAVDNTCELNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPSWRKSSLSESENATSLTTF CXCR5 mouse SEQ ID NO:34 MNYPLTLDMGSITYNMDDLYKELAFYSNSTEIPLQDSNFCSTVEGPLLTSFKAVFMPVAYSLIFLLGMMGNILVLVILERHRHTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKINFYCSSLLLACIAVDRYLAIVHAVHAYRRRRLLSIHITCTAIWLAGFLFALPELLFAKVGQPHNNDSLPQCTFSQEN EAETRAWFTSRFLYHIGGFLLPMLVMGWCYVGVVHRLLQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLERLKAVNSSCELSGYLSVAITLCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCAGPASLCQLFPNWRKSSLSESENATSLTTF VL 11G2 mice SEQ ID NO:35 DVVVTQSPDSLAVSLGQRATISCRAS ESVEYHGTS LMHWYQQKPGQPPKLLIY AASNVESG VPARFSGSGSGTDFSLNIHPVEEGDIAMYFC QQSRKVPWT FGGGTKLEIK VH 11G2 mice SEQ ID NO:36 EVKLVESGGGLVQPGGSLRLSCATS GFTFTDFY MSWVRQPPGRALEWLGF IRNKANGYT TEYSASVKGRFTISRDNSQSILYLQMNTLRAGDSATYYC VRVYGSTLHY WGQGTILTVSSA VL 41A10 mouse SEQ ID NO:37 QIVLTQSPAVMSASPGEKVTMTCSAS SSVNY IHWYQQKSGTSPKRWIY ETSRLASG VPVRFSGSGSGTSYSLTISTMEAEDAATYYC QQWSSNPLT FGAGTKLELK VH 41A10 mice SEQ ID NO:38 EVQLVESGGDLVKPGGSLKLSCAAS GFTFRSYG MSWVRQTPDKRLEWVAT ISSGGTY TFYPDILKGRITISRDNAKNTLYLQMSNLKSEDTAMYYC ARRGEDYRGALEH WGQGTSVTVSSA VL 5H7 mouse SEQ ID NO:39 DVVVTQSPDSLAVSLGQRATISCRAS ESVEYHGTSL MHWYQQKPGQPPKLLIY AASNVESG VPARFSGSGSGTDFSLNIHPVEEGDVSMFFC QQSRKVPWT FGGGTKLEIK VH 5H7 mice SEQ ID NO:40 EVKLVESGGGLVQPGDSLRLSCATS GFTFTDFY MSWVRQPPGRALEWLGF IRNKANGYTT EYSASVKGRFTISRDNSQSILYLQMNTLRAGDSATYYC VRVYGSTLHY WGQGTILTVSSA Light chain 2C9 (VL is indicated in uppercase letters and Cκ domain is indicated in lowercase letters) SEQ ID NO:41 DIVMTQSPDSLAVSLGERATINCKSS QSVLYSSNNKNY LAWYQQKPGQPPKLLIY WAS TRDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC HQYLSSYT FGGGTKVEIKrtvaapsvfIfppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstlt lskadyekhkvyacevthqglsspvtksfnrgec* Heavy chain 2C9 (VH is indicated in uppercase letters and constant region is indicated in lowercase letters) SEQ ID NO:42 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSTNA MNWVRQAPGKGLERVAR IRSKSNNYAT YYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYC VTMIPFAY WGQGTLVTVSSastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslss vvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtk nqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg* Light chain 16D7 SEQ ID NO:43 DIVMTQAAPSVAVTPGASVSISCRSS KSLLHSSGKTY LYWFLQRPGQSPQLLIY RLS SLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC MQHLEYPYT FGGGTKLEIKrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltls kadyekhkvyacevthqglsspvtksfnrgec* Heavy chain 16D7 (VH is indicated in uppercase letters and constant region is indicated in lowercase letters) SEQ ID NO:44 QVQLKESGPGLVAPSESLSITCTVS GFSLIDYG VNWIRQPPGKGLEWLGV IWGDGTT YYNPSLKSRLSISKDNSKSQVFLKVTSLTTDDTAMYYC ARIVY WGQGTLVTVSAastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvv tvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknq vsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg* Light chain 11A7 SEQ ID NO:45 qpvltqppsvskdlrqtatltctgn snnvgnqg atwlqqhqghppkllsy knn nrpsgiserfsasrsgntasltitglqpedeadYyc sawdsslsawv fgggtqltvlgqpkaapsvtlfppsseelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnkya assylsltpeqwkshrsyscqvthegstvektvaptecs* Heavy chain 11A7 SEQ ID NO:46 EVQLVESGGGLVQPGGSLRLSCAAS GFTVSSNY MSWVRQAPGKGLEWVSV IYSGGST YYADSVKGRFTISRHNSKNTLYLQMNSLRAEDTAVYYC ARGYVV WGQGTLVTVSSastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglysLss vvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpEvkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemt knqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg VL h11G2 (XC151) SEQ ID NO:47 DIQMTQSPSSLSASVGDRVTITCRAS ESVEYHGTS LMHWYQQKPGKAPKLLIY AASNVESG VPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSRKVPWT FGQGTKVEIK VL h11G2 (XC346) SEQ ID NO:48 DVQMTQSPSSLSASVGDRVTITCRAS ESVEYHGTS LMHWYQQKPGKAPKLLIY AASNVESG VPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSRKVPWT FGQGTKVEIK VL h11G2 (XC347) SEQ ID NO:49 DVQVTQSPSSLSASVGDRVTITCRAS ESVEYHGTS LMHWYQQKPGKAPKLLIY AASNVESG VPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSRKVPWT FGQGTKVEIK VL h11G2 (XC348) SEQ ID NO:50 DVQVTQSPSSLSASVGDRVTITCRAS ESVEYHGTS LMHWYQQKPGKAPKLLIY AASNVESG VPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQSRKVPWT FGQGTKVEIK VL h11G2 (XC349) SEQ ID NO:51 DIQVTQSPSSLSASVGDRVTITCRAS ESVEYHGTS LMHWYQQKPGKAPKLLIY AASNVESG VPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSRKVPWT FGQGTKVEIK VH h11G2 (XC152) SEQ ID NO:52 EVQLVESGGGLVKPGGSLRLSCAAS GFTFTDFY MSWVRQAPGKGLEWLGF IRNKANGYT TEYSAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC VRVYGSTLHY WGQGTLVTVSSAS VH h11G2 (XC350) SEQ ID NO:53 EVQLVESGGGLVQPGGSLRLSCAAS GFTFTDFY MSWVRQAPGKGLEWVAFIRNKANGYTTEYSASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC A RVYGSTLHY WGQGTLVTVSS VH 11G2 (XC351) SEQ ID NO:54 EVQLVESGGGLVQPGGSLRLSCATS GFTFTDFY MSWVRQAPGKGLEWLGF IRNKANGYT TEYSASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC VRVYGSTLHY WGQGTLVTVSS VH 11G2 (XC352) SEQ ID NO:55 EVQLVESGGGLVQPGGSLRLSCATS GFTFTDFY MSWVRQAPGKGLEWVAF IRNKANGYT TEYSASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC A RVYGSTLHY WGQGTLVTVSS VH h11G2 (XC353) SEQ ID NO:56 EVQLVESGGGLVQPGGSLRLSCAAS GFTFTDFY MSWVRQAPGKGLEWVAFIRNKANGYTTEYSASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC VRVYGSTLHY WGQGTLVTVSS VH h11G2 (XC354) SEQ ID NO:57 EVQLVESGGGLVQPGGSLRLSCAAS GFTFTDFY MSWVRQAPGKGLEWLGF IRNKANGYT TEYSASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC A RVYGSTLHY WGQGTLVTVSS VL h41A10 (XC143) SEQ ID NO:58 DIQMTQSPSSLSASVGDRVTITCSAS SSVNY IHWYQQKPGKAPKRWIY ETSRLASG VPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSNPLT FGQGTKVEIK VL h41A10 (XC144) SEQ ID NO:59 DIQLTQSPSSLSASVGDRVTITCSAS SSVNY IHWYQQKPGKAPKRLIY ETSRLASG VPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQWSSNPLT FGQGTKVEIK VL h41A10 (XC145) SEQ ID NO:60 diqltqspsslsasvgdrvtitcsas ssvny ihwyqqkpgkapkrwiy etsrlasg vpsrfsgsgsgTdytltisslqpedfatyyc qqwssnplt fgqgtkveik VL h41A10 (XC146) SEQ ID NO:61 diqmtqspsslsasvgdrvtitcsas ssvny ihwyqqkpgkapkrliy etsrlasg vpsrfsgsgsgtdftltisslqpedfatyyc qqwssnplt fgqgtkveik VL h41A10 (XC149) SEQ ID NO:62 diqmtqspsslsasvgdrvtitcsas ssvny ihwyqqkpgkapklliy etsrlasg vpsrfsgsgsgtdftltisslqpedfatyyc qqwssnpltf gqgtkveik VH h41A10 (XC150) SEQ ID NO:63 evqlvesggglvqpggslrlscaas gftfrsyg mswvrqapgkglewvat issggty tfypdsvkgrftisrdnaknslylqmnslraedtavyyc arrgedyrgaleh wgqgtlvtvssas Primer 5'VL1 SEQ ID NO:64 ATCACGCGGCCGCCTCACCATGAAGTTGCCTGTTAGGCTGTTGGTGCTG Primer 5' VL2 SEQ ID NO:65 ATCACGCGGCCGCCTCACCATGGAG(A/T)CAGACACACTCCTG(C/T)TATGGGT Primer 5' VL3 SEQ ID NO:66 ATCACGCGGCCGCCTCACCATGAGTGTGTGCTCACTCAGGTCCTGG(C/G)GTTG Primer 5' VL4 SEQ ID NO:67 ATCACGCGGCCGCCTCACCATGAGG(A/G)CCCTGCTCAG(A/T)TT(C/T)TTGG(A/C)(A/T)TCTTG Primer 5' VL5 SEQ ID NO:68 ATCACGCGGCCGCCTCACCATGGATTT(A/T)CAGGTGCAGATT(A/T)TCAGCTTC Primer 5' VL6 SEQ ID NO:69 ATCACGCGGCCGCCTCACCATGAGGT(G/T)C(C/T)(C/T)TG(C/T)T(G/C)AG(C/T)T(C/T)CTG(G/A)GG Primer 5' VL7 SEQ ID NO:70 ATCACGCGGCCGCCTCACCATGGGC(A/T)TCAAGATGGAGTCACA(G/T)(A/T)(C/T)(C/T)C(A/T)GG Primer 5' VL8 SEQ ID NO:71 ATCACGCGGCCGCCTCACCATGTGGGGA(C/T)CT(G/T)TTT(C/T)C(A/C)(A/C)TTTTTCAATTG Primer 5' VL9 SEQ ID NO:72 ATCACGCGGCCGCCTCACCATGGT(A/G)TCC(A/T)CA(C/G)CTCAGTTCCTTG Primer 5' VL10 SEQ ID NO:73 ATCCACGCGGCCGCCTCACCATGTATATATGTTTGTTGTCTATTTCT Primer 5' VL11 SEQ ID NO:74 ATCACGCGGCCGCCTCACCATGGAAGCCCCAGCTCAGCTTCTCTTCC Primer 3' VL SEQ ID NO:75 CAGTTGGTGCAGCATCCGTACGTTTGATTTCCAG Primer 5' VH1 SEQ ID NO:76 ACTAGCGGCCGCATGAAATGCAGCTGGGTCAT(C/G)TTCTTC Primer 5' VH2 SEQ ID NO:77 ACTAGCGGCCGCATGGGATGGAGCT(A/G)TATCAT(C/G)(C/T)TCT Primer 5' VH3 SEQ ID NO:78 ACTAGCGGCCGCATGAAG(A/T)TGTGGTTAAACTGGGTTTTTT Primer 5' VH4 SEQ ID NO:79 ACTAGCGGCCGCATG(A/G)ACTTTGGG(C/T)TCAGCTTG(A/G)TTT Primer 5' VH5 SEQ ID NO:80 ACTAGCGGCCGCATGGACTCCAGGCTCAATTTAGTTTTCCTT Primer 5' VH6 SEQ ID NO:81 ACTAGCGGCCGCATGGCTGTC(C/T)T(G/A)G(C/G)GCT(G/A)CTTCTTCTGC Primer 5' VH7 SEQ ID NO:82 ACTAGCGGCCGCATGG(A/G)ATGGAGC(G/T)GG(A/G)TCTTT(C/A)TCTT Primer 5' VH8 SEQ ID NO:83 ACTAGCGGCCGCATGAGAGTGCTGATTCTTTTGTG Primer 5' VH9 SEQ ID NO:84 ACTAGCGGCCGCATGG(A/C)TTGGGTGTGGA(A/C)CTTGCTATTCCTG Primer 5' VH10 SEQ ID NO:85 ACTAGCGGCCGCATGGATTTTGGGCTGATTTTTTTATTG Primer 5' VH11 SEQ ID NO:86 ACTAGCGGCCGCATGGGCAGACTTACATTCTCATTCCTG Primer 5' VH12 SEQ ID NO:87 ACTAGCGGCCGCATGATGGTGTTAAGTCTTCTGTACCTG Primer 3' VH SEQ ID NO:88 GGGGGTGTCGTCGACGCTG(A/C)(G/A)GAGAC(G/A)GTGA Human IgG1 constant region SEQ ID NO:89 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human kappa light chain constant region (Cκ) SEQ ID NO:90 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Human germline VH DP-54 showing a framework region (VH3 subgroup) with a JH4 segment SEQ ID NO:91 EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYW MSWVRQAPGKGLEWVAN IKQDGSE KYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARYFDY WGQGTLVTVSS JH4 SEQ ID NO:92 WGQGTLVTVSS human germline VL DPK9 framework (VKI subgroup) with JK4 segment SEQ ID NO:93 DIQMTQSPSSLSASVGDRVTITCRAS QSISSY LNWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPLT FGGGTKVEIK JK4 SEQ ID NO:94 FGGGTKVEIK Nucleic acid sequence encoding VL h11G2 (XC154) SEQ ID NO:95 GACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCTTCCGTGGGAGATAGGGTGACCATCACCTGCAGGGCCAGCGAGTCCGTGGAGTACCACGGCACCAGCCTGATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAACGTGGAGAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGAAGCGGCACCGACTTCACCCTG ACCATTAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGAGCAGGAAGGTGCCCTGGACCTTCGGCCAGGGCACCAAGGTCGAGATCAAG Nucleic acid sequence encoding VH h11G2 (XC155) SEQ ID NO:96 GAGGTGCAGCTGGTGGAGAGCGGAGGAGACTGGTGCAGCCTGGCGGAAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCACCTTTACCGACTTCTACATGAGCTGGGTGAGGCAGGCTCCCGGCAAAGGACTGGAGTGGGTGGGTTTCATCAGGAACAAGGCCAACGGCTACACCACGAGTATAGCCCTCCGTGAAGGGCAGGTTCACCATCAGC AGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCCAGAGTGTACGGCAGCACACTGCACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC Nucleic acid sequence encoding full length light chain (LC) h11G2 (XC154)
The constant domain is in lower case SEQ ID NO:97 GACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCTTCCGTGGGAGATAGGGTGACCATCACCTGCAGGGCCAGCGAGTCCGTGGAGTACCACGGCACCAGCCTGATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAACGTGGAGAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGAAGCGGCACCGACTTCACCCTG ACCATTAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGTCAGCAGAGCAGGAAGGTGCCCTGGACCTTCGGCCAGGGCACCAAGGTCGAGATCAAGcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaag tacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt tag Nucleic acid sequence encoding full length heavy chain (HC) h11G2 (XC155)
The constant domain is in lower case SEQ ID NO:98 gaggtgcagctggtggagagcggaggaggactggtgcagcctggcggaagcctgagactgagctgccgccgccagcggcttcacctttaccgacttctacatgagctgggtgaggcaggctcccggcaaaggactggagtgggtgggtttcatcaggaacaaggccaacggctacaccaccgagtatagcgcctccgtgaagggcagg ttcaccatcagcagggacaacgccaagaacagcctgtacctgcaagaacagcctgtacctgcagatgaacagcctgagggccgaggacaccgccgtgtactactgcgccagagtgtacggcagcacactgcactactggggccagggcaccctggtgaccgtgagcagcgcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggca cagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccag caacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtac gtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtg tacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcagggg aacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccggggttga Nucleic acid sequence encoding wild-type human IgG1 Fc SEQ ID NO:99 gcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtgg tgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggaccc ctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaaca aagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactaagaccacgcctcccgtgct ggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacgcagaagagcctctccctgtccccgggttga Nucleic acid sequence encoding wild-type human kappa chain (Cκ) constant domain SEQ ID NO:100 cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttcttcccagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagca gcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag

[000501] Although the ideas described are presented with reference to various uses, methods, kits, and compositions, it should be understood that various changes and modifications can be made without deviating from the gist of the present invention and the following claims. The above examples are provided to better illustrate the ideas described and are not intended to limit them. While the teachings of the present invention have been described in terms of these exemplary embodiments, those skilled in the art will appreciate that numerous variations and modifications to these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the teachings of the present invention.

[000502] All references cited herein, including patents, patent applications, articles, manuals, and the like, and references cited therein, are hereby incorporated herein by reference in their entirety for all purposes. In the event that one or more of the included literature and similar materials differ or conflict with this application, including, but not limited to, certain terms, use of terms, methods described, or the like, this application takes precedence.

[000503] Specialists in this field it is obvious that you can make various modifications and changes of the present invention without deviating from the scope or essence of the invention. Other embodiments of the invention will become apparent to those skilled in the art upon consideration of the present description and the practice of the invention presented herein. It is to be understood that the present description and examples are to be considered illustrative only, with the true scope and spirit of the invention being set forth in the following claims.

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SEQUENCE LISTING

<110> Pfizer Inc.

DZE REGENTS OF DZE UNIVERSITY OF CALIFORNIA

GROT, Rachel

SNYDER, William, Brian.

Cao, Xianjun

DUNN, Robert, Joseph

DAL PORTO, Joseph

CARIN, Michael

<120> ANTIBODIES AGAINST CXCR5 AND THEIR COMPOSITIONS AND USE

<130> 2420-562964EN

<140> EN 2020121591

<141> 2018-11-29

<150> PCT/US2018/062829

<151> 2018-11-28

<150> 62/732.985

<151> 2018-09-18

<150> 62/593.830

<151> 2017-12-01

<160> 100

<170>PatentIn version 3.5

<210> 1

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 1

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 2

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 2

Glu Ser Val Glu Tyr His Gly Thr Ser

15

<210> 3

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 3

Ala Ala Ser Asn Val Glu Ser Gly

15

<210> 4

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 4

Gln Gln Ser Arg Lys Val Pro Trp Thr

15

<210> 5

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 6

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 7

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 7

Gly Phe Thr Phe Thr Asp Phe Tyr

15

<210> 8

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 8

Ile Arg Asn Lys Ala Asn Gly Tyr Thr

15

<210> 9

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 9

Ala Arg Val Tyr Gly Ser Thr Leu His Tyr

1.5 10

<210> 10

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 11

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 11

Val Arg Val Tyr Gly Ser Thr Leu His Tyr

1.5 10

<210> 12

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 13

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

Glu Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 14

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 14

Ser Ser Val Asn Tyr

15

<210> 15

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 15

Glu Thr Ser Arg Leu Ala Ser Gly

15

<210> 16

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 16

Gln Gln Trp Ser Ser Asn Pro Leu Thr

15

<210> 17

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Thr Tyr Thr Phe Tyr Pro Asp Ile Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Glu Asp Tyr Arg Gly Ala Leu Glu His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 18

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Thr Tyr Thr Phe Tyr Pro Asp Ile Leu

50 55 60

Lys Gly Arg Ile Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Glu Asp Tyr Arg Gly Ala Leu Glu His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 19

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 19

Gly Phe Thr Phe Arg Ser Tyr Gly

15

<210> 20

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 20

Ile Ser Ser Gly Gly Thr Tyr

15

<210> 21

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 21

Ala Arg Arg Gly Glu Asp Tyr Arg Gly Ala Leu Glu His

1.5 10

<210> 22

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 22

Asp Val Val Val Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1.5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Gly Asp Ile Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 23

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 23

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Arg Ala Leu Glu Trp Leu

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Gly Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Val Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Ile Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 24

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 24

Gln Ile Val Leu Thr Gln Ser Pro Ala Val Met Ser Ala Ser Pro Gly

1.5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Glu Thr Ser Arg Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 25

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 25

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1.5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Thr Tyr Thr Phe Tyr Pro Asp Ile Leu

50 55 60

Lys Gly Arg Ile Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Asn Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Glu Asp Tyr Arg Gly Ala Leu Glu His Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 26

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 26

Asp Val Val Val Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1.5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Gly Asp Val Ser Met Phe Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 27

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 27

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Arg Ala Leu Glu Trp Leu

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Gly Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Val Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Ile Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 28

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 29

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 30

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 30

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1.5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 31

<211> 329

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 31

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1.5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 32

<211> 372

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 32

Met Asn Tyr Pro Leu Thr Leu Glu Met Asp Leu Glu Asn Leu Glu Asp

1.5 10 15

Leu Phe Trp Glu Leu Asp Arg Leu Asp Asn Tyr Asn Asp Thr Ser Leu

20 25 30

Val Glu Asn His Leu Cys Pro Ala Thr Glu Gly Pro Leu Met Ala Ser

35 40 45

Phe Lys Ala Val Phe Val Pro Val Ala Tyr Ser Leu Ile Phe Leu Leu

50 55 60

Gly Val Ile Gly Asn Val Leu Val Leu Val Ile Leu Glu Arg His Arg

65 70 75 80

Gln Thr Arg Ser Ser Thr Glu Thr Phe Leu Phe His Leu Ala Val Ala

85 90 95

Asp Leu Leu Leu Val Phe Ile Leu Pro Phe Ala Val Ala Glu Gly Ser

100 105 110

Val Gly Trp Val Leu Gly Thr Phe Leu Cys Lys Thr Val Ile Ala Leu

115 120 125

His Lys Val Asn Phe Tyr Cys Ser Ser Leu Leu Leu Ala Cys Ile Ala

130 135 140

Val Asp Arg Tyr Leu Ala Ile Val His Ala Val His Ala Tyr Arg His

145 150 155 160

Arg Arg Leu Leu Ser Ile His Ile Thr Cys Gly Thr Ile Trp Leu Val

165 170 175

Gly Phe Leu Leu Ala Leu Pro Glu Ile Leu Phe Ala Lys Val Ser Gln

180 185 190

Gly His His Asn Asn Ser Leu Pro Arg Cys Thr Phe Ser Gln Glu Asn

195 200 205

Gln Ala Glu Thr His Ala Trp Phe Thr Ser Arg Phe Leu Tyr His Val

210 215 220

Ala Gly Phe Leu Leu Pro Met Leu Val Met Gly Trp Cys Tyr Val Gly

225 230 235 240

Val Val His Arg Leu Arg Gln Ala Gln Arg Arg Pro Gln Arg Gln Lys

245 250 255

Ala Val Arg Val Ala Ile Leu Val Thr Ser Ile Phe Phe Leu Cys Trp

260 265 270

Ser Pro Tyr His Ile Val Ile Phe Leu Asp Thr Leu Ala Arg Leu Lys

275 280 285

Ala Val Asp Asn Thr Cys Lys Leu Asn Gly Ser Leu Pro Val Ala Ile

290 295 300

Thr Met Cys Glu Phe Leu Gly Leu Ala His Cys Cys Leu Asn Pro Met

305 310 315 320

Leu Tyr Thr Phe Ala Gly Val Lys Phe Arg Ser Asp Leu Ser Arg Leu

325 330 335

Leu Thr Lys Leu Gly Cys Thr Gly Pro Ala Ser Leu Cys Gln Leu Phe

340 345 350

Pro Ser Trp Arg Arg Ser Ser Leu Ser Glu Ser Glu Asn Ala Thr Ser

355 360 365

Leu Thr Thr Phe

370

<210> 33

<211> 372

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 33

Met Asn Tyr Pro Leu Met Leu Glu Met Asp Leu Glu Asn Leu Glu Asp

1.5 10 15

Leu Phe Leu Glu Phe Asp Lys Phe Asp Asn Tyr Asn Asp Thr Ser Leu

20 25 30

Val Glu Asn His Leu Cys Pro Ala Thr Glu Gly Pro Leu Met Ala Ser

35 40 45

Phe Lys Ala Val Phe Val Pro Val Ala Tyr Ser Leu Ile Phe Leu Leu

50 55 60

Gly Val Ile Gly Asn Val Leu Val Leu Val Ile Leu Glu Arg His Arg

65 70 75 80

Gln Thr Arg Ser Ser Thr Glu Thr Phe Leu Phe His Leu Ala Val Ala

85 90 95

Asp Leu Leu Leu Val Phe Ile Leu Pro Phe Ala Val Ala Glu Gly Ser

100 105 110

Val Gly Trp Val Leu Gly Thr Phe Leu Cys Lys Thr Val Ile Ala Leu

115 120 125

His Lys Val Asn Phe Tyr Cys Ser Ser Leu Leu Leu Ala Cys Ile Ala

130 135 140

Val Asp Arg Tyr Leu Ala Ile Val His Ala Val His Ala Tyr Arg His

145 150 155 160

Arg Arg Leu Leu Ser Ile His Ile Thr Cys Gly Thr Ile Trp Leu Val

165 170 175

Gly Phe Leu Phe Ala Leu Pro Glu Ile Leu Phe Ala Lys Val Ser Gln

180 185 190

Ala His Pro Asn Asn Ser Leu Pro Arg Cys Thr Phe Ser Gln Glu Asn

195 200 205

Gln Ala Glu Thr His Ala Trp Phe Thr Ser Arg Phe Leu Tyr His Val

210 215 220

Ala Gly Phe Leu Leu Pro Met Leu Val Met Gly Trp Cys Tyr Val Gly

225 230 235 240

Val Val His Arg Leu Arg Gln Ala Gln Arg Arg Pro Gln Arg Gln Lys

245 250 255

Ala Val Arg Val Ala Ile Leu Val Thr Ser Ile Phe Phe Leu Cys Trp

260 265 270

Ser Pro Tyr His Ile Val Ile Phe Leu Asp Thr Leu Val Arg Leu Lys

275 280 285

Ala Val Asp Asn Thr Cys Glu Leu Asn Gly Ser Leu Pro Val Ala Ile

290 295 300

Thr Met Cys Glu Phe Leu Gly Leu Ala His Cys Cys Leu Asn Pro Met

305 310 315 320

Leu Tyr Thr Phe Ala Gly Val Lys Phe Arg Ser Asp Leu Ser Arg Leu

325 330 335

Leu Thr Lys Leu Gly Cys Thr Gly Pro Ala Ser Leu Cys Gln Leu Phe

340 345 350

Pro Ser Trp Arg Lys Ser Ser Leu Ser Glu Ser Glu Asn Ala Thr Ser

355 360 365

Leu Thr Thr Phe

370

<210> 34

<211> 374

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 34

Met Asn Tyr Pro Leu Thr Leu Asp Met Gly Ser Ile Thr Tyr Asn Met

1.5 10 15

Asp Asp Leu Tyr Lys Glu Leu Ala Phe Tyr Ser Asn Ser Thr Glu Ile

20 25 30

Pro Leu Gln Asp Ser Asn Phe Cys Ser Thr Val Glu Gly Pro Leu Leu

35 40 45

Thr Ser Phe Lys Ala Val Phe Met Pro Val Ala Tyr Ser Leu Ile Phe

50 55 60

Leu Leu Gly Met Met Gly Asn Ile Leu Val Leu Val Ile Leu Glu Arg

65 70 75 80

His Arg His Thr Arg Ser Ser Thr Glu Thr Phe Leu Phe His Leu Ala

85 90 95

Val Ala Asp Leu Leu Leu Val Phe Ile Leu Pro Phe Ala Val Ala Glu

100 105 110

Gly Ser Val Gly Trp Val Leu Gly Thr Phe Leu Cys Lys Thr Val Ile

115 120 125

Ala Leu His Lys Ile Asn Phe Tyr Cys Ser Ser Leu Leu Leu Ala Cys

130 135 140

Ile Ala Val Asp Arg Tyr Leu Ala Ile Val His Ala Val His Ala Tyr

145 150 155 160

Arg Arg Arg Arg Leu Leu Ser Ile His Ile Thr Cys Thr Ala Ile Trp

165 170 175

Leu Ala Gly Phe Leu Phe Ala Leu Pro Glu Leu Leu Phe Ala Lys Val

180 185 190

Gly Gln Pro His Asn Asn Asp Ser Leu Pro Gln Cys Thr Phe Ser Gln

195 200 205

Glu Asn Glu Ala Glu Thr Arg Ala Trp Phe Thr Ser Arg Phe Leu Tyr

210 215 220

His Ile Gly Gly Phe Leu Leu Pro Met Leu Val Met Gly Trp Cys Tyr

225 230 235 240

Val Gly Val Val His Arg Leu Leu Gln Ala Gln Arg Arg Pro Gln Arg

245 250 255

Gln Lys Ala Val Arg Val Ala Ile Leu Val Thr Ser Ile Phe Phe Leu

260 265 270

Cys Trp Ser Pro Tyr His Ile Val Ile Phe Leu Asp Thr Leu Glu Arg

275 280 285

Leu Lys Ala Val Asn Ser Ser Cys Glu Leu Ser Gly Tyr Leu Ser Val

290 295 300

Ala Ile Thr Leu Cys Glu Phe Leu Gly Leu Ala His Cys Cys Leu Asn

305 310 315 320

Pro Met Leu Tyr Thr Phe Ala Gly Val Lys Phe Arg Ser Asp Leu Ser

325 330 335

Arg Leu Leu Thr Lys Leu Gly Cys Ala Gly Pro Ala Ser Leu Cys Gln

340 345 350

Leu Phe Pro Asn Trp Arg Lys Ser Ser Leu Ser Glu Ser Glu Asn Ala

355 360 365

Thr Ser Leu Thr Thr Phe

370

<210> 35

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 35

Asp Val Val Val Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1.5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Gly Asp Ile Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 36

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 36

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Arg Ala Leu Glu Trp Leu

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Gly Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Val Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Ile Leu Thr Val Ser Ser Ala

115 120

<210> 37

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 37

Gln Ile Val Leu Thr Gln Ser Pro Ala Val Met Ser Ala Ser Pro Gly

1.5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Glu Thr Ser Arg Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 38

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1.5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Thr Tyr Thr Phe Tyr Pro Asp Ile Leu

50 55 60

Lys Gly Arg Ile Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Asn Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Glu Asp Tyr Arg Gly Ala Leu Glu His Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala

115 120

<210> 39

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 39

Asp Val Val Val Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1.5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Gly Asp Val Ser Met Phe Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 40

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 40

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Arg Ala Leu Glu Trp Leu

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Gly Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Val Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Ile Leu Thr Val Ser Ser Ala

115 120

<210> 41

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 41

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1.5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Asp Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln

85 90 95

Tyr Leu Ser Ser Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 42

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Asn

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val

35 40 45

Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Thr Met Ile Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 43

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 43

Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Ala Val Thr Pro Gly

1.5 10 15

Ala Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Ser Gly Lys Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Leu Ser Ser Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 44

<211> 440

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 44

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Glu

1.5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Asp Tyr

20 25 30

Gly Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Asp Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Val Thr Ser Leu Thr Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Ile Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala

100 105 110

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

115 120 125

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

130 135 140

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

145 150 155 160

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

165 170 175

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

180 185 190

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

195 200 205

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

210 215 220

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

225 230 235 240

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

245 250 255

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

260 265 270

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

275 280 285

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

290 295 300

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

305 310 315 320

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

325 330 335

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met

340 345 350

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

355 360 365

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

370 375 380

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

385 390 395 400

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

405 410 415

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

420 425 430

Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 45

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 45

Gln Pro Val Leu Thr Gln Pro Ser Val Ser Lys Asp Leu Arg Gln

1.5 10 15

Thr Ala Thr Leu Thr Cys Thr Gly Asn Ser Asn Asn Val Gly Asn Gln

20 25 30

Gly Ala Thr Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu Leu

35 40 45

Ser Tyr Lys Asn Asn Asn Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser

50 55 60

Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Leu Gln

65 70 75 80

Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Glyn

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 46

<211> 441

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Tyr Val Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

115 120 125

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

130 135 140

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

145 150 155 160

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

165 170 175

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

180 185 190

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

195 200 205

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 47

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 47

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 48

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 48

Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 49

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 49

Asp Val Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 50

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 50

Asp Val Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 51

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 51

Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr His

20 25 30

Gly Thr Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 52

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 52

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser

115 120

<210> 53

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 53

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 54

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 54

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 55

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 55

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 56

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 56

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 57

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 57

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Tyr Gly Ser Thr Leu His Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 58

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 58

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr

35 40 45

Glu Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 59

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 59

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

Glu Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 60

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 60

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr

35 40 45

Glu Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 61

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 61

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr

35 40 45

Glu Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 62

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 62

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Glu Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 63

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 63

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Thr Tyr Thr Phe Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Glu Asp Tyr Arg Gly Ala Leu Glu His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser

115 120

<210> 64

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 64

atcacgcggc cgcctcacca tgaagttgcc tgttaggctg ttggtgctg 49

<210> 65

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other attribute

<222> (26)..(26)

<223> n can be a or t

<220>

<221> other attribute

<222> (41)..(41)

<223> n can be c or t

<400> 65

atcacgcggc cgcctcacca tggagncaga cacactcctg ntatgggt 48

<210> 66

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (45)..(45)

<223> n can be c or g

<400> 66

atcacgcggc cgcctcacca tgagtgtgct cactcaggtc ctggngttg 49

<210> 67

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other attribute

<222> (26)..(26)

<223> n can be a or g

<220>

<221> other attribute

<222> (38)..(38)

<223> n can be a or t

<220>

<221> other sign

<222> (41)..(41)

<223> n can be c or t

<220>

<221> other attribute

<222> (46)..(46)

<223> n can be a or c

<220>

<221> other sign

<222> (47)..(47)

<223> n can be a or t

<400> 67

atcacgcggc cgcctcacca tgaggncccc tgctcagntt nttggnntct tg 52

<210> 68

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other attribute

<222> (28)..(28)

<223> n can be a or t

<220>

<221> other attribute

<222> (41)..(41)

<223> n can be a or t

<400> 68

atcacgcggc cgcctcacca tggatttnca ggtgcagatt ntcagcttc 49

<210> 69

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other attribute

<222> (27)..(27)

<223> n can be g or t

<220>

<221> other attribute

<222> (29)..(29)

<223> n can be c or t

<220>

<221> other sign

<222> (30)..(30)

<223> n can be c or t

<220>

<221> other sign

<222> (33)..(33)

<223> n can be c or t

<220>

<221> other sign

<222> (35)..(35)

<223> n can be g or c

<220>

<221> other sign

<222> (38)..(38)

<223> n can be c or t

<220>

<221> other sign

<222> (40)..(40)

<223> n can be c or t

<220>

<221> other sign

<222> (44)..(44)

<223> n can be g or a

<400> 69

atcacgcggc cgcctcacca tgaggtncnn tgntnagntn ctgngg 46

<210> 70

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (26)..(26)

<223> n can be a or t

<220>

<221> other attribute

<222> (43)..(43)

<223> n can be g or t

<220>

<221> other attribute

<222> (44)..(44)

<223> n can be a or t

<220>

<221> other sign

<222> (45)..(45)

<223> n can be c or t

<220>

<221> other sign

<222> (46)..(46)

<223> n can be c or t

<220>

<221> other sign

<222> (48)..(48)

<223> n can be a or t

<400> 70

atcacgcggc cgcctcacca tgggcntcaa gatggagtca cannnncngg 50

<210> 71

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other attribute

<222> (29)..(29)

<223> n can be c or t

<220>

<221> other sign

<222> (32)..(32)

<223> n can be g or t

<220>

<221> other attribute

<222> (36)..(36)

<223> n can be c or t

<220>

<221> other attribute

<222> (38)..(38)

<223> n can be a or c

<220>

<221> other sign

<222> (39)..(39)

<223> n can be a or c

<400> 71

atcacgcggc cgcctcacca tgtgggganc tntttncnnt ttttcaattg 50

<210> 72

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (25)..(25)

<223> n can be a or g

<220>

<221> other sign

<222> (29)..(29)

<223> n can be a or t

<220>

<221> other sign

<222> (32)..(32)

<223> n can be c or g

<400> 72

atcacgcggc cgcctcacca tggtntccnc anctcagttc cttg 44

<210> 73

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 73

atccacgcgg ccgcctcacc atgtatatat gtttgttgtc tatttct 47

<210> 74

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 74

atcacgcggc cgcctcacca tggaagcccc agctcagctt ctcttcc 47

<210> 75

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 75

cagttggtgc agcatccgta cgtttgattt ccag 34

<210> 76

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (33)..(33)

<223> n can be c or g

<400> 76

actagcggcc gcatgaaatg cagctggggtc atnttcttc 39

<210> 77

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (26)..(26)

<223> n can be a or g

<220>

<221> other sign

<222> (33)..(33)

<223> n can be c or g

<220>

<221> other sign

<222> (34)..(34)

<223> n can be c or t

<400> 77

actagcggcc gcatgggatg gagctntatc atnntct 37

<210> 78

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (19)..(19)

<223> n can be a or t

<400> 78

actagcggcc gcatgaagnt gtggttaaac tgggtttttt 40

<210> 79

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (16)..(16)

<223> n can be a or g

<220>

<221> other attribute

<222> (25)..(25)

<223> n can be c or t

<220>

<221> other sign

<222> (34)..(34)

<223> n can be a or g

<400> 79

actagcggcc gcatgnactt tgggntcagc ttgnttt 37

<210> 80

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 80

actagcggcc gcatggactc caggctcaat ttagttttcc tt 42

<210> 81

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (22)..(22)

<223> n can be c or t

<220>

<221> other sign

<222> (24)..(24)

<223> n can be g or a

<220>

<221> other sign

<222> (26)..(26)

<223> n can be c or g

<220>

<221> other sign

<222> (30)..(30)

<223> n can be g or a

<400> 81

actagcggcc gcatggctgt cntngngctn ctcttctgc 39

<210> 82

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (17)..(17)

<223> n can be a or g

<220>

<221> other sign

<222> (25)..(25)

<223> n can be g or t

<220>

<221> other sign

<222> (28)..(28)

<223> n can be a or g

<220>

<221> other sign

<222> (34)..(34)

<223> n can be c or a

<400> 82

actagcggcc gcatggnatg gagcnggntc ttntctt 38

<210> 83

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 83

actagcggcc gcatgagagt gctgattctt ttgtg 35

<210> 84

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (17)..(17)

<223> n can be a or c

<220>

<221> other sign

<222> (29)..(29)

<223> n can be a or c

<400> 84

actagcggcc gcatggnttg ggtgtgganc ttgctattcc tg 42

<210> 85

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 85

actagcggcc gcatggattt tgggctgatt tttttattg 39

<210> 86

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 86

actagcggcc gcatgggcag acttacattc tcattcctg 39

<210> 87

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 87

actagcggcc gcatgatggt gttaagtctt ctgtacctg 39

<210> 88

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> other sign

<222> (20)..(20)

<223> n can be a or c

<220>

<221> other sign

<222> (21)..(21)

<223> n can be g or a

<220>

<221> other sign

<222> (27)..(27)

<223> n can be g or a

<400> 88

gggggtgtcg tcgacgctgn ngagacngtg a 31

<210> 89

<211> 330

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 89

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1.5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 90

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 90

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1.5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 91

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 91

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 92

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 92

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1.5 10

<210> 93

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 93

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 94

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 94

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

1.5 10

<210> 95

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 95

gacatccaga tgacccagag cccttccagc ctgagcgctt ccgtgggaga tagggtgacc 60

atcacctgca gggccagcga gtccgtggag taccacggca ccagcctgat gcactggtac 120

cagcagaagc ccggcaaggc ccccaagctg ctgatctacg ccgccagcaa cgtggagagc 180

ggcgtgccta gcagattcag cggcagcgga agcggcaccg acttcaccct gaccattagc 240

agcctgcagc ccgaggactt cgccacctac tactgtcagc agagcaggaa ggtgccctgg 300

accttcggcc agggcaccaa ggtcgagatc aag 333

<210> 96

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 96

60

agctgcgccg ccagcggctt cacctttacc gacttctaca tgagctggggt gaggcaggct 120

cccggcaaag gactggagtg ggtgggtttc atcaggaaca aggccaacgg ctacaccacc 180

gagtatagcg cctccgtgaa gggcaggttc accatcagca gggacaacgc caagaacagc 240

ctgtacctgc agatgaacag cctgagggcc gaggacaccg ccgtgtacta ctgcgccaga 300

gtgtacggca gcacactgca ctactggggc cagggcaccc tggtgaccgt gagcagc 357

<210> 97

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 97

gacatccaga tgacccagag cccttccagc ctgagcgctt ccgtgggaga tagggtgacc 60

atcacctgca gggccagcga gtccgtggag taccacggca ccagcctgat gcactggtac 120

cagcagaagc ccggcaaggc ccccaagctg ctgatctacg ccgccagcaa cgtggagagc 180

ggcgtgccta gcagattcag cggcagcgga agcggcaccg acttcaccct gaccattagc 240

agcctgcagc ccgaggactt cgccacctac tactgtcagc agagcaggaa ggtgccctgg 300

accttcggcc agggcaccaa ggtcgagatc aagcgtacgg tggctgcacc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600

acccatcagg gcctgagctc gcccgtcaca aaggcttca acaggggaga gtgttag 657

<210> 98

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 98

60

agctgcgccg ccagcggctt cacctttacc gacttctaca tgagctggggt gaggcaggct 120

cccggcaaag gactggagtg ggtgggtttc atcaggaaca aggccaacgg ctacaccacc 180

gagtatagcg cctccgtgaa gggcaggttc accatcagca gggacaacgc caagaacagc 240

ctgtacctgc agatgaacag cctgagggcc gaggacaccg ccgtgtacta ctgcgccaga 300

gtgtacggca gcacactgca ctactggggc cagggcaccc tggtgaccgt gagcagcgcg 360

tcgaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1260

cagggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtcccc gggttga 1347

<210> 99

<211> 990

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 99

gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag caccctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagagagcc tctccctgtc ccggggttga 990

<210> 100

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 100

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg ttag 324

<---

Claims (97)

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to the C-X-C chemokine receptor 5 (CXCR5), where the antibody is at least one of the antibodies selected from the group consisting of: a) an antibody containing a CDR-L1 containing the amino acid sequence of SEQ ID NO: 2; CDR-L2 containing the amino acid sequence of SEQ ID NO: 3; CDR-L3 containing the amino acid sequence of SEQ ID NO: 4; CDR-H1 containing the amino acid sequence of SEQ ID NO: 7; CDR-H2 containing the amino acid sequence of SEQ ID NO: 8; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 9; b) an antibody containing a CDR-L1 containing the amino acid sequence of SEQ ID NO: 2; CDR-L2 containing the amino acid sequence of SEQ ID NO: 3; CDR-L3 containing the amino acid sequence of SEQ ID NO: 4; CDR-H1 containing the amino acid sequence of SEQ ID NO: 7; CDR-H2 containing the amino acid sequence of SEQ ID NO: 8; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 11; c) an antibody containing a CDR-L1 containing the amino acid sequence of SEQ ID NO: 14; CDR-L2 containing the amino acid sequence of SEQ ID NO: 15; CDR-L3 containing the amino acid sequence of SEQ ID NO: 16; CDR-H1 containing the amino acid sequence of SEQ ID NO: 19; CDR-H2 containing the amino acid sequence of SEQ ID NO: 20; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21; d) an antibody containing a VL containing the amino acid sequence of SEQ ID NO: 1 and a VH containing the amino acid sequence of SEQ ID NO: 6; e) an antibody containing a VL containing the amino acid sequence of SEQ ID NO: 13 and a VH containing the amino acid sequence of SEQ ID NO: 18; f) an antibody containing a VL containing the amino acid sequence of SEQ ID NO: 47 and a VH containing the amino acid sequence of SEQ ID NO: 52; g) an antibody containing a VL containing the amino acid sequence of SEQ ID NO: 5 and a VH containing the amino acid sequence of SEQ ID NO: 6; h) an antibody containing a VL containing the amino acid sequence of SEQ ID NO: 5 and a VH containing the amino acid sequence of SEQ ID NO: 10; i) an antibody containing a VL containing the amino acid sequence of SEQ ID NO: 13 and a VH containing the amino acid sequence of SEQ ID NO: 17; j) an antibody containing a VL containing the amino acid sequence of SEQ ID NO: 1 and a VH containing the amino acid sequence of SEQ ID NO: 12; k) an antibody containing an LC containing the amino acid sequence of SEQ ID NO: 22 and an HC containing the amino acid sequence of SEQ ID NO: 23; l) an antibody containing an LC containing the amino acid sequence of SEQ ID NO: 24 and an HC containing the amino acid sequence of SEQ ID NO: 25; m) an antibody containing an LC containing the amino acid sequence of SEQ ID NO: 26 and an HC containing the amino acid sequence of SEQ ID NO: 27; And n) an antibody containing an LC containing the amino acid sequence of SEQ ID NO: 28 and an HC containing the amino acid sequence of SEQ ID NO: 29. 2. An antibody or antigen-binding fragment thereof according to claim 1, wherein said antibody or antigen-binding fragment exhibits at least one of the following biological activities: a) binds to human CXCR5+ B cells with an apparent EC50 affinity of less than about 26 x 10 ^-12 mol (pM), but does not bind to cells expressing mouse, rat or rabbit CXCR5 orthologues; b) has antagonist activity against CXCL13 inhibition of cAMP release triggered by forskolin; c) triggers ADCC of CXCR5-expressing cells in human donor and cynomolgus monkey PBMCs and in human donor TMCs; d) binds to human CXCR5 but does not bind to human chemokine receptors CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 or XCR1; e) depletes peripheral blood B cells; f) depletes Tfh-like cells in peripheral blood; g) depletes true Tfh cells in the spleen; And h) impairs the humoral response of immune memory. 3. An antibody or antigen-binding fragment thereof according to claim 1 or 2, containing a VL containing the amino acid sequence of SEQ ID NO: 1, and a VH containing the amino acid sequence of SEQ ID NO: 6. 4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, containing an LC containing the amino acid sequence of SEQ ID NO: 28, and an HC containing the amino acid sequence of SEQ ID NO: 29. 5. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is afucosylated. 6. An isolated antibody that specifically binds to the C-X-C chemokine receptor 5 (CXCR5) containing an LC consisting of the amino acid sequence of SEQ ID NO: 28 and an HC consisting of the amino acid sequence of SEQ ID NO: 29. 7. An isolated antibody that specifically binds to the C-X-C chemokine receptor 5 (CXCR5) containing an LC containing the amino acid sequence of SEQ ID NO: 28 and an HC containing the amino acid sequence of SEQ ID NO: 29. 8. An isolated antibody that specifically binds to the C-X-C chemokine receptor 5 (CXCR5), containing an antibody VL region containing the amino acid sequence of SEQ ID NO: 1, and an antibody VH region containing the amino acid sequence of SEQ ID NO: 6. 9. An isolated antibody that specifically binds to the C-X-C chemokine receptor 5 (CXCR5), containing: an antibody VL region containing the first, second and third CDRs from the VL region containing the amino acid sequence of SEQ ID NO: 1; And the VH region of the antibody containing the first, second and third CDRs from the VH region containing the amino acid sequence of SEQ ID NO: 6. 10. An isolated antibody that specifically binds to the C-X-C chemokine receptor 5 (CXCR5), containing: a) an antibody VL region containing the first, second and third CDRs from the VL region containing the amino acid sequence of SEQ ID NO: 1; the VH region of the antibody containing the first, second and third CDRs from the VH region containing the amino acid sequence of SEQ ID NO: 6; a light chain constant region (CL) of an antibody comprising the amino acid sequence of SEQ ID NO: 30; And heavy chain constant region (CH) antibodies containing the amino acid sequence of SEQ ID NO: 31; or b) an antibody VL region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 and contains a CDR-L1 containing the amino acid sequence of SEQ ID NO: 2; CDR-L2 containing the amino acid sequence of SEQ ID NO: 3; CDR-L3 containing the amino acid sequence of SEQ ID NO: 4; and a VH region of an antibody that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6 and contains a CDR-H1 containing the amino acid sequence of SEQ ID NO: 7; CDR-H2 containing the amino acid sequence of SEQ ID NO: 8; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 9. 11. Selected antibody that specifically binds to the receptor C-X-C-chemokine 5 (CXCR5), where the specified antibody contains a CDR-L1 containing the amino acid sequence of SEQ ID NO: 2; CDR-L2 containing the amino acid sequence of SEQ ID NO: 3; CDR-L3 containing the amino acid sequence of SEQ ID NO: 4; CDR-H1 containing the amino acid sequence of SEQ ID NO: 7; CDR-H2 containing the amino acid sequence of SEQ ID NO: 8; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 9. 12. An isolated antibody that specifically binds to CXCR5, according to any one of claims 6-11, wherein the antibody is afucosylated. 13. The isolated antibody or antigen-binding fragment thereof according to claim 11 or 12, which specifically binds to the C-X-C chemokine receptor 5 (CXCR5), where the antibody or antigen-binding fragment is at least one antibody selected from the group consisting of: a) an antibody or antigen-binding fragment thereof that binds to hCXCR5 expressed on human B cells with an apparent EC50 affinity of approximately 6.60 x 10 ^-12 moles (pM) with a standard deviation of approximately plus or minus 2.33 pM; b) an antibody or antigen-binding fragment thereof that binds to hCXCR5 expressed on circulating human follicular T helper cells with an apparent EC50 affinity of about 5.89 pM with a standard deviation of about plus or minus 1.40 pM; c) an antibody or antigen-binding fragment thereof that binds to hCXCR5 expressed on human follicular T helper (Tfh) cells with an apparent affinity EC50 of about 10.6 pM; d) an antibody or antigen-binding fragment thereof that binds to cynoCXCR5 expressed on cynomolgus monkey B cells with an apparent EC50 affinity of approximately 1.32 pM; e) an antibody or antigen-binding fragment thereof that binds to cynoCXCR5 expressed on Tfh-like cynomolgus monkey cells with an apparent EC50 affinity of approximately 10.5 pM; f) an antibody or antigen-binding fragment thereof that has antagonistic activity for CXCR5-CXCL13 signal transduction in a cAMP reporter assay with an EC50 of approximately 961 pM; g) an antibody or antigen-binding fragment thereof exhibiting ADCC activity against human B cells expressing hCXCR5 with an EC50 of approximately 2.01 pM with a standard deviation of approximately plus or minus 2.28 pM; h) an antibody or antigen-binding fragment thereof exhibiting ADCC activity against human Tfh-like cells expressing hCXCR5 with an EC50 of approximately 4.28 pM with a standard deviation of approximately plus or minus 2.88 pM; i) an antibody or antigen-binding fragment thereof that exhibits ADCC activity against human Tfh cells expressing hCXCR5 with an EC50 of approximately 0.11 pM; j) an antibody, or antigen-binding fragment thereof, exhibiting ADCC activity against cynoCXCR5 expressing cynoCXCR5-expressing cynomolgus monkey B cells with an EC50 of approximately 15.3 pM with a standard deviation of approximately plus or minus 11.7 pM; k) an antibody or its antigen-binding fragment that binds to hCXCR5 but does not detectably bind to the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4 , CXCR6, CXCR7 and XCR1 human; l) an antibody or antigen-binding fragment thereof that inhibits the binding of CXCR5 to CXCL13; m) an antibody or antigen-binding fragment thereof that binds to human CXCR5+ B cells with an apparent EC50 affinity of less than about 26 pM, but does not bind to cells expressing murine, rat, or rabbit CXCR5 orthologues; n) an antibody, or an antigen-binding fragment thereof, antagonistic to CXCL13 inhibition of cAMP release triggered by forskolin; o) an antibody or antigen-binding fragment thereof that triggers ADCC of CXCR5-expressing cells in human-donor and cynomolgus PBMCs and human-donor TMCs; p) an antibody or antigen-binding fragment thereof that binds to human CXCR5 but does not bind to the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4 , CXCR6, CXCR7 or XCR1 human; q) an antibody or antigen-binding fragment thereof that depletes B cells in peripheral blood; r) an antibody or antigen-binding fragment thereof that depletes Tfh-like cells in peripheral blood; s) an antibody or antigen-binding fragment thereof that depletes true Tfh cells in the spleen; And t) an antibody or its antigen-binding fragment that disrupts the humoral anamnestic immune response. 14. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-13. 15. The isolated nucleic acid according to claim 14, containing the nucleic acid sequence of SEQ ID NO: 95. 16. The isolated nucleic acid according to claim 14, containing the nucleic acid sequence of SEQ ID NO: 96. 17. The isolated nucleic acid of claim 14, comprising the nucleic acid sequence of SEQ ID NO: 95 and SEQ ID NO: 96. 18. The isolated nucleic acid according to claim 14, containing the nucleic acid sequence of SEQ ID NO: 97. 19. The isolated nucleic acid according to claim 14, containing the nucleic acid sequence of SEQ ID NO: 98. 20. The isolated nucleic acid of claim 14, comprising the nucleic acid sequence of SEQ ID NO: 97 and SEQ ID NO: 98. 21. An expression vector containing a nucleic acid according to any one of claims 14-20. 22. A host cell for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, containing an expression vector according to claim 21. 23. The host cell of claim 22, wherein said host cell is a mammalian cell selected from the group consisting of Chinese hamster ovary (CHO) cell, monkey kidney (COS) cell, human embryonic kidney cell line 293 (HEK-293 ), BALB/c NS0 mouse myeloma cell lines, PER.C6® human retinoblast cells, or Sp2.0 cells. 24. The host cell of claim 23, wherein said cell lacks a functional alpha-1,6-fucosyltransferase (FUT8). 25. The host cell of claim 24, wherein said cell is a Potelligent® CHOK1SV cell or a Lec13 CHO cell. 26. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing a Potelligent® CHOK1SV cell according to claim 25 under conditions in which said antibody or antigen-binding fragment is expressed by said host cell and is afucosylated. 27. The method of claim 26, further comprising isolating said antibody or antigen-binding fragment thereof. 28. An antibody or antigen-binding fragment thereof according to any one of claims 6-13, wherein the antibody is afucosylated and exhibits increased antibody-dependent cellular cytotoxicity (ADCC) compared to an otherwise identical antibody or antigen-binding fragment thereof that is fucosylated. 29. A pharmaceutical composition for the treatment or prevention of an immune disease, disorder or condition mediated by the C-X-C chemokine receptor 5 (CXCR5), comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-13 and 28 and a pharmaceutically acceptable carrier, or excipient. 30. An antibody or antigen-binding fragment thereof according to any one of claims 1-13, 28 and a pharmaceutical composition according to claim 29 for use in the treatment of an immunological disease, disorder or condition. 31. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1-13, 29 or a pharmaceutical composition according to claim 29 for the treatment of an immunological disease, disorder or condition. 32. A method of treating or preventing an immunological disease, disorder, or condition mediated by the C-X-C chemokine receptor 5 (CXCR5) in a patient, comprising administering to an individual an effective amount of a pharmaceutical composition according to claim 29, wherein said disease, disorder, or condition is selected from the group consisting of inflammatory responses such as inflammatory skin diseases including psoriasis and dermatitis; dermatomyositis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease; respiratory distress syndrome; dermatitis; meningitis; encephalitis; uveitis; colitis; gastritis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions including T cell infiltration and chronic inflammatory responses; atherosclerosis; lack of adhesion of leukocytes; rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); diabetes; multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and immune responses associated with acute and delayed hypersensitivity reactions mediated by cytokines and T-lymphocytes, usually found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; Wegener's granulomatosis; pernicious anemia; diseases, including diapedesis of leukocytes; inflammatory disorders of the central nervous system (CNS); multiple organ failure syndrome; hemolytic anemia; myasthenia gravis; diseases mediated by antigen-antibody complexes; anti-BMC antibody disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; myasthenic Lambert-Eaton syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; vitiligo; Reiter's disease; muscle stiffness syndrome; Behçet's disease; giant cell arteritis; immunocomplex nephritis; IgA nephropathy; IgM polyneuropathies; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic diseases; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveoretinitis; Guillain-Barré syndrome; Goodpasture's syndrome; mixed connective tissue disease; autoimmune-associated infertility; nodous polyarteritis; alopecia areata; idiopathic myxedema; graft-versus-host reactions; muscular dystrophy and malignant tumors expressing CXCR5. 33. The method of claim 32 wherein the dermatitis is atopic dermatitis. 34. The method of claim 32 wherein the inflammatory bowel disease is Crohn's disease and ulcerative colitis. 35. The method of claim 32 wherein the respiratory distress syndrome is adult respiratory distress syndrome (ARDS). 36. The method of claim 32, wherein the diabetes mellitus is type I diabetes mellitus or insulin dependent diabetes mellitus. 37. The method of claim 32 wherein the pernicious anemia is associated with Addison's disease. 38. The method of claim 32 wherein the hemolytic anemia is cryoglobulinemia or autoimmune hemolytic anemia with incomplete heat agglutinins. 39. The method of claim 32 wherein the muscular dystrophy is Duchenne dystrophy, Becker dystrophy, myotonic dystrophy, limb girdle dystrophy, scapulohumeral facial dystrophy, congenital dystrophy, oculopharyngeal dystrophy, distal dystrophy, Emery-Dreyfus dystrophy. 40. The method of claim 32, wherein the CXCR5-expressing cancer cells are pancreatic cancer, colon cancer, bladder cancer, T-cell leukemia, and B-cell leukemia. 41. The method of claim 32, wherein said disease is SLE or rheumatoid arthritis. 42. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or 28 for the preparation of a medicament for the treatment of an immunological disease, disorder or condition. 43. A method for detecting the C-X-C chemokine 5 receptor (CXCR5) in a sample, tissue, or cell using an antibody or antigen-binding fragment thereof according to any one of claims 1-13 or 28, including bringing the sample, tissue, or cell into contact with antibody and detection of CXCR5 in the sample, tissue, cell by detecting the antibody. 44. A method for reducing the biological activity of the C-X-C chemokine 5 receptor (CXCR5) in a patient, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-13 or 28 or a pharmaceutical composition according to claim 29. 45. The method of claim 44, wherein the antibody mediates depletion of at least one CXCR5 expressing cell selected from the group consisting of a Tfh cell in the spleen, a B cell in the peripheral blood, and a Tfh-like cell in the peripheral blood. 46. A method for inhibiting a humoral immune response in a patient, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-13 or 28 or a pharmaceutical composition according to claim 29. 47. The method of claim 46 wherein the antibody mediates depletion of at least one CXCR5 expressing cell selected from the group consisting of a follicular T helper (Tfh) cell in the spleen, a B cell in the peripheral blood, and a Tfh-like cell in peripheral blood.

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