Anti-CD79 antibodies and use of the same

Abstract

To provide anti-CD79 antibodies, and a method for treating arthritis by using the antibodies.SOLUTION: Provided are humanized and affinity matured antibodies (mAb) and fragments thereof that specifically bind to CD79 at high affinity. The anti-CD79 mAb and fragments thereof may be used to treat antibody-associated conditions, including autoimmune diseases, allergies, transplant rejection, or immune-mediated rejection of a therapeutics optionally in combination with an additional therapeutic agent. Furthermore, the anti-CD79 mAbs and fragments thereof can be used for diagnostic purposes, including to detect CD79 cells in biological samples and to diagnose B-cell associated disorders.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Description of Research with Federal Government Funds

[0001] This invention was made with government support under Grant No. 1R43AI120433-01 awarded by the government agency National Institute of Allergy and Infectious Diseases (NIAID). The government has certain rights in this invention.

[0002] Reference to a Sequence Listing, Table, or Computer Program

[0002] An official copy of the sequence listing is being submitted simultaneously with the specification via EFS-Web as an ASCII text file named "PRI011_ST25.txt", created on September 30, 2019, and having a size of 73 kilobytes. The sequence listing submitted via EFS-Web is part of the specification and is hereby incorporated by reference in its entirety.

Background Art

[0003]

[0003] B cells play an important role in the development of many autoimmune disorders, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis, and type I diabetes (T1D), as demonstrated by the efficacy of B cell-targeted therapies, such as rituximab, in these diseases. Unfortunately, current therapies are based on B cell depletion, which is problematic from a safety perspective. As a result of the resulting immunosuppression, long-term profound B cell depletion causes harmful effects of existing standard treatments, particularly opportunistic infections and activation from viral latency.

[0004]

[0004] CD79 (Cluster of Differentiation 79) is a transmembrane protein that forms a complex with the B cell receptor (BCR) and generates a signal after antigen recognition by the BCR. CD79 is composed of two distinct chains called CD79A and CD79B (previously known as Ig-alpha and Ig-beta). These form a heterodimer stabilized by disulfide bonds on the surface of B cells. Both CD79a and CD79b are members of the immunoglobulin superfamily. CD79 has been used as a pan-B cell marker and can be used for the detection of B cell neoplasms.

[0005]

[0005] Unlike anti-CD20 mAbs, the protective effect of CD79-targeted mAbs does not require cell depletion. Rather, they act by inducing a reversible state of unresponsiveness or anergy and thus do not participate in the generation of an immune response. In animal models, anti-CD79 antibodies have brought about immunosuppression and reduction of inflammation by inducing anergy in B cells.

Summary of the Invention

Means for Solving the Problems

[0006]

[0006] The present disclosure provides an antibody comprising a fragment thereof that specifically binds to CD79 with high affinity. The antibody may be monoclonal, chimeric or humanized. The chimeric anti-CD79 antibody comprising the fragment thereof may have non-human (e.g., mouse) complementarity-determining regions (CDRs) and non-human framework regions, and optionally one or more human constant domains. The non-human heavy and light chain variable regions include SEQ ID NOs: 1-2. The humanized anti-CD79 antibody comprising the fragment thereof may have non-human (e.g., mouse) CDRs and human framework regions, and optionally non-human framework amino acid residues adjacent to the CDRs and optionally one or more human constant domains. The non-human CDRs include, for example, VH CDR1-3 of SEQ ID NOs: 3-5 and VL-CDR1-3 of SEQ ID NOs: 6-8. The non-human CDRs include, for example, VH CDR1-3 of SEQ ID NOs: 3-5 and VL-CDR1-3 of SEQ ID NOs: 6-8.

[0007]

[0007] The disclosed humanized antibodies are anti-CD79 antibodies obtained by grafting the CDRs of SEQ ID NOs: 3-8 together with a selected number of framework residues of a mouse antibody into a human framework of the heavy chain and a human framework of the light chain. Anti-CD79 humanized antibodies can be prepared by combining nine variable regions of the humanized heavy chain (SEQ ID NOs: 9-17) and six variable regions of the humanized light chain (SEQ ID NOs: 18-23). The anti-CD79 antibodies disclosed herein also include those obtained from an affinity maturation library made from a humanized anti-CD79 antibody. The anti-CD79 antibody can be prepared using a heavy chain variable region selected from SEQ ID NOs: 9-17, 24-27, 32-41, 71, 72, and 75-77, and a light chain variable region selected from SEQ ID NOs: 18-23, 28-31, 42-56, and 73-74. The anti-CD79 antibody may also include a heavy chain variable region having 99%, 95%, 90%, 80% or 70% sequence identity with one of SEQ ID NOs: 9-17, 24-27, 32-41, 71, 72, and 75-77, and a light chain variable region having 99%, 95%, 90%, 80% or 70% sequence identity with one of SEQ ID NOs: 18-23, 28-31, 42-56, and 73-74. The anti-CD79 antibody can bind to CD79 with an affinity of 2.0-5.1 nM, or 45 nM to 300 nM, or 2.0 to 300 nM. The anti-CD79 antibody can bind with an affinity of at least 300 nM, or at least 140 nM, or at least 100 nM, or at least 5.1 nM, at least 3.8 nM, or at least 2.4 nM.

[0008]

[0008] The anti-CD79 antibodies described herein may include modifications that confer desired properties to the antibody. For example, the modification can extend the serum half-life of the antibody, or the modification can shorten the serum half-life. The modification can also enhance or decrease the effector function of the antibody. The modification will reduce immunogenicity or other undesirable side effects or adverse events caused by the anti-CD79 antibody.

[0009]

[0009] The anti-CD79 antibodies described herein can induce an anergic state in the B cells of a subject and can thus be used to treat certain autoimmune diseases. For example, autoimmune diseases associated with anti-self antibody responses can be treated with the anti-CD79 antibodies because the induced anergic state will prevent the production of anti-self antibodies. The anti-CD79 antibodies described herein can also be used to induce an anergic state in any condition having an unwanted antibody response. The anti-CD79 antibodies described herein can be used to induce an anergic state in B cells. The anti-CD79 antibodies described herein can be used to inhibit the proliferation of B cells. The anti-CD79 antibodies described herein can be used to prevent infusion reactions resulting from existing anti-drug antibodies. The anti-CD79 antibodies described herein can be used to prevent the formation of anti-drug antibodies after injection of biological therapies. Examples of unwanted antibody-related conditions can include, for example, autoimmune diseases, certain allergies (antibody-related allergies), certain types of type I diabetes, and the like. Autoimmune diseases that can be treated with the anti-CD79 antibodies can include, for example, systemic lupus erythematosus (SLE), inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), rheumatoid arthritis, multiple sclerosis, Graves' disease, CREST syndrome, systemic sclerosis, celiac disease, and the like. Other autoimmune diseases can include, for example, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid antibody syndrome, autoimmune angioedema , autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune ovaritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy (AMAN), Balo disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis with polyangiitis (EGPA), cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackievirus myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid gestationis (PG), hidradenitis suppurativa (HS) (inverse acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing diseases, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, Lyme disease, Ménière's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, cicatricial ocular pemphigoid, optic neuritis, relapsing rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), ParryRomberg syndrome, peripheral uveitis (peripheral iridocyclitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndromes types I, II, and III, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's disease, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease are mentioned. These undesirable antibody-related conditions can be treated by administering one or more of the anti-CD79 antibodies described herein to a subject suffering from the undesirable antibody-related condition.

[0010]

[0010] The anti-CD79 antibodies described herein can be used to treat CD79-positive hematopoietic malignancies such as lymphoma and leukemia. They can also be used in chimeric antigen receptors (CARs) to generate immune cells having an anti-CD79 CAR. Examples of such anti-CD79 CAR immune cells include T cells or natural killer cells having an anti-CD79 CAR. Anti-CD79 CAR T cells and / or natural killer cells can be used to treat diseases in which the causative cells of the disease present CD79. Such diseases include, for example, CD79-positive hematopoietic malignancies such as lymphoma and leukemia.

[0011] The following detailed description, which describes exemplary embodiments of the present disclosure, and the accompanying drawings will provide a better understanding of the features and advantages of the present disclosure.

Brief Description of the Drawings

[0012]

Figure 1

[0012] FIG. 1 is a graph showing the onset of type 1 diabetes over time in VH125NOD mice.

Figure 2

[0013] FIG. 2 is a graph showing the binding of cells expressing an anti-CD79 CAR-T construct of soluble CD79 antigen.

Figure 3a

[0014] FIG. 3a is a graph showing the competitive binding between the antibody LB517 / LB519 (hCur14 FALA) and Curly-14 regarding binding to B cells.

Figure 3b

Figure 3c

Figure 4a

[0015] FIG. 4a is a graph showing calcium influx in B cells after treatment with various anti-hCD79 antibodies.

Figure 4b

Figure 5

[0016] FIG. 5 is a graph showing the effect of treatment with an anti-hCD79 antibody on the onset of arthritis in an arthritis model system.

Figure 6

[0017] FIG. 6 is a diagram showing the effect of treatment with an anti-hCD79 antibody on the onset of lupus in a lupus model system.

Figure 7

[0018] FIG. 7 is a graph showing the effect of treatment with anti-hCD79 antibody on the onset of multiple sclerosis in a model system of MS.

DETAILED DESCRIPTION OF THE INVENTION

[0013]

[0019] Although various embodiments of the present disclosure are described herein, it will be apparent to those skilled in the art that such embodiments are presented by way of example only. Numerous modifications and variations to the embodiments described herein, as well as equivalents thereof and substitutes therefor, will be apparent to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments described herein may be utilized in practicing the present disclosure. It should also be understood that all embodiments of the present disclosure may optionally be combined with one or more of any other embodiments described herein that are consistent with that embodiment.

[0014]

[0020] When elements are presented in a list format (e.g., Markush group), it is understood that each possible subgroup of those elements is also disclosed and that any one or more elements may be removed from the list or group.

[0015]

[0021] Unless clearly indicated to the contrary, in any method described or claimed herein that includes two or more acts or steps, the order of the acts or steps of the method is not necessarily limited to the order in which the acts or steps of the method are recited, although the present disclosure also encompasses embodiments in which the order is so limited.

[0016]

[0022] In general, when embodiments of the present specification or claims are referred to as including one or more features, it is further understood that the present disclosure also encompasses embodiments consisting of or consisting essentially of such features.

[0017]

[0023] It is also understood that any embodiment of the present disclosure, for example, any embodiment found within the scope of the prior art, can be explicitly excluded from the claims, regardless of whether specific exclusions are enumerated herein.

[0018]

[0024] Any reference in this specification to an antibody or a peptide, polypeptide or protein such as a fragment thereof is further understood to include its pharmaceutically acceptable salts, unless specifically stated otherwise or the context clearly indicates otherwise. Such salts may have a positive net charge, a negative net charge, or no net charge.

[0019]

[0025] This specification includes headings for reference purposes and to help find specific sections. The headings are not intended to limit the scope and concepts of the embodiments described in the sections under such headings, and those embodiments and concepts may be applicable in other sections throughout the present disclosure.

[0020]

[0026] All patent documents and all non-patent documents cited in this specification are hereby incorporated by reference in their entirety to the same extent as if each patent document or non-patent document had been specifically and individually indicated to be incorporated herein by reference in its entirety.

[0021] Definitions

[0027] All technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, unless otherwise defined by their use in this specification or clearly indicated otherwise.

[0022]

[0028] As used in this specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" are to be construed to include both the singular and the plural referents unless specifically stated otherwise or the context clearly indicates otherwise.

[0023]

[0029] The terms "about" or "approximately" mean an acceptable error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within one standard deviation. In some embodiments, where no specific limit of error (e.g., standard deviation relative to a given average value in a graph or table of data) is recited, the term "about" or "approximately" means the range that would include the recited value, and also the range that would be included by rounding up or down the decimal portion of the recited value, taking into account significant figures. In certain embodiments, the term "about" or "approximately" means within ±10%, 5%, 4%, 3%, 2% or 1% of a particular value. Whenever the term "about" or "approximately" precedes the first numerical value in a series of two or more numerical values or ranges of numerical values, the term "about" or "approximately" applies to each of the numerical values or ranges of numerical values in that series.

[0024]

[0030] The term "antibody" is defined functionally as a binding protein and structurally as a protein that includes an amino acid sequence recognized as being derived from a framework region of an immunoglobulin-encoding gene. An antibody may consist of one or more polypeptides substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which as a result defines the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0025]

[0031] A common gamma immunoglobulin (antibody) structural unit is known to include a tetramer. Each tetramer is composed of two identical sets of polypeptide chains, each set having one "light" chain (about 25 kD) and one "heavy" chain (about 50 - 70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily involved in antigen recognition. The terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.

[0026]

[0032] Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments. Thus, for example, pepsin digests the antibody below the disulfide bonds in the hinge region to produce F(ab)’2, a dimer of Fab’, which itself is a light chain originally linked to VH-CH1-hinge by a disulfide bond. F(ab)’2 may be reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby converting the (Fab’)2 dimer to Fab’ monomers. Fab’ monomers are essentially Fab with a portion of the hinge region (see Fundamental Immunology, edited by W. E. Paul, Raven Press, N.Y. (1993) for a more detailed description of other antibody fragments). Although various antibody fragments are defined with respect to digestion of intact antibodies, one of ordinary skill in the art will understand that the fragments may be newly synthesized either chemically or by using recombinant DNA methods. Thus, the term antibody, as used herein, includes any antibody fragment produced by modification of the whole antibody or synthesized using recombinant DNA techniques. Preferred antibodies include single-chain antibodies (antibodies that exist as a single polypeptide chain), for example, single-chain Fv antibodies (sFv or scFv) in which the variable heavy and variable light regions are joined together (either directly or via a peptide linker) to form a continuous polypeptide, including V H -V L dimers. Single-chain Fv antibodies may be expressed from nucleic acids containing the V H and V L coding sequences joined together either directly or via a peptide linker to form a covalently linked V H -V L heterodimer (e.g., Huston et al., Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988, which is hereby incorporated by reference in its entirety). V H and V L are each linked as a single polypeptide chain, but V H and VL The domains interact non-covalently. Alternatively, the antibody may be another fragment. Other fragments may be formed, including the use of recombinant techniques. For example, if one of the (heavy or light) chains is fused to the g3 capsid protein and the complementary chain is secreted as a soluble molecule into the periplasm, the Fab molecule may be presented on the phage. The two chains may be encoded by the same or different replicons, and the two antibody chains in each Fab molecule assemble after translation, and the dimer is incorporated into the phage particle by binding to one of its chains to g3p (see, for example, U.S. Patent No. 5,733,743, which is incorporated herein by reference in its entirety). scFv antibodies, as well as numerous other structures that convert the polypeptide light and heavy chains of antibody V regions that would otherwise aggregate but are chemically separated into molecules that fold into a three-dimensional structure substantially similar to that of the antigen-binding site, are known to those of skill in the art (see, for example, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778, the entireties of all patents of which are incorporated herein by reference). Particularly preferred antibodies include any that are presented on phage or formed by recombinant techniques using vectors, in which case the chains are secreted as soluble proteins, such as scFv, Fv, Fab, (Fab’)2. Antibodies can also include diabodies and minibodies.

[0027]

[0033] Antibodies also include heavy chain dimers, such as antibodies from camelids. In camelids, the V H regions of the heavy chain dimer IgG do not need to make hydrophobic interactions with the light chain, so regions of the heavy chain that normally contact the light chain are changed to hydrophilic amino acid residues in camelids. The V H domains of the heavy chain dimer IgG are called V HH domains.

[0028]

[0034] In camelids, the diversity of the antibody repertoire is in V H or V HHIt is determined by the complementarity-determining regions (CDRs) 1, 2, and 3 of the domain. Camel V HH The CDR3 of the region is characteristically relatively long, averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9):1129, which is incorporated herein by reference in its entirety). This is in contrast to the CDR3 regions of antibodies of many other species. For example, mouse V H has an average of 9 amino acids in its CDR3.

[0029]

[0035] A library of antibody variable regions derived from camels, which maintains in vivo diversity of the variable regions of camels, can be prepared, for example, by the method disclosed in U.S. Patent Application Publication No. 20050037421, published on February 17, 2005, which is incorporated herein by reference in its entirety.

[0030]

[0036] As used herein, the term "binding specificity" of an antibody refers to the identity of the antigen to which the antibody binds, preferably the identity of the epitope to which the antibody binds.

[0037] As used herein, the term "chimeric polynucleotide" means that the polynucleotide contains wild-type regions and mutated regions. In some cases, it may mean that the polynucleotide contains wild-type regions from one polynucleotide and wild-type regions from another related polynucleotide.

[0031]

[0038] As used herein, the terms "complementary determining region" or "CDR" refer to terms recognized in the art as exemplified by Kabat and Chothia. CDRs are also known as hypervariable regions or hypervariable loops (Chothia and Lesk (1987) J Mol. Biol. 196: 901; Chothia et al., (1989) Nature 342: 877; E. A. Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) (1987); and Tramontano et al. (1990) J Mol. Biol. 215: 175, all of which are incorporated herein by reference in their entirety). "Framework region" or "FR" refers to the regions of the V domain adjacent to the CDRs. The positions of the CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, the international ImMunoGeneTics database (IMGT), and AbM (e.g., Johnson et al., supra; Chothia and Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions, Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). The definition of the antigen-binding site is also described below: Ruiz et al., IMGT, the international ImmunoGenetics database. Nucleic Acids Res., 28, 219 - 221 (2000); and Lefranc, M.-P. IMGT, the international ImmunoGenetics database. Nucleic Acids Res. Jan 1;29( 1):207 - 9 (2001); MacCallum et al., Antibody - antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262(5), 732 - 745 (1996); and Martin et al., Proc. Natl Acad. Sci. USA, 86, 9268 - 9272 (1989); Martin et al., Methods Enzymol., 203, 121 - 153, (1991); Pedersen et al., Immunomethods, 1, 126 (1992); and Rees et al., In Sternberg M. J. E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141 - 172, 1996, the entire contents of all of these references are hereby incorporated by reference).

[0032]

[0039] Whenever the term "at least" or "greater than" precedes the first of a series of two or more numerical values, the term "at least" or "greater than" applies to each of the numerical values in that series.

[0033]

[0040] The term "of heterologous origin" refers to an amino acid or nucleotide sequence that is not naturally associated with the amino acid or nucleotide sequence to which it is related.

[0041] Whenever the term "not exceeding" or "less than" precedes the first of a series of two or more numerical values, the term "not exceeding" or "less than" applies to each of the numerical values in that series.

[0034]

[0042] The term "polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include nucleotides with non-naturally occurring bases, nucleotides that are linked to other nucleotides by bonds other than the naturally occurring phosphodiester bond, and / or bases linked by bonds other than the phosphodiester bond. Non-limiting examples of nucleotide analogs include phosphorothioate, phosphorodithioate, phosphorotriester, phosphoramidate, boranophosphate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotide, peptide nucleic acid (PNA), and the like. Such polynucleotides may be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid molecule" generally refers to a larger polynucleotide. The term "oligonucleotide" generally refers to a shorter polynucleotide. In certain embodiments, an oligonucleotide comprises no more than about 50 nucleotides. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it is understood that this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T".

[0035]

[0043] The term "polypeptide" refers to a polymer composed of natural and / or non-natural amino acid residues, naturally occurring structural variants thereof, and / or synthetic non-natural analogs thereof, linked via peptide bonds. Synthetic polypeptides may be synthesized, for example, using an automated polypeptide synthesizer. Polypeptides may also be produced recombinantly in cells that express a nucleic acid sequence encoding the polypeptide. The term "protein" generally refers to a larger polypeptide. The term "peptide" generally refers to a shorter polypeptide. In certain embodiments, a peptide comprises no more than about 50, 40, or 30 amino acid residues. Polypeptides include antibodies and fragments thereof. Conventional notation is used herein to depict polypeptide sequences, with the left-hand terminus of the polypeptide sequence being the amino (N) terminus and the right-hand terminus of the polypeptide sequence being the carboxyl (C) terminus.

[0036]

[0044] A polypeptide may contain one or more modifications that may occur during the synthesis or cellular production of the polypeptide, such as one or more post-translational modifications, whether or not such modifications are intentional. Modifications can include, but are not limited to, glycosylation (e.g., N-linked and O-linked glycosylation), lipidation, phosphorylation, sulfation, acetylation (e.g., N-terminal acetylation), amidation (e.g., C-terminal amidation), hydroxylation, methylation, formation of intra- or intermolecular disulfide bonds, formation of a lactam between two side chains, formation of pyroglutamate, and ubiquitination. As another example, a polypeptide may be conjugated to a natural polymer (e.g., a polysaccharide) or a synthetic polymer (e.g., polyethylene glycol [PEG]), lipidated (e.g., acylated with an acyl group), or labeled with a detectable agent (e.g., a radionuclide, a fluorescent dye, or an enzyme). Pegylation can increase protease resistance, stability, and half-life, enhance solubility, and reduce aggregation of the polypeptide. 20 An acyl group), or may be labeled with a detectable agent (e.g., a radionuclide, a fluorescent dye, or an enzyme). Pegylation can increase protease resistance, stability, and half-life, enhance solubility, and reduce aggregation of the polypeptide.

[0037]

[0045] The term "conservative substitution" refers to the substitution of an amino acid in a polypeptide with a functionally, structurally, or chemically similar natural or non-natural amino acid. In certain embodiments, the following groups contain natural amino acids that are each conservative substitutions for one another. 1) Glycine (Gly / G), Alanine (Ala / A); 2) Isoleucine (Ile / I), Leucine (Leu / L), Methionine (Met / M), Valine (Val / V); 3) Phenylalanine (Phe / F), Tyrosine (Tyr / Y), Tryptophan (Trp / W); 4) Serine (Ser / S), Threonine (Thr / T), Cysteine (Cys / C); 5) Asparagine (Asn / N), Glutamine (Gln / Q); 6) Aspartic acid (Asp / D), Glutamic acid (Glu / E); and 7) Arginine (Arg / R), Lysine (Lys / K), Histidine (His / H)

[0046] In further embodiments, the following groups contain natural amino acids that are each conservative substitutions for one another. 1) Nonpolar: Ala, Val, Leu, Ile, Met, Pro (Proline / P), Phe, Trp; 2) Hydrophobic: Val, Leu, Ile, Phe, Tyr, Trp; 3) Aliphatic: Ala, Val, Leu, Ile; 4) Aromatic: Phe, Tyr, Trp, His; 5) Uncharged polar or hydrophilic: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gln, Tyr (tyrosine may also be considered a hydrophobic amino acid with a polar side chain); 6) Aliphatic hydroxyl or sulfhydryl containing: Ser, Thr, Cys; 7) Amide containing: Asn, Gln; 8) Acidic: Asp, Glu; 9) Basic: Lys, Arg, His; and 10) Small: Gly, Ala, Ser, Cys

[0047] In other embodiments, the amino acids may be grouped as follows. 1) Hydrophobic: Val, Leu, Ile, Met, Phe, Trp, Tyr; 2) Aromatic: Phe, Tyr, Trp, His; 3) Neutral hydrophilic: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gln; 4) Acidic: Asp, Glu; 5) Basic: Lys, Arg, His; and 6) Residues affecting main chain orientation: Pro, Gly

[0048] Polypeptides having one or more modifications to the parent polypeptide may optionally be referred to as "analogs", "derivatives" or "variants" of the parent polypeptide.

[0038]

[0049] The present disclosure encompasses pharmaceutically acceptable salts of polypeptides, including those having a positive net charge, those having a negative net charge, and those having no net charge.

[0050] The term "pharmaceutically acceptable" refers to substances (e.g., active ingredients or excipients) that are suitable for use in contact with the tissues and organs of a subject without undue irritation, allergic reactions, immunogenicity, and toxicity, and that provide a reasonable benefit / risk ratio and are effective for the intended use. A "pharmaceutically acceptable" excipient or carrier of a pharmaceutical composition is also compatible with the other components of the composition.

[0039]

[0051] The term "stringent hybridization conditions" refers to hybridization at 50% formamide, 5×SSC, at a temperature of 42°C and washing of the filter at 0.2×SSC, 60°C. (1×SSC is 0.15M NaCl, 0.015M sodium citrate.) Stringent hybridization conditions include washing, for example, 0.015M sodium chloride / 0.0015M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; washing in 0.2×SSC (sodium chloride / sodium citrate) at 42°C and washing in 50% formamide at 55°C, followed by washing in a high stringency wash solution consisting of 0.1×SSC containing EDTA at 55°C, formamide at 42°C, for example, 50% (v / v) formamide containing 50 mM sodium phosphate buffer, pH 6.5, containing 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 750 mM sodium chloride, 75 mM sodium citrate; or 50% formamide at 42°C, 5×SSC (0.75M NaCl, 0.075M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate, and also include low ionic strength and high temperature for hybridization using denaturing agents such as these.

[0040]

[0052] The term "subject" refers to an animal including, but not limited to, mammals such as primates (e.g., human, chimpanzee or monkey), rodents (e.g., rat, mouse, guinea pig, gerbil or hamster), lagomorphs (e.g., rabbit), suids (e.g., pig), equids (e.g., horse), canids (e.g., dog) or felids (e.g., cat).

[0041]

[0053] The terms "substantially identical" or "substantially the same" as related to two polypeptides or polynucleotides refer to two or more sequences or subsequences having at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid or nucleic acid residue identity when compared and aligned for maximum correspondence, as determined using a sequence comparison algorithm or by visual verification. The terms "substantially identical" or "substantially the same" may mean at least about 70% amino acid or nucleic acid residue identity. The terms "substantially identical" or "substantially the same" may mean at least about 85% amino acid or nucleic acid residue identity. Substantial correspondence or identity may exist over a region of a sequence that is at least about 20, 30, 40, 50, 100, 150 or 200 residues in length. The sequences may be substantially identical or the same over the entire length of either or both of the compared biopolymers.

[0042]

[0054] Optimal alignment of sequences for comparison may be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988); by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wisconsin), or by visual verification.

[0043]

[0055] An example of a useful algorithm is PILEUP. PILEUP creates multiple sequence alignments from a group of related sequences using progressive pairwise alignment to show relationships and percent sequence identity. This also shows a tree or phylogenetic tree indicating the cluster relationships used to create the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng and Doolittle, J. Mol. Evol., 35:351-360 (1987). The method used is similar to the method described by Higgins and Sharp, CABIOS, 5:151-153 (1989). This program can align up to about 300 sequences, each having a maximum length of about 5,000 nucleotides or amino acids. The multiple alignment procedure starts with a pairwise alignment of the two most similar sequences to generate a cluster of two aligned sequences. This cluster is then aligned with the next most related sequence or cluster of sequences to be aligned. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. This program is run by specifying the amino acid or nucleotide coordinates for a particular sequence and region of sequence comparison as well as by specifying program parameters. For example, a reference sequence may be compared with other test sequences to determine the percent sequence identity relatedness using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gap. Another algorithm useful for forming multiple alignments of sequences is Clustal W (see, e.g., Thompson et al., Nucleic Acids Research, 22:4673-4680

[1994] ).

[0044]

[0056] Another example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol., 215:403-410 (1990). Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. This algorithm begins by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, 1990). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. Subsequently, the word hits are extended in both directions along each sequence as far as possible while the cumulative alignment score increases. For nucleotide sequences, the parameter M (for pairs of matching residues) A cumulative score is calculated using a reward score (always >0) and an N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of a word hit in each direction is stopped in the following cases: when the cumulative alignment score decreases by an amount X from the maximum achieved value; when the cumulative score becomes zero or less due to the accumulation of one or more negatively scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, for example, a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses, by default, for example, a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915

[1989] ).

[0045]

[0057] In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787

[1993] ). One measure of similarity indicated by the BLAST algorithm is the minimum total probability [P(N)] that indicates the probability that a match between two nucleotide or amino acid sequences occurs by chance. In certain embodiments, a polynucleotide is considered to be similar to a reference sequence if the minimum total probability in the comparison of a test polynucleotide to a reference polynucleotide is less than about 0.1, 0.01, or 0.001.

[0046]

[0058] If two polypeptides differ only by conservative amino acid substitutions, the polypeptides may be substantially identical or identical to the second polypeptide. If two polynucleotides hybridize to each other under the stringent conditions described herein or under highly stringent conditions, the two nucleic acid sequences may be substantially identical or identical.

[0047]

[0059] The term "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject, prevents the medical condition being treated, reduces the risk that it will develop, delays its onset, slows its progression, or causes its regression, or alleviates to some extent the medical condition or one or more symptoms or complications of that condition. The term "therapeutically effective amount" also refers to an amount of a compound sufficient to elicit a biological or medical response in a cell, tissue, organ, system, animal, or human that is sought by a researcher, veterinarian, physician, or clinician.

[0048]

[0060] The terms "treat", "treating", and "treatment" include alleviating, improving, or suppressing a medical condition or one or more symptoms or complications associated with that condition, and alleviating, improving, or eradicating one or more causes of that condition. Reference to "treatment" of a medical condition includes prevention of that condition. The terms "prevent", "preventing", and "prevention" include preventing a medical condition or one or more symptoms or complications associated with that condition, reducing the risk that it will develop, and delaying its onset.

[0049] Anti-CD79 antibody

[0061] The antibodies described herein are specific for CD79 and include all of the above forms. The antibodies may be engineered for use in a particular organism. The organism may be a human, a canine, or a commercially useful domestic animal such as a pig, a horse, a dog, a cat, a chicken, or other fowl. Such engineering of the antibodies may be performed, for example, by the art Including CDR splicing, humanization, humaneering, chimerization, or isolated human (or other organism) antibodies using any repertory technology or monoclonal technology known in the art.

[0050]

[0062] The anti-CD79 antibody may include a heavy chain variable region selected from SEQ ID NOs: 9-17, 24-27, and 32-41, and a light chain variable region selected from SEQ ID NOs: 18-23, 28-31, and 42-56. For example, the heavy chain having the variable region of SEQ ID NO: 9 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 71, 72, 75, 76, or 77. The heavy chain having the variable region of SEQ ID NO: 10 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 11 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 12 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 13 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 14 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74.The heavy chain having the variable region of SEQ ID NO: 15 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 16 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 17 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 24 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 25 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 26 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 27 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 28 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 4. It may be combined with a light chain having a variable region of 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 33 may be combined with a light chain having a variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 34 may be combined with a light chain having a variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 35 may be combined with a light chain having a variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 36 may be combined with a light chain having a variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 37 may be combined with a light chain having a variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 38 may be combined with a light chain having a variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 39 may be combined with a light chain having a variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74.The heavy chain having the variable region of SEQ ID NO: 40 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 41 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 71 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 72 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 75 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 76 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74. The heavy chain having the variable region of SEQ ID NO: 77 may be combined with a light chain having the variable region of SEQ ID NO: 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 73, or 74.

[0051]

[0063] The anti-CD79 antibody may also be SEQ ID NOs: 9-17, 24-27, 32-41, 71, 7 2. It may include a heavy chain variable region having 99%, 95%, 90%, 80% or 70% sequence identity with one of 75-77, and a light chain variable region selected from SEQ ID NOs: 18-23, 28-31, 42-56, and 73-74. The anti-CD79 antibody may also include a heavy chain variable region having 99%, 95%, 90%, 80% or 70% sequence identity with one of SEQ ID NOs: 9-17, 24-28, and 33-43, and a light chain variable region having 99%, 95%, 90%, 80% or 70% sequence identity with one of SEQ ID NOs: 18-23, 28-31, 42-56, and 73-74. The anti-CD79 antibody may also include a heavy chain variable region selected from SEQ ID NOs: 9-17, 24-27, 32-41, 71, 72, and 75-77, and a light chain variable region having 99%, 95%, 90%, 80% or 70% sequence identity with one of SEQ ID NOs: 18-23, 28-31, 42-56, and 73-74. The anti-CD79 antibody can bind to CD79 with an affinity of 2.0-5.1 nM, or 45 nM to 300 nM, or 2.0 to 300 nM. The anti-CD79 antibody can bind with an affinity of at least 300 nM, or at least 140 nM, or at least 100 nM, or at least 5.1 nM, at least 3.8 nM, or at least 2.4 nM.

[0052]

[0064] Affinity maturation is used with the antibodies disclosed herein to obtain anti-CD79 antibodies with the desired affinity. When anti-CD79 antibodies are obtained from animals (e.g., transgenic animals having a human antibody repertoire), the antibodies produced in the transgenic animals may undergo affinity maturation. Alternatively, antibodies from transgenic animals, or other techniques (such as display techniques), may be affinity matured using screening and / or selection for antibodies with greater affinity following chain shuffling approaches and / or mutagenesis of the nucleic acids encoding VH and VL.

[0053]

[0065] The most widely used method for retaining specificity and affinity while minimizing the immunogenicity of non-human antibodies involves grafting the CDRs of non-human antibodies onto a human framework generally selected with respect to structural homology to the non-human framework (Jones et al., 1986, Nature 321:522-5; U.S. Patent No. 5,225,539, both of which are incorporated herein by reference in their entirety). The affinity of CDR-grafted antibodies can be improved by including some non-human residues at key positions in the framework (Bajorath et al., 1995, J Biol Chem 270:22081-4; Martin et al., 1991, Methods Enzymol. 203:121-53; Al-Lazikani, 1997, J Mol Biol 273:927-48, all of which are incorporated herein by reference in their entirety). An exemplary method for humanizing antibodies by CDR grafting is disclosed, for example, in U.S. Patent No. 6,180,370, which is incorporated herein by reference in its entirety.

[0054]

[0066] Improvements to conventional CDR grafting approaches use various hybrid selection approaches, where in successive iterations of selection for antigen binding, portions of the non-human antibody are combined with a library of complementary human antibody sequences, and in this process most of the non-human sequences are gradually replaced with human sequences. For example, in chain shuffling technology (Marks et al., 1992, Biotechnology 10:779-83, which is incorporated herein by reference in its entirety), one chain of a non-human antibody is combined with a naive human repertoire of the other chain on the basis of a rationale that the affinity of the non-human chain will be sufficient to constrain the selection of a human partner for the same epitope on the antigen. The selected human partner is then used to guide the selection of the human counterpart for the remaining non-human chain.

[0055]

[0067] Another technique is chain replacement technology, in which the non-human CDR3 is retained Rather, only the remaining portions of the V regions, including the framework and CDR1 and 2, were individually replaced in a stepwise fashion (e.g., U.S. Patent Application Publication No. 20030166871; Rader et al., Proc Natl Acad Sci USA 95:8910-15, 1998; Steinberger et al., J. Biol. Chem. 275:36073-36078, 2000; Rader et al., J. Biol. Chem. 275:13668-13676, 2000, the entire contents of all of these references are incorporated herein by reference).

[0056]

[0068] These techniques can be used to generate antibodies suitable for use in non-human subjects by placing the CDRs into the framework regions of the target species using an approach similar to the CDR grafting method used to generate antibodies for use in humans.

[0057]

[0069] Anti-CD79 antibodies may be generated using a starting anti-CD79 antibody and grafting portions of the variable region of the starting anti-CD79 antibody (e.g., CDRs) into a desired variable domain framework. The murine variable regions were as follows.

[0058]

Chemical Formula

[0059] (Heavy chain variable region, SEQ ID NO: 1)

[0060]

Chemical Formula

[0061] (Light chain variable region, SEQ ID NO: 2)

[0070] Anti-CD79 humanized antibodies were generated, for example, using the following CDRs.

[0062]

Chemical Formula

[0063]

[0071] Three VH CDRs were placed in the framework sequence of the human variable region for the heavy chain, creating the following VH chains.

[0064]

Chem.

[0065]

[0072] This VH was designated as H1. Substitutions were incorporated into the framework region of SEQ ID NO: 9, creating the following two additional VH chains.

[0066]

Chem.

[0067] SEQ ID NO: 10 was designated as H2.

[0068]

Chem.

[0069]

[0073] SEQ ID NO: 11 was designated as H3. Further changes may be incorporated into the framework region of SEQ ID NO: 9 and CDR2 in order to increase affinity and / or to stabilize the antibody against, for example, oxidation, deamination, and / or protease cleavage. Examples of such heavy chain sequences are as follows.

[0070]

Chem.

[0071]

[0074] SEQ ID NO: 12 was designated as H4.

[0072]

Chem.

[0073]

[0075] SEQ ID NO: 13 was designated as H5.

[0074]

Chem.

[0075]

[0076] Sequence number 14 is designated as H6.

[0076]

Chem.

[0077]

[0077] Sequence number 15 is designated as H7.

[0078]

Chem.

[0079]

[0078] Sequence number 16 is designated as H8.

[0080]

Chem.

[0081]

[0079] Sequence number 17 is designated as H9.

[0080] Three VL CDRs were arranged in the framework sequence for the human variable region of the light chain to create the following VL chain.

[0082]

Chem.

[0083]

[0081] Sequence number 18 was designated as L1. A substitution was incorporated into the framework region of sequence number 18 to produce the following additional VL chain.

[0084]

Chem.

[0085]

[0082] SEQ ID NO: 19 was designated as L2.

[0086]

Chem.

[0087]

[0083] SEQ ID NO: 20 was designated as L3. Further modifications may be incorporated into SEQ ID NO: 18 of the framework region and CDR1 in order to enhance affinity and / or to stabilize the antibody against, for example, oxidation, deamination, and / or protease cleavage. Examples of such light chain sequences are as follows.

[0088]

Chem.

[0089]

[0084] SEQ ID NO: 21 was designated as L4.

[0090]

Chem.

[0091]

[0085] SEQ ID NO: 22 was designated as L5.

[0092]

Chem.

[0093]

[0086] SEQ ID NO: 23 was designated as L6.

[0087] The three VH chains (H1 - H3) were combined with the three VL chains (L1 - L3) by a combinatorial approach, and each antibody was recombinantly expressed and isolated. The H1 - H3 and L1 - L3 antibody chains have related framework regions that differ at a small number of positions, which will improve affinity. These candidates were tested for affinity against CD79. These candidates had K values in the range of 45 nM to 300 nM. dIt bound to CD79. From these results, H1L2 (LB495 / PRI47, SEQ ID NOs: 9 and 19) was selected.

[0094]

[0088] The present disclosure includes pharmaceutically acceptable salts of anti-CD79 antibodies including those having a positive net charge, those having a negative net charge, and those having no net charge, and includes, but is not limited to, salts of anti-CD79 antibodies including the compound, in a pharmaceutical composition, for its therapeutic and diagnostic use, and its fragments in its preparation.

[0095] Affinity maturation

[0089] The humanized antibody H1L2 (SEQ ID NOs: 9 and 19) was affinity matured. Two phage display libraries were made from the heavy chain H1 (LB495) and two from the light chain L2 (PRI47). Saturation mutagenesis was performed on the CDR3 of the heavy chain (H1) and the light chain (L2). The mutagenized heavy chain was combined with L2, and the mutagenized light chain was combined with H1. Error-prone PCR was used to randomly mutagenize the heavy chain (H1) and the light chain (L2), and the mutagenized heavy chain was combined with L2, and the mutagenized light chain was combined with H1. Four libraries were made in phage display format for further screening.

[0096]

[0090] Each library was panned against CD79 in competitive binding, which included the humanized antibody H1L2. Clones were selected from the library in competitive panning, and the humanized H1L2 antibody was added to the competitive panning. The clones had to compete with the humanized H1L2 antibody to attach to CD79 on the substrate. Thirty-seven (37) clones were obtained from the competitive panning of the four libraries (five (5) from the CDR3 heavy chain library, four (4) from the CDR3 light chain library, eleven (11) from the heavy chain library, and 17 from the light chain library). The sequences of these five clones from the CDR3 heavy chain library were as follows.

[0097]

Chem.

[0098]

[0091] SEQ ID NO: 24 was designated as LB509 - A7.

[0099]

Chem.

[0100]

[0092] SEQ ID NO: 25 was designated as LB509 - C2.

[0101]

Chem.

[0102]

[0093] SEQ ID NO: 26 was designated as LB509 - C10.

[0103]

Chem.

[0104]

[0094] SEQ ID NO: 27 was designated as LB509 - G2.

[0105]

Chem.

[0106]

[0095] SEQ ID NO: 71 was also designated as LB509 - H1.

[0096] The sequences of four clones from the CDR3 light chain library were as follows.

[0107]

Chem.

[0108]

[0097] SEQ ID NO: 28 was designated as LB511-A9.

[0109]

Chem.

[0110]

[0098] SEQ ID NO: 29 was designated as LB511-B6.

[0111]

Chem.

[0112]

[0099] SEQ ID NO: 30 was designated as LB511-F6.

[0113]

Chem.

[0114]

[0100] SEQ ID NO: 31 was designated as LB511-F11.

[0101] The sequences of 11 clones from the heavy chain library were as follows.

[0115]

Chem.

[0116]

[0102] SEQ ID NO: 32 was designated as LB510-B5.

[0117]

Chem.

[0118]

[0103] SEQ ID NO: 33 was designated as LB510-C7.

[0119]

Chem.

[0120]

[0104] SEQ ID NO: 34 was designated as LB510-C8.

[0121]

Chem.

[0122]

[0105] SEQ ID NO: 35 was designated as LB510-F10.

[0123]

Chem.

[0124]

[0106] SEQ ID NO: 36 was designated as LB510-G4.

[0125]

Chem.

[0126]

[0107] SEQ ID NO: 37 was designated as LB510-G5.

[0127]

Chem.

[0128]

[0108] SEQ ID NO: 38 was designated as LB510-G6.

[0129]

Chem.

[0130]

[0109] SEQ ID NO: 39 was designated as LB510-G7.

[0131]

Chem.

[0132]

[0110] The sequence number 72 was also designated as LB510-H4.

[0133]

Chem.

[0134]

[0111] The sequence number 40 was designated as LB510-H7.

[0135]

Chem.

[0136]

[0112] The sequence number 41 was designated as LB510-H11.

[0113] The sequences of 17 clones from the light chain library were as follows.

[0137]

Chem.

[0138]

[0114] The sequence number 42 was designated as LB512-A7.

[0139]

Chem.

[0140]

[0115] The sequence number 43 was designated as LB512-A10.

[0141]

Chem.

[0142]

[0116] The sequence number 44 was designated as LB512-B8.

[0143]

Chem.

[0144]

[0117] The sequence number 45 was designated as LB512 - B10.

[0145]

Chemical formula

[0146]

[0118] The sequence number 46 was designated as LB512 - C2.

[0147]

Chemical formula

[0148]

[0119] The sequence number 47 was designated as LB512 - E2.

[0149]

Chemical formula

[0150]

[0120] The sequence number 48 was designated as LB512 - E5.

[0151]

Chemical formula

[0152]

[0121] The sequence number 49 was designated as LB512 - E8.

[0153]

Chemical formula

[0154]

[0122] The sequence number 50 was designated as LB512 - E10.

[0155]

Chemical formula

[0156]

[0123] The sequence number 73 was also designated as LB512-F7.

[0157]

Chem.

[0158]

[0124] The sequence number 51 was designated as LB512-F11.

[0159]

Chem.

[0160]

[0125] The sequence number 52 was designated as LB512-G2.

[0161]

Chem.

[0162]

[0126] The sequence number 74 was also designated as LB512-G5.

[0163]

Chem.

[0164]

[0127] The sequence number 53 was designated as LB512-H5.

[0165]

Chem.

[0166]

[0128] The sequence number 54 was designated as LB512-H7.

[0167]

Chem.

[0168]

[0129] The accession number 55 was designated as LB512-H8.

[0169]

Chemical formula

[0170]

[0130] The accession number 56 was designated as LB512-H11.

[0131] These 37 clones were screened in a competitive ELISA Clones that bound better than the patent antibody (H1L2) were selected. Nine clones with even better affinity were obtained from competitive ELISA. Two from the heavy chain CDR3 library (LB509-C2, SEQ ID NO: 25; and LB509-G2, SEQ ID NO: 27), four from the light chain CDR3 library (LB511-A9, SEQ ID NO: 28; LB511-B6, SEQ ID NO: 29; LB511-F6, SEQ ID NO: 30; LB511-F11, SEQ ID NO: 31), two from the heavy chain library (LB510-C7, SEQ ID NO: 33; LB510-G5, SEQ ID NO: 37), and one from the light chain library (A7, SEQ ID NO: 42). These clones were subjected to a confirmatory dilution ELISA, and five clones that showed even better binding were selected (heavy chain CDR clone LB509-C2, SEQ ID NO: 25; light chain CDR clones LB511-A9, SEQ ID NO: 28, LB511-B6, SEQ ID NO: 29, LB511-F6, SEQ ID NO: 30, and LB511-F11, SEQ ID NO: 31). Heavy chain CDR clone C2 had a change from Y to L in CDR3, and light chain clones A9, B6, F6, and F11 had amino acid changes from F to L, V, I, or R at the same position in CDR3. Two heavy chains with the H1 sequence, designated LB495 (SEQ ID NO: 10) or the change from Y to L designated LB517 (SEQ ID NO: 25) were prepared, and five light chains with the L2 sequence, designated PRI47 (SEQ ID NO: 19), or the change from F to I in the CDR3 of L2 designated LB518 (SEQ ID NO: 28), the change from F to L in the CDR3 of L2 designated LB519 (SEQ ID NO: 30), the change from F to R in the CDR3 of L2 designated LB520 (SEQ ID NO: 31), or the change from F to V in the CDR3 of L2 designated LB521 (SEQ ID NO: 29) were prepared. Each of these two heavy chains was individually combined with the five light chains by a combinatorial approach, and these antibodies were tested for binding kinetics.

[0171]

[0132] The affinity of binding to CD79 of all combinations of affinity matured antibodies was higher than that of the parental antibody H1L2. The parental antibody had a K of 5.1 nM for CD79, and the parental d ​ The binding affinity matured antibody had a K of 3.6 nM to 2.0 nM against CD79 d .

[0172] Anti-CD79 antibody modification

[0133] The anti-CD79 antibody may include a portion that extends the half-life (T 1 / 2 ) or / and the duration of action of the antibody. The portion may extend the circulating T 1 / 2 , blood T 1 / 2 , plasma T 1 / 2 , serum T 1 / 2 , elimination phase T 1 / 2 , biological T 1 / 2 , excretion T 1 / 2 or functional T 1 / 2 of the antibody, or any combination thereof.

[0173]

[0134] The anti-CD79 antibody may be modified by a single portion. Alternatively, the anti-CD 79 antibody may be modified by two or more substantially similar or identical portions or two or more portions of the same type. The anti-CD79 antibody may include two or more portions of different types, or two or more different types of portions. Two or more anti-CD79 antibodies may be bound to one portion. The bond between the anti-CD79 antibody and its portion may be covalent or non-covalent.

[0174]

[0135] The polypeptide portion may be recombinantly fused to the N-terminus or C-terminus of the heavy or light chain of the anti-CD79 antibody , optionally via a linker. The linker may include about 4 to 30 amino acid residues. The linker may include about 6 or 8 amino acid residues to about 20 amino acid residues, or about 6 or 8 amino acid residues to about 15 amino acid residues.

[0175]

[0136] The extension portion may be human serum albumin (HSA) or the fetal Fc receptor (F It may be a part thereof (e.g., domain III) that binds to cRn). HSA or its FcRn-binding portion can optionally have one or more mutations that confer beneficial properties or effects. In some embodiments, HSA or its FcRn-binding portion has one or more mutations such as K573P and / or E505G / V547A that improve HSA binding to pH-dependent FcRn and / or extend the HSA half-life. The extension portion may be an unstructured polypeptide.

[0176]

[0137] The extension portion may be a carboxy-terminal peptide (CTP) derived from the β-subunit of human chorionic gonadotropin (hCG). In the human body, the 4th, 5th, 7th, and 8th serine residues of the 34aa CTP of hCG-β are generally conjugated to O-glycans ending in sialic acid residues.

[0138] The extension portion may be one, two, three, four, five or more portions of a synthetic polymer.

[0177] The synthetic polymer may be biodegradable or non-biodegradable. Examples of biodegradable polymers useful as the extension portion include, but are not limited to, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA). Examples of non-biodegradable polymers useful as the extension portion include, but are not limited to, poly(ethylene glycol) (PEG), polyglycerol, poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), polyoxazoline, and poly(N-vinylpyrrolidone) (PVP). The synthetic polymer may be poly(ethylene glycol) (PEG). Pegylation may be performed by chemical or enzymatic, site-specific ligation, or by random ligation.

[0139] The individual mass (e.g., average molecular weight) of one or more synthetic polymer portions, or the total mass

[0178]

[0139] The individual mass (e.g., average molecular weight) of one or more synthetic polymer portions, or the total mass The amount may be about 10 - 50, 10 - 20, 20 - 30, 30 - 40 or 40 - 50 kDa, or may be about 10, 20, 30, 40 or 50 kDa. The individual mass (e.g., average MW) or total mass of one or more synthetic polymer moieties may also be about 50 - 100, 50 - 60, 60 - 70, 70 - 80, 80 - 90 or 90 - 100 kDa, or about above about 50 kDa, such as about 60, 70, 80, 90 or 100 kDa. Further, the mass of an individual synthetic polymer moiety (e.g., average MW) may be less than about 10 kDa, such as about 1 - 5 or 5 - 10 kDa, or about 5 kDa. The individual mass (e.g., average MW) or total mass of one or more synthetic polymer (e.g., PEG) moieties may be about 20 or 40 kDa.

[0179] Pharmaceutical composition

[0140] A further embodiment of the present disclosure relates to a pharmaceutical composition comprising an anti - CD79 antibody, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more pharmaceutically acceptable excipients or carriers. The composition may optionally contain an additional therapeutic agent. Generally, a pharmaceutical composition contains a therapeutically effective amount of an anti - CD79 antibody or fragment thereof, one or more pharmaceutically acceptable excipients or carriers and optionally a therapeutically effective amount of an additional therapeutic agent, and is formulated for administration to a subject for therapeutic use.

[0180]

[0141] Pharmaceutical compositions are generally prepared in accordance with current Good Manufacturing Practice (GMP) recommended or required by, for example, Federal Food, Drug, and Cosmetic Act §501(a)(2 )(B) and the International Conference on Harmonization Q7 guidelines.

[0181]

[0142] Pharmaceutical compositions / formulations can be prepared in a sterile form. For example, for injection or Parenteral dosage forms for administration by injection are generally sterilized. Sterile pharmaceutical compositions / formulations are formulated or manufactured according to pharmaceutical-grade sterilization standards known to those of skill in the art, such as those disclosed in or required by chapters 797, 1072, and 1211 of the United States Pharmacopeia and Title 21, Code of Federal Regulations, Part 211.

[0182]

[0143] Pharmaceutically acceptable excipients and carriers include pharmaceutically acceptable substances, materials, vehicles. Non-limiting examples of types of excipients include liquid and solid diluents, diluents, binders, lubricants, glidants, surfactants, dispersants, disintegrants, emulsifiers, wetting agents, suspending agents, thickening agents, solvents, isotonic agents, buffers, pH adjusters, absorption retardants, stabilizers, antioxidants, preservatives, antibacterial agents, antifungal agents, chelating agents, adjuvants, sweeteners, flavorants, colorants, encapsulating materials, and coating materials. The use of such excipients in pharmaceutical formulations is known in the art. For example, conventional vehicles and carriers include oils (e.g., vegetable oils such as olive oil and sesame oil), aqueous solvents {e.g., saline, buffered saline (e.g., phosphate buffered saline [PBS]), and isotonic solutions (e.g., Ringer's solution)}, and organic solvents (e.g., dimethyl sulfoxide [DMSO] and alcohols [e.g., ethanol, glycerol, and propylene glycol]), but are not limited thereto. The present disclosure encompasses the use of conventional excipients and carriers in formulations containing an anti-CD79 antibody or fragment thereof, provided that any conventional excipient or carrier is not incompatible with the anti-CD79 antibody or fragment thereof. For example, Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins (Philadelphia, Pennsylvania) (2005); Handbook of Pharmaceutical Excipients, 5th Edition, edited by Rowe et al., The Pharmaceutical Press and the See American Pharmaceutical Association (2005); Handbook of Pharmaceutical Additives, 3rd Edition, edited by Ash and Ash, Gower Publishing Co. (2007); and Pharmaceutical Pre-formulation and Formulation, edited by Gibson, CRC Press (Boca Raton, Florida) (2004).

[0183]

[0144] Suitable formulations are determined by various factors such as the selected route of administration. Possible routes of administration of a pharmaceutical composition containing an anti-CD79 antibody or a fragment thereof include oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial, intraperitoneal, intramedullary, intrathecal, and topical), intracavitary, and topical (including epidermal / on the skin, transdermal, mucosal, transmucosal, intranasal [e.g., by nasal spray or nasal drops], intraocular [e.g., by eye drops], pulmonary [e.g., by oral or nasal inhalation], buccal, sublingual, rectal [e.g., by suppository], and vaginal [e.g., by suppository]), but are not limited thereto. Topical formulations can be designed to provide local or systemic therapeutic effects. In certain embodiments, the anti-CD79 antibody or a fragment thereof is administered parenterally (e.g., intravenously, subcutaneously, intramuscularly, or intraperitoneally) by injection or by infusion over a period of time (e.g., as a bolus administration).

[0184]

[0145] Excipients and carriers that can be used to prepare parenteral formulations include , solvents (e.g., aqueous solvents such as water, saline, physiological saline, buffered saline [e.g., phosphate buffered saline], balanced salt solutions [e.g., Ringer's BSS] and aqueous dextrose solutions), isotonic / iso-osmotic agents (e.g., salts [e.g., NaCl, KCl and CaCl2] and sugars [e.g., sucrose]), buffers and pH adjusters (e.g., sodium dihydrogen phosphate / disodium hydrogen phosphate, citric acid / sodium citrate and L-histidine / L-histidine HCl), and emulsifiers (e.g., nonionic surfactants such as polysorbates [e.g., polysorbate 20 and 80] and poloxamers [e.g., poloxamer 188]) may be mentioned, but are not limited thereto. For protein formulations and delivery systems, see, for example, A. J. Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 3rd Edition, CRC Press (Boca Raton, Florida) (2015).

[0185]

[0146] The excipient may optionally enhance the stability of the protein or increase the solubility of the protein It may also include one or more substances that dissolve, inhibit protein aggregation, or reduce the solution viscosity, or any combination or all of them. Examples of such substances include hydrophilic amino acids (e.g., arginine and histidine), polyols (e.g., myo-inositol, mannitol, and sorbitol), saccharides {e.g., glucose (including D-glucose [dextrose]), lactose, sucrose, and trehalose}, osmolytes (e.g., trehalose, taurine, amino acids [e.g., glycine, sarcosine, alanine, proline, serine, β-alanine, and γ-aminobutyric acid], and betaines [e.g., trimethylglycine and trimethylamine N-oxide]), and nonionic surfactants {e.g., alkyl polyglycosides, ProTek® alkyl saccharides (e.g., monosaccharides [e.g., glucose] or disaccharides [e.g., maltose or sucrose] linked to long-chain fatty acids or the corresponding long-chain alcohols), and polypropylene glycol / polyethylene glycol block copolymers (e.g., poloxamers [e.g., Pluronic® F-68], and Genapol® PF-10 and its variants)}, but are not limited thereto. Such substances may be used to increase the solubility of the protein and to increase the protein concentration in the formulation. A higher protein concentration in the formulation is particularly advantageous for subcutaneous administration where the volume of rapid administration is limited (e.g., ≦ about 1.5 mL). Further, such substances may be used to stabilize the protein during the preparation, storage, and reconstitution of the lyophilized protein.

[0186]

[0147] For parenteral (e.g., intravenous, subcutaneous, or intramuscular) administration, one or more excipients A sterile solution or suspension of an anti-CD79 antibody in an aqueous solvent containing an agent may be prepared in advance and provided, for example, as a prefilled syringe. Alternatively, the anti-CD79 antibody may be dissolved or suspended in an aqueous solvent that may optionally contain one or more excipients prior to lyophilization (freeze-drying). The lyophilized anti-CD79 antibody stored in a suitable container (e.g., vial) may be reconstituted immediately prior to parenteral administration using, for example, sterile water that may optionally contain one or more excipients. When the anti-CD79 antibody is administered by injection (e.g., intravenously), the reconstituted solution or suspension of the anti-CD79 antibody may be added to an infusion bag containing, for example, sterile saline (e.g., about 0.9% NaCl) and diluted therein.

[0187]

[0148] Excipients for improving the transmucosal penetration of smaller proteins include cyclodex trin, alkyl saccharides (e.g., alkyl glycosides and alkyl maltosides [e.g., tetradecyl maltoside]), and bile acids (e.g., cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, chenodeoxycholic acid, and dehydrocholic acid), but are not limited thereto.

[0188]

[0149] Excipients for improving the trans-epithelial or transdermal penetration of smaller proteins include chemical penetration enhancers (CPEs, including fatty acids [e.g., oleic acid]), cell-penetrating peptides {CPPs, including arginine-rich CPPs [e.g., polyarginine, e.g., R6~R 11(For example, R6 and R9) and TAT-related CPPs, such as TAT(49-57), and amphiphilic CPPs [such as Pep-1 and penetratin], and skin-permeable peptides (SPPs, such as skin-permeable and cell-invasive peptides) are included, but not limited thereto. The transdermal penetration of smaller proteins can be further enhanced by the use of physical enhancement techniques, such as iontophoresis, cavitation or non-cavitational ultrasound, electroporation, thermal ablation, high frequency, microdermabrasion, microneedles or jet injection. US2007 / 0269379 provides an extensive list of CPEs. F. Milletti, Drug Discov. Today, 17: 850-860 (2012) is an overview of CPPs. R. Ruan et al., Ther. Deliv., 7: 89-100 (2016) describes CPPs and SPPs for the transdermal delivery of macromolecules, and M. Prausnitz and R. Langer, Nat. Biotechnol., 26: 1261-1268 (2008) describes various transdermal drug delivery methods.

[0189]

[0150] The anti-CD79 antibody can be delivered from a sustained-release composition. As used herein the term "sustained-release composition" includes sustained-release, extended-release, long-term release, slow-release and controlled-release compositions, systems and devices. Protein delivery systems are described, for example, in Banga (supra). The sustained-release composition can deliver a therapeutically effective amount of the anti-CD79 antibody over a long period of time. In some embodiments, the sustained-release composition delivers the anti-CD79 antibody for at least about 3 days, 1 week, 2 weeks, 3 weeks, 1 month (4 weeks), 6 weeks, 2 months, 3 months or longer. The sustained-release composition may be administered, for example, parenterally (e.g., intravenously, subcutaneously or intramuscularly).

[0190]

[0151] Sustained-release compositions of proteins include, for example, particulate systems, lipid or oily compositions, It may also be in the form of an implant. Examples of particulate systems include, but are not limited to, nanoparticles, nanospheres, nanocapsules, microparticles, microspheres and microcapsules. Nanoparticulate systems generally have a diameter or equivalent dimension of less than about 1 μm. In certain embodiments, the nanoparticles, nanospheres or nanocapsules have a diameter or equivalent dimension not exceeding about 500, 400 or 300 nm, or not exceeding about 200, 150 or 1 00 nm. In some embodiments, the microparticles, microspheres or microcapsules have a diameter or equivalent dimension of about 1-200, 100-200 or 50-150 μm, or about 1-100, 1-50 or 50-100 μm. Nano- or microcapsules generally contain a therapeutic agent in a central core, although the therapeutic agent is generally dispersed throughout the nano- or microparticles or spheres. In certain embodiments, the nanoparticle system is administered intravenously, while the microparticle system is administered subcutaneously or intramuscularly.

[0191]

[0152] In some embodiments, the sustained release particulate system or implant is made of a biodegradable polymer and / or hydrogel. In certain embodiments, the biodegradable polymer includes lactic acid and / or glycolic acid [e.g., L-lactic acid-based copolymers such as poly(L-lactide-co-glycolide) or poly(L-lactic acid-co-D,L-2-hydroxyoctanoic acid)]. Non-limiting examples of polymers from which hydrogels can be formed include polyvinyl alcohol, acrylate polymers (e.g., sodium polyacrylate), and other homopolymers and copolymers having a relatively large number of hydrophilic groups (e.g., hydroxyl and / or carboxylate groups). The biodegradable polymer of the particulate system or implant may be selected such that the polymer substantially completely degrades around the time when the treatment period is expected to end and the degradation by-products of the polymer are biocompatible like the polymer.

[0192] Alternatively, the sustained release composition of the protein may be composed of a non-biodegradable polymer. Examples of non-biodegradable polymers include, but are not limited to, poloxamers (e.g., poloxamer 407). The sustained release composition of the protein may also be composed of other natural or synthetic substances or materials, such as hydroxyapatite.

[0193]

[0154] The sustained release lipid or oily composition of the protein may be in the form of, for example, liposomes, micelles (e.g., those composed of biodegradable natural or / and synthetic polymers, such as lactosomes), and emulsions in oil. For example, it may be in the form of liposomes, micelles (e.g., those composed of biodegradable natural or / and synthetic polymers, such as lactosomes), and emulsions in oil.

[0194]

[0155] The sustained release composition may be formulated or designed as a depot that may be injected subcutaneously or intramuscularly, or implanted. The depot may be in the form of, for example, a polymeric particle system, a polymeric implant, or a lipid or oily composition. The depot formulation may contain a protein in a biocompatible solvent system and a mixture of, for example, a biodegradable polymer [e.g., poly(lactide-co-glycolide)] or a semi-biodegradable polymer (e.g., a block copolymer of lactic acid and PEG), whether or not such a mixture forms a particle system or an implant. For example, it may be formulated or designed as a depot that may be injected subcutaneously or intramuscularly, or implanted. The depot may be in the form of, for example, a polymeric particle system, a polymeric implant, or a lipid or oily composition. The depot formulation may contain a protein in a biocompatible solvent system and a mixture of, for example, a biodegradable polymer [e.g., poly(lactide-co-glycolide)] or a semi-biodegradable polymer (e.g., a block copolymer of lactic acid and PEG), whether or not such a mixture forms a particle system or an implant.

[0195]

[0156] The pharmaceutical composition may be presented in unit dosage form as a single dose, in which case all active and inactive ingredients are combined in a suitable system, and the components do not necessarily need to be mixed to form the administered composition. The unit dosage form generally contains an effective amount of the therapeutic agent. Representative examples of unit dosage forms are prefilled syringes for parenteral (e.g., intravenous, subcutaneous, or intramuscular) injection of the therapeutic agent, and single-use pens containing a needle and a needle cover. For example, it may be presented in unit dosage form as a single dose, in which case all active and inactive ingredients are combined in a suitable system, and the components do not necessarily need to be mixed to form the administered composition. The unit dosage form generally contains an effective amount of the therapeutic agent. Representative examples of unit dosage forms are prefilled syringes for parenteral (e.g., intravenous, subcutaneous, or intramuscular) injection of the therapeutic agent, and single-use pens containing a needle and a needle cover.

[0196]

[0157] Alternatively, the pharmaceutical composition may be presented as a kit, in which case the therapeutic The agent, excipient and carrier (e.g., solvent) are provided in two or more separate containers (e.g., ampoules, vials, tubes, bottles or syringes) and need to be combined to form the composition to be administered. The kit may include instructions regarding the storage, preparation and administration of the composition (e.g., a solution to be administered intravenously or subcutaneously).

[0197]

[0158] The kit can contain all active and inactive ingredients in unit dosage form, or the active and inactive ingredients in two or more separate containers, and may include instructions regarding the administration or use of a pharmaceutical composition for treating a medical condition.

[0198]

[0159] In some embodiments, the kit includes an anti-CD79 antibody or a pharmaceutical composition containing the same, and instructions regarding the administration or use of the anti-CD79 antibody or a pharmaceutical composition containing the same for treating an antibody-related condition.

[0199] Use of the anti-CD79 antibody

[0160] The above anti-CD79 antibody is for an antibody-related condition (e.g., disease, disorder and / or ​It may be administered to a subject suffering from a syndrome. When the subject is a human, the anti-CD79 antibody may be a chimeric mouse-human antibody or a humanized antibody. Such chimeric or humanized antibodies are described above. Examples of antibody-related conditions include, for example, autoimmune diseases, certain allergies (antibody-related allergies), certain types of type I diabetes, etc. Autoimmune diseases that can be treated with anti-CD79 antibodies include, for example, systemic lupus erythematosus (SLE), inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), rheumatoid arthritis, multiple sclerosis, Graves' disease, CREST syndrome, systemic sclerosis, celiac disease, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid antibody syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy (AMAN), Balo disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilicGranulomatosis with polyangiitis (GPA), cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackievirus myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid gestationis (PG), hidradenitis suppurativa (HS) (acne inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing diseases, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, Lyme disease, Ménière's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, scarring ocular pemphigoid, optic neuritis, relapsing polychondritis, Raynaud's disease, reactive arthritis, reflex sympathetic dystrophy, recurrent polychondritis, lower limb stasis Restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, episcleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and the like. Other antibody-related conditions that can be treated with anti-CD79 antibodies include, for example, allergies (antibody-related allergies), amyloidosis, certain forms of transplant rejection, and the like. These and other undesirable antibody-related conditions can be treated by administering one or more of the anti-CD79 antibodies described herein to a subject suffering from the undesirable antibody-related condition.

[0200]

[0161] The anti-CD79 antibodies described herein can induce an anergic state in the B cells of a subject and can thus be used to treat certain autoimmune diseases. For example, autoimmune diseases associated with anti-self antibody responses can be treated with anti-CD79 antibodies because the induced anergic state will prevent the production of anti-self antibodies. The anti-CD79 antibodies described herein can also be used to induce an anergic state in any condition having an undesirable antibody response. The anti-CD79 antibodies described herein can be used to induce an anergic state in B cells. The anti-CD79 antibodies described herein can be used to inhibit the proliferation of B cells.

[0201]

[0162] The anti-CD79 antibodies described herein can be used to detect the presence and / or amount of B cells in a sample and / or subject. It can be used to identify and / or isolate B cells. For example, the anti-CD79 antibodies described herein can be used to diagnose B cell malignancies or other lymphoproliferative disorders and / or can be used as a vehicle for selectively delivering drugs to B cell malignancies.

[0202]

[0163] The anti-CD79 antibodies described herein can be used to directly treat B cell malignancies and / or can be used to construct cytotoxic T cells that express a chimeric T cell receptor (CAR-T) for the treatment of CD79-positive B cell malignancies. The chimeric antigen receptor (CAR) can be prepared by using the antigen-binding portion of the anti-CD79 antibody described herein as the antigen-binding domain / portion of the CAR. These anti-CD79 CARs may be disposed on immune cells such as T cells or natural killer cells, and the anti-CD79 immune cells can be used to treat diseases caused by cells expressing CD79. Such diseases include, for example, CD79-positive hematopoietic cancers (e.g., lymphoma, leukemia, myeloma).

[0203]

[0164] The anti-CD79 antibodies disclosed herein for treating antibody-related conditions The therapeutically effective amount and frequency of administration, and the length of treatment thereby, may be determined by the treating physician and may be affected by various factors including the nature and severity of the condition, the potency of the antibody, the mode of administration, the age, weight, general health, sex and diet of the subject, and the response of the subject to the treatment. The therapeutically effective amount of an antibody (e.g., anti-CD79 antibody LB517 / LB519) for the treatment of an antibody-related condition may be from about 1, 5 or 10 mg to about 200 mg, from about 1, 5 or 10 mg to about 150 mg, from about 1, 5 or 10 mg to about 100 mg, or from about 1, 5 or 10 mg to about 50 mg, or as deemed appropriate by the treating physician, and may be administered as a single dose or divided doses. The therapeutically effective amount of the antibody may be about 1 - 5 mg, 5 - 10 mg, 10 - 20 mg, 20 - 30 mg, 30 - 40 mg, 40 - 50 mg, 50 - 100 mg, 100 - 150 mg or 1 50 - 200 mg. The therapeutically effective amount of the antibody may be about 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150 or 200 mg. The therapeutically effective amount of the antibody may be about 1 - 5 mg, 5 - 10 mg or 10 - 50 mg. The therapeutically effective amount of an antibody (e.g., anti-CD79 antibody LB517 / LB519) for the treatment of an antibody-related condition may be about 0.01 - 0.1 mg / kg, 0.1 - 0.5 mg / kg, 0.5 - 1 mg / kg, 1 - 2 mg / kg or 2 - 3 mg / kg body weight, or as deemed appropriate by the treating physician. The therapeutically effective amount of the antibody may be about 0.01 - 0.1 mg / kg, 0.1 - 0.5 mg / kg or 0.5 - 1 mg / kg body weight.

[0204]

[0165] The anti-CD79 antibody may be administered at any frequency suitable for treating an antibody-related condition The antibody (e.g., anti-CD79 antibody LB517 / LB519) may be administered once a day, once every two days, once every three days, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every six weeks, once every two months or once every three months, or as deemed appropriate by the treating physician. The antibody may also be administered once a week or once every two weeks.

[0205] Similarly, anti-CD79 antibodies can be used at any suitable time to treat an antibody-associated condition. The antibody (e.g., anti-CD79 antibody LB517 / LB519) may be administered for a period of at least about 1 week, 2 weeks, 1 month (4 weeks), 6 weeks, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or more, or as deemed appropriate by the treating physician. The antibody-associated condition may be a chronic condition. The chronic condition may exist, for example, for at least about 6 weeks or 2 months or more. The antibody may be administered for a period of at least about 6 weeks, 2 months, 3 months or 6 months. One, two, three, four, five or six doses of the antibody (e.g., anti-CD79 antibody LB517 / LB519) may be administered during the entire treatment regimen. One, two or three doses of the antibody may be administered during the entire treatment regimen.

[0206] Anti-CD79 antibodies (e.g., anti-CD79 antibody LB517 / LB519) also The antibody or fragment thereof may be administered in an irregular manner to treat the antibody-associated condition. For example, the antibody or fragment thereof may be administered in an irregular manner 1, 2, 3, 4, 5 or more times in a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months or 3 months. In addition, the anti-CD79 antibody (e.g., anti-CD79 antibody LB517 / LB519) may be used ad-hoc (as needed) to treat the antibody-associated condition. For example, the antibody may be administered in a regular or irregular manner 1, 2, 3, 4, 5 or more times until the blood pressure falls to a certain level for the treatment of hypertension. Once the blood pressure falls to a certain level, the administration of the antibody may be optionally discontinued. When the blood pressure reaches or exceeds a certain level, the administration of the antibody may be resumed, whether in a regular or irregular manner. The appropriate dosage of the antibody, the frequency of administration and the length of treatment therewith may be determined by the treating physician.

[0207]

[0168] To more rapidly establish a therapeutic level of an anti-CD79 antibody, at least one loading dose of the antibody or a fragment thereof may be administered prior to the maintenance dose. As deemed appropriate by the treating physician, a loading dose of the antibody (e.g., anti-CD79 antibody LB517 / LB519), followed by (i) one or more additional loading doses and thereafter, one or more therapeutically effective maintenance doses may be administered, or (ii) one or more therapeutically effective maintenance doses may be administered without additional loading doses. The loading dose of the drug may be more than the subsequent maintenance dose (e.g., about 1.5, 2, 3, 4, or 5 times more), and is designed to establish the therapeutic level of the drug more rapidly. One or more therapeutically effective maintenance doses may be any of the therapeutically effective amounts described herein. The loading dose may be about 2 or 3 times more than the maintenance dose. The loading dose of the anti- body may be administered on day 1, and thereafter, for example, once a week or once every two weeks, a maintenance dose of the antibody may be administered during the course of treatment. The antibody (e.g., anti-CD79 antibody LB517 / LB519) may be administered at a loading dose of about 2-10 mg, 10-20 mg, or 20-100 mg, or about 3-15 mg, 15-30 mg, or 30-150 mg on day 1, followed by once a week or once every two weeks, at a maintenance dose of about 1-5 mg, 5-10 mg, or 10-50 mg during the course of treatment (e.g., for at least about 2, 3, or 6 months), where the loading dose is about 2 or 3 times more than the maintenance dose, and the antibody or a fragment thereof may be administered parenterally (e.g., intravenously, subcutaneously, or intramuscularly).

[0208]

[0169] Two (or more) loading doses of the antibody may be administered prior to the maintenance dose. The first loading An antibody or fragment thereof in a loading dose may be administered on day 1, and for example, a second loading dose may be administered about 1 or 2 weeks later, and thereafter, a maintenance dose may be administered, for example, once a week or once every two weeks, during the treatment period. The first loading dose may be about 3 or 4 times more than the maintenance dose, and the second loading dose may be about 2 times more than the maintenance dose. The antibody (e.g., anti-CD79 antibody LB517 / LB519) is administered in a first loading dose of about 3 - 15 mg, 15 - 30 mg or 30 - 150 mg, or about 4 - 20 mg, 20 - 40 mg or 40 - 200 mg on day 1, in a second loading dose of about 2 - 10 mg, 10 - 20 mg or 20 - 100 mg about 1 or 2 weeks later, and subsequently, once a week or once every two weeks, in a maintenance dose of about 1 - 5 mg, 5 - 10 mg or 10 - 50 mg during the treatment period (e.g., for at least about 2, 3 or 6 months), where the first loading dose may be about 3 or 4 times more than the maintenance dose, and the second loading dose may be about 2 times more than the maintenance dose, and the antibody or fragment thereof may be administered parenterally (e.g., intravenously, subcutaneously or intramuscularly).

[0209] Combination therapy with additional therapeutic agents

[0170] The present disclosure provides a method of treating an antibody-related condition, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an anti-CD79 antibody described herein, optionally in combination with an additional therapeutic agent. The present disclosure further provides a composition comprising an anti-CD79 antibody described herein, or an anti-CD79 antibody described herein in combination with an optionally additional therapeutic agent for use as a medicament. Furthermore, the present disclosure provides the use of an anti-CD79 antibody described herein in the preparation of a medicament, optionally in combination with an additional therapeutic agent. In some embodiments, the medicament is used for the treatment of an antibody-related condition.

[0210]

[0171] One or more additional therapeutic agents may optionally be anti-C for treating an antibody-related condition It may be used in combination with a D79 antibody (e.g., anti-CD79 antibodies LB517 / LB519). Any additional therapeutic agent may be administered to the subject simultaneously with the administration of the antibody (e.g., as the same composition as the antibody or its fragment or as a separate composition) or sequentially before or after the administration of the antibody.

[0211]

[0172] Any additional therapeutic agent may be selected from immunosuppressants, anti-inflammatory agents, allergy medications, and combinations thereof. One or more immunosuppressants may be used in combination with an anti-CD769 antibody (e.g., anti-CD79 antibodies LB517 / LB519) to treat an antibody associate condition. Such immunosuppressants include, for example, anti-CD20 antibodies (e.g., rituximab), calcineurin inhibitors (e.g., tacrolimus, cyclosporine, etc.), proliferation inhibitors or IDMH inhibitors (e.g., mycophenolate mofetil, mycophenolate sodium, azathioprine, leflunomide, etc.), mTOR inhibitors (e.g., sirolimus, everolimus, etc.), steroids (e.g., corticosteroids such as prednisone, budesonide, prednisolone, etc.), and biologic agents (e.g., abatacept, adalimumab, anakinra, certolizumab, etanercept, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, daclizumab, muromonab). Biologic agents also include, for example, CTLA4 fusion proteins, anti-TNFα antibodies, IL-1 receptor antagonist proteins, TNF receptor fusion proteins, anti-IL17A antibodies, anti-α4 integrin antibodies, anti-IL6 receptor antibodies, anti-p40 subunit of IL12 / IL23 antibodies, anti-α4β7 integrin antibodies, anti-CD25 antibodies, and anti-CD3 antibodies.

[0212]

[0173] One or more anti-inflammatory agents are used to treat an antibody-related condition having an inflammatory component, It may be used in combination with an anti-CD79 antibody (for example, anti-CD79 antibodies LB517 / LB519). Examples of one or more anti-inflammatory agents include, for example, inhibitors of inflammatory cytokines or their receptors or their production (for example, TNF-α or / and IL-6 or IL-6R). Other anti-inflammatory agents include, for example, the following: non-steroidal anti-inflammatory drugs (NSAIDs), immunomodulators, immunosuppressants, anti-inflammatory cytokines and compounds that increase their production, inhibitors of inflammatory cytokines or their receptors, inhibitors of the production of inflammatory cytokines or their receptors, inhibitors of pro-inflammatory transcription factors or their activation or expression, pro-inflammatory prostaglandins (for example, prostaglandin E2 [PGE2]) or their receptors (for example, EP3) or inhibitors of their production, leukotrienes or their receptors or inhibitors of their production, inhibitors of phospholipase A2 (for example, secreted and cytoplasmic PLA2), suppressors of C-reactive protein (CRP) activity or levels, mast cell stabilizers, phosphodiesterase inhibitors, specialized pro-resolving mediators (SPM), other types of anti-inflammatory agents, as well as their analogs, derivatives, fragments and salts.

[0213]

[0174] Examples of non-steroidal anti-inflammatory drugs (NSAIDs) include the following Non-limiting examples include acetic acid derivatives, anthranilic acid derivatives (phenamates), enolic acid derivatives (oxicams), propionic acid derivatives, salicylates, COX-2-selective inhibitors, other types of NSAIDs, such as monoterpenoids (e.g., eucalyptol and phenols [e.g., carvacrol]), anilinopyridine carboxylic acids (e.g., clonixin), sulfonanilides (e.g., nimesulide), and dual inhibitors of lipoxygenase (e.g., 5-LOX) and cyclooxygenase (e.g., COX-2) (e.g., chebulagic acid, licofelone, 2-(3,4,5-trimethoxyphenyl)-4-(N-methylindol-3-yl)thiophene, and di-tert-butylphenolic compounds [e.g., DTPBHZ, DTPINH, DTPNHZ, and DTPSAL]); and analogs, derivatives, and salts thereof.

[0214]

[0175] The glucocorticoid class of corticosteroid drugs has anti-inflammatory and immune properties. Glucocorticoids include, but are not limited to, hydrocortisone-type, halogenated steroids, carbonates, and analogs, derivatives and salts thereof.

[0215]

[0176] Any additional therapeutic agent may be administered independently in any suitable manner. Potential modes of administration include, but are not limited to, oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial, intraperitoneal, intramedullary, intrathecal and topical), intracavitary, and topical (including dermal / epidermal, transdermal, mucosal, transmucosal, intranasal (e.g., by nasal spray or nasal drops), intraocular (e.g., by eye drops), pulmonary (e.g., by oral or nasal inhalation), buccal, sublingual, rectal (e.g., by suppository) and vaginal (e.g., by suppository). In some embodiments, any additional therapeutic agent is administered independently orally. Administration is by mouth or parenterally (eg, intravenously, subcutaneously, or intramuscularly).

[0216]

[0177] One or more anti-allergic agents may be used in combination with an anti-CD79 antibody (e.g., anti-CD79 antibodies LB517 / LB519) for treating antibody-related conditions. Such anti-allergic agents include, for example, antihistamines (e.g., cetirizine, fexofenadine, levocetirizine, loratadine, brompheniramine, chlorpheniramine, clemastine, diphenhydramine, ketotifen, naphazoline, pheniramine, desloratadine, azelastine, epinastine, olopatadine), decongestants (e.g., pseudoephedrine, phenylephrine, oxymetazoline), steroids (e.g., beclomethasone, ciclesonide, fluticasone furoate, mometasone, budesonide, triamcinolone, dexamethasone, loteprednol, prednisone epocrate), mast cell stabilizers (e.g., sodium cromoglycate, lodoxamide tromethamine, nedocromil, pemirolast), and leukotriene modifiers (e.g., montelukast). The anti-allergic agent may be used in combination with an anti-CD79 antibody (e.g., anti-CD79 antibodies LB517 / LB519) for treating antibody-related conditions. Such anti-allergic agents include, for example, antihistamines (e.g., cetirizine, fexofenadine, levocetirizine, loratadine, brompheniramine, chlorpheniramine, clemastine, diphenhydramine, ketotifen, naphazoline, pheniramine, desloratadine, azelastine, epinastine, olopatadine), decongestants (e.g., pseudoephedrine, phenylephrine, oxymetazoline), steroids (e.g., beclomethasone, ciclesonide, fluticasone furoate, mometasone, budesonide, triamcinolone, dexamethasone, loteprednol, prednisone epocrate), mast cell stabilizers (e.g., sodium cromoglycate, lodoxamide tromethamine, nedocromil, pemirolast), and leukotriene modifiers (e.g., montelukast).

[0217]

[0178] One or more immunosuppressive agents for transplantation may be used in combination with an anti-CD79 antibody (e.g., anti-CD79 antibodies LB517 / LB519) for treating a subject after a transplantation procedure. Such immunosuppressive agents include, for example, calcineurin inhibitors, proliferation inhibitors or IDMH inhibitors, mTOR inhibitors, and steroids. One or more immunosuppressive agents for transplantation may be used in combination with an anti-CD79 antibody (e.g., anti-CD79 antibodies LB517 / LB519) for treating a subject after a transplantation procedure. Such immunosuppressive agents include, for example, calcineurin inhibitors, proliferation inhibitors or IDMH inhibitors, mTOR inhibitors, and steroids.

[0218]

[0179] Any additional therapeutic agent, independently, includes, but is not limited to, daily (once a day) It may be administered two or more times, every two or three days, twice a week, once a week, every two weeks, every three weeks, once a month, every two months or every three months, or in an irregular pattern, or as appropriate as needed. The dosing frequency may depend, for example, on the selected mode of administration. The length of treatment with any additional therapeutic agent may be determined by the treating physician and may independently be, for example, at least about 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks (1 month), 6 weeks, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or more.

[0219] Diagnostic use of anti-CD79 antibodies

[0180] The anti-CD79 antibodies disclosed herein are useful for the diagnosis and prognosis assessment of B cell-related disorders. Furthermore, such antibodies can be used to facilitate treatment decisions.

[0220]

[0181] Anti-CD79 antibodies may be utilized to detect the presence of B cell lineage cells in an original or processed sample obtained from a subject. Biological samples may include, for example, blood, plasma, serum, urine, cerebrospinal fluid (CSF), cells or tissues. Samples may be analyzed directly, extracted prior to analysis, or the volume may be expanded by the addition of a suitable solvent.

[0221]

[0182] Biological samples may be contacted with anti-CD79 antibodies, and the samples is screened to detect binding of an antibody or its fragment to B cells. Detection of such binding indicates the presence of B cells in the sample. The anti-CD79 antibody may be labeled with a detectable agent (e.g., a fluorescent dye) such that binding of the antibody to B cells induces a signal. B cells in a biological sample may be immobilized on the surface prior to introduction of the labeled anti-CD79 antibody (direct assay), and the amount of signal corresponding to the amount of labeled antibody or its fragment bound to B cells is correlated with the amount of B cells in the sample. B cells in a biological sample may be captured by an unlabeled first antibody immobilized on the surface, and then detected by a labeled second antibody that binds to the captured B cells and generates a signal proportional to the amount of captured B cells (sandwich assay), where the unlabeled first antibody and the labeled second antibody bind to different epitopes on the B cells, and the unlabeled first antibody or / and the labeled second antibody may independently be an anti-CD79 antibody disclosed herein. is detected by a labeled second antibody that generates a signal (sandwich assay), in which case the unlabeled first antibody and the labeled second antibody bind to different epitopes on the B cells, and the unlabeled first antibody or / and the labeled second antibody may independently be an anti-CD79 antibody disclosed herein.

[0222]

[0183] B cells in a biological sample may be detected in a competitive assay. A sample (optionally suspended in a buffer) may be mixed with a labeled anti-CD79. The resulting mixture may then be contacted with a B cell marker-coated substrate. The greater the number of B cells in the sample, the more antibody / B cell complexes are formed, and the less unbound (free) antibody available to bind to the B cell marker on the substrate ("competition"), and thus the lower the resulting signal. A sample (optionally suspended in a buffer) may be mixed with a labeled anti-CD79. The resulting mixture may then be contacted with a B cell marker-coated substrate. The greater the number of B cells in the sample, the more antibody / B cell complexes are formed, and the less unbound (free) antibody available to bind to the B cell marker on the substrate ("competition"), and thus the lower the resulting signal.

[0223]

[0184] In the above direct, sandwich and competitive assays, the anti-CD79 primary antibody It may be labeled with a detectable agent. Alternatively, directly in sandwich and competitive assays, the anti-CD79 primary antibody may not be labeled, and a labeled secondary antibody (e.g., one that binds to the Fc region of the primary antibody) may bind after the primary antibody binds to B cells. When the secondary antibody is conjugated to an enzyme, the addition of the enzyme's substrate results in an enzyme / substrate reaction that produces a signal (e.g., a chromogenic, fluorescent, or electrochemical signal). To determine the presence and number of B cells in a sample, the absorbance, fluorescence, or electrochemical signal (e.g., current) of a solid support (e.g., a plate well or beads) is measured. Non-limiting examples of substrates for horseradish peroxidase (HRP) include 3-amino-9-ethylcarbazole (AEC), 3,3'-diaminobenzidine (DAB), and 3,3',5,5'-tetramethylbenzidine (TMB), and an example of a substrate for alkaline phosphatase is 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and an example of a substrate for β-glucuronidase is 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc). Such assays are called enzyme-linked immunosorbent assays (ELISA).

[0224]

[0185] Detectable agents include chromophores {e.g., dyes, pigments, and chromogens (e.g., 3-amino-9-ethylcarbazole [AEC], 5-bromo-4-chloro-3-indolyl phosphate [BCIP], 3,3'-diaminobenzidine [DAB], and 3,3',5,5'-tetramethylbenzidine [TMB])}, fluorophores / fluorescent dyes (e.g., fluorescent dyes such as fluorescein, fluorescein isothiocyanate, and rhodamine), chemiluminescent compounds (e.g., luciferin and luminol), radioisotopes (e.g., 3 H, 14 C, 32 P, 35 S, and 125I), radioactive elements (e.g., technetium), high electron density compounds, magnetic substances and particles (e.g., paramagnetic and ferromagnetic ones), magnetic resonance imaging (MRI) contrast agents (e.g., those containing gadolinium), enzymes (e.g., horseradish peroxidase [HRP], alkaline phosphatase, luciferase, β-glucuronidase and β-galactosidase), haptens and toxins, but not limited to these.

[0225]

[0186] The anti-CD79 antibodies described herein can be used in various immunometric assays ( immunometric assay). Such assays include, but are not limited to, chromogenic, fluorescent, chemiluminescent, light scattering, radiolabeled, electrochemical, enzymatic, precipitation, agglutination, coagulation, Western blot, grid blot, tissue blot, dot blot, dipstick, and biosensor assays. See, for example, Principles and Practice of Immunoassays, C. Price and D. Newman (eds.), Stockton Press (1997); and The Immunoassay Handbook, 2nd ed., D. Wild (ed.), Nature Publishing Group (2001 year). Furthermore, the anti-CD79 antibodies can be used for imaging, such as by MRI.

[0226]

[0187] The detection and measurement of the amount of B cells in a biological sample is useful for the diagnosis of B cell-related disorders 、may facilitate prognosis and treatment. In some embodiments, the disorder is associated with elevated B cell levels, and elevated B cell levels in a sample from a subject compared to B cell levels in a corresponding sample from other subjects without the disorder indicate a diagnosis of the disorder in the subject. A reference B cell level for diagnosis of the disorder may be determined, for example, based on a comparison of B cell levels in corresponding samples from a statistically or epidemiologically significant number of subjects without the disorder and B cell levels in corresponding samples from a statistically or epidemiologically significant number of subjects with the disorder. Similarly, B cell levels in a sample from a subject for treatment and prognosis prediction may be compared, for example, to a scale of B cell levels correlated with the severity of a B cell-related disorder, to determine the current severity of the disorder and to predict the expected course or outcome (e.g., progression or regression) of the disorder.

[0227]

[0188] Furthermore, detection and measurement of the amount of B cells in a biological sample can facilitate treatment decisions. In some embodiments, the amount of anti-CD79 antibody administered to a subject and / or the frequency of administration of the antibody to the subject is maintained or adjusted (increased or decreased), or administration of the antibody or a fragment thereof to the subject is discontinued based on the presence, amount or level of B cells in a sample from the subject.

[0228]

[0189] In some embodiments, the kit may optionally be labeled with a detectable agent and comprises an anti-CD79 antibody and instructions for using the anti-CD79 antibody, optionally for diagnostic use (e.g., in an immunoassay). In some embodiments, the kit may optionally be labeled with a detectable agent and comprises an anti-CD79 antibody and instructions for using the anti-CD79 antibody, optionally for diagnostic use (e.g., in an immunoassay).

[0229] Production of anti-CD79 antibody

[0190] The present disclosure provides a polynucleotide comprising a nucleic acid sequence encoding the anti-CD79 antibody described herein. The polynucleotide encodes the V domain of the anti-CD79 mAb or / and the V H domain or / and the V LIt may contain a nucleic acid sequence encoding a domain. The polynucleotide may contain a nucleic acid sequence encoding the heavy chain and / or light chain of an anti-CD79 mAb.

[0230]

[0191] The present disclosure further provides a construct (sometimes referred to as an expression or cloning construct) containing the nucleic acid sequence encoding the anti-CD79 antibody described herein. Suitable constructs include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes, yeast artificial chromosomes, lambda phages (e.g., those lacking the lysogeny gene), and viruses. The construct may exist as an episome in the cell or may be integrated into the chromosome (either as the construct and still being a construct, plasmid, or vector in any way).

[0231]

[0192] Various construct systems may be utilized. One class of constructs utilizes DNA elements derived from animal viruses such as adenovirus, baculovirus, bovine papillomavirus, polyomavirus, SV40 virus, vaccinia virus, and retroviruses (e.g., MMTV, MOMLV, and Rous sarcoma virus). Another class of constructs utilizes RNA elements derived from RNA viruses such as eastern equine encephalitis virus, flavivirus, and Semliki Forest virus.

[0232]

[0193] The construct may, for example, in addition to the nucleic acid sequence encoding the V H domain or / and V domain of the anti-CD79 mAb, or the heavy chain and / or light chain, contain mRNA L domain, or the nucleic acid sequence encoding the heavy chain and / or light chain, contain mRNA A variety of other elements can be included for optimal expression of A. For example, the construct can include a transcriptional promoter, a promoter plus operator, an enhancer, an open reading frame with or without introns and / or exons, a termination signal, a splicing signal, a secretion signal sequence, or a selection marker (e.g., a gene that confers resistance to an antibiotic or cytotoxic drug), or any combination or all of these.

[0233] The present disclosure also provides constructs encoding the anti-CD79 antibodies described herein. Also provided is a host cell that contains or expresses the tract.Suitable host cells include, but are not limited to, eukaryotic cells, mammalian cells (e.g., BHK, CHO, COS, HEK293, HeLa, MDCKII and Vero cells), insect cells (e.g., Sf9 cells), yeast cells and bacterial cells (e.g., E. coli cells).Host cells can be mammalian cells (e.g., CHO cells or HEK293 cells).

[0234] The host cells are immunized with anti-CD79 mAb V H Domain or V L Domain, also The host cell may contain or express constructs encoding the heavy or light chains of the anti-CD79 mAb. H Domains and V L The same or separate host cells may contain or express the V domain of an anti-CD79 mAb, or a single construct encoding the heavy and light chains. H Constructs encoding domains or heavy chains and V of said mAbs. L The antibody may comprise or express a separate construct encoding the light chain or a separate construct encoding the light chain.

[0235] The constructs can be transfected into host cells by any method known in the art. It may be transfected or introduced. Examples of transfection agents and methods include, but are not limited to, calcium phosphate, cationic polymers (e.g., DEAE-dextran and polyethyleneimine), dendrimers, fugene, cationic liposomes, electroporation, sonoporation, cell squeezing, gene gun, viral transfection, and retroviral transduction.

[0236]

[0197] For culturing the transfected host cells and recovering the recombinantly produced Methods and conditions for recovering the anti-CD79 antibody are known in the art and may vary or be optimized, for example, depending on the specific expression vector and / or host cell utilized. The V H domain and / or V L domain, or the heavy chain and / or light chain may be recombinantly produced. The entire heavy and light chains of anti-CD79 IgG1, IgG2, or IgG4, or the heavy and light chains of an anti-CD79 Fab fragment optionally fused to an extension, are recombinantly produced.

[0237]

[0198] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials in connection with which the publications are cited.

[0238]

[0199] The following examples are intended to illustrate the present disclosure only. Other assays, research, processes, protocols, procedures, techniques, reagents, and conditions may be used instead as needed. Research, processes, protocols, procedures, techniques, reagents, and conditions may be used instead as needed.

Examples

[0239] Example 1. Preparation of chimeric anti-CD79 antibody

[0200] A chimeric antibody HcLc was prepared from a murine antibody that binds CD79 with high specificity and high affinity. The V domain and V H domain (SEQ ID NOs: 1 and 2) were fused to the human IgG2 C L 1, C H 2, and C H 3 domains or the human kappa C H domain, respectively. L

[0240] Example 2. Preparation of humanized anti-CD79 antibody

[0201] The murine antibody that binds CD79 was also humanized. The CDRs (IgG1) of the heavy chain and the light chain (kappa) SEQ ID NOs: 3-8 were grafted into the acceptor human framework sequences. These three CDRs (CDR-L1, CDR-L2, and CDR-H2) contain amino acid motifs (DG, DS, and NG) that may be undesirable.

[0241]

Chemical modification

[0242] Using various CDR-L1s having A RIYPENGDTNY S GKFKG (SEQ ID NO: 58), or RIYPE A GDTNY

[0243]

[0202] The murine framework sequences were used with a database of human framework sequences​​ Human framework sequences were selected by aligning and finding the closest human homologs (generally about 65 - 70% sequence identity) for each chain. The human VH1 - 2 framework was used as the human acceptor framework that most closely matched the mouse framework sequence for the heavy chain, and VK2 - 30 was used as the human acceptor framework that most closely matched the mouse framework sequence for the light chain. Three different VL - FR2s, WFQQRPGQSPRRLIY (SEQ ID NO: 60), W L QQRPGQSPRRLIY (SEQ ID NO: 61), or W L QQRPGQSP K RLIY (SEQ ID NO: 62) were used. Two amino acid changes were made in VH - FR1 to QVQLVQSGAEVKKPGASVKVSCKASGY A F S (SEQ ID NO: 63). Three different combinations of VH - FR2 and VH - FR3, VH - FR2 WVRQAPGQGLEWMG (SEQ ID NO: 64) and VH - FR3 RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR (SEQ ID NO: 65), VH - FR2 WVRQAPGQGLEWMG (SEQ ID NO: 64) and VH - FR3 RVTMT A DTSISTAYMELSRLRSDDTAVYYCAR (SEQ ID NO: 66), and VH - FR2 WVRQAPGQGLEW I G (SEQ ID NO: 67) and VH - FR3 RVT L T A D K SISTAYMELSRLRSDDTAVYYCAR (SEQ ID NO: 68) were used.

[0244]

[0203] Humanized heavy chains H1 - H3 (SEQ ID NOs: 9 - 11) and L1 - L3 (SEQ ID NOs: 18 ~20) were prepared, mixed by a combinatorial approach, and combined to create a library of humanized clones (H1 - H3 were mixed with L1 - L3 by a combinatorial approach). Individual members were isolated and tested for affinity binding to CD79. The binding data obtained are listed in Table 1 below.

[0245] [Table 1]

[0246]

[0204] Lead antibody H1L2 (LB495 / PRI47, SEQ ID NOs: 9 and 19) was selected. I chose. Example 3. Affinity maturation of humanized antibody H1L2 (LB495 / PRI47, SEQ ID NOs: 9 and 19) The humanized antibody H1L2 (SEQ ID NOs: 9 and 19) was affinity matured. Four phage display libraries were generated, two from CDR1 (SEQ ID NO: 10) and two from light chain L2 (SEQ ID NO: 20). Saturation mutagenesis was performed on CDR3 to generate CDR3 heavy and CDR3 light chain libraries, and error-prone PCR was also used to randomly mutagenize the respective H1 heavy and L2 light chains to generate heavy and light chain libraries. Each heavy chain library was paired with the parent light chain L2 (SEQ ID NO: 19) and each light chain library was paired with the parent heavy chain H1 (SEQ ID NO: 9) to generate a phage display library of candidates for affinity maturation. Each library was panned against CD79 in a competition assay with the parent H1L2 antibody, and binding clones obtained from panning were tested for affinity in ELISA. A VH clone (SEQ ID NO: 25) with a mutation in CDR3 was selected, and a VL clone (SEQ ID NO: 28-31) with a mutation at the same position in CDR3 was selected. These heavy and light chains were rearranged into full-length IgG, produced from 293 cells, purified, and then tested for binding. The binding data for these full-length IgGs is shown in Table 2 below.

[0247] [Table 2]

[0248] PRI 47 is the light chain L2 (SEQ ID NO: 19), and PRI 43 and LB 495 is the heavy chain H1 (SEQ ID NO: 9), LB517 is the heavy chain LB509-C2 (SEQ ID NO: 25), LB518 is the light chain LB511-A9 (SEQ ID NO: 28), LB519 is the light chain LB511-F6 (SEQ ID NO: 30), LB520 is the light chain LB511-F11 (SEQ ID NO: 31), and LB521 is the light chain LB511-B6 (SEQ ID NO: 29).

[0249]

[0207] The affinity matured antibody showed higher affinity than the parental humanized antibody. Example 4. Development of antibody cell lines

[0208] CHO cells (Invitrogen) were cultured in serum-free medium (CD FortiCHO , Invitrogen) and co-transfected with individual plasmids encoding the heavy and light chains of the anti-CD79 antibody using Freestyle Max transfection reagent (Invitrogen). The antibody expression plasmid was linearized by restriction digestion with Sca I prior to transfection. Antibody expression in the conditioned medium was measured by ELISA.

[0250]

[0209] The transfected cells were subjected to 2 weeks of stable selection with 10 μg / ml puromycin and 50 0 μg / ml G418. After single cell cloning, high-producing CHO cell clones were screened and isolated. Antibody production was evaluated to be >500 mg / L in shake flask culture.

[0251] Example 5. Recombinant production of humanized anti-CD79 antibody

[0210] HEK293F cells (Invitrogen) were cultured in serum-free medium and co-transfected with the plasmid encoding LB517 and the plasmid encoding LB519, or plasmids expressing other antibody variants. On the 5th day after transfection, the cell culture supernatant was collected and subjected to protein A chromatography for antibody purification.

[0252] Example 6. Human CD79a / 79b knock-in mice

[0211] C57BL / 6 embryonic stem cells were manipulated to replace the mouse CD79a and CD79b gene loci with nucleic acids encoding human CD79a and human CD79b. The manipulated C57BL / 6 ES cells were transplanted into female C57BL / 6 mice to obtain C57BL / 6 offspring that were positive for human CD79a / b. The expression of human CD79a and CD79b on B cells was confirmed using FACS together with anti-human CD79 antibody.

[0253] Example 7. Anti-CD79 antibody induced B cell anergy in human CD79 C57BL / 6 mice

[0212] Human CD79 knock-in mice were treated with humanized anti-CD79 antibody LB517 / LB519 for 18 hours before assay. Ex vivo, subsequently, RBC-lysed splenocytes (1E6 / 100 μL) were stained with anti-B220 and fluorescently labeled anti-hCD79, or PTEN, or B cell receptor. LB517 / LB519 (hCur14 FALA) was able to bind to B cells and compete with Curly-14 (Figure 3a). The intracellular levels of PTEN and cell surface BCR were also characterized by staining with appropriate antibodies. LB517 / LB519 was able to induce PTEN expression (Figure 3b) and down-regulate BCR expression (Figure 3c).

[0254]

[0213] The B cells of these mice were assayed for calcium upon activation via the B cell receptor ​​RBC-lysed splenocytes (1E7 / mL) were stained with anti-B220 (B cells) and loaded with calcium detection dye (Indo-1 AM) for 1 h prior to flow analysis. Changes in intracellular Ca2+ levels were recorded as the ratio of fluorescence emission at 405 nm and 485 nm measurements in a FORTESSA (BD Bioscience) running Flow-Jo software (Tree Star). Baseline calcium was obtained for 30 s before adding stimulation with 100 μL of medium. Basal calcium measurements were subtracted from post-stimulation AUC to generate acute stimulation quantification. LB517 / LB519 stimulated calcium influx (Figure 4a) and desensitized the B cell receptor (Figure 4b).

[0255] B cells from C57BL / 6 mice also express tyrosine lysine upon B cell receptor stimulation. These studies demonstrated that pretreatment of B cells with the humanized anti-CD79 antibodies LB517 / LB519 desensitized B cells to activation via the B cell receptor, as tyrosine phosphorylation was inhibited in anti-CD79 antibody-treated B cells.

[0256] Example 8. Treatment of Type I Diabetes IgM heavy chain transgene VH1 on NOD and C57BL / 6 backgrounds Mice expressing VH125.C57BL / 6 and VH281 were obtained. VH125.C57BL / 6 mice were backcrossed to C57BL / 6-H2g7 (JAX) to generate VH125.C57BL / 6.H2g7. Two consecutive blood glucose measurements >250 mg / dL (One Touch) identified VH125.C57BL / 6.H2g7 mice as diabetic.

[0257]

[0216] VH who is prediabetic (serial blood glucose readings >150 to <200 mg / dL) 125NOD mice were treated with anti-CD79 antibody or physiological saline. Figure 1 shows that the physiological saline-treated mice developed type I diabetes (80% by 5 weeks). Figure 1 also shows that the mice treated with anti-CD79 antibody had less development of type I diabetes (25 - 30% by 5 weeks).

[0258] Example 9. Treatment of type I diabetes with humanized anti-CD79 antibody LB517 / LB519

[0217] C57BL / 6 mice expressing human CD79a / b on B cells were prepared according to Example 6 . The hCD79a / b C57BL / 6 mice had PTPn22 R620W integrated into the ROSA26 locus with an intervening floxed stop cassette. Tamoxifen-inducible cre of the PTPn22 R620W autoimmune risk allele in combination with streptozotocin (STZ) treatment to damage pancreatic β-cells promoted the disease in adult mice. tam

[0259]

[0218] Mice at 6 - 8 weeks of age are injected intraperitoneally (i.p.) with 40 mg / kg of STZ (Sigma-Aldric h) for 4 consecutive days. Blood glucose levels are measured twice a week starting 2 weeks after the last STZ injection using a Bayer Contour Meter (Bayer). Diabetes is defined by an increase in glucose levels > 500 mg / dL for two consecutive tests.

[0260]

[0219] PTPn22 R620W hCD79a / b C57BL / 6 mice are treated with humanized anti-CD79 antibody LB517 / LB519 or physiological saline before / during / after treatment of the mice with STZ. The mice are observed based on the blood glucose levels measured twice a week. Diabetes is defined by an increase in glucose levels > 500 mg / dL for two consecutive tests.

[0261] Example 10. Treatment of arthritis ​​

[0220] C57BL / 6 mice were immunized on day 0 with bovine or chicken type II collagen (CII) emulsified in complete Freund's adjuvant (CFA). Twenty-one days later, the mice were boost-immunized with CII emulsified in incomplete Freund's adjuvant (IFA). 1 mg of anti-CD79 or isotype control immunoglobulin was administered subcutaneously (s.c.) on day 0. Two hours after the mAb injection, the mice were immunized with collagen. After boost-immunization of individual legs, clinical scores were evaluated by applying a scale ranging from 0 to 4 as previously described (Hardy, 2014). Anti-CD79 significantly suppressed the onset of arthritis (Figure 5).

[0262]

[0221] hCD79a / b C57BL / 6 mice expressing human CD79a / b on B cells were generated according to Example 6. hCD79a / b C57BL / 6 mice were immunized on day 0 with bovine or chicken type II collagen (CII) emulsified in complete Freund's adjuvant (CFA). Twenty-one days later, the mice were boost-immunized with CII emulsified in incomplete Freund's adjuvant (IFA).

[0263]

[0222] Anti-mouse CD79 D265A, anti-human CD79 (humanized anti-CD79 antibody LB517 / LB519), anti-CD20 (18B12) or isotype control immunoglobulin was administered subcutaneously (s.c.) on day 0. Two hours after the mAb injection, the mice were immunized with collagen.

[0264]

[0223] After boost-immunization of individual legs, clinical scores were evaluated by applying a scale ranging from 0 to 4 as previously described (Hardy, 201 4). Example 11. Treatment of systemic lupus

[0224] MRL / lpr, MRL / lpr-Thy1.1 mice were given 0.5 ​​​Injections of mg of anti-CD79 were given for 6 to 17 weeks. Anti-CD79 reduced inflammation in the kidneys and submandibular salivary glands and improved survival at week 17 with once-weekly dosing (Figure 6).

[0265]

[0225] hCD79a / b C57BL / 6 mice expressing human CD79a / b on B cells were prepared according to Example 6. hCD79a / b C57BL / 6 mice were treated with short-term tamoxifen to deplete SH2-containing inositol lipid phosphatase (SHIP-1) and / or inositol lipid phosphatase PTEN in B cells. Getahun et al., J Exp Med. May 2, 2016;213(5):751-69, which is incorporated by reference in its entirety for all purposes.

[0266]

[0226] Starting at 8 weeks of age (after autoantibody appearance) in tamoxifen-treated human CD79 C57BL6 mice, humanized anti-CD79 antibodies LB517 / LB519, anti-CD20 (18B12), or isotype control immunoglobulin are administered subcutaneously once a week. Production of anti-chromatin autoantibodies, glomerular deposition, and mouse health status are observed.

[0267] Example 12. Epitope mapping of humanized anti-CD79 antibodies

[0227] The binding epitope of the anti-CD79 antibody is mapped by competition and structural analysis of various fragments of the CD79 antigen.

[0268] Example 13. Construction of anti-CD79 CAR-T for cancer treatment

[0228] The anti-CD79 antibody L1H2 was converted to a scFv antibody and in vector LB586 ​​​The plasmid LB586 was transfected into CHO cells and selected with the appropriate antibiotic for 2 weeks. Stable cells were stained with soluble biotinylated CD79 antigen followed by streptavidin PE complex. CD79 antigen binding was confirmed by flow cytometry analysis (Figure 2). The nucleic acid sequence of the anti-CD79 chimeric antigen receptor was:

[0269] [ka]

[0270]

[0229] The amino acid sequence of the anti-CD79 chimeric antigen receptor was as follows:

[0271] [ka]

[0272]

[0230] Anti-CD79 CAR-T vectors can be used to treat CD79-positive cancers. The antibody produces cytotoxic T cells to Example 14. Treatment of Multiple Sclerosis

[0231] 200 μL of complete Freund's adjuvant (CFA) emulsified in C57BL6 / J mice were immunized according to standard protocols using 100 mg of myelin oligodendrocyte glycoprotein (MOG) 35-55. The emulsion was injected subcutaneously at two sites, followed by two intraperitoneal (ip) injections of 200 ng of pertussis toxin (PTX) in phosphate-buffered saline (PBS), the first 1-2 h after MOG 35-55 and the second 24 h after. 1 mg of anti-CD79 was administered once a week starting on day 19. EAE scores and body weights were assessed daily to evaluate disease severity and stage. Anti-CD79 treatment mitigated the progression of the disease model (Figure 7).

[0273] Example 15. Engineering of affinity matured humanized antibody LB517 / 519

[0232] Two that are correlated with a high Asn deamidation tendency leading to complex problems in production of the "NG" motifs are present in VH CDR2 (SEQ ID NO: 4). When the mutation did not adversely affect antigen interaction, it was possible to mutate the Asn residue to eliminate the deamidation risk. The first NG motif was engineered to NS (SEQ ID NO: 75) and cloned into the expression vector LB630. The second NG motif was engineered to NA (SEQ ID NO: 76) and cloned into the expression vector LB631. Both NG motifs were engineered (SEQ ID NO: 77) and cloned into the expression vector LB632.

[0274]

Chem.

[0275]

[0233] SEQ ID NO: 75 was designated as LB630.

[0276]

Chem.

[0277]

[0234] SEQ ID NO: 76 was designated as LB631.

[0278]

Chem.

[0279]

[0235] SEQ ID NO: 77 was designated as LB632.

[0236] After being paired with the light chain expression vector LB519, the engineered antibody was produced from 293 cells by transient transfection and purified. The antigen-binding kinetics were characterized and shown in Table 3 below. The antigen-binding kinetics were characterized and shown in Table 3 below.

[0280]

Table 3

[0281]

[0237] Of the three antibodies engineered, LB631 / 519 and LB632 / 519 showed slightly weaker binding compared to LB517 / 519, while LB630 / 519 showed much improved antigen binding.

[0282]

[0238] The biological activities of these engineered antibodies were characterized as in Example 7. The engineered antibodies showed similar B cell desensitization effects compared to LB517 / 519 (Figs. 3 and 4). compared to LB517 / 519 (Figs. 3 and 4).

[0283]

[0239] While specific embodiments have been shown and described, various modifications may be made thereto, which is understood to be intended herein. It is also understood that the present disclosure is not limited by the specific examples provided herein. It is not intended that the description and examples of the embodiments and examples of the present disclosure herein be construed in a limiting sense. It is further understood that all aspects of the present disclosure are not limited to the specific depictions, configurations or relative proportions described herein, which may be affected by various conditions and variables. Various modifications and variations in the form and details of the embodiments and examples of the present disclosure will be apparent to those skilled in the art. Accordingly, it is intended that the present disclosure also include any and all such modifications, variations and equivalents. While specific embodiments have been shown and described, various modifications may be made thereto, which is understood to be intended herein. It is also understood that the present disclosure is not limited by the specific examples provided herein. It is not intended that the description and examples of the embodiments and examples of the present disclosure herein be construed in a limiting sense. It is further understood that all aspects of the present disclosure are not limited to the specific depictions, configurations or relative proportions described herein, which may be affected by various conditions and variables. Various modifications and variations in the form and details of the embodiments and examples of the present disclosure will be apparent to those skilled in the art. Accordingly, it is intended that the present disclosure also include any and all such modifications, variations and equivalents. While specific embodiments have been shown and described, various modifications may be made thereto, which is understood to be intended herein. It is also understood that the present disclosure is not limited by the specific examples provided herein. It is not intended that the description and examples of the embodiments and examples of the present disclosure herein be construed in a limiting sense. It is further understood that all aspects of the present disclosure are not limited to the specific depictions, configurations or relative proportions described herein, which may be affected by various conditions and variables. Various modifications and variations in the form and details of the embodiments and examples of the present disclosure will be apparent to those skilled in the art. Accordingly, it is intended that the present disclosure also include any and all such modifications, variations and equivalents.

Claims

1. An anti-CD79 antibody comprising a heavy chain having a variable region selected from the group consisting of SEQ ID NOs: 9-17, 24-27, 32-41, 71, 72, and 75-77, and a light chain having a variable region selected from the group consisting of SEQ ID NOs: 18-23, 28-31, 42-56, and 73-74.

2. The anti-CD79 antibody according to any one of claims 1, wherein the variable region of the heavy chain is selected from the group consisting of SEQ ID NOs: 24-27 and 75-77, and the variable region of the light chain is selected from the group consisting of SEQ ID NOs: 28-31.

3. The antibody according to claim 2, wherein the variable region of the heavy chain is SEQ ID NO: 25, and the variable region of the light chain is SEQ ID NO:

30.

4. The antibody according to claim 2, wherein the variable region of the heavy chain is SEQ ID NO: 75, and the variable region of the light chain is SEQ ID NO:

30.

5. The antibody according to claim 2, wherein the variable region of the heavy chain is SEQ ID NO: 76, and the variable region of the light chain is SEQ ID NO:

30.

6. The antibody according to claim 2, wherein the variable region of the heavy chain is SEQ ID NO: 77, and the variable region of the light chain is SEQ ID NO:

30.

7. An anti-CD79 antibody comprising a heavy chain having a variable region chain selected from the group consisting of SEQ ID NOs: 9-17, and a light chain having a variable region selected from the group consisting of SEQ ID NOs: 18-23.

8. A method for treating arthritis, comprising the steps of preparing a mammal having arthritis, and administering to the mammal the anti-CD79 antibody according to any one of claims 1-7.

9. The method according to claim 8, wherein the variable region of the heavy chain is SEQ ID NO: 25, and the variable region of the light chain is SEQ ID NO:

30.

10. The method according to claim 8, wherein the variable region of the heavy chain is SEQ ID NO: 75, and the variable region of the light chain is SEQ ID NO:

30.

11. The method according to claim 8, wherein the variable region of the heavy chain is SEQ ID NO: 76, and the variable region of the light chain is SEQ ID NO:

30.

12. The method according to claim 8, wherein the variable region of the heavy chain is SEQ ID NO: 77, and the variable region of the light chain is SEQ ID NO:

30.

13. A method for treating systemic lupus, comprising the steps of preparing a mammal having systemic lupus, and administering to the mammal the anti-CD79 antibody according to any one of claims 1-7.

14. The method according to claim 13, wherein the variable region of the heavy chain is SEQ ID NO: 25 and the variable region of the light chain is SEQ ID NO:

30.

15. The method according to claim 13, wherein the variable region of the heavy chain is SEQ ID NO: 75 and the variable region of the light chain is SEQ ID NO: 30 therein.

16. The method according to claim 13, wherein the variable region of the heavy chain is SEQ ID NO: 76 and the variable region of the light chain is SEQ ID NO:

30.

17. The method according to claim 13, wherein the variable region of the heavy chain is SEQ ID NO: 77 and the variable region of the light chain is SEQ ID NO:

30.

18. A method for treating type 1 diabetes, comprising the steps of preparing a mammal having type 1 diabetes and administering to the mammal the anti-CD79 antibody according to any one of claims 1 to 7.

19. The method according to claim 18, wherein the variable region of the heavy chain is SEQ ID NO: 25 and the variable region of the light chain is SEQ ID NO:

30.

20. The method according to claim 18, wherein the variable region of the heavy chain is SEQ ID NO: 75 and the variable region of the light chain is SEQ ID NO:

30.

21. The method according to claim 18, wherein the variable region of the heavy chain is SEQ ID NO: 76 and the variable region of the light chain is SEQ ID NO:

30.

22. The method according to claim 18, wherein the variable region of the heavy chain is SEQ ID NO: 77 and the variable region of the light chain is SEQ ID NO:

30.

23. A method for treating multiple sclerosis, comprising the steps of preparing a mammal having multiple sclerosis and administering to the mammal the anti-CD79 antibody according to any one of claims 1 to 7.

24. The method according to claim 23, wherein the variable region of the heavy chain is SEQ ID NO: 25 and the variable region of the light chain is SEQ ID NO:

30.

25. The method according to claim 23, wherein the variable region of the heavy chain is SEQ ID NO: 75 and the variable region of the light chain is SEQ ID NO:

30.

26. The method according to claim 23, wherein the variable region of the heavy chain is SEQ ID NO: 76 and the variable region of the light chain is SEQ ID NO:

30.

27. The method according to claim 23, wherein the variable region of the heavy chain is SEQ ID NO: 77 and the variable region of the light chain is SEQ ID NO:

30.

28. A method for treating an autoimmune disease, comprising the steps of preparing a mammal having an autoimmune disease and administering to the mammal an anti-CD79 antibody according to any one of claims 1 to 7.

29. The method according to claim 28, wherein the autoimmune disease is related to B cells.

30. A method for treating an allergy, comprising the steps of preparing a mammal having an allergy and administering to the mammal an anti-CD79 antibody according to any one of claims 1 to 7.

31. A method for preventing transplant rejection, comprising the steps of preparing a mammal that has received a transplanted organ and administering to the mammal an anti-CD79 antibody according to any one of claims 1 to 7.

32. A method for preventing an immune response to a therapeutic agent, comprising the steps of preparing a mammal that has received a therapeutic agent and administering to the mammal an anti-CD79 antibody according to any one of claims 1 to 7.

33. A T cell comprising an antigen-binding portion composed of a heavy-chain variable region selected from the group consisting of SEQ ID NOs: 9 to 17, 24 to 27, 32 to 41, 71, 72, and 75 to 77 and a light chain having a variable region selected from the group consisting of SEQ ID NOs: 18 to 23, 28 to 31, 42 to 56, and 73 to 74.

34. The T cell according to claim 33, wherein the chimeric antigen receptor is SEQ ID NO:

70.

35. A method for treating a subject having CD79-positive cancer, comprising administering to the subject the T cell according to claim 33, whereby the CD79-positive cancer is treated.

36. The method according to claim 35, wherein the chimeric antigen receptor of the T cell is SEQ ID NO: 70.

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