Anti-human CD19 antibody

Abstract

This disclosure relates to antibodies that bind to human CD19 (anti-human CD19 antibodies or anti-human CD19 antibodies), compositions comprising such anti-human CD19 antibodies, and methods of using such anti-human CD19 antibodies.

Description

Technical Field

[0001] This disclosure relates to antibodies that bind to human CD19 (“anti-human CD19 antibody” or “anti-CD19 antibody”), compositions comprising such anti-human CD19 antibody, and methods of using such anti-human CD19 antibody. Background Technology

[0002] B cells produce antibodies against various antigens and therefore play a crucial role in humoral immune responses. In addition, B cells act as antigen-presenting cells (APCs) and secrete cytokines. B cells express B cell receptors (BCRs) on their cell membranes; and BCRs allow B cells to bind to specific antigens, which trigger antibody responses. Abnormal regulation of B cells is associated with a variety of diseases.

[0003] Human B-lymphocyte antigen CD19 (also known as Cluster of Differentiation 19, B-lymphocyte surface antigen B4, T-cell surface antigen Leu-12, or CVID3) is a transmembrane protein that is widely expressed throughout all stages of B cell development until its eventual differentiation into plasma cells. Therefore, human CD19 is a pan-B-cell marker. CD19 has been found to be associated with complement receptor CD21 and tetraspan family member CD81 (Carter et al., Immunol. Res. 26: 45-54, 2002). CD19 is a co-receptor for the BCR and acts as an adaptor protein to recruit cytoplasmic signaling proteins to the BCR complex. CD19 is required for normal B cell function and is involved in a wide variety of B cell responses, including B cell survival, proliferation, activation, and differentiation.

[0004] Anti-human CD19 antibodies have been previously described and are being tested in clinical trials. Many known anti-human CD19 antibodies are B-cell depletion antibodies; for example, inebilizumab (also known as MEDI-551) and tafasitamab (also known as MOR208 or XmAb5574) in phase III clinical trials, loncastuximab in phase II clinical trials, and 4G7SDIE and DI-B4 in phase I clinical trials have all been reported as B-cell depletion antibodies (Cree et al., Lancet. 394(10206):1352-1363, 2019; Kellner et al., Leukemia. 27(7):1595–1598, 2013; Kaplon et al., MAbs. 12(1): 1703531, 2020; Seidel et al., Mol.Ther. 24(9):1634-43, 2016; WO2007076950).

[0005] Obexelimab (also known as XmAb5871) is an Fc-modified antibody that binds to both CD19 and FcγRIIb and inhibits B cell function by participating in inhibitory FcγRIIb receptor signaling (Szili et al., MAbs. 6(4): 991–999, 2014). However, the high affinity binding of obexelimab to FcγRIIb can lead to nonspecific binding to other cell types. In human patients, obexelimab has been reported to have a short half-life, T 1 / 2 The duration was 3.5 ± 1.0 days; and it reduced peripheral human B cell counts, by an average of approximately 30-40% of baseline levels (Jaraczewska-Baumann et al., European League Against Rheumatism (EULAR) 2015 Annual Meeting Poster). A Phase 1b / 2a Study of the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of XmAb®5871 in Patients with Rheumatoid Arthritis (Available at https: / / investors.xencor.com / static-files / 0017dcf1-deb2-46eb-93ff-90ba164ec50c on June 12, 2015).

[0006] Therefore, alternative anti-human CD19 antibodies that specifically bind to and inhibit human B cells without depleting them are still needed for the treatment of B-cell-related diseases. Summary of the Invention

[0007] This article provides antibodies that bind to human CD19 and inhibit B cell responses (e.g., proliferation, activation, and differentiation) without depleting B cells (i.e., "non-depleting anti-human CD19 antibodies"). Such non-depleting anti-human CD19 antibodies can be used to treat B cell-related conditions, such as autoimmune diseases, without destroying those important immune cells, thereby avoiding problematic concurrent immune compromise, prolonged immunosuppression, and other complications caused by B cell depletion. The anti-human CD19 antibodies provided herein possess one or more of the following properties for development and treatment of autoimmune diseases: 1) binding to human CD19 (and sometimes cynomolgus CD19) with desired binding affinity and / or association and dissociation rates; 2) specifically binding to human B cells and inhibiting primary human B cell proliferation, activation, and / or differentiation; 3) not depleting human B cells; 4) being internalized into human B cells; 5) low immunogenicity risk; 6) low hydrophobicity; and / or 7) good stability, solubility, viscosity, and pharmacokinetic characteristics. As described below, in a side-by-side comparison, one such anti-human CD19 antibody, CB3f, showed highly specific binding to human B cells in whole blood assays, while obemizole showed nonspecific binding to human neutrophils in addition to B cell binding. Since neutrophils are the most abundant type of white blood cell in humans, this nonspecific binding to neutrophils may explain the short half-life of obemizole observed in human patients. Furthermore, in in vitro B cell apoptosis assays, obemizole showed a dose-dependent induction of primary human B cell apoptosis, while the anti-human CD19 antibody CB3f did not induce apoptosis in primary human B cells in the same assay.

[0008] In one respect, this article provides novel non-depleted anti-human CD19 antibodies. In some embodiments, the anti-human CD19 antibody is a fully human antibody.

[0009] In some embodiments, this document provides an antibody that binds to human CD19, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions (HCDR) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions (LCDR) LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 29, HCDR2 comprises SEQ ID NO: 30, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 32, LCDR2 comprises SEQ ID NO: 33, and LCDR3 comprises SEQ ID NO: 34. In some embodiments, the anti-human CD19 antibody comprises a VH comprising SEQ ID NO: 37 and a VL comprising SEQ ID NO: 41.

[0010] In some embodiments, the anti-human CD19 antibody has a human IgG1 or IgG4 isotype. In some embodiments, the anti-human CD19 antibody has a human IgG4 isotype. In some embodiments, the anti-human CD19 antibody has a modified human IgG4 hinge region containing an S228P mutation (according to EU index number) that reduces in vivo IgG4 Fab-arm exchange (see Labrijn et al., Nat. Biotechnol. 2009, 27(8):767). In some embodiments, the anti-human CD19 antibody has a human IgG1 isotype. In some embodiments, the anti-human CD19 antibody has a modified human IgG1 Fc region that has reduced or eliminated Fc effector functions, such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Such antibodies are referred to as “IgG1-effector nullipopulation” antibodies.

[0011] In some embodiments, the anti-human CD19 antibody has a human IgG4 isotype. For example, in some embodiments, the antibody comprises a heavy chain (HC) comprising SEQ ID NO: 35 and a light chain (LC) comprising SEQ ID NO: 39. In some embodiments, the antibody has a human IgG1 isotype. For example, in some embodiments, the antibody comprises HC comprising SEQ ID NO: 50 and LC comprising SEQ ID NO: 39. In some embodiments, the antibody is an IgG1-effects inactive antibody. For example, in some embodiments, the antibody comprises HC comprising SEQ ID NO: 52 and LC comprising SEQ ID NO: 39.

[0012] In some embodiments, this document provides an antibody fragment (e.g., Fab or scFv) that binds to human CD19, wherein the antibody fragment comprises VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 29, HCDR2 comprises SEQ ID NO: 30, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 32, LCDR2 comprises SEQ ID NO: 33, and LCDR3 comprises SEQ ID NO: 34. In some embodiments, the antibody fragment comprises a VH comprising SEQ ID NO: 37 and a VL comprising SEQ ID NO: 41.

[0013] This document also provides antibodies that bind to human CD19, wherein the antibodies comprise VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 29, HCDR2 comprises SEQ ID NO: 30, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 43, LCDR2 comprises SEQ ID NO: 44, and LCDR3 comprises SEQ ID NO: 45. In some embodiments, the anti-human CD19 antibody comprises VH comprising SEQ ID NO: 37 and VL comprising SEQ ID NO: 48. In some embodiments, the antibody has a human IgG4 isotype. For example, in some embodiments, the antibody comprises HC comprising SEQ ID NO: 35 and LC comprising SEQ ID NO: 46. In some embodiments, the antibody has a human IgG1 isotype. For example, in some embodiments, the antibody comprises HC containing SEQ ID NO: 50 and LC containing SEQ ID NO: 46. In some embodiments, the antibody is an IgG1-effects inactive antibody. For example, in some embodiments, the antibody comprises HC containing SEQ ID NO: 52 and LC containing SEQ ID NO: 46.

[0014] In some embodiments, this document provides an antibody fragment (e.g., Fab or scFv) that binds to human CD19, wherein the antibody fragment comprises VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 29, HCDR2 comprises SEQ ID NO: 30, HCDR3 comprises SEQ ID NO: 31, LCDR1 comprises SEQ ID NO: 43, LCDR2 comprises SEQ ID NO: 44, and LCDR3 comprises SEQ ID NO: 45. In some embodiments, the antibody fragment comprises a VH comprising SEQ ID NO: 37 and a VL comprising SEQ ID NO: 48.

[0015] This document also provides antibodies that bind to human CD19, wherein the antibodies comprise VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 15, HCDR2 comprises SEQ ID NO: 16, HCDR3 comprises SEQ ID NO: 17, LCDR1 comprises SEQ ID NO: 18, LCDR2 comprises SEQ ID NO: 19, and LCDR3 comprises SEQ ID NO: 20. In some embodiments, the anti-human CD19 antibody comprises VH comprising SEQ ID NO: 23 and VL comprising SEQ ID NO: 27. In some embodiments, the antibody has a human IgG4 isotype. For example, in some embodiments, the antibody comprises HC comprising SEQ ID NO: 21 and LC comprising SEQ ID NO: 25. In some embodiments, the antibody has a human IgG1 isotype. In some embodiments, the antibody is an IgG1-effector null antibody. For example, in some embodiments, the antibody comprises HC containing SEQ ID NO: 54 and LC containing SEQ ID NO: 25.

[0016] In some embodiments, this document provides an antibody fragment (e.g., Fab or scFv) that binds to human CD19, wherein the antibody fragment comprises VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 15, HCDR2 comprises SEQ ID NO: 16, HCDR3 comprises SEQ ID NO: 17, LCDR1 comprises SEQ ID NO: 18, LCDR2 comprises SEQ ID NO: 19, and LCDR3 comprises SEQ ID NO: 20. In some embodiments, the antibody fragment comprises a VH comprising SEQ ID NO: 23 and a VL comprising SEQ ID NO: 27.

[0017] This document also provides an antibody binding to human CD19, wherein the antibody comprises VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO:1, HCDR2 comprises SEQ ID NO:2, HCDR3 comprises SEQ ID NO:3, LCDR1 comprises SEQ ID NO:4, LCDR2 comprises SEQ ID NO:5, and LCDR3 comprises SEQ ID NO:6. In some embodiments, the anti-human CD19 antibody comprises VH comprising SEQ ID NO:9 and VL comprising SEQ ID NO:13. In some embodiments, the antibody comprises HC comprising SEQ ID NO:7 and LC comprising SEQ ID NO:11.

[0018] In some embodiments, this document provides an antibody fragment (e.g., Fab or scFv) that binds to human CD19, wherein the antibody fragment comprises VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 1, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 3, LCDR1 comprises SEQ ID NO: 4, LCDR2 comprises SEQ ID NO: 5, and LCDR3 comprises SEQ ID NO: 6. In some embodiments, the antibody fragment comprises a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 13.

[0019] In another respect, this article provides a nucleic acid encoding a heavy chain or light chain or VH or VL of the novel anti-human CD19 antibody described herein, and a vector containing such nucleic acid.

[0020] In some embodiments, this document provides nucleic acids encoding a heavy or light chain of the anti-human CD19 antibody described herein. In some embodiments, this document provides nucleic acids comprising a sequence encoding SEQ ID NO: 35, 52, 50, 39, 46, 21, 54, 25, 7, or 11. In some embodiments, this document provides nucleic acids comprising a sequence encoding an antibody heavy chain, said antibody heavy chain comprising SEQ ID NO: 35, 52, 50, 21, 54, or 7. For example, the nucleic acid may comprise a sequence selected from SEQ ID NO: 36, 53, 51, 22, 55, or 8. In some embodiments, this document provides nucleic acids comprising a sequence encoding an antibody light chain, said antibody light chain comprising SEQ ID NO: 39, 46, 25, or 11. For example, the nucleic acid may comprise a sequence selected from SEQ ID NO: 40, 47, 26, or 12.

[0021] This document also provides nucleic acids encoding VH or VL of the anti-human CD19 antibody described herein. In some embodiments, this document provides nucleic acids comprising sequences encoding SEQ ID NO: 37, 23, 27, 9, 41, 48, 13. In some embodiments, this document provides nucleic acids comprising sequences encoding antibody VH, said antibody VH comprising SEQ ID NO: 37, 23, 27, 9. For example, the nucleic acid may comprise sequences selected from SEQ ID NO: 38, 24, 28, 10. In some embodiments, this document provides nucleic acids comprising sequences encoding antibody VL, said antibody VL comprising SEQ ID NO: 41, 48, 13. For example, the nucleic acid may comprise sequences selected from SEQ ID NO: 42, 49, 14.

[0022] This document also provides vectors containing nucleic acid sequences encoding the heavy or light chain of an antibody. For example, such vectors may contain nucleic acid sequences encoding SEQ ID NO: 35, 52, 50, 39, 46, 21, 54, 25, 7, or 11. In some embodiments, the vector contains SEQ ID NO: 36, 53, 51, 40, 47, 22, 55, 26, 8, or 12.

[0023] This document also provides vectors containing nucleic acid sequences encoding antibodies VH or VL. For example, such vectors may contain nucleic acid sequences encoding SEQ ID NO: 37, 23, 27, 9, 41, 48, or 13. In some embodiments, the vector contains SEQ ID NO: 38, 24, 28, 10, 42, 49, or 14.

[0024] This document also provides vectors comprising a first nucleic acid sequence encoding an antibody heavy chain and a second nucleic acid sequence encoding an antibody light chain. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 35, 50, or 52 and a second nucleic acid sequence encoding SEQ ID NO: 39 or 46. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 21 or 54 and a second nucleic acid sequence encoding SEQ ID NO: 25.

[0025] In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 35 and a second nucleic acid sequence encoding SEQ ID NO: 39. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 52 and a second nucleic acid sequence encoding SEQ ID NO: 39. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 50 and a second nucleic acid sequence encoding SEQ ID NO: 39. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 35 and a second nucleic acid sequence encoding SEQ ID NO: 46. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 52 and a second nucleic acid sequence encoding SEQ ID NO: 46. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 50 and a second nucleic acid sequence encoding SEQ ID NO: 46. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 21 and a second nucleic acid sequence encoding SEQ ID NO: 25. In some embodiments, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 54 and a second nucleic acid sequence encoding SEQ ID NO: 25. In some implementations, the vector comprises a first nucleic acid sequence encoding SEQ ID NO: 7 and a second nucleic acid sequence encoding SEQ ID NO: 11.

[0026] Also provided are compositions comprising a first vector comprising a nucleic acid sequence encoding an antibody heavy chain and a second vector comprising a nucleic acid sequence encoding an antibody light chain. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 35, 52, or 50 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 39 or 46. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 21 or 54 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 25.

[0027] In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 35 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 39. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 52 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 39. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 50 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 39. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 35 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 46. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 52 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 46. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 50 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 46. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 21 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 25. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 54 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 25. In some embodiments, the composition comprises a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 7 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 11.

[0028] The nucleic acids disclosed herein can be expressed in host cells, for example, after the nucleic acid has been operatively linked to an expression control sequence. Expression control sequences capable of expressing the nucleic acid operatively linked thereto are well known in the art. The expression vector may contain a sequence encoding one or more signal peptides that promote the secretion of one or more polypeptides from the host cell. Expression vectors containing the nucleic acid of interest (e.g., nucleic acids encoding the heavy or light chain of an antibody) can be transferred into host cells by well-known methods, such as stable or transient transfection, transformation, transduction, or infection. Furthermore, the expression vector may contain one or more selection markers, such as tetracycline, neomycin, and dihydrofolate reductase, to aid in the detection of host cells transformed with the desired nucleic acid sequence.

[0029] In another aspect, this document provides cells, such as host cells, that contain the nucleic acids, vectors, or nucleic acid compositions described herein. Host cells can be cells stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or part of the antibodies described herein. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing HC and LC peptides of antibodies of this disclosure. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with a first vector expressing an HC peptide of an antibody described herein and a second vector expressing an LC peptide of an antibody described herein. Such host cells, for example, mammalian host cells, can express the anti-human CD19 antibodies described herein. Known mammalian host cells capable of expressing antibodies include CHO cells, HEK293 cells, COS cells, and NSO cells.

[0030] In some embodiments, the cell, such as a host cell, comprises a vector that includes a first nucleic acid sequence encoding SEQ ID NO: 35, 50, or 52 and a second nucleic acid sequence encoding SEQ ID NO: 39 or 46. In some embodiments, the cell, such as a host cell, comprises a vector that includes a first nucleic acid sequence encoding SEQ ID NO: 21 or 54 and a second nucleic acid sequence encoding SEQ ID NO: 25.

[0031] In some embodiments, the cell, for example, a host cell, comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO: 35, 52, or 50, and a second vector containing a nucleic acid sequence encoding SEQ ID NO: 39 or 46. In some embodiments, the cell, for example, a host cell, comprises a first vector containing a nucleic acid sequence encoding SEQ ID NO: 21 or 54, and a second vector containing a nucleic acid sequence encoding SEQ ID NO: 25.

[0032] This disclosure further provides a method for producing the anti-human CD19 antibody described herein by culturing the aforementioned host cells, such as mammalian host cells, under such conditions to induce antibody expression and recovering the expressed antibody from the culture medium. The culture medium in which the antibody has been secreted can be purified using conventional techniques. Various protein purification methods can be used, and these methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0033] Antibodies produced by any of the methods described herein are also provided.

[0034] In another aspect, this document provides pharmaceutical compositions comprising the antibodies, nucleic acids, or carriers described herein. Such pharmaceutical compositions may also comprise one or more pharmaceutically acceptable excipients, diluents, or carriers. The pharmaceutical compositions can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy , 22nd edition (2012), A. Loyd et al., Pharmaceutical Press.

[0035] The anti-human CD19 antibodies, nucleic acids, vectors, or pharmaceutical compositions described herein can be used to treat B-cell-related diseases. Due to their crucial role in regulating the immune system, abnormal regulation of B cells is associated with a variety of diseases. B-cell-related diseases include autoimmune diseases caused by the activation of autoreactive B cells and T cells, as well as lymphomas and leukemias caused by excessive and / or uncontrolled B-cell proliferation. Examples of B-cell-related autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, Sjögren's syndrome, idiopathic thrombocytopenic purpura, type 1 diabetes mellitus, pemphigus vulgaris, neuromyelitis optica, ANCA vasculitis (anti-neutrophil cytoplasmic antibody-associated vasculitis), and myasthenia gravis.

[0036] The anti-human CD19 antibodies described herein are known to not deplete B cells, offering advantages over B-cell-depleting antibodies for the treatment of autoimmune diseases and avoiding problematic complications such as immune compromise, prolonged immunosuppression, and other complications arising from B-cell depletion. As shown below, the anti-human CD19 antibodies described herein are internalized in primary human B cells. Therefore, the anti-human CD19 antibodies described herein can also be used to deliver other therapeutic agents into human B cells.

[0037] In some embodiments, this document provides a method for treating B-cell-related conditions, such as autoimmune diseases, in a subject (e.g., a human patient) by administering a therapeutically effective amount of an anti-human CD19 antibody as described herein, a nucleic acid encoding such an anti-human CD19 antibody, a vector containing such a nucleic acid, or a pharmaceutical composition containing such an anti-human CD19 antibody, nucleic acid, or vector. The antibody, nucleic acid, vector, or pharmaceutical composition described herein can be administered via a parenteral route (e.g., subcutaneously and intravenously). In some embodiments, the B-cell-related condition is selected from systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, idiopathic thrombocytopenic purpura, type 1 diabetes mellitus, pemphigus vulgaris, neuromyelitis optica, ANCA vasculitis, and myasthenia gravis.

[0038] The disclosure also provides the anti-human CD19 antibodies, nucleic acids, vectors, or pharmaceutical compositions described herein for therapeutic use. Furthermore, this disclosure provides the anti-human CD19 antibodies, nucleic acids, vectors, or pharmaceutical compositions described herein for the treatment of B-cell-related conditions, such as, for example, autoimmune diseases, such as systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, idiopathic thrombocytopenic purpura, type 1 diabetes mellitus, pemphigus vulgaris, neuromyelitis optica, ANCA vasculitis, and myasthenia gravis.

[0039] This article also provides the use of the anti-human CD19 antibody, nucleic acid, carrier, or pharmaceutical composition described herein in the preparation of a medicament for the treatment of B-cell-related conditions, such as autoimmune diseases, including systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, idiopathic thrombocytopenic purpura, or type 1 diabetes, pemphigus vulgaris, neuromyelitis optica, ANCA vasculitis, and myasthenia gravis.

[0040] As used herein, the terms “a” and “an” and “the” and similar terms used in the context of this disclosure (especially in the context of the claims) shall be interpreted to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0041] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, or conjugated antibodies. Antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0042] An exemplary antibody is an immunoglobulin G (IgG) antibody comprising four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked by interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of approximately 100-125 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. Each light chain contains a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further subdivided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0043] The VH and VL regions can be further subdivided into highly denatured regions called complementarity-determining regions (CDRs), which are dotted with more conserved framework regions (FRs). CDRs are exposed on the protein surface and are important regions in antibodies specific for antigen binding. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this paper, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." CDRs contain most of the residues that specifically interact with the antigen. The allocation of amino acid residues to CDRs can be performed according to well-known schemes, including Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., “Canonical structures for the hypervariable regions of immunoglobulins”, Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), and North (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, Those described in 228-256 (2011) or IMGT (the international ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212). The North CDR definition is used for the anti-human CD19 antibodies described herein.

[0044] Exemplary embodiments of the antibodies disclosed herein also include antibody fragments or antigen-binding fragments comprising at least a portion of an antibody that retains the ability to specifically interact with an antigen, such as Fab, Fab', F(ab')2, Fv fragments, scFv, scFab, disulfide-linked Fvs (sdFv), Fd fragments, and linear antibodies.

[0045] Unless otherwise indicated, the terms “bind” and “binds” as used herein are intended to mean the ability of a protein or molecule to form a chemical bond or an attractive interaction with another protein or molecule, resulting in the two proteins or molecules coming into close proximity, as determined by commonly known methods in the art.

[0046] Unless otherwise stated, the term "CD19" as used herein refers to human B-lymphocyte antigen CD19 (also known as differentiation cluster 19, B-lymphocyte surface antigen B4, T-cell surface antigen Leu-12, or CVID3). The amino acid sequence of human CD19 is known in the art, for example, NCBI reference sequences NP_001171569.1 (isotype 1, SEQ ID NO: 56) or NP_001761.3 (isotype 2, SEQ ID NO: 57). Isotype 2 is a splice variant of isotype 1 and is one amino acid shorter than isotype 1 in its intracellular domain. The term "CD19" as used herein generally refers to all known human CD19 isotypes and polymorphic forms.

[0047] As used herein, the term "Fc region" refers to a region of the antibody that contains the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region may contain a portion or the entire hinge region of the antibody heavy chain.

[0048] As used herein, the term "non-depleting antibody" refers to an antibody that does not significantly reduce the number of B cells in the host body after treatment, as compared to the pre-treatment B cell count. The B cell count can be measured using well-known assays, such as those described in the examples. Non-depleting antibodies generally do not induce antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or B cell apoptosis.

[0049] As may be used interchangeably herein, the terms “nucleic acid” or “polynucleotide” refer to polymers of nucleotides, including single-stranded and / or double-stranded nucleotide-containing molecules such as DNA, cDNA, and RNA molecules, incorporating natural or modified nucleotides and / or nucleotide analogs. The polynucleotides of this disclosure may also include substrates incorporated therein, for example, by DNA or RNA polymerases or synthetic reactions.

[0050] As used herein, the term "subject" refers to a mammal, including, but not limited to, humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Preferably, the subject is humans.

[0051] As used herein, the term "therapeuticly effective amount" refers to the amount of a protein, nucleic acid, carrier, or composition that will elicit a biological or medical response in the subject, such as a reduction or inhibition of enzyme or protein activity, or improvement of symptoms, alleviation of condition, slowing or delaying disease progression, or prevention of disease, etc. In a non-limiting embodiment, the term "therapeuticly effective amount" refers to the amount of a protein, nucleic acid, carrier, or composition that, when applied to the subject, effectively at least partially alleviates, inhibits, prevents, and / or improves the condition, symptom, or disease.

[0052] As used herein, “treatment” or “treating” refers to all processes that may involve slowing, controlling, delaying, or stopping the progression of the condition or disease disclosed herein, or improving the symptoms of the condition or disease, but does not necessarily mean the complete elimination of all symptoms of the condition or disease. Treatment includes the administration of proteins or nucleic acids or carriers or compositions for the treatment of a disease or condition, particularly in humans. Attached Figure Description

[0053] Figure 1A-1B The parental antibody C323 against human CD19 was shown. Figure 1A ) and the optimized anti-human CD19 antibody CB3f ( Figure 1B Surface hydrophobicity distribution diagram.

[0054] Figure 2 This shows the binding of anti-human CD19 antibody to human CD19 in the ELISA assay.

[0055] Figure 3 The study showed that anti-human CD19 mAb CB3f inhibited the proliferation of primary B cells.

[0056] Figure 4 The study showed that anti-human CD19 mAb CB3f inhibited the activation of B cells in whole blood.

[0057] Figure 5 The study showed that anti-human CD19 mAb CB3f inhibited the differentiation of B cells into plasmablasts.

[0058] Figures 6A-6B The results showed a deficiency of anti-human CD19 mAb CB3f in CDC ( Figure 6A ) and ADCC (Figure 6B) activity.

[0059] Figure 7The binding specificity of anti-human CD19 mAb CB3f and obbelimumab to human whole blood cells was demonstrated.

[0060] Figure 8 This study demonstrates the difference in the induction of B cell apoptosis by obbelimumab and anti-human CD19 mAb CB3f.

[0061] Figures 9A-9B Showed on the 6th day after treatment ( Figure 9A ) and the 10th day ( Figure 9B The decrease in human IgM in NSG mice treated with anti-human CD19 mAb CB3f.

[0062] Figure 10 This study showed a decrease in CD86 expression on human B cells in NSG mice treated with anti-human CD19 mAb CB3f.

[0063] Figure 11 This shows the frequency of B cells in NSG mice treated with anti-human CD19 mAb CB3f.

[0064] Figure 12 This study demonstrates the internalization of anti-human CD19 mAb1 (CB3f) and anti-human CD19 mAb2 (C323.C1) in primary human B cells.

[0065] Figures 13A-13B This demonstrates a comparison of the efficacy of non-depleting CD19-substituted Ab and depleting CD20-substituted Ab in a mouse model of collagen-induced arthritis (CIA). Figure 13A The results show that in a mouse CIA model, treatment with non-depleted CD19 replacing Ab in a semi-established mode reduced clinical scores more significantly than treatment with depleted CD20 replacing Ab. Clinical scores of mice between days 21 and 42 of the study (n = 12 / group except for the disease-free control group (n = 5) receiving isotype control treatment). Symbols represent group means, and error bars represent the standard error (SEM) of the means. Dosing began on day 19. Figure 13B The results showed that in a mouse CIA model, treatment with non-depleted CD19 replacing Ab resulted in a greater reduction in clinical score AUC (days 24–42) than treatment with depleted CD20 replacing Ab. Clinical score AUCs of mice between day 24 and day 42 of the study (n = 12 / group except for the disease-free control group (n = 5) receiving isotype control treatment). Bars represent group means, and error bars represent the standard error (SEM) of the means. Mice were administered starting on day 19. Bars not sharing common letters were significantly different from each other (p < .05). One-way ANOVA and Tukey's post-hoc analysis were also performed.

[0066] Figure 14 In a NOD model of type 1 diabetes, treatment with non-depleted CD19 replacing Ab in a half-treatment modality significantly delayed and reduced the incidence of diabetes compared to depleted CD20 replacing Ab. Diabetes incidence (mice with blood glucose levels above 240 mg / dL were considered diabetic > 0.05, n = 10 / group except for the untreated group n = 9). Dosing was initiated in animals at 12 weeks of age.

[0067] Figure 15 This study showed that in a mouse EAE model, treatment with a half-treatment regimen of non-depleted CD19 replacing Ab resulted in a greater reduction in clinical scores than treatment with depleted CD20 replacing Ab. Clinical scores of mice between day 6 and day 42 of the study (n = 12 / group). Symbols represent group means, and error bars represent the standard error (SEM) of the means. Dosing was initiated on day 6. * p < 0.05 for same-day, same-type controls. Detailed Implementation Example

[0068] The following embodiments are provided to illustrate, but do not limit, the claimed invention.

[0069] Example 1. Generate an antibody that binds to human CD19 (anti-human CD19 antibody).

[0070] Anti-human CD19 antibody C323 was discovered from a phage display library using cell-based panning against human embryonic kidney (HEK) cells co-transfected with human CD19 (SEQ ID NO: 56) and its co-receptor CD21. Negative panning against parental HEK-293 cells was used to remove non-specific cell binders. CD19-specific binding was confirmed by ELISA using the CD19 extracellular domain (ECD) protein. After conversion to IgG form and purification, cell binding was confirmed by FACS using the Daudi human Burkitt lymphoma cell line and isolated primary human B cells.

[0071] Hydrophobic interaction chromatography (HIC) is a technique commonly used to separate proteins for characterizing antibody variants; and the retention time of the protein of interest to the HIC column reflects its total hydrophobicity (J Pharm Biomed Anal. 2016 130:3-18). C323 is considered hydrophobic due to its prolonged HIC column retention time. Antibody hydrophobicity can lead to manufacturing problems such as poor expression and protein aggregation. Directed and random mutagenesis were used to enhance the biophysical properties of C323 and increase its affinity and titer. Homology models of the variable region of the C323 anti-human CD19 parent mAb were generated. A spatial aggregation tendency algorithm was then applied to the model to identify surface-exposed hydrophobic plaques. The method identified eight surface-exposed hydrophobic residues within LCDR1, HCDR2, and HCDR3: LCDR1: Y31, Y32; HCDR2: I52, I53, and F54; HCDR3: F97, Y99, and Y100a (Kabat number) ( Figure 1A These residues were targeted for mutagenesis. A library was generated using VVK-based mutagenic oligonucleotides to include more hydrophilic (polar or charged) amino acids, and this was incorporated into a uracil-containing single-stranded DNA template encoding a primary C323 parent antibody with a deleted CDR sequence using Kunkel mutagenesis. Phage-expressed Fab was screened using biotinylated human CD19 ECD antigen. Affinity-neutral mutations identified in this manner were sequenced from DNA, and unique clones were obtained from *E. coli* (…). E. coli The amino acids were expressed as periplasmic Fab and analyzed by titration ELISA. The method identified three amino acid substitutions with neutral but more hydrophilic affinity: LCDR1: Y31H, HCDR2: F54Y, and HCDR3: Y99K (Kabat number).

[0072] Simultaneously, optimization was performed using NNK codon-based mutagenic oligonucleotides targeting all six CDRs to increase affinity, and these were incorporated into a single-stranded DNA template containing uracil with the selected CDRs missing using Kunkel mutagenesis. Screening for phage-expressed Fab variants was performed by capture-lift (Anal Biochem. 1998 256(2):169-77) and ELISA using biotinylated human CD19 ECD antigen. In this manner, CDR substitutions leading to increased affinity were identified: HCDR1: F29I, I34Y; HCDR2: G55D, HCDR3: G100bA; LCDR1: G27aK, A34H, LCDR2: S52R, A55P, LCDR3: N93Q (Kabat number), and their combination resulted in the production of C323.C1.

[0073] The three hydrophilic substitutions (Y31H, F54Y, and Y99K) were added to C323.C1 to generate a Fab template, which was used for the final round of CDR randomization using NNK-based mutagenesis. Beneficial CDR mutations were combined in the library, allowing all beneficial mutations to be randomly combined or reverted to wild-type sequences. The method identified a high-affinity variant called CB3. CB3 contains additional CDR residue substitutions: HCDR1: G27H, HCDR2: G50D, I53A, D55G, T56S, A57P; LCDR1: K27aH, H34A, LCDR3: L95Q (Kabat number).

[0074] Using CB3 as a template, additional CDR alterations were made to revert specific residues back to the germline individuality of human IGKV3-20. One residue in LCDR1, N29S, and five residues in LCDR2, A50G, T51A, R52S, T53S, and P55A, were simultaneously reverted with minimal impact on antibody function. This result produced the final molecule, CB3f.

[0075] Several forms of CB3f were produced, including (1) an IgG4 isotype comprising HC of SEQ ID NO: 35 and LC of SEQ ID NO: 39; (2) an IgG1 isotype comprising HC of SEQ ID NO: 50 and LC of SEQ ID NO: 39; and (3) an IgG1 effector null antibody comprising HC of SEQ ID NO: 52 and LC of SEQ ID NO: 39. Unless otherwise specified, “CB3f” refers to the IgG4 isotype comprising HC of SEQ ID NO: 35 and LC of SEQ ID NO: 39.

[0076] Figure 1A-1B The improved surface hydrophobicity profile of CB3f compared to parental antibody C323 is shown. The retention time of CB3f on the HIC column is reduced to less than half that of parental antibody C323. Simultaneously, the antibody titer of CB3f from transient CHO expression is more than doubled compared to the titer of parental C323 antibody.

[0077] Figure 2The improved binding of affinity-modified antibodies CB3 and CB3f to human CD19 was demonstrated in the ELISA assay compared to C323 and C323.C1. The ELISA assay was performed as follows: 96-well microtiter plates were coated overnight at 4°C with 50 µL / well of goat anti-human κ polyclonal antibody diluted to 5 µg / mL in phosphate-buffered saline (PBS). After overnight incubation, the plates were aspirated and blocked for 1 hour at room temperature with 200 µL of casein buffer. The plates were washed three times with PBST (PBS with 0.1% Tween). Anti-human CD19 antibody was diluted to 5 µg / mL in PBS casein and 50 µL was added to each column of the anti-human κ coated casein-blocked plates at 37°C for 1 hour. The plates were washed three times with PBST. Biotinylated human CD19 extracellular domain protein was serially diluted from 20 µg / mL to 9 ng / mL, and 50 µL was added to a plate and incubated at 37°C for 1 hour. The plate was washed three times with PBST and then transferred to a beaker containing 1 L of PBST and incubated overnight (approximately 16 hours) at 37°C with stirring. The wash buffer was aspirated, and 50 µL of a 1:1000 dilution of neutral avidin alkaline phosphatase conjugate in casein buffer was added and incubated at 37°C for 1 hour. The plate was washed three times with 0.1% Tween PBS. 50 µL of a 1:35 dilution of AMP-PMP substrate in deionized water was added, and the absorbance was read at 560 nm using a Spectramax plate reader.

[0078] As shown below, CB3f exhibits high affinity binding to CHO cells expressing human CD19 and, unexpectedly, also binds to CHO cells expressing cynomolgus monkey CD19. CB3f shares 92% and 96% germline identity with human IGHV1-69 and IGKV3-20, respectively. Therefore, the anti-human CD19 antibody CB3f possesses high affinity, low hydrophobicity, and a high percentage of human germline identity, i.e., low immunogenicity risk.

[0079] The anti-human CD19 antibodies described herein, including but not limited to CB3f, can be expressed in mammalian cell lines such as HEK293 or CHO, transiently or stably transfected with an expression system used to secrete antibodies using an optimal predetermined HC:LC vector ratio or a single vector system encoding both HCs and LCs. The clarified medium into which antibodies have been secreted can be purified using common techniques. After these chromatographic steps, the purity of the antibody can reach a value greater than 99.0% (monomer).

[0080] Example 2. Characterization of anti-human CD19 antibody.

[0081] Binding affinity with CD19

[0082] Solution-phase equilibrium binding affinity of anti-human CD19 mAb CB3f to membrane-bound human CD19 and cynomolgus monkey CD19 stably expressed on CHO cells was determined by solution-equilibrium titration (MSD-SET) at 37 °C. Furthermore, the monovalent affinity and kinetics of CB3fFab fragment binding to CD19 were measured by surface plasmon resonance (SPR) at 37 °C.

[0083] The MSD SI6000 instrument (Meso Scale Discovery, Rockville, MD) was used to read MSD plates. MSD assay plates were prepared as follows: Multiarray 96-well plates (Meso Scale Discovery, P / N L15XA-3) were coated overnight at 4°C with a solution of 1 μg / mL goat anti-human Fc capture antibody (Jackson ImmunoResearch, P / N 109-005-098) in PBS. After coating, the plates were washed 3X times in TBS + 0.1% Tween-20 (TBST).

[0084] CHO cells stably expressing human and cynomolgus monkey CD19 were fixed at room temperature in 1% paraformaldehyde in PBS for 5 minutes. The paraformaldehyde was removed by washing with PBS, and the cells were stored at 4°C in 1% Blocker A (diluted from 3% Blocker A, Meso Scale Discovery, P / N R93AA-1) containing 0.05% (w / v) sodium azide.

[0085] For human CD19 affinity measurements, samples were prepared in duplicate at fixed antibody concentrations of 400 pM, 80 pM, 16 pM, and 3.2 pM. For cynomolgus monkey CD19 affinity measurements, samples were prepared in duplicate at fixed antibody concentrations of 10 nM, 2 nM, 400 pM, and 80 pM. Fixed human CD19 CHO cells were precipitated by centrifugation and then subjected to a 22 x 10⁻⁶ ppm solution. 6 Cells / mL were resuspended in 3% Blocker A solution. Fixed cynomolgus macaque CD19 CHO cells were precipitated by centrifugation and then resuspended at 200 x 10⁻⁶ cells / mL. 6Cells were resuspended at 100 cells / mL in 3% Blocker A solution. Cells were serially diluted 2.5-fold in 3% Blocker A in 96-well conical plates to approximately 900 cells / mL for human CD19 and 8,400 cells / mL for cynomolgus monkey CD19, for a total of 12 cell dilutions each. These were mixed 1:1 with previously prepared antibody dilutions. For human CD19, the final antibody concentrations were 200 pM, 40 pM, 8 pM, and 1.6 pM, with a final cell density of 11 x 10⁻⁶ cells / mL. 6 The concentrations decreased from 100 cells / mL to approximately 450 cells / mL. For cynomolgus monkey CD19, the final antibody concentrations were 5 nM, 1 nM, 200 pM, and 40 pM, with a final cell density of 100 x 10⁻⁶ cells / mL. 6 The number of cells / mL decreased to approximately 4,200 cells / mL. Plates were incubated on a plate shaker at 37°C for 3–4 days for human CD19 and for 2 days for cynomolgus monkey CD19 to allow binding to reach equilibrium.

[0086] After incubation, cells were precipitated by centrifugation. 100 μL of the clarified supernatant from the plate was transferred to a prepared MSD plate and incubated at room temperature on a plate shaker for 60 minutes. After incubation, the plate was washed 3X with TBST, and then 100 μL of 1 μg / mL biotinylated goat anti-human IgG primary antibody (Southern Biotech, catalog 2010-08) in 1% Blocker A was added to all wells. This was incubated at room temperature on a plate shaker for 60 minutes. The plate was washed 3X with TBST, and then 100 µL of 1 µg / mL SULFO-TAG streptavidin (Meso ScaleDiscovery, P / NR92TC-1) in 1% Blocker A was added to all wells. This was incubated at room temperature on a plate shaker for 60 minutes. The plate was washed 3X with TBST, and then immediately before reading the plate, 1X read buffer T (Meso ScaleDiscovery, P / NR92TC-1) was added. Using nonlinear regression in GraphPad Prism to dissociate the constant (K) DThe least common multiple (LCM) of unknown antigen concentrations on cells was used to globally fit the MSD-SET data to the equilibrium binding equation (see Darling and Brault, 2005, Kinetic Exclusion Assay Technology: Characterization of Molecular Interactions. Assay and Drug Development Technologies 2: 647–657).

[0087] A Biacore T200 instrument (GE Healthcare Life Sciences), reagents, and Biacore T200 evaluation software Ver 3.1 were used for surface plasmon resonance analysis of the binding of CD19 to the Fab fragment of CB3f in humans, mice, and cynomolgus monkeys. Recombinant CD19-Fc protein was purchased from R&D Systems. An A protein sensor chip (GE A protein chip P / N 2912755) was used. The running buffer was 1X HBS-EP+ (Teknova P / N H8022), and the running temperature was 37°C.

[0088] The CD19 Fc fusion protein was diluted to 3 μg / mL in run buffer, and approximately 60 RU of human, cynomolgus monkey, and mouse proteins were captured in flow cells (Fc) 2, 3, and 4, respectively. The CB3f Fab fragment was diluted to 1000 nM in run buffer and then serially diluted 5-fold to 1.6 nM in run buffer for a total of 5 dilutions. Fab or buffer blanks were injected at 50 µL / min for 300 seconds, followed by a dissociation phase of 900 seconds. Regeneration was performed by injecting 10 mM glycine at pH 1.5 at 50 µL / min for 30 seconds on all Fc cells. Reference subtraction data were collected for Fc2-Fc1, Fc3-Fc1, and Fc4-Fc1, and then reference subtraction data were obtained by subtracting blanks. Binding rate (kb) on ) and dissociation rate (k off The "1:1 binding" model was used for fitting. The affinity (K) was calculated from the binding kinetics based on the following relationship. D ): K D = k off / k on .

[0089] CB3f has affinity (K) of 3.35 pM and 45.2 pM, respectively. D It binds to human and cynomolgus monkey CD19 expressed on CHO cells (Table 1).

[0090] The Fab fragment of CB3f is 3.20 x 10 6 M -1 s -1 binding rate (k on ), 2.35 x 10 -4 s -1 dissociation rate (k off ) and an affinity of 76.8 pM (K D () Combined with human CD19 (Table 2). The Fab fragment of CB3f is 1.04 x 10 6 M -1 s -1 binding rate (k on ), 9.78 x 10 -2 s -1 dissociation rate (k off ) and an affinity of 94.5 nM (K D Binding to cynomolgus monkey CD19 (Table 2). No binding to mouse CD19 was observed at a 1 µM Fab concentration.

[0091] Table 1: In vitro binding of CB3f to fixed CHO cells expressing human or cynomolgus monkey CD19.

[0092] [Measured by MSD-SET at 37°C. Results are reported as K from 3 independent replicate experiments.] D The geometric mean. The error estimate is calculated as the geometric mean x standard deviation log. 10 K D x ln 10]

[0093] Species mAb affinity (pM) Human CD19 3.35 ± 0.56 Crab-eating macaque CD19 45.2 ± 2.8

[0094] Table 2: In vitro binding parameters of the Fab fragment of CB3f to human, cynomolgus monkey and mouse CD19 Fc fusion proteins.

[0095] [Measured at 37°C by surface plasmon resonance (SPR). Results are reported as the mean ± standard deviation of three independent replicates.]

[0096]

[0097] stability

[0098] The stability of CB3f was assessed at a high concentration (approximately 100 mg / mL) in 5 mM histidine buffer (pH 6.0) with excipients. The concentrated samples were incubated at 5°C and 35°C for 4 weeks. After incubation, the percentage of high molecular weight (%HMW) of the samples was analyzed using size exclusion chromatography (SEC), fragmentation was analyzed by capillary electrophoresis (CE-SDS), and chemical modifications (e.g., deamidation, isomerization, or oxidation) were analyzed by LC-MS peptide mapping. After 4 weeks at 35°C, CB3f showed 0.5% ∆%HMW, 0.6% ∆% fragmentation, and no CDR chemical modifications greater than 0.2%.

[0099] Freeze / thaw stability under identical conditions was evaluated using three repeated slow, controlled temperature cycles simulating the freeze / thaw conditions of a large bulk drug substance placed at -70°C. After three freeze-thaw cycles, CB3f showed a ∆%HMW of 1.9%, as measured by SEC. Other excipients could further reduce the %HMW increase (data not shown).

[0100] These results indicate that CB3f possesses good physical and chemical stability.

[0101] solubility

[0102] Solubility was assessed by concentrating 100 mg CB3f to approximately 0.5 mL using a 30 kDa molecular weight cutoff centrifugal filter (e.g., Amicon UC filter, Millipore, catalog # UFC903024). The final concentration of the sample was measured using a Solo VPE spectrophotometer (C Technologies, Inc.) via ultraviolet absorbance at 280 nm.

[0103] CB3f exhibits a solubility greater than or equal to 183 mg / mL in 5 mM histidine pH 6 buffer and greater than or equal to 170 mg / mL in PBS (phosphate-buffered saline) pH 7.4. These results indicate that CB3f exhibits high solubility.

[0104] Viscosity

[0105] The viscosity of CB3f was analyzed at 15 °C at a concentration of approximately 125 mg / mL in 5 mM histidine at pH 6.0 with different excipients. Viscosity measurements were performed using a VROC Initium (RheoSense). CB3f showed viscosities of 14.2 cP at 131 mg / mL in 5 mM histidine + 280 mM mannitol at pH 6, 9.5 cP in 5 mM histidine + 150 mM sodium chloride at pH 6.0, and 6.1 cP in 5 mM histidine at pH 6.0 + 280 mM arginine. These results indicate that CB3f exhibits low viscosity, which allows for high-concentration dosing.

[0106] Pharmacokinetics (PK)

[0107] The pharmacokinetic (PK) properties of CB3f were investigated in cynomolgus monkeys following single subcutaneous administration of 0.03, 0.3, 1, and 10 mg / kg CB3f or single intravenous bolus administration of 1 mg / kg CB3f. CB3f showed linear PK across the examined subcutaneous dose range, while the final half-life (T0) was relatively short. 1 / 2 The time range was from 182 to 301 hours. The time following administration of 1 mg / kg CB3f via rapid intravenous perfusion was T... 1 / 2 It lasts for 324 hours.

[0108] According to reports, obeliximab has a mean T3 of 3.5 ± 1.0 days. 1 / 2 This is equivalent to 60-108 hours (Jaraczewska-Baumann et al., European League Against Rheumatism (EULAR) 2015 Annual Meeting Poster). A Phase 1b / 2a Study of the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of XmAb®5871 in Patients with Rheumatoid Arthritis (June 12, 2015, available at https: / / investors.xencor.com / static-files / 0017dcf1-deb2-46eb-93ff-90ba164ec50c). Therefore, CB3f has a better T than obbelimumab. 1 / 2 .

[0109] Example 3. In vitro functional characterization of anti-human CD19 antibody.

[0110] In vitro inhibition of B cell proliferation by anti-human CD19 mAb CB3f.

[0111] The ability of anti-human CD19 mAb CB3f to inhibit the proliferation of primary human B cells was tested in an in vitro B cell proliferation assay.

[0112] Primary human B cells were isolated from healthy donor PBMCs using a B cell isolation kit (Stemcell Technologies) via negative selection. Primary human B cells were isolated at 1 x 10-1 6 Cells were resuspended at 100 cells / mL and cultured at 37°C in RPMI-1640 complete medium (containing 10% fetal bovine serum, 1x MEM-nonessential amino acids, 1 mM sodium pyruvate, 1x penicillin-streptomycin solution (both from Corning), 1x Glutamax (Gibco), and 0.1% β-mercaptoethanol (Life Technologies)) on 96-well, U-bottom polystyrene plates. Cells were pretreated with anti-human CD19 antibody for 1 hour and cultured at 37°C. 0 The mice were stimulated for 2 days with 5% CO2 using mouse anti-human IgM (2 μg / mL - Southern Biotech) and rabbit anti-mouse IgG (12 μg / mL - Thermo Fisher). Then [ 3 H]-thymidine (1µCi thymidine / pore, PerkinElmer, Boston, MA Pulsed cells were used for 18 hours of cell culture. 3 The incorporation level of H]-thymidine was measured using a 2450 microplate counter (MicroBeta² serial number: 5129186, PerkinElmer, Boston, MA) and expressed as cell counts per minute (ccpm).

[0113] Anti-human CD19 mAb CB3f inhibited the proliferation of primary human B cells in a dose-dependent manner, while the isotype control showed no inhibition at any tested concentration. Figure 3 The inhibitory function of CB3f was tested using B cells obtained from 12 different donors, and the mean IC50 of CB3f was measured. 50 The value was 0.007 nM (Table 3). The data showed that anti-human CD19 mAb CB3f could inhibit the proliferation of human primary B cells in a dose-dependent manner, while B cell proliferation was not affected by the allotype control antibody.

[0114] Table 3. Inhibition of primary B cell proliferation by anti-human CD19 mAb CB3f

[0115] <![CDATA[IC 50 Value (nM) Donor 1 0.006 Donor 2 0.009 Donor 3 0.003 Donor 4 0.006 Donor 5 0.004 Donor 6 0.003 Donor 7 0.003 Donor 8 0.005 Donor 9 0.005 10 donors 0.028 Donor 11 0.004 12 donors 0.005 average value 0.007

[0116] In vitro inhibition of B cell activation in human whole blood

[0117] The ability of anti-human CD19 mAb CB3f to inhibit the activation of primary human B cells in whole blood was tested in an in vitro whole blood activation assay.

[0118] Human whole blood treated with EDTA (healthy donors, TSRI Normal Blood Donor Services, San Diego, CA) was cultured in polystyrene 96-well, U-bottom plates and pre-incubated at 37°C and 5% CO2 for 30 min to 1 h with CB3f or allotype control antibody. 7 nM of each antibody was used as the highest concentration, and was simultaneously 4-fold diluted and titrated 12-point in complete medium (RPMI-1640 containing 10% fetal bovine serum, 1x MEM-nonessential amino acids, 1 mM sodium pyruvate, 1x penicillin-streptomycin solution (both from Corning), and 1x Glutamax (Gibco), 0.1% β-mercaptoethanol (Life Technologies). Whole blood was stimulated at 37°C and 5% CO2 for 24 h with 2.5 µg / mL TLR9 ligand (CpG ODN 7909 (InvivoGen, San Diego, CA)). B cell activation distribution was measured by flow cytometry, and data were analyzed using FlowJo software.

[0119] Flow cytometry was performed as follows. Processed and activated human whole blood was lysed with RBC lysis buffer (Fisherscientific, USA). Cells were stained at 4°C for 30 minutes with an appropriate combination of fluorescently conjugated antibodies to identify B cell activation markers: CD69 BV605 (cat #310938) from BioLegend. Cells were also stained with CD3FITC (cat #300306), CD19 APC (cat #363006), CD20 PerCP-Cy5.5 (cat #560736), and the fixable viability dye eFluor™ 780 (eBioscience), all from BioLegend. At least 25,000–50,000 live-cell gating events were analyzed for each sample. Samples were acquired on a BDFortessa X-20 and results were analyzed using FlowJo software.

[0120] Anti-human CD19 mAb CB3f inhibited the activation of primary human B cells in a dose-dependent manner, as measured by a reduction in CD69+ B cells. The assay was performed using blood obtained from three different donors, and representative results were shown... Figure 4 In the aforementioned determination, the average IC50 of CB3f was... 50The concentration was 0.008 nM (Table 4). The allotype control antibody did not show inhibition of CD69 expression at any tested concentration. Data showed that anti-human CD19 mAb CB3f inhibited the activation of primary human B cells in whole blood in a dose-dependent manner, while B cell activation was unaffected by the allotype control antibody.

[0121] Table 4. Anti-human CD19 mAb CB3f inhibits B cell activation in whole blood.

[0122] <![CDATA[IC 50 Value (nM) Donor 1 0.015 Donor 2 0.0006 Donor 3 0.008 average value 0.008

[0123] In vitro inhibition of B cell differentiation into plasmablasts

[0124] Memory B cells were isolated from healthy donor PBMCs using a memory B cell isolation kit (Miltenyi Biotec). Primary human memory B cells were isolated at a ratio of 1 x 10-1. 6 Cells were resuspended at 100 cells / mL and cultured at 37°C in 96-well, U-bottom polystyrene plates in complete medium (RPMI-1640 containing 10% fetal bovine serum, 1x MEM-nonessential amino acids, 1 mM sodium pyruvate, 1x penicillin-streptomycin solution (all from Corning), 1x Glutamax (Gibco), and 0.1% β-mercaptoethanol (Life Technologies). Cells were pretreated with anti-human CD19 mAb CB3f for 1 hour and stimulated for 5 days with 50 ng / mL anti-CD40, 200 ng / mL BAFF, 1 ng / mL IL-2, and 100 ng / mL IL-21 (all from R&D). Cells were washed and stained at 4°C with an antibody combination conjugated with an appropriate fluorescent dye for 30 minutes to identify memory B cells differentiating into plasmablasts: all from BioLegend CD38 PE, CD3 FITC (cat # 300306), CD19... APC (cat # 363006), CD20 PerCP-Cy5.5 from BDPharminogen (cat # 560736), and the fixable viability dye eFluor™ 780, eBioscience. At least 25,000–50,000 live-cell gating events were analyzed for each sample. Samples were acquired on a BDFortessa X-20 and results were analyzed using FLowJo software. Plasma blastocyte percentage was defined as CD38. 亮 / CD20 低 B cells%.

[0125] Anti-human CD19 mAb CB3f inhibited the differentiation of primary human memory B cells into plasmablasts in a dose-dependent manner. Figure 5The experiment was repeated three times, and representative data are presented. The allotype control antibody did not show inhibition of plasmablast differentiation at any tested concentration. The data show that CB3f can inhibit the differentiation of primary human memory B cells into plasmablasts in a dose-dependent manner, and differentiation is unaffected by the allotype control antibody.

[0126] CD19 mAb CB3f is a non-depleting mAb and lacks activity in in vitro CDC and ADCC assays.

[0127] Anti-human CD19 mAb CB3f is a B-cell inhibitory antibody designed to suppress B-cell function without causing B-cell depletion. In vitro assays confirmed that CB3f lacks complement-dependent cytotoxic (CDC) and antibody-dependent cytotoxic (ADCC) activity, meaning it does not exhibit depletion function.

[0128] Wil2-s cell lines expressing CD19 and CD20 were used as target cells, and Jurkat cell lines (Eli Lilly and Company) expressing functional FcγRIIIa (V158)-NFAT-Luc were used as effector cell lines. CB3f was tested, and IgG1 antibody, a known effective inducer of ADCC and CDC, was used as a positive control.

[0129] CB3f was serially diluted three times, starting at 10 μg / mL for CDC and 1 μg / mL for ADCC assays. Add 50 μL / well of the test compound or assay buffer to a 96-well plate (Costar 3916). Wil2-s cells were diluted to 1x10⁻⁶. 6 A concentration of 100 cells / mL was obtained, and 50 μL / well was added to the plate. CDC and ADCC plates were incubated at 37°C for 1 hour. Immediately afterwards, Jurkat V158 cells were diluted to 3 x 10⁻⁶ cells / mL. 6 Cells / mL concentrations were added to ADCC plates at 50 μL / well, or pre-diluted complement (Quidel A113) from human serum to CDC plates at 50 μL / well. CDC plates were incubated at 37°C for 2 hours, followed by the addition of 100 μL / well Cell-Titre Glo (Promega G7571). ADCC plates were incubated at 37°C for 4 hours, followed by the addition of 100 μL / well ONE-Glo (Promega E8130). The contents of the plates were mixed at low speed using a plate shaker, and the luminescence signal was read on an Envision 11 multimode plate reader using an integration of 0.2 cps. Data were analyzed using GraphPad Prism v8.2.

[0130] Results from CDC and ADCC measurements showed Figures 6A-6B (Representative results from three independent plate runs). All reaction levels were classified relative to the positive control IgG1 antibody. Anti-human CD19 mAb CB3f at the concentrations shown showed neither CDC activity ( Figure 6A ) also lacks ADCC activity ( Figure 6B ).

[0131] The specificity of anti-human CD19 mAb binding to B cells in human whole blood.

[0132] Anti-human CD19 mAb CB3f is a B-cell inhibitory antibody that works by suppressing B-cell function. Obeliskinea is an antibody that binds to both CD19 and FcγRIIb. However, the high affinity binding of obeliskinea to FcγRIIb can lead to binding to other cell types in a CD19-independent manner. Therefore, the binding specificity of obeliskinea and anti-human CD19 mAb CB3f was compared using a human whole blood binding assay.

[0133] EDTA-treated human whole blood (healthy donors, TSRI Normal Blood Donor Services, San Diego, CA) was seeded into polystyrene 96-well plates and stained with appropriate combinations of extracellular antibodies conjugated with fluorescent dyes at different concentrations of Alexa Flour® 647-conjugated CB3f, Alexa Flour® 647-conjugated and Alexa Flour® 647-conjugated isotype controls, and fluorescent dye-conjugated extracellular antibodies to detect lymphocyte / granulocyte populations. 7 nM of each antibody was used as the highest concentration, and the plates were prepared in 1x Ca2+-free DPBS supplemented with 2% fetal bovine serum (FBS-heat-inactivated) from Corning®. 2+ Mg-free 2+The mixture was 3-fold diluted and titrated at 8 points in Dulbecco's phosphate-buffered saline. The mixture included CD20 PerCP-Cy5.5 (cat # 560736), CD45BV421 (cat # 563879), and CD66 FITC (cat # 555724) from BD Biosciences; CD3 BV605 (cat # 317322) and CD11b PE-Cy7 (cat # 101216) from BioLegend; and the fixable viability dye eFluor™ 780 (cat # 65-0865-14) from eBioscience. Whole blood was stained with antibodies at 4°C in the dark for 30 minutes. Dead cells were excluded by the viability dye, and at least 25,000–50,000 live-cell gating events were analyzed. Samples were obtained on a BD Fortessa X-20 and the results were analyzed using FlowJo software.

[0134] Flow cytometry analysis confirmed that, at all tested concentrations, CB3f bound only to B cells in human whole blood. Figure 7 Obelimab showed binding to a population of human B cells as well as CD20-negative cells (i.e., non-B cells) that express CD66 and CD11b and are therefore identified as neutrophils. Figure 7 Therefore, the data indicate that anti-human CD19 mAb CB3f binds highly specifically to human B cells in whole blood, while obelicumab, in addition to binding to B cells, also shows non-specific binding to human neutrophils. Since neutrophils are the most abundant type of white blood cell in humans, this non-specific binding to neutrophils may explain the short half-life of obelicumab observed in human patients. The experiments were repeated using blood from four different donors, and representative results were shown... Figure 7 middle.

[0135] In vitro apoptosis assay.

[0136] As mentioned above, during clinical studies, obeliximab was shown to reduce B-cell counts in human patients (Jaraczewska-Baumann et al., EULAR 2015 Annual Meeting Poster: Phase 1b / 2a Study of the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of XmAb®5871 in Patients with Rheumatoid Arthritis (June 12, 2015)

[0137] The ability of anti-human CD19 mAb CB3f and obbelimumab to induce apoptosis in primary human B cells was tested using an in vitro B cell apoptosis assay. Primary human B cells were isolated from healthy donor PBMCs using a B cell isolation kit (Stemcell Technologies) via negative selection. Primary human B cells were isolated at a rate of 1 x 102 6 Cells were resuspended at 100 cells / mL and cultured at 37°C in RPMI-1640 complete medium (containing 10% fetal bovine serum, 1x MEM-nonessential amino acids, 1 mM sodium pyruvate, 1x penicillin-streptomycin solution (both from Corning), 1x Glutamax (Gibco), and 0.1% β-mercaptoethanol (Life Technologies)) in 96-well, U-bottom polystyrene plates. Cells were treated with CB3f, obbelimumab, or allotype control antibody at indicated concentrations for 24 hours at 37°C and 5% CO2. B-cell apoptosis was measured by flow cytometry using annexin V staining combined with viability dyes. Cells were stained at 4°C for 30 minutes with antibody conjugated to appropriate fluorescent dyes to identify B-cell activation markers: CD19 APC (Biolegend), CD20 PerCP-Cy5.5 (BD Pharminogen), annexin V (Invitrogen), and the immobilizable viability dye eFluor™. 780 (eBioscience). Apoptotic cells are defined as living annexin V cells. + Cells. Samples were obtained on a BD Fortessa X-20 and the results were analyzed using FlowJo software.

[0138] Repeated experiments were conducted using B cells obtained from three different donors, and representative data showed that... Figure 8 In contrast, oxaberitumab induces B-cell apoptosis in a dose-dependent manner (Figure 8), which may explain the reduced B-cell counts observed in human patients in clinical studies. In comparison, CB3f induces very little to no apoptosis in human B cells when compared to isotype controls. Figure 8 The data indicate that, compared to obelicumab, anti-human CD19 mAb CB3f does not induce apoptosis in primary human B cells first in vitro and then in vivo, suggesting a different mechanism of action for CB3f. The data further suggest that anti-human CD19 mAb CB3f exerts its effect by inhibiting B cell function without depleting B cells or significantly reducing B cell numbers.

[0139] Example 4. In vivo functional characterization of anti-human CD19 antibody.

[0140] Female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, JAX Labs, Stock #05557) were housed in cages of three at 72°C under a 12-hour light:dark cycle, with free access to food and water (n=33). Human peripheral blood mononuclear cells (PBMCs) were isolated from LRS tubes obtained from the San Diego Blood Bank (San Diego, CA) using SepMate 50 Ficol preparation tubes, following the manufacturer's instructions (StemCell Technologies, Vancouver, BC). Freshly isolated PBMCs were incubated at 1.2 e 8 Cells / mL were suspended in PBS and intravenously transferred into mice on day 0 with 100 µL of PBMC suspension (1.2 e). 7 / mice, n=29); 4 mice were not administered PBMCs as a non-transfer control. On day 1, mice were randomly assigned to three weight-matched groups and subcutaneously administered 0.01 or 1.0 mg / kg human IgG4 isotype control or CB3f (200 µL / mouse, n = 10, 10, and 9, respectively). Dosing continued weekly for the remainder of the experiment. Regular health checks and weight measurements were performed. On days 6 and 10, blood was collected in heparin-coated capillaries by tail clipping. On day 15, blood was collected in EDTA tubes by cardiac puncture under isoflurane anesthesia for FACS analysis and clarified by centrifugation for plasma analysis. Spleens were harvested and processed into single-cell suspensions for FACS analysis.

[0141] Mice were weighed in a BSL2 enclosure, and clinical signs of adverse stress were assessed 2–3 times per week. Common clinical signs in the model were ruffled fur, hunched shoulders, emaciation, and difficulty breathing or moving. Weight change was calculated as a percentage of baseline weight: (weight on day (x) / weight on day 0) * 100.

[0142] Blood from a cardiac puncture was collected in EDTA-coated tubes, clarified by centrifugation, and the resulting plasma was stored at -80°C for future processing. Plasma IgM levels were measured using the Mesoscale Discovery Human Isotyping panel (Rockville Maryland) according to the manufacturer's instructions.

[0143] Single-cell suspensions of mouse spleen were used for FACS analysis. Cells were seeded in 96-well plates and stained at 4°C for 30 min with an appropriate combination of fluorescently conjugated antibodies to identify B-cell activation markers: hCD45-BV421, CD86 BV650, CD3 APC, CD19 FTIC, CD20 PerCP-Cy5.5, and the fixable viability dye eFluor™ 780, all derived from BioLegend. At least 250,000 live-cell gating events were analyzed for each sample. Samples were obtained on a BD Fortessa X-20 and results were analyzed using FlowJo software. Data were plotted and statistics were calculated using Prism software (GraphPad, San Diego, CA). Differences in weight between groups were determined by a two-factor RM-ANOVA with Tukey post-hoc analysis test. Differences in plasma IgM levels were determined by a 1-factor ANOVA with Tukey post-hoc analysis test, and were considered significant if p < 0.05.

[0144] Injection of human PBMCs into NSG mice resulted in significant translocation of functional B cells, as measured by human IgM secretion into the periphery. On days 6 and 10 post-translocation, human IgM levels in IgG4 isotype control animals rapidly increased from undetectable to 1.3 ± 0.1 and 48.8 ± 6.2 µg / mL, respectively. CB3f administration at 0.01 mg / kg / wk significantly reduced IgM secretion by 60% and 78% on day 6, respectively. Figures 9A-9B As shown in the figure (p<0.01 for both doses). A 57% reduction in circulating IgM was also observed on day 10 with CB3f at a dose of 1.0 mg / kg / wk (p<0.05). There was no difference in mouse weight between groups, and no mice showed signs of GvHD.

[0145] Activation of human B cells in NSG mice was measured by the expression of the activation marker CD86. For example... Figure 10 As confirmed in the study, treatment with anti-human CD19 mAb CB3f reduced the expression of CD86 on human B cells in a dose-dependent manner.

[0146] Treatment with CB3f resulted in a slight decrease in the percentage of spleen B cells, which may be attributed to reduced B cell activation and proliferation. Nevertheless, B cells were present in the spleen at a substantially unchanged number (>50% of spleen cells), indicating the non-depletion properties of the anti-human CD19 mAb CB3f. Figure 11 ).

[0147] Example 5. Internalization of anti-human CD19 antibody in primary human B cells

[0148] Peripheral blood mononuclear cells (PBMCs) were collected from LRS-WBCs of healthy volunteers using standard density gradient centrifugation. Human primary B cells were isolated from healthy donor PBMCs using a B cell isolation kit via negative selection. For internalization studies, B cells were collected at 4 x 10⁻⁶ cells / mL. 6 Cells were resuspended at 100 cells / mL and 50 µL was added to each well of a 96-well plate. A labeled F(ab')2 targeting the human Ig Fcγ fragment (F(ab')2-TAMRA-QSY7) was used as a probe to track internalization. The test antibody and probe were incubated at 4°C for 30 min to form a complex, and 50 µL was added to each well of B cells. The final concentration of the test antibody was 2 µg / mL. Cells were cultured in a CO2 incubator at 37°C for 24 h. Cells were then washed twice with 2% FBS PBS and resuspended in 2% FBS PBS with a viability dye (SYTOX Green, Invitrogen). Data were collected on a BD Fortessa X-20 and analyzed in FlowJo.

[0149] Both anti-human CD19 mAb1 (CB3f) and anti-human CD19 mAb2 (C323.C1) showed internalization in primary human B cells at 24 hours. Figure 12 The IgG1 effector ineffective isotype control antibody was used as a negative control and did not show any significant internalization, as evidenced by the low percentage of TAMRA+ cells. Figure 12 The positive control IgG4 mAb showed similar internalization to the tested anti-human CD19 mAbs. Figure 12 ).

[0150] Example 6. Comparison of the efficacy of non-depleted anti-mouse CD19 substitute antibody and B-cell depleted anti-mouse CD20 substitute antibody in animal models.

[0151] In vivo function of non-depleted CD19 replacement antibody and B cell depleted CD20 replacement antibody in a mouse CIA model effect

[0152] In the study, non-depleted anti-mouse CD19 replacement antibody (CD19 replacement Ab) and B-cell depleted anti-mouse CD20 replacement antibody (CD20 replacement Ab) were tested in a mouse collagen-induced arthritis (CIA) model with a semi-established disease pattern, along with an allotype control antibody (i.e., antibodies were introduced after disease induction but before the animals developed any detectable clinical score).

[0153] Starting on day 25, mice immunized with collagen and complete Freund's adjuvant (CFA) developed observable signs of joint inflammation, while disease-free mice treated with the same type did not exhibit observable joint inflammation throughout the study. Figure 13A Treatment with CD20 replacing Ab (10 mg / kg SC, once weekly) starting on day 19 improved the mean clinical score, while CD19 replacing Ab (5 mg / kg SC, twice weekly) even further reduced the mean clinical score. Figure 13A Compared with the isotype control, CD20 substitution with Ab did not significantly reduce the clinical score AUC (). Figure 13B On the other hand, compared with both the isotype control and CD20-substituted Ab, CD19-substituted Ab significantly reduced the clinical score AUC (). Figure 13B These results suggest that CD19 substitution Ab reduces disease severity more significantly than CD20 substitution Ab, as assessed by clinical score AUC in a mouse model of CIA arthritis performed in a semi-established pattern.

[0154] Non-depleted CD19-substituted Abs and B-cell depleted CD20-substituted Abs in a mouse model of NOD with type 1 diabetes Internal effects.

[0155] The NOD / ShiLtJ mouse strain (commonly referred to as non-obese diabetic mice) is a polygenic model of autoimmune type 1 diabetes (T1D). Diabetes in NOD mice is characterized by hyperglycemia and insulitis, with leukocyte infiltration of the islets. Disease prevalence is highest in female mice.

[0156] In the study, non-depleted CD19 substitutes were tested in a mouse NOD model of type 1 diabetes in a half-treatment disease modality, together with depleted CD20 substitutes (i.e., the antibody was introduced just before the control animals began to develop clinical scores).

[0157] Female NOD-LTJ mice were untreated and administered either a CD19-alternative Ab (5 mg / kg / BIW) or a CD20-alternative Ab starting at 12 weeks of age. Blood glucose levels in all mice were measured weekly using an AccuCheck Aviva glucometer (Roche cat# 06870287001) and test strips (Roche cat# 06908373001 lot# 497064). Mice with blood glucose levels above 240 mg / dL were considered to have diabetes. Untreated mice developed diabetes starting at 14 weeks of age. Treatment with a CD20-alternative Ab (5 mg / kg SC, twice weekly) showed very mild protection against disease progression, while a CD19-alternative Ab (5 mg / kg SC, twice weekly) delayed and reduced the incidence of diabetes. Figure 14These results suggest that non-depleted CD19-substituted Abs can delay and reduce the incidence of type 1 diabetes in NOD mice more significantly than depleted anti-CD20-substituted Abs.

[0158] In vivo efficacy of non-depleting CD19-substituted Ab and B-cell depleting CD20-substituted Ab in a mouse EAE model

[0159] Experimental autoimmune encephalomyelitis (EAE) in mice has been widely used as a model of inflammatory neurodegeneration and demyelinating diseases, such as multiple sclerosis (MS).

[0160] In the study, non-depleted anti-CD19 substitutes were tested in a half-treatment disease modality with depleted CD20 substitutes in a protein lipoprotein (PLP)-induced remission-relapse (RR) demyelinating EAE model (i.e., antibodies were introduced after disease induction but before the animals began to develop any detectable clinical fraction).

[0161] For this study, female 9-10 week old SJL mice (Jackson Labs) with an average weight of 18-21 g were used. Mice were immunized with Hooke Kit™ PLP139-151 / CFA emulsion (catalog number EK-0120, HookeLaboratories, Lawrence MA) and 75 ng of pertussis toxin (PTX). Animals were randomized to study groups based on weight, with 15 mice in each group. Starting from day 8, mice were subcutaneously administered 5 mg / kg of non-depleted CD19-alternative Ab, depleted CD20-alternative Ab, or isotype mouse IgG1 control twice weekly. EAE scores were collected daily in increments of 0.5 units from grade 0 to 6. Mice were sacrificed on day 42. Compared with the isotype control, treatment with non-depleted CD19-alternative Ab significantly reduced EAE scores on days 35-41; 51% of mice showed a reduction in EAE scores on the last day. Figure 15 At any time point in the test, treatment with depleted CD20 replacing Ab did not show a significant reduction in disease score compared to isotype control. Figure 15 These results suggest that non-depleted CD19 replacement Abs can reduce disease severity more significantly than depleted CD20 replacement Abs, as assessed by clinical scores in the EAE model of multiple sclerosis in a treatment-half-treatment modality.

[0162] sequence list

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] CB3f / CB3-IgG1 effector ineffective

[0175] SEQ ID NO: 52 CB3f / CB3 IgG1-Effector Invalid HC (LC same as CB3f / CB3) (underlined for changes in IgG1 WT)

[0176]

[0177]

[0178] SEQ ID NO: 53 CB3f / CB3 IgG1-effecton null HC DNA (LC is the same as CB3f / CB3)

[0179]

[0180] C323.C1-IgG1 effector ineffective

[0181] SEQ ID NO: 54 C323.C1 IgG1-Effective inactive HC (LC same as CB3.C1) (underlined for changes in IgG1 WT)

[0182]

[0183] SEQ ID NO: 55 C323.C1 IgG1-effecton null HC DNA

[0184]

[0185]

[0186] SEQ ID NO: 56-person CD19 isotype 1 (NCBI reference sequence: NP_001171569.1)

[0187]

[0188] SEQ ID NO: 57 human CD19 isotype 2 (NCBI reference sequence: NP_001761.3)

[0189] sequence list <110> Eli Lilly <120> Anti-human CD19 antibody <130> X22706 <150> 62 / 983,093 <151> 2020-02-28 <160> 57 <170> PatentIn version 3.5 <210> 1 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 1 Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 2 Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 3 Ala Arg Glu Asp Phe Gly Tyr Asn Tyr Gly Met Asp Val 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 4 Arg Ala Ser Gln Gly Val Asn Ser Tyr Tyr Leu Ala 1 5 10 <210> 5 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 5 Tyr Ala Thr Ser Thr Arg Ala Thr 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 6 Gln His Tyr Gly Asn Ser Leu Phe Thr 1 5 <210> 7 <211> 446 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Phe Gly Tyr Asn Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 <210> 8 <211> 1338 <212> DNA <213> artificial sequence <220> <223> synthetic construction <400> 8 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaggac 300 ttcggctaca attacggcat ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360 gcttctacca agggcccatc ggtcttcccg ctagcgccct gctccaggag cacctccgag 420 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 600 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 660 aaatatggtc ccccatgccc accctgccca gcacctgagt ttctgggggg accatcagtc 720 ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 780 tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 960 tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1140 tgggaaagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 1260 aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 1320 ​​​​​​​​​​​​​​​​​ 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Phe Gly Tyr Asn Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 10 <211> 360 <212> DNA <213> Artificial sequence <​​​​​​​​cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaggac 300 ttcggctaca attacggcat ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360 <210> 11 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 1'0 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Asn Ser Tyr 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 [[ID=)4]]Met Tyr Ala Thr Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 It should be noted that there seems to be an error in the original text at "Met Tyr Ala Thr Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser" where "Thr" is repeated in the sequence. This might affect the correct understanding and translation. Please double-check the original text for accuracy.Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Asn Ser Leu 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 12 <211> 645 <212> DNA <213> artificial sequence <220> <223> synthetic construction <400> 12 gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gggtgttaac agctactact tagcctggta ccagcagaaa 120ctctcctgca gggccagtca gggtgttaac agctactact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatgtat gctacatcca ccagggccac tggcatccca 180cctggccagg ctcccaggct cctcatgtat gctacatcca ccagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cactatggta actcactatt cactttcggc 300cctgaagatt ttgcagtgta ttactgtcag cactatggta actcactatt cactttcggc 300<00008​​​​​​​​​​​​​​​​​<223> Synthetic constructs <400> 13 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Asn Ser Tyr 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Met Tyr Ala Thr Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Asn Ser Leu 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 100 105 <210> 14 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 14 gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gggtgttaac agctactact tagcctggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatgtat gctacatcca ccagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cactatggta actcactatt cactttcggc 300 cctgggacca aggtggagat caaa 324 <210> 15 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 15 Lys Ala Ser Gly Gly Thr Ile Ser Ser Tyr Ala Tyr Ser 1 5 10 <210> 16 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 16 Gly Ile Ile Pro Ile Phe Asp Thr Ala Asn 1 5 10 <210> 17 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 17 Ala Arg Glu Asp Phe Gly Tyr Asn Tyr Ala Met Asp Val 1 5 10 <210> 18 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 18 Arg Ala Ser Gln Lys Val Asn Ser Tyr Tyr Leu His 1 5 10 <210> 19 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 19 Tyr Ala Thr Arg Thr Arg Pro Thr 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 20 Gln His Tyr Gly Gln Ser Leu Phe Thr 1 5 <210> twenty one <211> 446 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> twenty one Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Ile Ser Ser Tyr 20 25 30 Ala Tyr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Asp Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Phe Gly Tyr Asn Tyr Ala Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 <210> 22 <211> 1338 <212> DNA <213> artificial sequence <220> <223> synthetic construction <400> 22 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggagg caccattagc agctatgctt acagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttgacac agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaggac 300 ttcggctaca attacgcgat ggacgtctgg ggccaaggga cccttgtcac cgtctcctca 360 gcttctacca agggcccatc ggtcttcccg ctagcgccct gctccaggag cacctccgag 420 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 600 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 660 aaatatggtc ccccatgccc accctgccca gcacctgagt ttctgggggg accatcagtc 720 ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 780 tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 960 tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1140 tgggaaagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 1260 aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 1320 ctctccctgt ctctgggt 1338 <210> 23 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Ile Ser Ser Tyr 20 25 30 Ala Tyr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Asp Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Phe Gly Tyr Asn Tyr Ala Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 24 <211> 360[[ID=​​​​​​​​​​​​​​cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttgacac agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaggac 300 ttcggctaca attacgcgat ggacgtctgg ggccaaggga cccttgtcac cgtctcctca 360 <210> 25 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 25 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Lys Val Asn Ser Tyr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Ala Thr Arg Thr Arg Pro Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Gln Ser Leu 85 90 95 Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 26 <211> 645 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 26 gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gaaagttaac agctactact tacattggta ccagcagaaa 120ctctcctgca gggccagtca gaaagttaac agctactact tacattggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat gctacaagaa ccaggccaac tggcatccca 180cctggccagg ctcccaggct cctcatctat gctacaagaa ccaggccaac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag​​​​​​​​​​​​​​​​​​​​​​<223> Synthetic constructs <400> 27 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Lys Val Asn Ser Tyr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Ala Thr Arg Thr Arg Pro Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Gln Ser Leu 85 90 95 Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 28 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 28 gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gaaagttaac agctactact tacattggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat gctacaagaa ccaggccaac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cactatggtc agtcactatt cactttcggc 300 caggggacca aggtggagat caaa 324 <210> 29 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 29 Lys Ala Ser Gly His Thr Ile Ser Ser Tyr Ala Tyr Ser 1 5 10 <210> 30 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 30 Asp Ile Ile Pro Ala Tyr Gly Ser Pro Asn 1 5 10 <210> 31 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 31 Ala Arg Glu Asp Phe Gly Lys Asn Tyr Ala Met Asp Val 1 5 10 <210> 32 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 32 Arg Ala Ser Gln His Val Ser Ser His Tyr Leu Ala 1 5 10 <210> 33 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 33 Tyr Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 34 Gln His Tyr Gly Gln Ser Gln Phe Thr 1 5 <210> 35 <211> 446 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 35 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly His Thr Ile Ser Ser Tyr 20 25 30 Ala Tyr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ala Tyr Gly Ser Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Phe Gly Lys Asn Tyr Ala Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 <210> 36 <211> 1338 <212> DNA <213> artificial sequence <220> <223> synthetic construction <400> 36 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggaca taccattagc agctatgctt acagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagac atcatccctg cctatggctc accaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaggac 300 ttcggcaaga attacgcgat ggacgtctgg ggccaaggga cccttgtcac cgtctcctca 360 gcttctacca agggcccatc ggtcttcccg ctagcgccct gctccaggag cacctccgag 420 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 600 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 660 aaatatggtc ccccatgccc accctgccca gcacctgagt ttctgggggg accatcagtc 720 ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 780 tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 960 tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1140 tgggaaagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 1260 aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc​​​​​​​​​​​​​​​​​​ 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly His Thr Ile Ser Ser Tyr 20 25 30 Ala Tyr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ala Tyr Gly Ser Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Phe Gly Lys Asn Tyr Ala Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <21l> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 38 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggaca taccattagc agctatgctt acagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagac atcatccctg cctatggctc accaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaggac 300 ttcggcaaga attacgcgat ggacgtctgg ggccaaggga cccttgtcac cgtctcctca 360 <210> 39 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 39 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Ser His 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Gln Ser Gln 85 90 95 Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175<​​​​​​​​​​​​​​​​​​​​​​​​​ gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gcatgttagc agccactact tagcttggta ccagcagaaa 120ctctcctgca gggccagtca gcatgttagc agccactact tagcttggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 24 cctgaagatt ttgcagtgta ttactgtcag cactatggtc agtcacagtt cactttcggc 300 cctgaagatt ttgcagtgta ttactgtcag cactatggtc agtcacagtt cactttcggc 300 caggggacca aggtggagat caaacggacc gtggctgcac catctgtctt catcttcccg 360 caggggacca aggtggagat caaacggacc gtggctgcac catctgtctt catcttcccg 360 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgc 645 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgc 645 <210>\ 41<210>\ 41 <211>\ 108<211>\ 108 <21<223> Synthetic constructs <400> 41 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Ser His 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Gln Ser Gln 85 90 95 Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 42 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 42 gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gcatgttagc agccactact tagcttggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cactatggtc agtcacagtt cactttcggc 300 caggggacca aggtggagat caaa 324 <210> 43 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 43 Arg Ala Ser Gln His Val Asn Ser His Tyr Leu Ala 1 5 10 <210> 44 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 44 Tyr Ala Thr Arg Thr Arg Pro Thr 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 45 Gln His Tyr Gly Gln Ser Gln Phe Thr 1 5 <210> 46 <211> 215 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 46 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Asn Ser His 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Ala Thr Arg Thr Arg Pro Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Gln Ser Gln 85 90 95 Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 47 <211> 645 <212> DNA [[ID=зо]]<213> Artificial Sequence <220> <223> Synthetic Construct <400> 47 gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gcatgttaac agccactact tagcttggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat gctacaagaa ccaggccaac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cactatggtc agtcacagtt cactttcggc 300 caggggacca aggtggagat caaacggacc gtggctgcac catctgtctt catcttcccg 360 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgc 645 <210> 48 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct[[ID=2,7]] <400> 48 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Asn Ser His 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Ala Thr Arg Thr Arg Pro Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Gln Ser Gln 85 90 95 Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 49 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct [[ID=3II]]<400> 49 gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gcatgttaac agccactact tagcttggta ccagcagaaa 120 cctggccagg ctcccaggct cctcatctat gctacaagaa ccaggccaac tggcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcacccctcag 240 It should be noted that there seems to be a misspelling in the original text at the end of line where "ctcaccatcag cagactggag" is likely incorrect and should be something like "ctcaccatcag cagactggag" as it doesn't make sense as is. I've translated it as it is in the provided text but this might need to be corrected in the original source. Also, there is a typo in the translation of line [[ID=3II]] which should be .cctgaagatt ttgcagtgta ttactgtcag cactatggtc agtcacagtt cactttcggc 300 caggggacca aggtggagat caaa 324 <210> 50 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 50 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly His Thr Ile Ser Ser Tyr 20 25 30 Ala Tyr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ala Tyr Gly Ser Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Phe Gly Lys Asn Tyr Ala Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly <210> 51 <211> 1347 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 51 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggaca taccattagc agctatgctt acagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggagac atcatccctg cctatggctc accaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaggac 300 ttcggcaaga attacgcgat ggacgtctgg ggccaaggga cccttgtcac cgtctcctca 360 gcttctacca agggcccatc ggtcttcccg ctagcaccct cctccaagag cacctctggg 420 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggacgag 1080 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gccccccgtg 1200 ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 1320 cagaagagcc tctccctgtc tccgggt 1347 <210> 52 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 52 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly His Thr Ile Ser Ser Tyr 20 25 30 Ala Tyr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Ile Pro Ala Tyr Gly Ser Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Phe Gly Lys Asn Tyr Ala Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 53 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 53 caggtgcaac ttgtccaaag cggagctgag gttaagaaac caggttcatc tgtgaaggtg 60 tcatgtaagg caagcggcca caccatttca tcttacgcat actcctgggt tcgacaagct 120 ccaggccagg gattggaatg gatgggagac ataatcccag catacggatc acctaactac 180 gcacagaagt ttcaggggag agtgacaatt acagccgacg agtctactag cactgcttac 240 atggagttgt cttcacttcg gtcagaggat acagcagttt actattgtgc cagggaggat 300 ttcgggaaaa attatgctat ggatgtatgg ggtcagggca ccctggttac tgtatcatct 360 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 660 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgccggggga 720 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780 gaggtcacat gcgtggtggt gtccgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840 tatgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aagactggct gaatggcaag 960 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080 atgaccaaga accaagtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 ctggactccg acggctcctt cttcctctat tccaagctca ccgtggacaa gagcaggtgg 1260 ctggactccg acggctcctt cttcctctat tccaagctca ccgtggacaa gagcaggtgg 1260 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320 cagaagagcc tctccctgtc tccgggcaaa 1350 cagaagagcc tctccctgtc tccgggcaaa 1350 <210> 54<210> 54 <211> 450<211> 450 <212> PRT<212> PRT <213> 人工序列<213> Artificial sequence <220><220> <223> 合成构建体 <223> Synthetic construct <400> 54<400> 54 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Ile Ser Ser Tyr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Ile Ser Ser Tyr 20 25 30 20 25 30 Ala Tyr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Ala Tyr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 35 40 45 Gly Gly Ile Ile Pro Ile Phe Asp Thr Ala Asn Tyr Ala Gln Lys Phe Gly Gly Ile Ile Pro Ile Phe Asp Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Phe Gly Tyr Asn Tyr Ala Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 55 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 55 caggttcagc tcgtgcaaag tggagctgag gtgaaaaaac ctggttctag cgtcaaggtc 60 tcttgtaagg cctctggggg cactataagc tcttatgctt atagttgggt gcgccaggcc 120 ccaggacagg gcttggaatg gatgggcggc ataataccca tattcgacac agccaactac 180 gctcaaaaat ttcaggggag agtgactata actgcagacg agagcactag caccgcttac 240 atggagttga gtagtctccg cagtgaagac acagccgtct attattgcgc tagggaggac 300 tttggttaca actacgccat ggatgtctgg ggccagggta ctttggtcac cgtatctagt 360 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc 660 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgccggggga 720 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780 gaggtcacat gcgtggtggt gtccgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840 tatgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aagactggct gaatggcaag 960 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080 atgaccaaga accaagtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 ctggactccg acggctcctt cttcctctat tccaagctca ccgtggacaa gagcaggtgg 1260 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320 cagaagagcc tctccctgtc tccgggcaaa 1350 <210> 56 <211> 557 <212> PRT <213> Homo sapiens <400> 56 Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met 1 5 10 15 Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp 20 25 30​​​​​​​​​​​​​Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu 85 90 95 Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr 100 105 110 Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120 125 Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro 130 135 140 Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala 145 150 155 160 Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro 165 170 175 Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro 180 185 190 Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser 195 200 205 Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser 210 215 220 Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp 225 230 235 240 Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala 245 250 255 Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu 260 265 270 Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly 275 280 285 Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu 290 295 300 Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg 305 310 315 320 Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val 325 330 335 Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu 340 345 350 Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala 355 360 365 Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp 370 375 380 Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly 385 390 395 400 Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu 405 410 415 Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu 420 425 430 Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly 435 440 445 Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu 450 455 460 Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser 465 470 475 480 Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Ala 485 490 495 Gly Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro 500 505 510 Gln Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp 515 520 525 Ala Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala 530 535 540 Trp Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg 545 550 555 <210> 57 <211> 556 <212> PRT <213> Homo sapiens <400> 57 Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met 1 5 10 15 Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp 20 25 30 Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln 35 40 45 Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu 50 55 60 Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile 65 70 75 80 Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu 85 90 95 Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr 100 105 110 Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120 125 Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro 130 135 140 Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala 145 150 155 160 Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro 165 170 175 Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro 180 185 190 Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser 195 200 205 Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser 210 215 220 Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp 225 230 235 240 Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala 245 250 255 Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu 260 265 270 Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly 275 280 285 Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu 290 295 300 Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg 305 310 315 320 Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val 325 330 335 Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu 340 345 350 Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala 355 360 365 Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp 370 375 380 Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly 385 390 395 400 Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu 405 410 415 Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu 420 425 430 Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly 435 440 445 Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu 450 455 460 Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser 465 470 475 480 Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Gly 485 490 495 Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln 500 505 510 Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala 515 520 525 Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala Trp 530 535 540 Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg 545 550 555

Claims

1. An antibody that binds to human CD 19, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1 consists of SEQ ID NO: 29, the HCDR2 consists of SEQ ID NO: 30, the HCDR3 consists of SEQ ID NO: 31, the LCDR1 consists of SEQ ID NO: 32, the LCDR2 consists of SEQ ID NO: 33, and the LCDR3 consists of SEQ ID NO:

34.

2. The antibody of claim 1, wherein the VH consists of SEQ ID NO: 37 and the VL consists of SEQ ID NO:

41.

3. The antibody of claim 1 or 2, wherein the antibody comprises a heavy chain (HC) consisting of SEQ ID NO: 35 and a light chain (LC) consisting of SEQ ID NO:

39.

4. The antibody of claim 1 or 2, wherein the antibody comprises a heavy chain (HC) consisting of SEQ ID NO: 52 and a light chain (LC) consisting of SEQ ID NO:

39.

5. The antibody of claim 1 or 2, wherein the antibody is a non-depleting antibody, wherein the non-depleting antibody is an antibody that does not significantly reduce the number of B cells in a subject after treatment as compared to the number of B cells prior to treatment. ​ ​ ​ ​ ​ ​

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