Use of Anti-CXCR5 antibodies

Abstract

The invention provides method of using antibodies, and antigen-binding fragments thereof, that specifically bind to CXCR5. The method of using the antibodies include method of treating disease. The antibodies can be afucosylated and exhibit increased ADCC compared with the otherwise identical fucosylated antibodies. The invention includes uses, and associated methods of using the antibodies.

Description

[0001] USE OF ANTI-CXCR5 ANTIBODIES

[0002] BACKGROUND

[0003] C-X-C chemokine receptor type 5 (CXCR5), also known as CD185 or Burkitt lymphoma receptor 1 (BLR1), is a naturally occurring, G-protein-coupled receptor expressed by B cells, bona fide follicular T helper (Tfh) cells, 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 treating autoimmune diseases.

[0004] 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 immunoglobulin (Ig) class switching of the antibodies they produce. Bona fide Tfh cells provide instructive signals to aid this process, which culminates in the production of affinity-matured antibodies. Humoral “memory” of the GO reaction is maintained by long-lived plasma cells, which sustain antibody levels, and by memory B cells which, upon re-challenge with antigen, trigger secondary GO reactions with cognate help from memory Tfh cells that lead to the production of more high-affinity plasma cells (McHeyzer-Williams et al., Nature Rev. Immunol. (2011) 12(1):24-34). Circulating Tfh-like cells (hereinafter referred to as “Tfh-like” or “cTfh”) are thought to differentiate into bona fide Tfh cells upon re-encountering antigen to support these memory responses as well (Crotty et al., Annu. Rev. Immmunol. (2011) 29:621- 663).

[0005] Importantly, the generation of autoreactive B cells can also arise from GC reactions, with undesired consequences. Indeed, numerous chronic, systemic autoimmune diseases, such as SLE, RA, myositis, Sjogren’s syndrome, ANCA-associated vasculitis, and scleroderma, show evidence of autoreactive humoral responses. For example, many of the autoantibodies that are hallmarks of these diseases are high-affinity, somatically mutated, and Ig-switched, suggesting that they arose from autoreactive GC reactions (Vinuesa et al., Nature Rev. Immunol. (2009) 9(12):845-857). In addition, increased frequencies of circulating Tfh-like cells have been detected in the peripheral blood of patients with many of these autoimmune diseases, the levels of which often correlate with autoantibody titers and / or disease severity (Tangye et al., Nature Rev. Immunol. (2013) 13(6) :412-426). Taken together, these data highlight B cells, bona fide Tfh cells, and circulating Tfh-like cells as potential therapeutic targets for many systemic autoimmune diseases. CXCR5 is expressed by B cells, bona fide Tfh cells, and circulating Tfh-like cells and mediates their trafficking to and participation in GC reactions along a gradient of the CXCR5 ligand, 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). As such, targeting CXCR5 may have therapeutic benefit for treatment of autoimmune diseases. In addition, targeting CXCR5 may also have therapeutic benefit in cancers characterized by the proliferation of cells expressing CXCR5, such as cancers of the pancreas, colon, bladder, T-cell leukemia, and B-cell leukemia.

[0006] SUMMARY OF THE INVENTION

[0007] This application discloses methods of using isolated antibodies, and antigen-binding fragments thereof, which specifically bind CXCR5 (C-X-C chemokine receptor type 5). In certain aspects, methods of use of an anti-CXCR5 antibody includes methods of treating or preventing disease. In certain aspects, methods of use of an anti-CXCR5 antibody include methods of treating or preventing autoimmune disease. In certain aspects, methods of use of an anti- CXCR5 antibody include methods of treating or preventing immune or inflammatory disease, including for example, primary immune thrombocytopenia (ITP) (also referred to as immune thrombocytopenic purpura and immune thrombocytopenia), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ANCA-vasculitis, pemphigus vulgaris, myasthenia gravis (MG) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In other aspects, methods of use of an anti-CXCR5 antibody include methods of treating or preventing immune or inflammatory disease, including for example, Immunoglobulin G4 related disease (lgG4 RD). In some embodiments, methods of use of an anti-CXCR5 antibody comprises methods of treating diseases associated with or mediated by CXCR5 expression and / or binding to CXCL13, including, but not limited to, inflammatory and immune diseases. In some embodiments, methods of treating or preventing disease comprise administering an anti- CXCR5 antibody that is fucosylated and / or afucosylated and can exhibit altered effector function.

[0008] In certain aspects, methods of use comprise methods of treating or preventing disease by administering antibodies, and antigen-binding fragments thereof, that bind CXCR5 and decrease CXCR5 binding to CXCL13. In other aspects, methods of use comprise methods of treating or preventing disease by administering antibodies, and antigen-binding fragments thereof, that bind CXCR5, are afucosylated and exhibit increased antibody-dependent cellular cytotoxicity (ADCC) compared to otherwise identical but fucosylated antibodies, and antigenbinding fragments thereof.

[0009] In certain aspects, the present disclosure provides methods of use comprising methods of treating or preventing disease by administering antibodies, and antigen-binding fragments thereof, that specifically bind CXCR5, wherein the antibody, and antigen-binding fragment thereof, binds to an epitope comprising leucine (Leu; L) at amino acid residue number 11 (L11) according to the numbering of SEQ ID NO: 15. In another aspect, the disclosure provides methods of use comprising methods of treating or preventing disease by administering antibodies, or antigen-binding fragments thereof, that specifically bind CXCR5, wherein the antibody, and antigen-binding fragment thereof, binds to an epitope comprising an aspartate (Asp; D) at amino acid residue number 22 according to the numbering of SEQ I D NO: 15.

[0010] In some aspects, the present disclosure provides methods of treating a subject with an immune or inflammatory disease comprising subcutaneous administration of a unit dose of at least is 6 mg, 10 mg, 18 mg or 50 mg of an anti-CXCR5 antibody comprising a variable region of the light chain (VL) comprising the amino acid sequence of SEQ ID NO:1 and a variable region of the heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:5, wherein the unit dose is administered once a month. In some embodiments, the immune or inflammatory disease is selected from the group consisting of primary immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ANCA- vasculitis, pemphigus vulgaris, myasthenia gravis (MG) and chronic inflammatory demyelinating polyradiculoneuropathy (Cl DP). In some embodiments, the immune or inflammatory disease is Immunoglobulin G4 related disease (lgG4 RD). In some embodiments, a unit dose is 18 mg. In some embodiments, a unit dose is 50 mg. In some embodiments, a subject has ITP. In some embodiments, the method further comprises i) measuring the absolute platelet count in the subject, and ii) if the absolute platelet count is greater than 50 x 103platelets per pL of blood, the absolute platelet count is at least 2-fold greater than the baseline value of the absolute platelet count for the subject, and the subject was not exposed to splenectomy or rescue medication before the measuring step, assigning a responder status to the subject on the modified overall response scale. In some embodiments, the method further comprises i) measuring the absolute platelet count in the subject, and ii) if the absolute platelet count is greater than 100 x 103platelets per pL of blood, and the subject was not exposed to splenectomy or rescue medication before the measuring step, assigning a responder status to the subject on the complete response scale.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention there are shown in the drawings embodiment(s). It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

[0013] FIG. 1 is an exemplary schema for the 6 mg dose cohort.

[0014] FIG. 2 is an exemplary schema for the 18 mg dose cohort. DETAILED DESCRIPTION

[0015] Disclosed herein are method of using antibodies that specifically bind to CXCR5 and further, antibodies that antagonize CXCR5 activity or its interaction with CXCL13. Use of the anti-CXCR5 antibodies include methods of treating and preventing disease. In some embodiments, the disease includes immune thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ANCA-vasculitis, pemphigus vulgaris, myasthenia gravis (MG) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In some embodiments, methods of treating or preventing a disease comprise administering to a subject (e.g., a patient) an effective amount of the anti-CXCR5 antibody.

[0016] Fucosylated and afucosylated antibodies that bind CXCR5 are provided along with methods of use. In some embodiments, afucosylated antibody heavy chains and light chains that are capable of forming antibodies that bind CXCR5 are also provided. In some embodiments, afucosylated antibodies, heavy chains, and light chains comprising one or more particular complementarity determining regions (CDRs) are provided. In some embodiments, afucosylated anti-CXCR5 antibodies have altered effector function. In some embodiments, the antibodies have enhanced ADCC activity relative to otherwise identical fucosylated anti-CXCR5 antibodies.

[0017] Polynucleotides encoding antibodies that bind CXCR5, or antigen-binding fragments thereof, are provided. Polynucleotides encoding antibody heavy chains or light chains are also provided. Host cells that express fucosylated and / or afucosylated anti-CXCR5 antibodies are provided. Methods of treatment using afucosylated and fucosylated antibodies to CXCR5 are provided. Such methods include, but are not limited to, methods of treating diseases associated with or mediated by CXCR5 expression and / or binding to CXCL13, including, but not limited to, inflammatory and immune diseases.

[0018] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0019] All references cited herein, including patent applications, patent publications, and Genbank Accession numbers are herein incorporated by reference, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety.

[0020] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies 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)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 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)); The 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 updated versions thereof.

[0021] 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; leukocyte adhesion deficiency; rheumatoid arthritis; diabetes mellitus (e. g. Type I diabetes mellitus or insulin dependent diabetes mellitis); multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; ANCA vasculitis; Wegener’s disease; pernicious anemia (Addison's disease); diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder; multiple organ injury syndrome; hemolytic anemia (including, but not limited to cryoglobinemia or Coombs positive anemia); myasthenia gravis; antigen-antibody complex mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous; pemphigus vulgaris; autoimmune polyendocrinopathies; vitiligo; Reiter's disease; stiff-person syndrome; Bechet disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathies; primary immune thrombocytopenia (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic diseases; Hashimoto’s thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveoretinitis; Guillain-Barre syndrome; Goodpasture's syndrome; mixed connective tissue disease; autoimmune-associated infertility; polyarteritis nodosa; alopecia areata; idiopathic myxedema; graft versus host disease; muscular dystrophy (Duchenne, Becker, Myotonic, Limb-girdle, Facioscapulohumeral, Congenital, Oculopharyngeal, Distal, Emery-Dreifuss), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and controlling the proliferation of cancer cells expressing CXCR5 such as cancers of the pancreas, colon, bladder, T-cell leukemia, and B-cell leukemia as would be appreciated by one skilled in the art provided with the teachings disclosed herein. i„ DEFINITIONS

[0022] The present invention may be understood more readily by reference to the following detailed description of exemplary embodiments of the invention and the examples included therein.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

[0024] It is understood that aspect and embodiments of the invention described herein include “consisting” and / or “consisting essentially of aspects and embodiments. As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise.

[0025] In the context of a multiple dependent claim, the use of "or" refers back to more than one preceding independent or dependent claim.

[0026] “About” or “approximately,” when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater. Numeric ranges are inclusive of the numbers defining the range.

[0027] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g. 1 to 6.1 , and ending with a maximum value of 10 or less, e.g., 5.5 to 10.

[0028] Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.

[0029] It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of’ are also provided.

[0030] Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.

[0031] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0032] Polypeptide or antibody “fragments” or “portions” according to the invention may be made by truncation, e.g. by removal of one or more amino acids from the N and / or C-terminal ends of a polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids may be removed from the N and / or C terminal in this way. Fragments or portions may also be generated by one or more internal deletions.

[0033] A variant antibody may comprise 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 from the specific sequences and fragments discussed above. "Deletion" variants may comprise the 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 the deletion of specific amino acid domains or other features. "Insertion" variants may comprise the insertion of individual amino acids, insertion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or insertion of larger amino acid regions, such as the insertion of specific amino acid domains or other features. "Substitution" variants preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, 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 which can be used to select suitable substituents are as follows.

[0034] Substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but framework alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of “conservative substitutions.” If such substitutions result in a change in biological activity, then more substantial changes, denominated “exemplary substitutions” shown below, or as further described below in reference to amino acid classes, may be introduced and the products screened. TABLE 1

[0035] Amino Acids and Substitutions

[0036] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a betasheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: i. Non-polar: Norleucine, Met, Ala, Vai, Leu, lie; ii. Polar without charge: Cys, Ser, Thr, Asn, Gin; iii. Acidic (negatively charged): Asp, Glu; iv. Basic (positively charged): Lys, Arg; v. Residues that influence chain orientation: Gly, Pro; and vi. Aromatic: Trp, Tyr, Phe, His.

[0037] Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

[0038] One type of substitution, for example, that may be made is to change one or more cysteines in the antibody, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine. For example, there can be a substitution of a non-canonical cysteine. The substitution can be made in a 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 proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0039] An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen-binding fragment thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen-binding fragment, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. Antigen-binding fragments include, for example, Fab, Fab’, F(ab’)2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), fragments including complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and polypeptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, I g E, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, lgG2, IgGs, lgG4, IgAi and lgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0040] The terms "antigen-binding portion" or “antigen-binding fragment” of an antibody (or simply "antibody portion"), as used interchangeably herein, refers to one or more fragments of an antibody 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 encompassed within 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) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR), disulfide-linked Fvs (dsFv), and anti-idiotypic (anti-ld) antibodies and intrabodies. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)); see e.g., 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 encompassed within the term "antigen-binding fragment" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. Proc. Natl.

[0041] Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure 2:1121-1123).

[0042] Antibodies may be derived 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 (e.g., sharks) and camelids (e.g., llamas).

[0043] A “variable region” of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three “complementarity determining regions (CDRs)” also known as hypervariable regions (HVR), and contribute to the formation of the antigen binding site of antibodies. If variants of a subject variable region are desired, particularly with substitution in amino acid residues outside of a CDR region (i.e. , in the framework region), appropriate amino acid substitution, preferably, conservative amino acid substitution, can be identified by comparing the subject variable region to the variable regions of other antibodies which contain CDR1 and CDR2 sequences in the same canonical class as the subject variable region (Chothia and Lesk, J. Mol. Biol. 196(4): 901-917, 1987).

[0044] In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, and the conformational definition.

[0045] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., 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 positions of certain structural loop regions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., 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. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198.

[0046] The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as the “conformational definition” of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined in accordance with any of Kabat, Chothia, extended, AbM, contact, and / or conformational definitions.

[0047] “Contact residue” as used herein with respect to an antibody or the antigen specifically bound thereby, refers to an amino acid residue present on an antibody / antigen comprising at least one heavy atom (i.e. , not hydrogen) that is within 4 A or less of a heavy atom of an amino acid residue present on the cognate antibody / antigen.

[0048] “Framework” (FR) residues are antibody variable domain residues other than the CDR residues. A VH or VL domain framework comprises four framework sub-regions, FR1, FR2, FR3 and FR4, interspersed with CDRs in the following structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.

[0049] As stated previously herein, residues in a variable domain are typically numbered according Kabat, which is a numbering system used for heavy chain variable domains or light chain variable domains of the compilation 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 actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Various algorithms for assigning Kabat numbering are available. The algorithm implemented in the version 2.3.3 release of Abysis (wvAv.abvsis.org) can be used to assign Kabat numbering to variable regions CDR-L1 , CDR-L2, CDR-L3, CDR-H2, and CDR-H3, and the AbM definition can then be used for CDR-H1.

[0050] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example. As used herein, "humanized" antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a 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 comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

[0051] The antibody, or antigen-binding fragment thereof, of the invention may be affinity matured. For example, an affinity matured antibody can be produced by procedures 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 W02004 / 058184).

[0052] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.

[0053] The term “chimeric antibody” is intended to refer to antibodies 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 derived from a human antibody or vice versa. The term also encompasses an antibody comprising a V region from one individual from one species (e.g., a first mouse) and a constant region from another individual from the same species (e.g., a second mouse).

[0054] The term “antigen (Ag)” refers to the molecular entity used for immunization of an immunocompetent vertebrate to produce the antibody (Ab) that recognizes the Ag or to screen an expression library (e.g., phage, yeast or ribosome display library, among others). Herein, Ag is termed more broadly and is generally intended to include target molecules that are specifically recognized by the Ab, thus including fragments or mimics of the molecule used in an immunization process for raising the Ab or in library screening for selecting the Ab. Thus, for antibodies of the invention binding to CXCR5, full-length CXCR5 from mammalian species (e.g., human, monkey, mouse and rat CXCR5), including monomers and multimers, such as dimers, trimers, etc. thereof, as well as truncated and other variants of CXCR5, are referred to as an antigen.

[0055] Generally, the term “epitope” refers to the area or region of an antigen (e.g., a protein, nucleic acid, carbohydrate, or lipid, etc.) to which an antibody specifically binds, i.e. , an area or region in physical contact with the antibody. Thus, the term “epitope” refers to that portion of a molecule capable of being recognized by 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 a surface grouping 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 can be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. A “nonlinear epitope” or “conformational epitope” comprises noncontiguous polypeptides (or amino acids) within the antigenic protein to which an antibody specific to the epitope binds. The term “antigenic epitope” as used herein, is defined as a portion of an antigen to which an antibody can specifically bind as determined by any method well known in the art, for example, by conventional immunoassays. Alternatively, during the discovery process, the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to conduct competition and cross-competition studies to find antibodies that compete or cross-compete with one another for binding to CXCR5, e.g., the antibodies compete for binding to the antigen.

[0056] An antibody that “preferentially binds” or “specifically binds” (used interchangeably herein) to an epitope is a term well understood in the art, and methods to determine 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, with greater duration and / or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. Also, an antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration to that target in a sample than it binds to other substances present in the sample. For example, an antibody that specifically or preferentially binds to an CXCR5 epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and / or with greater duration than it binds to other CXCR5 epitopes or non- CXCR5 epitopes. It is also understood by reading this definition, for example, that an antibody (or moiety or epitope) which specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. “Specific binding” or “preferential binding” includes a compound, e.g., a protein, a nucleic acid, an antibody, and the like, which recognizes and binds to a specific molecule, but does not substantially recognize or bind other molecules in a sample. For instance, an antibody or a peptide receptor which recognizes and binds to a cognate ligand or binding partner (e.g., an anti-CXCR5 antibody that binds CXCR5) in a sample, but does not substantially recognize or bind other molecules in the sample, specifically binds to that cognate ligand or binding partner. Thus, under designated assay conditions, the specified binding moiety (e.g., an antibody or an antigen-binding fragment thereof or a receptor or a ligand binding fragment thereof) binds preferentially to a particular target molecule and does not bind in a significant amount to other components present in a test sample. A variety of assay formats may be used to select an antibody or peptide that specifically binds a molecule of interest. For example, solid-phase ELISA immunoassay, immunoprecipitation, Biacore™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescence- activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA) and Western blot analysis are among many assays that may be used to identify an antibody that specifically reacts with an antigen or a receptor, or ligand binding fragment thereof, that specifically binds with a cognate ligand or binding partner. Typically, a specific or selective reaction will be at least twice the background signal or noise, more typically more than 10 times background, even more typically, more than 50 times background, more typically, more than 100 times background, yet more typically, more than 500 times background, even more typically, more than 1000 times background, and even more typically, more than 10,000 times background. Additionally, an antibody is said to “specifically bind” an antigen when the equilibrium dissociation constant (KD) is < 1 pM, preferably < 100 nM, more preferably < 10 nM, even more preferably, < 100 pM, yet more preferably, < 10 pM, and even more preferably, < 1 pM. In some embodiments, an antibody is said to “specifically bind” an antigen when the equilibrium dissociation constant (KD) is < 7 nM.

[0057] The term “binding affinity” is herein used as a measure of the strength of a non- covalent interaction between two molecules, e.g., and antibody, or fragment thereof, and an antigen. The term “binding affinity” is used to describe monovalent interactions (intrinsic activity).

[0058] Additionally, to determine the binding affinity of CXCR5 antibodies to CXCR5-expressing cells, cell binding experiments can be performed to determine the apparent affinity. The apparent affinity of antibody binding to cells expressing the target can be calculated as the EC50 of equilibrium binding titration curves in which the geometric mean fluorescence intensity (gMFI) of the antigen binding population is quantified by flow cytometry.

[0059] Binding affinity between two molecules, e.g., an antibody, or fragment thereof, and an antigen, through a monovalent interaction may be quantified by determination of the dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation using, e.g., the surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka(or kon) and dissociation rate constant kd(or kotf), respectively. KDis related to kaand kdthrough the equation KD = kdI ka. The value of the dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al. (1984, Byte 9: 340-362). For example, the Ko may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays to evaluate the binding ability of ligands such as antibodies towards target antigens are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis, and other assays exemplified elsewhere herein. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g., by using a Biacore™ system, or KinExA.

[0060] A competitive binding assay can be conducted in which the binding of the antibody to the antigen is compared to the binding of the target by another ligand of that target, such as another antibody or a soluble receptor that otherwise binds the target. The concentration at which 50% inhibition occurs is known as the Kj. Under ideal conditions, the Kj is equivalent to KD. The Ki value will never be less than the KD, SO measurement of Kj can conveniently be substituted to provide an upper limit for KD.

[0061] Following the above definition, binding affinities associated with different molecular interactions, e.g., comparison of the binding affinity of different antibodies for a given antigen, may be compared by comparison of the KD values for the individual antibody / antigen complexes. KD values for antibodies or other binding partners can be determined using methods well established in the art. One method for determining the KDis by using surface plasmon resonance, typically using a biosensor system such as a Biacore® system.

[0062] Similarly, the specificity of an interaction may be assessed by determination and comparison of the KD value for the interaction of interest, e.g., a specific interaction between an antibody and an antigen, with the KD value of an interaction not of interest, e.g., a control antibody known not to bind CXCR5.

[0063] An antibody that specifically binds its target may bind its target with a high affinity, that is, exhibiting a low KDas discussed above, and may bind to other, non-target molecules with a lower affinity. For example, the antibody may bind to non-target molecules with a KDof 1 x 10'6M or more, more preferably 1 x 10'5M or more, more preferably 1 x 10'4M or more, more preferably 1 x 10'3M or more, even more preferably 1 x 10'2M or more. An antibody of the invention is preferably capable of binding to its target with an affinity that is at least two-fold, 10- fold, 50-fold, 100-fold 200-fold, 500-fold, 1,000-fold or 10,000-fold or greater than its affinity for binding to another non-CXCR5 molecule.

[0064] The term “compete”, as used herein with regard to an antibody, means that a first antibody, or an antigen-binding fragment thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody, or an antigen-binding fragment thereof, such that the result of binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are encompassed by 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 portion thereof), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and / or crosscompeting antibodies are encompassed and can be useful for the methods disclosed herein.

[0065] Standard competition assays may be used to determine whether two antibodies compete with each other. One suitable assay for antibody competition involves the use of the Biacore technology, which can measure the extent of interactions using surface plasmon resonance (SPR) technology, typically using a biosensor system (such as a BIACORE® system). For example, 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 for measuring antibody competition uses an ELISA-based approach.

[0066] Furthermore, a high throughput process for “binning” antibodies based upon their competition is described in International Patent Application No. WO2003 / 48731 . Competition is present if one antibody (or fragment) reduces the binding of another antibody (or fragment) to CXCR5. For example, a sequential binding competition assay may be used, with different antibodies being added sequentially. The first antibody may be added to reach binding that is close to saturation. Then, the second antibody is added. If the binding of second antibody to CXCR5 is not detected, or is significantly reduced (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% reduction) as compared to a parallel assay in the absence of the first antibody (which value can be set as 100%), the two antibodies are considered as competing with each other.

[0067] In addition, an exemplary antibody epitope binning assay using domain swapping between human and mouse CXCR5 proteins to assess potential epitopes among several antibodies is provided in Example 8 in WO2019 / 108639, US 10,875915 and US 11 ,958,901. The skilled artisan would appreciate, armed with the teachings provided herein, that there are a wide variety of assays known in the art that can be used to determine the binding to a target of at least two antibodies relative to each other, and such assays are included herein.

[0068] CXCR5 antibodies may be characterized using methods well known in the art. For example, one method 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 solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, 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 an additional example, epitope mapping can be used to determine the sequence to which a CXCR5 antibody binds. Epitope mapping is commercially available from various sources, for example, Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch. Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with CXCR5 antibody.

[0069] In addition, the epitope to which the CXCR5 antibody binds can be determined in a systematic screening by using overlapping peptides derived from the CXCR5 sequence and determining binding by the antibody. According to the gene fragment expression assays, the open reading frame encoding CXCR5 can be fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of CXCR5 with the antibody to be tested is determined. The gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled CXCR5 fragments is then determined by immunoprecipitation and gel electrophoresis.

[0070] Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) or yeast (yeast display). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, mutagenesis of an antigen, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, alanine scanning mutagenesis experiments can be performed using a mutant CXCR5 in which various residues of the CXCR5 polypeptide have been replaced with alanine. By assessing binding of the antibody to the mutant CXCR5, the importance of the particular CXCR5 residues to antibody binding can be assessed.

[0071] Yet another method which can be used to characterize a CXCR5 antibody is to use competition assays with other antibodies known to bind to the same antigen, i.e., various fragments on CXCR5, to determine if the CXCR5 antibody binds to the same epitope as other antibodies. Competition assays are well known to those of skill in the art. Further, the epitope for a given antibody / antigen binding pair can be defined and characterized at different levels of detail using a variety of experimental and computational epitope mapping methods. The experimental methods include mutagenesis, X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, hydrogen / deuterium exchange Mass Spectrometry (H / D-MS) and various competition binding methods well-known in the art. As each method relies on a unique principle, the description of an epitope is intimately linked to the method by which it has been determined. Thus, the epitope for a given antibody / antigen pair will be defined differently depending on the epitope mapping method employed.

[0072] At its most detailed level, the epitope for the interaction between the Ag and the Ab can be defined by the spatial coordinates defining the atomic contacts present in the Ag-Ab interaction, as well as information about their relative contributions to the binding thermodynamics. At a less detailed level the epitope can be characterized by the spatial coordinates defining the atomic contacts between the Ag and Ab. At a further less detailed level the epitope can be characterized by the amino acid residues that it comprises as defined by a specific criterion, e.g., by distance between atoms (e.g., heavy, i.e., non-hydrogen atoms) in the Ab and the Ag. At a further less detailed level the epitope can be characterized through function, e.g. by competition binding with other Abs. The epitope can also be defined more generically as comprising amino acid residues for which substitution by another amino acid will alter the characteristics of the interaction between the Ab and Ag (e.g. using alanine scanning).

[0073] From the fact that descriptions and definitions of epitopes, dependent on the epitope mapping method used, are obtained at different levels of detail, it follows that comparison of epitopes for different Abs on the same Ag can similarly be conducted at different levels of detail.

[0074] Epitopes described at the amino acid level, e.g., determined from an 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 shared by the epitopes. Epitopes are said to be separate (unique) if no amino acid residue is shared by the epitopes.

[0075] Epitopes characterized by competition binding are said to be overlapping if the binding of the corresponding antibodies are mutually exclusive, i.e., binding of one antibody excludes simultaneous or consecutive binding of the other antibody. The epitopes are said to be separate (unique) if the antigen is able to accommodate binding of both corresponding antibodies simultaneously.

[0076] The definition of the term “paratope” is derived from the above definition of “epitope” by reversing the perspective. Thus, the term “paratope” refers to the area or region on the antibody which specifically binds an antigen, i.e., the amino acid residues on the antibody which make contact with the antigen (CXCR5) as “contact” is defined elsewhere herein. The epitope and paratope for a given antibody / antigen pair may be identified by routine methods. For example, the general location of an epitope may be determined by assessing the ability of an antibody to bind to different fragments or variant CXCR5 polypeptides. The specific amino acids within CXCR5 that make contact with an antibody (epitope) and the specific amino acids in an antibody that make contact with CXCR5 (paratope) may also be determined using routine methods, such as those described in the examples. For example, the antibody and target molecule may be combined and the antibody / antigen complex may be crystallized. The crystal structure of the complex may be determined and used to identify specific sites of interaction between the antibody and its target.

[0077] An antibody according to the current invention may bind to the same epitope or domain of CXCR5 as the antibodies of the invention that are specifically disclosed herein. Analyses and assays that may be used for the purpose of such identification include assays assessing the competition for binding of CXCR5 between the antibody of interest and CXCR5 receptor, in biological activity assays as described in Examples 1-10 of WO 2019 / 108639, and in analysis of the crystal structure of the antibody.

[0078] An antibody of the invention may have the ability 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 antibodies described herein for binding to CXCR5, or to a suitable fragment or variant of CXCR5 that is bound by the antibodies disclosed herein.

[0079] That is, if a first antibody competes with a second antibody for binding to CXCR5, but it does not compete where the second antibody is first bound to CXCR5, it is deemed to “compete” with the second antibody (also referred to as unidirectional competition). Where an antibody competes with another antibody regardless of which antibody is first bound to CXCR5, 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 e.g. by using a Biacore™ system, ELISA assays or flow cytometry may be used to demonstrate competition / cross-competition. Such competition / cross-competition may suggest that the two antibodies bind to identical, overlapping or similar epitopes.

[0080] An antibody of the invention may therefore be identified by a method that comprises a binding assay which assesses whether or not a test antibody is able to compete / cross- compete with a reference antibody for a binding site on the target molecule. Methods for carrying out competitive binding assays are disclosed herein and / or are well known in the art. For example they may involve 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 may then be exposed to a test / second antibody and the extent to which the test antibody is able to displace the reference antibody of the invention from antibody / target complexes may be assessed. An alternative method may involve contacting a test antibody with a target molecule under conditions that allow for antibody binding, then adding a reference antibody of the invention that is capable of binding that target molecule and assessing the extent to which the reference antibody of the invention is able to displace the test antibody from antibody / target complexes or to simultaneously bind to the target (i.e. , non-competing antibody).

[0081] The ability of a test antibody to inhibit the binding of a reference antibody of the invention to the target demonstrates that the test antibody can compete with a 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 that is identified as competing with a reference antibody of the invention in such a method is also an antibody of the present invention. The fact that the test antibody can bind CXCR5 in the same region as a 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 may therefore mimic the action of the reference antibody and is, thus, an antibody of the invention. This can be confirmed by comparing the activity of CXCR5 in the presence of the test antibody with the activity of CXCR5 in the presence of the reference antibody under otherwise identical conditions, using an assay as more fully described elsewhere herein.

[0082] The reference antibody of the invention may be an antibody as described herein, such as 11G2, 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 described herein or any variant or portion thereof as described herein that retains the ability to bind to CXCR5.

[0083] As stated previously elsewhere herein, specific binding may be assessed with reference to binding of the antibody to a molecule that is not the target. This comparison may be made by comparing the ability of an antibody to bind to the target and to another molecule. This comparison may be made as described above in an assessment of KDor Kj. 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.

[0084] The other molecule used to determine specific binding may be unrelated in structure or function to the target. For example, the other molecule may be an unrelated material or accompanying material in the environment.

[0085] The other molecule used to determine specific binding may be another molecule involved in the same in vivo pathway as the target molecule, i.e., CXCR5. By ensuring that the antibody of the invention has specificity for CXCR5 over another such molecule, unwanted in vivo cross- reactivity may be avoided.

[0086] The antibody of the invention may retain the ability to bind to some molecules that are related to the target molecule.

[0087] Alternatively, the antibody of the invention may have specificity for a particular target molecule. For example, it may bind to one target molecule as described herein, but may not bind, or may bind with significantly reduced affinity to a different target molecule as described herein. For example, a full length mature human CXCR5 may be used as the target, but the antibody that binds to that target may be unable to bind to or may bind with lesser affinity to, e.g. other CXCR5 proteins from other species, such as other mammalian CXCR5. In some embodiments, the antibody binds to both human and mouse CXCR5.

[0088] A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a 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 a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% sequence identity with a native sequence Fc region and / or with an Fc region of a parent polypeptide, and most preferably, at least about 90% sequence identity therewith, more preferably, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.

[0089] As known in the art, a “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0090] The terms “IgG Fc region”, “Fc region”, “Fc domain” and “Fc”, as interchangeably used herein refer to the portion of an IgG molecule that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule. As used herein, the terms relate to the constant region of an antibody excluding the first constant region immunoglobulin domain and further relates to portions of that region. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains, or portions thereof. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cy2 and Cy3 (C gamma 2 and C gamma 3) and the hinge between Cy1 (C gamma 1) and Cy2 (C gamma 2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the Ell index of Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 as described in Kabat et al., 1991. Typically, the Fc domain comprises from about amino acid residue 236 to about 447 of the human I gG 1 constant domain. An exemplary human wild type IgG 1 Fc domain (including the CH1 and hinge) amino acid sequence is set forth in SEQ ID NO:13 and SEQ ID NO:14. Fc polypeptide may refer to this region in isolation, or this region in the context of an antibody, or an antigenbinding fragment thereof, or Fc fusion protein.

[0091] The heavy chain constant domain comprises the Fc region and further comprises the CH1 domain and hinge as well as the CH2 and CH3 (and, optionally, CH4 of IgA and IgE) domains of the IgG heavy chain.

[0092] A “functional Fc region” possesses at least one effector function of a native sequence Fc region. Exemplary “effector functions” include C1 q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; down-regulation of cell surface receptors (e.g. B cell receptor), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody variable domain or antigen-binding fragment thereof) and can be assessed using various assays known in the art for evaluating such antibody effector functions.

[0093] A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human lgG1 Fc region (non-A and A allotypes); native sequence human lgG2 Fc region; native sequence human lgG3 Fc region; and native sequence human lgG4 Fc region as well as naturally occurring variants thereof.

[0094] A "variant Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification.

[0095] “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcyR is a native human FcR. In some embodiments, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine- based activation motif (IT AM) in its cytoplasmic domain Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in 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 those to be identified in the future, are encompassed by the term “FcR” herein.

[0096] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et ah, J. Immunol. 117:587 (1976) and Kim et ah, J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et ah, Nature Biotechnology, 15(7):637- 640 (1997); Hinton et ah, J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al).

[0097] “Effector functions” refer to biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibodydependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

[0098] "Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcyRIII and perform ADCC effector function(s). Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils. The effector cells may be isolated from a native source, e.g., from blood.

[0099] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Pat. Nos. 5,500,362 or 5,821 ,337 or U.S. Pat. No. 6,737,056 (Presta), may be performed. Useful effector cells for such assays include PBMC and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed 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, e.g., in U.S. Pat. No. 7,923,538, and U.S. Pat. No. 7,994,290.

[0100] An antibody having an "enhanced ADCC activity" refers to an antibody that is more effective at mediating ADCC in vitro or in vivo compared to the parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect, and when the amounts of such antibody and parent antibody used in the assay are essentially the same. In some embodiments, the antibody and the 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 the in vitro ADCC assay as herein disclosed, but other assays or methods for determining ADCC activity, e.g. in an animal model etc., are contemplated. In some embodiments, an antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA. In some embodiments, an antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA (V158). In some embodiments, an antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA (F158).

[0101] An antibody with “altered” FcR binding affinity or ADCC activity is one which has either enhanced or diminished FcR binding activity and / or ADCC activity compared to a parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect. An antibody that “displays increased binding” to an FcR binds at least one FcR with better affinity than the parent antibody. An antibody that “displays decreased binding” to an FcR, binds at least one FcR with lower affinity than a parent antibody. Such antibodies that display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0-20 percent binding to the FcR compared to a native sequence IgG Fc region.

[0102] "Enhanced affinity for Fc gamma RIIIA" refers to an antibody that has greater affinity for Fc gamma RIIIA (also referred to, in some instances, as CD 16a) than a parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is afucosylated while the parent antibody is fucosylated. Any suitable method for determining affinity for Fc gamma RIIIA may be used. In some embodiments, affinity for Fc gamma RIIIA is determined by a method described herein. In some embodiments, an antibody with enhanced affinity for Fc gamma RIIIA has enhanced ADCC activity. In some embodiments, an antibody with enhanced affinity for Fc gamma RIIIA has enhanced affinity for Fc gamma Rl IIA(V158). In some embodiments, an antibody with enhanced affinity for Fc gamma RIIIA has enhanced affinity for Fc gamma RII IA(F158).

[0103] L-fucose, also referred to as 6-deoxy-L-galactose, is a monosaccharide that is a component of some N- and O-linked glycans and glycolipids in animals. See Becker and Lowe, Glycobiology 13:41 R-51 R (2003). Fucose is typically added as a terminal modification to glycans, including glycans attached to blood group antigens, selectins and antibodies. Fucose can be attached to glycans via a(1 ,2)-, a(1 ,3)-, a(1,4)- and a(1 ,6)-linkages by specific fucosyltransferases. A(1 ,2)-fucose linkages are typically associated with the H-blood group antigens. A(1,3)- and a(1 ,4)-fucose linkages are associated with modification of LewisX antigens. A(1,6)-fucose linkages are associated with N-linked GIcNAc molecules, such as those on antibodies.

[0104] The carbohydrate moieties of the present invention will be described with reference to commonly used nomenclature for the description of oligosaccharides. A review of carbohydrate chemistry which uses this nomenclature is found in Hubbard and Ivatt (1981) Ann. Rev. Biochem. 50:555-583. This nomenclature includes, for instance, Man, which represents mannose; GIcNAc, which represents 2-N-acetylglucosamine; Gal which represents galactose; Fuc for fucose; and Glc, which represents glucose. Sialic acids are described by the shorthand notation 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).

[0105] The carbohydrate structures of the present invention occur on the protein expressed as N-linked oligosaccharides. “N-linked glycosylation” refers to the attachment of the carbohydrate moiety via GIcNAc to an asparagine residue in a polypeptide chain. The N-linked carbohydrates all contain a common Man 1-6(Man1-3)Manpi-4GlcNAcpi-4GlcNAcp-R core structure. Therefore, in the core structure described, R represents an asparagine residue of the produced glycoprotein. The sequence of the protein produced will contain an asparagine-X- serine, asparagine-X-threonine, and asparagine-X-cysteine, wherein X is any amino acid except proline (Asn-Xaa-Ser / Thr). “O-linked” carbohydrates, by contrast are characterized by a common core structure, which is the GalNAc attached to the hydroxyl group of a threonine or serine but no consensus sequence is required. Of the N-linked carbohydrates the most important are the “complex” N-linked carbohydrates such as the “bi-antennary” structures described herein.

[0106] The skilled artisan will recognize that the glycoprotein immunoglobulin G (IgG) is associated with three types of complex biantennary structures containing zero, one or two galactose residues (Wormland et al., 1997, Biochemistry 36:1370-1380) commonly known as GO, G1 and G2, respectively. With respect to human antibody molecules of the IgG class each has an N-linked oligosaccharide attached at the amide side chain of Asn 297 of the p-4 bend of the inner face 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 moiety attached at Asn 297 of the IgG CH2 domain is of the complex biantennary type having the identified hexasaccharide core structure and variable outer sugar residues (see Jefferis et al., 1997, supra; Wyss and Wagner, 1996, Current Opinions in Biotech. 7:409-416). The core structure (GlcNAc2Man3GlcNAc) is typical of biantennary oligosaccharides and is represented schematically in FIG. 1.

[0107] Since each core structure may have a bisecting N-acetylglucoseamine, core fucose and either galactose or sialic acid outer saccharides, there are a total of 36 structurally unique oligosaccharides which may occupy the Asn 297 site (Jefferis and Lund, supra). It will also be recognized that within a particular CH2 domain, glycosylation at Asn 297 may be asymmetric owing to different oligosaccharide chains attached at either Asn 297 residue within the two chain Fc domain. For example, while the heavy chain synthesized within a single antibodysecreting cell may be homogeneous in its amino acid sequence, it is generally differentially glycosylated resulting in a large number of structurally unique Ig glycoforms.

[0108] The major types of complex oligosaccharide structures, also referred to as “glycoforms,” found in the CH2 domain of the IgG are depicted in International Patent Publication No. WO 99 / 22764 at page 7.

[0109] According to the present invention GO refers to a biantennary structure wherein no terminal sialic acids (NeuAcs) or Gals are present, G1 refers to a biantennary structure having one Gal and no NeuAcs and G2 refers to a biantennary structure with two terminal Gals and no NeuAcs. See, e.g., FIGs. 2A-2G of WO 2019 / 108639, depicting exemplary structures of GO, G1 , G-1 and G2.

[0110] “Afucosylated” antibody or an antibody “lacking fucose” refers to an IgGI or lgG3 isotype antibody that lacks fucose in its constant region glycosylation. Glycosylation of human IgGI or lgG3 occurs at Asn297 as core fucosylated biantennary complex oligosaccharide glycosylation terminated with up to 2 Gal residues. In some embodiments, an afucosylated antibody lacks fucose at Asn297. These structures are designated as GO, G1 (a 1 ,6 or a 1 ,3) or G2 glycan residues, depending on the amount of terminal Gal residues. See, e.g., Raju, T. S., BioProcess Int. 1 : 44-53 (2003). CHO type glycosylation of antibody Fc is described, e.g., in Routier, F. FL, Glycoconjugate J. 14: 201-207 (1997). Various antibody glycoforms are shown in FIGs. 2A-2G of WO 2019 / 108639.

[0111] In some embodiments, the “fucosyl” or “afucosylated”, as used interchangeably herein, antibody refers to an antibody that has been glycoengineered to lack core fucose. Antibodies with reduced fucose content in glycan moieties have increased affinity to FcyRllla (CD16), and as a result, possess enhanced activity-dependent cellular cytotoxicity (ADCC) activity. Afucosyl antibodies can be produced using the Potelligent CHOK1SV cell line (Lonza Biologies), which lacks both alleles of the gene responsible for fucose addition (a1 ,6- fucosyltransferase). Afucosyl or reduced fucose antibodies can also be generated by modifying the oligosaccharide biosynthesis activities in various ways. For example, overexpression of N- acetylglucosamine-transferase III (GnTIII) in the Golgi apparatus of the production cell line generates bisected 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 generation of afucosylated lgG1 glycoforms and resulting enhanced ADCC activity. Fucosylation can also be decreased in cell culture by use of sugar analogs, such as, but not limited to, fucose analogs as described in WO 2012 / 019165. Thus, afucosylated, or reduced fucose, antibodies can be produced using a wide variety of methods well-known in the art.

[0112] In some embodiments, an afucosylated antibody has enhanced affinity for Fc gamma RII IA. In some embodiments, an afucosylated antibody has enhanced affinity for Fc gamma RII IA(V158). In some embodiments, an afucosylated antibody has enhanced affinity for Fc gamma RII IA(F158).

[0113] “Glycoform” refers to a complex oligosaccharide structure comprising linkages of various carbohydrate units. Such structures are described in, e.g., Essentials of Glycobiology Varki et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999), which also provides a review of standard glycobiology nomenclature. Such glycoforms include, but are not limited to, G2, G1 , GO, G-1 , and G-2 (see, e.g., International Patent Publication No. WO 99 / 22764).

[0114] “Glycosylation pattern” is defined as the pattern of carbohydrate units that are covalently attached to a protein (e.g., the glycoform) as well as to the site(s) to which the glycoform(s) are covalently attached to the peptide backbone of a protein, more specifically to an immunoglobulin protein.

[0115] In some embodiments, at least 85 percent of a batch of antibodies recombinantly expressed in non glycomodified CHO host cells are fucosylated at Asn297. When referring to a composition comprising a plurality of antibodies, the antibodies are considered to be afucosylated if less than about 5 percent of the antibodies in the composition comprise fucose at at least one Asn297. Even more preferably, the level of afucosylation is about 100%, that is, no fucose is detected on either heavy chain Asn297 glycoform using standard methods for measuring fucosylation of an antibody. Methods of measuring fucose include any methods known in the art, including the methods described herein. In some embodiments, fucose is undetectable in a composition comprising a plurality of afucosylated antibodies. In some embodiments, an afucosylated antibody has enhanced ADCC activity.

[0116] “Complement dependent 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 the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass), which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed. Antibodies with altered Fc region amino acid sequences and increased or decreased Clq binding capability are described, e.g., in U.S. Pat. No. 6, 194,551 B I, U.S. Pat. No. 7,923,538, U.S. Pat. No. 7,994,290 and WO 1999 / 51642. See also, e.g., Idusogie et al, J.

[0117] 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 or nucleic acids that occurs naturally within a certain population (e.g., human, mouse, rats, cell, etc.).

[0118] “C-X-C chemokine receptor type 5” or “CXCR5,”used interchangeably herein, also referred to in the art as “CD185” and “Burkitt lymphoma receptor 1 (BLR1),” is a G-protein- coupled receptor expressed on certain cells. The term CXCR5 includes CXCR5 homologs and orthologs, including human, cynomolgus monkey, rat, rabbit, and mouse, among others. As used herein, “CXCR5” refers to a mammalian CXCR5, such as human, rat or mouse, as well as non-human primate, bovine, ovine, or porcine CXCR5. A nonlimiting exemplary example of CXCR5 is human (see, e.g., Genbank Accession Number P60568, SEQ ID NO:15). The term “CXCR5” also encompasses fragments, variants, isoforms, and other homologs of such CXCR5 molecules. Variant CXCR5 molecules will generally be characterized by having the same type of activity as naturally occurring CXCR5, such as the ability to bind CXCR5 receptor, the ability to induce receptor-mediated activity, and the ability to bind, or not, the antibody, or antigenbinding fragment thereof, of the invention.

[0119] The 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 surface accessible residues of CXCR5. Where the CXCR5 comprises a homomultimeric form of CXCR5, the target 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 surface accessible residues of a first subunit of CXCR5, 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 surface accessible residues of a second subunit of CXCR5. The target molecule may comprise a known epitope from CXCR5.

[0120] As outlined elsewhere herein, certain positions of the antibody molecule can be altered. By “position” as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index and Kabat index can be used to number amino acid residues of an antibody. For example, position 297 is a position in the human antibody lgG1. Corresponding positions are determined as outlined above, generally through alignment with other parent sequences.

[0121] By "residue" as used herein is meant a position in a protein and its associated amino acid identity. For example, Asparagine 297 (also referred to as Asn297, also referred to as N297) is a residue in the human antibody lgG1.

[0122] The term “Tfh cell” or “Tfh” or “bona fide Tfh” or “germinal center Tfh cell” or “GO Tfh cell,” as used interchangeably herein, refers to follicular helper T cells found within the germinal center (GO), which is a structure consisting of GO Tfh cells, GO B cells, follicular dendritic cells (FDCs), 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 instructive signals to B cells to guide isotype switching, somatic hypermutations, and rapid cellular division to seed germinal centers.

[0123] The terms “Tfh-like cells,” “circulating Tfh cells” and “cTfh”, used interchangeably herein, refer to Tfh cells that have exited 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. In comparison to germinal center Tfh cells (i.e. , “bona fide 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 maintain the ability to stimulate Ab production and Ig class switching in B cells in vitro upon reactivation with cognate antigens.

[0124] As known in the art, “polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5’ and 3’ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are 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 abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”), (O)NR2(“amidate”), P(O)R, P(O)OR’, CO or CH2(“formacetal”), in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0125] As used herein, “vector” means a construct, which is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0126] A “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected and / or transformed in vivo with a polynucleotide of this invention.

[0127] Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells.

[0128] Any host cell susceptible to cell culture, and to expression of protein or polypeptides, may be utilized in accordance with the present invention. In certain embodiments, the host cell is mammalian. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). Nonlimiting exemplary mammalian cells include, but are not limited to, NSO cells, HEK 293 and Chinese hamster ovary (CHO) cells, and their derivatives, such as 293-6E and CHO DG44 cells, CHO DXB11, and Potelligent CHOK1SV cells (BioWa / Lonza, Allendale, NJ). Mammalian host cells also include, but are not limited to, human cervical carcinoma cells (HeLa, ATCC CCL 2), baby hamster kidney (BHK, ATCC CCL 10) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e.g., Hep G2). Other non-limiting examples of mammalian cells that may be used in accordance with the present invention include human retinoblasts (PER.C6; CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney 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); canine 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 (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; a human hepatoma line (Hep G2); and numerous myeloma cell lines, including, but not limited to, BALB / c mouse myeloma line (NS0 / 1 , ECACC No: 85110503), NS0 cells and Sp2 / 0 cells.

[0129] Additionally, any number of commercially and non-commercially available cell lines that express polypeptides or proteins may be utilized in accordance with the present invention. One skilled in the art will appreciate that different cell lines might have different nutrition requirements and / or might require different culture conditions for optimal growth and polypeptide or protein expression, and will be able to modify conditions as needed.

[0130] The invention includes any eukaryotic expression system known in the art or disclosed herein for production of proteins of interest, such as expression in an insect cell system, a yeast expression system, or a mammalian cell system, such as, but not limited to, CHO cells.

[0131] As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: improved survival rate (reduced mortality), reduction in inflammatory response to the disease, reduction in the amount of tissue fibrosis, improvement in the appearance of the disease lesions, limitation of the pathological lesions to focal sites, decreased extent of damage from the disease, decreased duration of the disease, and / or reduction in the number, extent, or duration of symptoms related to the disease. In some embodiments, beneficial or desired clinical results include: an increase in absolute platelet count, an increase in platelet count over time, a decrease in absolute B cell count, a decrease in B cell count over time, a decrease in absolute cTfh cell count, a decrease in cTfh cell count over time, or a combination thereof, when a subject has ITP and is administered an anti-CXCR5 antibody.

[0132] The term “treatment” includes the administration of the compounds or agents of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[0133] “Ameliorating” means a lessening or improvement of one or more symptoms as compared to not administering an anti-CXCR5 antibody. “Ameliorating” also includes shortening or reduction in duration of a symptom.

[0134] As used herein, an “effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired results. In more specific aspects, an effective amount prevents, alleviates or ameliorates symptoms of disease or infection, and / or prolongs the survival of the subject being treated. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms of a CXCR5-mediated disease, disorder or condition, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and / or delaying the progression of the disease of patients. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. In some embodiments, an effective dosage of an anti- CXCR5 antibody results in an increase in absolute platelet count, an increase in platelet count over time, a decrease in absolute B cell count, a decrease in B cell count over time, a decrease in absolute cTfh cell count, a decrease in cTfh cell count over time, or a combination thereof, when a subject has ITP and is administered an anti-CXCR5 antibody. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective dosage” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved. An “individual” or a “subject” is a mammal, more preferably, a human. Mammals also include, but are not limited to, farm animals (e.g., 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 for a disease, disorder or condition mediated by or associated with CXCR5 binding to its receptor and signaling mediated thereby. In certain embodiments, the subject has an autoimmune disease, disorder or condition, such as type 1 diabetes. In certain embodiments, the subject is in need of immunosuppression therapy. In some embodiments, a subject has immune thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ANCA-vasculitis, pemphigus vulgaris, myasthenia gravis (MG) or chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In some embodiments, a subject is a patient.

[0135] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical acceptable excipient" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions comprising 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).

[0136] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0137] II. ANTI-CXCR5 ANTIBODIES

[0138] The present invention relates to antibodies that bind to CXCR5 and in particular to those disclosed in WO 2019 / 108639 (incorporated herein in its entirety). Preferably, the antibodies specifically bind to CXCR5, i.e. , they bind to CXCR5 but they do not detectably bind, or bind at a lower affinity, to other molecules. The invention further relates to anti-CXCR5 antibodies that exhibit an altered effector function. In some embodiments, the altered effector function is increased ADCC. In some embodiments, the antibodies lack, or contain detectably decreased levels of, fucose (i.e., they are afucosylated). The invention also relates to compositions comprising such antibodies as well as uses for such antibodies, including therapeutic and pharmaceutical uses. In some embodiments, the disclosure provides any of the following, or compositions (including pharmaceutical compositions) comprising, an antibody having a light chain sequence, or a fragment thereof, and a heavy chain, or a fragment thereof, derived from 11G2. In some embodiments, the anti-CXCR5 antibody is a monoclonal antibody.

[0139] The CXCR5 antibodies of the invention may be made by any method known in the art. General techniques for production of human and mouse antibodies are known in the art and / or are described herein.

[0140] In some embodiments, an antibody, or antigen-binding fragment thereof that binds CXCR5, comprises a CDR-L1 , a CDR-L2, and a CDR-L3 as set forth in the amino acid sequence of SEQ ID NO:1.

[0141] In some embodiments, an antibody, or antigen-binding fragment thereof that binds CXCR5, comprises a CDR-H 1 , a CDR-H2, and a CDR-H3 as set forth in the amino acid sequence of SEQ ID NO:5.

[0142] In some embodiments, an antibody, or antigen-binding fragment thereof that binds CXCR5, comprises a CDR-L1 , a CDR-L2, a CDR-L3 as set forth in the amino acid sequence of SEQ ID NO:1, and a CDR-H1 , a CDR-H2, and a CDR-H3 as set forth in the amino acid sequence of SEQ ID NO:5.

[0143] In some embodiments, an antibody, or antigen-binding fragment thereof that binds CXCR5, comprises a CDR-L1 , a CDR-L2, and a CDR-L3 as set forth in the amino acid sequence encoded by the insert of the plasmid deposited with the ATCC having the Accession number PTA-124324.

[0144] In some embodiments, an antibody, or antigen-binding fragment thereof that binds CXCR5, comprises a CDR-H1 , a CDR-H2, and a CDR-H3 as set forth in the amino acid sequence encoded by the insert of the plasmid deposited with the ATCC having Accession number PTA-124323.

[0145] In some embodiments, an antibody, or antigen-binding fragment thereof that binds CXCR5, comprises a CDR-L1 , a CDR-L2, and a CDR-L3 amino acid sequence encoded by the insert of the plasmid deposited with the ATCC having the Accession number PTA-124324, and a CDR-H1 , a CDR-H2, and a CDR-H3 amino acid sequence encoded by the insert of the plasmid deposited with the ATCC having Accession number PTA-124323.

[0146] In some embodiments, an antibody, or antigen-binding fragment thereof that binds CXCR5, comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, an antibody, or antigen-binding fragment thereof that binds CXCR5, comprises the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences as set forth in the sequence of SEQ ID NO:9.

[0147] In some embodiments, an antibody, or antigen-binding fragment thereof that binds CXCR5, comprises the CDR-H1 , CDR-H2, and CDR-H3 amino acid sequences as set forth in the sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0148] In some embodiments, an antibody, or antigen-binding fragment thereof that binds CXCR5, comprises the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences as set forth in the sequence of SEQ ID NO:9 and comprises the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences as set forth in the sequence of SEQ ID NQ:10 or SEQ ID NO:11.

[0149] In some embodiments, antibody, or antigen-binding fragment thereof that binds CXCR5, comprises a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, a VH comprises 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. In some embodiments, a VH comprises the amino acid sequence of SEQ ID NO:5.

[0150] In some embodiments, an antibody or antigen-binding fragment that binds CXCR5 comprises a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1. In some embodiments, a VL comprises an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to the amino acid sequence SEQ ID NO:1. In some embodiments, a VL may comprise the amino acid sequence of SEQ ID NO:1.

[0151] In some embodiments, an antibody or antigen-binding fragment that binds CXCR5 comprises a VL comprising or consisting of an amino acid sequence of SEQ ID NO:1 and a VH comprising or consisting of the amino acid sequence of SEQ ID NO:5.

[0152] In some embodiments, an afucosylated antibody or antigen-binding fragment thereof that binds CXCR5 comprises a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO:5, and further comprising an IgG 1 constant domain (SEQ ID NO:13 or SEQ ID NO:14).

[0153] In some embodiments, an antibody variant comprises 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 to the full length heavy chain. In a further embodiment, a variant shares at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a full length heavy chain, and wherein the antibody or antigen-binding fragment specifically binds CXCR5. In some embodiments, an afucosylated antibody or antigen-binding fragment thereof that binds CXCR5 comprises a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO:1 , and further comprising a light chain constant domain (SEQ ID NO: 12). In some embodiments, an afucosylated antibody light chain constant domain can be selected from a CK or CA constant region, for example the CK constant region of SEQ ID NO:12.

[0154] In some embodiments, an antibody variant comprises 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 to the full length light chain. In a further embodiment, a variant shares at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a full length light chain, and wherein the antibody or antigen-binding fragment specifically binds CXCR5.

[0155] In some embodiments, an antibody or antigen-binding fragment that binds CXCR5 comprises a heavy chain (HC) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to the amino acid sequence of SEQ ID NQ:10 or SEQ ID NO:11. In some embodiments, a HC comprises or consists of the amino acid sequence of SEQ ID NQ:10 or SEQ ID NO:11. Preferably, the antibody is afucosylated.

[0156] In some embodiments an antibody or antigen-binding fragment that binds CXCR5 comprises a light (LC) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:9. In some embodiments, a LC comprises or consists of the amino acid sequence of SEQ ID NO:9. Preferably, the antibody is afucosylated.

[0157] In some embodiments, an antibody or antigen-binding fragment that binds CXCR5 comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NQ:10 or SEQ ID NO:11 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:9, and optionally, the antibody is afucosylated.

[0158] Preferably, an antibody, or antigen-binding fragment thereof, of the invention is afucosylated. Even more preferably, the antibody, or antigen-binding fragment thereof, is afucosylated and exhibits increased ADCC effector function compared to an otherwise identical antibody that is fucosylated.

[0159] Epitope Mapping

[0160] Also provided by the invention is an antibody, or antigen-binding fragment thereof, that binds to the same human CXCR5 epitope as an antibody, or antigen-binding fragment thereof, described herein. For example, antibody competition assays (and overlapping epitope analysis) can be assessed by SPR, flow cytometry, and any other assay known in the art. In one aspect, the invention encompasses an antibody, or antigen-binding fragment thereof, that binds human CXCR5, and cynomolgus CXCR5, but does not bind mouse CXCR5.

[0161] The invention also includes an antibody, or antigen-binding fragment thereof, that binds the N-terminus region (“N”) of human CXCR5. The N-terminus of CXCR5 comprises amino acid residue numbers 1-51 based on the numbering of the amino acid sequence of SEQ ID NO:15.

[0162] The invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 at an epitope comprising leucine (L) at amino acid residue position number 11 according to the numbering of the amino acid sequence of SEQ ID NO: 15.

[0163] In one aspect, the invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 at an epitope comprising aspartate (D) at amino acid residue position number 22 according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0164] In one aspect, the invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 at an epitope comprising leucine at amino acid residue position number 11 and aspartate at amino acid residue position number 22 according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0165] The invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 where the amino acid residue at position number 11 is leucine according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0166] The invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 where the amino acid residue at position number 11 is leucine, but does not bind CXCR5 wherein the amino acid residue at position number 11 is threonine, according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0167] In one aspect, the invention includes an antibody, or antigen-binding fragment thereof, that specifically binds wild type human CXCR5 but does not bind where amino acid residue position number 11 , according to the numbering of the amino acid sequence of SEQ ID NO:15, is not leucine.

[0168] The invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 where the amino acid residue at position number 22 is aspartate according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0169] In one aspect, the invention includes an antibody, or antigen-binding fragment thereof, that specifically binds wild type human CXCR5 but does not bind where the amino acid residue at position number 22 is not aspartate according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0170] In one aspect, the invention includes an antibody, or antigen-binding fragment thereof, that specifically binds wild type human CXCR5 but does not bind where amino acid residue position number 22 is alanine, according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0171] The invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 where the amino acid residue at position number 11 is leucine and the amino acid residue at position number 22 is aspartate, according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0172] In one aspect, the invention includes an antibody, or antigen-binding fragment thereof, that specifically binds human CXCR5 but does not bind CXCR5 where the amino acid residue at position number 11 is not leucine and the amino acid residue at position number 22 is not aspartate, according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0173] In one aspect, the invention includes an antibody, or antigen-binding fragment thereof, that specifically binds human CXCR5 but does not bind where the amino acid residue at position number 11 is threonine and the amino acid residue at position number 22 is alanine, according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0174] The skilled artisan would appreciate, armed with the teachings of the present invention, that the amino acid residue at position number 11 and / or at position number 22, according to the numbering of the amino acid sequence of SEQ ID NO:15, are critical for the binding of an antibody of the invention. More specifically, these amino acid residues are crucial for binding to CXCR5 by antibody 11G2, or antigen-binding fragment thereof. Thus, the skilled artisan would understand that the invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 wherein 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. Certain substitutions of the amino acid residue at position 11 may preserve binding, but not substitution of that (leucine) amino acid residue with threonine. Similarly, certain substitutions or deletion of the amino acid residue at position 22 may preserve binding, but not substitution of aspartate at that amino acid residue number with alanine. Therefore, a feature of the 11G2 antibody, or antigen-binding fragment thereof, or an antibody that competes for binding therewith, is that the antibody binds human CXCR5 wherein leucine 11 and aspartate 22 are present according to the numbering of the amino acid sequence of SEQ ID NO: 15, but the antibody does not bind where leucine 11 is replaced with threonine, or another amino acid residue, and where the antibody does not bind where aspartate 22 is replaced with alanine, or another amino acid residue. Testing for loss of binding after amino acid substitution at amino acid residue position number 11 and / or number 22 can be performed using a wide variety of methods well-known in the art, including binding analysis using point mutation polypeptides as per the methods exemplified in WO 2019 / 108639. Based upon the teachings provided herein, one skilled in the art would appreciate that an antibody of the invention can compete with, e.g., 11G2, and yet not comprise an epitope comprising leucine at amino acid residue number 11 and / or aspartate at amino acid residue number 22 according to the numbering of the amino acid sequence of SEQ ID NO: 15. That is, an antibody can compete with an antibody of invention for binding to CXCR5, but the competing antibody binding is not affected where the leucine at amino acid residue number 11 or the aspartate at amino acid residue number 22 are substituted with a different amino acid (e.g., threonine for leucine and / or alanine for aspartate). Thus, the antibody of the invention competes for binding to CXCR5 and also does not bind CXCR5 where amino acid residue number 11 is not leucine, and more specifically, it is threonine, and / or where amino acid residue number 22 is not aspartate, and more specifically, it is alanine. As stated previously elsewhere herein, production of mutant CXCR5 proteins and assays for assessing antibody competition binding are well-known in the art, including those methods described in WO 2019 / 108639 Thus, based upon the teachings provided herein, the skilled artisan would be readily capable of identifying antibodies that bind CXCR5, compete for binding with an antibody of the invention, and lose the ability to bind CXCR5 muteins where certain amino acids have been substituted, e.g., leucine 11 , aspartate 22, or both, all according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0175] In another aspect, the invention includes an antibody, or antigen-binding fragment thereof, that competes for binding with antibody 11G2, or antigen-binding fragment thereof, where the antibody does not bind CXCR5 where amino acid residue number 11 is not leucine, according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0176] In another aspect, the invention includes an antibody, or antigen-binding fragment thereof, that competes for binding with antibody 11G2, or antigen-binding fragment thereof, where the antibody does not bind CXCR5 where amino acid residue number 22 is not aspartate, according to the numbering of the amino acid sequence of SEQ ID NO: 15.

[0177] In another aspect, the invention includes an antibody, or antigen-binding fragment thereof, that competes for binding with antibody 11G2, or antigen-binding fragment thereof, where the antibody does not bind CXCR5 where amino acid residue number 11 is not leucine and amino acid residue number 22 is not aspartate, according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0178] In another aspect, the invention includes an antibody, or antigen-binding fragment thereof, that competes for binding with antibody 11G2, or antigen-binding fragment thereof, where the antibody does not bind CXCR5 where amino acid residue number 11 is threonine, according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0179] In another aspect, the invention includes an antibody, or antigen-binding fragment thereof, that competes for binding with antibody 11G2, or antigen-binding fragment thereof, where the antibody does not bind CXCR5 where amino acid residue number 22 is alanine, according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0180] In another aspect, the invention includes an antibody, or antigen-binding fragment thereof, that competes for binding with antibody 11G2, or antigen-binding fragment thereof, where the antibody does not bind CXCR5 where amino acid residue number 11 is threonine and where amino acid residue number 22 is alanine, according to the numbering of the amino acid sequence of SEQ ID NO:15.

[0181] Biological Activity of anti-CXCR5 Antibodies

[0182] In addition to binding an epitope on CXCR5, the antibody, or antigen-binding fragment thereof, of the invention can mediate a biological activity. That is, the invention includes an isolated antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 and mediates at least one detectable activity selected from the following: (a) binds CXCR5+ cells with high apparent affinity, but does not bind cells expressing CXCR5 mouse, rat or rabbit orthologs; (b) antagonizes CXCL13 inhibition of cAMP release triggered by forskolin; (c) triggers ADCC of CXCR5-expressing cells in human donor and cynomolgus PBMCs and human donor TMCs; (d) binds human CXCR5 but does not bind human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1 , CXCR1 , CXCR2, CXCR3, CXCR4, CXCR6, CXCR7, or XCRI ; I depletes B cells in the peripheral blood and / or secondary lymphoid organs (i.e., lymph nodes, spleen, Peyer’s patches, and mucosa-associated lymphoid tissue); (f) depletes Tfh-like cells in the peripheral blood; (g) depletes bona fide Tfh cells in the secondary lymphoid organs; and (h) impairs a humoral immune memory response.

[0183] In one aspect, the invention includes an antibody, or binds CXCR5+ cells with high apparent affinity, but does not bind cells expressing CXCR5 mouse, rat or rabbit orthologs. Apparent affinity binding can be assessed using flow cytometry to detect antibody binding to cells expressing the target protein (e.g., CXCR5). The cells can be transiently or stably transfected with a nucleic acid encoding CXCR5. Alternatively, the cells can be cells that naturally express CXCR5 on their surface. Regardless of the sources of CXCR5+ cells, the binding of the antibody to the cells can be readily assessed using a variety of art-recognized methods. The antibody, or antigen-binding fragment thereof, bind human CXCR5, or cyno CXCR5, but do not detectably bind, or bind to a much lesser extent, mouse, rat or rabbit CXCR5.

[0184] The invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 and antagonizes an activity mediated by CXCL13 binding to CXCR5. There are many assays known in the art to determine the inhibition of an activity mediated by CXCR5-CXCL13 signaling. One such assay is a cAMP reporter assay. In such an assay, forskolin induces cAMP production which is inhibited by CXCR5-CXCL13 signaling in a cell stably expressing CXCR5. The ability of the anti-CXCR5 antibody to bind CXCR5 and antagonize the effect of CXCR5-CXCL13 signaling is therefore 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 EC50 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, about 900 pM, about 950 pM, about 960 pM, about 970 pM, about 980 pM, about 990 pm, or about 1000 pM. More preferably, the antibody, or antigenbinding fragment thereof, inhibits CXCL13 inhibition of cAMP triggered by forskolin with an EC50 of about 961 pM.

[0185] The invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 and that triggers ADCC of CXCR5-expressing cells in human donor and cynomolgus PBMCs and human donor tonsillar mononuclear cells (TMCs). Many ADCC assays can be used to assess the ADCC activity of an antibody. The invention includes an antibody, or antigen-binding fragment thereof, that exhibits an ADCC activity on human B cells, human Tfh-like cells, human Tfh cells and cynomolgus monkey B cells with an EC50 of about 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 EC50 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 Tfh cells, and about 15.3 ± 11.7 pM against cyno B cells. Even more preferably, the antibody, or antigen-binding fragment thereof, is afucosylated.

[0186] The invention encompasses an antibody, or antigen-binding fragment thereof, that binds human CXCR5 but does not detectably bind human proteins CCR1 , CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1 , CXCR3R1 , CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7, or XCRI .

[0187] The invention encompasses an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 and exhibits dose-dependent depletion of B cells in the peripheral blood. The depletion can be permanent, or more preferably, the depletion is transient and / or reversible. In one aspect, the antibody dose sufficient to mediate B cell depletion can range from 0.001 to about 0.2 mg, wherein 0.0001 mg / kg can mediate a depletion of B cells and Tfh- like cells in the peripheral blood of cynomolgus monkeys of about 50% compared with the percentage of B cells or Tfh-like cells in the peripheral blood where the antibody is not administered. In addition, the antibody can mediate maximal depletion of B cells and Tfh-like cells in the peripheral blood of a cynomolgus monkey administered at a dose of less than 5 mg / kg administered intravenously (IV). Preferably, partial to full recovery of B cells, Tfh-like and Tfh cells occurs following administration of the antibody.

[0188] The invention includes an antibody, or antigen-binding fragment thereof, that specifically binds CXCR5 and impairs humoral immune memory response. In one aspect the humoral memory response is assessed using a vaccine recall assay. That is the antibody is administered to a subject that has been vaccinated previously with an antigen, e.g., tetanus toxoid (TT). The subject is given a second administration of the antigen and the immune response, e.g., IgM and IgG titers, is compared with the immune response in an otherwise identical subject to which the antibody is not administered. The skilled artisan would appreciate once provided the teachings disclosed herein, that many assays to determine a humoral memory response can be used to assess the ability of the antibody, or antigen-binding fragment thereof, to impair a humoral memory response. Further, the skilled artisan would appreciate that the ability to impair a humoral memory response is a desirable characteristic for an antibody, or antigen-binding fragment thereof, to be used as a therapeutic to treat or prevent an immune disease in a subject in need thereof.

[0189] The invention encompasses an antibody, or antigen-binding fragment thereof, that exhibits at least one, preferably, two, even more preferably, three, even more preferably, four, yet more preferably, five, even more preferably, six, more preferably, seven, and even more preferably, all of the above discussed biological activities.

[0190] III. AFUCOSYLATED ANTI-CXCR5 ANTIBODIES

[0191] In one embodiment, antibodies are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region (i.e. , afucosylated antibodies). For example, the amount of fucose in a composition comprising a plurality of such antibodies may 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 comprising a plurality of such antibodies comprises at least 80 percent, more preferably, at least 85 percent, yet more preferably, at least 90 percent, even more preferably, at least 95 percent afucosylated antibodies, yet 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; that is, fucose is not detected at Asn297 using an art-recognized method for detecting fucose in an antibody. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures). In some embodiments, the level of fucosylation is no more than 0.5%, which is based on the limit of quantification (LOQ) for the test method. Thus, in some embodiments, the level of afucosylation is greater than or equal to 99.5%. The N-linked oligosaccharide profile method can be used to determine the level of fucosylation, sialylation, mannosylation, and terminal glactosylation in a sample. The N-linked Oligosaccharide method can be used to evaluate N-linked glycans. Briefly, N-linked glycans are enzymatically released from the protein with peptide-N-glycosidase F. The glycans are then derivatized by a fluorescent agent and analyzed using hydrophilic interaction liquid chromatography and fluorescence detection. The chromatographic profile is then compared to that of the reference material. This method and many other methods are known in the art for assessing fucosylation of an antibody, and can be used to determine the level of fucosylation present in the antibody of the present invention.

[0192] Non-limiting exemplary methods of detecting fucose in an antibody include MALDI- TOF mass spectrometry (see, e.g., WO 2008 / 077546), HPLC measurement of released fluorescently labeled oligosaccharides (see, e.g., Schneider et al, “N-Glycan analysis of monoclonal antibodies and other glycoproteins using LIHPLC 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 measurement of released fluorescently labeled oligosaccharides (see, e.g., Ma et al., Anal. Chem., 71: 5185-5192 (1999)), and HPLC with pulsed amperometric detection to measure monosaccharide composition (see, e.g., Hardy, et al, Analytical Biochem., 170: 54-62 (1988)). Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about plus or minus 3 amino acids upstream or downstream of position 297, i.e. , between positions 294 and 300, due to minor sequence variations in antibodies. In a CXCR5 antibody described herein, Asn297 is found in the sequence QYNST, and is in bold and underlined in Table 4 (e.g., SEQ ID NO:13 or SEQ ID NO:14 of wild type human lgG1 Fc domain).

[0193] Fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "afucosylated" or "fucose-deficient" antibodies include: US 2003 / 0157108; WO 2000 / 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; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; W02005 / 053742; W02002 / 031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing afucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 Al, Presta, L; and WO 2004 / 056312, Adams et al., especially at Example 11), and knockout cell lines, such as cell lines lacking a functional alpha- 1 ,6-fucosyltransferase gene, FLIT8, e.g., knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng, 94(4):680-688 (2006); and W02003 / 085107).

[0194] Antibodies are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GIcNAc. Such antibodies may have reduced fucosylation and / or improved ADCC function. Examples of such antibodies are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibodies may have improved CDC function. Such antibodies are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0195] In some embodiments of the invention, an afucosylated antibody mediates ADCC in the presence of human effector cells more effectively than a parent antibody that comprises fucose. Generally, ADCC activity may be determined using the in vitro ADCC assay as herein disclosed, but other assays or methods for determining ADCC activity, e.g. in an animal model etc., are contemplated.

[0196] In some embodiments, afucosylated anti-CXCR5 antibodies have enhanced ADCC activity in vitro and / or in vivo. In some embodiments, afucosylated anti-CXCR5 antibodies have enhanced ADCC activity in vitro. In some embodiments, ADCC activity in vitro is determined by a method described herein. Briefly, serial dilutions of anti-CXCR5 antibodies or an isotype control are incubated with peripheral blood mononuclear cells (PBMCs) from healthy human donors or cynomolgus monkeys. In this assay, the PBMCs are the source of the natural killer (NK) effector cells and the target CXCR5+ B and Tfh-like cells. Flow cytometry is used to quantify the number of B and Tfh-like cells remaining after approximately 20 hr. The cytotoxicity titration curves were generated by plotting the percentage of cytotoxicity of the antigen binding population against the log of PF-06835375 antibody concentration. ECso values were determined using GraphPad Prism® (version 6.0, GraphPad Software, Inc, San Diego, CA) nonlinear-regression curve fits and a sigmoidal log of agonist dose-response model, according to the following equation:

[0197] Log (agonist) vs. response - variable slope (four parameters)

[0198] Y = Bottom + (Top - Bottom I (1 + 10A((LogEC5o - X)*HillSlope))

[0199] Where Y is the percentage of cytotoxicity, X is antibody concentration, Top is the maximum Y- value corresponding to the upper plateau of the sigmoidal curve, Bottom is the minimum Y- value corresponding to the lower plateau of the sigmoidal curve (constrained to 0), and LogECso is the log of the concentration of antibody at the inflection point of the curve. The EC50 values were summarized across experiments using average and standard deviations (STDEV). In some embodiments, the ability of the humanized mAbs to induce ADCC of bona fide Tfh cells from human tonsil was assessed similarly using CD4+ T cells isolated from tonsillar mononuclear cells with the addition of NK cells isolated from PBMCs. In some embodiments, Ba / F3 cells that express CXCR5 are used as target cells. In some embodiments, cytotoxicity is determined by quantifying LDH release using CytoTox Non- Radioactive Cytotoxicity Assay (Promega, Madison, Wl).

[0200] In some embodiments, maximal lysis is determined using 5 percent Triton X-100 and spontaneous release is determined in the absence of antibody. In some embodiments, the percentage of specific lysis may be determined using the formula: (experimental - spontaneous release) I (maximal - spontaneous release) x 100 = percent specific lysis. In some embodiments, an afucosylated anti-CXCR5 antibody having enhanced ADCC activity results in 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 greater than specific lysis with the same amount of a fucosylated antibody, at at least one concentration of antibody tested. In some embodiments, an afucosylated anti-CXCR5 antibody having enhanced ADCC activity results in 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 greater than specific lysis with a fucosylated antibody, where each antibody is at a concentration of between 0.01 and 1 microg / ml and the target cells are Ba / F3 cells expressing CXCR5. In some embodiments, the antibodies are tested at concentrations ranging from 0.000005 microg / ml to 5 microg / ml

[0201] In some embodiments, afucosylated anti-CXCR5 antibodies have enhanced affinity for Fc gamma RIIIA. In some embodiments, afucosylated anti-CXCR5 antibodies have enhanced affinity for Fc gamma RII IA(V158). In some embodiments, afucosylated anti-CXCR5 antibodies have enhanced affinity for Fc gamma RII IA(F158). In some embodiments, antibody affinity for Fc gamma RIIIA is determined using surface plasmon resonance and / or as follows, which is described with reference to Fc gamma RIIIA(V158), but which is also suitable for determining affinity for Fc gamma Rl IIA(F158). Briefly, in some embodiments, fucosylated or afucosylated anti-CXCR5 antibody is captured on a protein A-coated dextran chip. Fc gamma RIIIA (V158) (available from, e.g., R and D Systems) is injected at various concentrations. The association constant, dissociation constant, and affinity of Fc gamma RIIIA (V158) for fucosylated and afucosylated anti-CXCR5 antibody may be determined, e.g., using software provided with the surface plasmon resonance system (for example, Biacore T200 Evaluation Software 1 :1 binding model). In some embodiments, an afucosylated anti-CXCR5 antibody can have enhanced affinity for Fc gamma RIIIA (such as Fc gamma RII IA(V158) or Fc gamma Rl IIA(F158)) and can bind to Fc gamma RIIIA with at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 17-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, or at least 1000-fold greater affinity than a fucosylated anti-CXCR5 antibody.

[0202] IV. ANTI-CXCR5 ANTIBODY EXPRESSION AND PRODUCTION

[0203] NUCLEIC ACIDS ENCODING ANTI-CXCR5 ANTIBODIES

[0204] The invention also provides polynucleotides encoding any of the antibodies, including antibody fragments and modified antibodies described herein. The invention also provides a method of making any of the polynucleotides described herein. Polynucleotides can be made and expressed by procedures known in the art.

[0205] The sequence of a desired antibody, defined antibody fragment, or antigen-binding fragment thereof, and nucleic acid encoding such antibody, or fragment thereof, can be determined using standard sequencing techniques. A nucleic acid sequence encoding a desired antibody, defined antibody fragment, or antigen-binding fragment thereof, may be inserted into various vectors (such as cloning and expression vectors) for recombinant production and characterization. A nucleic acid encoding the heavy chain, defined antibody fragment, or an antigen-binding fragment of the heavy chain, and a nucleic acid encoding the light chain, defined antibody fragment, or an antigen-binding fragment of the light chain, can be cloned into the same vector, or different vectors.

[0206] In one aspect, the invention provides polynucleotides encoding the amino acid sequence of the CXCR5 antibody and antigen-binding fragments thereof: h11G2 VH (XC155) and h11G2 VH (XC156). In some embodiments, the polynucleotide encoding the amino acid sequences, encodes an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and more preferably identical to, the amino acid sequence of the antibodies, or antigen-binding fragment thereof, of the present invention as disclosed herein.

[0207] The invention provides a polynucleotide comprising one or both of the nucleic acid sequence of the DNA insert of the plasmid deposited with the ATCC and having Accession No. PTA-124323, and Accession No. PTA-124324.

[0208] The invention provides a polynucleotide comprising the nucleic acid sequence of the insert in the plasmid deposited with the ATCC and having Accession No. PTA-124323.

[0209] The invention provides a polynucleotide comprising the nucleic acid sequence of the insert in the plasmid deposited with the ATCC and having Accession No. PTA-124324.

[0210] The invention provides a polynucleotide comprising the nucleic acid sequence of the insert of the plasmid deposited with the ATCC and having Accession No. PTA-124323, and Accession No. PTA-124324. The invention provides cells comprising one or more nucleic acid molecules as set forth in one or more of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.

[0211] The invention provides cells comprising one or more nucleic acid molecules as set forth in SEQ ID NO:16 and SEQ ID NO:17. In some embodiments, a VL of an anti-CXCR5 antibody is encoded by a nucleic acid molecule comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, a VH of an anti-CXCR5 antibody is encoded by a nucleic acid molecule comprising or consisting of the nucleic acid sequence of SEQ ID NO:17.

[0212] The invention provides cells comprising one or more nucleic acid molecules as set forth in SEQ ID NQ:20 and SEQ ID NO:21. In some embodiments, a heavy chain constant region of an anti-CXCR5 antibody is encoded by a nucleic acid molecule comprising or consisting of the nucleic acid sequence of SEQ ID NQ:20. In some embodiments, a light chain constant region of an anti-CXCR5 antibody is encoded by a nucleic acid molecule comprising or consisting of the nucleic acid sequence of SEQ ID NO:21.

[0213] The invention provides cells comprising one or more nucleic acid molecules as set forth in SEQ ID NO:18 and SEQ ID NO:19. In some embodiments, a LC of an anti-CXCR5 antibody is encoded by a nucleic acid molecule comprising or consisting of the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, a HC of an anti-CXCR5 antibody is encoded by a nucleic acid molecule comprising or consisting of the nucleic acid sequence of SEQ ID NO:19.

[0214] In another aspect, the invention provides polynucleotides and variants thereof encoding an anti-CXCR5 antibody, wherein such variant polynucleotides share at least 70%, at least 75%, at least 80%, at least 85%, 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 to any of the specific nucleic acid sequences disclosed herein. These amounts are not meant to be limiting, and increments between the recited percentages are specifically envisioned as part of the disclosure.

[0215] In one embodiment, the VH and VL domains, or antigen-binding fragment thereof, or full length HC or LC, are encoded by separate polynucleotides. Alternatively, both VH and VL, or antigen-binding fragment thereof, or HC and LC, are encoded by a single polynucleotide.

[0216] Polynucleotides complementary to any such sequences are also encompassed by the present disclosure. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present disclosure, and a polynucleotide may, but need not, be linked to other molecules and / or support materials.

[0217] Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes an antibody or a portion thereof) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions such that the immunoreactivity of the encoded polypeptide is not diminished, relative to a native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. In some embodiments, variants exhibit 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 to a polynucleotide sequence that encodes a native antibody or a portion thereof. These amounts are not meant to be limiting, and increments between the recited percentages are specifically envisioned as part of the disclosure.

[0218] Two polynucleotide or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positi“ns, usually 30 to”about 75, or 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0219] Optimal alignment of sequences for comparison may be conducted using the MegAlign® program in the Lasergene® suite of bioinformatics software (DNASTAR®, Inc., Madison, Wl), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In 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.

[0220] In some embodiments, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

[0221] Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0222] Suitable “moderately stringent conditions” include prewashing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50 °C-65 °C, 5X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

[0223] As used herein, “highly stringent conditions” or “high stringency conditions” are those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 °C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1 % polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 °C; or (3) employ 50% formamide, 5X SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt’s solution, sonicated salmon sperm DNA (50 pg / 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. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

[0224] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present disclosure. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present disclosure. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and / or database sequence comparison).

[0225] The polynucleotides of this disclosure can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence.

[0226] For preparing polynucleotides using recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides may be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known within the art. See, e.g., Sambrook et al., 1989.

[0227] Alternatively, PCR allows reproduction of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

[0228] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those of skill in the art, as set forth in Sambrook et al., 1989, for example.

[0229] In some embodiments, a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain. In some embodiments, the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a mole- or mass-ratio of between 5: 1 and 1 :5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1 : 1 and 1:5 for the vector encoding the heavy chain and the vector encoding the light chain is used. In some embodiments, a mass ratio of 1 :2 for the vector encoding the heavy chain and the vector encoding the light chain is used. Vectors

[0230] In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al, Biotechnol. Prog. 20:880-889 (2004).

[0231] Suitable cloning and expression vectors can include a variety of components, such as promoter, enhancer, and other transcriptional regulatory sequences. The vector may also be constructed to allow for subsequent cloning of an antibody variable domain into different vectors. Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and / or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1 , pCR1 , RP4, phage DNAs, 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. Expression vectors are further provided. Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide according to the disclosure. It is implied that an expression vector must be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87 / 04462. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.

[0232] The vectors containing the polynucleotides of interest and / or the polynucleotides themselves, can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on features of the host cell. Host Cells

[0233] The antibody, or antigen-binding fragment thereof, may be made recombinantly using a suitable host cell. A nucleic acid encoding the antibody or antigen-binding fragment thereof can be cloned into an expression vector, which can then be introduced into a host cell, such as E. coli cell, a yeast cell, an insect cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell where the cell does not otherwise produce an immunoglobulin protein, to obtain the synthesis of an antibody in the recombinant host cell. Preferred host cells include a CHO cell, a Human embryonic kidney HEK-293 cell, or an Sp2.0 cell, among 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. A polypeptide fragment of an antibody, especially shorter polypeptides up to about 50 amino acids, can be conveniently made by chemical synthesis. Methods of chemical synthesis for proteins and peptides are known in the art and are commercially available.

[0234] In various embodiments, anti-CXCR5 heavy chains (e.g., comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11 or both) and / or anti-CXCR5 light chains (e.g., comprising the amino acid sequence of SEQ ID NO:9) may 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 may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells, and FLIT8 CHO cells; PER.C6 cells (Crucell); and NSO cells. In some embodiments, anti-CXCR5 heavy chains and / or anti-CXCR5 light chains may be expressed in yeast. See, e.g., U.S. Publication No. US 2006 / 0270045 Al. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the anti-CXCR5 heavy chains and / or anti-CXCR5 light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0235] Introduction of one or more nucleic acids into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Nonlimiting exemplary methods are described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rded. Cold Spring Harbor Laboratory Press (2001). Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.

[0236] In some embodiments afucosylated anti-CXCR5 antibodies are produced in cells capable of producing afucosylated antibodies, such as Lec3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108; and WO 2004 / 056312, especially at Example 11), and knockout cell lines, such as cell lines lacking a functional alpha-1 , 6-fucosyltransferase gene, FUT8, e.g., knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda et al, Biotechnol. Bioeng, 94(4):680-688 (2006); and W02003 / 085107). In some embodiments, afucosylated anti-CXCR5 antibodies are produced in CHO cells lacking a functional FUT8 gene. In some embodiments, afucosylated anti-CXCR5 antibodies are produced in Potelligent CHOK1SV cells (BioWa / Lonza, Allendale, NJ).

[0237] Anti-CXCR5 antibodies may 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 antibody constant regions. For example, a Protein A, Protein G, Protein A / G, or an antibody affinity column may be used to bind the constant region and to purify an anti-CXCR5 antibody. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying some polypeptides. Many methods of purifying polypeptides are known in the art.

[0238] In some embodiments, an anti-CXCR5 antibody is produced in a cell-free system. Non-limiting exemplary cell- free systems are described, e.g., 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).

[0239] V. USES AND MEDICAL THERAPIES

[0240] In some embodiments, the present disclosure provides for therapeutic methods for removing, inhibiting or reducing CXCR5 activity or signaling using an anti-CXCR5 antibody or antigen-binding fragment thereof, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-CXCR5 antibody or antigen-binding fragment thereof. A disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of CXCR5 activity or signaling (e.g., ITP, CIDP, RA, SLE, MG; pemphigus vulgaris, ANCA vasculitis).

[0241] In certain aspects, methods of use of an anti-CXCR5 antibody includes methods of treating or preventing disease. In certain aspects, methods of use of an anti-CXCR5 antibody include methods of treating or preventing autoimmune disease and comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-CXCR5 antibody or antigen-binding fragment thereof. In certain aspects, methods of use of an anti- CXCR5 antibody include methods of treating or preventing immune or inflammatory disease, including for example, primary immune thrombocytopenia (ITP) (also referred to as immune thrombocytopenic purpura and immune thrombocytopenia), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ANCA vasculitis, pemphigus vulgaris, myasthenia gravis and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-CXCR5 antibody or antigen-binding fragment thereof. In some embodiments, methods of use of an anti- CXCR5 antibody comprises methods of treating diseases associated with or mediated by CXCR5 expression and / or binding to CXCL13, including, but not limited to, inflammatory and immune diseases.

[0242] In some aspects, the present disclosure provides an anti-CXCR5 antibody, or antigen-binding fragment thereof, for use in removing, inhibiting or reducing CXCR5 activity or signaling. In some embodiments, the use may comprise 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 may treat any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition or reduction of CXCR5 activity or signaling. In some aspects, the present disclosure provides an anti-CXCR5 antibody, or antigen-binding fragment thereof, for use in a method of treating or preventing disease as described herein. In some aspects, the present disclosure provides the use of an anti-CXCR5 antibody, or antigen-binding fragment thereof, in the manufacture of a medicament for a method of treating or preventing disease as described herein.

[0243] Such diseases, disorders or conditions include, but are not limited to, inflammatory responses such as systemic lupus erythematosus (SLE); chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; diabetes mellitus (e. g. Type I diabetes mellitus or insulin dependent diabetes mellitis); multiple sclerosis; Reynaud’s syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjogren’s syndrome; juvenile onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; ANCA vasculitis; Wegener’s disease; pernicious anemia (Addison’s disease); diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder; multiple organ injury syndrome; hemolytic anemia (including, but not limited to cryoglobinemia or Coombs positive anemia); myasthenia gravis; antigen-antibody complex mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves’ disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous; pemphigus vulgaris; autoimmune polyendocrinopathies; vitiligo; Reiter’s disease; stiffperson syndrome; Bechet disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathies; immune thrombocytopenia (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic diseases; Hashimoto’s thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveoretinitis; Guillain-Barre syndrome; Goodpasture’s syndrome; mixed connective tissue disease; autoimmune-associated infertility; polyarteritis nodosa; alopecia areata; idiopathic myxedema; graft versus host disease; muscular dystrophy (Duchenne, Becker, Myotonic, Limb-girdle, Facioscapulohumeral, Congenital, Oculopharyngeal, Distal, Emery-Dreifuss); chronic inflammatory demyelinating polyradiculoneuropathy (Cl DP); and controlling the proliferation of cancer cells expressing CXCR5 such as cancers of the pancreas, colon, bladder, T-cell leukemia, and B-cell leukemia as would be appreciated by one skilled in the art provided with the teachings disclosed herein.

[0244] The present disclosure provides novel methods of treating disease (e.g., ITP) by administering anti-CXCR5 antibodies that are capable of neutralizing CXCR5 functional activity. These antibodies may be used advantageously to increase absolute platelet count when used as a treatment for ITP. In some embodiments, absolute platelet count in a subject treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased to at least 1 x 102platelets per pL blood, 10 x 102platelets per pL blood, 25 x 102platelets per pL blood, 50 x 102platelets per pL blood, 75 x 102platelets per pL blood, 10 x 103platelets per pL blood, 25 x 103platelets per pL blood, 50 x 103platelets per pL blood, 75 x 103platelets per pL blood, 10 x 104platelets per pL blood, 25 x 104platelets per pL blood, 50 x 104platelets per pL blood, 75 x 10 platelets per pL blood, 10 x 105platelets per pL blood, 25 x 105platelets per pL blood, 50 x 105platelets per pL blood, 75 x 105platelets per pL blood or more. In some embodiment, an increase in absolute platelet count is measured at least after one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti-CXCR5 antibody, or antigen binding fragment thereof. In some embodiments, an increase in absolute platelet count is measured after every dose or any combination of doses (e.g., after every other dose) of an anti-CXCR5 antibody, or antigen binding fragment thereof.

[0245] In some embodiments, absolute platelet count in a subject treated with an anti- CXCR5 antibody, or antigen-binding fragment thereof, is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to absolute platelet count in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, absolute platelet count in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to absolute platelet count in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, absolute platelet count in a subject treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by at least 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10-fold or more as compared to absolute platelet count in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, absolute platelet count in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by at least 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10-fold or more as compared to absolute platelet count in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, absolute platelet count in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is not significantly different from absolute platelet count in a subject, or population of subjects, who do not have ITP. In some embodiments, absolute platelet count is measured over days, weeks, months or years. In some embodiments, absolute platelet count is measured after at least one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti-CXCR5 antibody, or antigen binding fragment thereof.

[0246] In some embodiments, a subject treated with an anti-CXCR5 antibody, or antigenbinding fragment thereof exhibits a modified overall response (mOR). In some embodiments, a subject with ITP treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof exhibits a modified overall response (mOR) which comprises i) an absolute platelet count of 50 x 103platelets or more per pL blood, ii) an absolute platelet count that is at least 2-fold larger than the baseline value of platelet count and iii) the subject has not had a splenectomy or been administered a rescue medication. In some embodiments, a mOR is measured after at least one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti-CXCR5 antibody, or antigen binding fragment thereof.

[0247] In some embodiments, a subject treated with an anti-CXCR5 antibody, or antigenbinding fragment thereof exhibits a complete response (CR). In some embodiments, a subject with ITP treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof exhibits a complete response (CR) which comprises i) an absolute platelet count of 100 x 103platelets or more per pL blood, and iii) the subject has not had a splenectomy or been administered a rescue medication. In some embodiments, a CR is measured after at least one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti-CXCR5 antibody, or antigen binding fragment thereof. In some embodiments, platelet count overtime in a subject treated with an anti- CXCR5 antibody, or antigen-binding fragment thereof, is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to platelet count over time in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, platelet count over time in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to platelet count over time in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, platelet count over time in a subject treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by at least 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10-fold or more as compared to platelet count over time in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, platelet count over time in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by at least 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5- fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10-fold or more as compared to platelet count over time in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, platelet count over time in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is not significantly different from platelet count over time in a subject, or population of subjects, who do not have ITP. In some embodiments, platelet count over time is measured over days, weeks, months or years. In some embodiments, platelet count over time is measured after at least one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti-CXCR5 antibody, or antigen binding fragment thereof.

[0248] In some embodiments, time to a bleeding event in a subject treated with an anti- CXCR5 antibody, or antigen-binding fragment thereof, is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to time to a bleeding event in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, time to a bleeding event in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to time to a bleeding event in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, time to a bleeding event in a subject treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by at least 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10-fold or more as compared to time to a bleeding event in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, time to a bleeding event in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by at least 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5- fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10-fold or more as compared to time to a bleeding event in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, time to a bleeding event in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is not significantly different from time to a bleeding event in a subject, or population of subjects, who do not have ITP. In some embodiments, time to a bleeding event is measured over days, weeks, months or years. In some embodiments, time to a bleeding event is measured after at least one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti-CXCR5 antibody, or antigen binding fragment thereof.

[0249] In some embodiments, time to require rescue medication in a subject treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to time to require rescue medication in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, time to require rescue medication in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to time to require rescue medication in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, time to require rescue medication in a subject treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by at least 1.5-fold, 2.0- fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10-fold or more as compared to time to require rescue medication in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, time to require rescue medication in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is increased by at least 1.5- fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10-fold or more as compared to time to require rescue medication in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, time to require rescue medication in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is not significantly different from time to require rescue medication in a subject, or population of subjects, who do not have ITP. In some embodiments, time to require rescue medication is measured over days, weeks, months or years. In some embodiments, time to require rescue medication is measured after at least one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti-CXCR5 antibody, or antigen binding fragment thereof.

[0250] In some embodiments, absolute B-cell count in a subject treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to absolute B-cell count in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, absolute B-cell count in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to absolute B-cell count in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, absolute B-cell count in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is not significantly different from absolute B-cell count in a subject, or population of subjects, who do not have ITP. In some embodiments, absolute B-cell count is measured over days, weeks, months or years. In some embodiments, absolute B-cell count is measured after at least one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti-CXCR5 antibody, or antigen binding fragment thereof.

[0251] In some embodiments, B-cell count over time in a subject treated with an anti- CXCR5 antibody, or antigen-binding fragment thereof, is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to B-cell count over time in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, B-cell count over time in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to B-cell count over time in the same subject prior to treatment with an anti- CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, B-cell count over time in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen- binding fragment thereof, is not significantly different from B-cell count over time in a subject, or population of subjects, who do not have ITP. In some embodiments, B-cell count over time is measured over days, weeks, months or years. In some embodiments, B-cell count over time is measured after at least one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti-CXCR5 antibody, or antigen binding fragment thereof.

[0252] In some embodiments, absolute cTfh cell count in a subject treated with an anti- CXCR5 antibody, or antigen-binding fragment thereof, is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to absolute cTfh cell count in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, absolute cTfh cell count in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to absolute cTfh cell count in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, absolute cTfh cell count in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is not significantly different from absolute cTfh cell count in a subject, or population of subjects, who do not have ITP. In some embodiments, absolute cTfh cell count is measured over days, weeks, months or years. In some embodiments, absolute cTfh cell count is measured after at least one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti- CXCR5 antibody, or antigen binding fragment thereof.

[0253] In some embodiments, cTfh cell count over time in a subject treated with an anti- CXCR5 antibody, or antigen-binding fragment thereof, is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to cTfh cell count over time in a subject, or group of subjects, (e.g., patient(s) with ITP) who are not treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, cTfh cell count over time in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to cTfh cell count over time in the same subject prior to treatment with an anti-CXCR5 antibody, or antigen-binding fragment thereof. In some embodiments, cTfh cell count over time in a subject (e.g., a patient with ITP) treated with an anti-CXCR5 antibody, or antigen-binding fragment thereof, is not significantly different from cTfh cell count over time in a subject, or population of subjects, who do not have ITP. In some embodiments, cTfh cell count over time is measured over days, weeks, months or years. In some embodiments, cTfh cell count over time is measured after at least one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine dose, 10 doses or more doses of an anti- CXCR5 antibody, or antigen binding fragment thereof.

[0254] The anti-CXCR5 antibodies, or antigen-binding fragments thereof, of the present invention may also be used to detect and / or measure CXCR5, or CXCR5-expressing cells in a sample, e.g., for diagnostic purposes. For example, an anti-CXCR5 antibody, or fragment thereof, may be used to diagnose a condition or disease characterized by aberrant expression (e.g., over-expression, under-expression, lack of expression, etc.) of CXCR5. Exemplary diagnostic assays for CXCR5 may comprise, e.g., 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.

[0255] The following embodiments, designated EMB1 to EMB51 , represent specific embodiments of the present invention.

[0256] EMB1 is a method of treating a subject with an immune or inflammatory disease comprising subcutaneous administration of a unit dose of at least 6 mg, 10 mg, 18 mg or 50 mg of an anti-CXCR5 antibody comprising a variable region of the light chain (VL) comprising the amino acid sequence of SEQ ID NO:1 and a variable region of the heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:5, wherein the unit dose is administered once a month.

[0257] EMB2 is the method of EMB1 , wherein the immune or inflammatory disease is selected from the group consisting of primary immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ANCA-vasculitis, pemphigus vulgaris, myasthenia gravis (MG) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).

[0258] EMB 3 is the method of EMB1 or EMB2, wherein the dose is 18 mg.

[0259] EMB4 is the method of EMB1 or EMB2, wherein the dose is 50 mg.

[0260] EMB5 is the method of any one of EMB1 to EMB4, wherein in the subject has ITP. EMB 6 is the method of EMB5, further comprising i) measuring the absolute platelet count in a sample of blood from the subject, and ii) if the absolute platelet count is greater than 50 x 103platelets per pL of blood, the absolute platelet count is at least 2-fold greater than the baseline value of the absolute platelet count for the subject, and the subject was not exposed to splenectomy or rescue medication before the measuring step, assigning a responder status to the subject on the modified overall response scale.

[0261] EMB7 is the method of EMB5, further comprising i) measuring the absolute platelet count in a sample of blood from the subject, and ii) if the absolute platelet count is greater than 100 x 103platelets per pL of blood, and the subject was not exposed to splenectomy or rescue medication before the measuring step, assigning a responder status to the subject on the complete response scale.

[0262] EMB8 is a method of treating a subject with Immunoglobulin G4 related disease (lgG4- RD) comprising administration of an anti-CXCR5 antibody comprising a variable region of the light chain (VL) comprising the amino acid sequence of SEQ ID NO:1 and a variable region of the heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:5.

[0263] EMB9 is the method of EMB8 wherein the anti-CXCR5 antibody is administered subcutaneously.

[0264] EMB10 is the method of EMB8 wherein the anti-CXCR5 antibody is administered intravenously.

[0265] EMB11 is the method of any one of EMB8 to EMB10 wherein a dose of about 6 mg, about 10 mg, about 18 mg or about 50 mg of an anti-CXCR5 antibody is administered.

[0266] EMB12 is the method of EMB11 wherein the anti-CXCR5 antibody is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every month, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every two months, once every 12 weeks, once every three months, once every 16 weeks, once every four months, once every 20 weeks, once every five months, once every 24 weeks, once every six months, once every 26 weeks or once a year.

[0267] EMB13 is the method of EMB11 wherein 18 mg or 50 mg of the anti-CXCR5 antibody is administered on day one, week four, week eight and week twelve and optionally on week sixteen, week twenty and week twenty four of a twenty-four week treatment period.

[0268] EMB14 is the method of any one of EMB8 to EMB10 wherein one or more additional therapeutic agents are administered.

[0269] EMB15 is the method of EMB14 wherein the additional therapeutic agents are selected from the group consisting of glucocorticoids, disease-modifying anti-rheumatic drugs (DMARDs) and B-cell depleting agents.

[0270] EMB16 is the method of EMB15 wherein the additional therapeutic agents are selected from the group consisting of prednisone, methotrexate, sulfasalazine, hydroxychloroquine, leflunomide, azathioprine, apremilast, cyclophosphamide, cyclosporine, mycophenolic acid, adalimumab, belimumab, infliximab, etanercept, tocilizumab, sarilumab, golimumab, canakinumab, guselkumab, 64stekinumab64, secukinumab, 64stekinumab, certolizumab pegol, secukinumab, ixekizumab, anakinra, tofacitinib, baricitinib.upacitinib, ruxolitinib, abrocitinib, ritlecitinib, inebilizumab and rituximab.

[0271] EMB17 is the method of EMB16 wherein a dose of about 6 mg, about 10 mg, about 18 mg or about 50 mg of an anti-CXCR5 antibody is administered. EMB18 is the method of EMB17 wherein the anti-CXCR5 antibody is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every month, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every two months, once every twelve weeks, once every three months, once every sixteen weeks, once every four months, once every twenty weeks, once every five months, once every twenty four weeks, once every six months, once every twenty six weeks or once a year.

[0272] EMB19 is the method of EMB17 wherein 18 mg or 50 mg of the anti-CXCR5 antibody is administered on day one, week four, week eight and week twelve and optionally on week sixteen, week twenty and week twenty four of a twenty-four week treatment period.

[0273] EMB20 is the method of EMB18 wherein the anti-CXCR5 antibody is administered subcutaneously.

[0274] EMB21 is the method of EMB18 wherein the anti-CXCR5 antibody is administered intravenously.

[0275] EMB22 is an anti-CXCR5 antibody comprising a variable region of the light chain (VL) comprising the amino acid sequence of SEQ ID NO:1 and a variable region of the heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:5 for use in the treatment of Immunoglobulin G4 related disease (lgG4-RD) in a subject.

[0276] EMB23 is the anti-CXCR5 antibody for use of EMB22 wherein the anti-CXCR5 antibody is used subcutaneously.

[0277] EMB24 is the anti-CXCR5 antibody for use of EMB22 wherein the anti-CXCR5 antibody is used intravenously.

[0278] EMB25 is the anti-CXCR5 antibody for use of any one of EMB22 to EMB24 wherein the amount of anti-CXCR5 antibody is a dose of about 6 mg, about 10 mg, about 18 mg or about 50 mg.

[0279] EMB26 is the anti-CXCR5 antibody for use of EMB25 wherein use of the anti-CXCR5 antibody is once a week, once every two weeks, once every three weeks, once every four weeks, once every month, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every two months, once every twelve weeks, once every three months, once every sixteen weeks, once every four months, once every twenty weeks, once every five months, once every twenty four weeks, once every six months, once every twenty six weeks or once a year.

[0280] EMB27 is the anti-CXCR5 antibody for use of EMB25 wherein the use of 18 mg or 50 mg of the anti-CXCR5 antibody is on day one, week four, week eight and week twelve and optionally on week sixteen, week twenty and week twenty four of a twenty-four week treatment period. EMB28 is the anti-CXCR5 antibody for use of any one of EMB22 to EMB24 wherein one or more additional therapeutic agents are used.

[0281] EMB29 is the anti-CXCR5 antibody for use of EMB28 wherein the additional therapeutic agents are selected from the group consisting of glucocorticoids, disease-modifying antirheumatic drugs (DMARDs) and B-cell depleting agents.

[0282] EMB30 is the anti-CXCR5 antibody for use of EMB28 wherein the additional therapeutic agents are selected from the group consisting of prednisone, methotrexate, sulfasalazine, hydroxychloroquine, leflunomide, azathioprine, apremilast, cyclophosphamide, cyclosporine, mycophenolic acid, adalimumab, belimumab, infliximab, etanercept, tocilizumab, sarilumab, golimumab, canakinumab, guselkumab, 66stekinumab66, secukinumab, 66stekinumab, certolizumab pegol, secukinumab, ixekizumab, anakinra, tofacitinib, baricitinib.upacitinib, ruxolitinib, abrocitinib, ritlecitinib, inebilizumab and rituximab.

[0283] EMB31 is the anti-CXCR5 antibody for use of claim 30 wherein the amount of anti- CXCR5 antibody is a dose of about 6 mg, about 10 mg, about 18 mg or about 50 mg.

[0284] EMB32 is the anti-CXCR5 antibody for use of EMB31 wherein the anti-CXCR5 antibody is used once a week, once every two weeks, once every three weeks, once every four weeks, once every month, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every two months, once every twelve weeks, once every three months, once every sixteen weeks, once every four months, once every twenty weeks, once every five months, once every twenty four weeks, once every six months, once every twenty six weeks or once a year.

[0285] EMB33 is the anti-CXCR5 antibody for use of EMB31 wherein 18 mg or 50 mg of the anti-CXCR5 antibody is administered on day one, week four, week eight and week twelve and optionally on week sixteen, week twenty and week twenty four of a twenty-four week treatment period.

[0286] EMB34 is the anti-CXCR5 antibody for use of EMB32 wherein the anti-CXCR5 antibody is used subcutaneously.

[0287] EMB35 is the anti-CXCR5 antibody for use of EMB33 wherein the anti-CXCR5 antibody is used intravenously.

[0288] EMB36 is an anti-CXCR5 antibody for use to treat a subject according to the method of any one of EMB1 to EMB21.

[0289] EMB37 is the use of an anti-CXCR5 antibody in the manufacture of a medicament for use in treating a subject according to the method of any one of EMB1 to EMB21.

[0290] EMB38 is a method of treating a subject with an immune or inflammatory disease comprising subcutaneous administration of a dose of about 6 mg, about 10 mg, about 18 mg or about 50 mg of an anti-CXCR5 antibody comprising a variable region of the light chain (VL) comprising the amino acid sequence of SEQ ID NO:1 and a variable region of the heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:5, wherein the dose is administered every four weeks.

[0291] EMB39 is the method of EMB38, wherein the immune or inflammatory disease is selected from the group consisting of primary immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ANCA-vasculitis, pemphigus vulgaris, myasthenia gravis (MG) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).

[0292] EMB40 is the method of EMB38 or EMB39, wherein the dose is 18 mg.

[0293] EMB41 is the method of EMB38 or EMB39, wherein the dose is 50 mg.

[0294] EMB42 is the method of any one of EMB38 to EMB41 , wherein the subject has ITP.

[0295] EMB43 is the method of EMB42, further comprising i) measuring the absolute platelet count in a blood sample obtained from the subject, and ii) if the absolute platelet count is greater than 50 x 103platelets per pL of blood, the absolute platelet count is at least 2-fold greater than the baseline value of the absolute platelet count for the subject, and the subject was not exposed to splenectomy or rescue medication before the measuring step, assigning a responder status to the subject on the modified overall response scale.

[0296] EMB44 is the method of EMB42, further comprising i) measuring the absolute platelet count in a blood sample obtained from the subject, and ii) if the absolute platelet count is greater than 100 x 103platelets per pL of blood, and the subject was not exposed to splenectomy or rescue medication before the measuring step, assigning a responder status to the subject on the complete response scale.

[0297] EMB45 is an anti-CXCR5 antibody comprising a variable region of the light chain (VL) comprising the amino acid sequence of SEQ ID NO:1 and a variable region of the heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:5 for use in the treatment of an immune or inflammatory disease in a subject wherein a dose of about 6 mg, about 10 mg, about 18 mg or about 50 mg of the anti-CXCR5 antibody is used subcutaneously every four weeks.

[0298] EMB46 is the use of EMB45 wherein the immune or inflammatory disease is selected from the group consisting of primary immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ANCA-vasculitis, pemphigus vulgaris, myasthenia gravis (MG) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).

[0299] EMB47 is the use of EMB45 or EMB46, wherein the dose is 18 mg.

[0300] EMB48 is the use of EMB45 or EMB46, wherein the dose is 50 mg.

[0301] EMB49 is the use of any one of EMB45 to EMB48, wherein the subject has ITP.

[0302] EMB50 is the use of EMB49 further comprising i) measuring the absolute platelet count in a blood sample obtained from the subject, and ii) if the absolute platelet count is greater than 50 x 103platelets per pL of blood, the absolute platelet count is at least 2-fold greater than the baseline value of the absolute platelet count for the subject, and the subject was not exposed to splenectomy or rescue medication before the measuring step, assigning a responder status to the subject on the modified overall response scale.

[0303] EMB51 is the use of EMB49 further comprising i) measuring the absolute platelet count in a blood sample obtained from the subject, and ii) if the absolute platelet count is greater than 100 x 103platelets per pL of blood, and the subject was not exposed to splenectomy or rescue medication before the measuring step, assigning a responder status to the subject on the complete response scale.

[0304] VI. COMPOSITIONS

[0305] The disclosure also provides pharmaceutical compositions comprising an effective amount of an CXCR5 antibody, or antigen-binding fragment thereof, described herein. The pharmaceutical compositions can be admininstered in a specified dose such as a 6 mg, 18 mg or 50 mg dose (which can also be referred to as a unit dose). Examples of such compositions, as well as how to formulate, are also described herein. In some embodiments, the composition comprises one or more CXCR5 antibodies. In other embodiments, the CXCR5 antibody is a human antibody. In other embodiments, the CXCR5 antibody is a humanized antibody. In some embodiments, the CXCR5 antibody comprises a constant region that is capable of triggering a desired immune response, such as antibody-mediated lysis orADCC. In other embodiments, the CXCR5 antibody comprises a constant region that is afucosylated and provides enhanced ADCC compared with an otherwise identical antibody that is fucosylated. In other embodiments, the CXCR5 antibody comprises one or more CDR(s) of the antibody (such as one, two, three, four, five, or, in some embodiments, all six CDRs).

[0306] It is understood that the compositions can comprise more than one CXCR5 antibody, or antigen-binding fragment thereof. In one embodiment, a composition (e.g., a pharmaceutical composition) comprises an antibody comprising a VL comprising or consisting of an amino acid sequence of SEQ ID NO:1 and a VH comprising or consisting of an amino acid sequence of SEQ ID NO:5. In one embodiment, a composition (e.g., a pharmaceutical composition) comprises i) an anti-CXCR5 antibody comprising a light chain comprising or consisting of SEQ ID NO:9 and a heavy chain comprising or consisting of SEQ ID NQ:10 and ii) an anti-CXCR5 antibody comprising a light chain comprising or consisting of SEQ ID NO:9 and a heavy chain comprising or consisting of SEQ ID NO:11.

[0307] The composition used in the present disclosure can further comprise pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and practice of Pharmacy 20thEd., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may comprise 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 counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0308] The CXCR5 antibody, antigen-binding fragment thereof, and compositions thereof can also be used in conjunction with other agents that serve to enhance and / or complement the effectiveness of the agents.

[0309] The disclosure also provides compositions, including pharmaceutical compositions, comprising any of the polynucleotides of the disclosure. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding the antibody as described herein. In other embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies described herein. In some embodiments, the composition comprises an expression vector comprising a polynucleotide comprising or consisting of a nucleic acid sequence of SEQ ID NO:16 and SEQ ID NO:17. In some embodiments, the composition comprises an expression vector comprising a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO:18 and SEQ ID NO:19.

[0310] In still other embodiments, the composition comprises either or both of the polynucleotides encoding the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 5, either or both of the polynucleotides encoding the amino acid sequence shown in SEQ ID NO:9 and SEQ ID NO: 10 or either or both of the polynucleotides encoding the amino acid sequence shown in SEQ ID NO:9 and SEQ ID NO:11.

[0311] In another aspect, the polynucleotide can encode the VH, VL and / or both VH and VL of the antibody of the disclosure (e.g., the amino acid sequence of SEQ ID NO:1 and SEQ ID NO:5). That is, the composition comprises a single polynucleotide or more than one polynucleotide encoding the antibody, or antigen-binding fragment thereof, or the disclosure.

[0312] Pharmaceutical compositions of the disclosure also can be administered in combination therapy, such as, combined with other agents. For example, the combination therapy can include CXCR5 antibody, or antigen-binding fragment thereof, of the present disclosure combined with at least one other therapy wherein the therapy may be surgery, immunotherapy, or drug therapy.

[0313] The pharmaceutical compounds of the disclosure may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenylsubstituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N’-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0314] A pharmaceutical composition of the disclosure also may include a pharmaceutically acceptable anti-oxidant. 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) oil-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, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0315] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the disclosure 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, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0316] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by 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 into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0317] Pharmaceutical compositions typically 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 to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be suitable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0318] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.

[0319] Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0320] A pharmaceutical composition of the present disclosure may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1 %-1.0% (w / w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.

[0321] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the disclosure are known in the art and described, for example in Remington’s Pharmaceutical Sciences, Genaro, ed., Mack Publishing Co., Easton, PA (1985), which is incorporated herein by reference.

[0322] In one embodiment, the 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 ranging from about 5 to 6. In some embodiments, the intravenous formulation is a sterile aqueous solution containing 5 or 10 mg / mL of antibody, with 20 mM sodium acetate, 0.2 mg / mL polysorbate 80, and 140 mM sodium chloride at pH 5.5. Further, a solution comprising an antibody, or antigen-binding fragment thereof, can comprise, among many other compounds, histidine, mannitol, sucrose, trehalose, glycine, poly(ethylene) glycol, EDTA, methionine, and any combination thereof, and many other compounds known in the relevant art.

[0323] In some embodiments, a pharmaceutical composition of the present disclosure comprises 50 mg / mL of an anti-CXCR5 antibody or antigen-binding fragment of the present disclosure, 20 mM histidine, 8.5% sucrose, 0.02% polysorbate 80, and 0.005% EDTA at pH 5.8.

[0324] In some embodiments, a pharmaceutical composition for subcutaneous administration comprises 50 mg / mL of a CXCR5 antibody or antigen-binding fragment of the present disclosure, 20 mM histidine, 8.5% sucrose, 0.02% polysorbate 80, and 0.005% EDTA at pH 5.8. In some embodiments, a pharmaceutical composition for subcutaneous administration comprises 50 mg / mL of a CXCR5 antibody comprising a VL of the amino acid sequence of SEQ ID NO:1 and a VH of the amino acid sequence of SEQ ID NO:5, 20 mM histidine, 8.5% sucrose, 0.02% polysorbate 80, and 0.005% EDTA at pH 5.8.

[0325] In some embodiments, a pharmaceutical composition for treatment of a subject with ITP by subcutaneous administration comprises 50 mg / mL of a CXCR5 antibody comprising a VL comprising the amino acid sequence of SEQ ID NO:1 and a VH comprising the amino acid sequence of SEQ ID NO:5, 20 mM histidine, 8.5% sucrose, 0.02% polysorbate 80, and 0.005% EDTA at pH 5.8.

[0326] In some embodiments, a pharmaceutical composition for treatment of a subject with ITP by subcutaneous administration comprises 50 mg / mL of a CXCR5 antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:8, 20 mM histidine, 8.5% sucrose, 0.02% polysorbate 80, and 0.005% EDTA at pH 5.8.

[0327] In some embodiments, a pharmaceutical composition for subcutaneous administration comprises 50 mg / mL of a CXCR5 antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:8, 20 mM histidine, 8.5% sucrose, 0.02% polysorbate 80, and 0.005% EDTA at pH 5.8.

[0328] In some embodiments, a pharmaceutical composition for treatment of a subject with an immune or inflammatory disease (e.g., CIDP, RA, SLE, MG; pemphigus vulgaris, ANCA vasculitis) by subcutaneous administration comprises 50 mg / mL of a CXCR5 antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:8, 20 mM histidine, 8.5% sucrose, 0.02% polysorbate 80, and 0.005% EDTA at pH 5.8.

[0329] In some embodiments, a pharmaceutical composition of the present invention comprises the following components: 100 mg / mL CXCR5 antibody or antigen-binding fragment of the present disclosure, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80 at pH 5.8. A composition may be provided as a liquid formulation or as a lyophilized powder. When a powder is reconstituted at full volume, the composition retains the same formulation. Alternatively, a powder may be reconstituted at half volume, in which case the composition comprises 100 mg CXCR5 antibody or antigen-binding fragment thereof of the present disclosure, 20 mM histidine, 10% sucrose, and 0.02% polysorbate 80 at pH 5.8.

[0330] In one embodiment, part of the dose is administered by an intravenous bolus and the rest by infusion of the antibody formulation. For example, a 0.01 mg / kg intravenous injection of the CXCR5 antibody, or antigen-binding fragment thereof, may be given as a bolus, and the rest of the antibody dose may be administered by intravenous injection. A predetermined dose of the CXCR5 antibody, or antigen-binding fragment thereof, may be administered, for example, over a period of an hour and a half to two hours to five hours.

[0331] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0332] In one embodiment the compositions of the disclosure are pyrogen-free formulations which are substantially free of endotoxins and / or related pyrogenic substances. Endotoxins include toxins that are confined inside a microorganism and are released when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions. The Food and Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (Ell) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1 ):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight it is advantageous to remove even trace amounts of endotoxin. In one embodiment, endotoxin and pyrogen levels 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, endotoxin and pyrogen levels 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.

[0333] In one embodiment, the disclosure comprises administering a composition wherein said administration is oral, parenteral, intramuscular, intranasal, vaginal, rectal, lingual, sublingual, buccal, intrabuccal, intravenous, cutaneous, subcutaneous or transdermal.

[0334] In another embodiment the disclosure further comprises administering a composition in combination with other therapies, such as surgery (e.g., splenectomy), chemotherapy, hormonal therapy, biological therapy, immunotherapy or radiation therapy.

[0335] VII. DOSING / ADMINISTRATION

[0336] To prepare pharmaceutical or sterile compositions including an CXCR5 antibody, or antigen-binding fragment thereof of the disclosure, the antibody is mixed with a pharmaceutically acceptable carrier or excipient. Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman’s The Pharmacological 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.).

[0337] Selecting an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (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).

[0338] Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.

[0339] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0340] Compositions comprising CXCR5 antibodies or antigen-binding fragments thereof, of the disclosure can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week. Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation. A specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose may be at least 0.05 pg / kg body weight, at least 0.2 pg / kg, at least 0.5 pg / kg, at least 1 pg / kg, at least 10 pg / kg, at least 100 pg / kg, at least 0.2 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, or at least 50 mg / kg (see, e.g., 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 pg, at least 20 pg, at least 25 pg, at least 30 pg, at least 35 pg, at least 40 pg, at least 45 pg, at least 50 pg, at least 55 pg, at least 60 pg, at least 65 pg, at least 70 pg, at least 75 pg, at least 80 pg, at least 85 pg, at least 90 pg, at least 95 pg, or at least 100 pg. The doses administered to a subject may number at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12, or more.

[0341] For CXCR5 antibodies or antigen-binding fragments thereof of the disclosure, the dosage administered to a patient may be 0.0001 mg / kg to 100 mg / kg of the patient’s body weight. The dosage may be between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 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 0.01 to 0.10 mg / kg of the patient’s body weight.

[0342] The dosage of the CXCR5 antibody or antigen-binding fragment thereof may be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg / kg. The dosage of the antibodies of the disclosure may be 150 pg / kg or less, 125 pg / kg or less, 100 pg / kg or less, 95 pg / kg or less, 90 pg / kg or less, 85 pg / kg or less, 80 pg / kg or less, 75 pg / kg or less, 70 pg / kg or less, 65 pg / kg or less, 60 pg / kg or less, 55 pg / kg or less, 50 pg / kg or less, 45 pg / kg or less, 40 pg / kg or less, 35 pg / kg or less, 30 pg / kg or less, 25 pg / kg or less, 20 pg / kg or less, 15 pg / kg or less, 10 pg / kg or less, 5 pg / kg or less, 2.5 pg / kg or less, 2 pg / kg or less, 1.5 pg / kg or less, 1 pg / kg or less, 0.5 pg / kg or less, or 0.1 pg / kg or less of a patient's body weight.

[0343] Unit dose of the CXCR5 antibodies or antigen-binding fragments thereof of the disclosure may be 0.1 mg to 200 mg, 0.1 mg to 175 mg, 0.1 mg to 150 mg, 0.1 mg to 125 mg, 0.1 mg to 100mg, 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.

[0344] In some embodiments, a unit dose of an anti-CXCR5 antibody is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg or more. In some embodiments, the unit dose is 6 mg, 10 mg, 18 mg or 50 mg.

[0345] In some embodiments, a unit dose of an anti-CXCR5 antibody comprising a VL amino acid sequence of SEQ ID NO:1 and a VH amino acid sequence of SEQ ID NO:5 is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg,

[0346] 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg,

[0347] 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg or more. In some embodiments, the unit dose is 6 mg, 10 mg, 18 mg or 50 mg. In some embodiments, the unit dose is 6 mg, 10 mg, 18 mg or 50 mg.

[0348] In some embodiments, a unit dose for treatment of a subject by subcutaneous administration of an anti-CXCR5 antibody comprising a VL amino acid sequence of SEQ ID N0:1 and a VH amino acid sequence of SEQ ID NO:5 is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg or more. In some embodiments, the unit dose is 6 mg, 10 mg, 18 mg or 50 mg.

[0349] In some embodiments, a unit dose for treatment of a subject with ITP by subcutaneous administration of an anti-CXCR5 antibody comprising a VL amino acid sequence of SEQ ID NO:1 and a VH amino acid sequence of SEQ ID NO:5 is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg or more. In some embodiments, the unit dose is 6 mg, 10 mg, 18 mg or 50 mg.

[0350] In some embodiments, a unit dose for treatment of a subject with an immune or inflammatory disease (e.g., CIDP, RA, SLE, MG; pemphigus vulgaris, ANCA vasculitis) by subcutaneous administration of an anti-CXCR5 antibody comprising a VL amino acid sequence of SEQ ID NO:1 and a VH amino acid sequence of SEQ ID NO:5 is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg or more. In some embodiments, the unit dose is 6 mg, 10 mg, 18 mg or 50 mg.

[0351] In some embodiments, a unit dose of an anti-CXCR5 antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:8 is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg,

[0352] 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg or more. In some embodiments, the unit dose is 6 mg, 10 mg, 18 mg or 50 mg.

[0353] In some embodiments, a unit dose treatment of a subject by subcutaneous administration of an anti-CXCR5 antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:8 is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg or more. In some embodiments, the unit dose is 6 mg, 10 mg, 18 mg or 50 mg.

[0354] In some embodiments, a unit dose for treatment of a subject with ITP by subcutaneous administration of an anti-CXCR5 antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:8 is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg,

[0355] 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg or more. In some embodiments, the unit dose is 6 mg, 10 mg, 18 mg or 50 mg.

[0356] In some embodiments, a unit dose for treatment of a subject with an immune or inflammatory disease (e.g., CIDP, RA, SLE, MG; pemphigus vulgaris, ANCA vasculitis) by subcutaneous administration of an anti-CXCR5 antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:8 is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg,

[0357] 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg,

[0358] 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg or more. In some embodiments, the unit dose is 6 mg, 10 mg, 18 mg or 50 mg.

[0359] In some embodiments, a unit dose for treatment of a subject with ITP by subcutaneous administration of an anti-CXCR5 antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:8 is at least is 6 mg, 10 mg, 18 mg or 50 mg.

[0360] The dosage of the CXCR5 antibodies or antigen-binding fragments thereof of the disclosure may achieve a serum titer of at least 0.1 pg / mL, at least 0.5 pg / mL, at least 1 pg / mL, at least 2 pg / mL, at least 5 pg / mL, at least 6 pg / mL, at least 10 pg / mL, at least 15 pg / mL, at least 20 pg / mL, at least 25 pg / mL, at least 50 pg / mL, at least 100 pg / mL, at least 125 pg / mL, at least 150 pg / mL, at least 175 pg / mL, at least 200 pg / mL, at least 225 pg / mL, at least 250 pg / mL, at least 275 pg / mL, at least 300 pg / mL, at least 325 pg / mL, at least 350 pg / mL, at least 375 pg / mL, or at least 400 pg / mL in a subject. Alternatively, the dosage of the antibodies of the disclosure may achieve a serum titer of at least 0.1 pg / mL, at least 0.5 pg / mL, at least 1 pg / mL, at least, 2 pg / mL, at least 5 pg / mL, at least 6 pg / mL, at least 10 pg / mL, at least 15 pg / mL, at least 20 pg / mL, at least 25 pg / mL, at least 50 pg / mL, at least 100 pg / mL, at least 125 pg / mL, at least 150 pg / mL, at least 175 pg / mL, at least 200 pg / mL, at least 225 pg / mL, at least 250 pg / mL, at least 275 pg / mL, at least 300 pg / mL, at least 325 pg / mL, at least 350 pg / mL, at least 375 pg / mL, or at least 400 pg / mL in the subject.

[0361] Doses of CXCR5 antibodies, or antigen-binding fragments thereof of the disclosure may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 28 days, 29 days, 30 days, 31 days, 1 month, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

[0362] In some embodiments, a method of treating a subject with ITP comprises administering a unit dose of at least is 6 mg, 10 mg, 18 mg or 50 mg of an anti-CXCR5 antibody comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO:2, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO:4, a CDR-H1 comprising the amino acid sequence of SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:8 once a month.

[0363] In some embodiments, a method of treating a subject with ITP comprises subcutaneous administration of a unit dose of at least is 6 mg, 10 mg, 18 mg or 50 mg of an anti-CXCR5 antibody comprising a VL comprising the amino acid sequence of SEQ ID NO:1 and a VH comprising the amino acid sequence of SEQ ID NO:5 once a month.

[0364] In some embodiments, a method of treating a subject with an immune or inflammatory disease (e.g., CIDP, RA, SLE, MG; pemphigus vulgaris, ANCA vasculitis) comprises subcutaneous administration of a unit dose of at least is 6 mg, 10 mg, 18 mg or 50 mg of an anti-CXCR5 antibody comprising a VL comprising the amino acid sequence of SEQ ID NO:1 and a VH comprising the amino acid sequence of SEQ ID NO:5 once a month.

[0365] An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects (see, e.g., Maynard, et al., 1996, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fia.; Dent, 2001 , Good Laboratory and Good Clinical Practice, llrch Publ, London, UK).

[0366] The route of administration may be by, e.g., topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by sustained release systems or an implant (see, e.g., 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. Pat. Nos. 6,350466 and 6,316,024). Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference their entirety. In one embodiment, the CXCR5 antibody, or antigen-binding fragment thereof, or a composition of the disclosure is administered using Alkermes AIR™ pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).

[0367] A composition of the present disclosure may also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. Selected routes of administration for antibodies of the disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, a composition of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0368] If the CXCR5 antibodies, or antigen-binding fragments thereof, of the disclosure are administered in a controlled release or sustained release system, a pump may be used to achieve controlled or sustained release (see, Langer, supra; Sefton, 1987, CRC Grit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:501 ; Saudek et al., 1989, N. Engl. J. Med. 321:514).

[0369] Polymeric materials can be used to achieve controlled or sustained release of the therapies of the disclosure (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fia. (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 ; see also 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. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), polyvinyl alcohol), polyacrylamide, polyethylene glycol), polylactides (PLA), polyoeactide-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. A controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[0370] Controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies of the disclosure or conjugates thereof. See, e.g., U.S. Pat. No. 4,526,938, International Patent Publication Nos. WO 91 / 05548, WO 96 / 20698, Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a 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 their entirety.

[0371] If the CXCR5 antibody, or antigen-binding fragment thereof, of the disclosure is administered topically, it can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington’s Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19thed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some instances, greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in some instances, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.

[0372] If the compositions comprising CXCR5 antibodies, or antigen-binding fragments thereof, are administered intranasally, it can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0373] Methods for co-administration or treatment with a second therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation, are well known in the art (see, e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10 th 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.). An effective amount of therapeutic may decrease the symptoms by at least 10 percent; by at least 20 percent; at least about 30 percent; at least 40 percent, or at least 50 percent.

[0374] Additional therapies (e.g., prophylactic or therapeutic agents), which can be administered in combination with the CXCR5 antibodies, or antigen-binding fragments of the disclosure, may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart from the antibodies of the disclosure. The two or more therapies may be administered within one same patient visit.

[0375] The CXCR5 antibodies, or antigen-binding fragments thereof, of the disclosure and the other therapies may be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, optionally, followed by the administration of a third therapy (e.g., prophylactic or therapeutic agent) for a period of time and so forth, and repeating this sequential administration, i.e. , the cycle in order to reduce the development of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and / or to improve the efficacy of the therapies.

[0376] In one embodiment, the CXCR5 antibodies of the disclosure can be co-administered with compositions for treating autoimmune diseases and disorders, including, but not limited to, 84driamycin, azathiopurine, busulfan, cyclophosphamide, cyclosporine A, Cytoxan, fludarabine, 5-fluorouracil, methotrexate, mycophenolate mofetil, 6-mercaptopurine, a corticosteroid, a nonsteroidal anti-inflammatory, 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, anti-thymocyte globulin and Mg, and others, which are known to persons skilled in the art.

[0377] In one embodiment, the CXCR5 antibodies of the disclosure can be co-administered with compositions for treating diabetes, including, but not limited to, biguanides (e.g., buformin, metformin, and phenform), hormones and analogs thereof (amylin, insulin, insulin aspart, insulin detemir, insulin glargine, insulin glulisine, insulin lispro, liraglutide, and pramlintide), sulfonylurea derivatives (acetohexamide, carbutamide, chlorpropamide, glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glisoxepid, glyburide, glybuthiazole, glybuzole, glyhexamide, glymidine, tolazamide, tolbutamide, and tolcyclamide), thiazolidinediones (pioglitazone, rosiglitazone, and troglitazone), acarbose, exenatide, miglitol, mitiglinide, muraglitazar, nateglinide, repaglinide, sitagliptin, tesaglitazar, vildagliptin, and voglibose.

[0378] In certain embodiments, the CXCR5 antibodies, or antigen-binding fragments thereof of the disclosure can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the disclosure cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811 ; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade, 1989, J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016); mannosides (Umezawa et al., Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman et al., 1995, FEBS Lett. 357: 140; M. Owais et al., 1995, Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen, 1994, FEBS Lett. 346:123; Killion; Fidler, 1994; Immunomethods 4:273.

[0379] The disclosure provides protocols for the administration of pharmaceutical composition comprising CXCR5 antibodies, or antigen-binding fragments thereof, of the disclosure alone or in combination with other therapies to a subject in need thereof. The therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the present disclosure can be administered concomitantly or sequentially to a subject. The therapy (e.g., prophylactic or therapeutic agents) of the combination therapies of the present disclosure can also be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the therapies (e.g., agents) to avoid or reduce the side effects of one of the therapies (e.g., agents), and / or to improve, the efficacy of the therapies.

[0380] The therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the disclosure can be administered to a subject concurrently. The term “concurrently” is not limited to the administration of therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather it is meant that a pharmaceutical composition comprising CXCR5 antibodies, or antigen-binding fragments thereof, of the disclosure are administered to a subject in a sequence and within a time interval such that the antibodies of the disclosure or conjugates thereof can act together with the other therapy(ies) to provide an increased benefit than if they were administered otherwise. For example, each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to a subject separately, in any appropriate form and by any suitable route. In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered to a subject less than 15 minutes, less than 30 minutes, less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart. In other embodiments, two or more therapies (e.g., prophylactic or therapeutic agents) are administered to a within the same patient visit.

[0381] The prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

[0382] VIII. KITS

[0383] The disclosure also provides kits comprising the antibodies described herein. Kits of the disclosure include one or more containers comprising an CXCR5 antibody described herein and instructions for use in accordance with any of the methods of the disclosure described herein. Generally, these instructions comprise a description of administration of the antibody for the above described therapeutic treatments. In some embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing an applicator, e.g., single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes), are included. The instructions relating to the use of a CXCR5 antibody generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0384] In some embodiments, a kit includes a unit dose of 6 mg, 10 mg, 18 mg, or 50 mg of an anti-CXCR5 antibody comprising a VL comprising the amino acid sequence of SEQ ID NO:1 and a VH comprising the amino acid sequence of SEQ ID NO:5.

[0385] In some embodiments, a kit includes a unit dose of 6 mg, 10 mg, 18 mg, or 50 mg of an anti-CXCR5 antibody comprising a VL comprising the amino acid sequence of SEQ ID NO:1 and a VH comprising the amino acid sequence of SEQ ID NO:5 in a pharmaceutical composition comprising 50 mg / mL of the anti-CXCR5 antibody 20 mM histidine, 8.5% sucrose, 0.02% polysorbate 80 and 0.005% EDTA at pH 5.8.

[0386] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a CXCR5 antibody of the disclosure. The container may further comprise a second pharmaceutically active agent.

[0387] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.

[0388] The disclosure also provides diagnostic kits comprising any or all of the antibodies described herein. The diagnostic kits are useful for, for example, detecting 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 put them at risk of developing CXCR5-mediated disease, disorder or condition or a CXCR5 deficiency 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 suspected of having a CXCR5 mediated disease or a CXCR5 deficiency disease, disorder or condition. Diagnostic kits of the disclosure include one or more containers comprising an CXCR5 antibody described herein and instructions for use in accordance with any of the methods of the disclosure described herein. Generally, these instructions comprise a description of use of the CXCR5 antibody to detect the presence of CXCR5 in individuals at risk for, or suspected of having, a CXCR5 mediated disease or a CXCR5 deficiency disease, disorder or condition. In some embodiments, an exemplary diagnostic kit can be configured to contain reagents such as, for example, a CXCR5 antibody, a negative control sample, a positive control sample, and directions for using the kit.

[0389] IX. EQUIVALENTS

[0390] The foregoing description and following Examples detail certain specific embodiments of the disclosure and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the disclosure may be practiced in many ways and the disclosure should be construed in accordance with the appended claims and any equivalents thereof.

[0391] Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein and the claimed disclosure below. The following examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings.

[0392] X. GENERAL TECHNIQUES

[0393] It is to be understood that this invention is not limited to specific synthetic methods of making that may of course vary. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

[0394] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0395] XI. BIOLOGICAL DEPOSIT

[0396] Representative materials of the present invention were deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, on July 26, 2017. Vector h11G2-VH (XC155), having ATCC Accession No. PTA-124323, comprises a DNA insert encoding the heavy chain variable region of antibody hi 1G2 (XC155), and vector h11G2-VL (XC154), having ATCC Accession No. PTA-124324, comprises a DNA insert encoding the light chain variable region of antibody h11G2 (XC154). The deposits were made under the provisions of the Budapest T reaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Pfizer Inc. and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. Section 122 and the Commissioner’s rules pursuant thereto (including 37 C.F.R. Section 1.14 with particular reference to 886 OG 638).

[0397] The owner of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

[0398] Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein and the claimed invention below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings.

[0399] All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated by reference in their entirety for all purposes. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0400] EXAMPLES

[0401] The disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0402] Example 1 : Treatment of adult patients with moderate to severe Primary Immune Thrombocytopenia (ITP) with an anti-CXCR5 antibody, PF-06835375.

[0403] Description of the antibody tested

[0404] In one embodiment, the antibody provided herein is an anti-CXCR5 antibody designated as PF-06835375. PF-06835375 is a humanized, afucosyl Ig G1 antibody against CXCR5, a surface receptor that is expressed on B cells, bona fide Tfh cells, and cTfh cells. Bona fide Tfh cells are located within lymphoid organs (e.g., spleen, lymph nodes, tonsils, Peyer’s patches, and mucosa-associated lymphoid tissue). cTfh cells are located in the peripheral blood.

[0405] PF-06835375 is a selective antibody directed against the CXCR5 receptor that has 2 distinct mechanisms of action: (1) depletion of CXCR5-positive cells through ADCC, and (2) antagonism of CXCL13-dependent signaling. By utilizing an antibody that is also antagonistic, cells that escape killing may still be inhibited from trafficking to and participating in GC immune responses. As such, by targeting CXCR5 through a combination of depletion and antagonism, PF-06835375 will effectively impair autoreactive GC reactions, including humoral memory responses, thereby suppressing autoantibody maturation and production known to, in part, underlie autoimmune disease pathology.

[0406] The PF-06825275 antibody comprises a light chain amino acid sequence of SEQ ID NO:9 and a heavy chain amino acid sequence of SEQ ID NO:10 or SEQ ID NO:11 (including C- terminal lysine). In some embodiments, a pharmaceutical composition comprising an anti- CXCR5 antibody comprises an antibody comprising i) a light chain amino acid sequence of SEQ ID N0:9 and a heavy chain amino acid sequence of SEQ ID NO:10, ii) a light chain amino acid sequence of SEQ ID NO:9 and a heavy chain amino acid sequence of SEQ ID NO:11 , or iii) a combination thereof.

[0407] PF-06835375 is supplied as a sterile solution, packaged in a single use, 6 mL clear glass vial sealed with a coated serum stopper and an aluminum overseal. Each vial contains 1.35 mL PF-06835375 at a nominal concentration of 50 mg / mL.

[0408] Primary ITP

[0409] Primary ITP is an acquired autoimmune disorder characterized by platelet counts <100 x 103 / pL and normal other blood cell counts in the absence of another cause (Kuter et al., Br. J. Haematol. (2019) 185(3):503-13). The primary clinical concern with ITP is an increased risk of bleeding but an increased risk of thrombosis has also been reported (Rodeghiero et al., Am. J. Hematol. (2016) 91(1):39-45). Plasma derived Ig is approved to raise platelet counts to treat or prevent bleeding or to allow a patient to undergo an invasive procedure. Similar to plasma derived Ig, the therapeutic goal of the PF-06835375 treatment will be to increase platelet levels in patients with ITP either transiently or in more sustained manner by reducing or potentially eliminating the pathogenic antiplatelet antibodies (Lucchini et al., Haematologica (2019) 104(6):1124-1135).

[0410] As has been shown with treatment of B cells depleting agent such as rituximab, (Lucchini et al., Haematologica (2019) 104(6): 1124-1135; Ghanima et al., Lancet (2015) 385 (9978): 1653-1661) treatment with PF-06835375 in patients with ITP is anticipated to increase the platelet levels. However, long-term follow-up data showed that only 20-30% of patients remained in remission (Lucchini et al., Haematologica (2019) 104(6):1124-1135; Patel et al., Blood (2012) 119(25):5989-5995). It has been demonstrated that CXCR5+ CD4+Tfh cells expression were significantly higher in the patients with ITP than in healthy controls (Xie et al., Int. J. Biol. Sci. (2015) 11(2):220-229). The additional effects of PF-06835375 on bona fide Tfh cells, and cTfh cells may provide a longer lasting effect on the production of anti-platelet autoantibody. This effect differentiates PF-06835375 from other B-cell depleting agents. Therefore this may provide a more sustained benefit than B cell depletion alone and may result in longer duration of remission in patients treated with PF-06835375.

[0411] Additional therapies for ITP include corticosteroids, I Vlg, thrombopoeitin receptor agonists such as avotrombopag (Doptelet®), eltrombopag (Promacta®), fostamatinib (Tavalisse®), and romiplostim (Nplate®), anti-Rh D agents (WinRho®), and splenectomy. Immunosuppressive therapy such as rituximab has been used off-label (Arnold et al., Ann. Internal Med. (2007)146(1):25-3). While these therapies are effective measures to increase peripheral blood platelet counts, the duration of overall response once the therapy is discontinued is limited (Lucchini et al., Haematologica (2019) 104(6):1124-1135; Deshayes et al., Rev. Med. Intern. (2021) 42(1):32-37); Stasi et al. Blood (2001) 98(4):952-957; Cooper et al., Br. J. Haematol. (2004) 125(2):232-239; Vital et al., Arthritis Rheum. (2011) 63.3:603-608). Patient Population

[0412] This is a Phase 2, open-label, multicenter, safety and efficacy study to investigate PF-06835375 in adult participants with moderate to severe, persistent or chronic primary ITP. This study will focus on participants with persistent (3 to 12 months) or chronic (>12 months) ITP.

[0413] The original study design required 40 participants to be enrolled (with 30 completing Week 12) into the study and exposed to the 6 mg dose of PF-06835375. Each participant in the 6 mg cohort received one subcutaneous injection at Day 1 , Week 4, and Week 8 visits. Interim analysis recommended early stopping for futility of the 6 mg cohort and to progress with a higher and additional dose with a longer treatment period in a second cohort. A second interim analysis recommended an additional higher dose in a third cohort.

[0414] Accordingly, an additional cohort of approximately 30 participants to be exposed to a higher dose of 18 mg subcutaneous (SC) PF-06835375 was enrolled. Each participant in the second cohort will receive one 18 mg subcutaneous injection at the Day 1 , Week 4, Week 8, and Week 12 visits. A third cohort of approximately 30 participants to be exposed to a higher dose of 50 mg subcutaneous (SC) PF-06835375 is enrolled. Each participant in the third cohort will receive one 50 mg subcutaneous injection at the Day 1, Week 4, Week 8, and Week 12 visits.

[0415] At the time of interim analysis, 31 participants were exposed to the 6 mg dose. Therefore, approximately 91 participants cumulatively will be enrolled and exposed to the study interventions.

[0416]

[0002] Inclusion Criteria: Participants (males and females) between the ages of 18 (or the minimum country-specific age of consent if >18) and 70 years, inclusive, at Screening with a diagnosis of ITP in accordance with established guidelines:

[0417] • Ongoing ITP (platelet counts <50 x 103 / .L) [No severe bleeding within 1 month or during screening], AND

[0418] • Persistent ITP (3 to 12 months) or Chronic ITP >12 months. AND

[0419] • Failed initial therapy or require alternative therapy for ITP, in the opinion of the Investigator.

[0420] Additional criteria include BMI 17.5 to 40 kg / m2, and minimum weight >40 kg (88 pounds) and informed consent.

[0421] Treatment duration

[0422] The study includes three cohorts of participants. The duration of the first cohort of the study was approximately 24 weeks, including up to a 28-day screening period. The 6 mg dose SC injection of PF-06835375 was administered once monthly for 3 months. Dosing occurred in the clinic on Day 1 , Week 4 and Week 8. Platelet counts were assessed at Baseline, Weeks 1 , 4, 5, 8, 9, 12, 16, and 20.

[0423] The duration of the second and third cohorts of the study will be approximately 28 weeks, including up to a 28-day screening period. The 18 mg dose and 50 mg dose SC injection of PF-06835375 will be administered once monthly for 4 months. Dosing will occur in the clinic on Day 1 , Week 4, Week 8 and Week 12. Platelet counts will be assessed at Baseline, Weeks 1 , 4, 5, 8, 9, 12, 13, 16, 20, and 24.

[0424] The cohorts include additional Follow-up visits (at the discretion of the investigator) starting at Week 28 and occurring every 8 weeks for participants who do not demonstrate stable or increasing concentrations of B cells by Week 20 for the 6 mg cohort and by Week 24 for the 18 mg and 50 mg cohorts.

[0425] Administration: All participants will be administered a 40 mg dose of oral prednisolone or equivalent approximately 2 hours before study intervention administration. Within 1 hour before study intervention administration, participants will be administered a 500 mg dose of acetaminophen and 25 mg of oral diphenhydramine. At approximately 4 hours post dose participants will receive a 20 mg dose of oral prednisolone or equivalent see 10.9 and a 500 mg dose of acetaminophen. At approximately 6 hours post dose, a third 500 mg dose of acetaminophen maybe administered prior to discharge, at investigator’s discretion. Additionally, at approximately 10 hours post dose, participant may self-administer an additional 500 mg of acetaminophen, NSAID medication or other medications deemed appropriate at investigator’s discretion. These treatment medications should be reported as concomitant medications.

[0426] The preferred body location of the SC injections is in the abdomen. If abdominal injections are not possible, arm or thigh locations may also be used. If an arm is used for the SC injection, the opposite arm should be used for the blood sample collections for PK analysis. Interim Analysis

[0427] Interim analysis data from the 6 mg cohort study in ITP patients were available for 17 participants. The mean B cell counts dropped to less than 10 cells / pd through Day 64 and was around 12.7 cells / pd at Week 12 with gradual repletion over the next few weeks through Week 20. The mean platelet count increased to approximately 83000 cells / pL at Week 1 post dose but was not sustained during the dosing interval (4 weeks). A similar trend of increase in platelets was seen at Week 5 (1 week post second dose) and Week 9 (1 week post third dose). Overall, B cell depletion was not as prolonged as expected and the increase in platelet count was not sustained. Interim analysis of the 6 mg SC once every 4 weeks for 3 months (Q4Wx3) dose suggested futility for efficacy.

[0428] Thus, a second cohort with a higher dose of 18 mg SC given once every 4 weeks for 4 months (Q4Wx4) may provide prolonged B and cTfh cell depletion and thereby may lead to sustained increase in platelet counts. This 3-fold increase in dose was selected to provide sufficient separation in PK from the 6 mg SC interim PK data which showed high variability. Additionally, B / cTfh cell depletion data from the Phase 1 study did not show a difference between the 6 mg and 10 mg SC cohorts.

[0429] Based on modeling, the mean anticipated exposures for 18 mg SC Q4Wx4 are 269 ng / mL and 133 ng / mL for Cmax and Cavrespectively. These predicted exposures are well below the stopping criteria and the safety margins for Cmax and Cavare 7063.2 and 6690.8 fold, respectively.

[0430] A third cohort with a higher dose of 50 mg SC given Q4W x 4 may provide even prolonged B and cTfh cell depletion and thereby may lead to sustained increase in platelet counts for longer duration. This approx. 3-fold increase in dose was selected to provide sufficient separation in PK from the 18 mg SC exposures observed at interim analysis.

[0431] A PK-PD model developed based on study PK and B cell data was able to adequately describe the observed PK data in this study at 6 mg and 18 mg Q4W doses. Based on this modeling, the mean anticipated exposures for 50 mg SC Q4W x 4 are 750 ng / mL and 380 ng / mL for Cmax and Cav respectively. These predicted exposures are well below the stopping criteria and the safety margins for Cmax and Cav are 2533.3 and 2341.8 fold respectively.

[0432] Efficacy Assessments

[0433] Platelet counts measurements will be used for different evaluations of treatment efficacy.

[0434] The absolute value of platelet count of treated participants at week 12 will be evaluated as the primary objective. The proportion of participants with modified overall response (mOR) and complete response (CR) at week 12 will be determined as key secondary objectives.

[0435] The World Health Organization (WHO) Bleeding Scale (Fogarty et al., Curr. Med. Res. Opin (2012)28(1 ):79-87) is a single clinical investigator-determined classification of bleeding on a simple 5-point scale:

[0436] Table 2. WHO Bleeding Scale

[0437] On the Day 1 visit, the investigator asks the participant, “Have you experienced any bruising or bleeding within the last 7 days?” At each subsequent visit, the investigator asked, “Have you experienced any bruising or bleeding since I saw you last?”

[0438] Safety assessments will include medical history (e.g., complete medical history, ITP disease history (including disease duration, extent of disease, and prior treatments), cardiovascular disease history, venous thromboembolic event history, alcohol and tobacco use history), chest radiography, IGRA tuberculin test, screening for progressive multifocal leukoencephalopathy (PML) caused by John Cunningham Virus (JCV), physical examination (e.g., assessments of the cardiovascular, respiratory, gastrointestinal, skin and neurological systems), vital signs (e.g., temperature, systolic and diastolic blood pressure, and pulse rate and respiratory rate), pulse oximetry, electrocardiograms, laboratory assessments (including IB- cell counts), injection site reactions (ISR) (e.g., itching redness, swelling, pain), cytokine release syndrome (CRS), monitoring for infections (e.g., viral, bacterial, fungal), viral surveillance (e.g., cytomegalovirus, Epstein-Barr virus, herpes simplex virus type 1 & 2, varicella-zoster), hepatitis B and C (e.g., HBsAg, HBcAB, HCVAb), pregnancy testing, suicidal ideation and behavior risk,

[0439] Adverse events (AEs) may arise from symptoms or other complaints reported to the investigator by the participant (or, when appropriate, by a caregiver, surrogate, or the participant's legally authorized representative), or they may arise from clinical findings of the Investigator or other healthcare providers (clinical signs, test results, etc.).

[0440] Adverse events of special interest include 1) / njection site reaction, 2) bleeding events and 3) CRS.

[0441] Pharmacokinetics (PK): Blood samples of approximately 3 mL will be collected for measurement of serum concentrations of PF-06835375 as specified in the Schedule of Activities. Instructions for the collection and handling of biological samples will be provided in the laboratory manual or by the sponsor. The actual date and time of each sample will be recorded.

[0442]

[0003] The actual times may change, but the number of samples will remain the same. All efforts will be made to obtain the samples at the exact nominal time relative to dosing. Collection of samples up to and including 4 hours after dose administration that are obtained within 10% of the nominal time relative to dosing (e.g., within 6 minutes of a 60-minute sample) will not be captured as a protocol deviation, as long as the exact time of the collection is noted on the source document and the CRF. Collection of samples more than 10 hours after dose administration that are obtained <1 hour away from the nominal time relative to dosing will not be captured as a protocol deviation, as long as the exact time of the collection is noted on the source document and the CRF. This protocol deviation window does not apply to samples to be collected more than 10 hours after dose administration at outpatient / follow-up visits with visit windows.

[0443] Samples will be used to evaluate the PK of PF-06835375. Samples collected for analyses of PF-06835375 serum concentration may also be used to evaluate safety or efficacy aspects related to concerns arising during or after the study, for metabolite identification and / or evaluation of the bioanalytical method, or for other internal exploratory purposes.

[0444] Samples collected for measurement of serum concentrations of PF-06835375 will be analyzed using a validated analytical method in compliance with applicable SOPs.

[0445] Biomarkers: Blood samples will be collected for biomarker research including protein and cell phenotyping analyses. The samples will be analyzed for the following biomarkers:

[0446] • Leukocyte cell surface expression markers and immune cell populations including

[0447] B cells and Tfh cells and subsets (transitional B cells, Naive B cells, memory B cells, lgD+ CD27+ B cells, plasmablasts, plasma cells) will be assessed via flow cytometry.

[0448] • Cytokines, including but not limited to IL-6, TNFa, and IFNy.

[0449] • Complement products, including but not limited to C3a, C4 and CH50.

[0450] • Immunoglobulins.

[0451] • Anti-platelet antibodies.

[0452] • Autoantibodies that may include but is not limited to ANA, ENA, anti-dsDNA, and antiphospholipid antibody (APA).

[0453] • Exploratory auto-antibodies by protein array.

[0454] Immunogenicity Assessments: Blood samples of approximately 5 mL, to provide a minimum of 3 mL of serum, will be collected for determination of anti-drug antibodies (ADA) and neutralizing antibodies (Nab).

[0455] Objectives and Endpoints

[0456] Table 3. Objectives and Endpoints Table 3. Objectives and Endpoints Table 3. Objectives and Endpoints

[0457] Primary endpoints: The primary estimand is characterized as follows. Population includes all treated participants. The endpoint is the observed value of the log-transformed value of normalized platelet count LPC (Iog2 (Platelet counts / 103per .L)). The (hypothetical) treatment condition of interest is the treatment without splenectomy or rescue medication. Population-level summary is the mean value of outcome as Week 12 (6 mg cohort) and Week 16 (18 mg cohort).

[0458] Key secondary endpoints: The key secondary endpoints will evaluate the proportion of participants with mOR or CR at Week 12 (6 mg cohort) and Week 16 (18 mg and 50 mg cohorts). A participant is assigned a responder status on the modified overall response (mOR) scale at any visit if all of the following conditions are satisfied at that visit.

[0459] • The value of platelet count per .L exceeds the target value of 50 x103.

[0460] • This value is at least 2-fold larger than the baseline value of platelet count.

[0461] • The participant was not exposed to splenectomy or rescue medication before or at the visit.

[0462] If any of these conditions is not satisfied, then the participant’s mOR status is a non-res ponder.

[0463] A participant is assigned a responder status on the complete response (CR) scale at any visit if all of the following conditions are satisfied at that visit.

[0464] • The value of platelet count per .L exceeds the target value of 100 x103.

[0465] • The participant was not exposed to splenectomy or rescue medication before or at the visit.

[0466] If any of these conditions is not satisfied, then the participant’s CR status is a non-responder. Note that for each of these outcomes (mOR or CR) the missing value leads to the assignment of a non-responder’s status.

[0467] Safety endpoints: safety endpoints for the study include:

[0468] • T reatment-emergent AEs and SAEs.

[0469] • Withdrawals from active treatment due to Aes.

[0470] • Serious infections, defined as any infection (viral, bacterial, and fungal) requiring hospitalization or parenteral antimicrobials.

[0471] • Safety laboratory tests (e.g., hematology [including coagulation panel], chemistry and lipid profiles).

[0472] • Vital signs.

[0473] • ECG.

[0474] Secondary endpoints: the secondary endpoints / estimands will include evaluation of expected value (with 90% confidence intervals) for means of log2(Platelet counts) and proportions of mOR and CR responders over time. Censoring of the Iog2 (Platelet counts), definitions of mOR and CR responses will be similar and methods for the analysis (MM RM and Wald) will be the same as the methods described for the key secondary endpoints.

[0475] E1 (for mean concentration) and E4 (for absolute values and change from baseline of concentration) will be used on B and cTfh cells. The E1 approach will be based on the application of mixed mode for repeated measures (MM RM) analysis to the observations collected at all visits. The MMRM analysis will contain log2(Concentration) ofB and cTfH cells as an outcome and the scheduled study visit as a covariate. The model will use an unstructured covariance matrix. The observations collected, after / at the time of splenectomy or exposure to rescue medication will be censored in the E1 analysis but not in the E4 analysis.

[0476] Tertiary / exploratory endpoints: Distributions of time to mOR and CR will be summarized by the Kaplan-Meier curves.

[0477] PF-06835375 concentrations and ADA / Nab incidence at timepoints as specified in the Schedule of Activities (SoA) will be summarized by descriptive statistics. Concentrations of antibodies will be explored by descriptive statistics and analyzed.

[0478] The PK concentration population is defined as all enrolled participants who received at least one dose of PF-06835375 and in whom at least one concentration value is reported.

[0479] PK concentrations will be summarized and presented with summary statistics. A population PK model may be developed for the purpose of estimating PK parameters. Any population PK model developed to characterize the PK data will be reported separately.

[0480] Example 2: Efficacy of PF-06835375 in reducing the risk of a disease flare in patients with lgG4-RD Primary Objectives and Endpoints

[0481] Primary Objective

[0482] To evaluate the efficacy of PF-06835375 in reducing the risk of a disease flare in patients with lgG4-RD.

[0483] Primary Endpoint

[0484] Time to disease flare, defined as the time in days from Day 1 (dosing) to the date of the first treated and AC-determined (adjudication committee determined) lgG4 RD flare within the 52- week randomized-controlled period (RCP). The date of disease flare is defined as the date of initiation of any flare treatment (new or increased glucocorticoid (GO) treatment, other immunotherapy, or interventional procedure) deemed necessary by the Investigator for the flare.

[0485] Secondary Objectives and Endpoints

[0486] Secondary Objectives

[0487] To evaluate the safety and tolerability of PF-06835375 in patients with lgG4-RD.

[0488] To evaluate the effect of PF-06835375 on other measures of disease activity.

[0489] Secondary Endpoints

[0490] Incidence of treatment-emergent adverse events (TEAEs), TESAEs, and treatment-emergent adverse events of special interest (AESIs) during the 52-week RCP.

[0491] The incidence of anti-drug antibodies (ADAs) directed against PF-06835375 during the RCP. Annualized flare rate for treated and AC-determined flares during the RCP.

[0492] Annualized flare rate for AC-determined flares, whether or not treated, during the RCP.

[0493] The proportion of subjects achieving flare-free complete remission at Week 52. Complete remission is defined as an lgG4-RD Responder Index (Rl) score of 0 at Week 52, no AC- determined flare during the RCP, and no treatment for flare or disease control except the required 8-week GC taper (see Wallace, Z.S. et al. Arthritis & Rheumatology, January 2020, 72(1), 7-19).

[0494] Time to initiation of first treatment (medication or procedure) for new or worsening disease activity by the Investigator within the RCP, regardless of AC determination of flare. Glucocorticoid use, calculated as the cumulative GC dose taken for the purpose of lgG4-RD disease control during the RCP.

[0495] Study Design

[0496] This is a multicenter, randomized, double-blind (Investigator, subject, and Sponsor will be blinded to treatment assignment), placebo-controlled, parallel-cohort study to evaluate the efficacy and safety of PF-06835375 for prevention of disease flare in adults with active lgG4-RD who are at high risk of recurrent flare. The study is to be conducted at one or more sites in one or more countries. The expected duration of each subject’s participation in this study is up to 400 days (screening and RCP) or, for eligible subjects who enroll in the optional open label period (OLP), up to 813 days (screening, RCP, interval between RCP and OLP, and OLP).

[0497] Subjects will be stratified by first or subsequent lgG4-RD manifestation (i.e. , newly diagnosed vs. recurrent) and randomized 1 :1 :1 : 1 by an interactive voice / web response system (IXRS) to one of four blinded treatment groups:

[0498] • PF-06835375 groups: Subjects will receive a subcutaneous (SC) injection of PF-06835375 (6 mg, 18 mg or 50 mg) on Day One, Week Four, Week Eight and Week Twelve.

[0499] • Placebo group: Subjects will receive an SC injection of placebo on Day 1 , Week Four, Week Eight and Week Twelve.

[0500] A total of 320 subjects (each group having 80 subjects) will be employed in this study.

[0501] All subjects will receive daily oral prednisone (or equivalent) from Day 1 through Week 8 during RCP, according to a tapering dose schedule:

[0502] • 20 mg / day for Weeks 1-2

[0503] • 15 mg / day for Weeks 3-4

[0504] • 10 mg / day for Weeks 5-6

[0505] • 5 mg / day for Weeks 7-8.

[0506] Descriptive summary on total prednisone dose (mg) received based on above tapering schedule and daily dose (mg / day) per subject will be provided.

[0507] During the 52-week RCP, subjects will attend monthly visits for study assessments and procedures. Suspicion of flare will trigger diagnostic assessments by the Investigator. The Investigator will determine if the event meets protocol-defined criteria for flare, and, independent of the flare determination, will decide if the event requires treatment. The same data reviewed by the Investigator will also be evaluated by a central, independent, and blinded AC (blinded to treatment allocation and to investigator determination of meeting flare criteria or requiring treatment) that will determine whether protocol-defined flare criteria are met.

[0508] The end of the RCP is defined, per subject, as the date of completion of the Week 52 RCP visit. All subjects should be followed through the end of the RCP. A subject who decides to withdraw from the RCP should be asked to complete the final visit (early discontinuation visit [EDV]) for safety follow-up.

[0509] In an optional one-year OLP, all eligible subjects who choose to participate will receive PF-06835375. Subjects assigned to the placebo group during the RCP will receive 3 PF-06835375 SC injections (OLP Day 1 , Week 4, Week 8 and Week 12), while those assigned to the PF-06835375 groups in the RCP will receive SC PF-06835375 on OLP Day 1 and Week 26 and a placebo infusion on Day 15. The IXRS will assign the OLP Day 15 treatments, maintaining the blind for the RCP treatments.

[0510] Primary efficacy analysis

[0511] The estimand with a treatment policy strategy is defined by the following:

[0512] • Target population: Subjects in the FAS

[0513] • Variable: Time in days from Day 1 (dosing) to the date of the first treated and AC-determined lgG4-RD flare within the 52-week RCP. Date of flare onset is defined as date of onset of treatment (medication or procedure).

[0514] • Intercurrent event: All data captured during the 52-week RCP will be used for analysis. Subjects who do not complete the RCP and who have not had a treated and AC-determined flare during the RCP will be censored at the time of discontinuation.

[0515] • Population-level summary: Hazard ratio (HR) between PF-06835375 versus placebo and its associated 95% Cl.

[0516] To assess the robustness towards violations of the assumption of non-informative censoring of subjects who do not complete the RCP, the following sensitivity analysis will be performed:

[0517] • Target population: Subjects in the FAS

[0518] • Variable: Time in days from Day 1 (dosing) to the date of the first treated and AC-determined lgG4-RD flare within the 52-week RCP. Date of flare onset is defined as date of onset of treatment (medication or procedure).

[0519] • Intercurrent event: All data captured during the 52-week RCP will be used for analysis. Subjects who do not complete the RCP due to lgG4-RD related death or due to subject perception of lack of efficacy and who have not had a treated and determined flare will be considered as having a flare with onset at the time of discontinuation I withdrawal from the RCP. Subjects who do not complete the RCP due to other reasons and who have not had a treated and AC-determined flare will be censored at the time of discontinuation I withdrawal from RCP.

[0520] • Population-level summary: HR between PF-06835375 versus placebo and its associated 95% Cl.

[0521] The hazard rate in the PF-06835375 groups will be compared to that in the placebo group using the Cox proportional hazards model with the treatment indicator (PF-06835375 or placebo) and the stratification factor as the explanatory variables. The HR of PF-06835375 versus placebo will be estimated together with its associated 95% Cl. SAS PROC PH REG will be used for fitting this model.

[0522] Additional Analysis on the primary efficacy endpoint

[0523] The estimand of the additional analysis is defined by the following:

[0524] • Target population: Subjects in the FAS • Variable: Time in days from Day 1 (dosing) to the date of the first treated and AC-determined lgG4-RD flare within the 52-week RCP. Date of flare onset is defined as date of onset of treatment (medication or procedure).

[0525] • Intercurrent event:

[0526] Subjects who do not complete the RCP and who have not had a treated and AC-determined flare will be censored at the time of discontinuation;

[0527] Subjects, who 1) receive any treatment for lgG4-RD, including GC or immunosuppressive treatment (other than GC tapered in accordance with the protocol-specified schedule during the first 8 weeks of the RCP) and / or 2) receive any prohibited GC or immunosuppressive treatment prior to experiencing a treated and AC-determined flare, will be censored at the time when relevant treatment was first received.

[0528] • Population-level summary: HR between PF-06835375 versus placebo and its associated 95% Cl.

[0529] The robustness towards violations of the assumption of non-informative censoring on subjects who do not complete the RCP and I or subjects who received prohibited GC or immunosuppressive treatment prior to experiencing a treated and AC-determined flare will be assessed with the following sensitivity analysis:

[0530] • Target population: Subjects in the FAS

[0531] • Variable: Time in days from Day 1 (dosing) to the date of the first treated and AC-determined lgG4-RD flare within the 52-week RCP. Date of flare onset is defined as date of onset of treatment (medication or procedure).

[0532] • Intercurrent event:

[0533] Subjects who do not complete the RCP due to lgG4-RD related death or due to subject perception of lack of efficacy and who have not had a treated and AC-determined flare will be considered as having a flare at the time of discontinuation I withdrawal from RCP. Subjects who do not complete the RCP due to other reasons and who have not had a treated and AC- determined flare will be censored at the time of discontinuation I withdrawal from RCP.

[0534] Subjects who receive the treatment for lgG4-RD, including GC or immunosuppressive treatment (other than GC tapered in accordance with the protocol-specified schedule during the first 8 weeks of the RCP) related to lgG4-RD prior to experiencing a treated and AC-determined flare will be considered as having a flare at the time of treatment.

[0535] Subjects who receive any prohibited GC or immunosuppressive treatment not related to lgG4- RD prior to experiencing a treated and AC-determined flare will be censored at the time when those treatment were first received.

[0536] Population-level summary: HR between PF-06835375 versus placebo and its associated 95% Cl. To assess the impact of immortal time bias, the following analysis will be performed:

[0537] • Target population: Subjects in the FAS

[0538] • Variable: Time in days from Day 1 (dosing) to the date of the first treated and AC-determined lgG4-RD flare within the 52-week RCP. Date of flare onset is defined as earliest date of subject reported new / worsening symptoms, new / worsening physical exam findings, new / worsening laboratory findings, incidental finding on imaging, or other collected on flare assessment page.

[0539] • Intercurrent event: All data captured during the 52-week RCP will be used for analysis. Subjects who do not complete the RCP and who have not had a treated and AC-determined flare during the RCP will be censored at the time of discontinuation. • Population-level summary: Hazard ratio...

Claims

CLAIMSWhat is claimed is:1 . A method of treating a subject with an immune or inflammatory disease comprising subcutaneous administration of a unit dose of at least 6 mg, 10 mg, 18 mg or 50 mg of an anti- CXCR5 antibody comprising a variable region of the light chain (VL) comprising the amino acid sequence of SEQ ID NO:1 and a variable region of the heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:5, wherein the unit dose is administered once a month.

2. The method of claim 1 , wherein the immune or inflammatory disease is selected from the group consisting of primary immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ANCA-vasculitis, pemphigus vulgaris, myasthenia gravis (MG) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).

3. The method of claim 1 or 2, wherein the dose is 18 mg.

4. The method of claim 1 or 2, wherein the dose is 50 mg.

5. The method of any one of claims 1-4, wherein in the subject has ITP.

6. The method of claim 5, further comprising i) measuring the absolute platelet count in the subject, and ii) if the absolute platelet count is greater than 50 x 103platelets per pL of blood, the absolute platelet count is at least 2-fold greater than the baseline value of the absolute platelet count for the subject, and the subject was not exposed to splenectomy or rescue medication before the measuring step, assigning a responder status to the subject on the modified overall response scale.

7. The method of claim 5, further comprising i) measuring the absolute platelet count in the subject, and ii) if the absolute platelet count is greater than 100 x 103platelets per pL of blood, and the subject was not exposed to splenectomy or rescue medication before the measuring step, assigning a responder status to the subject on the complete response scale.

8. A method of treating a subject with Immunoglobulin G4 related disease (lgG4-RD) comprising administration of an anti-CXCR5 antibody comprising a variable region of the light chain (VL) comprising the amino acid sequence of SEQ ID NO:1 and a variable region of the heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:5.

9. The method of claim 8 wherein the anti-CXCR5 antibody is administered subcutaneously.

10. The method of claim 8 wherein the anti-CXCR5 antibody is administered intravenously.

11. The method of any one of claims 8 to 10 wherein a dose of about 6 mg, about 10 mg, about 18 mg or about 50 mg of an anti-CXCR5 antibody is administered.

12. The method of claim 11 wherein the anti-CXCR5 antibody is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every month, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every two months, once every 12 weeks, once every three months, once every 16 weeks, once every four months, once every 20 weeks, once every five months, once every 24 weeks, once every six months, once every 26 weeks or once a year.

13. The method of claim 11 wherein 18 mg or 50 mg of the anti-CXCR5 antibody is administered on day one, week four, week eight and week twelve and optionally on week sixteen, week twenty and week twenty four of a twenty-four week treatment period.

14. The method of any one of claims 8 to 10 wherein one or more additional therapeutic agents are administered.

15. The method of claim 14 wherein the additional therapeutic agents are selected from the group consisting of glucocorticoids, disease-modifying anti-rheumatic drugs (DMARDs) and IB- cell depleting agents.

16. The method of claim 15 wherein the additional therapeutic agents are selected from the group consisting of prednisone, methotrexate, sulfasalazine, hydroxychloroquine, leflunomide, azathioprine, apremilast, cyclophosphamide, cyclosporine, mycophenolic acid, adalimumab, belimumab, infliximab, etanercept, tocilizumab, sarilumab, golimumab, canakinumab, guselkumab, risankizumab, secukinumab, ustekinumab, certolizumab pegol, secukinumab,ixekizumab, anakinra, tofacitinib, baricitinib.upacitinib, ruxolitinib, abrocitinib, ritlecitinib, inebilizumab and rituximab.

17. The method of claim 16 wherein a dose of about 6 mg, about 10 mg, about 18 mg or about 50 mg of an anti-CXCR5 antibody is administered.

18. The method of claim 17 wherein the anti-CXCR5 antibody is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every month, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every two months, once every twelve weeks, once every three months, once every sixteen weeks, once every four months, once every twenty weeks, once every five months, once every twenty four weeks, once every six months, once every twenty six weeks or once a year.

19. The method of claim 17 wherein 18 mg or 50 mg of the anti-CXCR5 antibody is administered on day one, week four, week eight and week twelve and optionally on week sixteen, week twenty and week twenty four of a twenty-four week treatment period.

20. The method of claim 18 wherein the anti-CXCR5 antibody is administered subcutaneously.

21. The method of claim 18 wherein the anti-CXCR5 antibody is administered intravenously.

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