CD19 Antibodies and Methods of Using Them

Abstract

The present disclosure generally relates to immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that can bind to the CD19 protein. The antibodies of the present technology are useful in methods for detecting and treating CD19-associated autoimmune diseases or CD19-associated cancers in subjects in need thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 831,123, filed April 8, 2019, the entire contents of which are incorporated herein by reference. The present technology generally relates to the preparation and use of immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to the CD19 protein. In particular, the present technology relates to the preparation of CD19-binding antibodies and their use in the detection and treatment of CD19-associated cancers and CD19-associated autoimmune diseases. [Background technology]

[0002] The following description of the background of the present technology is provided merely as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology. Non-Hodgkin's lymphoma (NHL) is a heterogeneous disease that includes more than 30 types of B- and T-lymphocyte malignancies and accounts for 4.3% of all cancers diagnosed in the United States. B-cell malignancies include non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), and acute lymphocytic leukemia (ALL). B-cell lymphoma accounts for 85% of all NHL, 30% for diffuse large B-cell lymphoma (DLBCL), and 20% for follicular lymphoma, resulting in nearly 19,000 deaths. In the United States, CLL accounts for one-third of leukemias and is responsible for 4,600 deaths annually (Jemal et al., J Natl Cancer Inst 109 (9), djx030 (2017)). Summary of the Invention

[0003] In one aspect, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) V Hcomprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12, and / or (b) V L provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. The antibody may further comprise an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments, the antibody comprises an IgG4 constant region comprising an S228P mutation. In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', scF v and F v In some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment binds to a CD19 polypeptide that contains an Ig-like C2 loop comprising the amino acid sequence EE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP FLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGS (SEQ ID NO:82) (corresponding to amino acid residues 29-118 of SEQ ID NO:60 or SEQ ID NO:61). Additionally or alternatively, in certain embodiments, the antibody or antigen-binding fragment binds to a conformational epitope comprising amino acid residues corresponding to positions 29-118 of SEQ ID NO:60 or SEQ ID NO:61.

[0004] In one aspect, the disclosure provides an antibody comprising a heavy chain (HC) comprising an amino acid sequence comprising SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:45, SEQ ID NO:47, or a variant thereof with one or more conservative amino acid substitutions, and / or a light chain (LC) amino acid sequence comprising SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:44, SEQ ID NO:46, or a variant thereof with one or more conservative amino acid substitutions.

[0005] In some embodiments, the antibody comprises an HC amino acid sequence and an LC amino acid sequence, respectively, selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:20 (chFMC63xCD3 BsAb); SEQ ID NO:26 and SEQ ID NO:24 (BC250- hFMC63 VL-2 / VH-lbxCD3 BsAb); SEQ ID NO:29 and SEQ ID NO:28 (mFMC63xmC825 BsAb); SEQ ID NO:31 and SEQ ID NO:30 (mFMC63xhC825 BsAb); SEQ ID NO:45 and SEQ ID NO:44 (hFMC63 VL-2VH-lbxmC825); and SEQ ID NO:47 and SEQ ID NO:46 (hFMC63 VL-2VH-lbxhC825).

[0006] In one aspect, the disclosure provides an antibody comprising: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 17, 18, or 19; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, or 12. In another aspect, the disclosure provides an antibody comprising (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the LC sequence present in any one of SEQ ID NOs: 20, 24, 28, 30, 44, or 46, and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the HC sequence present in any one of SEQ ID NOs: 22, 26, 29, 31, 45, or 47.

[0007] In any of the above embodiments, the antibody is a chimeric antibody, a humanized antibody, or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. In certain embodiments, the antibody of the present technology comprises an IgG4 constant region comprising an S228P mutation. In any of the above embodiments, the antibody or antigen-binding fragment binds to a CD19 polypeptide comprising an Ig-like C2 loop comprising the amino acid sequence EE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP FLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGS (SEQ ID NO: 82) (corresponding to amino acid residues 29-118 of SEQ ID NO: 60 or SEQ ID NO: 61). Additionally or alternatively, in certain embodiments, the antibody or antigen-binding fragment binds to a conformational epitope comprising amino acid residues corresponding to positions 29-118 of SEQ ID NO: 60 or SEQ ID NO: 61. Additionally or alternatively, in some embodiments, the antibodies of the present technology lack α-1,6-fucose modifications. In one aspect, the disclosure provides a bispecific antibody or antigen-binding fragment comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from any one of SEQ ID NOs: 32-43, or 48-59. In certain embodiments, the bispecific antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 32-43, or 48-59.

[0008] In one aspect, the disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain, the first polypeptide chain comprising, from N-terminal to C-terminal, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (iii) a light chain variable domain of the first immunoglobulin; (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4; (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (vii) a light chain variable domain of the second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembly disassembly sequence. and a SEQ ID NO:16 (SEQ ID NO:16) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0009] In another aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain, the first polypeptide chain comprising, from N-terminal to C-terminal, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (iii) a heavy chain variable domain of the first immunoglobulin; (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4; (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; and (vi) an amino acid sequence comprising the heavy chain variable domain of a second immunoglobulin. (vii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (vii) a light chain variable domain of a second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembly degradation (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12; and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0010] In certain embodiments of the bispecific antigen-binding fragments disclosed herein, the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain. In some embodiments, the SADA polypeptide comprises the tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, and CBFA2T1. Additionally or alternatively, in some embodiments, the bispecific antigen-binding fragment comprises an amino acid sequence selected from SEQ ID NOs: 32-43, or 48-59.

[0011] In one aspect, the disclosure provides a bispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other, and (a) each of the first and fourth polypeptide chains comprises, from N-terminal to C-terminal, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) light and heavy chain variable domains of a second immunoglobulin capable of specifically binding to a second epitope and comprising the amino acid sequence (GGGGS)6. and (b) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a heavy chain variable domain of a second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, linked together via a linker to form a single-chain variable fragment, wherein each of the second and third polypeptide chains comprises, from N-terminal to C-terminal, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; and (ii) a heavy chain constant domain of the first immunoglobulin; wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12; and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In certain embodiments, the second immunoglobulin binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD19, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.

[0012] In one aspect, the present disclosure provides a recombinant nucleic acid sequence encoding any of the antibodies or antigen-binding fragments described herein. In some embodiments, the recombinant nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 21, 23, 25, and 27. In another aspect, the present disclosure provides a host cell or vector comprising any of the recombinant nucleic acid sequences disclosed herein. In one aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment of the present technology and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to a substance selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

[0013] In some embodiments of the bispecific antibodies or antigen-binding fragments of the present technology, the bispecific antibodies bind to T cells, B cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in some embodiments, the bispecific antibodies or antigen-binding fragments bind to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD19, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.DOTA-Bn DOTA-Bn DOTA-Phe-Lys(HSG)- D-Tyr-Lys(HSG)-NH2、Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-N H2、DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2;DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、DOTA-D-Tyr-D-Lys(HSG-)-L-D- ys(HSG)-NH2、DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2、Ac-D-D-PheDO-D -Tyr-D-Lys(DOTA)-NH2、Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2、Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DPA-LY)-AcH2 (HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2、DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2、(Phe-D-D-Cys) -Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2、Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、(Tscg-CysD)-D-ys-Glu D-Glu-D-Lys(HSG)-NH2、Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2、Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)AH2、 c-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2、およびAc-D- The formation of Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH

[0014] In another aspect, the present disclosure provides a method of treating a CD19-associated cancer or a CD19-associated autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of any one of the antibodies or antigen-binding fragments disclosed herein. In certain embodiments, the antibody comprises an HC amino acid sequence and an LC amino acid sequence, respectively, selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:20 (chFMC63xCD3 BsAb); SEQ ID NO:26 and SEQ ID NO:24 (BC250- hFMC63 VL-2 / VH-1bxCD3 BsAb); SEQ ID NO:29 and SEQ ID NO:28 (mFMC63xmC825 BsAb); SEQ ID NO:31 and SEQ ID NO:30 (mFMC63xhC825 BsAb); SEQ ID NO:45 and SEQ ID NO:44 (hFMC63 VL-2VH-1bxmC825); and SEQ ID NO:47 and SEQ ID NO:46 (hFMC63 VL-2VH-1bxhC825), and the antibody specifically binds to CD19. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs:32-43, or 48-59.

[0015] Examples of CD19-associated cancers include acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, bilineage leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B-lymphoblastic acute leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, or marginal zone B-cell non-Hodgkin's lymphoma.

[0016] Examples of CD19-associated autoimmune diseases include multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus, paraneoplastic syndromes, pemphigus vulgaris, type 2 diabetes, or graft-versus-host disease. Additionally or alternatively, in some embodiments of the method, the antibody or antigen-binding fragment is administered to a subject separately, sequentially, or simultaneously with an additional therapeutic agent. Examples of additional therapeutic agents for treating cancer include one or more of alkylating agents, platinum agents, taxanes, vinca agents, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, and bisphosphonate therapy agents. Examples of additional therapeutic agents for treating autoimmune diseases include one or more of nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs (DMARDs), anti-TNF biologics, abatacept, tocilizumab, anakinra, and rituximab.

[0017] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of an antibody or antigen-binding fragment of the present technology, wherein the antibody or antigen-binding fragment is configured to localize to a tumor expressing CD19 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody or antigen-binding fragment that is higher than a reference value. In some embodiments, the subject has been diagnosed with or is suspected of having cancer. The level of radioactivity emitted by the antibody or antigen-binding fragment can be detected using positron emission tomography or single-photon emission computed tomography.

[0018] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising the antibody or antigen-binding fragment of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle-emitting isotopes include: 86 Y, 90 Y,89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu and 67 In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., by an N297A mutation in the Fc region resulting in aglycosylation).

[0019] Also disclosed herein is a kit for detecting and / or treating CD19-associated cancer or CD19-associated autoimmune disease, comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein), or a functional variant thereof (e.g., a substitution variant), and instructions for use. In certain embodiments, the immunoglobulin-related composition is coupled to one or more detectable labels. In one embodiment, the one or more detectable labels comprise a radioactive label, a fluorescent label, or a chromogenic label. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the anti-CD19 immunoglobulin-related compositions described herein. In some embodiments, the secondary antibody is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label. In another aspect, the present disclosure provides a method for selecting a subject for pretargeted radioimmunotherapy, the method comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that binds to the radiolabeled DOTA hapten and the CD19 antigen, wherein the conjugate is configured to localize to a tumor that expresses the CD19 antigen recognized by the bispecific antibody or antigen-binding fragment of the conjugate; (b) detecting the level of radioactivity emitted by the conjugate; and (c) selecting the subject for pretargeted radioimmunotherapy if the level of radioactivity emitted by the conjugate is higher than a reference value.

[0020] In one aspect, the present disclosure provides a method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with a CD19-associated cancer, the method comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that recognizes and binds to the radiolabeled DOTA hapten and the CD19 antigen target, wherein the conjugate is configured to localize to tumors that express the CD19 antigen target recognized by the bispecific antibody or antigen-binding fragment of the conjugate. In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that recognizes and binds to the radiolabeled DOTA hapten and a CD19 antigen target, wherein the conjugate is configured to localize to tumors that express the CD19 antigen target recognized by the bispecific antibody or antigen-binding fragment of the conjugate.

[0021] In any of the above embodiments of the methods disclosed herein, the conjugate is administered intravenously, intramuscularly, intraarterially, intrathecally, intravesically, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In some embodiments of the methods disclosed herein, the subject is a human. Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten is 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, 255 Fm, 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu,67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th or 64 It contains Cu and may contain an alpha particle radioisotope, a beta particle radioisotope or an Auger emitter.

[0022] In one aspect, the present disclosure provides a method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with a CD19-associated cancer, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody or antigen-binding fragment of the present technology, wherein the anti-DOTA bispecific antibody or antigen-binding fragment is configured to localize to tumors expressing the CD19 antigen target, and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody or antigen-binding fragment. In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody or antigen-binding fragment of the present technology, wherein the anti-DOTA bispecific antibody or antigen-binding fragment is configured to localize to tumors expressing the CD19 antigen target, and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody or antigen-binding fragment. In some embodiments, the methods of the present technology further comprise administering to the subject an effective amount of a clarifying agent prior to administration of the radiolabeled DOTA hapten.

[0023] Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten is 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, 255 Fm, 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th or 64 Cu and may include an alpha particle-emitting isotope, a beta particle-emitting isotope or an Auger emitter. In any of the above embodiments of the methods disclosed herein, the subject is a human. [Brief explanation of the drawings]

[0024] [Figure 1A] Figure 1A is a schematic diagram showing the structure of the modular IgG-scFv CD19-BsAb. CH1 to CH3 are the constant domains of the heavy chain of the first antibody. CL is the constant domain of the light chain of the first antibody. The C-terminus of CL is fused to a single-chain Fv fragment (scFv) derived from the second antibody. [Figure 1B]Figure 1B is a chromatogram generated using size-exclusion high-performance liquid chromatography (SEC-HPLC) demonstrating the purity of the CD19-BsAb, BC250, of the present technology. The CD19-BsAb was passed through a size-exclusion column, and proteins in the eluate were detected based on absorbance of ultraviolet light at a wavelength of 280 nm. Fractions were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which showed that the CD19-BsAb eluted in peak 3 at 15.656 minutes on the chromatogram. [Figure 2] Figure 2 shows flow cytometry profiles demonstrating binding of CD19-BsAb BC250 to the CD19(+) acute lymphoblastic leukemia (ALL) cell line NALM6. The indicated amounts of CD19-BsAb BC250 or control BsAb were used for immunostaining. Antibody binding to NALM6 cells was detected using flow cytometry. While increasing concentrations of control BsAb did not lead to an increase in fluorescence, increasing concentrations of CD19-BsAb BC250 led to a proportional increase in fluorescence, demonstrating binding of CD19-BsAb BC250 to NALM6 cells. [Figure 3] Figure 3 shows that CD19-BsAb BC250 mediates antigen-specific T cell cytotoxicity in acute lymphoblastic leukemia (ALL). To assess whether CD19-BsAb can redirect T cells to kill ALL cells, NALM6 ALL cells were loaded with 51Cr. The indicated concentrations of CD19-BsAb BC250 or control BsAb were incubated with 51Cr-loaded NALM6 cells along with T cells. A standard 4-hour 51Cr release assay was performed, and the amount of released 51Cr was plotted as a function of BsAb concentration. Incubation with the control BsAb released low baseline levels of 51Cr (see inverted triangles). Incubation with the BC250 clone showed increased 51Cr release compared to the control BsAb, demonstrating cancer cell killing (compare circles with inverted triangles). Based on these data, the EC50 of CD19-BsAb BC250 for cytolysis of NALM6 cells was calculated to be 42 fM. [Figure 4] Figure 4 shows the change in binding affinity of the CD19 antibody of the present technology by altering the amino acids in the antibody's VL and / or VH framework regions without manipulating the amino acid sequence of the complementarity-determining regions (CDRs). Eight humanized VH sequences and three humanized VL sequences based on the murine FMC63 antibody were developed (see Figures 12-13 below). The humanized VH and VL sequences were paired with each other to generate 24 different humanized versions of the murine FMC63 antibody. To assess their affinity for CD19, a single dose of each antibody was used to stain NALM6 ALL cells (CD19 positive). After staining, the cells were washed five times. After each wash, an aliquot of cells was removed and incubated with a fluorochrome-conjugated anti-human secondary antibody. Binding of the CD19 antibody to NALM6 cells was detected using flow cytometry. As shown in Figure 4, the binding of some antibodies withstood repeated washing, demonstrating their high affinity for their target. These humanized clones exhibited a spectrum of affinity ranging from low to high. Because the humanized VH and VL sequences had identical CDR sequences to the FMC63 parent, these data demonstrate that the affinity of a humanized CD19 antibody can be tuned by altering the sequence of the antibody framework regions while retaining the CDR sequences of the parent antibody. [Figure 5A] Figure 5A shows that the potency of anti-CD19 BsAbs correlates with their binding affinity to CD19. To assess the effect of antibody affinity on cytotoxic potency, the four indicated humanized BsAb clones were selected from 24 humanized clones based on their affinity for CD19 (as determined by flow cytometry). T cell cytotoxicity against CD19(+) NALM6 ALL cells in the presence of different doses of the four BsAbs was measured by a standard 4-hour 51Cr release assay. EC50 values ​​were calculated and plotted as a function of the mean fluorescence intensity (MFI) of NALM6 ALL cell staining, an indicator of BsAb affinity. Clones with higher affinity for CD19 showed greater potency (lower EC50) in killing ALL cells. [Figure 5B] FIG. 5B shows a graph of the cytotoxicity of various anti-CD19 BsAbs plotted as a function of BsAb concentration. [Figure 5C] Figure 5C shows a comparison of the EC50 of various anti-CD19 BsAbs relative to their corresponding affinities to CD 19. The potency of anti-CD19 BsAbs correlates with their affinity to CD19, with BC250 (VL-2, VH-1b) exhibiting the highest cytotoxic potency. [Figures 6A-6C] Figures 6A-6C show the in vivo efficacy of CD19-BsAb (BC250) against ALL NALM6 cells in a xenograft mouse model. For in vivo studies, one million NALM6-luciferase-expressing ALL cells were intravenously injected into NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice on day 0. After 3 days, mice were imaged (bioluminescence imaging, BLI) and divided into seven treatment groups: (1) activated T cells alone, (2) activated T cells plus 100 ng BC119 (a GD2 × CD3 control BsAb that does not bind to NALM6 cells), (3) activated T cells plus BC250 (0.01 ng), (4) activated T cells plus BC250 (0.1 ng), (5) activated T cells plus BC250 (1 ng), (6) activated T cells plus BC250 (10 ng), and (7) activated T cells plus BC250 (100 ng). Treatment began on day 3 once leukemia was established. Mice received weekly injections of 10 million activated T cells for 3 weeks. BsAb was administered retroorbitally twice weekly: one injection mixed with activated T cells and the other BsAb injection alone. After the final dose of activated T cells, antibody treatment was continued for four more doses and then stopped. To support in vivo T cell survival, 1000 IU IL2 was administered subcutaneously twice a week. Figure 6A shows leukemia progression monitored by BLI. Figure 6B shows a plot of total luminous flux from mice as a function of days after injection of NALM6-luciferase ALL cells. Figure 6C shows the percentage of mice surviving as a function of time after injection of NALM6-luciferase ALL cells. [Figure 7A-7C] Figures 7A-7C show the in vivo efficacy of CD19-BsAb (BC250) against human Burkitt's lymphoma cells in a xenograft mouse model. One million Daudi cells were intravenously injected into NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice on day 0. Three days later, mice were imaged (bioluminescence imaging, BLI) and divided into three treatment groups: (1) no treatment (tumor only), (2) activated T cells (T cells) plus 100 ng BC119 (a GD2 x CD3 control BsAb that does not bind to Daudi cells), and (3) activated T cells plus BC250 (100 ng). Once lymphoma was established, treatment began on day 14. Mice received weekly injections of 20 million activated T cells for three weeks. BsAb was administered retroorbitally twice weekly: one dose mixed with activated T cells, and the other BsAb dose was injected alone. After the final dose of activated T cells, antibody treatment was continued two more times and then stopped. To support in vivo T cell survival, 1000 IU IL2 was administered subcutaneously twice weekly. Figure 7A shows lymphoma progression monitored by BLI. Figure 7B shows a plot of total flux from mice as a function of days after injection of Daudi cells. Figure 7C shows photographs of kidneys from representative mice from different treatment groups (top panel) and corresponding flow cytometry images of kidney homogenates stained with anti-CD19 and anti-human CD45 antibodies (bottom panel). The clusters of cells along the diagonal line are Daudi cells. [Figures 8A-8D]Figures 8A-8D show the in vivo efficacy of CD19-BsAb (BC250) against human chronic myeloid leukemia blast crisis (CML) BV173 cells in a xenograft mouse model. One million BV173-luciferase-expressing cells were intravenously injected into NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice on day 0. Three days later, mice were imaged (bioluminescence imaging, BLI) and divided into three treatment groups: (1) no treatment (tumor only), (2) activated T cells (T cells) plus 100 ng BC119 (a GD2 × CD3 control BsAb that does not bind to BV173 cells), and (3) activated T cells plus BC250 (100 ng). Treatment began on day 14 once leukemia was established. Mice received a single injection of 8.8 million activated T cells. BsAb was administered retroorbitally twice weekly: one dose mixed with activated T cells, and the other BsAb was injected alone. After a single T cell dose, antibody treatment was continued for five additional doses and then stopped. To support in vivo T cell survival, 1000 IU IL2 was administered subcutaneously twice weekly. Leukemia progression was monitored by BLI. Figure 8A shows leukemia progression monitored by BLI. Figure 8B shows the percentage of mice surviving as a function of time after injection of BV173-luciferase cells. Figure 8C shows a plot of total luminous flux from mice as a function of days after injection of BV173 cells. Figure 8D shows photographs of representative mouse livers from different treatment groups (upper panels) and corresponding flow cytometry images of liver homogenates stained with anti-CD19 and anti-human CD45 antibodies (lower panels). The livers of mice from the group that did not receive BC250 had multiple visible metastases, whereas mice treated with ATC / BC250 showed no liver metastases. [Figure 9A-9B]Figures 9A-9B show the efficacy of CD19-BsAb (BC250) against human Burkitt lymphoma cells in a xenograft mouse model. One million Raji-luciferase Burkitt lymphoma cells were injected intravenously into NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice on day 0. Three days later, mice were imaged (bioluminescence imaging, BLI) and divided into three treatment groups: (1) no treatment (tumor only), (2) activated T cells (T cells) plus 100 ng BC119 (GD2 × CD3 control BsAb), and (3) activated T cells plus BC250 (100 ng). Once lymphoma was established, treatment began on day 3. Over a three-week period, mice received three injections of an average of 23 million activated T cells on days 3, 7, and 10. BsAb was mixed with activated T cells and administered retroorbitally. Two additional doses of BC250 were administered on days 14 and 20. 1000 IU IL2 was administered subcutaneously twice weekly to support in vivo T cell survival. Figure 9A shows lymphoma progression monitored by BLI. Figure 9B shows a plot of total luminous flux from mice as a function of days after injection of Raji cells. [Figures 10A-10B]Figures 10A-10B show a comparison of the cytotoxic properties of the present technology's CD19-BsAb (BC250) and the bispecific T-cell engager (BiTE) blinatumomab (the only FDA-approved BsAb for leukemia). Figure 10A shows the in vitro cytotoxicity of increasing doses of CD19-BsAb (BC250) and blinatumomab. Figure 10B shows the in vivo efficacy of CD19-BsAb (BC250) and blinatumomab against NALM6 cells in a xenograft mouse model. NSG mice were intravenously injected with 1 million NALM6-luciferase-expressing ALL cells on day 0. After 3 days, mice were imaged (bioluminescence imaging, BLI) and divided into seven treatment groups: (1) T cells alone, (2) T cells plus 5 femtomolar BC250, (3) T cells plus 50 femtomolar BC250, (4) T cells plus 500 femtomolar BC250, (5) T cells plus 10 femtomolar bulinatumomab, (6) T cells plus 100 femtomolar bulinatumomab, and (7) T cells plus 1000 femtomolar bulinatumomab. Treatment began on day 4 once leukemia was established. Over a 3-week period, mice received three injections of 10 million activated T cells on days 4, 11, and 18. BsAb was administered 5 days per week. After the final dose of activated T cells, the antibody was administered eight more times and then stopped. To support T cell survival in vivo, 1000 IU IL2 was administered subcutaneously twice a week. Leukemia progression was monitored by BLI. [Figures 11A-11F]Figures 11A-11F show a comparison of tumor burden and survival in NALM6 xenograft mice treated with CD19-BsAb (BC250) vs. blinatumomab. Figure 11A shows a plot of total luminous flux as a function of time in mice treated with ATC alone or in combination with 10 femtomolar bulinatumomab or 5 femtomolar BC250. Figure 11B shows percent survival in mice treated with ATC alone or in combination with 10 femtomolar bulinatumomab or 5 femtomolar BC250. Figure 11C shows a plot of total luminous flux as a function of time in mice treated with ATC alone or in combination with 100 femtomolar bulinatumomab or 50 femtomolar BC250. Administration of 50 femtomoles / dose of BC250 reduced leukemia burden by 11-fold and 68-fold at 11 and 17 days, respectively, compared with 100 femtomoles / dose of blinatumomab. Figure 11D shows the percent survival of mice treated with ATC alone or in combination with 100 femtomoles blinatumomab or 50 femtomoles BC250. Figure 11E shows a plot of total luminous flux as a function of time in mice treated with ATC alone or in combination with 1000 femtomoles blinatumomab or 500 femtomoles BC250. Figure 11F shows the percent survival of mice treated with ATC alone or in combination with 1000 femtomoles blinatumomab or 500 femtomoles BC250. The p-values ​​shown in Figures 11A, 11C, and 11E are based on the statistical difference between ATC / blinatumomab vs. ATC / BC250 at days 11 and 17. The p-values ​​shown in Figures 11B, 11D, and 11F are based on the statistical difference between ATC / blinatumomab vs. ATC / BC250. [Figures 11G-11I]Figures 11G-11I show a comparison of the in vivo efficacy of BC250 and blinatumomab. The experiment described in Figure 10B was repeated with 10 mice per group. Activated T cells were injected into leukemia mice (NALM6 xenograft mice) along with the bispecific antibody. Human interleukin-2 (IL2) was injected subcutaneously to support T cell engraftment. The treatment protocol is shown in Figure 11H. These results demonstrate the superior efficacy of BC250 compared to blinatumomab. [Figures 11J-11K] Figures 11J-11K show that BC250 was superior to blinatumomab for treating ALL xenografts in vivo. To compare the efficacy of BC250 and Blincyto in in vivo T cell arming experiments, NSG mice transplanted with NALM6 human ALL cells were treated with activated T cells. Activated T cells were obtained by incubating T cells with BsAb for 20 minutes and then washing them to remove unbound antibody. These results demonstrate that BC250-armed T cells reduced leukemia growth and improved mouse survival more potently than blinatumomab. [Figure 12] Figure 12 shows the amino acid sequences of the murine and humanized FMC63 heavy chain variable domains (SEQ ID NOS: 1 and 5-12). CDR sequences are shown in underlined bold font. The amino acid sequences of the VHCDR1, VHCDR2, and VHCDR3 regions of the murine FMC63 VH domain are GVSLPDYG (SEQ ID NOS: 2), IWGSETT (SEQ ID NOS: 3), and AKHYYYGGSYAMDY (SEQ ID NOS: 4), respectively. VH-1b (SEQ ID NOS: 5), VH-2b (SEQ ID NOS: 6), VH-3 (SEQ ID NOS: 7), VH-4 (SEQ ID NOS: 8), VH-5b (SEQ ID NOS: 9), VH-6b (SEQ ID NOS: 10), VH-7b (SEQ ID NOS: 11), and VH-8b (SEQ ID NOS: 12) are humanized versions of the murine FMC63 VH domain. [Figure 13]Figure 13 shows the amino acid sequences of the murine and humanized FMC63 light chain variable domains (SEQ ID NOS: 13 and 17-19). CDR sequences are shown in underlined bold font. The amino acid sequences of the VLCDR1, VLCDR2, and VLCDR3 regions of the murine FMC63 VL domain are QDISKY (SEQ ID NOS: 14), HTS (SEQ ID NOS: 15), and QQGNTLPYT (SEQ ID NOS: 16), respectively. VL-1 (SEQ ID NOS: 17), VL-2 (SEQ ID NOS: 18), and VL-3 (SEQ ID NOS: 19) are humanized versions of the murine FMC63 VL domain. [Figures 14A-14B] Figures 14A and 14B show the amino acid and nucleotide sequences of the light chain of chimeric BsAb (chFMC63), represented as SEQ ID NOs: 20 and 21, respectively. Figures 14C and 14D show the amino acid and nucleotide sequences of the heavy chain of chimeric BsAb (chFMC63), represented as SEQ ID NOs: 22 and 23, respectively. The signal peptide is underlined, the variable domains of the chimeric anti-CD19 antibody are shown in italics, and the linker sequence is bold and underlined. [Figures 15A-15B] Figures 15A-15B show the amino acid and nucleotide sequences of the light chain of BsAb BC250 (hFMC63 L2HC1b), represented as SEQ ID NOs: 24 and 25, respectively. Figures 15C-15D show the amino acid and nucleotide sequences of the heavy chain of BsAb BC250 (hFMC63 L2HC1b), represented as SEQ ID NOs: 26 and 27, respectively. The signal peptide is underlined, the variable domains of anti-CD19 BsAb BC250 (hFMC63 L2HC1b) are shown in italics, and the linker sequence is bold and underlined. [Figures 16A-16B] 16A-16B show the amino acid sequences of the light and heavy chains of the murine FMC63×murine C825 (anti-DOTA) BsAb, respectively, represented as SEQ ID NOs: 28 and 29. The signal peptide is underlined, the variable domains of the murine FMC63×murine C825 (anti-DOTA) BsAb are shown in italics, and the linker sequence is bold and underlined. [Figures 17A-17B] 17A-17B show the amino acid sequences of the light and heavy chains of murine FMC63×humanized C825 (anti-DOTA) BsAb, respectively, represented as SEQ ID NOs: 30 and 31. The signal peptide is underlined, the variable domains of murine FMC63×humanized C825 (anti-DOTA) BsAb are shown in italics, and the linker sequence is bold and underlined. [Figures 18A-18B] 18A-18B depict the amino acid sequences of two murine FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 32 and 33). The signal peptide is underlined, the variable domains of the murine FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p53 tetramerization domain is shown in bold. [Figures 19A-19B] 19A-19B depict the amino acid sequences of two murine FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 34 and 35). The signal peptide is underlined, the variable domains of the murine FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p63 tetramerization domain is shown in bold. [Figures 20A-20B] 20A-20B depict the amino acid sequences of two murine FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 36 and 37). The signal peptide is underlined, the variable domains of the murine FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p73 tetramerization domain is shown in bold. [Figures 21A-21B]21A-21B depict the amino acid sequences of two murine FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 38 and 39). The signal peptide is underlined, the variable domains of the murine FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p53 tetramerization domain is shown in bold. [Figures 22A-22B] 22A-22B depict the amino acid sequences of two murine FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 40 and 41). The signal peptide is underlined, the variable domains of the murine FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p63 tetramerization domain is shown in bold. [Figures 23A-23B] 23A-23B depict the amino acid sequences of two murine FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 42 and 43). The signal peptide is underlined, the variable domains of the murine FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p73 tetramerization domain is shown in bold. [Figures 24A-24B] 24A-24B show the amino acid sequences of the light and heavy chains of the humanized FMC63×mouse C825 (anti-DOTA) IgG-scFv BsAb, respectively, represented as SEQ ID NOs: 44 and 45. The signal peptide is underlined, the variable domains of the humanized FMC63×mouse C825 (anti-DOTA) BsAb are shown in italics, and the linker sequence is bold and underlined. [Figures 25A-25B]25A-25B show the amino acid sequences of the light and heavy chains of the humanized FMC63 x humanized C825 (anti-DOTA) IgG-scFv BsAb, respectively, represented as SEQ ID NOs: 46 and 47. The signal peptide is underlined, the variable domains of the humanized FMC63 x humanized C825 (anti-DOTA) BsAb are shown in italics, and the linker sequence is bold and underlined. [Figures 26A-26B] 26A-26B depict the amino acid sequences of two humanized FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 48 and 49). The signal peptide is underlined, the variable domains of the humanized FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p53 tetramerization domain is shown in bold. [Figures 27A-27B] 27A-27B depict the amino acid sequences of two humanized FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 50 and 51). The signal peptide is underlined, the variable domains of the humanized FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p63 tetramerization domain is shown in bold. [Figures 28A-28B] 28A-28B depict the amino acid sequences of two humanized FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 52 and 53). The signal peptide is underlined, the variable domains of the humanized FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p73 tetramerization domain is shown in bold. [Figures 29A-29B]29A-29B depict the amino acid sequences of two humanized FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 54 and 55). The signal peptide is underlined, the variable domains of the humanized FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p53 tetramerization domain is shown in bold. [Figure 30A-30B] 30A-30B depict the amino acid sequences of two humanized FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 56 and 57). The signal peptide is underlined, the variable domains of the humanized FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p63 tetramerization domain is shown in bold. [Figure 31A-31B] 31A-31B depict the amino acid sequences of two humanized FMC63 x humanized C825 (anti-DOTA) single-chain bispecific tandem fragment variable (scBsTaFv) immunoglobulin-related compositions of the present technology (SEQ ID NOs: 58 and 59). The signal peptide is underlined, the variable domains of the humanized FMC63 x humanized C825 (anti-DOTA) scBsTaFvs are shown in italics, the linker or spacer sequence is shown in bold underlined font, and the p73 tetramerization domain is shown in bold. [Figure 32] FIG. 32 shows stability data, EC50 data, and MFI data for each CD19-CD3 IgG-scFv bispecific antibody clone against several cell lines. [Figure 33A-33B]Figure 33A shows the pharmacokinetic profile of BC250 in individual animals (n=5). Figure 33B shows the mean pharmacokinetic activity of BC250 for the animals shown in Figure 33A. The pharmacokinetics of BC250 were determined by injecting mice with antibody and bleeding the mice starting 4 hours post-injection and continuing for up to 96 hours. Blood BC250 levels were determined using an ELISA assay. [Figures 34A-34C] Figures 34A-34C show a comparison of two bispecific antibodies with N297A / K322A (BC250) and L234A / L235A (BC258) substitutions in T cell-mediated cytotoxicity assays against the CD19(+) leukemia cell lines Daudi, NALM6, and Raji, respectively. BC258 and BC250 have identical VL and VH sequences (i.e., VL-2, VH-1b) but different Fc domain sequences. BC258 has L234A / L235A (LALA) mutations in its Fc domain, while BC250 has N297A / K322A mutations in its Fc domain. Without wishing to be bound by theory, it is believed that silencing the Fc of bispecific antibodies may prevent unwanted killing of Fc receptor-bearing immune cells or T cells by complement activation. BC250 is more potent at lysing leukemia cells than BC258 in at least two of the three CD19(+) leukemia cell lines tested. [Figures 35A-35C] Figures 35A-35C show a comparison of two bispecific antibodies with N297A / K322A (BC250) and L234A / L235A (BC258) substitutions in a leukemia NALM6 xenograft mouse model. Leukemia mice were treated with activated T cells (ATC) mixed with different doses of BC250 or BC258, and tumor growth was measured by bioluminescence imaging. Both bispecific antibodies showed comparable efficacy in vivo. DETAILED DESCRIPTION OF THE INVENTION

[0025] It will be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in varying levels of detail in order to provide a substantial understanding of the present technology.

[0026] The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) capable of specifically binding to a CD19 polypeptide. The immunoglobulin-related compositions of the present technology are useful in methods for detecting or treating CD19-associated cancer or CD19-associated autoimmune disease in subjects in need thereof. Accordingly, various aspects of the present methods relate to the preparation, characterization, and manipulation of anti-CD19 antibodies. The immunoglobulin-related compositions of the present technology are useful alone or in combination with additional therapeutic agents for treating cancer. In some embodiments, the immunoglobulin-related composition is a humanized antibody, a chimeric antibody, or a bispecific antibody.

[0027] The practice of the present method employs many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; US Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring levels of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0028] definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, a reference to a "cell" includes a combination of two or more cells, etc. In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry, and hybridization described below, are well known and commonly used in the art.

[0029] As used herein, the term "about" in connection with a number is generally interpreted as including numbers within 1%, 5% or 10% in either direction (more or less) of the number, unless otherwise stated or clear from the context (except where such number is less than 0% or more than 100% of the possible value). As used herein, "administration" of an agent or drug to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration can be performed by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally, or topically. Administration includes self-administration and administration by another. "Adjuvant" refers to one or more substances that stimulate the immune system. In this context, adjuvants are used to enhance the immune response to one or more vaccine antigens or antibodies. Adjuvants can be administered to a subject before, in combination with, or after administration of a vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponin), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, and glycoproteins), hormones, cytokines, and costimulatory factors.

[0030] As used herein, the term "antibody" collectively refers to immunoglobulin or immunoglobulin-like molecules, including, by way of example and not limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response in any vertebrate, e.g., mammals such as humans, goats, rabbits, and mice, as well as in non-mammalian species, such as shark immunoglobulins. As used herein, "antibody" (including intact immunoglobulins) and "antigen-binding fragments" specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to substantially exclude binding to other molecules (e.g., with a binding constant at least 10 times higher than the binding constant for other molecules in a biological sample). 3 M -1 Greater than, at least 10 4 M -1 Greater than or at least 10 5 M -1 (Antibodies and antibody fragments having a larger binding constant for a molecule of interest). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), etc. Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd See also Ed., W.H. Freeman & Co., New York, 1997.

[0031] More specifically, an antibody refers to a polypeptide ligand that contains at least a light chain immunoglobulin variable region or a heavy chain immunoglobulin variable region and specifically recognizes and binds to an epitope of an antigen. An antibody is composed of a heavy chain and a light chain, each of which contains a heavy chain variable region (V H ) region and the light chain variable (V L ) region. H Area and V LThese regions are involved in binding to the antigen recognized by the antibody. Immunoglobulins typically have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind to antigens. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, incorporated herein by reference). The Kabat database is currently maintained online. The sequences of the framework regions of various light or heavy chains are relatively conserved within a species. The framework regions of antibodies, which are the combined framework regions of the constituent light and heavy chains, primarily adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions act to form a scaffold that positions the CDRs in the correct orientation through interchain, non-covalent interactions.

[0032] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are usually identified by the chain in which the individual CDR is located. Thus, V H CDR3 is located in the variable domain of the antibody heavy chain in which it is found, V LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to the CD19 protein have a specific V H Area and V L Each CDR has a specific CDR sequence and therefore a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although the CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity-determining residues (SDRs). As used herein, "immunoglobulin-related compositions" refer to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) as well as antibody fragments. An antibody or its antigen-binding fragment specifically binds to an antigen.

[0033] As used herein, the term "antibody-related polypeptide" refers to an antigen-binding antibody fragment, including a single-chain antibody, which may contain a variable region alone or in combination with all or part of the following polypeptide elements: hinge region, CH1, CH2, and CH3 domains of an antibody molecule. Also encompassed by the present technology are any combinations of variable regions and hinge regions, CH1, CH2, and CH3 domains. Antibody-related molecules useful in the present method include, but are not limited to, Fab, Fab', and F(ab'), Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L or V H Examples include: (i) Fab fragments, V L , V H , C L and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H(vi) dAb fragments consisting of domains (Ward et al., Nature 341: 544-546, 1989), and (vi) isolated complementarity-determining regions (CDRs). As such, an "antibody fragment" or "antigen-binding fragment" can contain a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0034] "Bispecific antibody" or "BsAb" as used herein refers to an antibody that can simultaneously bind to two targets with distinct structures, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen. A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding portion in a bispecific antibody is a V H and / or V L In some such embodiments, the V H and / or V L The V region is one found in a particular monoclonal antibody. In some embodiments, a bispecific antibody contains two antigen-binding portions, each derived from a different monoclonal antibody. H and / or V L In some embodiments, a bispecific antibody contains two antigen-binding moieties, one of which contains a V region containing the CDRs from a first monoclonal antibody. H and / or V L The other antigen-binding portion comprises an immunoglobulin molecule having a V region containing CDRs derived from a second monoclonal antibody. H and / or V L These include antibody fragments having regions (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.).

[0035] As used herein, a "clearing agent" is a substance that binds to excess bispecific antibodies present in the subject's blood compartment to promote rapid clearance by the kidney. The use of a clearing agent prior to hapten administration (e.g., DOTA) promotes a better tumor-to-background ratio in pretargeted radioimmunotherapy (PRIT) systems. Examples of clearing agents include 500kD-dextran-DOTA-Bn(Y) (Orcutt et al., Mol Cancer Ther. 11(6): 1365-1372 (2012)), 500kD aminodextran-DOTA conjugates, antibodies against pretargeting antibodies, and the like. As used herein, the term "conjugated" refers to the association of two molecules by any method known to those skilled in the art.Suitable types of association include chemical bonds and physical bonds.Chemical bonds include, for example, covalent bonds and coordinate bonds.Physical bonds include, for example, hydrogen bonds, dipolar interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions and aromatic stacking.

[0036] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which fragments contain a light chain variable domain (V L ) connected to the heavy chain variable domain (V H )Includes(V H V L (The term "diabodies" is used interchangeably with "diabodies" and is used interchangeably with "diabodies" in the context of "antibody" or "antibody-specific antibodies"). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and generate two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0037] As used herein, the term "single chain antibody" or "single chain Fv (scFv)" refers to an Fv fragment, V L and V HA single-chain antibody molecule may comprise a polymer having several individual molecules, such as a dimer, trimer, or other polymer. v Fragment, V L and V H The two domains are encoded by separate genes, but they are L and V H The domains pair to form a monovalent molecule (single-chain F v (scF v The antibodies may be joined using recombinant methods by synthetic linkers that allow them to be produced as a single protein chain forming a single chain (known as a single chain antibody). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Such single-chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies. Any of the above antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as are intact antibodies.

[0038] As used herein, "antigen" refers to a molecule to which an antibody (or antigen-binding fragment thereof) can selectively bind. The target antigen can be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen can be a polypeptide (e.g., a CD19 polypeptide). The antigen can be administered to an animal to generate an immune response in the animal. The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that contains the portion of the polypeptide that is involved in binding to the antigen. Examples of antigen-binding fragments useful in the present technology include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2. "Binding affinity" refers to the strength of the overall non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain antibodies that tend to dissociate easily from the antigen, whereas high affinity complexes generally contain antibodies that tend to remain bound to the antigen for extended periods of time. As used herein, the term "biological sample" refers to a sample material derived from living cells.Biological samples can include tissues, cells, cell protein or membrane extracts and biological fluids (such as ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present in a subject.Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsil, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostatic fluid, semen, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph and tears. Biological samples can also be obtained from biopsies of internal organs or cancer.Biological samples can be obtained from subjects for diagnosis or research, or can be obtained from non-diseased individuals as controls or for basic research.Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy.In certain embodiments, biological samples are blood samples or samples derived from bone marrow aspiration and biopsy. As used herein, the term "CDR-grafted antibody" refers to an antibody in which at least one CDR of an "acceptor" antibody has been replaced by a CDR "graft" from a "donor" antibody with the desired antigen specificity.

[0039] As used herein, the term "chimeric antibody" refers to an antibody in which the Fc constant region of a monoclonal antibody derived from one species (e.g., a murine Fc constant region) has been replaced, using recombinant DNA technology, with the Fc constant region derived from an antibody of another species (e.g., a human Fc constant region). In general, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO86 / 01533; Cabilly et al. al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 0125,023; Better et al., Science 240: 1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al. See Proc. Natl. Acad. Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Wood et al., Nature 314: 446-449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559, 1988.

[0040] As used herein, the term "consensus FR" refers to the framework (FR) antibody region in the consensus immunoglobulin sequence. The FR region of an antibody does not contact the antigen. As used herein, "control" refers to a substitute sample used in an experiment for comparison purposes.Control can be "positive" or "negative".For example, when the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a specific type of disease, a positive control (a compound or composition known to exhibit desired therapeutic effect) and a negative control (a subject or sample that does not receive therapy or receives a placebo) are usually used.

[0041] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention or reduction of a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In therapeutic or prophylactic applications, the amount of a composition administered to a subject will vary depending on the composition, the extent, type, and severity of the disease, and individual characteristics, such as general health, age, sex, weight, and tolerance to drugs. Those skilled in the art can determine the appropriate dosage depending on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, a therapeutic composition may be administered to a subject with one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition that ameliorates or eliminates the physiological effects of the disease or condition. A therapeutically effective amount may be administered in one or more administrations.

[0042] As used herein, the term "effector cell" refers to an immune cell involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and possess specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as neutrophils capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcαR are involved in the specific killing of target cells and in presenting or binding to antigen-presenting cells to other components of the immune system.

[0043] As used herein, the term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, but the binding to the latter is not lost. In some embodiments, the "epitope" of the CD19 protein is a region of the protein to which the anti-CD19 antibody of the present technology specifically binds. In some embodiments, the epitope is a conformational epitope or a nonconformational epitope. To screen for anti-CD19 antibodies that bind to the epitope, a conventional cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), may be performed. This assay can be used to determine whether an anti-CD19 antibody binds to the same site or epitope as the anti-CD19 antibody of the present technology. Alternatively, or in addition, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutated by alanine scanning or the like to identify contact residues. In a different method, peptides corresponding to different regions of the CD19 protein can be used in a competitive assay using the test antibody, or using the test antibody and an antibody with a characterized or known epitope. As used herein, "expression" includes one or more of the following, as necessary for proper expression and function: transcription of a gene into precursor mRNA, splicing and other processing of the precursor mRNA to produce a mature mRNA, mRNA stability; translation of the mature mRNA into a protein (including codon usage and tRNA availability), and glycosylation and / or other modifications of the translation product.

[0044] As used herein, the term "gene" refers to a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns and other untranslated regions that control expression. "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage of "sequence identity" to another sequence (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), meaning that that percentage of bases (or amino acids) are identical when aligned in a comparison of the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. Notably, programs include BLASTN and BLASTP, using the following default parameters: genetic code=standard, filter=none, strand=both, cutoff=60, expect=10, matrix=BLOSUM62, description=50 sequences, sorted=HIGH SCORE, database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that share a specified percent homology and encode polypeptides with the same or similar biological activity. Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% identity or less than 25% identity with each other.

[0045] As used herein, "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which recipient hypervariable region residues are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance, such as binding affinity. Generally, a humanized antibody comprises substantially all of at least one, and usually two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv), in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are of human immunoglobulin consensus FR sequences, although the FR regions may contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FRs is usually no more than six in the heavy chain and no more than three in the light chain. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, for example, Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).

[0046] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., V LApproximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in H Approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the H1 sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from the "hypervariable loops" (e.g., V L Residues 26–32 (L1), 50–52 (L2), and 91–96 (L3) in V H Among these, 26-32 (H1), 52A-55 (H2), and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)) are included.

[0047] As used herein, the terms "identical" or percent "identity," when used in reference to two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence of an antibody described herein)) when compared and aligned for maximum correspondence over a comparison window or designated region as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (e.g., the NCBI website). Such sequences are then said to be "substantially identical." The term can also refer to or apply to the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as those that have substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0048] As used herein, the term "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or V H The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (herein abbreviated as LCVR or V L The light chain constant region consists of one domain, C L It consists of: V H and V LThe regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are separated by more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0049] As used herein, the terms "individual," "patient," or "subject" can refer to an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including, but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0050] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulation are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0051] As used herein, the term "polyclonal antibody" refers to a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. Use of this term includes at least two (2) antibody preparations containing antibodies that specifically bind to different epitopes or regions of an antigen. As used herein, the term "polynucleotide" or "nucleic acid" refers to any RNA or DNA, and may be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more usually, double-stranded or a mixture of single-stranded and double-stranded regions. Furthermore, polynucleotide refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other reasons.

[0052] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer comprising two or more amino acids joined together by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic textbooks and in more detailed laboratories, as well as in a voluminous research literature. As used herein, "PRIT" or "pretargeted radioimmunotherapy" refers to a multi-step process that overcomes the slow blood clearance of tumor-targeting antibodies, which contributes to undesirable toxicity to normal tissues such as bone marrow. In pretargeting, a radionuclide or other diagnostic or therapeutic agent is attached to a small hapten. A pretargeting bispecific antibody bearing binding sites for the hapten and the target antigen is administered first. Unbound antibody is then allowed to clear from the circulation, followed by administration of the hapten.

[0053] As used herein, the term "recombinant," for example, when used in reference to a cell or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all. As used herein, the term "separate" therapeutic use refers to the administration of at least two active ingredients simultaneously or substantially simultaneously by different routes. As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, and the administration routes are the same or different. More specifically, sequential use refers to the complete administration of one of the active ingredients before the start of the administration of another active ingredient(s). Thus, one of the active ingredients can be administered over several minutes, hours, or days, and then the other active ingredient(s) can be administered. In this case, there is no simultaneous treatment.

[0054] As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize or bind to other molecules. As used herein, the term "specific binding," "specifically binds to" or "is specific for" a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), refers to, for example, a binding affinity of about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 K for molecules that bind to M D The term "specifically binds" can also refer to binding of a molecule (e.g., an antibody or antigen-binding fragment thereof) to a particular polypeptide (e.g., a CD19 polypeptide) or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0055] As used herein, the term "concurrent" therapeutic use refers to the administration of at least two active ingredients by the same route at the same time or substantially the same time. As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, provides a desired therapeutic effect in a subject in need thereof. "Treating" or "treatment," as used herein, refers to the treatment of a disease or disorder described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., halting its development, (ii) alleviating the disease or disorder, i.e., causing regression of the disorder, (iii) slowing the progression of the disorder, and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treating means that the symptoms associated with the disease are, for example, alleviated, reduced, cured, or placed in remission.

[0056] It should also be understood that the various modes of treatment for disorders as described herein include not only complete treatment, but also less-than-complete treatment, and are intended to mean "substantial" in which some biologically or medically relevant result is achieved. Treatment can be continuous long-term treatment for chronic diseases or a single or two or three administrations for treating acute conditions. Amino acid sequence modification(s) of the anti-CD19 antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of anti-CD19 antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion from and / or insertion into and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to obtain the antibody of interest, as long as the resulting antibody possesses the desired properties. Modifications also include changes in the glycosylation pattern of the protein. Hypervariable regions are the most popular sites for substitutional mutagenesis, although FR changes are also contemplated. "Conservative substitutions" are shown in the table below.

[0057] [Table 1]

[0058] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis may be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants may be subjected to screening as described herein to select antibodies with similar or superior properties in one or more relevant assays for further development. CD19

[0059] CD19 (also known as cluster of differentiation 19, B lymphocyte surface antigen B4 (or simply B4), T cell surface antigen Leu-12, or common variable immunodeficiency syndrome-3 (CVID3)) is a central positive response regulator in B cells. Human CD19 is approximately 556 amino acids long and is encoded by the 7.41 kilobase CD19 gene located on the short arm of chromosome 16. The CD19 gene contains at least 15 exons encoding extracellular, transmembrane, and cytoplasmic domains. At least five isoforms of the CD19 protein exist in nature. The amino acid sequence of human CD19 isoform 2 precursor (NCBI Reference Sequence: NP_001761.3; SEQ ID NO: 60) is set forth below: 1 MPPPRLLFFL LFLTPMEVRP EEPLVVKVEE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP 61 FLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGSGE 121 LFRWNVSDLG GLGCGLKNRS SEGPSSPSGK LMSPKLYVWA KDRPEIWEGE PPCLPPRDSL 181 NQSLSQDLTM APGSTLWLSC GVPPDSVSRG PLSWTHVHPK GPKSLLSLEL KDDRPARDMW 241 VMETGLLLPR ATAQDAGKYY CHRGNLTMSF HLEITARPVL WHWLLRTGGW KVSAVTLAYL 301 IFCLCSLVGI LHLQRALVLR RKRKRMTDPT RRFFKVTPPP GSGPQNQYGN VLSLPTPTSG 361 LGRAQRWAAG LGGTAPSYGN PSSDVQADGA LGSRSPPGVG PEEEEGEGYE EPDSEEDSEF 421 YENDSNLGQD QLSQDGSGYE NPEDEPLGPE DEDSFSNAES YENEDEELTQ PVARTMDFLS 481 PHGSAWDPSR EATSLGSQSY EDMRGILYAA PQLRSIRGQP GPNHEEDADS YENMDNPDGP 541 DPAWGGGGRM GTWSTR

[0060] The amino acid sequence of human CD19 isoform 1 precursor (NCBI Reference Sequence: NP_001171569.1; SEQ ID NO: 61) is set forth below: 1 MPPPRLLFFL LFLTPMEVRP EEPLVVKVEE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP 61 FLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGSGE 121 LFRWNVSDLG GLGCGLKNRS SEGPSSPSGK LMSPKLYVWA KDRPEIWEGE PPCLPPRDSL 181 NQSLSQDLTM APGSTLWLSC GVPPDSVSRG PLSWTHVHPK GPKSLLSLEL KDDRPARDMW 241 VMETGLLLPR ATAQDAGKYY CHRGNLTMSF HLEITARPVL WHWLLRTGGW KVSAVTLAYL 301 IFCLCSLVGI LHLQRALVLR RKRKRMTDPT RRFFKVTPPP GSGPQNQYGN VLSLPTPTSG 361 LGRAQRWAAG LGGTAPSYGN PSSDVQADGA LGSRSPPGVG PEEEEGEGYE EPDSEEDSEF 421 YENDSNLGQD QLSQDGSGYE NPEDEPLGPE DEDSFSNAES YENEDEELTQ PVARTMDFLS 481 PHGSAWDPSR EATSLAGSQS YEDMRGILYA APQLRSIRGQ PGPNHEEDAD SYENMDNPDG 541 PDPAWGGGGR MGTWSTR

[0061] Three additional shorter isoforms of the CD19 protein are the 468 amino acid long CD19 isoform X1 (NCBI Reference Sequence: XP_006721166.1; SEQ ID NO: 62), the 467 amino acid long CD19 isoform X2 (NCBI Reference Sequence: XP_016879382.1; SEQ ID NO: 63), and the 282 amino acid long CD19 isoform X3 (NCBI Reference Sequence: XP_011544283.1; SEQ ID NO: 64). CD19 is a 95 kDa type I transmembrane glycoprotein of the immunoglobulin superfamily (IgSF) with two extracellular C2-type Ig-like domains and a large, highly conserved cytoplasmic tail. The cytoplasmic domain is 240 amino acids long and contains nine conserved tyrosine residues. Upon tyrosine phosphorylation, CD19 functions as a specialized adaptor protein involved in intrinsic and antigen receptor-induced signal transduction.

[0062] CD19 is a crucial coreceptor for B cell antigen receptor (BCR) signaling. Through modulation of BCR signaling, CD19 regulates B lymphocyte activation and differentiation, controlling antigen-dependent B cell development and immunoglobulin-induced B lymphocyte activation to optimize immune responses.

[0063] CD19 is expressed at all stages of B cell development through terminal differentiation and on follicular dendritic cells. CD19 surface expression coincides with B cell lineage commitment from hematopoietic stem cells during immunoglobulin (Ig) gene rearrangement. CD19 is then expressed on early pro-B cells, late pro-B cells, memory B cells, plasmablasts, and some plasma cells. CD19 expression is observed in B cell leukemias, lymphomas, and other cancers. CD19 is expressed in >90% of ALL, B-NHL, and CLL. CD19 has a broader expression profile than CD20 and is retained even after CD20 downregulation or loss. Overexpression of CD19 in mice can lead to autoimmune disease. Immunoglobulin-related compositions of the present technology

[0064] The anti-CD19 immunoglobulin-related compositions of the present disclosure may be useful in the diagnosis or treatment of CD19-associated cancers and CD19-associated autoimmune diseases. Anti-CD19 immunoglobulin-related compositions within the scope of the present technology include, for example, but are not limited to, monoclonal, chimeric, humanized, bispecific antibodies and diabodies that specifically bind to target polypeptides, as well as homologs, derivatives, or fragments thereof. The present disclosure also provides antigen-binding fragments of any of the anti-CD19 antibodies disclosed herein, including Fab, F(ab)'2, Fab', scF v and F v The present technology discloses an anti-CD19 bispecific antibody format that addresses the existing problems of poor tumor antigen binding avidity, short in vivo half-life, and toxicity. In one aspect, the present technology provides chimeric and humanized variants of FMC63, including multispecific immunoglobulin-related compositions (e.g., bispecific antibody agents).

[0065] In one aspect, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L (a) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12, and / or (b) V L provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In some embodiments, the antibody further comprises an Fc domain of any isotype, such as, but not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM and IgY. Non-limiting examples of constant region sequences include the following:

[0066] Human IgD constant region, Uniprot:P01880 (SEQ ID NO: 65) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK Human IgG1 constant region, Uniprot:P01857 (SEQ ID NO: 66) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 constant region, Uniprot:P01859 (SEQ ID NO: 67) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0067] Human IgG3 constant region, Uniprot:P01860 (SEQ ID NO: 68) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0068] Human IgM constant region, Uniprot:P01871 (SEQ ID NO: 69) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0069] Human IgG4 constant region, Uniprot:P01861 (SEQ ID NO:70) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Human IgA1 constant region, Uniprot:P01876 (SEQ ID NO:71) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0070] Human IgA2 constant region, Uniprot:P01877 (SEQ ID NO:72) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 73) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs: 65-72. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 73. In some embodiments, the antibody or antigen-binding fragment binds to a CD19 polypeptide that comprises an Ig-like C2 loop comprising the amino acid sequence EE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP FLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGS (SEQ ID NO: 82) (corresponding to amino acid residues 29-118 of SEQ ID NO: 60 or SEQ ID NO: 61). Additionally or alternatively, in certain embodiments, the antibody or antigen-binding fragment binds to a conformational epitope comprising amino acid residues corresponding to positions 29-118 of SEQ ID NO:60 or SEQ ID NO:61.

[0071] In another aspect, the disclosure provides an isolated immunoglobulin-related composition (e.g., an antibody or antigen-binding fragment thereof) comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:45, SEQ ID NO:47, or a variant thereof with one or more conservative amino acid substitutions.

[0072] Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain (LC) amino acid sequence comprising SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:44, SEQ ID NO:46, or a variant thereof with one or more conservative amino acid substitutions.

[0073] In some embodiments, the immunoglobulin-related compositions of the present technology comprise an HC amino acid sequence and an LC amino acid sequence, respectively, selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:20 (chFMC63xCD3 BsAb); SEQ ID NO:26 and SEQ ID NO:24 (BC250- hFMC63 VL-2 / VH-lbxCD3 BsAb); SEQ ID NO:29 and SEQ ID NO:28 (mFMC63xmC825 BsAb); SEQ ID NO:31 and SEQ ID NO:30 (mFMC63xhC825 BsAb); SEQ ID NO:45 and SEQ ID NO:44 (hFMC63 VL-2VH-lbxmC825); and SEQ ID NO:47 and SEQ ID NO:46 (hFMC63 VL-2VH-lbxhC825).

[0074] In any of the above embodiments of immunoglobulin-related compositions, the HC and LC immunoglobulin variable domain sequences form an antigen-binding site that binds to a CD19 polypeptide comprising an Ig-like C2 loop comprising the amino acid sequence EE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP FLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGS (SEQ ID NO:82) (corresponding to amino acid residues 29-118 of SEQ ID NO:60 or SEQ ID NO:61). In some embodiments, the epitope is a conformational epitope. Additionally or alternatively, in certain embodiments, the antibody or antigen-binding fragment binds to a conformational epitope comprising amino acid residues corresponding to positions 29-118 of SEQ ID NO:60 or SEQ ID NO:61.

[0075] In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody. In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one CD19 polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one CD19 polypeptide. -3M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10 -15 Dissociation constant (K D ) binds to at least one CD19 polypeptide. In certain embodiments, the immunoglobulin-related composition is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In some embodiments, the antibody comprises a human antibody framework region.

[0076] In certain embodiments, the immunoglobulin-related compositions comprise one or more of the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 17, 18, or 19, and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, or 12. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted with another amino acid. The substitution may be a "conservative substitution" as defined herein. In some embodiments, the immunoglobulin-related composition comprises (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the LC sequence present in any one of SEQ ID NOs: 20, 24, 28, 30, 44, or 46, and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the HC sequence present in any one of SEQ ID NOs: 22, 26, 29, 31, 45, or 47.

[0077] In one aspect, the disclosure provides an immunoglobulin-related composition comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 32-43, or 48-59. In certain embodiments, the immunoglobulin-related composition of the disclosure comprises an amino acid sequence selected from SEQ ID NOs: 32-43, or 48-59.

[0078] In one aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain, the first polypeptide chain comprising, from N-terminal to C-terminal, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (iii) a light chain variable domain of the first immunoglobulin; (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4; (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; and (vi) an amino acid sequence comprising the heavy chain variable domain of a second immunoglobulin. (vii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (vii) a light chain variable domain of a second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembly degradation (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0079] In another aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain, the first polypeptide chain comprising, from N-terminal to C-terminal, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (iii) a heavy chain variable domain of the first immunoglobulin; (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4; (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; and (vi) an amino acid sequence comprising the heavy chain variable domain of a second immunoglobulin. (vii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (vii) a light chain variable domain of a second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembly degradation (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.

[0080] In certain embodiments of the bispecific antigen-binding fragments disclosed herein, the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain. In some embodiments, the SADA polypeptide comprises the tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, and CBFA2T1. Additionally or alternatively, in some embodiments, the bispecific antigen-binding fragment comprises an amino acid sequence selected from SEQ ID NOs: 32-43, or 48-59.

[0081] In one aspect, the disclosure provides a bispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first and second polypeptide chains are covalently linked to each other, the second and third polypeptide chains are covalently linked to each other, and the third and fourth polypeptide chains are covalently linked to each other, and (a) each of the first and fourth polypeptide chains comprises, from N-terminal to C-terminal, (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain of the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) light and heavy chain variable domains of a second immunoglobulin capable of specifically binding to a second epitope and comprising the amino acid sequence (GGGGS)6. and (b) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a heavy chain variable domain of a second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, linked together via a linker to form a single-chain variable fragment, wherein the second and third polypeptide chains each comprise, from N-terminal to C-terminal, (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; and (ii) a heavy chain constant domain of the first immunoglobulin, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NOs: 17, 18, and 19. In certain embodiments, the second immunoglobulin binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD19, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.

[0082] In certain embodiments, the immunoglobulin-related composition contains an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments, the immunoglobulin-related composition contains an IgG4 constant region comprising a S228P mutation.

[0083] In some embodiments, the anti-CD19 immunoglobulin-related compositions described herein contain structural modifications to promote rapid binding and cellular uptake and / or slow release. In some embodiments, the anti-CD19 immunoglobulin-related compositions (e.g., antibodies) of the present technology may contain deletions in the CH2 constant heavy chain region to promote rapid binding and cellular uptake and / or slow release. In some embodiments, Fab fragments are used to promote rapid binding and cellular uptake and / or sustained release. In some embodiments, F(ab)'2 fragments are used to promote rapid binding and cellular uptake and / or sustained release. In one aspect, the present technology provides a recombinant nucleic acid sequence encoding any one of the immunoglobulin-related compositions described herein. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 21, 23, 25, and 27.

[0084] In another aspect, the technology provides host cells that express any nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein.

[0085] The immunoglobulin-related compositions (e.g., anti-CD19 antibodies) of the present technology may be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of one or more CD19 polypeptides, or may be specific for both a CD19 polypeptide and a heterologous composition, such as a heterologous polypeptide or solid support material. See, e.g., WO93 / 17715, WO92 / 08802, WO91 / 00360, WO92 / 05793, Tutt et al., J. Immunol. 147: 60-69 (1991), U.S. Patent Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648, and 6,106,835, and Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related composition is chimeric. In certain embodiments, the immunoglobulin-related composition is humanized. The immunoglobulin-related composition of the present technology can also be recombinantly fused to heterologous polypeptides at N-terminus or C-terminus, or chemically conjugated to polypeptides or other compositions (including covalent and non-covalent conjugation).For example, the immunoglobulin-related composition of the present technology can be recombinantly fused to or conjugated to molecules that are useful as labels and effector molecules, such as heterologous polypeptides, drugs or toxins, in detection assays.For example, see WO92 / 08495, WO91 / 14438, WO89 / 12624, U.S. Patent No. 5,314,995 and EP0396387. In any of the above embodiments of the immunoglobulin-related composition of the present technology, the antibody or antigen-binding fragment may be conjugated to a substance selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. For chemical or physical binding, a functional group on the immunoglobulin-related composition typically associates with a functional group on the substance. Alternatively, a functional group on the substance associates with a functional group on the immunoglobulin-related composition.

[0086] The functional groups on the substance and the immunoglobulin-related composition can be directly associated. For example, a functional group (e.g., a sulfhydryl group) on the substance can associate with a functional group (e.g., a sulfhydryl group) on the immunoglobulin-related composition to form a disulfide. Alternatively, the functional groups can associate via a crosslinker (i.e., a linker). Some examples of crosslinkers are described below. The crosslinker can be attached to either the substance or the immunoglobulin-related composition. The number of substances or immunoglobulin-related compositions in the conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of substances that can be associated with the conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions that can be associated with the substance depends on the number of functional groups present on the substance.

[0087] In yet another embodiment, the conjugate comprises one immunoglobulin-related composition associated with one substance. In one embodiment, the conjugate comprises at least one substance chemically bound (e.g., conjugated) to at least one immunoglobulin-related composition. The substance can be chemically bound to the immunoglobulin-related composition by any method known to those skilled in the art. For example, a functional group on the substance can be directly attached to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate, and hydroxyl.

[0088] Substances can also be chemically linked to the immunoglobulin-related composition by cross-linking agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Inc., Rockford, Illinois. The Pierce Biotechnology, Inc. website can be helpful. Additional cross-linking agents include the platinum cross-linkers described in U.S. Patent Nos. 5,580,990, 5,985,566, and 6,133,038 to Kreatech Biotechnology, BV, Amsterdam, The Netherlands. Alternatively, the functional groups on the substance and the immunoglobulin-related composition may be identical. Homobifunctional crosslinkers are typically used to crosslink identical functional groups. Examples of homobifunctional crosslinkers include EGS (i.e., ethylene glycol bis[succinimidyl succinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (i.e., 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane), and BMH (i.e., bis-maleimidohexane). Such homobifunctional crosslinkers are also available from Pierce Biotechnology, Inc.

[0089] In other cases, it may be beneficial to cleave the substance from the immunoglobulin-related composition. The Pierce Biotechnology, Inc. website mentioned above can also assist those skilled in the art in selecting a suitable crosslinker that can be cleaved by an enzyme, for example, in a cell. Thus, the substance can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl).

[0090] In another embodiment, the conjugate comprises at least one substance physically bound to at least one immunoglobulin-related composition. Any method known to those skilled in the art can be used to physically bind a substance to the immunoglobulin-related composition. For example, the immunoglobulin-related composition and the substance can be mixed together by any method known to those skilled in the art. The order of mixing is not important. For example, the substance can be physically mixed with the immunoglobulin-related composition by any method known to those skilled in the art. For example, the immunoglobulin-related composition and the substance can be placed in a container and agitated, for example, by shaking the container, to mix the immunoglobulin-related composition and the substance. The immunoglobulin-related compositions can be modified by any method known to those of skill in the art, for example, the immunoglobulin-related compositions can be modified with crosslinking agents or functional groups, as described above.

[0091] A. Methods of Preparing the Anti-CD19 Antibodies of the Present Technology General Overview. First, a target polypeptide against which an antibody of the present technology can be produced is selected. For example, antibodies can be produced against the full-length CD19 protein, a CD19 protein lacking the cytoplasmic domain, the CD19 extracellular and transmembrane domains, or a portion of the extracellular domain of the CD19 protein (e.g., a region containing the two Ig-like C2 loops of CD19, or a constant region 2 (C2) type Ig-like loop containing amino acid residues corresponding to positions 29-118 of SEQ ID NO: 60 or SEQ ID NO: 61). Techniques for producing antibodies against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, display libraries, xeno- or human-based mice, hybridomas, etc. Target polypeptides within the scope of the present technology include any polypeptide derived from the CD19 protein that contains an extracellular domain capable of eliciting an immune response (e.g., the Ig-like C2 loop encoded by exon 2 of CD19).

[0092] It should be understood that recombinantly engineered antibodies and antibody fragments, eg, antibody-related polypeptides, directed against the CD19 protein and fragments thereof are suitable for use in accordance with the present disclosure. Anti-CD19 antibodies that can be subjected to the techniques described herein include monoclonal and polyclonal antibodies and antibody fragments, such as Fab, Fab', F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains, and / or antibody fragments. Methods useful for the high-yield production of antibody Fv-containing polypeptides, such as Fab' and F(ab')2 antibody fragments, have been described. See U.S. Patent No. 5,648,237. Generally, the antibody is obtained from the original species. More specifically, the nucleic acid or amino acid sequence of the variable part of the light chain, heavy chain or both of the original species antibody having specificity for the target polypeptide antigen is obtained. The original species can be any species that has been useful for producing the antibody or antibody library of the present technology, such as rat, mouse, rabbit, chicken, monkey, human, etc. Phage or phagemid display technology is a useful technique for deriving the antibody of the present technology.Techniques for producing and cloning monoclonal antibodies are well known to those skilled in the art.The expression of the sequence encoding the antibody of the present technology can be carried out in E. coli.

[0093] Due to the degeneracy of nucleic acid encoding sequences, other sequences encoding substantially the same amino acid sequence as that of a naturally occurring protein may be used in the practice of the present technology. These include, but are not limited to, nucleic acid sequences comprising all or part of the nucleic acid sequences encoding the above polypeptides, altered by the substitution of different codons encoding functionally equivalent amino acid residues within the sequence, thus resulting in a silent change. It is understood that the nucleotide sequences of the immunoglobulins of the present technology allow for up to 25% sequence homology variation, as calculated by standard methods (see "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.), as long as such variants form functional antibodies that recognize the CD19 protein. For example, one or more amino acid residues within a polypeptide sequence may be substituted with another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent change. Substitutes for amino acids within the sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar natural amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Proteins or fragments or derivatives thereof that are differentially modified during or after translation, for example, by glycosylation, proteolytic cleavage, or linkage to antibody molecules or other cellular ligands, are also within the scope of the present technology.Additionally, nucleic acid sequences encoding immunoglobulins can be mutated in vitro or in vivo to create and / or destroy translation, initiation and / or termination sequences to generate variations in the coding region and / or to create new restriction endonuclease sites or destroy existing ones, further facilitating in vitro modifications. Any technique for mutagenesis known in the art may be used, including, but not limited to, in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, the use of Tab linkers (Pharmacia), etc.

[0094] Preparation of Polyclonal Antisera and Immunogens. The method for producing antibodies or antibody fragments of the present technology typically involves immunizing a subject (generally a non-human subject such as a mouse or rabbit) with purified CD19 protein or a fragment thereof, or with cells expressing the CD19 protein or a fragment thereof. An appropriate immunogenic preparation may contain, for example, recombinantly expressed CD19 protein or a chemically synthesized CD19 peptide. The extracellular domain of the CD19 protein, or a portion or fragment thereof (e.g., a portion or fragment containing amino acid residues corresponding to positions 29-118 of SEQ ID NO: 60 or SEQ ID NO: 61), can be used as an immunogen to generate anti-CD19 antibodies that bind to the CD19 protein or a portion or fragment thereof using standard techniques for polyclonal and monoclonal antibody preparation. The full-length CD19 protein or a fragment thereof is useful as an immunogen. In some embodiments, the CD19 fragment contains amino acid residues corresponding to positions 29-118 of SEQ ID NO: 60 or SEQ ID NO: 61, such that antibodies raised against the peptide form specific immune complexes with the CD19 protein. A polypeptide encoded by exon 2 of CD19 can also be used as an immunogen.

[0095] In some embodiments, the antigenic CD19 peptide comprises at least 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90 amino acid residues. Depending on the application and methods known to those skilled in the art, longer antigenic peptides are sometimes more desirable than shorter antigenic peptides. Multimers of a given epitope are sometimes more effective than monomers.

[0096] If necessary, the immunogenicity of the CD19 protein (or fragment thereof) can be increased by fusion or conjugation with a hapten such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such haptens are known in the art. To enhance a subject's immune response to the polypeptide, the CD19 protein target may be combined with a conventional adjuvant, such as Freund's complete or incomplete adjuvant. Various adjuvants used to enhance immunological responses include, but are not limited to, Freund's (complete and incomplete), inorganic gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptide or oil emulsions, dinitrophenol, etc.), human adjuvants such as bacillus Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0097] In describing the present technology, an immune response can be described as a "primary" or "secondary" immune response. A primary immune response, also referred to as a "protective" immune response, refers to an immune response generated in an individual as a result of several initial exposures (e.g., an initial "immunization") to a particular antigen, such as a CD19 protein. In some embodiments, the immunization can result from vaccination of an individual with a vaccine containing an antigen. For example, the vaccine can be a CD19 vaccine containing one or more CD19 protein-derived antigens. The primary immune response can weaken or become attenuated over time, and can even disappear, or at least become so attenuated that it cannot be detected. Thus, the present technology also relates to a "secondary" immune response, also referred to herein as a "memory immune response." The term secondary immune response refers to an immune response induced in an individual after a primary immune response has already been generated. Thus, a secondary immune response can be elicited to boost a pre-existing immune response that has become weakened or attenuated, or to reconstitute a previous immune response that has disappeared or is no longer detectable. A secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs upon stimulation of memory B cells generated upon initial presentation of the antigen. A delayed-type hypersensitivity (DTH) response is initiated by CD4 + It is a type of cell-mediated secondary or memory immune response mediated by T cells. Initial exposure to an antigen primes the immune system, and further exposure results in DTH.

[0098] After appropriate immunization, anti-CD19 antibodies can be prepared from the subject's serum. If desired, antibody molecules directed against the CD19 protein can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as polypeptide A chromatography, to obtain the IgG fraction. Monoclonal antibody. In one embodiment of the present technology, the antibody is an anti-CD19 monoclonal antibody. For example, in some embodiments, the anti-CD19 monoclonal antibody can be a human or mouse anti-CD19 monoclonal antibody. To prepare monoclonal antibodies against CD19 protein or its derivatives, fragments, analogs or homologs, any technology that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, hybridoma technology (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497), trioma technology, human B cell hybridoma technology (see, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72), and EBV hybridoma technology for producing human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). In practicing the present technology, human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids encoding regions of an antibody may be isolated. PCR may be used to amplify sequences encoding portions of antibodies from the population, using primers derived from sequences encoding conserved regions of the antibody, and DNA encoding the antibody or a fragment thereof, e.g., the variable domain, is then reconstructed from the amplified sequences.Such amplified sequences can also be fused to DNA encoding other proteins, such as bacteriophage coat or bacterial cell surface proteins, for expression and display of the fusion polypeptide in phage or bacteria. The amplified sequences can then be expressed and further selected or isolated based on, for example, the affinity of the expressed antibody or fragment thereof for an antigen or epitope present in the CD19 protein. Alternatively, hybridomas expressing anti-CD19 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject's spleen using routine methods. See, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomas using standard methods yields monoclonal antibodies of varying specificity (i.e., for various epitopes) and affinity. Selected monoclonal antibodies with desired properties, such as CD19 binding, can be used as expressed by hybridomas, conjugated to molecules such as polyethylene glycol (PEG) to alter their properties, or the cDNA encoding them can be isolated, sequenced, and manipulated in various ways. To enhance the immunogenic properties of the CD19 protein, synthetic dendritic trees can be added to reactive amino acid side chains, such as lysine. CPG dinucleotide methods can also be used to enhance the immunogenic properties of the CD19 protein. Other manipulations include substituting or deleting specific aminoacyl residues that contribute to antibody instability during storage or after administration to a subject, and affinity maturation methods to improve the affinity of antibodies for the CD19 protein. Hybridoma technology. In some embodiments, the antibody of the present technology is an anti-CD19 monoclonal antibody produced by a hybridoma comprising B cells obtained from a transgenic non-human animal, such as a transgenic mouse, whose genome comprises a human heavy chain transgene and a human light chain transgene, fused to an immortalized cell. Hybridoma technology is known in the art and includes those taught in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0099] Phage display technology. As described above, the antibodies of this technology can be produced by applying recombinant DNA and phage display technology. For example, anti-CD19 antibodies can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. Phages with the desired binding properties are selected from repertoire or combinatorial antibody libraries (e.g., human or murine) by direct selection using antigens, usually antigens bound or captured to a solid surface or bead. The phages used in these methods are usually filamentous phages, including fd and M13, which have Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Furthermore, methods for the construction of Fab expression libraries may be adapted to allow rapid and efficient identification of monoclonal Fab fragments having the desired specificity for a CD19 polypeptide, e.g., a polypeptide or a derivative, fragment, analog or homolog thereof (see, e.g., Huse, et al., Science 246: 1275-1281, 1989).Other examples of phage display methods that can be used to generate antibodies of the present technology include those described in Huston et al., Proc. Natl. Acad. Sci USA, 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994, PCT / GB91 / 01134, WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, WO96 / 06213, WO92 / 01047 (Medical Research Council al.), WO97 / 08320 (Morphosys), WO92 / 01047 (CAT / MRC), WO91 / 17271 (Affymax), and those disclosed in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, and 5,733,743. A method useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides through disulfide bonds is described by Lohning, US Pat. No. 6,753,136.As described in the above references, after phage selection, the antibody coding region obtained from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can also be used using methods known in the art, such as those disclosed in WO92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.

[0100] Generally, hybrid antibodies or hybrid antibody fragments cloned into a display vector can be selected against an appropriate antigen to identify variants that maintain good binding activity, since the antibody or antibody fragment is presented on the surface of a phage or phagemid particle. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector formats can be used for this process, such as cloning an antibody fragment library into a lytic phage vector for selection and / or screening (modified T7 or lambda Zap systems).

[0101] Expression of Recombinant Anti-CD19 Antibodies. As described above, the antibodies of the present technology can be produced by applying recombinant DNA technology. Recombinant polynucleotide constructs encoding the anti-CD19 antibodies of the present technology typically contain expression control sequences operably linked to the coding sequences of the anti-CD19 antibody chains, including naturally associated or heterologous promoter regions. As such, another embodiment of the present technology includes vectors containing one or more nucleic acid sequences encoding the anti-CD19 antibodies of the present technology. For recombinant expression of one or more polypeptides of the present technology, nucleic acids containing all or part of the nucleotide sequence encoding the anti-CD19 antibody are inserted into an appropriate cloning or expression vector (i.e., a vector containing the elements necessary for transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as described in detail below. Methods for producing a diverse collection of vectors are described by Lerner et al., U.S. Patent Nos. 6,291,160 and 6,680,192.

[0102] In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this disclosure, "plasmid" and "vector" can be used interchangeably, as plasmids are the most common form of vector. However, this technology is intended to include other forms of expression vectors that are not technically plasmids but serve equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses). Such viral vectors enable infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence encoding the anti-CD19 antibody and for the collection and purification of the anti-CD19 antibody, e.g., a cross-reactive anti-CD19 antibody. See generally U.S. Patent Application Publication No. 2002 / 0199213. These expression vectors are typically replicable in the host organism either as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors contain a selectable marker, such as ampicillin resistance or hygromycin resistance, to allow detection of cells transformed with the desired DNA sequence. The vector may also encode a signal peptide, such as pectate lyase, useful for directing the secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0103] The recombinant expression vector of the present technology contains a nucleic acid encoding a protein having CD19 binding properties in a form suitable for expression in a host cell, which means that the recombinant expression vector contains one or more regulatory sequences selected based on the host cell to be used for expression, operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be understood by those skilled in the art that the design of an expression vector can vary depending on factors such as the choice of host cell to be transformed, the level of expression of the desired polypeptide, and the like. Typical regulatory sequences useful as promoters for recombinant polypeptide expression (e.g., anti-CD19 antibodies) include, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization. In one embodiment, a polynucleotide encoding the anti-CD19 antibody of the present technology is operably linked to the ara B promoter and can be expressed in a host cell. See U.S. Patent No. 5,028,530.The expression vectors of the present technology can be introduced into host cells to thereby produce polypeptides or peptides (such as, for example, anti-CD19 antibodies) comprising fusion polypeptides encoded by nucleic acids as described herein.

[0104] Another aspect of the present technology relates to host cells expressing anti-CD19 antibodies, containing nucleic acids encoding one or more anti-CD19 antibodies. The recombinant expression vectors of the present technology can be designed for expression of anti-CD19 antibodies in prokaryotic or eukaryotic cells. For example, anti-CD19 antibodies can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, e.g., yeast, yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, recombinant expression vectors can be transcribed and translated, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods useful for preparing and screening polypeptides with predetermined properties, such as anti-CD19 antibodies, by expressing stochastically generated polynucleotide sequences have previously been described. See U.S. Patent Nos. 5,763,192, 5,723,323, 5,814,476, 5,817,483, 5,824,514, 5,976,862, 6,492,107, and 6,569,641.

[0105] Expression of polypeptides in prokaryotes is most often carried out using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides in Escherichia coli (E. coli). Fusion vectors add several amino acids to the polypeptide encoded herein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) to increase expression of the recombinant polypeptide, (ii) to increase the solubility of the recombinant polypeptide, and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Fusion expression vectors often incorporate a proteolytic cleavage site at the junction of the fusion moiety and the recombinant polypeptide to allow separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Such enzymes and their cognate recognition sequences include factor X, thrombin, and enterokinase. Common fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[0106] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). A method for targeted assembly of separate active peptides or protein domains to obtain multifunctional polypeptides by polypeptide fusion has been described by Pack et al., U.S. Pat. Nos. 6,294,353 and 6,692,935. One strategy for maximizing recombinant polypeptide expression in E. coli, such as anti-CD19 antibodies, is to express the polypeptide in a host bacterium with an impaired ability to proteolytically cleave the recombinant polypeptide. For example, see Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into expression vector so that each individual codon of each amino acid is preferentially used in expression host, for example, E. coli (see, for example, Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). This modification of the nucleic acid sequence of this technology can be carried out by standard DNA synthesis technology.

[0107] In another embodiment, the anti-CD19 antibody expression vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (Invitrogen Corp, San Diego, CA). Alternatively, anti-CD19 antibodies may be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expressing polypeptides, such as anti-CD19 antibodies, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0108] In yet another embodiment, the nucleic acid encoding the anti-CD19 antibody of the present technology is expressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6: 187-195, 1987).When used in mammalian cells, the control function of expression vectors is often provided by viral regulatory elements.For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells suitable for expressing the anti-CD19 antibodies of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0109] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev. 1: 268-277, 1987), lymphocyte-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), promoters of T-cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983), neuron-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1988). 1989), pancreatic-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., whey promoters; U.S. Pat. No. 4,873,316 and European Patent Application Publication No. 264,166). Developmentally regulated promoters are also included, such as mouse hox promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989).

[0110] Another aspect of the present method relates to a host cell into which the recombinant expression vector of the present technology is introduced.The terms "host cell" and "recombinant host cell" are used interchangeably herein.It is understood that these terms refer not only to the specific target cell, but also to the progeny or potential progeny of such a cell.Since certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term herein.

[0111] Host cells can be any prokaryotic or eukaryotic cell. For example, anti-CD19 antibodies can be expressed in bacterial cells such as E. coli, insect cells, yeast, or mammalian cells. Mammalian cells are suitable hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Exemplary expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. Co et al., J Immunol. 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

[0112] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various art-recognized techniques for introducing foreign nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics, or viral-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., Molecular Cloning). Suitable methods for transducing or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

[0113] For stable transfection of mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants, a gene encoding a selection marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selection markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selection marker can be introduced into the host cells on the same vector as that encoding the anti-CD19 antibody, or on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have integrated the selection marker gene survive, while other cells die).

[0114] Host cells containing the anti-CD19 antibody of the present technology, such as prokaryotic or eukaryotic host cells in culture, can be used to produce (i.e., express) a recombinant anti-CD19 antibody. In one embodiment, the method includes culturing the host cells (into which a recombinant expression vector encoding the anti-CD19 antibody has been introduced) in a suitable medium such that the anti-CD19 antibody is produced. In another embodiment, the method further includes isolating the anti-CD19 antibody from the medium or the host cells. Once expressed, the anti-CD19 antibody, e.g., a collection of anti-CD19 antibodies or anti-CD19 antibody-related polypeptides, is purified from the culture medium and host cells. The anti-CD19 antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like. In one embodiment, the anti-CD19 antibody is produced in a host organism by the method of Boss et al., U.S. Pat. No. 4,816,397. Typically, the anti-CD19 antibody chain is expressed with a signal sequence, thus releasing it into the culture medium. However, if the anti-CD19 antibody chains are not naturally secreted by the host cells, they can be released by treatment with mild detergents. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0115] Polynucleotides encoding anti-CD19 antibodies, for example, anti-CD19 antibody coding sequences, can be incorporated into transgenes for introduction into the genome of transgenic animals and subsequent expression in the milk of transgenic animals.See, for example, U.S. Patent Nos. 5,741,957, 5,304,489 and 5,849,992.Suitable transgenes include light and / or heavy chain coding sequences operably linked to the promoter and enhancer derived from mammary gland-specific genes, such as casein or β-lactoglobulin.To produce transgenic animals, transgenes can be microinjected into fertilized oocytes or integrated into the genome of embryonic stem cells, and the nuclei of such cells are transferred into enucleated oocytes.

[0116] Single-chain antibody. In one embodiment, the anti-CD19 antibody of the present technology is a single-chain anti-CD19 antibody. According to the present technology, technology can be adopted to produce single-chain antibodies specific to CD19 protein (see, for example, U.S. Patent No. 4,946,778). Examples of the technology that can be used to produce single-chain Fv and antibodies of the present technology include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.

[0117] Chimeric and humanized antibodies. In one embodiment, the anti-CD19 antibody of the present technology is a chimeric anti-CD19 antibody. In one embodiment, the anti-CD19 antibody of the present technology is a humanized anti-CD19 antibody. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies derived from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), and in this case, the resulting CDR-grafted antibody is called a "humanized" antibody.

[0118] Recombinant anti-CD19 antibodies, such as chimeric and humanized monoclonal antibodies containing both human and non-human portions, can be produced using standard recombinant DNA technology and are within the scope of the present technology. For some uses, including the in vivo use of the anti-CD19 antibodies of the present technology in humans and the use of these materials in in vitro detection assays, chimeric or humanized anti-CD19 antibodies may be used. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA technology known in the art. Such useful methods include, but are not limited to, those described in International Application No. PCT / US86 / 02269, U.S. Pat. No. 5,225,539, European Patent No. 184187, European Patent No. 171496, European Patent No. 173494, PCT International Publication No. WO86 / 01533, U.S. Pat. Nos. 4,816,567, 5,225,539, European Patent No. 125023, Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987. Cancer Res. 47: 999-1005; Wood, et al., 1985. Nature 314: 446-449; Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229: 1202-1207; Oi, et al. (1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321: 552-525; Verhoeyan, et al., 1988.For example, antibodies can be humanized using a variety of techniques, including CDR grafting (EP 0239400, WO 91 / 09967, U.S. Pat. Nos. 5,530,101, 5,585,089, 5,859,205, 6,248,516, EP 460167), veneering or resurfacing (EP 0592106, EP 0519596, Padlan EA, Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91: 969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332).In one embodiment, cDNA encoding a murine anti-CD19 monoclonal antibody is digested with restriction enzymes specifically selected to remove sequences encoding the Fc constant region, and the equivalent portion of cDNA encoding a human Fc constant region is substituted (Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; U.S. Patent No. 6,180,370; U.S. Patent No. 6,300,064;

[0119] In one embodiment, the present technology provides for the construction of humanized anti-CD19 antibodies that are less likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response, while still possessing effective antibody effector functions. As used herein, the terms "human" and "humanized" in relation to antibodies refer to any antibody that is predicted to induce a therapeutically acceptable, weak immunogenic response in human subjects. In one embodiment, the present technology provides humanized anti-CD19 antibodies, heavy and light chain immunoglobulins.

[0120] CDR antibody. In some embodiments, the anti-CD19 antibody of the present technology is an anti-CD19 CDR antibody. Generally, the donor and acceptor antibodies used to generate anti-CD19 CDR antibodies are monoclonal antibodies derived from different species, and the acceptor antibody is usually a human antibody (to minimize antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody. Grafting is performed by grafting a single V of the acceptor antibody. H or V L or V H and V L The CDRs may be from multiple CDRs (or portions thereof) in one or both of the variable domains of the acceptor antibody. Although only the number of CDRs required to allow the resulting CDR-grafted antibody to properly bind to the CD19 protein needs to be replaced, all three CDRs of all variable domains of the acceptor antibody are often replaced with the corresponding donor CDRs. Methods for producing CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; and Winter U.S. Patent No. 5,225,539; and EP0682040. H and V LMethods useful for preparing polypeptides are taught by Winter et al., U.S. Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP 0451216; and EP 0120694.

[0121] After selecting suitable framework region candidates from the same family and / or family members, either or both of the heavy and light chain variable regions are produced by grafting CDRs from the original species into the hybrid framework regions. For any of the above embodiments, assembly of a hybrid antibody or hybrid antibody fragment having a hybrid variable chain region can be achieved using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on CDRs from the target species and the original species) can be produced by oligonucleotide synthesis and / or PCR. Nucleic acids encoding CDR regions can also be isolated from the original species antibody using appropriate restriction enzymes and ligated into the target species framework by ligation using appropriate ligation enzymes. Alternatively, the framework regions of the variable chains of the original species antibody can be altered by site-directed mutagenesis.

[0122] Since hybrids are constructed by selecting from multiple candidates corresponding to each framework region, there are numerous combinations of sequences that are amenable to construction according to the principles described herein. Thus, a library of hybrids can be assembled, with members having different combinations of individual framework regions. Such a library can be an electronic database collection or a physical collection of hybrid sequences.

[0123] This process usually does not alter the FRs of the acceptor antibody that flank the grafted CDRs. However, one skilled in the art can sometimes improve the antigen-binding affinity of the resulting anti-CD19 CDR-grafted antibody by replacing specific residues in a given FR to create a similar FR with the corresponding FR of the donor antibody. Suitable positions for substitution include amino acid residues adjacent to or capable of interacting with the CDR (see, e.g., U.S. Pat. No. 5,585,089, especially columns 12-16). Alternatively, one skilled in the art can start with the donor FR and modify it to be more similar to the acceptor FR or human consensus FR. Techniques for making these modifications are known in the art. In particular, if the resulting FR matches or is at least 90% or more identical to the human consensus FR at that position, doing so may not significantly increase the antigenicity of the resulting modified anti-CD19 CDR-grafted antibody compared to the same antibody with fully human FRs. Bispecific antibodies (BsAbs). Bispecific antibodies are antibodies that can simultaneously bind to two targets with distinct structures, such as two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen. BsAbs can be generated, for example, by combining heavy and / or light chains that recognize different epitopes on the same or different antigens. In some embodiments, a bispecific binding agent functions by binding to one antigen (or epitope) on one of its two binding arms (one VH / VL pair) and a different antigen (or epitope) on its second arm (a different VH / VL pair). By this definition, a bispecific binding agent has two distinct antigen-binding arms (both in terms of specificity and CDR sequence) and is monovalent for each antigen it binds to.

[0124] The bispecific antibodies (BsAbs) and bispecific antibody fragments (BsFabs) of the present technology have, for example, at least one arm that specifically binds to CD19 and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of B cells, T cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in certain embodiments, the second target antigen is selected from the group consisting of CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD19, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, and KIR. In certain embodiments, the BsAb can bind to tumor cells that express the CD19 antigen on their cell surface. In some embodiments, the BsAb is genetically engineered to promote tumor cell killing by directing (or recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include those having a first antigen-binding site specific for CD19 and a second antigen-binding site specific for a small molecule hapten (e.g., DTPA, IMP288, DOTA, DOTA-Bn, DOTA-desferrioxamine, other DOTA chelates described herein, biotin, fluorescein, or those disclosed in Goodwin, D A. et al, 1994, Cancer Res. 54(22):5937-5946).

[0125] A variety of bispecific fusion proteins can be produced using molecular engineering. For example, BsAbs have been constructed that utilize either a complete immunoglobulin framework (e.g., IgG), a single-chain variable fragment (scFv), or a combination thereof. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and another scFv fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, e.g., comprising an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs, composed of two scFv units in tandem, have proven to be a clinically successful bispecific antibody format. In some embodiments, the BsAb comprises two single-chain variable fragments (scFvs) in tandem, in which an scFv that binds to a tumor antigen (e.g., CD19) is linked to an scFv that engages T cells (e.g., by binding to CD3). In this way, T cells are recruited to the tumor site, whereby they can mediate cytotoxic killing of tumor cells. See, for example, Dreier et al., J. Immunol. 170:4397-4402 (2003); Bargou et al., Science 321:974-977 (2008)). In some embodiments, the BsAb of the present technology comprises two single-chain variable fragments (scFvs) in tandem, in which an scFv that binds to a tumor antigen (e.g., CD19) is linked to an scFv that binds to the small molecule DOTA hapten.

[0126] Recent methods for producing BsAbs include engineered recombinant monoclonal antibodies with additional cysteine ​​residues that cross-link more strongly than more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins by linking two or more different single-chain antibody or antibody fragment segments with the required dual specificities. See, e.g., Coloma et al., Nature Biotech. 15:159-163 (1997). Using molecular engineering, a variety of bispecific fusion proteins can be produced.

[0127] Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in a similar manner. Recombinant methods can be used to produce various fusion proteins. In certain embodiments, the BsAb of the present technology comprises an immunoglobulin comprising a heavy chain, a light chain, and an scFv. In certain embodiments, the scFv is linked to the C-terminus of the heavy chain of any CD19 immunoglobulin disclosed herein. In certain embodiments, the scFv is linked to the C-terminus of the light chain of any CD19 immunoglobulin disclosed herein. In various embodiments, the scFv is linked to the heavy or light chain via a linker sequence. The appropriate linker sequence required for in-frame connection of the heavy chain Fd to the scFv is extracted by PCR reaction. L and V kappa The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and transformed into the V domain of the CD19 antibody. H The resulting vector is ligated into a vector containing DNA sequences encoding the bispecific fusion protein. The resulting vector can be used to transfect an appropriate host cell, e.g., a mammalian cell, for expression of the bispecific fusion protein.

[0128] In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, the linker is characterized in that it does not adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., the first and / or second antigen-binding site). In some embodiments, linkers are used in the BsAbs described herein based on the specific properties they confer to the BsAb, such as increased stability. In some embodiments, the BsAbs of the present technology comprise a G4S linker. In some specific embodiments, the BsAb of the present technology is (G4S) n linker, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0129] Self-assembly and disassembly (SADA) conjugates. In some embodiments, the anti-CD19 antibodies of the present technology comprise one or more SADA domains. The SADA domains can be designed and / or adapted to achieve environment-dependent multimerization with beneficial kinetic, thermodynamic, and / or pharmacological properties. For example, it has been recognized that the SADA domain can be part of a conjugate that enables effective delivery of a payload to a target site of interest while minimizing the risk of off-target interactions. The anti-CD19 antibodies of the present technology can comprise a SADA domain linked to one or more binding domains. In some embodiments, such conjugates are characterized in that they multimerize to form complexes of a desired size under appropriate conditions (e.g., in a solution where the conjugate is present above a threshold concentration or pH and / or when the conjugate is present at a target site characterized by an appropriate level or density of receptors for the payload), and disassemble into smaller forms under other conditions (e.g., in the absence of an appropriate environmental multimerization trigger).

[0130] SADA conjugates may have improved characteristics compared to conjugates lacking the SADA domain. In some embodiments, the improved characteristics of multimeric conjugates include increased target avidity / binding, increased specificity for target cells or tissues, and / or increased initial serum half-life. In some embodiments, improved characteristics include SADA conjugates exhibiting reduced nonspecific binding, decreased toxicity, and / or improved renal clearance upon dissociation into smaller states (e.g., dimers or monomers). In some embodiments, SADA conjugates exhibit at least 75% identity with the amino acid sequence of a human homomultimerizing polypeptide and one or more multimerization dissociation constants (K D ) SADA polypeptides having an amino acid sequence characterized by:

[0131] In some embodiments, the SADA conjugate is constructed and arranged to adopt a first multimerization state and one or more higher order multimerization states. In some embodiments, the first multimerization state is less than about 70 kDa in size. In some embodiments, the first multimerization state is a non-multimerized state (e.g., a monomer or a dimer). In some embodiments, the first multimerization state is a monomer. In some embodiments, the first multimerization state is a dimer. In some embodiments, the first multimerization state is a multimerized state (e.g., a trimer or tetramer). In some embodiments, the higher order multimerization state is a homotetramer or higher order homomultimer greater than 150 kDa in size. In some embodiments, the conjugate comprises a SADA polypeptide K. D In some embodiments, the higher order homomultimerized conjugates are stable in aqueous solution when present at concentrations above SADA polypeptide K. D below which the SADA conjugate transitions from the higher order multimerization state to the first multimerization state under physiological conditions.

[0132] In some embodiments, the SADA polypeptide is covalently linked to the binding domain via a linker. Any suitable linker known in the art may be used. In some embodiments, the SADA polypeptide is linked to the binding domain via a polypeptide linker. In some embodiments, the polypeptide linker is a Gly-Ser linker. In some embodiments, the polypeptide linker is or comprises the sequence (GGGGS)n, where n represents the number of repeated GGGGS units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more. In some embodiments, the binding domain is directly fused to the SADA polypeptide. In some embodiments, the SADA domain is a human polypeptide or a fragment and / or derivative thereof. In some embodiments, the SADA domain is substantially non-immunogenic in humans. In some embodiments, the SADA polypeptide is stable as a multimer. In some embodiments, the SADA polypeptide lacks unpaired cysteine ​​residues. In some embodiments, the SADA polypeptide does not have large exposed hydrophobic surfaces. In some embodiments, the SADA domain has, or is predicted to have, a structure comprising helical bundles that can associate in a parallel or anti-antiparallel orientation. In some embodiments, the SADA polypeptide is capable of reversible multimerization. In some embodiments, the SADA domain is a tetramerization domain, a heptamerization domain, a hexamerization domain, or an octamerization domain. In certain embodiments, the SADA domain is a tetramerization domain. In some embodiments, the SADA domain consists of multimerization domains, each consisting of helical bundles that associate in a parallel or anti-antiparallel orientation. In some embodiments, the SADA domain is selected from one of the following human proteins: p53, p63, p73, heterogeneous nuclear ribonucleoprotein C (hnRNPC), the N-terminal domain of synaptosomal-associated protein 23 (SNAP-23), stefin B (cystatin B), potassium voltage-gated channel subfamily KQT member 4 (KCNQ4), or cyclin D-associated protein (CBFA2T1). Examples of suitable SADA domains are described in PCT / US2018 / 031235, the entire contents of which are incorporated herein by reference. Provided below are polypeptide sequences of exemplary SADA domains.

[0133] Human p53 tetramerization domain amino acid sequence (321-359) KPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEP (SEQ ID NO: 74) Human p63 tetramerization domain amino acid sequence (396-450) RSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQQQHQHLLQKQ (SEQ ID NO: 75) Human p73 tetramerization domain amino acid sequence (348-399) RHGDEDTYYLQVRGRENFEILMKLKESLELMELVPQPLVDSYRQQQQLLQRP (SEQ ID NO: 76) Human HNRNPC tetramerization domain amino acid sequence (194-220) QAIKKELTQIKQKVDSLLENLEKIEKE (SEQ ID NO: 77) Human SNAP-23 tetramerization domain amino acid sequence (23-76) STRRILGLAIESQDAGIKTITMLDEQKEQLNRIEEGLDQINKDMRETEKTLTEL (SEQ ID NO: 78) Human stefin B tetramerization domain amino acid sequence (2-98) MCGAPSATQPATAETQHIADQVRSQLEEKENKKFPVFKAVSFKSQVVAGTNYFIKVHVGDEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF (SEQ ID NO: 79) KCNQ4 tetramerization domain amino acid sequence (611-640) DEISMMGRVVKVEKQVQSIEHKLDLLLGFY (SEQ ID NO: 80) CBFA2T1 tetramerization domain amino acid sequence (462-521) TVAEAKRQAAEDALAVINQQEDSSESCWNCGRKASETCSGCNTARYCGSFCQHKDWEKHH (SEQ ID NO: 81)

[0134] In some embodiments, the SADA polypeptide is or comprises the tetramerization domain of p53, p63, p73, heterogeneous nuclear ribonucleoprotein C (hnRNPC), the N-terminal domain of synaptosomal-associated protein 23 (SNAP-23), stefin B (cystatin B), potassium voltage-gated channel subfamily KQT member 4 (KCNQ4), or cyclin D-associated protein (CBFA2T1). In some embodiments, the SADA polypeptide is or comprises a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 74-81.

[0135] Fc Modifications. In some embodiments, the anti-CD19 antibodies of the present technology comprise a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification compared to a wild-type Fc region (or parent Fc region) such that the affinity of the molecule for an Fc receptor (e.g., FcγR) is altered. This is provided, however, that the variant Fc region does not have substitutions at positions that directly contact an Fc receptor, based on crystallographic and structural analyses of Fc-Fc receptor interactions, e.g., those disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that directly contact an Fc receptor, e.g., FcγR, include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G loop).

[0136] In some embodiments, the anti-CD19 antibodies of the present technology having a variant Fc region with one or more amino acid modifications have altered affinity for activating and / or inhibitory receptors, wherein the one or more amino acid modifications are an N297 substitution with alanine or a K322 substitution with alanine. Glycosylation modifications. In some embodiments, the anti-CD19 antibodies of the present technology have an Fc region with variant glycosylation compared to the parent Fc region. In some embodiments, the variant glycosylation comprises the absence of fucose, and in some embodiments, the variant glycosylation results from expression in GnT1-deficient CHO cells.

[0137] In some embodiments, the antibodies of the present technology may have glycosylation sites that are modified relative to a suitable reference antibody that binds to an antigen of interest (e.g., CD19) without altering the functionality of the antibody, e.g., its binding activity to the antigen. As used herein, a "glycosylation site" includes any particular amino acid sequence in an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) is specifically and covalently attached. Oligosaccharide side chains are usually linked to the antibody backbone by either N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, such as serine or threonine. For example, an Fc-glycoform (hCD19-IgGln) lacking certain oligosaccharides containing fucose and terminal N-acetylglucosamine can be produced in certain CHO cells and exhibit enhanced ADCC effector function.

[0138] In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, for example, U.S. Patent No. 6,218,149, EP 0359096 B1, U.S. Patent Publication No. US 2002 / 0028486, International Patent Application Publication No. WO 03 / 035835, U.S. Patent Publication No. 2003 / 0115614, U.S. Patent No. 6,218,149, and U.S. Patent No. 6,472,511, all of which are incorporated herein by reference in their entireties. In some embodiments, the carbohydrate content of an antibody (or a relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In some specific embodiments, the present technology involves deleting a glycosylation site in the Fc region of an antibody by modifying position 297 from asparagine to alanine.

[0139] Engineered glycoforms can be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycoforms can be made by any method known to those of skill in the art, for example, by using engineered or variant expression strains, by co-expression with one or more enzymes, such as N-acetylglucosaminyltransferase III (GnTIII), by expressing molecules comprising an Fc region in different organisms or cell lines derived from different organisms, or by modifying the carbohydrate(s) after the molecule comprising an Fc region has been expressed. Methods for producing engineered glycoforms are known in the art, including, but not limited to, Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-3473, U.S. Patent No. 6,602,684, U.S. Patent Application No. 10 / 277,370, U.S. Patent Application No. 10 / 113,929, International Patent Application Publication Nos. WO00 / 61739A1, WO01 / 292246A1, WO02 / 311140A1, WO02 / 30954A1, POTILLEGENT™ technology (Biowa, Inc. Princeton, New Jersey), GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland), each of which is incorporated herein by reference in its entirety. See, for example, International Patent Application Publication No. WO 00 / 061739, U.S. Patent Application Publication No. 2003 / 0115614, Okazaki et al., 2004, JMB, 336: 1239-49.

[0140] Fusion Protein. In one embodiment, the anti-CD19 antibody of the present technology is a fusion protein. When fused to a second protein, the anti-CD19 antibody of the present technology can be used as an antigenic tag. Examples of domains that can be fused to a polypeptide include heterologous signal sequences as well as other heterologous functional regions. The fusion does not necessarily have to be direct, but can occur via a linker sequence. Furthermore, the fusion protein of the present technology can also be genetically engineered to improve the characteristics of the anti-CD19 antibody. For example, additional amino acids, particularly a region of charged amino acids, can be added to the N-terminus of the anti-CD19 antibody to improve stability and durability during purification from host cells or subsequent handling and storage. Peptide moieties can also be added to the anti-CD19 antibody to facilitate purification. Such regions can be removed prior to final preparation of the anti-CD19 antibody. The addition of peptide moieties to facilitate handling of polypeptides is well known and routine in the art. The anti-CD19 antibody of the present technology can also be fused to a marker sequence, such as a peptide, to facilitate purification of the fused polypeptide. In selected embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in pQE vectors (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available. For example, hexahistidine provides convenient purification of the fusion protein, as described by Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

[0141] Thus, any of these above fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology. Also, in some embodiments, the fusion proteins described herein exhibit increased half-life in vivo. Fusion proteins with disulfide-bonded dimeric structures (such as IgG) can be more effective at binding and neutralizing other molecules than monomeric secreted proteins or protein fragments alone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

[0142] Similarly, EP-AO 464533 (Canadian counterpart 2045869) discloses fusion proteins containing various portions of the constant region of immunoglobulin molecules together with another human protein or fragment thereof. In many cases, the Fc portion in the fusion protein is beneficial in therapy and diagnosis, and can therefore, for example, result in improved pharmacokinetic properties. See EP-A 0232262. Alternatively, it may be desirable to delete or modify the Fc portion after the fusion protein has been expressed, detected, and purified. For example, the Fc portion may interfere with therapy and diagnosis when the fusion protein is used as an antigen for immunization. In drug discovery, for example, human proteins such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify hIL-5 antagonists. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

[0143] Labeled anti-CD19 antibody. In one embodiment, the anti-CD19 antibody of the present technology is coupled with a labeling moiety, i.e., a detectable group. The particular label or detectable group conjugated to the anti-CD19 antibody is not a critical aspect of the technology, as long as it does not significantly interfere with the specific binding of the anti-CD19 antibody of the present technology to the CD19 protein. The detectable group can be any material with a detectable physical or chemical property. Such detectable labels have been well developed in the fields of immunoassays and imaging. Generally, almost any label useful in such methods can be applied to the present technology. Thus, the label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the implementation of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radiolabels (e.g., 3 H, 14 C. 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc), other contrast agents such as microbubbles (for ultrasound imaging), 18 F, 11 C. 15 O (for positron emission tomography), 99m T.C., 111In (for single photon emission computed tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference in its entirety for all purposes. Also, see Handbook of Fluorescent Probes and Research Chemicals (6 th See also Molecular Probes, Inc., Eugene, OR).

[0144] The label is coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on factors such as the sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal regulations. Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule that is inherently detectable or covalently bound to a signal system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Several ligands and anti-ligands can be used. When the ligand has a neutral anti-ligand, such as biotin, thyroxine, and cortisol, it can be used in conjunction with a labeled, naturally occurring anti-ligand. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, such as an anti-CD19 antibody.

[0145] Molecules can also be directly conjugated to signal-generating compounds, for example, by conjugation with enzymes or fluorophores. Enzymes of interest as labels are primarily hydrolases, particularly phosphatases, esterases, and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moieties include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties include, but are not limited to, luciferin and 2,3-dihydrophthalazinediones, such as luminol. For a summary of various labeling or signal-generating systems that can be used, see U.S. Patent No. 4,391,904.

[0146] Means for detecting labels are well known to those skilled in the art. Thus, for example, if the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. If the label is a fluorescent label, it can be detected by exciting the fluorescent dye with the appropriate wavelength of light and detecting the resulting fluorescence. Fluorescence can be detected visually by photographic film, by using an electronic detector such as a charge-coupled device (CCD) or a photomultiplier tube, etc. Similarly, enzymatic labels can be detected by providing an appropriate substance to the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels can be easily detected by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold appears pink, while various conjugated beads appear the color of the bead. Some assay formats do not require the use of labeled components. For example, agglutination assays can be used to detect the presence of labeled antibodies, such as anti-CD19 antibodies. In this case, antigen-coated particles are agglutinated by a sample containing the target antibody. In this format, no components need to be labeled, and the presence of the labeled antibody is detected by simple visual inspection.

[0147] B. Identification and Characterization of the Anti-CD19 Antibodies of the Present Technology Methods for identifying and / or screening anti-CD19 antibodies of the present technology. Methods useful for identifying and screening antibodies against CD19 polypeptides with the desired specificity for the CD19 protein (e.g., those that bind to an epitope containing amino acid residues corresponding to positions 29-118 of SEQ ID NO: 60 or SEQ ID NO: 61, or the polypeptide encoded by exon 2 of CD19 mRNA) include any immunologically mediated method known in the art. For example, flow cytometry and immunofluorescence or immunohistochemistry can be used. Components of the immune response can be detected in vitro by a variety of methods well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes are incubated with radioactively labeled target cells, and the lysis of these target cells can be detected by the release of radioactivity; (2) helper T lymphocytes are incubated with antigen and antigen-presenting cells, and cytokine synthesis and secretion can be measured by standard methods (Windhagen A et al., Immunity, 2: 373-80, 1995); (3) antigen-presenting cells are incubated with whole protein antigen, and the presentation of that antigen on MHC can be detected by either T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4) mast cells are incubated with a reagent that crosslinks their Fc-epsilon receptors, and histamine release can be measured by enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983) and (5) enzyme-linked immunosorbent assay (ELISA).

[0148] Similarly, the products of an immune response in either a model organism (e.g., a mouse) or a human subject can also be detected by a variety of methods well known to those skilled in the art. For example, (1) the production of antibodies in response to vaccination can be easily detected by standard methods currently used in clinical laboratories, such as ELISA; (2) the migration of immune cells to sites of inflammation can be detected by scratching the surface of the skin and placing a sterile container over the scratch site to capture the migrating cells (Peters et al., Blood, 72:1310-5, 1988); (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactants can be detected by immunohistochemistry. 3 (4) phagocytosis of granulocytes, macrophages and other phagocytes in PBMCs can be measured by placing PBMCs in wells with labeled particles (Peters et al., Blood, 72: 1310-5, 1988); and (5) differentiation of immune system cells can be measured by labeling PBMCs with antibodies against CD molecules such as CD4 and CD8 and measuring the fraction of PBMCs that express these markers.

[0149] In one embodiment, the anti-CD19 antibody of the present technology is selected by displaying CD19 protein on the surface of replicable genetic packaging.For example, see U.S. Patent Nos. 5,514,548, 5,837,500, 5,871,907, 5,885,793, 5,969,108, 6,225,447, 6,291,650, 6,492,160, EP585287, EP605522, EP616640, EP1024191, EP589877, EP774511, EP844306.Methods useful for producing / selecting filamentous bacteriophage particles containing phagemid genomes encoding binding molecules with desired specificity have been described. See, for example, EP 774511, US 5,871,907, US 5,969,108, US 6,225,447, US 6,291,650, US 6,492,160.

[0150] In some embodiments, the anti-CD19 antibodies of the present technology are selected using display of CD19 protein on the surface of yeast host cells. A method useful for isolating Fv polypeptides by yeast surface display is described by Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10. In some embodiments, the anti-CD19 antibody of the present technology is selected using ribosome display.A useful method for identifying a ligand in a peptide library using ribosome display is described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-26, 1994 and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997.

[0151] In certain embodiments, the anti-CD19 antibody of the present technology is selected using tRNA display of CD19 peptide. A useful method for in vitro selection of ligands using tRNA display is described by Merryman et al., Chem. Biol., 9: 741-46, 2002. In one embodiment, the anti-CD19 antibody of the present technology is selected using RNA display.A method useful for selecting peptides and proteins using RNA display libraries is described by Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al., FEBS Lett., 414: 405-8, 1997.A method useful for selecting peptides and proteins using non-natural RNA display libraries is described by Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

[0152] In some embodiments, the anti-CD19 antibodies of the present technology are expressed in the periplasm of Gram-negative bacteria and mixed with labeled CD19 protein. See WO 02 / 34886. Clones expressing recombinant polypeptides with affinity for CD19 protein increase the concentration of labeled CD19 protein that binds to the anti-CD19 antibody, allowing the cells to be isolated from the rest of the library, as described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Patent Publication No. 2004 / 0058403.

[0153] After selection of the desired anti-CD19 antibody, it is contemplated that the antibody can be produced in large quantities by any technique known to those skilled in the art, such as, for example, prokaryotic or eukaryotic expression. For example, but not limited to, an anti-CD19 antibody that is an anti-CD19 hybrid antibody or fragment may be produced using conventional techniques to construct an expression vector encoding an antibody heavy chain, wherein the CDRs and, optionally, the minimum portion of the variable region framework necessary to retain the original species' antibody binding specificity (as genetically engineered according to the techniques described herein) are derived from the original species' antibody, and the remainder of the antibody is derived from a target species immunoglobulin engineered as described herein, thereby generating a vector for expression of the hybrid antibody heavy chain.

[0154] Measuring CD19 Binding. In some embodiments, a CD19 binding assay refers to an assay format in which a CD19 protein and an anti-CD19 antibody are mixed under conditions suitable for binding between the CD19 protein and the anti-CD19 antibody, and the amount of binding between the CD19 protein and the anti-CD19 antibody is assessed. The amount of binding is compared to a suitable control, which can be the amount of binding in the absence of CD19 protein, the amount of binding in the presence of a nonspecific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assays include, for example, ELISA, radioimmunoassay, scintillation proximity assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for direct measurement of CD19 protein binding to an anti-CD19 antibody include, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), etc. Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. If the specific binding of the candidate anti-CD19 antibody is at least 1 percent greater than the binding observed in the absence of the candidate anti-CD19 antibody, the candidate anti-CD19 antibody is useful as an anti-CD19 antibody of the present technology. In some embodiments, the CD19 protein is a CD19 protein containing an extracellular domain (e.g., the two Ig-like C2 loops of CD19) or a CD19 polypeptide containing amino acid residues corresponding to positions 29-118 of SEQ ID NO:60 or SEQ ID NO:61.

[0155] Use of the anti-CD19 antibody of the present technology General. The anti-CD19 antibodies of the present technology are useful in methods known in the art for localizing and / or quantitating CD19 protein (e.g., for use in measuring the level of CD19 protein in an appropriate physiological sample, for use in diagnostic methods, for use in polypeptide imaging, etc.). The antibodies of the present technology are useful for isolating or detecting CD19 protein by standard techniques such as affinity chromatography, immunofluorescence, flow cytometry, immunohistochemistry, or immunoprecipitation. The anti-CD19 antibodies of the present technology can facilitate the purification of native immunoreactive CD19 protein from biological samples, such as mammalian serum or cells, and recombinantly produced immunoreactive CD19 protein expressed in a host system. Furthermore, the anti-CD19 antibodies of the present disclosure can be used to detect immunoreactive CD19 protein (e.g., in plasma, cell lysates, or cell supernatants) to assess the abundance and pattern of expression of the immunoreactive polypeptide. The anti-CD19 antibodies of the present technology can be used diagnostically to monitor immunoreactive CD19 protein levels in tissues as part of a clinical trial procedure, for example, to determine the effectiveness of a given treatment regimen. As described above, detection can be facilitated by coupling (i.e., physically linking) the anti-CD19 antibodies of the present technology to a detectable substance.

[0156] Detection of CD19 protein. An exemplary method for detecting the presence or absence of immunoreactive CD19 protein in a biological sample includes obtaining a biological sample from a test subject and contacting the biological sample with an anti-CD19 antibody of the present technology that can detect immunoreactive CD19 protein, thereby detecting the presence of immunoreactive CD19 protein in the biological sample. Detection can be achieved by a detectable label attached to the antibody. The term "labeled," with respect to anti-CD19 antibodies, is intended to encompass direct labeling of an antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of an antibody by reactivity with another compound, such as a secondary antibody, which is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected using fluorescently labeled streptavidin.

[0157] In some embodiments, the anti-CD19 antibodies disclosed herein are conjugated to one or more detectable labels. For such use, the anti-CD19 antibodies can be detectably labeled by covalent or non-covalent attachment of chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent or other labels. Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6 phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0158] Examples of suitable radioisotope labels include: 3 H, 111 In, 125 I, 131 I, 32 P, 35 S, 14 C. 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 EU, 90 Y,67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Examples include Pd. 111 In is due to the liver 125 I or 131 I-labeled CD19-binding antibodies avoid the problem of dehalogenation, making this an exemplary isotope when in vivo imaging is used. Furthermore, this isotope has a more convenient gamma-emission energy for imaging (Perkins et al., Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, I-labeled CD19-binding antibodies coupled to monoclonal antibodies using 1-(P-isothiocyanatobenzyl)-DPTA are useful. 111 In shows little uptake in non-tumor tissues, especially the liver, enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870(1987)). Examples of suitable non-radioactive isotope labels include: 157 Gd, 55 Mn, 162 Dy, 52 Tr and 56 Fe is one example.

[0159] Examples of suitable fluorescent labels include: 152 Examples of suitable toxin labels include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin. Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels, and aequorin labels. Examples of nuclear magnetic resonance imaging agents include heavy metal nuclei such as Gd, Mn, and iron.

[0160] The detection method of the present technology can be used to detect immunoreactive CD19 protein in biological samples in vitro and in vivo. In vitro methods for detecting immunoreactive CD19 protein include enzyme-linked immunosorbent assay (ELISA), flow cytometry, Western blot, immunohistochemistry, immunoprecipitation, radioimmunoassay, and immunofluorescence. Furthermore, in vivo techniques for detecting immunoreactive CD19 protein include introducing labeled anti-CD19 antibodies into a subject. For example, anti-CD19 antibodies can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In one embodiment, a biological sample contains CD19 protein molecules from a test subject. Immunoassays and imaging. The anti-CD19 antibodies of the present technology can be used to assay immunoreactive CD19 protein levels in biological samples (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be studied using classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101: 976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase and radioisotopes or other radioactive substances, such as iodine ( 125 I,121 I, 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In) and technetium ( 99 mTc) and fluorescent labels such as fluorescein, rhodamine and green fluorescent protein (GFP) and biotin.

[0161] In addition to assaying the immunoreactive CD19 protein level in biological samples, the anti-CD19 antibody of the present technology can be used for in vivo imaging of CD19.The antibodies useful for this method include those that can be detected by X-ray imaging, NMR or ESR.For X-ray imaging, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not obviously harmful to subjects.Suitable markers for NMR and ESR include those with detectable characteristic spins, such as deuterium, which can be incorporated into anti-CD19 antibody by labeling the nutrients of related scFv clones.

[0162] Suitable detectable imaging moieties, such as radioisotopes (e.g., 131 I, 112 In, 99 An anti-CD19 antibody labeled with a radiopaque substance or a material detectable by nuclear magnetic resonance (mTc) is introduced into a subject (e.g., parenterally, subcutaneously, or intraperitoneally). It will be understood in the art that the size of the subject and the imaging system used will dictate the amount of imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is typically about 5-20 millicuries. 99 The range of mTc is reached.Labeled anti-CD19 antibody then accumulates at the location of the cells that contain specific target polypeptide.For example, labeled anti-CD19 antibody of the present technology accumulates in the cells and tissues that CD19 protein is localized in the subject.

[0163] Thus, the present technology provides a method for diagnosing a medical condition, comprising: (a) assaying the expression of immunoreactive CD19 protein in an individual's cells or bodily fluids by measuring binding of an anti-CD19 antibody of the present technology; and (b) comparing the amount of immunoreactive CD19 protein present in the sample with a standard reference, wherein an increase or decrease in the level of immunoreactive CD19 protein compared to the standard indicates the medical condition. Affinity purification. The anti-CD19 antibody of the present technology can be used to purify immunoreactive CD19 protein from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins such as polyacrylamide and latex beads. The technology for coupling antibodies to such solid supports is well known in the art (Weir et al., "Handbook of Experimental Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, NY (1974)). The simplest method for binding an antigen to an antibody-support matrix is ​​to collect beads in a column and allow the antigen solution to flow through the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using a slow flow rate. The immobilized antibody captures the antigen as it flows past. Alternatively, the antigen can be contacted with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or shaking the slurry, which allows for maximum contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed through the column to recover the beads. The beads are washed using an appropriate wash buffer, and then the pure or substantially pure antigen is eluted.

[0164] The target antibody or polypeptide can be conjugated to a solid support such as beads.In addition, the first solid support such as beads can also be conjugated to a second solid support, which can be a second bead or other support, if necessary, by any suitable means, including those disclosed herein for the conjugation of polypeptide with support.Therefore, any of the conjugation methods and means disclosed herein for the conjugation of polypeptide with solid support can be applied to the conjugation of the first support with the second support, and the first and second solid support can be the same or different.

[0165] Suitable linkers that can be used as cross-linking agents to conjugate polypeptides to solid supports include various substances that can react with functional groups present on the surface of the support, with the polypeptide, or with both. Reagents useful as cross-linking agents include homobifunctional and, in particular, heterobifunctional reagents. Useful bifunctional cross-linking agents include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. Cross-linking agents can be selected to provide a selectively cleavable bond between the polypeptide and the solid support. For example, photolabile cross-linking agents, such as 3-amino-(2-nitrophenyl)propionic acid, can be used as a means to cleave the polypeptide from the solid support (Brown et al., Mol. Divers, pp. 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996) and U.S. Patent No. 5,643,722). Other cross-linking reagents are well known in the art (see, eg, Wong (1991), supra; and Hermanson (1996), supra).

[0166] Antibodies or polypeptides can be immobilized on solid supports, such as beads, via a covalent amide bond formed between a carboxyl-functionalized bead and the amino terminus of the polypeptide, or conversely, via a covalent amide bond formed between an amino-functionalized bead and the carboxyl terminus of the polypeptide. Furthermore, a bifunctional trityl linker can be attached to a support, such as a 4-nitrophenyl active ester on a resin, such as a Wang resin, via the amino or carboxyl groups on the resin. Using the bifunctional trityl approach, the solid support may require treatment with a volatile acid, such as formic acid or trifluoroacetic acid, to ensure that the polypeptide can be cleaved and removed. In such cases, the polypeptide can be deposited on the bottom of a well on the solid support or as a bead-free patch on the flat surface of the solid support. After adding a matrix solution, the polypeptide can be released into MS.

[0167] Hydrophobic trityl linkers can also be used as acid-labile linkers by using a volatile acid or an appropriate matrix solution, such as a matrix solution containing 3-HPA, to cleave the amino-linked trityl group from the polypeptide. Acid lability can also be modified. For example, trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl can be modified with an appropriate p-substituted or more acid-labile tritylamine derivative of the polypeptide, i.e., trityl ether and tritylamine bonds are formed on the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker by, for example, disrupting the hydrophobic attraction, or, if necessary, by cleaving the trityl ether or tritylamine bond under acidic conditions, including under typical MS conditions where a matrix such as 3-HPA acts as an acid.

[0168] Orthogonally cleavable linkers can also be useful for binding a first solid support, such as beads, to a second solid support, or for binding a target polypeptide to a solid support.Using such linkers, the first solid support, such as beads, can be selectively cleaved from the second solid support without cleaving the polypeptide from the support, and then the polypeptide can be cleaved from the beads at a later time.For example, a disulfide linker that can be cleaved using a reducing agent such as DTT can be used to bind beads to the second solid support, and an acid-cleavable bifunctional trityl group can be used to immobilize the polypeptide to the support.Optionally, for example, the link between the first and second supports can remain intact, and the link between the polypeptide and the solid support can be cleaved first.The trityl linker can provide covalent or hydrophobic conjugation, but regardless of the nature of the conjugation, the trityl group is easily cleaved under acidic conditions.

[0169] For example, beads can be attached to a second support by a linking group that can be selected to have a length and chemical properties that promote high-density binding of the beads to the solid support or high-density binding of the polypeptide to the beads. Such linking groups can have, for example, a "tree-like" structure, thereby providing a variety of functional groups per attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, pentaerythrol, and tris-hydroxy-aminomethane. Non-covalent association. By non-covalent interaction, an antibody or polypeptide can be conjugated to a solid support, or a first solid support can also be conjugated to a second solid support. For example, magnetic beads made of ferromagnetic materials that can be magnetized can be attracted to a magnetic solid support and can be released from the support by removing the magnetic field. Alternatively, the solid support can be provided with ionic or hydrophobic moieties, which can respectively allow the ionic or hydrophobic moieties to interact with polypeptides, such as polypeptides containing attached trityl groups, or with a second solid support having hydrophobic properties.

[0170] The solid support may also be provided with a member of a specific binding pair and thus be conjugated to a polypeptide or a second solid support containing a complementary binding moiety. For example, avidin- or streptavidin-coated beads can be bound to a polypeptide incorporating a biotin moiety, or to a second solid support coated with biotin or a biotin derivative, such as iminobiotin. It should be understood that any of the binding members disclosed herein or otherwise known in the art may be reversed. Thus, biotin, for example, may be incorporated into either the polypeptide or the solid support, and conversely, avidin or other biotin-binding moiety is incorporated into the support or polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and specifically interacting antigens, and other such pairs known to those of skill in the art.

[0171] A. Diagnostic Uses of the Anti-CD19 Antibodies of the Present Technology General. The anti-CD19 antibodies of the present technology are useful in diagnostic methods. As such, the present technology provides methods for using antibodies in diagnosing CD19 activity in a subject. The anti-CD19 antibodies of the present technology can be selected to have any level of epitope binding specificity and extremely high binding affinity for the CD19 protein. Generally, the higher the binding affinity of the antibody, the more stringent washing conditions can be implemented in immunoassays to remove nonspecifically bound materials without removing the target polypeptide. Thus, the anti-CD19 antibodies of the present technology useful in diagnostic assays typically have a binding affinity of about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 or 10 12 M -1 Additionally, it is desirable for anti-CD19 antibodies used as diagnostic reagents to have a sufficient kinetic on-rate to reach equilibrium under standard conditions in at least 12 hours, at least five (5) hours, or at least one (1) hour.

[0172] Anti-CD19 antibodies can be used to detect immunoreactive CD19 protein in a variety of standard assay formats, including immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, See U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,879,262, 4,034,074, 3,791,932, 3,817,837, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, and 4,098,876. Biological samples can be obtained from any tissue or body fluid of a subject.In certain embodiments, the subject is in the early stage of cancer.In one embodiment, the early stage of cancer is determined by the level or expression pattern of CD19 protein in the sample obtained from the subject.In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF) and biopsied body tissue.

[0173] Immunoassays or sandwich assays are one format for the diagnostic method of this technology. See U.S. Patent Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use one antibody, for example, an anti-CD19 antibody or a population of anti-CD19 antibodies immobilized on a solid phase, and another anti-CD19 antibody or a population of anti-CD19 antibodies in solution. Typically, the solution anti-CD19 antibody or population of anti-CD19 antibodies is labeled. When an antibody population is used, the population may contain antibodies that bind to different epitope specificities within the target polypeptide. Thus, the same population can be used for both the solid phase and the solution antibody. When an anti-CD19 monoclonal antibody is used, first and second CD19 monoclonal antibodies with different binding specificities are used for the solid phase and the solution phase. The solid-phase (also called "capture") and solution (also called "detection") antibodies can be contacted with the target antigen in either order or simultaneously. If the solid-phase antibody is contacted first, the assay is called a forward assay. Conversely, if the solution antibody is contacted first, the assay is called a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is called a simultaneous assay. After contacting the CD19 protein with the anti-CD19 antibody, the sample is usually incubated for a period varying from about 10 minutes to about 24 hours, usually about 1 hour. A wash step is then performed to remove sample components that are not specifically bound to the anti-CD19 antibody being used as a diagnostic reagent. If the solid-phase and solution antibodies are bound in separate steps, washing may be performed after either or both binding steps. After washing, binding is usually quantified by detecting a label linked to the solid phase by binding of the labeled solution antibody. Usually, for a given pair of antibodies or antibody populations and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of target antigen. The concentration of immunoreactive CD19 protein in the sample being tested is then read by interpolation from the calibration curve (i.e., standard curve).The analyte can be determined from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of the bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of CD19 protein in the sample.

[0174] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, as well as particles such as agarose, dextran-based gels, dipsticks, microparticles, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway, NJ), and the like. Immobilization can be by absorption or covalent attachment. Anti-CD19 antibodies can be tethered to a linker molecule such as biotin for attachment to a surface-bound linker such as avidin. In some embodiments, the present disclosure provides an anti-CD19 antibody of the present technology conjugated to a diagnostic agent. The diagnostic agent may include a radioactive or non-radioactive label, a contrast agent (e.g., for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label may be a gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope. The diagnostic agent is an antibody moiety, i.e., a molecule conjugated to an antibody or antibody fragment or subfragment and administered, and is useful in diagnosing or detecting disease by determining the location of cells containing the antigen.

[0175] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (e.g., using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancement agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated to MRI enhancement agents, and is incorporated herein by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancement agents for use in magnetic resonance imaging, and fluorescent compounds. To load an antibody component with a radiometal or paramagnetic ion, it may be necessary to react it with a reagent having a long tail to which multiple chelating groups are attached to bind the ion. Such tails can be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains bearing pendant groups to which chelating groups, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and similar groups known to be useful for this purpose, can be attached. Chelates can be coupled to the antibodies of the present technology using standard chemistry. Chelates are usually linked to antibodies by groups that allow for the formation of bonds with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659. A particularly useful metal-chelate combination includes 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, which are used with diagnostic isotopes for radioimaging. The same chelates, when complexed with non-radioactive metals such as manganese, iron, and gadolinium, are useful for MRI when used with the CD19 antibodies of the present technology.

[0176] Macrocyclic chelates, such as NOTA (1,4,7-triaza-cyclononane-N,N',N"-triacetic acid), DOTA, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), are intended for use with a variety of metals and radiometals, such as radionuclides of gallium, yttrium, and copper, respectively. Such metal chelate complexes can be stabilized by tailoring the ring size to the metal of interest. Other examples of DOTA chelates include: (i) DOTA-Phe-Lys(HSG)-D-Tyr- Lys(HSG)-NH2;(ii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2;(iii)DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2;(iv)DOTA-D-Glu -D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(v)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(vi)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-N H2;(vii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2;(viii)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2;(ix)Ac-D-Phe-D-Lys(DTPA )-D-Tyr-D-Lys(DTPA)-NH2;(x)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2;(xi)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-C ys)-NH2;(xii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2;(xiii)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)- D-Lys(DOTA)-NH2;(xiv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(xv)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(xvi)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2;(xvii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2;(xviii)Ac -D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2; and (xix)Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2. ; For RAIT 223 Other ring-type chelates, such as macrocyclic polyethers, that are of interest for stably binding nuclides such as Ra are also contemplated.

[0177] B. Therapeutic Uses of Anti-CD19 Antibodies of the Present Technology The immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) of the present technology are useful for treating CD19-associated cancers and CD19-associated autoimmune diseases. Such treatments can be used in patients identified as having pathologically high levels of CD19 (e.g., those diagnosed by the methods described herein) or in patients diagnosed with a disease known to be associated with such pathological levels. In one aspect, the present disclosure provides a method for treating CD19-associated cancer or CD19-associated autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology. Examples of cancers that can be treated by the antibodies of the present technology include, but are not limited to, acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, bilineage leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B-lymphoblastic acute leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, marginal zone B-cell non-Hodgkin's lymphoma. Examples of autoimmune diseases that can be treated by the antibodies of the present technology include, but are not limited to, multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus, paraneoplastic syndromes, pemphigus vulgaris, type 2 diabetes, and graft-versus-host disease.

[0178] The composition of the present technology can be used with other therapeutic agents useful in the treatment of CD19-related cancer.For example, the antibody of the present technology can be administered separately, sequentially, or simultaneously with at least one additional therapeutic agent selected from the group consisting of alkylating agents, platinum agents, taxanes, vinca agents, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutic agents, and targeted biological therapeutic agents (for example, therapeutic peptides described in US6306832, WO2012007137, WO2005000889, WO2010096603, etc.).In some embodiments, at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapy agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, elbrigatran, riboflavin ... In some embodiments, the therapeutic agent may be a medicament for treating rheumatoid arthritis, such as bronchodilator, steroids, or other steroids. ... The compositions of the present technology may be administered to a subject in need thereof as a single bolus, or the dosing regimen may include multiple doses administered at various times after the appearance of the tumor.

[0179] The method of treating an autoimmune disease may further comprise administering to the subject, sequentially, separately, or simultaneously, at least one additional therapy selected from a nonsteroidal anti-inflammatory drug (NSAID), a glucocorticoid, a disease-modifying antirheumatic drug (DMARD), an anti-TNF biologic, abatacept, tocilizumab, anakinra, and rituximab. Examples of NSAIDs include: (1) salicylic acid derivatives such as acetylsalicylic acid (aspirin), diflunisal, and sulfasalazine; (2) para-aminophenol derivatives such as acetaminophen; (3) fenamic acids such as mefenamic acid, meclofenamic acid, and flufenamic acid; (4) propionic acid derivatives such as ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen, and oxaprozin; (5) enolic acid (oxicam) derivatives such as piroxicam and tenoxicam; (6) selective COX-2 inhibitors such as meloxicam, salicylic acid, and nimesulide; and (7) highly selective COX-2 inhibitors such as celecoxib, rofecoxib, valdecoxib, lumiracoxib, parecoxib, and etoricoxib. Examples of glucocorticoids include prednisone / prednisolone, methylprednisolone, and fluorinated glucocorticoids such as dexamethasone and betamethasone. Examples of DMARDs include methotrexate, leflunomide, gold compounds, sulfasalazine, azathioprine, cyclophosphamide, antimalarials, D-penicillamine, cyclosporine, hydroxychloroquine, and chloroquine. Examples of anti-TNF biologics include infliximab, etanercept, adalimumab, golimumab, and certolizumab pegol. Administration can be by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intracranially, intratumorally, intrathecally, or topically. Administration includes self-administration and administration by another. It should also be understood that the various modes of treatment of medical conditions as described include complete treatment as well as less-than-complete treatment, and are intended to mean "substantial" in that some biologically or medically relevant result is achieved. In some embodiments, the antibodies of the present technology comprise pharmaceutical formulations that can be administered to a subject in need thereof in one or more doses. Dosage regimens can be adjusted to provide the desired response (e.g., a therapeutic response).

[0180] Typically, an effective amount of the antibody composition of the present technology sufficient to achieve a therapeutic effect ranges from about 0.000001 mg / kilogram of body weight / day to about 10,000 mg / kilogram of body weight / day. Typically, the dosage range is from about 0.0001 mg / kilogram of body weight / day to about 100 mg / kilogram of body weight / day. For administration of an anti-CD19 antibody, the dosage ranges from about 0.0001 to 100 mg / kg of the subject's body weight, more usually 0.01 to 5 mg / kg weekly, every two weeks, or every three weeks. For example, the dosage can be within the range of 1 mg / kg or 10 mg / kg weekly, every two weeks, or every three weeks, or 1 to 10 mg / kg weekly, every two weeks, or every three weeks. In one embodiment, a single dose of the antibody ranges from 0.1 to 10,000 micrograms / kg of body weight. In one embodiment, the antibody concentration in the carrier ranges from 0.2 to 2000 micrograms per milliliter delivered. Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months. The anti-CD19 antibody may be administered multiple times. The interval between single doses may be hourly, daily, weekly, monthly, or yearly. The interval may be irregular, as indicated by measuring the blood level of the antibody in the subject. In some methods, the dosage is adjusted to achieve a serum antibody concentration in the subject of about 75 μg / mL to about 125 μg / mL, 100 μg / mL to about 150 μg / mL, about 125 μg / mL to about 175 μg / mL, or about 150 μg / mL to about 200 μg / mL. Alternatively, the anti-CD19 antibody may be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the subject. The dosage and frequency of administration vary depending on whether treatment is preventive or therapeutic.In preventive application, a relatively low dosage is administered at relatively infrequent intervals for a long period of time.In therapeutic application, a relatively high dosage is sometimes required at relatively short intervals until the progression of disease is reduced or terminated, or until the patient shows partial or complete alleviation of the symptoms of disease.Then, patient can be administered a preventive dosage regimen. In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology, wherein the antibody is configured to localize to a tumor expressing CD19 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a radioactivity level emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as injected dose per gram (%ID / g). The reference value can be calculated by measuring the radioactivity level present in non-tumor (normal) tissue and calculating the average radioactivity level present in the non-tumor (normal) tissue ± standard deviation by computer. In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0181] In some embodiments, the subject has been diagnosed with or is suspected of having cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0182] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle-emitting isotopes include: 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu and 67Examples of alpha particle radioactive isotopes include Cu. 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At and 255 Examples of Auger emitters include Fm. 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl and 203 Pb. In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., by the N297A mutation in the Fc region, which results in aglycosylation). The therapeutic efficacy of such immunoconjugates can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio by computer. In some embodiments, the immunoconjugate has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0183] In one aspect, the present disclosure provides a method for detecting a tumor in a subject in need thereof, the method comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and the CD19 antigen, wherein the conjugate is configured to localize to a tumor that expresses the CD19 antigen recognized by the bispecific antibody of the conjugate; and (b) detecting the presence of a solid tumor in the subject by detecting a level of radioactivity emitted by the conjugate that is higher than a reference value. In some embodiments, the subject is a human.

[0184] In another aspect, the present disclosure provides a method for selecting a subject for pretargeted radioimmunotherapy, the method comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and CD19 antigen, wherein the conjugate is configured to localize in the tumor that expresses the CD19 antigen that is recognized by the bispecific antibody of the conjugate; (b) detecting the radioactivity level emitted by the conjugate; and (c) selecting the subject for pretargeted radioimmunotherapy when the radioactivity level emitted by the conjugate is higher than the reference value.In some embodiments, the subject is human.

[0185] (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2;(ii)A c-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2;(iii)DOTA-D-Asp- D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2;(iv)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(v)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG )-NH2;(vi)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(vii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2;(viii)Ac-D-Phe-NH2; Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2;(ix)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2;(x)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(B z-DTPA)-NH2;(xi)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2;(xii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys scg-Cys)-NH2;(xiii)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2;(xiv)Tscg-D-Cys-D-Glu-D-Lys(GHlu-)-D D-Lys(HSG)-NH2;(xv)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(xvi)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)(DOTA) D-Cys-NH2;(xvii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2;(xviii)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH(xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2 and (xx)DOTA. The radiolabel can be an alpha particle radioisotope, a beta particle radioisotope, or an Auger emitter. Examples of radiolabels include: 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, 255 Fm, 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th or 64 Cu is an example.

[0186] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the conjugate is detected using positron emission tomography or single photon emission computed tomography. Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is diagnosed with acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, hodgkin's lymphoma, leukemia ... The subject is diagnosed with or suspected of having a CD19-associated cancer, such as Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, bilineage leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B-lymphoblastic acute leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, marginal zone B-cell non-Hodgkin's lymphoma, etc. Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is diagnosed with or suspected of having an autoimmune disease, such as multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus, paraneoplastic syndrome, pemphigus vulgaris, type 2 diabetes, or graft-versus-host disease.

[0187] Additionally or alternatively, in some embodiments of the methods disclosed herein, the conjugate is administered intravenously, intramuscularly, intraarterially, intrathecally, intraarticularly, intraorbitally, intradermally, intraperitoneally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In certain embodiments, the conjugate is administered into the cerebrospinal fluid or blood of the subject.

[0188] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the conjugate is detected between 2 and 120 hours after administration of the conjugate. In certain embodiments of the methods disclosed herein, the level of radioactivity emitted by the conjugate is expressed as a percentage of the injected dose per gram of tissue (%ID / g). A reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissue and computing the average level of radioactivity present in the non-tumor (normal) tissue ± the standard deviation. In some embodiments, the reference value is the standard uptake value (SUV). See Thie JA, J Nucl Med. 45(9):1431-4 (2004). In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0189] In another aspect, the present disclosure provides a method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with a CD19-associated cancer, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to tumors expressing the CD19 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. In some embodiments, the subject is a human. The anti-DOTA bispecific antibody is administered (e.g., according to a dosing regimen) under conditions and for a period sufficient to saturate tumor cells. In some embodiments, unbound anti-DOTA bispecific antibody is cleared from the bloodstream after administration of the anti-DOTA bispecific antibody. In some embodiments, a radiolabeled DOTA hapten is administered after a period that may be sufficient to allow clearance of unbound anti-DOTA bispecific antibody.

[0190] The radiolabeled DOTA hapten can be administered any time between 1 minute and 4 or more days after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range therein, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered any time after 4 or more days following administration of the anti-DOTA bispecific antibody.

[0191] Additionally or alternatively, in some embodiments, the method further comprises administering an effective amount of a clearing agent to the subject prior to administration of the radiolabeled DOTA hapten. The clearing agent can be any molecule (dextran, dendrimer, or polymer) that can be conjugated with the C825-hapten. In some embodiments, the clearing agent is 2000 kD, 1500 kD, 1000 kD, 900 kD, 800 kD, 700 kD, 600 kD, 500 kD, 400 kD, 300 kD, 200 kD, 100 kD, 90 kD, 80 kD, 70 kD, 60 kD, 50 kD, 40 kD, 30 kD, 20 kD, 10 kD, or 5 kD or less. In some embodiments, the clarifying agent is a 500 kD aminodextran-DOTA conjugate (such as 500 kD dextran-DOTA-Bn(Y), 500 kD dextran-DOTA-Bn(Lu), or 500 kD dextran-DOTA-Bn(In)).

[0192] In some embodiments, the clearing agent and the radiolabeled DOTA hapten are administered without further administration of the anti-DOTA bispecific antibody of the present technology.For example, in some embodiments, the anti-DOTA bispecific antibody of the present technology is administered according to a regimen that includes at least one cycle of: (i) administration of the anti-DOTA bispecific antibody of the present technology (which may result in saturation of associated tumor cells); (ii) administration of the radiolabeled DOTA hapten and optionally a clearing agent; (iii) any further administration of the radiolabeled DOTA hapten and / or the clearing agent without further administration of the anti-DOTA bispecific antibody.In some embodiments, the method can include multiple such cycles (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0193] Additionally or alternatively, in some embodiments of the method, the anti-DOTA bispecific antibody and / or radiolabeled DOTA hapten is administered intravenously, intramuscularly, intraarterially, intrathecally, intraarticularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, intratumorally, orally, or intranasally. In one aspect, the present disclosure provides a method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with CD19-related cancer, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to the radiolabeled DOTA hapten and a CD19 antigen target, wherein the conjugate is configured to localize to a tumor that expresses the CD19 antigen target recognized by the bispecific antibody of the conjugate. The conjugate can be administered intravenously, intramuscularly, intraarterially, intrathecally, intraarticularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In some embodiments, the subject is a human.

[0194] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to tumors expressing the CD19 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. The anti-DOTA bispecific antibody is administered (e.g., according to a dosing regimen) under conditions and for a period sufficient to saturate tumor cells. In some embodiments, unbound anti-DOTA bispecific antibody is removed from the bloodstream after administration of the anti-DOTA bispecific antibody. In some embodiments, the radiolabeled DOTA hapten is administered after a period that may be sufficient to allow clearance of the unbound anti-DOTA bispecific antibody. In some embodiments, the subject is human.

[0195] Thus, in some embodiments, the method further comprises administering to the subject an effective amount of a clarifying agent prior to administration of the radiolabeled DOTA hapten, which may be administered any time between 1 minute and 4 or more days after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range therein, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered any time after 4 or more days following administration of the anti-DOTA bispecific antibody.

[0196] The clearing agent can be a 500 kD aminodextran-DOTA conjugate (e.g., 500 kD dextran-DOTA-Bn(Y), 500 kD dextran-DOTA-Bn(Lu), or 500 kD dextran-DOTA-Bn(In), etc.). In some embodiments, the clearing agent and radiolabeled DOTA hapten are administered without further administration of an anti-DOTA bispecific antibody. For example, in some embodiments, the anti-DOTA bispecific antibody is administered according to a regimen comprising at least one cycle of: (i) administration of an anti-DOTA bispecific antibody of the present technology (which may result in saturation of associated tumor cells); (ii) administration of a radiolabeled DOTA hapten and optionally a clearing agent; (iii) any further administration of the radiolabeled DOTA hapten and / or clearing agent without further administration of the anti-DOTA bispecific antibody. In some embodiments, the method may include multiple such cycles (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0197] Also provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to the radiolabeled DOTA hapten and CD19 antigen target, wherein the conjugate is configured to localize in the tumor that expresses the CD19 antigen target that is recognized by the bispecific antibody of the conjugate.The therapeutic effectiveness of such a conjugate can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio by computer. In some embodiments, the conjugate has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0198] Toxicity. Optimally, an effective amount (e.g., dose) of an anti-CD19 antibody described herein provides a therapeutic benefit without causing substantial toxicity to the subject. Toxicity of the anti-CD19 antibodies described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., LD 50 (lethal dose for 50% of the population) or LD 100The therapeutic index can be determined by determining the lethal dose (the dose that is lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a non-toxic dosage range for use in humans. The dosage of the anti-CD19 antibody described herein lies within a range of circulating concentrations that includes the effective dose with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the subject's condition. See, for example, Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

[0199] Formulation of Pharmaceutical Compositions. According to the methods of the present technology, the anti-CD19 antibody can be incorporated into a pharmaceutical composition suitable for administration. The pharmaceutical composition generally comprises a recombinant or substantially purified antibody and a pharmaceutically acceptable carrier in a form suitable for administration to a subject. The pharmaceutically acceptable carrier is determined in part by the particular composition to be administered, as well as by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of pharmaceutical compositions for administering antibody compositions (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18 th (See, e.g., U.S. Pat. No. 6,1990.) Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0200] The terms "pharmaceutically acceptable," "physiologically acceptable," and their grammatical variations are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that the material can be administered to a subject without causing any undesirable physiological effects to the extent that they interfere with the administration of the composition. For example, a "pharmaceutically acceptable excipient" generally refers to a safe, non-toxic excipient that is useful in preparing the desired pharmaceutical composition, including excipients that are acceptable for veterinary use and for human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. "Pharmaceutically acceptable salts and esters" refer to salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the composition can react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, such as sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkane- and arene-sulfonic acids, e.g., methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the anti-CD19 antibody, e.g., C 1-6Examples include alkyl esters. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a mono-acid-mono-salt or ester or a di-salt or ester; similarly, when two or more acidic groups are present, some or all of such groups may be salified or esterified. The anti-CD19 antibodies named in this technology may exist in unsalted or unesterified form, or in salified and / or esterified form, and the naming of such anti-CD19 antibodies is intended to include both the original (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain embodiments of the present technology may exist in two or more stereoisomeric forms, and the naming of such anti-CD19 antibodies is intended to include all single stereoisomers and all mixtures (whether racemic or not) of such stereoisomers. Those skilled in the art will have no difficulty in determining the appropriate timing, sequence, and dosage of administration of particular drugs and compositions of the present technology.

[0201] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin.Non-aqueous vehicles such as liposomes and hardened oils can also be used.The use of such media and compounds for pharmaceutically active substances is well known in the art.Unless any conventional media or compound is incompatible with anti-CD19 antibody, its use in the composition is considered.Additional active compounds can also be incorporated into the composition.

[0202] The pharmaceutical composition of the present technology is formulated to be compatible with its intended administration route. The anti-CD19 antibody composition of the present technology can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intrathecally, intraperitoneally, intranasally, or intramuscularly, or as an inhalant. The anti-CD19 antibody can also be administered in combination with other drugs that are at least partially effective in treating various CD19-related cancers or CD19-related autoimmune diseases. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: sterile diluents such as water for injection, saline solution, hydrogenated oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetic acid, citric acid, or phosphate; and compounds for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0203] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is desirable to include isotonic compounds in the composition, for example, sugars, polyalcohols such as mannitol and sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be brought about by including in the composition a compound that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solution can be prepared by incorporating the anti-CD19 antibody of the present technology in a suitable solvent with one or a combination of the above-listed components as needed in the required amount, followed by filtration sterilization.Generally, dispersion is prepared by incorporating the anti-CD19 antibody into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed components.For the preparation of sterile powder for preparing sterile injectable solution, the method of preparation is vacuum drying and lyophilization, which can obtain a powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile-filtered.The antibody of the present technology can be administered in the form of depot injection or indwelling preparation, which can be formulated in a way that allows the sustained or pulsatile release of active ingredient.

[0204] Oral compositions generally contain an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the anti-CD19 antibody may be incorporated with an excipient and used in the form of a tablet, lozenge, or capsule. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash; the compound in the fluid carrier is applied orally, swished, and expectorated or swallowed. Pharmaceutically compatible binding compounds and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating compound such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin, or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the anti-CD19 antibodies are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0205] Systemic administration can be via transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, anti-CD19 antibodies are formulated into ointments, salves, gels, or creams as are commonly known in the art. Anti-CD19 antibodies can also be prepared as pharmaceutical compositions in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0206] In one embodiment, anti-CD19 antibodies are formulated with carriers that protect the anti-CD19 antibodies from rapid elimination from the body, such as controlled-release formulations, 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. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0207] C. Kit The present technology provides a kit for detecting and / or treating CD19-associated cancer or CD19-associated autoimmune disease, comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein), or a functional variant thereof (e.g., a substitution variant). The above-mentioned components of the kit of the present technology may be packaged in a suitable container and labeled for the diagnosis and / or treatment of CD19-associated cancer or CD19-associated autoimmune disease. The above-mentioned components may be stored in unit or multi-dose containers, such as sealed ampoules, vials, bottles, syringes, and test tubes, as aqueous, preferably sterile solutions, or as lyophilized, preferably sterile, formulations for reconstitution. The kit may further comprise a second container holding a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients of the pharmaceutical composition and saline solution. Additionally, the kit may include instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The container may be formed from a variety of materials, such as glass or plastic, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper that can be pierced by a hypodermic injection needle). The kit may further include more containers containing pharmaceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and culture media for one or more suitable hosts. The kit may also include instructions customarily included in commercially available packaging of therapeutic or diagnostic agents, containing information regarding, for example, the indications, usage, dosage, manufacture, administration, contraindications, and / or warnings regarding the use of such therapeutic or diagnostic agents.

[0208] The kit is useful for detecting the presence of immunoreactive CD19 protein in any biological sample, including, but not limited to, serum, plasma, lymph, cyst fluid, urine, stool, cerebrospinal fluid, ascites, or blood, and in any bodily fluid, including a biopsy sample of bodily tissue. For example, the kit may include one or more humanized, chimeric, or bispecific anti-CD19 antibodies (or antigen-binding fragments thereof) of the present technology that can bind to CD19 protein in a biological sample, a means for determining the amount of CD19 protein in the sample, and a means for comparing the amount of immunoreactive CD19 protein in the sample with a standard. One or more of the anti-CD19 antibodies may be labeled. The kit components (e.g., reagents) may be packaged in a suitable container. The kit may further include instructions for using the kit to detect immunoreactive CD19 protein. For antibody-based kits, the kits may include, for example, 1) a first antibody, e.g., a humanized, chimeric, or bispecific CD19 antibody (or antigen-binding fragment thereof) of the present technology attached to a solid support that binds to the CD19 protein, and optionally, 2) a second, different antibody that binds to either the CD19 protein or the first antibody and is conjugated to a detectable label. The kit may also include, for example, a buffer, a preservative, or a protein stabilizer. The kit may further include components necessary for detecting the detectable label, such as an enzyme or a substrate. The kit may also include a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kit may be enclosed in an individual container, and all of the various containers may be in a single package along with instructions for interpreting the results of the assay performed using the kit. The kit of the present technology may include a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, for example, to detect CD19 protein in vitro or in vivo, or to treat CD19-related cancer or CD19-related autoimmune disease in a subject in need thereof. In certain embodiments, the use of the reagents may follow the methods of the present technology. [Example]

[0209] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way. The following examples demonstrate the preparation, characterization, and use of exemplary anti-CD19 antibodies of the present technology. The following examples demonstrate the production of chimeric, humanized, and bispecific antibodies of the present technology, and the characterization of their binding specificity and in vitro and in vivo biological activity.

[0210] Example 1 Sequence and construction of CD19-CD3 bispecific antibodies A CD19-CD3 BsAb was designed using the IgG-scFv modular platform shown in Figure 1. The anti-CD3 humanized OKT3 (huOKT3) single-chain Fv fragment (scFv) was genetically fused to the carboxyl terminus of the light chain of FMC63 IgG1. A humanized version of the murine FMC63 antibody was generated. Sequence elements were linked using various linkers, spacers, etc. The sequences disclosed herein show some examples of modular sequence elements, linkers, spacers, etc. For example, CD19-BsAb was constructed by fusing humanized OKT3 scFv to the C-terminus of the light chain of a chimeric or humanized anti-CD19 antibody via a (G4S)3 linker, as previously described in Xu H et al., Cancer Immunology Research 3:266-277 (2015) and Lopez-Albaitero A et al., OncoImmunology 6:e1267891 (2017). N297A and K322A mutations were introduced into the Fc region of the antibody to eliminate FcR and complement binding activity, respectively (Shields RL et al., Journal of Biological Chemistry 276:6591-6604 (2001); Idusogie EE et al., Journal of Immunology 164:4178-4184 (2000)). When selecting antibodies, scFv fragments and the exact mechanism for joining them, the following factors were taken into consideration, amongst others: (1) Optimal size (100-200 kd) to maximize tumor uptake. See, e.g., Wittrup KD, Thurber GM, Schmidt MM, et al., Methods Enzymol 503:255-68 (2012); (2) bivalency for tumor targets to maintain avidity; (3) a scaffold that spontaneously assembles like any IgG (heavy and light chains) in CHO cells, which can be purified by standard protein A affinity chromatography; (4) a structural configuration that renders the anti-CD3 component functionally monovalent and therefore reduces nonspecific activation of T cells; and (5) A platform with proven tumor targeting efficiency in animal models.

[0211] Humanization of mouse FMC63. The CDRs of the heavy and light chains of FMC63 were transformed into V H Human framework IGHV4-4 * 08-IGHJ4 * 01, and V L Human framework IGKV1-33 * 01-IGKJ2 * Based on homology with .01, the huFMC63 variable heavy chain was grafted onto a human IgG1 framework. From eight heavy chain and three light chain designs, 24 versions of the huFMC63 gene were synthesized and expressed in DG44 cells. The amino acid sequences of the murine, chimeric, and humanized FMC63 variable heavy chains are shown in Figure 12 and Figures 14C-14D. The amino acid sequences of the murine, chimeric, and humanized FMC63 variable light chains are shown in Figure 13 and Figures 14A-14B.

[0212] Example 2 Design of CD19-specific BsAb Construction of CD19-BsAb. CD19-BsAb was designed as follows: the light chain polypeptide of the BsAb contains, from the N-terminus to the C-terminus, (1) a signal peptide, (2) the V domain of the mouse anti-CD19 antibody (FMC63) (or its humanized version),L domain, (3) human C L domain, (4) (G4S)3 linker, (5) V of humanized anti-CD3 antibody OKT3 H domain, (6) (G4S)6 linker, and (7) V of OKT3 L The heavy chain polypeptide of a BsAb contains, in order from the N-terminus to the C-terminus, (1) a signal peptide, (2) the V domain of mouse FMC63 (or its humanized version), H domain, and (3) human CH 1-3 domains. See, e.g., Figures 14A-D and 15A-15B.

[0213] Additional anti-CD19 BsAbs for pretargeted radioimmunotherapy. Two additional categories of CD19 BsAbs include (a) the IgG(L)-scFv platform for three-step pretargeted radioimmunotherapy (PRIT) (PCT / US2018 / 040911) and (b) the multimeric antibody platform for two-step targeting (SADA) (PCT / US2018 / 031235). Both platforms are versatile options for endocytosis of antigens such as HER2 (see, e.g., Cheal SM, Xu H, Guo HF, et al., Eur J Nucl Med Mol Imaging 43:925-37 (2016)) and can be used with different radionuclides, such as alpha-emitter lanthanides (PCT / 2018 / 0409011). Exemplary amino acid sequences of CD19-specific DOTA-binding bispecific antibodies or antigen-binding fragments are shown in Figures 16A-16B through 31A-31B.

[0214] Example 3 Expression and characterization of CD19-CD3-specific BsAb DNA constructs encoding both the heavy and light chains of the CD19-CD3 BsAb were inserted into mammalian expression vectors and transfected into CHO-S cells. Stable clones showing the highest levels of antibody production were selected. The selected stable clones were expanded in shaker flasks. The bispecific antibody was purified from the supernatant collected from the shaker flasks using one-step Protein A affinity chromatography. Purity of CD19-CD3 BsAb. The purity of the CD19-CD3 IgG-scFv bispecific antibody was assayed using size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC). Proteins in the eluate were detected based on the absorbance of ultraviolet light at a wavelength of 280 nm. Figure 1B shows the SEC-HPLC profile of the purified CD19-CD3 bispecific antibody. Fractions from SEC-HPLC were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). As shown in Figure 1B, CD19-BsAb eluted in peak 3 at 15.656 minutes on the chromatogram. Binding of CD19-CD3 bispecific antibodies to CD19(+) cells. Binding of CD19-CD3 bispecific antibodies to the CD19(+) acute lymphoblastic leukemia (ALL) cell line NALM6 was assayed using flow cytometry. An irrelevant bispecific antibody that binds globoH was used as a negative control. NALM6 cells were incubated with the indicated amounts of CD19-BsAb BC250 or control BsAb, followed by incubation with a fluorochrome-conjugated anti-human secondary antibody. Binding was detected using flow cytometry. As shown in Figure 2, increasing concentrations of the control BsAb did not result in an increase in fluorescence compared to the no-antibody control. In contrast, increasing concentrations of CD19-BsAb BC250 from 0.01 to 0.1 μg resulted in a proportional increase in fluorescence, demonstrating binding of CD19-BsAb BC250 to NALM6 cells. See Figure 2.

[0215] Thus, these results demonstrate that the antibodies or antigen-binding fragments of the present technology are useful in methods for detecting CD19 polypeptides in biological samples. The affinity of CD19 antibodies can be adjusted by altering the antibody framework region sequences without changing the CDR sequences. H and three humanized V L The sequence was developed. Humanized V H and V L The sequence has CDR sequences that are identical to the CDR sequences of mouse FMC63, but H or V L They differ from each other with respect to specific amino acid residues within the framework regions (see Figures 12-13).

[0216] Humanized V H and V L The sequences were paired together to generate 24 humanized versions of the CD19-CD3 IgG-scFv bispecific antibody. The affinity of the 24 humanized CD19-CD3 BsAbs for CD19 was assayed using flow cytometry. The V of the murine FMC63 antibody H and V LA CD19-CD3 IgG-scFv bispecific antibody containing the CD19 domain was used as a positive antibody control. The CD19-CD3 IgG-scFv bispecific antibody was incubated with NALM6 cells (CD19 positive). After binding, the cells were washed five times. After each wash, a cell aliquot was removed and stained with a fluorochrome-conjugated anti-human secondary antibody. Binding of the CD19 antibody to NALM6 cells was detected using flow cytometry, and normalized fluorescence intensity was calculated. As shown in Figure 4, there was a spectrum of affinity ranging from low to high for the various humanized CD19-CD3 IgG-scFv bispecific antibody clones. These data demonstrate that the affinity of the humanized CD19 antibody of this technology can be tuned by altering the antibody framework region sequence without changing the CDR sequence. Figure 32 shows the stability, EC50, and MFI data for each CD19-CD3 IgG-scFv bispecific antibody clone against several cell lines. Figures 33A-33B show the pharmacokinetic properties of the BC250 BsAb of the present technology.

[0217] These results demonstrate that the antibodies or antigen-binding fragments of the present technology are useful in methods for detecting CD19 polypeptide in biological samples.

[0218] Example 4 Cytotoxicity of CD19-CD3 IgG-scFv BsAb against CD19 target cells Cytotoxicity of the bispecific CD19-CD3 IgG-scFv antibody BC250. To assess whether the CD19-CD3 BsAb is cytotoxic, T cell cytotoxicity was measured in CD19(+) ALL cells using a standard 4-hour incubation. 51 The Cr release assay was used to test the activity of globoH. An irrelevant bispecific antibody that binds globoH was used as a negative control. NALM6 cells 51 Cr, and increasing concentrations of CD19-BsAb BC250 or control BsAb were administered together with activated T cells. 51 The released ATP was incubated with Cr-loaded NALM6 cells.51 The amount of Cr was quantified, which is an indicator of cell lysis. 51 Cr release was plotted as a function of BsAb concentration. Incubation with control BsAb resulted in a low baseline level of Cr release. 51 Cr was released (see inverted triangles in Figure 3). Incubation with the BC250 clone significantly reduced the release of Cr compared to the control BsAb. 51 Based on these data, the EC2 of CD19-BsAb BC250 for cytolysis of NALM6 cells was 0.01. 50 was calculated to be 42 fM.

[0219] The potency of anti-CD19 BsAbs correlates with their affinity for CD19. To evaluate the effect of antibody affinity on cytotoxic potency, humanized BsAb clones BC250, BC253, BC254, and BC255 were selected from 24 humanized CD19-CD3 IgG-scFv antibodies based on their affinity for CD19. T cell cytotoxicity in CD19(+)NALM6 ALL cells in the presence of different doses of the four BsAbs was measured using a standard 4-hour incubation period. 51 The cytotoxicity of different BsAbs was measured using a Cr release assay. The cytotoxicity of different BsAbs was plotted as a function of BsAb concentration. The mean fluorescence intensity (MFI) observed in NALM6 ALL cells containing the four BsAb clones was measured by flow cytometry. EC 50 Values ​​were calculated and plotted as a function of mean fluorescence intensity (MFI).

[0220] As shown in Figures 5B-5C, the potency of anti-CD19 BsAbs correlated with their affinity for CD19, with BC250 (VL-2, VH-1b) exhibiting the highest cytotoxic potency. Because the MFI value is an indicator of BsAb affinity, clones with higher affinity for CD19 exhibited more potent killing of ALL cells (lower EC 50) (See Figure 5A). Clone BC250 (VL-2+VH-1b) (1) had a higher affinity for CD19, (2) was more stable over time at 40°C (data not shown), and (3) L and V H The humanness of the sequence met the WHO criteria (>85%) and was therefore selected as the lead construct.

[0221] These results collectively demonstrate that the anti-CD19 antibodies or antigen-binding fragments of the present technology can detect CD19(+) tumors and inhibit tumor growth. These results demonstrate that the antibodies or antigen-binding fragments of the present technology are useful in methods for treating CD19-associated cancers in subjects in need thereof.

[0222] Example 5 In vivo therapeutic effect of this technology's CD19-CD3 IgG-scFv BsAb Efficacy of CD19-BsAb (BC250) against human ALL NALM6 in a xenograft mouse model. For in vivo studies, one million NALM6-luciferase-expressing ALL cells were transfected with NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were intravenously injected with NALM6 on day 0. Three days later, mice were imaged (bioluminescence imaging, BLI) and divided into seven treatment groups: (1) activated T cells alone, (2) activated T cells plus 100 ng BC119 (a GD2 × CD3 control BsAb that does not bind to NALM6 cells), (3) activated T cells plus BC250 (0.01 ng), (4) activated T cells plus BC250 (0.1 ng), (5) activated T cells plus BC250 (1 ng), (6) activated T cells plus BC250 (10 ng), and (7) activated T cells plus BC250 (100 ng). Treatment began on day 3 once leukemia was established. Mice received weekly injections of 10 million activated T cells for three weeks. BsAb was administered retroorbitally twice weekly: one dose was mixed with activated T cells, and the other BsAb was injected alone. After the final dose of activated T cells, antibody treatment continued four more times and then stopped. To support T cell survival in vivo, 1000 IU IL2 was administered subcutaneously twice weekly. Leukemia progression was monitored by BLI.

[0223] As shown in Figures 6A-6B, animals receiving ≥1 ng of BC250 BsAb showed a decrease in BLI signal and total flux compared to the group receiving the negative control BC119 BsAb. Therefore, BC250 BsAb can redirect activated T cells to reduce leukemia burden. As shown in Figure 6C, treatment with 0.001 μg (1 ng), 0.01 μg (10 ng), and 0.1 μg (100 ng) BC250 BsAb demonstrated a statistically significant improvement in survival compared to treatment with activated T cells alone (no BsAb control) (p = 0.0047 for activated T cells alone vs. activated T cells / BC250 1 ng). BC250 at 100 ng / dose was found to be curative for NALM6 cells in this in vivo model (p = 0.0082 for activated T cells alone vs. activated T cells / BC250 10 ng and 100 ng).

[0224] Efficacy of CD19-BsAb (BC250) against human Burkitt lymphoma (Daudi) in a xenograft mouse model. One million Daudi cells were transfected with NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were intravenously injected with Daudi cells on day 0. Three days later, mice were imaged (bioluminescence imaging, BLI) and divided into three treatment groups: (1) no treatment (tumor only), (2) activated T cells (T cells) plus 100 ng BC119 (a GD2 × CD3 control BsAb that does not bind to Daudi cells), and (3) activated T cells plus BC250 (100 ng). Once lymphoma was established, treatment began on day 14. Mice received weekly injections of 20 million activated T cells for three weeks. BsAb was administered retroorbitally twice weekly: one injection mixed with activated T cells and the other BsAb injection alone. After the final injection of activated T cells, antibody treatment continued two more times and then stopped. To support in vivo T cell survival, 1000 IU IL2 was administered subcutaneously twice weekly. Lymphoma progression was monitored by BLI. Total flux from mice was plotted as a function of days after injection of Daudi cells. As shown in Figure 7A, 100 ng / dose of BC250 antibody was curative for Daudi xenografts. Mice treated with 100 ng / dose of BC250 antibody showed a substantial reduction in tumor burden compared to control mice treated with a negative control antibody (BC119). See Figure 7B. Furthermore, mice in the group that did not receive BC250 exhibited multiple visible metastases in the kidneys, whereas mice treated with T cells / BC250 did not exhibit kidney metastases. See Figure 7C. Flow cytometry of kidney homogenates revealed extensive lymphoma growth in the kidneys of the control group, but kidneys from the T cell / BC250-treated group were free of lymphoma cells, thus confirming cure in the BC250-treated mice.

[0225] Efficacy of CD19-BsAb (BC250) against the human chronic myeloid leukemia blast crisis (CML) cell line BV173 in a xenograft mouse model. One million BV173-luciferase-expressing cells were transfected with NOD.Cg-Prkdcscid Il2rg tm1Wjl / SzJ(NSG) mice were intravenously injected with IgG on day 0. Three days later, mice were imaged (bioluminescence imaging, BLI) and divided into three treatment groups: (1) no treatment (tumor only), (2) activated T cells (T cells) plus 100 ng BC119 (a GD2 × CD3 control BsAb that does not bind to BV173 cells), and (3) activated T cells plus BC250 (100 ng). Once leukemia was established, treatment began on day 14. Mice received a single injection of 8.8 million activated T cells. BsAb was administered retroorbitally twice weekly: one injection mixed with activated T cells, and the other BsAb injection alone. After the single T cell injection, antibody treatment continued for five additional doses and then stopped. To support in vivo T cell survival, 1000 IU IL2 was administered subcutaneously twice weekly. Leukemia progression was monitored by BLI.

[0226] As shown in Figure 8A, 100 ng / dose of BC250 antibody was curative against BV173 xenografts. Mice treated with 100 ng / dose of BC250 antibody showed increased survival and a substantial reduction in tumor burden compared to control mice treated with a negative control antibody (BC119). See Figures 8B-8C. As shown in Figure 8D, mice in the group that did not receive BC250 exhibited multiple visible metastases in the liver, whereas mice treated with T cells / BC250 exhibited no liver metastases. Flow cytometry of liver homogenates revealed extensive leukemic proliferation in the livers of the control group, but livers from the T cell / BC250-treated group were devoid of leukemic cells, thus confirming a cure in the BC250-treated mice.

[0227] Efficacy of CD19-BsAb (BC250) against human Burkitt lymphoma (Raji) in a xenograft mouse model. One million Raji-luciferase Burkitt lymphoma cells were transfected with NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were intravenously injected on day 0. Three days later, mice were imaged (bioluminescence imaging, BLI) and divided into three treatment groups: (1) no treatment (tumor only), (2) activated T cells (T cells) plus 100 ng BC119 (GD2 × CD3 control BsAb), and (3) activated T cells plus BC250 (100 ng). Treatment began on day 3 once lymphoma was established. Over a 3-week period, mice received three injections of an average of 23 million activated T cells on days 3, 7, and 10. BsAb was mixed with the activated T cells and administered retroorbitally. Two additional doses of BC250 were administered on days 14 and 20. To support in vivo T cell survival, 1000 IU IL2 was administered subcutaneously twice weekly. Lymphoma progression was monitored by BLI.

[0228] As shown in Figures 9A-9B, 100 ng / dose of BC250 antibody reduced the Raji cell leukemia burden in this in vivo xenograft model compared to untreated (tumor only) or negative control BC119 BsAb-treated groups.

[0229] Thus, these results demonstrate that the antibodies or antigen-binding fragments of the present technology are useful in methods of treating CD19-associated cancers in subjects in need thereof.

[0230] Example 6 Superior in vivo therapeutic efficacy of our CD19-BsAb in ALL xenografts compared to the bispecific T cell engager (BiTE) blinatumomab Comparison of the in vitro anti-leukemic efficacy of BC250 BsAb with blinatumomab. Blinatumomab is the only FDA-approved BsAb for leukemia. To compare the efficacy of the bispecific CD19-CD3 IgG-scFv antibody BC250 with blinatumomab, in vitro cytotoxicity in the presence of activated T cells was assayed using CD19(+)NALM6 cells as target cells. Increasing concentrations of the CD19-BsAb BC250 or blinatumomab were administered in the presence of activated T cells. 51Cr-loaded NALM6 cells were incubated for 4 hours. 51 Cr release assay was performed. 51 The Cr release level was plotted as a function of BsAb concentration. As shown in Figure 10A, CD19-BsAb (BC250) BsAb and blinatumomab showed similar killing of CD19 leukemia cells in vitro. Comparison of the anti-leukemic efficacy of BC250 BsAb and blinatumomab in vivo. To compare the in vivo efficacy of CD19-BsAb (BC250) and blinatumomab against ALL xenografts, NSG mice were intravenously injected with 1 million NALM6-luciferase-expressing ALL cells on day 0. Three days later, mice were imaged (bioluminescence imaging, BLI) and divided into seven treatment groups: (1) T cells alone, (2) T cells plus 5 femtomolar BC250, (3) T cells plus 50 femtomolar BC250, (4) T cells plus 500 femtomolar BC250, (5) T cells plus 10 femtomolar blinatumomab, (6) T cells plus 100 femtomolar blinatumomab, and (7) T cells plus 1000 femtomolar blinatumomab. Blinatumomab was used at a 2x dose compared to BC250 to ensure that any observed differences were not due to a lack of the same number of available antigen-binding sites. Treatment began on day 4 once leukemia was established. Over a 3-week period, mice received three injections of 10 million activated T cells on days 4, 11, and 18. BsAb was administered 5 days per week. After the final dose of activated T cells, the antibody was administered 8 more times and then stopped. To support in vivo T-cell survival, 1000 IU IL2 was administered subcutaneously twice a week. Leukemia progression was monitored by BLI.

[0231] As shown in Figure 10B, mice treated with BC250 or blinatumomab showed reduced tumor burden compared to untreated animals in the "tumor only" group. Furthermore, the leukemia burden in mice treated with 5 femtomolar BC250 was reduced compared to mice treated with 10 femtomolar blinatumomab. Similarly, the leukemia burden in mice treated with 50 and 500 femtomolar BC250 was significantly reduced compared to mice treated with 100 and 1000 femtomolar blinatumomab, respectively. See Figure 10B. As shown in Figures 11A-11F, BC250 reduces leukemia burden and increases survival more potently than blinatumomab at all doses tested. Specifically, 50 fmol / dose of BC250 can effectively reduce leukemia burden by 11-fold and 68-fold at 11 and 17 days, respectively, compared to 100 fmol / dose of blinatumomab. Figures 11G-11K further demonstrate that BC250 was superior to blinatumomab for treating ALL xenografts in vivo.

[0232] Figures 34A-34C show a comparison of two bispecific antibodies with N297A / K322A (BC250) and L234A / L235A (BC258) substitutions in T cell-mediated cytotoxicity assays against the CD19(+) leukemia cell lines Daudi, NALM6, and Raji, respectively. Without wishing to be bound by theory, it is believed that silencing the Fc of bispecific antibodies may prevent unwanted killing of T cells by Fc receptor-bearing immune cells or complement activation. As shown in Figures 34A-34C, BC250 is more potent at lysing leukemia cells than BC258 in at least two of the three CD19(+) leukemia cell lines tested. Figures 35A-35C further demonstrate that both the BC250 and BC258 bispecific antibodies showed comparable efficacy in vivo in a mouse leukemia model.

[0233] Thus, these results demonstrate that the antibodies or antigen-binding fragments of the present technology are useful in methods of treating CD19-associated cancers in subjects in need thereof.

[0234] Example 7 Use of anti-CD19 BsAb in PRIT IgG-based CD19-C825 BsAb. CD19(+) leukemia cells are injected into animals via subcutaneous, intraperitoneal, intravenous, or other routes. Treatment will begin after tumor establishment (depending on tumor type and injection route). Treatment consists of one or more cycles. Each cycle involves the administration of the test BsAb (250 μg, intravenous) followed 24-48 hours later by injection of a clearing agent (DOTA-dextran or DOTA-dendrimer; dose is 5-15% of the BsAb dose; see Cheal SM et al., Mol Cancer Ther 13:1803-12, 2014). 4 hours later, DOTA- 177 Lu (up to 1.5 mCi) or DOTA- 225 Ac (1 μCi) will be injected intravenously. 225 Ac is DOTA- 177 It is more potent than Lu and may require fewer cycles to eradicate the tumor. Tetramerized BsAb. CD19(+) leukemia cells are injected into animals subcutaneously, intraperitoneally, intravenously, or by other routes, and treatment begins after tumor establishment (depending on the tumor type and injection route). Treatment consists of one or more cycles. Each cycle consists of administration of BsAb (250 μg, intravenous) followed 24-48 hours later by DOTA- 177 Lu (up to 1.5 mCi) or DOTA- 225 In general, the treatment consisted of an intravenous injection of DOTA-Ac (1 μCi). 225 Ac is DOTA- 177 It is more potent than Lu and may require fewer cycles to eradicate the tumor. These results demonstrate that the anti-CD19 antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastatic progression. Thus, the immunoglobulin-related compositions disclosed herein are useful for detecting and treating CD19-related cancers in subjects in need thereof.

[0235] Example 8 Use of the Anti-CD19 Antibodies of the Present Technology to Treat Autoimmune Diseases Transgenic mice expressing human CD3 on mouse T cells are mated with transgenic mice expressing human CD19 on mouse B cells. The resulting offspring are mated with New Zealand Black (NZB) mice, which naturally develop anemia due to autoantibodies aga...

Claims

1. Heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L and wherein: (a) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, and 12, and V L comprises the amino acid sequence of SEQ ID NO: 17; or (b) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 6, and V L comprises the amino acid sequence of SEQ ID NO: 18; or (c) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 10, 11 and 12, and V L comprises the amino acid sequence of SEQ ID NO: 19, and the antibody or antigen-binding fragment binds to a CD19 polypeptide comprising amino acid residues corresponding to positions 29 to 118 of SEQ ID NO: 60 or SEQ ID NO: 61; The antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody, or the antigen-binding fragment may be a Fab, F(ab') 2 , Fab', scF v and F v may be selected from the group consisting of The bispecific antibody may bind to T cells, B cells, myeloid cells, plasma cells or mast cells, or the bispecific antibody or antigen-binding fragment may bind to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD19, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten, an antibody or antigen-binding fragment thereof.

2. further comprising an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE; The IgG1 may comprise one or more amino acid substitutions selected from the group consisting of N297A and K322A; or The IgG4 may comprise a S228P mutation, or 10. The antibody or antigen-binding fragment of claim 1, wherein the antibody optionally lacks alpha-1,6-fucose modifications.

3. An antibody comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO:26, SEQ ID NO:45, or SEQ ID NO:47, and a light chain (LC) amino acid sequence comprising SEQ ID NO:24, SEQ ID NO:44, or SEQ ID NO:46, the antibody or antigen-binding fragment binds to a CD19 polypeptide comprising amino acid residues corresponding to positions 29-118 of SEQ ID NO:60 or SEQ ID NO:61; or The antibody may lack α-1,6-fucose modifications, or The antibody may be a chimeric antibody, a humanized antibody, or a bispecific antibody, where the bispecific antibody may bind to T cells, B cells, myeloid cells, plasma cells, or mast cells, and the bispecific antibody may bind to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD19, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten antibody.

4. a bispecific antibody or antigen-binding fragment comprising an amino acid sequence selected from any one of SEQ ID NOs: 48-59; or SEQ ID NO:26 and SEQ ID NO:24 (BC250- hFMC63 VL-2 / VH-1b x CD3 BsAb); SEQ ID NO:45 and SEQ ID NO:44 (hFMC63 VL-2VH-1bxmC825); and SEQ ID NO: 47 and SEQ ID NO: 46 (hFMC63 VL-2VH-1b x hC825) The antibody of claim 3, comprising an HC amino acid sequence and an LC amino acid sequence, each selected from the group consisting of:

5. A recombinant nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 4, The recombinant nucleic acid may be selected from the group consisting of SEQ ID NOs: 21, 23, 25 and 27.

6. A host cell or vector comprising the recombinant nucleic acid of claim 5.

7. 10. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 4 and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to a substance selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

8. 10. A pharmaceutical composition comprising the antibody of claim 4 for treating a CD19-associated cancer or a CD19-associated autoimmune disease in a subject in need thereof, The antibody specifically binds to CD19, The CD19-associated cancer may be acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, bilineage leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B-lymphoblastic acute leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, or marginal zone B-cell non-Hodgkin's lymphoma; or The CD19-associated autoimmune disease may be multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus, paraneoplastic syndromes, pemphigus vulgaris, type 2 diabetes, or graft-versus-host disease; or The antibody or antigen-binding fragment may be formulated for separate, sequential or simultaneous administration to a subject with an additional therapeutic agent, a pharmaceutical composition.

9. A pharmaceutical composition for detecting a tumor in a subject in vivo, comprising the antibody or antigen-binding fragment of any one of claims 1 to 4, the antibody or antigen-binding fragment is configured to localize to a tumor expressing CD19 and is labeled with a radioisotope; The subject may have been diagnosed with or be suspected of having a CD19-associated cancer, or The level of radioactivity emitted by the antibody or antigen-binding fragment may be detected using positron emission tomography or single photon emission computed tomography.

10. 10. The pharmaceutical composition of claim 9, the detecting further comprises administering to the subject an effective amount of an immunoconjugate comprising the antibody or antigen-binding fragment of any one of claims 1 to 4 conjugated to a radionuclide; The radionuclide may be an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger emitter, or any combination thereof, where the beta particle-emitting isotope is: 86 Y. 90 Y. 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu and 67 The pharmaceutical composition may be selected from the group consisting of Cu.

11. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 4 and instructions for use, The antibody or antigen-binding fragment of any one of claims 1 to 4 may be coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label; or The kit may further comprise a secondary antibody that specifically binds to the antibody of any one of claims 1 to 4.

12. 5. The bispecific antibody or antigen-binding fragment of claim 1, wherein the bispecific antibody binds to a radiolabeled DOTA hapten and a CD19 antigen.

13. 13. A conjugate comprising a radiolabeled DOTA hapten and the bispecific antibody or antigen-binding fragment of claim 12 for selecting a subject for pretargeted radioimmunotherapy, The complex is configured to localize to CD19-expressing tumors.

14. 13. A conjugate comprising a radiolabeled DOTA hapten and the bispecific antibody or antigen-binding fragment of claim 12 for increasing tumor sensitivity to radiation therapy in a subject diagnosed with a CD19-associated cancer or for treating cancer in a subject in need thereof, wherein the conjugate is configured to localize to CD19-expressing tumors.

15. 13. A pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment of claim 12 for increasing tumor sensitivity to radiation therapy in a subject diagnosed with a CD19-associated cancer or for treating cancer in a subject in need thereof, the method comprising: (a) administering an effective amount of a bispecific antibody or antigen-binding fragment, wherein the bispecific antibody or antigen-binding fragment is configured to localize to CD19-expressing tumors; (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the bispecific antibody or antigen-binding fragment. Including, The method may further comprise administering to the subject an effective amount of a clarifying agent prior to administration of the radiolabeled DOTA hapten, or the subject may be a human.

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