Anti-CD19 antibodies and uses thereof

Abstract

Antibodies, fragments and fusion proteins thereof, that specifically bind to CD19 are described, as well as methods of making and using such antibodies. Such antibodies, fusion proteins, and fragments thereof are useful for the treatment and diagnosis of various B-cell disorders, including B-cell malignancies and autoimmune diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the Chinese invention patent application with application number 202180030649.6, filing date April 23, 2021, and title “Anti-CD19 Antibodies and Uses Thereof.” The original application is a national phase application with international application number PCT / US2021 / 028880, which claims a filing date of April 23, 2021, the contents of which are incorporated herein by reference in their entirety. This application claims priority to U.S. Provisional Patent Application Serial No. 60 / 015,385, filed on April 24, 2020, which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which was submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on March 30, 2021, is named MIL-004WO_SL.txt, and is 38,765 bytes in size. Background Art

[0005] The CD19 antigen is a cell surface protein present on B cells. CD19 is expressed on both normal and malignant B cells, and its abnormal growth can lead to B-cell lymphomas. For example, CD19 is expressed on B-cell lineage malignancies, including but not limited to non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia. There is a need in the art for the development of anti-CD19 antibodies. Summary of the Invention

[0006] To address many of the problems associated with B cell disorders and their treatment, the present invention provides human anti-CD19 antibodies and fragments thereof for use in treating B cell lymphomas and leukemias, as well as autoimmune disorders. The antibodies and fragments thereof of the present invention can be used alone, as fusion proteins, or conjugated to at least one diagnostic and / or therapeutic agent, or in combination with other therapeutic modalities. Human CD19 combined with the anti-CD19 antibodies or fragments thereof described herein can exhibit ADCC activity, induction of apoptosis, and inhibition of B cell proliferation.

[0007] In one aspect, the present invention provides an antibody or fragment thereof that specifically binds to CD 19. In one aspect, the present invention provides a CD 19 antibody or fragment thereof, comprising a heavy chain variable complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3).

[0008] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGAVPIT (SEQ ID NO: 12) (LCDR3).

[0009] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQVDSLHPFT (SEQ ID NO: 13) (LCDR3).

[0010] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGGVPPLT (SEQ ID NO: 14) (LCDR3).

[0011] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVRSSYLA (SEQ ID NO: 8) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQLFDSPYT (SEQ ID NO: 15) (LCDR3).

[0012] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVRSSYLA (SEQ ID NO: 8) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGVPPLT (SEQ ID NO: 16) (LCDR3).

[0013] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGGVPPFT (SEQ ID NO: 17) (LCDR3).

[0014] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASNRAT (SEQ ID NO: 10) (LCDR2), and QQAGVFPFT (SEQ ID NO: 18) (LCDR3).

[0015] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASRRAT (SEQ ID NO: 11) (LCDR2), and QQAGIPPYT (SEQ ID NO: 19) (LCDR3).

[0016] In some embodiments, the CD19 antibody or fragment thereof comprises an immunoglobulin light chain variable (VL) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 20; and an immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 5.

[0017] In one aspect, the present invention provides a CD19 antibody or fragment thereof, comprising an immunoglobulin light chain variable (VL) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 20-27; and an immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 5.

[0018] In some embodiments, the VL region comprises an amino acid sequence that is at least 95% identical to SEQ ID NOs: 20-27.

[0019] In some embodiments, the VL region comprises an amino acid sequence identical to SEQ ID NOs: 20-27.

[0020] In some embodiments, the VH region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:5.

[0021] In some embodiments, the VH region comprises an amino acid sequence identical to SEQ ID NO:5.

[0022] In some embodiments, the VL region comprises an amino acid sequence identical to SEQ ID NOs: 20-27, and the VH region is identical to SEQ ID NO: 5.

[0023] In some embodiments, the CD19 antibody or fragment thereof is selected from the group consisting of: an IgA antibody, an IgG antibody, an IgE antibody, an IgM antibody, a bispecific or multispecific antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd' fragment, a Fd fragment, an isolated CDR or a collection thereof; a single-chain variable fragment (scFv), a polypeptide-Fc fusion, a single domain antibody, a camel antibody; a masked antibody, a small modular immunopharmaceutical ("SMIPsTM"), a single chain, a tandem diabody, a VHH, an Anticalin, a Nanobody, a minibody, a BiTE, an ankyrin repeat protein, a DARPIN, an Avimer, a DART, a TCR-like antibody, an Adnectin, an Affilin, a transmembrane antibody (Trans-body); an Affibody, a TrimerX, a MicroProtein, a Fynomer, a Centyrin; and a KALBITOR.

[0024] In some embodiments, the CD19 antibody or fragment thereof is a monoclonal antibody or a single-chain variable fragment (scFv).

[0025] In some embodiments, the CD19 antibody or fragment thereof is an antibody comprising an IgG constant region.

[0026] In some embodiments, the CD19 antibody or fragment thereof is a single-chain variable fragment (scFv).

[0027] In some embodiments, the CD19 scFv comprises a linker sequence comprising SEQ ID Nos: 36-39.

[0028] In some embodiments, the CD19 scFv comprises a signal peptide selected from SEQ ID NOs: 40-42.

[0029] In some embodiments, the antibody or fragment thereof binds CD19 with a KD of between about 8 nanomolar (nM) and about 242 nM.

[0030] In some embodiments, the antibody or fragment thereof binds CD19 on a target cell with an EC50 of between about 0.1 nM and about 2.7 nM.

[0031] In one aspect, the present invention provides a method of treating cancer, comprising administering the CD19 antibody or fragment thereof according to any one of the preceding claims to a subject in need of treatment.

[0032] In some embodiments, the cancer is selected from leukemia, lymphoma, or myeloma.

[0033] In one aspect, the present invention provides a pharmaceutical composition comprising a CD19 antibody or fragment thereof and a pharmaceutically acceptable carrier, wherein the CD19 antibody or fragment thereof comprises a heavy chain variable region having a complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2) and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3).

[0034] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGAVPIT (SEQ ID NO: 12) (LCDR3).

[0035] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQVDSLHPFT (SEQ ID NO: 13) (LCDR3).

[0036] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGGVPPLT (SEQ ID NO: 14) (LCDR3).

[0037] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVRSSYLA (SEQ ID NO: 8) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQLFDSPYT (SEQ ID NO: 15) (LCDR3).

[0038] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVRSSYLA (SEQ ID NO: 8) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGVPPLT (SEQ ID NO: 16) (LCDR3).

[0039] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGGVPPFT (SEQ ID NO: 17) (LCDR3).

[0040] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASNRAT (SEQ ID NO: 10) (LCDR2), and QQAGVFPFT (SEQ ID NO: 18) (LCDR3).

[0041] In some embodiments, the CD19 antibody or fragment thereof further comprises a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASRRAT (SEQ ID NO: 11) (LCDR2), and QQAGIPPYT (SEQ ID NO: 19) (LCDR3).

[0042] In one aspect, the present invention provides a method of treating cancer, comprising administering a CD19 antibody or an antigen-binding fragment thereof to a subject in need of treatment, wherein the CD19 antibody or fragment thereof comprises a heavy chain variable region having a complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3).

[0043] In some embodiments, the method of treating cancer comprises administering a CD19 antibody or fragment thereof, further comprising a light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGAVPIT (SEQ ID NO:12) (LCDR3).

[0044] In some embodiments, the method of treating cancer comprises administering a CD19 antibody or fragment thereof, further comprising a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQVDSLHPFT (SEQ ID NO:13) (LCDR3).

[0045] In some embodiments, the method of treating cancer comprises administering a CD19 antibody or fragment thereof, further comprising a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPLT (SEQ ID NO:14) (LCDR3).

[0046] In some embodiments, the method of treating cancer comprises administering a CD19 antibody or fragment thereof, further comprising a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVRSSYLA (SEQ ID NO: 8) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQLFDSPYT (SEQ ID NO: 15) (LCDR3).

[0047] In some embodiments, the method of treating cancer comprises administering a CD19 antibody or fragment thereof, further comprising a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGVPPLT (SEQ ID NO:16) (LCDR3).

[0048] In some embodiments, the method of treating cancer comprises administering a CD19 antibody or fragment thereof, further comprising a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPFT (SEQ ID NO:17) (LCDR3).

[0049] In some embodiments, the method of treating cancer comprises administering a CD19 antibody or fragment thereof, further comprising a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASNRAT (SEQ ID NO:10) (LCDR2), and QQAGVFPFT (SEQ ID NO:18) (LCDR3).

[0050] In some embodiments, the method of treating cancer comprises administering a CD19 antibody or fragment thereof, further comprising a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASRRAT (SEQ ID NO:11) (LCDR2), and QQAGIPPYT (SEQ ID NO:19) (LCDR3).

[0051] In one aspect, the invention provides a nucleic acid encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:5.

[0052] In one aspect, the invention provides a nucleic acid encoding an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 20-27.

[0053] In one aspect, the present invention provides a vector comprising a nucleic acid sequence encoding an amino acid sequence at least 90% identical to SEQ ID NO: 5 and / or encoding an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 20-27.

[0054] In one aspect, the invention provides an isolated cell comprising a vector comprising a nucleic acid sequence encoding an amino acid sequence at least 90% identical to SEQ ID NO: 5 and / or encoding an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 20-27.

[0055] definition

[0056] A: The article "a" or "an" is used herein to refer to one or more than one (ie, to at least one) of the grammatical object of the article. For example, "an" or "an" means one element or more than one element.

[0057] Affinity: As used herein, the term "affinity" refers to a characteristic of the binding interaction between a binding moiety (e.g., an antigen binding moiety (e.g., a variable domain described herein) and / or an Fc receptor binding moiety (e.g., an FcRn binding moiety described herein)) and a target (e.g., an antigen (e.g., CD19) and / or an FcR (e.g., FcRn)), and is indicative of the strength of the binding interaction. In some embodiments, the measure of affinity is expressed as a dissociation constant (K D In some embodiments, the binding moiety has a high affinity for the target (e.g., K D Less than about 10 -7 M, less than about 10 -8 M or less than about 10 -9 In some embodiments, the binding moiety has a low affinity for the target (e.g., K D Above about 10 -7 M, higher than about 10 -6 M, higher than about 10 -5 M, or above about 10 -4 M). In some embodiments, the binding moiety has a high affinity for the target at a first pH, a low affinity for the target at a second pH, and an intermediate affinity for the target at pH levels between the first pH and the second pH.

[0058] About or approximately: As used herein, the term "about" or "approximately" as applied to one or more target values ​​refers to a value similar to the stated reference value. In certain embodiments, unless otherwise indicated or apparent from the context, the term "about" or "approximately" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction of the stated reference value (greater than or less than the stated reference value) (except where such a number would exceed 100% of the possible value).

[0059] Antibody: As used herein, the term "antibody" refers to a polypeptide comprising at least one immunoglobulin variable region, for example, an amino acid sequence providing an immunoglobulin variable domain or an immunoglobulin variable domain sequence. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab, F(ab')2, Fd, Fv, and dAb fragments) as well as complete antibodies, such as complete immunoglobulin types IgA, IgG, IgE, IgD, IgM (and subtypes thereof). The light chain of an immunoglobulin may be κ-type or λ-type.

[0060] Binding moiety: As used herein, a "binding moiety" is any molecule or portion of a molecule that is capable of specifically binding to a target, e.g., a target of interest, e.g., an antigen (e.g., CD19) and / or an FcR (e.g., FcRn). Binding moieties include, for example, antibodies, antigen-binding fragments thereof, an Fc region or Fc fragment thereof, antibody mimetics, peptides, and aptamers.

[0061] An antigen binding fragment or its antigen fragment refers to a portion of an intact antibody. An antigen binding fragment or its antibody fragment refers to a portion of an intact antibody that binds to an antigen (e.g., CD19). An antigen binding fragment may comprise the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, antibody mimics, scFv and single-chain antibodies.

[0062] Complementarity Determining Region (CDR): A "CDR" of a variable domain is an amino acid residue within a variable region that is defined according to Kabat, Chothia, the accumulation of Kabat and Chothia, AbM, contact and / or conformational definitions, or any CDR determination method well known in the art. Antibody CDRs can be identified as the hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC. The positions of CDRs can also be identified as the structural loop structures originally described by Chothia and others. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other CDR identification methods include the "AbM definition," which is a compromise between Kabat and Chothia and is a method of defining CDRs using Oxford Molecular's AbM antibody modeling software (now ) or the “contact definition” of CDRs based on observed antigen contacts, as described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach referred to herein as the “conformational definition” of CDRs, the positions of CDRs can be identified as residues that contribute enthalpicly to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1 156-1166, 2008. There are other CDR boundary definitions that may not strictly follow one of the above methods, but will still overlap with at least a portion of the Kabat CDRs, but they will be shortened or extended based on predictions or experimental results that a particular residue or residue group, or even the entire CDR, has no significant effect on antigen binding. As used herein, CDRs may refer to CDRs defined by any method known in the art (including a combination of methods). The methods used herein can utilize CDRs defined according to any of these methods. For any given embodiment comprising more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extension, AbM, contact and / or conformational definitions.

[0063] Constant region: As used herein, the term "constant region" refers to a polypeptide corresponding to or derived from one or more constant region immunoglobulin domains of an antibody. The constant region can include any or all of the following immunoglobulin domains: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain (derived from IgA, IgD, IgG, IgE, or IgM), and a CH4 domain (derived from IgE or IgM).

[0064] Epitope: As used herein, "epitope" is a term in the art, referring to a local region of an antigen to which an antibody can specifically bind. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or continuous epitope), or an epitope can be, for example, jointly derived from two or more discontinuous regions (conformation, nonlinear, discontinuous or discontinuous epitopes) of one or more polypeptides. In certain embodiments, the epitope to which an antibody binds can be determined by, for example, NMR spectroscopy, X-ray crystallography, ELISA assays, hydrogen / deuterium exchange combined mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody: antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software known in the art, such as Refmac and Phenix. Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For descriptions of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al. (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.

[0065] Fc region: As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides", each of which comprises an antibody constant region excluding the first constant region immunoglobulin domain. In some embodiments, the "Fc region" comprises two Fc polypeptides connected by one or more disulfide bonds, chemical linkers, or peptide linkers. "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also include part or all of the flexible hinge N-terminal to these domains. For IgG, the "Fc polypeptide" comprises the immunoglobulin domains Cgamma2 (Cγ2) and Cgamma3 (Cγ3), and the lower portion of the hinge between Cgamma1 (Cγ1) and Cγ2. Although the boundaries of the Fc polypeptide may vary, the human IgG heavy chain Fc polypeptide is generally defined as comprising residues starting at T223 or C226 or P230 to its carboxyl terminus, where numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, VA). For IgA, the Fc polypeptide comprises immunoglobulin domains Calpha2 (Cα2) and Calpha3 (Cα3), and the lower portion of the hinge between Calpha1 (Cα1) and Cα2. The Fc region can be synthetic, recombinant, or produced from natural sources such as IVIG.

[0066] K a :As used herein, “K a ” refers to the rate at which a specific binding moiety associates with a target to form a binding moiety / target complex.

[0067] K d :As used herein, “K d ” refers to the dissociation rate of the specific binding moiety / target complex.

[0068] K D :As used herein, “K D ” refers to the dissociation constant, which is obtained from K d With K a The ratio (i.e., K d / K a ) and is expressed as molar concentration (M). K D The values ​​can be determined using methods well established in the art, for example, by using surface plasmon resonance, or using methods such as The system is determined by the biosensor system.

[0069] Reference: A "reference" entity, system, quantity, set of conditions, etc. is an entity, system, quantity, set of conditions, etc., to which a test entity, system, quantity, set of conditions, etc. as described herein is compared. For example, in some embodiments, a "reference" antibody is an unengineered control antibody as described herein.

[0070] Selective binding: As used herein, "selective binding," "selectively binds," "specifically binds," or "specifically binds" means that, with respect to a binding moiety and a target, the binding moiety preferentially associates with the target over binding to non-target entities. Some degree of non-specific binding may occur between the binding moiety and non-targets. In some embodiments, a binding moiety selectively binds a target if the binding between the binding moiety and the target is 2-fold, 5-fold, 10-fold, or 100-fold greater than the binding of the binding moiety to the non-target. In some embodiments, a binding moiety selectively binds a target if the binding affinity is less than about 10 -5 M, less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M, or less than about 10 -9 In some embodiments, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, typically with lower affinity, such as by, for example, immunoassays, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art.

[0071] Single-chain variable fragment (scFv): As used herein, the term "single-chain variable fragment" or "scFv" refers to a single-chain variable fragment covalently linked to form a V H ::VL heterodimer of immunoglobulin (eg, mouse or human) heavy chain (V H ) and light chain (V L ) variable region fusion protein. Heavy chain (V H ) and light chain ( VL ) directly or through a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids) that connects V H The N-terminus of V L C-terminus of the H The C-terminus of V LThe N-terminus of the polypeptide is connected. The linker is generally rich in glycine to improve flexibility, and serine or threonine to improve solubility. The linker can connect the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entirety.

[0072] Subject: As used herein, the term "subject" means any subject for whom diagnosis, prognosis, or treatment is desired. For example, the subject can be a mammal, such as a human or non-human primate (e.g., an ape, monkey, gorilla, or chimpanzee), dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, or cow.

[0073] Target: As used herein, a "target" is any molecule that is specifically bound by the binding portion of an antibody or antigen-binding fragment thereof. In some embodiments, the target is an antigen described herein (e.g., CD19). In some embodiments, the target is an FcR (e.g., FcRn). The terms "primary target" and "secondary target" are used herein to refer to molecules of two different molecular species, rather than two molecules of the same molecular species. For example, in some embodiments, the first target is a serum protein and the second target is FcRn.

[0074] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic molecule (e.g., an anti-CD19 antibody as described herein) that imparts a therapeutic effect to a treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives an indication of the effect they feel). In particular, a "therapeutically effective amount" refers to an amount of a therapeutic molecule or composition that effectively treats, improves, or prevents a specific disease or illness, or exhibits a detectable therapeutic or preventive effect, such as by improving the symptoms associated with the disease, preventing or delaying the onset of the disease, and / or also alleviating the severity or frequency of the disease symptoms. A therapeutically effective amount can be administered in a dosage regimen that can include multiple unit doses. For any particular therapeutic molecule, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosage regimen) can vary, for example, depending on the route of administration, in combination with other agents. In addition, the specific therapeutically effective amount (and / or unit dose) for any particular subject may depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific agent being used; the specific composition being used; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific therapeutic molecule being used; the duration of the treatment; and like factors well known in the medical arts.

[0075] Treatment: As used herein, the term "treatment" (treatment / treat / treating) refers to any partial or complete alleviation, improvement, alleviation, suppression of one or more symptoms or features of a specific disease, disorder and / or condition, delaying its onset, reducing its severity and / or reducing its incidence of therapeutic molecules (e.g., anti-CD19 molecules described herein). Such treatment can be for subjects who do not show signs of the relevant disease, disorder and / or condition and / or for subjects who only show early signs of the disease, disorder and / or condition. Alternatively or in addition, such treatment can be for subjects who show one or more established signs of the relevant disease, disorder and / or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The drawings are for illustration purposes only; they are not intended to be limiting.

[0077] Figure 1 Exemplary flow cytometry chromatograms showing binding of anti-CD19 antibodies to CD19-expressing Raji and NALM-6 cells (wild-type and CD19 knockout).

[0078] Figures 2A to 2C Shown are exemplary binding curves of anti-CD19 antibodies binding to CD19 on NALM-6 cells.

[0079] Figure 3 Shown are exemplary results of a competition assay used to bin and characterize anti-CD19 antibodies and antigen-binding fragments thereof by epitope. DETAILED DESCRIPTION

[0080] The present disclosure is based in part on the discovery of engineered antibodies and antigen-binding fragments thereof that exhibit binding to CD19 (e.g., human CD19). CD19 (also known as cluster of differentiation 19, B lymphocyte antigen CD19, B lymphocyte surface antigen B4, B4, CVID3, or differentiation antigen CD19) is a protein with a single transmembrane domain, a cytoplasmic C-terminus, and an extracellular N-terminus. CD19 is specifically expressed in normal and tumor B cells and follicular dendritic cells. The surface density of CD19 is highly regulated throughout the development and maturation of B cells until expression is lost during terminal plasma cell differentiation. In addition, CD19 is a surface biomarker of B lymphocytes and may therefore be a useful antigen for identifying cancer cells (e.g., B cell lymphomas) produced by such B cells.

[0081] Antibody

[0082] The anti-CD19 antibodies described herein are designed to specifically bind to CD19. In certain embodiments, the currently disclosed anti-CD19 antibodies and fragments thereof bind to human CD19. In certain embodiments, human CD19 comprises or consists of the amino acid sequence having NCBI Reference Number: NP_001171569.1 (SEQ ID NO: 4), or a fragment thereof.

[0083] SEQ ID NO:4 is provided below:

[0084] MPPPRLLFFL LFLTPMEVRP EEPLVVKVEE GDNAVLQCLK GTSDGPTQQL TWSRESPLKPFLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGSGE LFRWNVSDLGGLGCGLKNRS SEGPSSPSGK LMSPKLYVWA KDRPEIWEGE PPCLPPRDSL NQSLSQDLTM APGSTLWLSCGVPPDSVSRG PLSWTHVHPK GPKSLLSLEL KDDRPARDMW VMETGLLLPR ATAQDAGKYY CHRGNLTMSFHLEITARPVL WHWLLRTGGW KVSAVTLAYL IFCLCSLVGI LHLQRALLVLR RKRKRMTDPT RRFFKVTPPPGSGPQNQYGN VLSLPTPTSG LGRAQRWAAG LGGTAPSYGN PSSDVQADGA LGSRSPPGVG PEEEEGEGYEEPDSEEDSEF YENDSNLGQD QLSQDGSGYE NPEDEPLGPE DEDSFSNAES YENEDEELTQ PVARTMDFLSPHGSAWDPSR EATSLAGSQS YEDMRGILYA APQLRSIRGQ PGPNHEEDAD SYENMDNPDG PDPAWGGGGRMGTWSTR[SEQ ID NO:4]

[0085] In certain embodiments, human CD19 comprises or consists of the amino acid sequence having NCBI Reference Number: NP_001761.3 (SEQ ID NO: 45), or a fragment thereof.

[0086] SEQ ID NO:45 is provided below:

[0087] MPPPRLLFFL LFLTPMEVRP EEPLVVKVEE GDNAVLQCLK GTSDGPTQQL TWSRESPLKPFLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGSGE LFRWNVSDLGGLGCGLKNRS SEGPSSPSGK LMSPKLYVWA KDRPEIWEGE PPCLPPRDSL NQSLSQDLTM APGSTLWLSCGVPPDSVSRG PLSWTHVHPK GPKSLLSLEL KDDRPARDMW VMETGLLLPR ATAQDAGKYY CHRGNLTMSFHLEITARPVL WHWLLRTGGW KVSAVTLAYL IFCLCSLVGI LHLQRALLVLR RKRKRMTDPT RRFFKVTPPPGSGPQNQYGN VLSLPTPTSG LGRAQRWAAG LGGTAPSYGN PSSDVQADGA LGSRSPPGVG PEEEEGEGYEEPDSEEDSEF YENDSNLGQD QLSQDGSGYE NPEDEPLGPE DEDSFSNAES YENEDEELTQ PVARTMDFLSPHGSAWDPSR EATSLGSQSY EDMRGILYAA PQLRSIRGQP GPNHEEDADS YENMDNPDGP DPAWGGGGGRMGTWSTR[SEQ ID NO:45]

[0088] In certain embodiments, the anti-CD19 antibodies and antigen-binding fragments thereof described herein bind to the extracellular domain of CD19. In certain embodiments, the anti-CD19 antibodies and antigen-binding fragments thereof bind to the extracellular domain of human CD19. In certain embodiments, the extracellular domain of human CD19 comprises or consists of amino acids 20 to 291 of SEQ ID NO: 4. In certain embodiments, the extracellular domain of human CD19 comprises or consists of amino acids 20 to 291 of SEQ ID NO: 45.

[0089] In certain embodiments, CD19 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence of SEQ ID NO:4, or a fragment thereof.

[0090] In certain embodiments, CD19 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence of SEQ ID NO:45 or a fragment thereof.

[0091] Anti-CD19 antibodies described herein can be immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies or other protein scaffolds with antibody-like properties, as well as other immune binding moieties known in the art, including, for example, Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, Feb, scFv, SMIP, antibodies, double antibodies, three antibodies, four antibodies, mini antibodies, maximum antibodies (maxibody), tandab, DVD, BiTe, TandAb, etc., or any combination thereof. The subunit structure and three-dimensional configuration of different classes of antibodies are known in the art.

[0092] An antibody can be an immunoglobulin molecule having four polypeptide chains (e.g., two heavy (H) chains and two light (L) chains). The heavy chain can include a heavy chain variable domain and a heavy chain constant domain. The heavy chain constant domain can include CH1, hinge, CH2, CH3, and in some cases include a CH4 region. A suitable heavy chain constant region can be derived from any immunoglobulin (e.g., IgA, IgG, or IgE). In some embodiments, a suitable heavy chain constant region can be derived from IgG1, IgG2, or IgG4. In a specific embodiment, a suitable heavy chain constant region is derived from IgG1. The light chain can include a light chain variable domain and a light chain constant domain. The light chain constant domain can include a kappa light chain or a lambda light chain. The heavy chain variable domain of the heavy chain and the light chain variable domain of the light chain can generally be further subdivided into regions with variability, referred to as complementary determining regions (CDRs), interspersed with more conserved regions, referred to as framework regions (FRs). Such heavy and light chain variable domains can each include three CDRs and four framework regions, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, one or more of which can be engineered as described herein. The amino acid distribution of each domain conforms to the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987); Chothia et al. Nature 342: 878-883 (1989) definitions. As used herein, CDR refers to each of the heavy chain (HCDR1, HCDR2, HCDR3) and light chain (LCDR1, LCDR2, LCDR3).

[0093] Embodiments of the present invention include antibodies comprising the CDRs found in the vH and vL domains described herein, which are identified using conventional numbering systems such as the IMGT, Kabat, and Clothia numbering systems. Such numbering systems are well known in the art.

[0094] Heavy chain variable region

[0095] In some embodiments, the anti-CD19 antibodies or fragments thereof described herein comprise a common heavy chain variable region. In some embodiments, the anti-CD19 antibodies comprise a heavy chain variable region (vH) complementarity determining region (CDR) sequence:

[0096] vH CDR1: SYGMH (SEQ ID NO: 1)

[0097] vH CDR2:LIWYDGSNKYYADSVKG(SEQ ID NO:2)

[0098] vH CDR3: PVEGLLRGFDY (SEQ ID NO:3)

[0099] In certain embodiments, CDRs are identified according to the Kabat numbering system.

[0100] In some embodiments, the variable heavy chain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK GLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAKPVEGLLRGFDYWGQGTLVTVSS (SEQ ID NO: 5).

[0101] In some embodiments, the anti-CD 19 antibody comprises a heavy chain amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:5.

[0102] In some embodiments, the anti-CD19 antibody comprises a heavy chain amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5, and further comprising one or more of the vH CDR1, vHCDR2, and / or vHCDR3 sequences described herein.

[0103] In some embodiments, the engineered antibody comprises a heavy chain amino acid sequence identical to SEQ ID NO: 5. In certain embodiments, V H comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identity or homology to the amino acid sequence set forth in SEQ ID NO: 5. For example, V Hcomprising an amino acid sequence having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity or homology to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-CD 19 antibody comprises no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid substitutions relative to SEQ ID NO: 5.

[0104] In some embodiments, the anti-CD19 variable heavy chain is encoded by a polynucleotide comprising the following nucleic acid sequence:

[0105] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTGATATGGTATGATGGAAGTAATAAATACTATGC AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAGCCAGTGGAAGGACTATTAAGAGGATTCGATTACTGGGGACAGGGTACATTGGTCACCGTCTCCTCA(SEQ ID NO:6)

[0106] In some embodiments, the anti-CD 19 antibody comprises a heavy chain nucleic acid sequence that has at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 6. In some embodiments, the engineered antibody comprises a heavy chain nucleic acid sequence that is identical to SEQ ID NO: 6. In some embodiments, the anti-CD 19 antibody comprises a nucleic acid sequence that encodes an antibody that comprises no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid substitutions relative to SEQ ID NO: 5.

[0107] In some embodiments, the anti-CD19 antibody is encoded by a polynucleotide comprising a nucleic acid sequence encoding a heavy chain amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 6, and further comprising one or more of the vH CDR1, vHCDR2 and / or vHCDR3 sequences described herein.

[0108] As will be appreciated by those skilled in the art, any such heavy chain CDR sequences can be readily combined, for example, by molecular biology techniques, with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR or constant domain, or portion thereof, as disclosed herein or otherwise known in the art (such as may be present in any form of an antibody or antigen-binding fragment thereof disclosed herein or otherwise known in the art).

[0109] In various engineered antibodies as described herein, heavy chain constant domain can be any classification (or subclass). In various engineered antibodies as described herein, heavy chain constant domain can comprise the amino acid sequence of any one or more in IgG, IgM, IgA, IgD or IgE, comprises subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. In each embodiment, the constant domain of engineered antibodies as described herein can comprise a mixture of two or more classifications (or subclass) of immunoglobulin heavy chain constant domain. For example, anti-CD19 antibodies can comprise a first part of a constant domain with a sequence of an immunoglobulin constant domain selected from IgG, IgM, IgA, IgD or IgE class constant domains and a second part of a constant domain with a sequence of an immunoglobulin constant domain different from first and selected from IgG, IgM, IgA, IgD or IgE class constant domains. In some cases, the constant domains of the anti-CD19 antibodies described herein can include a mixture of two or more subclasses of a particular class of constant domains, such as a first portion of a constant domain having a sequence of an immunoglobulin constant domain selected from a constant domain of the IgG1, IgG2, IgG3, or IgG4 subclass, and a second portion of a constant domain having a sequence of an immunoglobulin constant domain different from the first and selected from a constant domain of the IgG1, IgG2, IgG3, or IgG4 subclass. In some specific embodiments, the constant domains include all or part of an IgG2 constant domain and all or part of an IgG4 constant domain.

[0110] In some cases, the anti-CD19 antibodies include an antibody constant region, Fc region, or Fc fragment that exhibits altered binding to one or more Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, or FcRn receptors) (e.g., compared to a reference constant region). In some embodiments, the constant region, Fc region, or Fc fragment is engineered to bind to a target (e.g., FcRn receptor) in an altered manner (e.g., in a pH-sensitive manner (e.g., in a stronger or weaker pH-sensitive manner) and / or with reduced or increased binding) relative to a reference constant region, Fc region, or Fc fragment. In some embodiments, the anti-CD19 antibodies include an antibody constant region, Fc region, or Fc fragment that exhibits reduced binding to one or more Fcγ receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, or FcγRIV) (e.g., compared to a reference constant region). In some embodiments, the anti-CD19 antibody comprises an antibody constant region, Fc region, or Fc fragment that exhibits increased binding to an FcRn receptor at serum pH and / or at intracellular pH (as compared to a reference constant region).

[0111] For example, an anti-CD19 antibody may include a constant region, Fc region, or Fc fragment of an IgG antibody engineered to include amino acid additions, deletions, or substitutions of one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436 (Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH)). Without wishing to be bound by theory, it is believed that one or more of these constant region, Fc region, or Fc fragment amino acids mediates interaction with an Fc receptor, such as FcRn. In some embodiments, one or more of these disclosed amino acids is substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine. In some embodiments, a non-histidine residue is substituted with a histidine residue. In some embodiments, a histidine residue is substituted with a non-histidine residue.

[0112] In some embodiments, the anti-CD19 antibody comprises an IgG antibody constant region, Fc region, or Fc fragment having an amino acid modification at one or more of positions 308, 309, 311, 312, and 314, more specifically, substituted with threonine, proline, serine, aspartic acid, and leucine, respectively, at one or more of positions 308, 309, 311, 312, and 314. In some embodiments, the residues at one or more of positions 308, 309, and 311 are substituted with isoleucine, proline, and glutamic acid, respectively. In other embodiments, the residues at one or more of positions 308, 309, 311, 312, and 314 are substituted with threonine, proline, serine, aspartic acid, and leucine, respectively.

[0113] In some embodiments, the anti-CD19 antibody comprises an IgG antibody constant region, Fc region, or Fc fragment having an amino acid modification at one or more of positions 251, 252, 254, 255, and 256, more specifically, a substitution at one or more of these positions. In some embodiments, residue 251 is substituted with leucine or arginine, residue 252 is substituted with leucine, tyrosine, phenylalanine, serine, tryptophan, or threonine, residue 254 is substituted with threonine or serine, residue 255 is substituted with leucine, glycine, isoleucine, or arginine, and / or residue 256 is substituted with serine, phenylalanine, arginine, glutamine, glutamic acid, aspartic acid, alanine, asparagine, or threonine. In some embodiments, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine or leucine, residue 254 is substituted with threonine or serine, and / or residue 255 is substituted with arginine. In other embodiments, residue 252 is substituted with phenylalanine and / or residue 256 is substituted with aspartic acid. In some embodiments, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine, residue 254 is substituted with threonine or serine, and / or residue 255 is substituted with arginine.

[0114] In some embodiments, the anti-CD19 antibody comprises an IgG constant region, Fc region, or Fc fragment having an amino acid modification at one or more of positions 428, 433, 434, 435, and 436, more specifically, a substitution at one or more of these positions. In some embodiments, residue 428 is substituted with methionine, threonine, leucine, phenylalanine, or serine, residue 433 is substituted with lysine, arginine, serine, isoleucine, proline, glutamine, or histidine, residue 434 is substituted with phenylalanine, tyrosine, or histidine, residue 435 is substituted with tyrosine, and / or residue 436 is substituted with histidine, asparagine, arginine, threonine, lysine, methionine, or threonine. In some embodiments, the residues at one or more positions 433, 434, 435, and 436 are substituted with lysine, phenylalanine, tyrosine, and histidine, respectively. In some embodiments, residue 428 is substituted with methionine and / or residue 434 is substituted with tyrosine.

[0115] In some embodiments, the anti-CD19 antibody comprises an IgG antibody constant region, Fc region, or Fc fragment having an amino acid modification at one or more of positions 385, 386, 387, and 389, more specifically, a substitution at one or more of these positions. In some embodiments, residue 385 is substituted with arginine, aspartic acid, serine, threonine, histidine, lysine, or alanine, residue 386 is substituted with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine, residue 387 is substituted with arginine, histidine, serine, threonine, alanine, or proline, and / or residue 389 is substituted with proline or serine. In some embodiments, the residues at one or more of positions 385, 386, 387, and 389 are substituted with arginine, threonine, arginine, and proline, respectively. In some embodiments, the residues at one or more of positions 385, 386, and 389 are substituted with aspartic acid, proline, and serine, respectively.

[0116] In some embodiments, the anti-CD19 antibody comprises a constant region, Fc region, or Fc fragment of an IgG antibody having one or more of the following substitutions: leucine at residue 251, tyrosine or leucine at residue 252, threonine or serine at residue 254, arginine at residue 255, threonine at residue 308, proline at residue 309, serine at residue 311, aspartic acid at residue 312, leucine at residue 314, arginine at residue 385, threonine at residue 386, arginine at residue 387, proline at residue 389, methionine at residue 428, lysine at residue 433, phenylalanine or tyrosine at residue 434, tyrosine at position 435, and / or tyrosine at position 436. Other amino acid substitutions that may be included in the constant region, Fc region, or Fc fragment include those described in, for example, US Patent Nos. 6,277,375; 8,012,476; and 8,163,881.

[0117] In some embodiments, the anti-CD19 antibodies described herein include a heavy chain constant domain comprising, for example, the Ala-Ala mutations described in PCT publications WO 94 / 28027 and WO 98 / 47531; and Xu et al. (2000) Cell Immunol 200:16-26. Thus, in some embodiments, anti-CD19 antibodies having one or more mutations in the heavy chain constant region comprising the Ala-Ala mutation have reduced effector function or lack effector function. According to these embodiments, the constant region of the anti-CD19 antibodies described herein may include a substitution of alanine at position 234 and / or a mutation of alanine at position 235 (EU numbering).

[0118] As will be appreciated by those skilled in the art, any such heavy chain constant region sequence can be readily combined, for example, by molecular biology techniques, with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR or constant domain or portion thereof as disclosed herein or otherwise known in the art (such as may be present in any form of antibody or antigen-binding fragment thereof disclosed herein or otherwise known in the art).

[0119] Light chain variable region

[0120] The present invention further provides a CD19 antibody or fragment thereof comprising various specific sequences in one or more light chain variable regions, contained in the light chain complementary determining regions LCDR1-3. In various embodiments, the molecules having specific light chain variable regions are provided with heavy chain sequences as discussed above. In certain embodiments, the CDRs are identified according to the Kabat numbering system.

[0121] Therefore, in one aspect, the present invention provides a CD19 antibody or fragment thereof, comprising a heavy chain variable complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2) and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3) and a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2) and QQAGAVPIT (SEQ ID NO: 12) (LCDR3).

[0122] In some embodiments, the CD19 antibody or fragment thereof comprises a heavy chain variable complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3), and a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQVDSLHPFT (SEQ ID NO: 13) (LCDR3).

[0123] In some embodiments, the CD19 antibody or fragment thereof comprises a heavy chain variable complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3), and a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGGVPPLT (SEQ ID NO: 14) (LCDR3).

[0124] In some embodiments, the CD19 antibody or fragment thereof comprises a heavy chain variable complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3), and a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVRSSYLA (SEQ ID NO: 8) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQLFDSPYT (SEQ ID NO: 15) (LCDR3).

[0125] In some embodiments, the CD19 antibody or fragment thereof comprises a heavy chain variable complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3), and a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVRSSYLA (SEQ ID NO: 8) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGVPPLT (SEQ ID NO: 16) (LCDR3).

[0126] In some embodiments, the CD19 antibody or fragment thereof comprises a heavy chain variable complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3), and a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASSRAT (SEQ ID NO: 9) (LCDR2), and QQAGGVPPFT (SEQ ID NO: 17) (LCDR3).

[0127] In some embodiments, the CD19 antibody or fragment thereof comprises a heavy chain variable complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3), and a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASNRAT (SEQ ID NO: 10) (LCDR2), and QQAGVFPFT (SEQ ID NO: 18) (LCDR3).

[0128] In some embodiments, the CD19 antibody or fragment thereof comprises a heavy chain variable complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3), and a light chain variable region having a complementarity determining region (CDR) sequence of RASQSVSSSYLA (SEQ ID NO: 7) (LCDR1), GASRRAT (SEQ ID NO: 11) (LCDR2), and QQAGIPPYT (SEQ ID NO: 19) (LCDR3).

[0129] In some embodiments, the CD19 antibody or fragment thereof comprises an immunoglobulin light chain variable (VL) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 20; and an immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 5.

[0130] In one aspect, the present invention provides a CD19 antibody or fragment thereof, comprising an immunoglobulin light chain variable (VL) region comprising an amino acid sequence at least 90% identical to SEQ ID NOs: 20-27; and an immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 5. In some embodiments, the VL region comprises an amino acid sequence at least 95% identical to SEQ ID NOs: 20-27.

[0131] In some embodiments, the anti-CD19 antibody or fragment thereof comprises a heavy chain variable complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3) and / or comprises a light chain variable region (vL) complementarity determining region (CDR) sequence shown in Table 1. In some embodiments, the anti-CD19 antibody or fragment thereof comprises a light chain variable region (vL) complementarity determining region (CDR) sequence shown in Table 1:

[0132] Table 1. Anti-CD19 light chain variable CDRs

[0133]

[0134] In some embodiments, the anti-CD 19 antibody or fragment thereof comprises a light chain variable region (vL) having the amino acid sequence shown in Table 2 or encoded by the nucleic acid sequence shown.

[0135] Table 2. Variable light chain sequences

[0136]

[0137]

[0138]

[0139] In some embodiments, the anti-CD 19 antibody comprises a light chain amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 20-27.

[0140] In some embodiments, the anti-CD19 antibody comprises a light chain amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 20-27, and further comprising one or more of the vL CDR1, vLCDR2 and / or vLCDR3 sequences described herein.

[0141] In some embodiments, the anti-CD 19 antibody or fragment thereof comprises a light chain amino acid sequence identical to SEQ ID NOs: 20-27. In some embodiments, the anti-CD 19 antibody comprises no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid substitutions relative to SEQ ID NOs: 20-27.

[0142] In some embodiments, the nucleic acid sequence of the invention encodes an anti-CD19 antibody comprising a light chain amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 20-27, and further comprising one or more of the vL CDR1, vLCDR2 and / or vLCDR3 sequences described herein.

[0143] As will be appreciated by those skilled in the art, any such light chain CDR sequences can be readily combined, for example, by molecular biology techniques, with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR or constant domain, or portion thereof, as disclosed herein or otherwise known in the art (such as may be present in any form of an antibody or antigen-binding fragment thereof disclosed herein or otherwise known in the art).

[0144] In some embodiments, the anti-CD19 antibodies described herein include a light chain comprising any light chain constant domain sequence, such as a light chain constant sequence known to those skilled in the art. As will be appreciated by those skilled in the art, the light chain constant domain can be a kappa light chain constant domain or a lambda light chain constant domain. In certain embodiments, the light chain constant domain as disclosed herein is a kappa light chain constant domain. In various embodiments, the anti-CD19 antibodies described herein include a light chain constant domain.

[0145] Exemplary antibodies

[0146] Engineered antibodies can include various heavy chains and light chains as described herein. In some embodiments, anti-CD19 antibodies can include two heavy chains and light chains. In various embodiments, the disclosure encompasses antibodies comprising at least one heavy chain and / or light chain as disclosed herein, at least one heavy chain and / or light chain framework domain as disclosed herein, at least one heavy chain and / or light chain CDR domain as disclosed herein, and / or any heavy chain and / or light chain constant domain as disclosed herein.

[0147] In various embodiments, the anti-CD19 antibodies disclosed herein are homodimeric monoclonal antibodies. In various embodiments, the anti-CD19 antibodies disclosed herein are heterodimeric antibodies. In various embodiments, the anti-CD19 antibodies are, for example, typical antibodies or diabodies, triabodies, tetrabodies, minibodies, maximal antibodies, tandabs, DVDs, BiTes, scFvs, TandAb scFvs, Fabs, Fab2s, Fab3s, F(ab')2s, etc., or any combination thereof.

[0148] In some embodiments, the present disclosure provides fusion proteins comprising one or more variable domains or engineered antibodies as described herein, or portions thereof, and one or more additional polypeptides.

[0149] Exemplary single-chain variable fragments

[0150] In some embodiments, the present disclosure provides single chain variable fragments. In some embodiments, the scFv is a human scFv. A "single chain variable fragment" or "scFv" refers to a single chain variable fragment covalently linked to form a V H ::VL heterodimer of immunoglobulin (eg, mouse or human) heavy chain (V H ) and light chain (V L ) variable region fusion protein. Heavy chain (V H ) and light chain (V L ) directly or through a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids) that connects V H The N-terminus of V L C-terminus of the H The C-terminus of V L The N-terminus of the polypeptide is connected. The linker is generally rich in glycine to increase flexibility, and serine or threonine to increase solubility. The linker can connect the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80 (6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entirety. In certain embodiments, the linker is a G4S linker (SEQ ID NO: 46).

[0151] Alternatively or additionally, scFv can be derived from Fab' (rather than from an antibody, e.g., obtained from a Fab library). In certain embodiments, the anti-CD19 antibody or its fragment is a Fab. In certain embodiments, the Fab is cross-linked. In certain embodiments, the anti-CD19 antibody or its fragment is a Fab2. Any of the aforementioned molecules can be included in a fusion protein with a heterologous sequence to form an anti-CD19 antigen antibody or its antigen-binding fragment.

[0152] In certain embodiments, the anti-CD19 antibody or fragment thereof is present in an amount of at least about 1 x 10 -6 M, at least about 1x 10 -7 M, at least about 1x 10 -8 M, at least about 1x 10 -9 M or at least about 1x -10 In certain embodiments, the anti-CD19 antibody or fragment thereof binds to CD19 (e.g., human CD19) with a dissociation constant (Kd) of at least about 2 x 10 -8 In certain embodiments, the anti-CD19 antibody or fragment thereof binds to CD19 (e.g., human CD19) with a dissociation constant (Kd) of about 2x 10-8 M with about 8x 10 -9 The dissociation constant (Kd) between M and CD19 (e.g., human CD19) is quantified.

[0153] In some embodiments, the anti-CD19 antibody or its fragment binds to CD19 (e.g., human CD19) with a dissociation constant (Kd) between about 1 nM and 50 nM, about 5 nM and 30 nM, about 5 nM and 25 nM, or about 8 nM and 20 nM. In some embodiments, the anti-CD19 antibody or its fragment binds to CD19 (e.g., human CD19) with a dissociation constant (Kd) of at least about 50 nM, at least about 40 nM, at least about 35 nM, at least about 30 nM, at least about 25 nM, at least about 20 nM, at least about 19 nM, at least about 18 nM, at least about 17 nM, at least about 16 nM, at least about 15 nM, at least about 14 nM, at least about 13 nM, at least about 12 nM, at least about 11 nM, at least about 10 nM, at least about 9 nM, at least about 8 nM, at least about 7 nM, at least about 6 nM, at least about 5 nM.

[0154] In some embodiments, the anti-CD19 scFv comprises a variable heavy chain comprising SEQ ID Nos: 1-4. In some embodiments, the anti-CD19 scFv comprises a variable light chain comprising one or more CDR sequences provided in Table 1. In some embodiments, the anti-CD19 scFv comprises a variable light chain comprising one or more light chain sequences provided in Table 2.

[0155] In some embodiments, the anti-CD19 scFv comprises a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 36 provided below:

[0156] GGGGSGGGGSGGGGS [SEQ ID NO: 36]

[0157] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 37 provided below:

[0158] GGGGSGGGGSGGGSGGGGS[SEQ ID NO:37]

[0159] In some embodiments, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 38 provided below:

[0160] GGGGSGGGGSGGGGSGGGSGGGGS[SEQ ID NO:38]

[0161] In some embodiments, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 39 provided below:

[0162] GGGGSGGGGSGGGGSGGGGSGGGSGGGGS[SEQ ID NO:39]

[0163] In some embodiments, anti-CD19 antibodies or fragments thereof comprise conservative sequence modifications (e.g., anti-CD19 antibodies or fragments thereof described herein). In some embodiments, conservative sequence modifications are amino acid modifications that do not significantly affect or change the binding characteristics of the currently disclosed anti-CD19 antibodies or fragments thereof (e.g., the antibodies or fragments thereof) comprising the amino acid sequence. Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications may be introduced into anti-CD19 antibodies or fragments thereof by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids may be classified according to their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue within the same group. For example, amino acids may be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine. Therefore, one or more amino acid residues in the CDR region can be replaced with other amino acid residues from the same group, and the modified antibody can be tested for retained function. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues in a specified sequence or CDR region are changed.

[0164] In some embodiments, the light chain and / or heavy chain of the anti-CD19 scFv comprises a signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous or identical to the amino acid sequence MDMRVPAQLLGLLLLWLPDTRC (SEQ ID NO: 40) or MEFGLSWVFLVALLRGVQC (SEQ ID NO: 41).

[0165] In some embodiments, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 42. EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGAVPITFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPVEGLLRGFDYWGQGTLVTVSS [SEQ ID NO: 42]

[0166] In some embodiments, the anti-CD19 scFv comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous or identical to SEQ ID NO:42.

[0167] In some embodiments, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO:43.

[0168] EIVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQLFDSPYTFGGGTKVEIKGGGGSGGGGSGGGGS QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPVEGLLRGFDYWGQGTLVTVSS[SEQ ID NO:43]

[0169] In some embodiments, the anti-CD19 scFv comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous or identical to SEQ ID NO:43.

[0170] In some embodiments, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO:44.

[0171] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVALIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPVEGLLRGFDYWGQGTLVTVSSGGG GSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGIPPYTFGGGTKVEIK[SEQ ID NO:44]

[0172] In some embodiments, the anti-CD19 scFv comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous or identical to SEQ ID NO:44.

[0173] Nucleotide sequence

[0174] The present disclosure includes encoding one or more heavy chains, heavy chain variable domains, heavy chain framework regions, heavy chain CDRs, heavy chain constant domains, light chains, light chain variable domains, light chain framework regions, light chain CDRs, light chain constant domains, or other immunoglobulin-like sequences, or nucleotide sequences of antibodies disclosed herein. In various embodiments, such nucleotide sequences may be present in a vector. In various embodiments, such nucleotide sequences may be present in a cell, such as a cell of a subject requiring treatment or a cell for producing an antibody, such as a mammalian cell for producing an antibody.

[0175] Engineered antibodies and fusion proteins

[0176] In some embodiments, the present disclosure provides fusion proteins comprising (i) one or more antigen binding regions described herein (e.g., antigen binding regions of immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds with antibody-like properties, as well as other antigen binding portions known in the art, including, for example, Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, Feb, scFv, SMIP, antibodies, diabodies, triabodies, tetrabodies, minibodies, maxibodies, tandab, DVD, BiTe, TandAb, etc.), such as one or more variable domains described herein or portions thereof (e.g., one or more CDRs described herein), and (ii) one or more additional polypeptides. For example, albumin is an abundant serum protein that is protected from degradation by pH-dependent cycling mediated by interaction with FcRn. In some embodiments, one or more variable domains or engineered antibodies as described herein, or portions thereof (e.g., one or more CDRs as described herein) are fused to albumin, portions thereof (e.g., portions of albumin that bind to FcRn), and / or engineered variants of albumin that bind to FcRn with increased affinity. In other cases, one or more variable domains or engineered antibodies as described herein, or portions thereof (e.g., one or more CDRs as described herein) are fused to an albumin-binding polypeptide to form a fusion protein-albumin complex, which in turn can bind to FcRn. In some embodiments, the albumin-binding polypeptide is a single-chain variable fragment (scFv). Albumin or a portion thereof can include one or more amino acid mutations that can alter the binding of the albumin fusion protein to FcRn. Such mutations are known in the art (see, e.g., Andersen et al., Nature Communications 3:610 doi:10.1038 / nocmms1607 (2012)). In other cases, one or more variable domains or engineered antibodies as described herein, or portions thereof (e.g., one or more CDRs as described herein) are fused to transferrin. Transferrin is recycled by binding to the transferrin receptor (see, eg, Widera et al., Adv. Drug Deliv. Rev. 55: 1439-66 (2003)).

[0177] pH-dependent binding of anti-CD19 antibodies to CD19 and / or Fc receptors

[0178] The engineered antibodies described herein can be engineered to exhibit pH dependence or enhanced pH dependence in affinity for CD19 (e.g., mediated by one or more variable domains described herein), and / or altered (e.g., increased, e.g., pH-dependent) affinity for FcRn (e.g., mediated by one or more constant domains described herein). For example, in some embodiments, an antibody capable of binding to CD19 or a variable domain capable of binding to CD19 binds to CD19 with a higher affinity at serum pH (e.g., at neutral pH or at a pH greater than 7.4) than at compartmental (e.g., endosome) pH (e.g., at acidic pH or at a pH equal to or less than pH 6.0). In various embodiments in which CD19 is bound by an antibody having pH-dependent CD19 binding, the shift in pH from serum pH to compartmental pH (e.g., from serum to endosome) promotes separation (i.e., "unbinding") of CD19 and the antibody at compartmental pH and / or in a specific compartment, such as an endosome. In various embodiments, this pH-dependent binding can mediate antibody recycling and / or CD19 degradation. In certain cases, the transition from serum pH to compartmental pH (e.g., from serum to endosomes) promotes the separation (i.e., "unbinding") of CD19 and antibody at compartmental pH and / or in a specific compartment such as an endosome, such that the antibody is recycled out by FcRn and the antigen is degraded in the lysosome. In some such cases, the pH dependence of CD19 binding increases the "processivity" of the antibody, at least in that upon recycling, the antibody returns to the serum and is free to bind to the target circulating CD19. In some cases, the recycling of antibodies exhibiting pH-dependent CD19 binding can continue until the antibody is ultimately degraded or degraded, at which time a single antibody molecule may have bound to and mediated the inactivation of multiple (rather than just one) CD19 molecules.

[0179] In certain embodiments, the anti-CD19 antibodies disclosed herein include a constant domain (e.g., an Fc domain) that exhibits increased affinity for an Fc receptor (e.g., FcRn) relative to a control. In some embodiments, this increased affinity relative to a control occurs at a serum pH (e.g., greater than 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, or a higher pH). In some embodiments, this increased affinity relative to a control occurs at a compartmental pH (e.g., less than 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, or a pH lower). In certain embodiments, the anti-CD19 antibodies disclosed herein include a constant domain (e.g., an Fc domain) that exhibits a pH dependency (or enhanced pH dependency) of affinity for an Fc receptor (such as FcRn) relative to a control. The neonatal Fc receptor (FcRn) is an MHC class I molecule that functions to protect IgG and albumin from catabolism, mediates transport of IgG across epithelial cells, and participates in antigen presentation by professional antigen-presenting cells. The IgG antibody subtype exhibits a longer serum half-life, which is mainly due to the clearance of antibodies from endosomes by FcRn, allowing IgG to recycle from cells.

[0180] In some embodiments, the serum half-life of the anti-CD19 antibodies is increased. For example, the combination of the anti-CD19 antibodies and FcRn increases the serum half-life of the antibody to about 4 days to about 45 days, such as about 5 days to about 30 days, about 10 days to about 30 days, or about 20 days to about 30 days. In certain embodiments, the serum half-life of the anti-CD19 antibodies described herein is about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days or longer.

[0181] In certain embodiments, the anti-CD19 antibodies described herein exhibit a pH-dependent change in affinity for CD19. Affinity can be measured as K D , the equilibrium dissociation constant between antibody and antigen; K D In various embodiments, the K of an anti-CD19 antibody as described herein for CD19 at serum pH (e.g., above 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2 or higher pH) or under serum conditions is negatively correlated. D is less than about 10 -4 , 10-5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 or 10 -15 M. In certain instances, the antibodies described herein have a K for CD19 at serum pH. D Between 0.001 and 1 nM, e.g., 0.001 nM, 0.005 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM. In some embodiments, the K for CD19 is between ..., at a compartmental pH (e.g., a pH below 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, or less) or under compartmental conditions. D Compared to the K of the same antibody against CD19 at serum pH or under serum conditions D fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold or more. In some embodiments, the K of CD19 is 5.0 or 6.5 at a compartmental pH (e.g., a pH below 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, or less) or under compartmental conditions. D It can be greater than 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7, 10 -6 , 10 -5 , 10 -4 or 10 -3 M. In certain instances, the anti-CD19 antibodies described herein have a K for CD19 at compartmental pH or under compartmental conditions. D It can be, for example, equal to or greater than 1 nM, such as 1 nM, 2nM, 3nM, 4nM, 5nM, 10nM, 20nM, 30nM, 40nM, 50nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1mM or more.

[0182] In some embodiments, anti-CD19 antibodies as described herein, when administered to a subject, for example, in the serum of the subject, show a longer half-life than a reference antibody (for example, the antibody that cross-competition CD19 is combined). In various embodiments, the half-life of a reference antibody (for example, the antibody that cross-competition CD19 is combined) in serum can be, for example, 250 to 300 hours. In various embodiments, the half-life of an anti-CD19 antibody as described herein in serum can be, for example, at least 250 hours, for example, at least 260, 270, 280, 290 or 300 hours. In certain embodiments, the half-life of an anti-CD19 antibody as described herein in serum can be at least 300 hours, for example, at least 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1,000 hours. In certain embodiments, the half-life of an anti-CD 19 antibody as described herein in serum can be at least 1,000 hours, e.g., at least 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, or 15,000 hours or longer. In various embodiments, the half-life of an anti-CD 19 antibody as described herein in serum can be at least 12 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. In various embodiments, the half-life of an anti-CD 19 antibody as described herein in serum can be increased by at least a factor, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold or more, compared to a reference antibody (e.g., an antibody that cross-competes for CD 19 binding).

[0183] In certain embodiments, the anti-CD19 antibodies described herein exhibit increased half-life in plasma, increased mean retention time in plasma, and / or increased CD19 clearance levels (e.g., antibodies that cross-compete CD19 binding). These parameters can be determined by methods known to those skilled in the art (e.g., as described in Nestorov et al., J. Clin. Pharmacol. 48:406-417 (2008); Leveque et al., Anticancer Research 25:2327-2344 (2005); Igawa et al., PLoS One 8:e63236.doi:10.1371 / journal.pone.0063236 (2013)). For example, an anti-CD 19 antibody described herein (e.g., a single dose of such an anti-CD 19 antibody) reduces the level of CD 19 in plasma by at least 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, 750-fold, 1000-fold, 1500-fold, or more relative to a reference antibody.

[0184] Engineered antibodies and their fragments

[0185] The CD19 antibodies and fragments thereof according to the present disclosure are engineered to include one or more binding moieties that specifically bind to one or more targets in a pH-dependent manner. The CD19 antibodies and fragments thereof encompass nucleic acids (e.g., RNA and DNA), proteins (e.g., antibodies), and combinations thereof. The pH-dependent binding moiety may be or include, for example, nucleic acids (e.g., RNA and DNA) and aptamers, polypeptides (e.g., antibodies or fragments thereof, albumin, receptors, ligands, signal peptides, avidin, and protein A), polysaccharides, biotin, hydrophobic groups, hydrophilic groups, drugs, and any organic molecule that binds to a receptor.

[0186] Antibodies or fragments thereof as binding moieties

[0187] In some embodiments, the antibodies or fragments thereof described herein are anti-CD19 antibodies. In some cases, one or more binding moieties described herein are or include antibodies, antigen-binding fragments thereof, and / or Fc regions (or Fc fragments) thereof. The basic structure of an IgG antibody consists of two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulfide bonds. The amino acid sequence of the first domain located at the amino terminus of each chain is variable and provides the antibody binding specificity found in each individual antibody. These are referred to as the variable heavy (VH) and variable light (VL) regions. The amino acid sequences of the other domains of each chain are relatively constant and are referred to as the constant heavy (CH) and constant light (CL) regions. For IgG antibodies, the light chain includes a variable region (VL) and a constant region (CL). The IgG heavy chain includes a variable region (VH), a first constant region (CH1), a hinge region, a second constant region (CH2), and a third constant region (CH3). In IgE and IgM antibodies, the heavy chain includes an additional constant region (CH4).

[0188] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibody light chain molecules comprising two heavy chains and two light chains, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affybodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to as "antibody mimetics"), and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations.

[0189] As used herein, the term "Fc fragment" refers to one or more fragments of the Fc region that retains Fc functions and / or activity (such as binding to Fc receptors) as described herein. As used herein, the term "antigen binding fragment" of an antibody refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen binding fragment" of an antibody include Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, scFv fragments, dAb fragments (Ward et al., (1989) Nature 341:544-546) and isolated complementary determining regions (CDRs). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the practicality of the fragment can be screened in the same manner as with intact antibodies.

[0190] In some aspects, the present invention provides antibodies or fragments thereof that bind to human CD19, comprising human heavy and / or light constant regions, wherein the human heavy constant region comprises an isotype variant of an Fc region comprising human IgG1, human IgG2, human IgG3, or human IgG4.

[0191] In another aspect, the present invention provides a humanized antibody or fragment thereof that binds to human CD19, wherein the antibody comprises a variant human IgG Fc region comprising an amino acid substitution S324N, i.e., the serine at amino acid position 324 of the parent antibody is substituted with asparagine, and the antibody comprising the variant human IgG Fc region exhibits improved complement dependent cytotoxicity (CDC) compared to the parent antibody.

[0192] Antibodies or fragments can be produced by any method known in the art for synthesizing antibodies (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods 182: 41-50; WO 92 / 22324; WO 98 / 46645). Chimeric antibodies can be produced using, for example, the method described in Morrison, 1985, Science 229: 1202, and humanized antibodies can be produced by the method described, for example, in U.S. Pat. No. 6,180,370.

[0193] Additional compositions and methods described herein are bispecific antibodies and multivalent antibodies as described in, for example, Segal et al., J. Immunol. Methods 248: 1-6 (2001); and Tutt et al., J. Immunol. 147: 60 (1991).

[0194] Engineered antigen-binding region

[0195] In some embodiments, the binding portion is or includes an antibody (e.g., an IgG antibody, such as an IgG1, IgG2, or IgG3 antibody) or an antigen-binding fragment that is engineered to bind to a target (i.e., an antigen) in an altered manner (e.g., in a pH-sensitive manner, such as in a stronger or weaker pH-sensitive manner) relative to a reference antibody or antigen-binding fragment. For example, the antibody can be engineered by modifying (e.g., by adding, deleting, or substituting) amino acids within one or more antibody CDRs and / or at positions encompassed by the antibody CDR structure. Exemplary non-limiting sites of antibodies that can be modified include the following (amino acid positions are indicated based on Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH)).

[0196] Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102

[0197] Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92 and L94.

[0198] In some embodiments, one or more of these disclosed amino acids can be replaced by histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine or glutamine. Without wishing to be bound by theory, it is believed that replacing the amino acid at one or more positions in these positions with histidine can produce antibodies with pH-dependent antigen-binding properties. In some embodiments, non-histidine residues are replaced by histidine residues. In some embodiments, histidine residues are replaced by non-histidine residues. Other engineered antigen-binding regions include those described in, for example, U.S. Publication No. 20110229489.

[0199] Engineered constant region

[0200] In some cases, the binding moiety is or comprises an antibody constant region, Fc region, or Fc fragment that binds one or more Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, or FcRn receptor). In some embodiments, the constant region, Fc region, or Fc fragment is engineered to bind to a target (e.g., an Fc receptor) in an altered manner (e.g., in a pH-sensitive manner, e.g., in a more or less pH-sensitive manner) relative to a reference constant region, Fc region, or Fc fragment.

[0201] In some cases, the binding portion can be or include the constant region, Fc region, or Fc fragment of an IgG antibody engineered to include amino acid additions, deletions, or substitutions of one or more of the amino acid residues described herein (e.g., 251-256, 285-290, 308-314, 385-389, and 428-436 (Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH))).

[0202] Production of CD19 antibodies and fragments

[0203] In some embodiments, antibodies as described herein or fragments thereof are engineered to include one or more binding moieties that exhibit pH sensitivity binding to one or more targets by mutagenesis using known techniques. For example, a sequence of a reference polypeptide (e.g., a therapeutic antibody or therapeutic fusion protein) can be obtained, and one or more amino acid residues can be added, deleted, or substituted. In some embodiments, one or more amino acid residues are replaced by histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine. In some embodiments, one or more amino acids are replaced by histidine. Without wishing to be bound by theory, it is believed that replacing amino acid residues with histidine can result in insertion of protonation sites, which can increase the pH sensitivity of the binding moiety. Polypeptides can be produced using standard methods and measured as described herein for binding to the target of interest. Other methods of increasing the pH sensitivity of a binding moiety are described in, for example, Sarkar et al., Nature Biotechnology 20:908-913 (2002); Murtaugh et al., Protein Science 20:1619-1631 (2011); and US Pub. No. 20110229489.

[0204] In some embodiments, a first target of interest is selected, and an antibody that selectively binds to the target is provided, obtained, and / or generated (e.g., using known methods as described herein). One or more amino acids in the antigen binding region and / or Fc region are substituted (e.g., with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine), and the pH sensitivity of binding to the target (and, additionally or alternatively, to FcRn) is determined.

[0205] In some embodiments, a polypeptide that naturally binds to a target of interest is provided, obtained, and / or produced. A known method is used to conjugate a polypeptide to an Fc region or Fc fragment as described herein (e.g., it binds to FcRn with a desired binding affinity). For example, the polypeptide and the Fc region or Fc fragment can be chemically or recombinantly expressed as a fusion protein. Additionally or alternatively, one or more amino acids of the polypeptide can be substituted (e.g., by histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine), and the pH sensitivity of the polypeptide in combination with the target is determined.

[0206] In some embodiments, the antibodies described herein or fragments thereof are engineered to include one or more binding moieties that are identified and / or selected by screening. For example, a library expressing an antigen binding portion, such as a phage library, can be used to identify antigen binding portions that bind to an antigen in a pH-sensitive manner. Such a library can be screened for antigen binding portions that have a first affinity for the antigen at a first pH (e.g., at pH 7.4) and a second affinity for the antigen at a second pH (e.g., at pH 5.5). The antibodies described herein or fragments thereof can be engineered to include such identified pH-sensitive antigen binding portions. Additionally and / or alternatively, a library can be used to identify FcRn binding portions that bind to FcRn in a pH-sensitive manner. Methods for screening recombinant antibody libraries are known (see, eg, Hoogenboom, Nature Biotech. 23: 1105-1116 (2005); US Patent No. 5,837,500; US Patent No. 5,571,698; WO 2012 / 044831).

[0207] PEGylation

[0208] In certain embodiments, anti-CD19 antibodies as described herein can be PEGylated to include single or multiple (e.g., 2-4) PEG moieties. Such PEGylated antibodies can exhibit increased half-lives compared to non-PEGylated reference antibodies, e.g., antibodies having the same amino acid sequence but having different, different amounts of PEGylation, or no PEGylation.

[0209] PEGylation can be performed by any suitable reaction known in the art. Methods for preparing PEGylated proteins generally include (a) reacting a polypeptide with polyethylene glycol (e.g., a reactive ester or aldehyde derivative of PEG) under conditions where the polypeptide is attached to one or more PEG groups; and (b) obtaining a reaction product. Generally, the conditions used for the reaction can be determined on a case-by-case basis based on known parameters and the desired outcome.

[0210] A variety of PEG attachment methods are available to those skilled in the art. For example, the steps of PEGylating an antibody or fragment thereof as described herein can be performed by acylation or alkylation with a reactive polyethylene glycol molecule.

[0211] Measuring the interaction of the binding moiety with the target

[0212] The binding properties of an antibody or fragment thereof as described herein (e.g., an anti-CD19 antibody as described herein) to a target (e.g., CD19 and / or FcRn) can be measured by methods known in the art, such as one of the following methods: BIACORE analysis, enzyme-linked immunosorbent assay (ELISA), X-ray crystallography, sequence analysis, and scanning mutagenesis. The binding interactions of the antibody to CD19 and / or FcRn can be analyzed using surface plasmon resonance (SPR). SPR or biomolecular interaction analysis (BIA) detects biospecific interactions in real time without labeling any interactants. The mass change (indicating a binding event) of the BIA chip binding surface causes the refractive index of light near the surface to change. The refractive index change can produce a detectable signal that is measured as an indication of a real-time reaction between biomolecules. Methods for using SPR are described, for example, in U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63: 2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5: 699-705 and online resources provided by BIAcore International AB (Uppsala, Sweden). Alternatively, SPR available from Sapidyne Instruments (Boise, supra) may be used. (Kinetic Exclusion Assay) determination.

[0213] Information from SPR can be used to provide the equilibrium dissociation constant (K) for the binding of a binding moiety to a target (e.g., an anti-CD19 antibody to CD19 and / or FcRn). D ) and kinetic parameters (including K on and K off ). Such data can be used to compare different molecules. Information from SPR can also be used to develop structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of a specific binding moiety to a target at various pH levels can be assessed. Binding partners with specific binding parameters, such as high affinity, low affinity, and slow K at specific pH levels, can be identified.off The variant amino acid at a given position is associated.

[0214] Treatment

[0215] In some embodiments, the antibodies or fragments thereof described herein (e.g., anti-CD19 antibodies described herein) are used in methods for treating one or more CD19-related disorders. In some embodiments, the antibodies or fragments thereof described herein (e.g., anti-CD19 antibodies described herein) are used as medicaments. CD19-related disorders may include, but are not limited to, disorders caused by symptoms including, including, symptoms caused in whole or in part by CD19 expression, or symptoms known to occur in conjunction with CD19 expression.

[0216] In some aspects, the present invention provides a method for treating cancer, the method comprising administering an agent that specifically binds to CD19 (e.g., an anti-CD19 antibody or fragment thereof as described herein). Cancer is a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth lead to the formation of malignant tumors, which invade adjacent tissues and may also be transferred to distant parts of the body through the lymphatic system or bloodstream. In some embodiments, "cancer" or "cancer tissue" includes solid tumors. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myelomas and other white blood cell malignancies. In some embodiments, the cancer is a B-cell lymphoma.

[0217] In some embodiments, the B-cell lymphoma is selected from the group consisting of acute lymphoblastic leukemia (ALL), AIDS-related lymphoma, ALK-positive large B-cell lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), classical Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, intravascular large B-cell lymphoma, HHV8-associated multicentric Castleman's disease), large B-cell lymphoma, lymphomatoid granuloma, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), nodular marginal zone B-cell lymphoma (NMZL), nodular lymphocyte-predominant Hodgkin lymphoma, non-Hodgkin lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, splenic marginal zone lymphoma (SMZL), and Waldenstrom's macroglobulinemia. In some embodiments, the B cell lymphoma is selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma (MCL), marginal zone B cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), and non-Hodgkin's lymphoma. In some embodiments, the B cell lymphoma is non-Hodgkin's lymphoma.

[0218] In various embodiments, administration of an antibody or fragment thereof described herein (e.g., an anti-CD 19 antibody or fragment thereof described herein) results in a decrease in the incidence, frequency, level, and / or amount of one or more symptoms or biomarkers of a CD 19-associated disorder, as described herein or otherwise known in the art, compared to a previously measured or reference value for the subject, e.g., a decrease in one or more symptoms or biomarkers by at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0219] In some embodiments, administration of an antibody or fragment thereof described herein (e.g., an anti-CD 19 antibody described herein) to a subject having cancer results in a greater reduction or improvement in one or more symptoms or biomarkers of cancer compared to a reference antibody (e.g., an antibody that cross-competes for CD 19 binding) under comparable conditions.

[0220] In some embodiments, the antibodies or fragments thereof described herein (e.g., anti-CD19 antibodies described herein) exhibit a reduced effective dose compared to a reference protein (e.g., an antibody that cross-competes for CD19 binding). For example, the effective dose of an anti-CD19 antibody as described herein can be, for example, less than 1,000 mg / agent, for example, less than 900 mg / agent, 800 mg / agent, 700 mg / agent, 600 mg / agent, 500 mg / agent, 550 mg / agent, 400 mg / agent, 350 mg / agent, 300 mg / agent, 200 mg / agent, 100 mg / agent, 50 mg / agent, 25 mg / agent or lower. In some cases, the effective dose of an anti-CD19 antibody as disclosed herein is lower than the effective or recommended or approved dose of a reference antibody, and the dose of the reference antibody can be, for example, 900 mg / agent or 600 mg / agent. Alternatively or in combination with dosages as disclosed herein, an anti-CD 19 antibody as described herein can be administered less than once a week, for example, less than once a week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or once a year. In some cases, an effective or useful frequency of administration of an anti-CD 19 antibody as disclosed herein is lower than the effective or recommended or approved frequency of administration of a reference antibody, which can be administered weekly (e.g., at a dosage of 300-600 mg, depending on the subject's weight) or biweekly (e.g., at a dosage of 300-1200 mg, depending on the subject's weight).

[0221] In some embodiments, the antibodies or fragments thereof described herein (e.g., anti-CD19 antibodies described herein) can be administered at a reduced dose as compared to a reference protein (e.g., an antibody that cross-competes for CD19 binding), while achieving equal, equally effective, comparably effective, or substantially effective results, wherein the anti-CD19 antibody is in the same, equivalent, or substantially equivalent formulation as the reference (e.g., an antibody that cross-competes for CD19 binding) and / or is administered by the same, equivalent, or substantially equivalent route of administration. In some embodiments, the anti-CD19 antibodies described herein can be administered at extended intervals as compared to a reference antibody (e.g., an antibody that cross-competes for CD19 binding), while achieving equal, equally effective, comparably effective, or substantially effective results, wherein the anti-CD19 antibody is in the same, equivalent, or substantially equivalent formulation as the reference and / or is administered by the same, equivalent, or substantially equivalent route of administration. In some embodiments, the anti-CD19 antibodies described herein can be administered with a reduced number of unit doses and / or a shortened treatment period, while achieving equivalent, equally effective, comparably effective, or substantially effective results, as compared to a reference antibody, wherein the anti-CD19 antibody is in the same, equivalent, or substantially equivalent formulation and / or is administered by the same, equivalent, or substantially equivalent route of administration as the reference (e.g., an antibody that cross-competes for CD19 binding).

[0222] According to some such embodiments, the anti-CD19 antibodies described herein are less likely to induce adverse reactions, such as adverse immune reactions, when administered to a subject compared to an effective dose of a reference antibody (e.g., an antibody that cross-competes for CD19 binding). Thus, in various embodiments, the anti-CD19 antibodies disclosed herein are less likely to induce adverse reactions or side effects per unit of active administration compared to a reference antibody. In various embodiments, the anti-CD19 antibodies disclosed herein are less likely to induce adverse reactions or side effects with a particular severity per unit of active administration compared to a reference antibody. In various embodiments, the anti-CD19 antibodies disclosed herein may induce one or more adverse reactions or side effects to a lesser extent or in fewer patients per unit of active administration compared to a reference antibody. Examples of adverse reactions or side effects that may be associated with the administration of antibodies that can bind to CD19 may include headache, nasopharyngitis, back pain, nausea, diarrhea, hypertension, upper respiratory tract infection, abdominal pain, vomiting, anemia, cough, peripheral edema, and / or urinary tract infection.

[0223] In some embodiments, after administration to a subject (e.g., as a single dose), the level of an antibody or fragment thereof described herein (e.g., an anti-CD19 antibody described herein) is measured in plasma at a defined time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days) after administration relative to the level of a control (e.g., an antibody that cross-competes for CD19 binding) at the same defined time. For example, at a defined time after administration of a single dose, the level of an anti-CD19 antibody described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% higher than the corresponding level of a reference antibody.

[0224] In some embodiments, the level of an antibody or fragment thereof described herein (e.g., an anti-CD19 antibody described herein) is measured in plasma at a defined time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days) after administration (e.g., a single dose) relative to the level of a control at the same defined time. For example, at a defined time after administration, the level of an anti-CD19 antibody described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% higher than the corresponding level of a reference antibody.

[0225] In some embodiments, the anti-CD19 antibodies described herein have an increased half-life (e.g., relative to a control, such as a reference antibody, such as an antibody that cross-competes for CD19 binding), and thus the anti-CD19 antibodies can be administered to a subject at increased intervals between doses. For example, the anti-CD19 antibodies can be administered once per week, every two weeks, every three weeks, every four weeks, every 6 weeks, every 8 weeks, or longer durations.

[0226] In some embodiments, the therapeutically effective amount of an antibody or fragment thereof described herein (e.g., an anti-CD19 antibody described herein) is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the effective amount of a reference therapeutic protein (e.g., an antibody that cross-competes for CD19 binding). In some embodiments, a single dose of an anti-CD19 antibody described herein achieves a therapeutic effect comparable to two or more doses of the reference antibody.

[0227] In some embodiments, an antibody or fragment thereof described herein (e.g., an anti-CD 19 antibody described herein) is administered at a dose that is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the concentration of the target antigen (e.g., CD 19) in a subject.

[0228] In some embodiments, any of various methods and delivery systems known to those skilled in the art can be used to physically introduce antibody as described herein or its fragment (for example, anti-CD19 antibody as described herein) into a subject. The exemplary route of administration of preparations disclosed herein includes intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. As used herein, phrase "parenteral administration" means the mode of administration except enteral and topical administration, usually administered by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, preparations are administered by non-parenteral route, and these approaches include local, epidermal or mucosal routes of administration, for example, intranasal, vaginal, rectal, sublingual or local. Administration can also be performed, for example, once, repeatedly, and / or through one or more extended periods.

[0229] In some embodiments, the antibodies or fragments thereof described herein (e.g., anti-CD19 antibodies described herein) can be used for a variety of diagnostic and therapeutic applications. For example, detectable labeled versions of engineered antibodies as described herein can be used to detect the presence or amount of CD19 in a sample (e.g., a biological sample). Engineered antibodies as described herein can be used in in vitro assays to study binding to CD19. In some embodiments, anti-CD19 antibodies as described herein can be used as positive controls in assays designed to identify other novel compounds that can be used to treat CD19-related disorders. For example, anti-CD19 antibodies as described herein can be used as positive controls in assays for identifying other compounds (e.g., small molecules, aptamers, or antibodies) that are bound to CD19.

[0230] The antibodies or antigen-binding fragments thereof described herein can be used to monitor subjects, such as subjects who have, are suspected of having, are at risk of developing, or are being treated for one or more CD19-related disorders. Monitoring can include determining the amount or activity of CD19 in a subject, such as in the serum of a subject. In some embodiments, after administering an anti-CD19 antibody as described herein, an assessment is performed for at least one (1) hour, such as at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days, or longer, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks, or longer. The subject can be assessed at one or more of the following times: before treatment begins; during treatment; or after administering one or more elements of treatment. The assessment can include assessing whether further treatment is needed, for example, assessing whether the dose, frequency of administration, or duration of treatment should be changed. It can also include assessing whether a selected treatment modality needs to be added or deleted, for example, adding or deleting any treatment for a CD19-related disorder described herein.

[0231] Formulation and administration

[0232] In various embodiments, the antibodies or antigen-binding fragments thereof described herein (e.g., anti-CD19 antibodies described herein) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions can be useful, for example, for preventing and / or treating diseases, such as CD19-related disorders. Pharmaceutical compositions can be formulated by methods known to those skilled in the art (e.g., as described in Remington's Pharmaceutical Sciences, 17th edition, Alfonso R. Gennaro, ed., Mack Publishing Company, Easton, Pa. (1985)).

[0233] Suitable mode of administration can be selected according to the age and condition of the experimenter. The single dose of the pharmaceutical composition containing antibody as herein described or its fragment (for example, anti-CD19 antibody as herein described) can be selected from the scope of 0.001 to 1000mg / kg body weight. On the other hand, dosage can be selected within the scope of 0.001 to 100000mg / body weight, but the disclosure is not limited to this type of scope. The dosage and method of administration vary according to the patient's weight, age, condition, etc., and those skilled in the art can appropriately select as needed.

[0234] In various embodiments, the pharmaceutical composition can be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, but are not limited to, any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The compositions of the present invention may contain pharmaceutically acceptable salts, such as acid addition salts or base addition salts.

[0235] In various embodiments, compositions comprising antibodies as described herein, such as sterile preparations for injection, can be formulated according to conventional pharmaceutical practice using distilled water for injection as a vehicle. For example, physiological saline or isotonic solutions containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as aqueous solutions for injection, optionally in combination with suitable solubilizers, for example, alcohols such as ethanol and polyols such as propylene glycol or polyethylene glycol, and nonionic surfactants such as polysorbate 80. TM , HCO-50, etc.

[0236] As disclosed herein, the pharmaceutical composition can be in any form known in the art. Such forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.

[0237] The selection of any particular form or use may depend in part on the mode of administration and therapeutic application of expectation.For example, the composition containing the composition expected for systemic or local delivery can be in the form of injectable or infusible solution.Therefore, composition can be formulated to be used by parenteral mode (for example, intravenous, subcutaneous, intraperitoneal or intramuscular injection).As used herein, parenteral administration refers to the mode of administration except enteral and topical application, usually by injection, and includes but is not limited to intravenous, intranasal, intraocular, through lung, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, through trachea, subcutaneous, subcutaneous, intraarticular, under capsule, subarachnoid, intraspinal, epidural, intracranial, intracarotid artery and intrasternal injection and infusion.

[0238] The administration route can be parenteral administration, such as by injection, nasal administration, pulmonary administration or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection.

[0239] In various embodiments, the pharmaceutical compositions of the present invention can be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating the desired amount of the compositions described herein into an appropriate solvent with one of the ingredients listed above, or a combination thereof, and then sterilizing by filtration as needed. In general, dispersions are prepared by incorporating the compositions described herein into a sterile vehicle containing an alkaline dispersion medium and the other ingredients required as listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods include vacuum drying and freeze drying, which obtain a powder of the compositions described herein plus any additional required ingredients (see below) from a previously sterile filtered solution thereof. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using a surfactant, the appropriate fluidity of the solution can be maintained. Prolonged absorption of the injectable composition can be achieved by including an agent that prolongs absorption (e.g., monostearate and gelatin) in the composition.

[0240] The pharmaceutical composition can be administered parenterally in the form of an injectable preparation of a sterile solution or suspension contained in water or another pharmaceutically acceptable liquid. For example, the pharmaceutical composition can be formulated in the following manner: the therapeutic molecule is appropriately combined with a pharmaceutically acceptable carrier or medium, such as sterile water and physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavor excipient, diluent, vehicle, preservative, adhesive, and then mixed into the unit dosage form required for generally accepted pharmaceutical practice. The amount of the active ingredient contained in the pharmaceutical preparation is such that a suitable dose within a specified range is provided. Non-limiting examples of oily liquids include sesame oil and soybean oil, and it can be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that can be included are buffers (such as phosphate buffer or sodium acetate buffer), soothing agents (such as procaine hydrochloride), stabilizers (such as benzyl alcohol or phenol) and antioxidants. The prepared injection can be packaged in a suitable ampoule.

[0241] In some embodiments, the compositions can be formulated for storage at temperatures below 0° C. (e.g., -20° C. or -80° C.). In some embodiments, the compositions can be formulated for storage at 2° C.-8° C. (e.g., 4° C.) for up to 2 years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years). Thus, in some embodiments, the compositions described herein are stable for storage at 2° C.-8° C. (e.g., 4° C.) for at least 1 year.

[0242] In certain cases, the pharmaceutical composition can be formulated as a solution. In some embodiments, the composition can be formulated as a buffered solution, for example, at an appropriate concentration and suitable for storage at 2°C-8°C (eg, 4°C).

[0243] Compositions comprising one or more engineered antibodies as described herein can be formulated in immunoliposome compositions. Such formulations can be prepared by methods known in the art. Liposomes with extended circulation time are disclosed, for example, in U.S. Patent No. 5,013,556.

[0244] In certain embodiments, the composition can be formulated to contain a carrier that will protect the compound from rapid release, such as a controlled release formulation including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are known in the art. See, for example, JR Robinson (1978) "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

[0245] In some embodiments, the composition can be formulated into a composition suitable for intrapulmonary administration (e.g., administered via an inhaler or nebulizer) to a mammal such as a human. Methods for preparing such compositions are well known in the art. Dry powder inhaler formulations and suitable systems for administering the formulations are also known in the art. Pulmonary administration can be oral and / or nasal. Examples of pharmaceutical devices for pulmonary delivery include metered dose inhalers, dry powder inhalers (DPIs), and nebulizers. For example, the compositions described herein can be administered to a subject's lungs via a dry powder inhaler. These inhalers are propellant-free devices that can deliver dispersible and stable dry powder formulations to the lungs. Dry powder inhalers are well known in the medical field and include, but are not limited to: (AstraZeneca; London, England) Inhaler ( Cambridge, Mass.); (GlaxoSmithKline; London, England); and ECLIPSE TM(Sanofi-Aventis; Paris, France). See also, for example, PCT Publication Nos. WO 04 / 026380, WO 04 / 024156, and WO 01 / 78693. DPI devices have been used for pulmonary administration of polypeptides such as insulin and growth hormone. In some embodiments, the compositions described herein can be administered intrapulmonary via a metered dose inhaler. These inhalers rely on a propellant to deliver discrete doses of the compound to the lungs. Other devices and methods of intrapulmonary administration are described, for example, in U.S. Patent Application Publication Nos. 20050271660 and 20090110679, the disclosures of each of which are incorporated herein by reference in their entirety.

[0246] In some embodiments, compositions can be formulated to be delivered to eyes in the form of, for example, a pharmaceutically acceptable solution, suspension, or ointment. Preparations for the treatment of eyes can be in the form of a sterile aqueous solution containing, for example, other ingredients, such as, but not limited to, preservatives, buffers, tonicity agents, antioxidants, and stabilizers, nonionic wetting agents, or clarifying agents, and tackifiers. Preparations as described herein can be administered by conventional methods, for example, in the form of drops, or by immersing eyes in therapeutic solutions, topically applied to the eyes of a subject (for example, a subject suffering from AMD) in need of treatment, and the drops or therapeutic solutions contain one or more compositions.

[0247] In certain embodiments, various devices for introducing drugs into the vitreous cavity of the eye may be suitable for administering the compositions described herein. For example, U.S. Publication No. 2002 / 0026176 describes a drug-containing plug that can be inserted through the sclera into the vitreous cavity, thereby delivering the drug to the vitreous cavity. In another example, U.S. Patent No. 5,443,505 describes an implantable device for introduction into the suprachoroidal space or avascular region to provide sustained release of the drug into the interior of the eye. U.S. Patent Nos. 5,773,019 and 6,001,386 each disclose an implantable drug delivery device that can be attached to the scleral surface of the eye. Other methods and devices for delivering therapeutic agents to the eye (e.g., transscleral patches and delivery through contact lenses) are described, for example, in Ambati and Adamis (2002) Prog Retin Eye Res 21(2):145-151; Ranta and Urtti (2006) Adv Drug Delivery Rev 58(11):1164-1181; Barocas and Balachandran (2008) Expert Opin Drug Delivery 5(1):1-10(10); Gulsen and Chauhan (2004) Invest Opthalmol Vis Sci 45:2342-2347; Kim et al. (2007) Ophthalmic Res 39:244-254; and PCT Publication No. WO 04 / 073551, the disclosures of which are incorporated herein by reference in their entirety.

[0248] In certain embodiments, the administration of antibodies as described herein is achieved by administering to the subject a nucleic acid encoding the antibody. Nucleic acids encoding therapeutic antibodies as described herein can be incorporated into gene constructs to be used as part of a gene therapy regimen to deliver nucleic acids that can be used for intracellular expression and production of antibodies. The expression constructs of such components can be administered in any therapeutically effective carrier, such as any preparation or composition capable of effectively delivering component genes to cells in vivo. The method includes inserting the subject gene into a viral vector, including recombinant retroviruses, adenoviruses, adeno-associated viruses, slow viruses, and herpes simplex virus-1 (HSV-1), or recombinant bacteria or eukaryotic plasmids. Viral vectors can directly transfect cells; plasmid DNA can be delivered by means of, for example, cationic liposomes (lipofectin) or derived polylysine conjugates, gramicidin S, artificial viral envelopes, or other such intracellular carriers, as well as direct injection of gene constructs or CaPO4 precipitation (see, for example, WO04 / 060407). Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM, known to those skilled in the art (see, e.g., Eglitis et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc Natl Acad Sci USA 85:6460-6464; Wilson et al. (1988) Proc Natl Acad Sci USA 85:3014-3018; Armentano et al. (1990) Proc Natl Acad Sci USA 87:6141-6145; Huber et al. (1991) Proc Natl Acad Sci USA 88:8039-8043; Ferry et al. (1991) Proc Natl Acad Sci USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc Natl Acad Sci USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc Natl Acad Sci USA 89:10892-10895; Hwu et al. (1993) J Immunol 150:4104-4115; U.S. Patent Nos. 4,868,116 and 4,980,286; and PCT Publication Nos. WO89 / 07136, WO89 / 02468, WO89 / 05345, and WO92 / 07573).Another viral gene delivery system utilizes vectors derived from adenovirus (see, e.g., Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155). Suitable adenoviral vectors derived from adenovirus Ad 5 type d1324 strain or other adenovirus strains (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Another viral vector system that can be used to deliver the subject gene is adeno-associated virus (AAV). See, e.g., Flotte et al. (1992) Am J Respir Cell Mol Biol 7:349-356; Samulski et al. (1989) J Virol 63:3822-3828; and McLaughlin et al. (1989) J Virol 62:1963-1973.

[0249] In various embodiments, subcutaneous administration can be accomplished by a device such as a syringe, a prefilled syringe, an automatic injector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, an ambulatory syringe infusion pump with a subcutaneous infusion set, or other devices for subcutaneous administration of the antibody drug.

[0250] The injection system of the present disclosure can adopt the delivery pen as described in U.S. Patent No. 5,308,341. Pen devices are well known in the art and are most commonly used for self-delivery of insulin by diabetics. Such devices can include at least one injection needle (e.g., a 31-gauge needle with a length of about 5 to 8 mm), usually pre-filled with one or more therapeutic unit doses of therapeutic solution, and can be used to quickly deliver the solution to the subject with as little pain as possible. A drug delivery pen includes a vial holder in which a vial of a therapeutic drug or other drug can be received. The pen may be a completely mechanical device, or it may be combined with an electronic circuit to accurately set and / or indicate the dose of drug injected into the user's body. See, for example, U.S. Patent No. 6,192,891. In some embodiments, the needle of the pen device is disposable and the kit includes one or more disposable replacement needles. Pen devices suitable for delivering any of the compositions characterized by the present invention are further described, for example, in U.S. Patent Nos. 6,277,099; 6,200,296; and 6,146,361, the disclosures of each of which are incorporated herein by reference in their entirety. A microneedle-based pen device is described, for example, in U.S. Patent No. 7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the precision pen injector (PPI) device MOLLY manufactured by Scandinavian Health Ltd. TM .

[0251] In some embodiments, the compositions described herein can be delivered therapeutically to a subject by topical administration. As used herein, "topical administration" or "local delivery" can refer to delivery that is independent of transporting the composition or agent to its intended target tissue or site via the vascular system. For example, the composition can be delivered by injection or implantation of the composition or agent or by injection or implantation of a device containing the composition or agent. In certain embodiments, after topical administration near a target tissue or site, the composition or agent or one or more components thereof can diffuse to the intended target tissue or site that is not the site of administration.

[0252] In some embodiments, the compositions provided herein are in unit dosage form, which can be suitable for self-administration. Such unit dosage forms can be provided in containers, which are typically, for example, vials, cartridges, prefilled syringes, or disposable pens. Dosage meters such as the dosage meter devices described in U.S. Patent No. 6,302,855 can also be used, for example, with the injection systems described herein.

[0253] The suitable dose of the compositions described herein (the dose that can treat or prevent the disease of the subject) can depend on a variety of factors, including, for example, the age, sex and weight of the subject to be treated and the specific inhibitor compound used. For example, the dosage of a composition comprising an antibody as described herein required for treating a subject with a CD19 related disorder may be different from the dosage of different preparations of the antibody. Other factors affecting the dosage applied to the subject include, for example, the type or severity of the disease. For example, a subject with a CD19 related disorder may need to apply a different dosage from a subject with another CD19 related disorder. Other factors may include, for example, other medical conditions that affect the subject at the same time or in the past, the subject's general health condition, the subject's genetic predisposition, diet, application time, excretion rate, drug combination, and any other therapeutic agent applied to the subject. It should also be understood that the specific dosage and treatment regimen of any particular subject can also be adjusted based on the judgment of the treating physician.

[0254] Compositions as described herein can be administered in fixed doses or in mg / kg (mg / kg) dosages. In some embodiments, dosage can also be selected to reduce or avoid producing antibodies or other host immune responses for one or more antibodies or its antigen-binding fragment in the composition. Although it is not intended to limit, the exemplary dosage of antibodies such as compositions as described herein includes, for example, 1-1000mg / kg, 1-100mg / kg, 0.5-50mg / kg, 0.1-100mg / kg, 0.5-25mg / kg, 1-20mg / kg and 1-10mg / kg. The exemplary dosage of compositions as described herein includes, but is not limited to, 0.1mg / kg, 0.5mg / kg, 1.0mg / kg, 2.0mg / kg, 4mg / kg, 8mg / kg or 20mg / kg.

[0255] The pharmaceutical solution may comprise a therapeutically effective amount of a composition as described herein. Such an effective amount can be easily determined by one of ordinary skill in the art based in part on the effect of the administered composition or the combined effect of the composition and one or more other active agents (if more than one agent is used). The therapeutically effective amount of the composition as described herein can also be varied according to factors such as the ability of the individual's disease state, age, sex, and body weight and the composition (and one or more other active agents) to induce a desired reaction (e.g., improvement of at least one disease parameter, such as improvement of at least one symptom of CD19-related disorders) in the individual. For example, a therapeutically effective amount of the composition as described herein can suppress a specific disorder (mitigate its severity or eliminate its occurrence) and / or prevent a specific disorder, and / or any symptom of a specific disorder known in the art or described herein. A therapeutically effective amount is also an amount in which any toxic or deleterious effect of the composition is exceeded by a therapeutically beneficial effect.

[0256] Suitable human doses of any of the compositions described herein can be further evaluated, for example, in a Phase I dose escalation study. See, for example, van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10):3499-3500.

[0257] The toxicity and therapeutic efficacy of the composition can be determined by known pharmaceutical procedures in cell culture or experimental animals (e.g., any animal model of a CD19-related disorder). These procedures can be used, for example, to determine the LD 50 (a dose lethal to 50% of the population) and ED 50 (The dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD 50 / ED 50 Compositions described herein that exhibit a high therapeutic index are preferred. Although compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets the compound to the site of the affected tissue and minimizes potential damage to normal cells and thereby reduces side effects.

[0258] Those skilled in the art will recognize that the data obtained from cell culture assays and animal studies can be used in formulating a dosage range for use in humans. Suitable dosages of the compositions described herein are generally in the range comprising ED 50 The circulating concentration range of the composition with little or no toxicity. The dosage may vary within this range depending on the dosage form adopted and the route of administration utilized. For the compositions described herein, the therapeutically effective dose may initially be estimated by cell culture assays. The dosage may be formulated in an animal model to achieve a circulating plasma concentration range including the I0 (i.e., the concentration of the antibody that achieves half-maximal inhibition of symptoms) as measured in cell culture. The information may be used to more accurately determine the useful dose in the human body. The levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, for example, where local administration is desired (e.g., administration to the eyes or joints), cell culture or animal model establishment may be used to determine the dosage required to achieve a therapeutically effective concentration in the local site.

[0259] Combination therapy

[0260] In various embodiments, anti-CD19 antibodies as described herein may be included in a therapeutic process that also includes administering at least one other agent to a subject. In various embodiments, the other agent administered in combination with anti-CD19 antibodies as described herein may be a chemotherapeutic agent. In various embodiments, the other agent administered in combination with antibodies as described herein may be an agent that suppresses inflammation.

[0261] In some embodiments, anti-CD19 antibodies are single-chain variable fragments (scFv) that are specific for human CD19. In some embodiments, anti-CD19 scFv can be conjugated (e.g., connected) to a therapeutic agent (e.g., a chemotherapeutic agent and a radioactive atom) to bind to cancer cells, deliver the therapeutic agent to the cancer cells, and kill cancer cells expressing human CD19. In some embodiments, anti-CD19 antibodies are connected to a therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent, a cytokine, a radioactive atom, siRNA, or a toxin. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the agent is a radioactive atom.

[0262] In some embodiments, the method can be combined with other therapies for CD19-related disorders. For example, the composition can be administered to the subject simultaneously with, before, or after chemotherapy. In some embodiments, the composition can be administered to the subject simultaneously with, before, or after adoptive therapy.

[0263] In various embodiments, the other agent used in combination with an anti-CD19 antibody as described herein can be used simultaneously with the anti-CD19 antibody, used on the same day with the anti-CD19 antibody, or used in the same week with the anti-CD19 antibody. In various embodiments, the other agent used in combination with an anti-CD19 antibody as described herein can be used in a single formulation with the anti-CD19 antibody. In certain embodiments, the other agent is used in a time-separated manner with the use of an anti-CD19 antibody as described herein, for example, before or after one or more hours, before or after one or more days, before or after one week or more weeks, or before or after one month or more months of use of the anti-CD19 antibody. In various embodiments, the frequency of administration of one or more other agents can be identical, similar or different with the frequency of administration of an anti-CD19 antibody as described herein.

[0264] Combination therapy encompasses treatment regimens that include the administration of two different antibodies as described herein and / or treatment regimens that include the administration of an antibody as described herein by multiple formulations and / or routes of administration.

[0265] In some embodiments, the composition can be formulated with one or more additional therapeutic agents, such as additional therapies for treating or preventing a subject's CD19 related disorder (e.g., cancer or autoimmune disorder). Additional agents for treating a subject's CD19 related disorder will vary depending on the specific disorder being treated, but may include, but are not limited to, rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, ifosfamide (osfamide), carboplatin, etoposide, dexamethasone, cytarabine, cisplatin, cyclophosphamide, or fludarabine.

[0266] Compositions as described herein can replace or supplement a therapy that was previously or currently being used. For example, after treatment with a composition as described herein, the use of one or more additional activating agents can be stopped or reduced, for example, with lower levels, such as after administering an anti-CD19 antibody as described herein with a lower level than a reference antibody that cross-competes CD19 binding. In some embodiments, the use of the previous therapy can be maintained. In some embodiments, the previous therapy will be maintained until the level of the composition reaches a level sufficient to provide a therapeutic effect. Two therapies can be administered in combination.

[0267] Recombinant gene technology

[0268] In light of the present disclosure, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are described in the following literature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed. 1985); Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0269] Recombinant expression of a gene (e.g., a nucleic acid encoding a polypeptide (e.g., an anti-CD19 antibody as described herein)) can include constructing an expression vector containing the nucleic acid encoding the polypeptide. Once the polynucleotide is obtained, a vector for producing the polypeptide can be generated by recombinant DNA technology using techniques known in the art. Known methods can be used to construct an expression vector comprising a polypeptide coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo genetic recombination.

[0270] The expression vector can be transferred to host cells by conventional techniques, and the transfected cells can then be cultured by conventional techniques to produce the polypeptide.

[0271] All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0272] The present invention also includes the following embodiments:

[0273] Embodiment 1. A CD19 antibody or an antigen-binding fragment thereof, comprising:

[0274] Heavy chain variable complementarity determining region (CDR) sequences of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3).

[0275] Embodiment 2. The CD19 antibody or antigen-binding fragment thereof according to embodiment 1, further comprising:

[0276] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGAVPIT (SEQ ID NO:12) (LCDR3).

[0277] Embodiment 3. The CD19 antibody or antigen-binding fragment thereof according to embodiment 1, further comprising:

[0278] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQVDSLHPFT (SEQ ID NO:13) (LCDR3).

[0279] Embodiment 4. The CD19 antibody or antigen-binding fragment thereof according to embodiment 1, further comprising:

[0280] A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPLT (SEQ ID NO:14) (LCDR3).

[0281] Embodiment 5. The CD19 antibody or antigen-binding fragment thereof according to embodiment 1, further comprising:

[0282] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQLFDSPYT (SEQ ID NO:15) (LCDR3).

[0283] Embodiment 6. The CD19 antibody or antigen-binding fragment thereof according to embodiment 1, further comprising:

[0284] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGVPPLT (SEQ ID NO:16) (LCDR3).

[0285] Embodiment 7. The CD19 antibody or antigen-binding fragment thereof according to embodiment 1, further comprising:

[0286] A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPFT (SEQ ID NO:17) (LCDR3).

[0287] Embodiment 8. The CD19 antibody or antigen-binding fragment thereof according to embodiment 1, further comprising:

[0288] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASNRAT (SEQ ID NO:10) (LCDR2), and QQAGVFPFT (SEQ ID NO:18) (LCDR3).

[0289] Embodiment 9. The CD19 antibody or antigen-binding fragment thereof according to embodiment 1, further comprising:

[0290] A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASRRAT (SEQ ID NO:11) (LCDR2), and QQAGIPPYT (SEQ ID NO:19) (LCDR3).

[0291] Embodiment 10. The CD19 antibody or antigen-binding fragment thereof according to embodiment 1, wherein the CD19 antibody or antigen-binding fragment thereof comprises:

[0292] An immunoglobulin light chain variable (V) sequence comprising an amino acid sequence at least 90% identical to SEQ ID NO: 20 L ) area; and

[0293] An immunoglobulin heavy chain variable (V) sequence comprising an amino acid sequence at least 90% identical to SEQ ID NO: 5 H )district.

[0294] Embodiment 11. A CD19 antibody or antigen-binding fragment thereof, comprising:

[0295] An immunoglobulin light chain variable (V) sequence comprising an amino acid sequence at least 90% identical to SEQ ID NOs: 20-27 L ) area; and

[0296] An immunoglobulin heavy chain variable (V) comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 5 H )district.

[0297] Embodiment 12. The CD19 antibody or antigen-binding fragment thereof according to embodiments 1-11, wherein the V L The region comprises an amino acid sequence that is at least 95% identical to SEQ ID NOs: 20-27.

[0298] Embodiment 13. The CD19 antibody or antigen-binding fragment thereof according to embodiment 12, wherein the V L The region comprises an amino acid sequence identical to SEQ ID NOs: 20-27.

[0299] Embodiment 14. The CD19 antibody or antigen-binding fragment thereof as described in any one of the preceding embodiments, wherein the V HThe region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:5.

[0300] Embodiment 15. The CD19 antibody or antigen-binding fragment thereof as described in any one of the preceding embodiments, wherein the V H The region comprises an amino acid sequence identical to SEQ ID NO:5.

[0301] Embodiment 16. The CD19 antibody or antigen-binding fragment thereof as described in any one of the preceding embodiments, wherein the V L The region comprises an amino acid sequence identical to SEQ ID NO: 20-27, and the V H The region is identical to SEQ ID NO:5.

[0302] Embodiment 17. The CD19 antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, wherein the CD19 antibody or fragment thereof is selected from the group consisting of: an IgA antibody, an IgG antibody, an IgE antibody, an IgM antibody, a bispecific or multispecific antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd' fragment, a Fd fragment, an isolated CDR or a collection thereof; a single-chain variable fragment (scFv), a polypeptide-Fc fusion, a single domain antibody, a camelid antibody; a masked antibody, a small modular immunopharmaceutical ("SMIPs™"), a single chain, a tandem diabody, a VHH, an anticalin, a nanobody, a minibody, a BiTE, an ankyrin repeat protein, a DARPIN, an Avimer, a DART, a TCR-like antibody, an Adnectin, an Affilin, a transmembrane antibody; an affibody, a TrimerX, a MicroProtein, a Fynomer, a Centyrin; and a KALBITOR.

[0303] Embodiment 18. The CD19 antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, wherein the CD19 antibody or fragment thereof is a monoclonal antibody or a single-chain variable fragment (scFv).

[0304] Embodiment 19. The CD19 antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, wherein the CD19 antibody or fragment thereof is an antibody comprising an IgG constant region.

[0305] Embodiment 20. The CD19 antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, wherein the CD19 antibody or fragment thereof is a single-chain variable fragment (scFv).

[0306] Embodiment 21. The CD19 antibody or antigen-binding fragment thereof of Embodiment 20, wherein the CD19scFv comprises a linker sequence comprising SEQ ID Nos: 36-39.

[0307] Embodiment 22. The CD19 antibody or antigen-binding fragment thereof of embodiment 20 or 21, wherein the CD19 scFv comprises a signal peptide selected from SEQ ID NO: 40 or 41.

[0308] Embodiment 23. The CD19 antibody or antigen-binding fragment thereof of any of the preceding embodiments, wherein the antibody or fragment thereof has a K between about 8 nanomolar (nM) and about 242 nM. D Binds to CD19.

[0309] Embodiment 24. The CD19 antibody or antigen-binding fragment thereof of any of the preceding embodiments, wherein the antibody or fragment thereof has an EC of between about 0.1 nM and about 2.7 nM. 50 Binds to CD19 on target cells.

[0310] Embodiment 25. A method of treating cancer, comprising administering the CD19 antibody or antigen-binding fragment thereof according to any one of the preceding embodiments to a subject in need of treatment.

[0311] Embodiment 26. The method of embodiment 25, wherein the cancer is selected from leukemia, lymphoma, or myeloma.

[0312] Embodiment 27. A pharmaceutical composition comprising a CD19 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, wherein the CD19 antibody or fragment thereof comprises:

[0313] A heavy chain variable region having a complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3).

[0314] Embodiment 28. The pharmaceutical composition of embodiment 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0315] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGAVPIT (SEQ ID NO:12) (LCDR3).

[0316] Embodiment 29. The pharmaceutical composition of embodiment 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0317] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQVDSLHPFT (SEQ ID NO:13) (LCDR3).

[0318] Embodiment 30. The pharmaceutical composition of embodiment 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0319] A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPLT (SEQ ID NO:14) (LCDR3).

[0320] Embodiment 31. The pharmaceutical composition of embodiment 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0321] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQLFDSPYT (SEQ ID NO:15) (LCDR3).

[0322] Embodiment 32. The pharmaceutical composition of embodiment 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0323] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGVPPLT (SEQ ID NO:16) (LCDR3).

[0324] Embodiment 33. The pharmaceutical composition of embodiment 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0325] A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPFT (SEQ ID NO:17) (LCDR3).

[0326] Embodiment 34. The pharmaceutical composition of embodiment 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0327] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASNRAT (SEQ ID NO:10) (LCDR2), and QQAGVFPFT (SEQ ID NO:18) (LCDR3).

[0328] Embodiment 35. The pharmaceutical composition of embodiment 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0329] A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASRRAT (SEQ ID NO:11) (LCDR2), and QQAGIPPYT (SEQ ID NO:19) (LCDR3).

[0330] Embodiment 36. A method of treating cancer, the method comprising administering to a subject in need of treatment a CD19 antibody or antigen-binding fragment thereof, wherein the CD19 antibody or fragment thereof comprises:

[0331] A heavy chain variable region having a complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3).

[0332] Embodiment 37. The method of embodiment 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0333] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGAVPIT (SEQ ID NO:12) (LCDR3).

[0334] Embodiment 38. The method of embodiment 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0335] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQVDSLHPFT (SEQ ID NO:13) (LCDR3).

[0336] Embodiment 39. The method of embodiment 36, wherein the CD19 antibody or fragment thereof further comprises:

[0337] A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPLT (SEQ ID NO:14) (LCDR3).

[0338] Embodiment 40. The method of embodiment 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0339] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQLFDSPYT (SEQ ID NO:15) (LCDR3).

[0340] Embodiment 41. The method of embodiment 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0341] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGVPPLT (SEQ ID NO:16) (LCDR3).

[0342] Embodiment 42. The method of embodiment 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0343] A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPFT (SEQ ID NO:17) (LCDR3).

[0344] Embodiment 43. The method of embodiment 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0345] A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASNRAT (SEQ ID NO:10) (LCDR2), and QQAGVFPFT (SEQ ID NO:18) (LCDR3).

[0346] Embodiment 44. The method of embodiment 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises:

[0347] A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASRRAT (SEQ ID NO:11) (LCDR2), and QQAGIPPYT (SEQ ID NO:19) (LCDR3).

[0348] Embodiment 45. A nucleic acid encoding an amino acid sequence at least 90% identical to SEQ ID NO:5.

[0349] Embodiment 46. A nucleic acid encoding an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 20-27.

[0350] Embodiment 47. A vector comprising the isolated nucleic acid sequence of any one of embodiments 45 or 46.

[0351] Embodiment 48. An isolated cell comprising the vector of embodiment 47.

[0352] Example

[0353] The following examples describe some preferred modes of making and practicing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0354] Example 1. Identification and characterization of anti-CD19 antibodies

[0355] This example shows the characterization of anti-CD19 antibodies. Human anti-CD19 antibodies are derived from and produced in Adimab yeast. Antigens are biotinylated using the EZ-Link Sulfo-NHS-Biotinylation kit (Thermo Scientific, catalog number 21425). The antigen is concentrated to approximately 1 mg / mL and the buffer is exchanged into PBS, followed by addition of a 1:7.5 molar ratio of biotinylation reagent. Before the buffer is exchanged again to remove free biotin in the solution, the mixture is maintained at 4°C overnight. Biotinylation is confirmed by binding to a streptavidin sensor of the protein labeled on ForteBio.

[0356] Eight naive synthetic yeast libraries were propagated as previously described, each with a diversity of approximately 10 9 (See, e.g., Y. Xu et al., PEDS 26(10), 663-70 (2013); WO2009036379; WO2010105256; and WO2012009568).

[0357] For the first two rounds of selection, magnetic bead sorting technology was performed using the Miltenyi MACS system, as previously described (see, e.g., Siegel et al., J Immunol Methods 286(1-2), 141-153(2004)). Briefly, yeast cells (approximately 1010 cells / library) were incubated with biotinylated antigen in 30°C wash buffer (phosphate buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) for 30 minutes. After washing once with 40 mL of ice-cold wash buffer, the cell pellet was resuspended in 20 mL of wash buffer, and Streptavidin MicroBeads (500 μl) were added to the yeast and incubated at 4°C for 15 minutes. Next, the yeast was pelleted, resuspended in 5 mL of wash buffer, and loaded onto a Miltenyi LS column. After loading 5 mL, the column was washed 3 times with 3 mL of wash buffer. The column was then removed from the magnetic field and the yeast were eluted with 5 mL of growth medium and then grown overnight.

[0358] The third round of selection was performed using flow cytometry (FACS). 7Yeast pellets were washed three times with wash buffer and incubated at 30°C with 100-200nM biotinylated antigen under equilibrium conditions. The fourth and fifth rounds of selection were performed by incubating biotinylated NALM-6 and Raji cells with selected yeast output from the 3rd round of FACS. After incubation, pre-washed M-280 Strepavidin Dynabeads (catalog number 60210) were added to the yeast / mammalian cell complex and incubated. Next, the complex was separated using a DynaMag-2 magnet and unbound supernatant was removed. The beads / cell complex was washed three times with 1mL of selection buffer. The captured complex was then transferred to a flask containing yeast growth medium for breeding. In the sixth round of selection, the NALM-6 / Raji cells of either round and additional round were selected for the bred yeast, selected using 100nM recombinant CD19 antigen, or negatively selected using a multispecific reagent (PSR) to remove non-specific antibodies.

[0359] For PSR depletion, the library was incubated with a 1:10 dilution of biotinylated PSR reagent as previously described (see, e.g., Y. Xu et al., PEDS 26(10), 663-70(2013)). The yeast was then washed twice with wash buffer and stained with a 1:100 dilution of goat F(ab')2 anti-human kappa-FITC (LC-FITC) (Southern Biotech, catalog number 2062-02) and a 1:500 dilution of Streptavidin-AF633 (SA-633) (Life Technologies, catalog number S21375) or a 1:50 dilution of Extravidin-phycoerthyrin (EA-PE) (Sigma-Aldrich, catalog number E4011) secondary reagent at 4°C for 15 minutes. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.3 mL of wash buffer and transferred to a strainer-capped sort tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sorting gates were determined to select antibodies with the desired characteristics. Selection rounds were repeated until a population with all the desired characteristics was obtained. After the final round of sorting, yeast was inoculated and individual colonies were picked for characterization.

[0360] Light chain diversification

[0361] The heavy chain from the initial output is used to prepare a light chain diversification library for additional selection rounds. Heavy chain plasmids are extracted from yeast, bred in Escherichia coli, subsequently purified therefrom, and converted into a light chain library with a diversity of 5 × 106. These libraries are selected as described above, i.e., one round of MACS is performed, two rounds of cell selection are performed with Raji or NALM-6 cells, and then the fourth round of FACS selection is performed using recombinant CD19 antigen. Specific to light chain diversification, Raji and NALM-6 cell selection is incorporated into the initial negative selection of engineered Raji and NALM-6 cells, which have undergone targeted gene knockout of the CD19 gene. After consuming CD19 knockout cells, positive selection is performed using engineered Raji and NALM-6 cells expressing endogenous CD19 and overexpressed. In different FACS selection rounds, the (multi-specific reagent) PSR binding, species cross-reactivity, and affinity pressure of the library are assessed by antigen titration. Sorting is performed to obtain a colony with the desired characteristics. Individual colonies from each of the above FACS selection rounds were picked for sequencing and characterization.

[0362] Antibody production and purification

[0363] Yeast clones were grown to saturation and then induced at 30°C with shaking for 48 hours. After induction, yeast cells were pelleted and the supernatant was harvested for purification. IgG was purified using a protein A column and eluted with acetic acid at pH 2.0.

[0364] ForteBio K D Measurement

[0365] The ForteBio Octet system (Octet RED384, generally as described previously (see, e.g., Estep et al., Mabs 5(2), 270-278(2013)). Briefly, ForteBio affinity measurements were performed by loading IgG online onto an AHC sensor. The antibodies were immobilized on anti-human IgG needles and bound to a soluble CD19-HSA fusion protein (CD19 extracellular domain fused to human serum albumin). The sensor was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The IgG-loaded sensor was exposed to 100 nM soluble CD19-HSA fusion protein antigen for 3 minutes and then transferred to assay buffer for 3 minutes for off-rate measurements. All kinetics were analyzed using a 1:1 binding model. A summary of the binding kinetics is provided in Table 3. The affinity of the exemplary antibodies ranged from 8 nM to 20 nM. The vH of all anti-CD19 antibodies shown in Table 3 includes the vH sequence of SEQ ID NO: 5.

[0366] Table 3. Binding kinetics of anti-CD19 antibodies

[0367]

[0368] Example 2. Cellular Binding of Anti-CD19 Antibodies

[0369] Anti-CD19 antibodies were assessed for cellular binding by flow cytometry on Raji and NALM-6 cell lines expressing endogenous CD 19. Each antibody was tested on the CD19-positive parental line and the corresponding CD19 knockout Raji and NALM-6 lines to confirm specific binding of the target on the cells. Figure 1 The flow cytometry chromatograms shown in show a 1-2 log shift on the CD19 positive lines compared to background binding on the corresponding knockout cell lines.

[0370] Figures 2A to 2C The serial dilution flow cytometry results shown in Figure 4 show saturated binding on NALM-6 cells. EC50 values ​​calculated from the curves are provided in Table 4. The exemplary anti-CD19 antibodies bind to CD19-positive NALM-6 cells with a high affinity of at least 0.2 nM. The higher affinity observed on CD19-positive cells compared to soluble protein suggests that the epitope bound by these antibodies may be more naturally presented on cells than in the CD19-HSA fusion protein.

[0371] Table 4. EC50 of anti-CD19 antibodies on NALM6 cells

[0372]

[0373] Example 3. Epitope Binning by Cross-Competition

[0374] Cross-competition epitope framing and assays were performed to determine if any anti-CD19 antibodies bound to a unique non-competitive epitope with the reference anti-CD19 antibody, Denintuzumab. Epitope framing and / or ligand blocking were performed using a sandwich cross-blocking assay. A control anti-target IgG was loaded onto the AHQ sensor, and unoccupied Fc binding sites on the sensor were blocked with an irrelevant human IgG1 antibody. The sensor was then exposed to 100 nM target antigen, followed by a second anti-target antibody or ligand. Additional binding of the second antibody or ligand after antigen association indicates that the epitope is not occupied (non-competitor), while no binding indicates epitope blocking (competitor or ligand blocking).

[0375] The anti-CD19 reference control, dinuximab, was captured on an anti-human IgG needle, and then loaded with soluble CD19 in liquid phase. Instead of a dissociation step, a second loading step was performed for each test antibody. Antibodies that bind to a non-competing epitope with dinuximab will reflect new binding events on the trace, while antibodies whose epitope is blocked by dinuximab will reflect a flat trace. Figure 3 The results shown in Figure 4 show that each antibody competes for binding to the epitope with dinotuzumab. These results indicate that these antibodies compete with the reference anti-CD19 antibody dinotuzumab for binding to a similar epitope on CD19.

[0376] Example 4. Characterization of Anti-CD19 scFv Binding to CD19

[0377] Relative competition for shared epitopes was assessed in a Biacore SPR assay. In this assay, scFvs containing the vH and vL of anti-CD19 antibody 2 (SEQ ID NO: 42) were evaluated for relative competitive binding to the CD19-T2 binders, SJ25c1 and FMC63 ("Tisagenlecleucel" from Kymriah and "Axicabtagene ciloleucel" from Yescarta). Antibody 2 scFv was immobilized on a Biacore CM5 chip and then bound by a mixture of 100 nM CD19-HSA-his and a titer of each competitor scFv (0, 50 nM, 100 nM, 200 nM, and 800 nM). After a 3-minute competitive binding step, a 5-minute dissociation step was performed in buffer HBS-EP (300 mM NaCl).

[0378] In this assay, CD19 antibody 1 scFv competed with all reference antibodies (data not shown). Even at the lowest concentration (50 nM), FMC63 scFv completely competed for CD19 binding. The assay was repeated to include 25 nM, where a small amount of binding to CD19 was retained, indicating that the binding affinity of these antibodies to CD19 was similar to but slightly higher than that of SJ25C1 scFv. These data indicate that all tested binding agents compete for binding to overlapping epitopes on CD19.

[0379] To further assess the potential of anti-CD19 antibodies to bind to nonspecific membrane proteins, scFvs derived from the CD19 antibodies described herein were evaluated in a surface membrane protein (SMP) assay. The SMP assay is an ELISA-based assay in which human HEK-293 or insect SF9 cell membranes are coated on a plate to test the nonspecific binding of these test antibodies to these membranes. Similar to the SPR assay, internal control high and low nonspecific binding antibodies (high nonspecific binding control sc209, and low nonspecific binding control 5f9) were included. The results obtained were consistent with the above-mentioned SPR assay, with low binding activity of the CD19 antibodies to HEK-293 or SF9 membranes.

[0380] Other implementation plans

[0381] Although many embodiments of the present invention are described herein, the disclosure and examples can be modified to provide other methods and compositions of the present invention. Therefore, it will be understood that the scope of the present invention is defined by the appended claims, in addition to the specific embodiments shown by way of example. All references cited herein are hereby incorporated by reference.

Claims

1. A CD19 antibody or an antigen-binding fragment thereof, comprising: Heavy chain variable complementarity determining region (CDR) sequences of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3).

2. The CD19 antibody or antigen-binding fragment thereof according to claim 1, further comprising: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGAVPIT (SEQ ID NO:12) (LCDR3).

3. The CD19 antibody or antigen-binding fragment thereof according to claim 1, further comprising: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQVDSLHPFT (SEQ ID NO:13) (LCDR3).

4. The CD19 antibody or antigen-binding fragment thereof according to claim 1, further comprising: A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPLT (SEQ ID NO:14) (LCDR3).

5. The CD19 antibody or antigen-binding fragment thereof according to claim 1, further comprising: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQLFDSPYT (SEQ ID NO:15) (LCDR3).

6. The CD19 antibody or antigen-binding fragment thereof according to claim 1, further comprising: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGVPPLT (SEQ ID NO:16) (LCDR3).

7. The CD19 antibody or antigen-binding fragment thereof according to claim 1, further comprising: A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPFT (SEQ ID NO:17) (LCDR3).

8. The CD19 antibody or antigen-binding fragment thereof according to claim 1, further comprising: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASNRAT (SEQ ID NO:10) (LCDR2), and QQAGVFPFT (SEQ ID NO:18) (LCDR3).

9. The CD19 antibody or antigen-binding fragment thereof according to claim 1, further comprising: A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASRRAT (SEQ ID NO:11) (LCDR2), and QQAGIPPYT (SEQ ID NO:19) (LCDR3).

10. The CD19 antibody or antigen-binding fragment thereof according to claim 1, comprising: An immunoglobulin light chain variable (V) sequence comprising an amino acid sequence at least 90% identical to SEQ ID NO: 20 L ) area; and An immunoglobulin heavy chain variable (V) comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 5 H )district.

11. A CD19 antibody or an antigen-binding fragment thereof, comprising: An immunoglobulin light chain variable (V) sequence comprising an amino acid sequence at least 90% identical to SEQ ID NOs: 20-27 L ) area; and An immunoglobulin heavy chain variable (V) comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 5 H )district.

12. The CD19 antibody or antigen-binding fragment thereof of claims 1-11, wherein the V L The region comprises an amino acid sequence that is at least 95% identical to SEQ ID NOs: 20-27.

13. The CD19 antibody or antigen-binding fragment thereof according to claim 12, wherein the V L The region comprises an amino acid sequence identical to SEQ ID NOs: 20-27.

14. The CD19 antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the V H The region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

5.

15. The CD19 antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the V H The region comprises an amino acid sequence identical to SEQ ID NO:

5.

16. The CD19 antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the V L The region comprises an amino acid sequence identical to SEQ ID NO: 20-27, and the V H The region is identical to SEQ ID NO:

5.

17. The CD19 antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the CD19 antibody or fragment thereof is selected from the group consisting of: an IgA antibody, an IgG antibody, an IgE antibody, an IgM antibody, a bispecific or multispecific antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd' fragment, a Fd fragment, an isolated CDR, or a collection thereof; a single-chain variable fragment (scFv), a polypeptide-Fc fusion, a single domain antibody, a camelid antibody; masked antibodies, small modular immunopharmaceuticals ("SMIPs™"), single chains, tandem diabodies, VHHs, Anticalins, Nanobodies, Minibodies, BiTEs, Ankyrin Repeat Proteins, DARPINs, Avimers, DARTs, TCR-like antibodies, Adnectins, Affilins, transmembrane antibodies; Affibodies, TrimerX, MicroProteins, Fynomers, Centyrins; and KALBITOR.

18. The CD19 antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the CD19 antibody or fragment thereof is a monoclonal antibody or a single-chain variable fragment (scFv).

19. The CD19 antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the CD19 antibody or fragment thereof is an antibody comprising an IgG constant region.

20. The CD19 antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the CD19 antibody or fragment thereof is a single-chain variable fragment (scFv).

21. The CD19 antibody or antigen-binding fragment thereof of claim 20, wherein the CD19 scFv comprises a linker sequence comprising SEQ ID Nos: 36-39.

22. The CD19 antibody or antigen-binding fragment thereof of claim 20 or 21, wherein the CD19 scFv comprises a signal peptide selected from SEQ ID NO: 40 or 41.

23. The CD19 antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or fragment thereof has a K between about 8 nanomolar (nM) and about 242 nM. D Binds to CD19.

24. The CD19 antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or fragment thereof has an EC of between about 0.1 nM and about 2.7 nM. 50 Binds to CD19 on target cells.

25. A method of treating cancer, comprising administering the CD19 antibody or antigen-binding fragment thereof according to any one of the preceding claims to a subject in need of treatment.

26. The method of claim 25, wherein the cancer is selected from leukemia, lymphoma, or myeloma.

27. A pharmaceutical composition comprising a CD19 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, wherein the CD19 antibody or fragment thereof comprises: A heavy chain variable region having a complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3).

28. The pharmaceutical composition of claim 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGAVPIT (SEQ ID NO:12) (LCDR3).

29. The pharmaceutical composition of claim 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQVDSLHPFT (SEQ ID NO:13) (LCDR3).

30. The pharmaceutical composition of claim 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPLT (SEQ ID NO:14) (LCDR3).

31. The pharmaceutical composition of claim 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQLFDSPYT (SEQ ID NO:15) (LCDR3).

32. The pharmaceutical composition of claim 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGVPPLT (SEQ ID NO:16) (LCDR3).

33. The pharmaceutical composition of claim 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPFT (SEQ ID NO:17) (LCDR3).

34. The pharmaceutical composition of claim 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASNRAT (SEQ ID NO:10) (LCDR2), and QQAGVFPFT (SEQ ID NO:18) (LCDR3).

35. The pharmaceutical composition of claim 27, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASRRAT (SEQ ID NO:11) (LCDR2), and QQAGIPPYT (SEQ ID NO:19) (LCDR3).

36. Use of a CD19 antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating cancer, wherein the CD19 antibody or the fragment thereof comprises: A heavy chain variable region having a complementarity determining region (CDR) sequence of SYGMH (SEQ ID NO: 1) (HCDR1), LIWYDGSNKYYADSVKG (SEQ ID NO: 2) (HCDR2), and PVEGLLRGFDY (SEQ ID NO: 3) (HCDR3).

37. The method of claim 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGAVPIT (SEQ ID NO:12) (LCDR3).

38. The method of claim 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQVDSLHPFT (SEQ ID NO:13) (LCDR3).

39. The method of claim 36, wherein the CD19 antibody or fragment thereof further comprises: A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPLT (SEQ ID NO:14) (LCDR3).

40. The method of claim 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQLFDSPYT (SEQ ID NO:15) (LCDR3).

41. The method of claim 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVRSSYLA (SEQ ID NO:8) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGVPPLT (SEQ ID NO:16) (LCDR3).

42. The method of claim 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASSRAT (SEQ ID NO:9) (LCDR2), and QQAGGVPPFT (SEQ ID NO:17) (LCDR3).

43. The method of claim 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having the complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASNRAT (SEQ ID NO:10) (LCDR2), and QQAGVFPFT (SEQ ID NO:18) (LCDR3).

44. The method of claim 36, wherein the CD19 antibody or antigen-binding fragment thereof further comprises: A light chain variable region having complementarity determining region (CDR) sequences of RASQSVSSSYLA (SEQ ID NO:7) (LCDR1), GASRRAT (SEQ ID NO:11) (LCDR2), and QQAGIPPYT (SEQ ID NO:19) (LCDR3).

45. A nucleic acid encoding an amino acid sequence at least 90% identical to SEQ ID NO:

5.

46. ​​A nucleic acid encoding an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 20-27.

47. A vector comprising the isolated nucleic acid sequence of any one of claims 45 or 46.

48. An isolated cell comprising the vector of claim 47.

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