CD19-specific antibodies and chimeric antigen receptors

Abstract

CD19 binding molecules are provided, including anti-CD19 antibodies, including antibody fragments (e.g., single-chain fragments), and chimeric receptors including the antibodies, such as chimeric antigen receptors (CARs). The antibodies include human antibodies, including those that compete with reference antibodies (e.g., mouse antibodies) for binding to CD19. In some embodiments, the antibodies exhibit functional properties similar to those of the reference antibodies, such as comparable binding affinity and / or competitive inhibition properties. Genetically engineered cells expressing the chimeric receptors are also provided, as well as the use of the binding molecules and cells in adoptive cell therapy.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 043,273, filed on August 28, 2014, entitled “CD19-Specific Antibodies and Chimeric Antigen Receptors,” and U.S. Provisional Application No. 62 / 078,942, filed on November 12, 2014, entitled “CD19-Specific Antibodies and Chimeric Antigen Receptors,” the contents of which are incorporated herein by reference in their entireties.

[0003] Incorporation by reference into the sequence listing

[0004] This application is submitted with a sequence listing in electronic format. The provided sequence listing is named 735042000740seqlist.tXt, was created on April 28, 2015, and has a file size of 215 kb. The electronic format of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates in some aspects to CD19 binding molecules, specifically, to anti-CD19 antibodies, including antibody fragments. The present invention also relates to recombinant receptors comprising the antibodies, including chimeric antigen receptors (CARs) containing the antibodies. The present invention also relates to genetically engineered cells expressing the receptors and antibodies, and their use in adoptive cell therapy. background

[0006] CD19 is expressed on normal B cells and is expressed by cells and tissues of various diseases and conditions, including most B cell malignancies. Most patients with B cell malignancies have not been cured by existing therapies, including therapies targeting CD19 and / or other B cell markers. Various CD19-binding molecules, including anti-CD19 antibodies, and chimeric antigen receptors containing anti-CD19 antibody portions, and cells expressing the chimeric receptors, are available. Improved CD19-binding molecules and engineered CD19-targeted cells are needed. For example, molecules and cells with reduced immunogenicity, and / or human antibodies, including antibody fragments that specifically bind to CD19, and chimeric receptors expressing the human antibodies, are needed for adoptive cell therapy. Embodiments that meet the above needs are provided. SUMMARY OF THE INVENTION

[0007] CD19-binding molecules are provided, including polypeptides, such as anti-CD19 antibodies, including antigen-binding antibody fragments, such as single-chain fragments including scFv fragments, and polypeptides comprising the antibodies, including fusion proteins, receptors, such as recombinant receptors, including chimeric receptors such as chimeric antigen receptors (CARs) containing the antibodies as antigen recognition portions. In a specific embodiment, the antibody is a human antibody, such as a human single-chain fragment including scFv.

[0008] Antibodies or antigen-binding fragments thereof are provided, including those that specifically bind to CD19. In some embodiments, the antibodies comprise specific complementary determining regions (CDRs), including heavy chain CDRs (CDR-H) and light chain CDRs (CDR-L). In some embodiments, the CDRs have or include the amino acid sequences of CDRs of a reference antibody or its chain or sequence.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the antibody, e.g., its VH region, comprises a heavy chain complementary determining region 3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the VH region has at least or about 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62, e.g., at least or about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the antibody or fragment comprises the CDR-H1 of SEQ ID NO: 18 and the CDR-H3 of SEQ ID NO: 20. In some embodiments, the antibody or fragment further comprises a CDR-H2 sequence comprising SEQ ID NO: 81, 82, 19, or 72.

[0010] In some embodiments, the antibody has CDR-H1, CDR-H2, and / or CDR-H3 comprising the amino acid sequences of CDR 1, 2, and 3 sequences contained in the heavy chain variable (VH) region of the reference antibody, respectively. In some embodiments, the VH region of the reference antibody has the amino acid sequence set forth in SEQ ID NO: 11 or 12. In some embodiments, it has the amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62.

[0011] In some embodiments, the antibody has, for example, further comprising, CDR-L1, CDR-L2 and / or CDR-L3, each comprising a light chain variable (V L) region. In some embodiments, the V L It has the amino acid sequence shown in SEQ ID NO: 13, 14, 15, 16 or 17. In some embodiments, the VL of the reference antibody has the amino acid sequence shown in SEQ ID NO: 13, 14, 15, 16, 17, 71, 65, 64, 66, 70, 69, 67, 90 or 91.

[0012] In some embodiments, the CDRs in the reference antibody, VH or VL refer to CDRs defined by any numbering scheme, e.g., those defined herein. In some embodiments, the CDRs in the reference antibody, VH or VL refer to CDRs defined by the Kabat numbering scheme described herein, CDRs defined by the Chothia scheme described herein, or CDRs defined by the Contact scheme described herein.

[0013] In some embodiments, the antibody comprises a VH chain comprising CDR-H1, CDR-H2 and / or CDR-H3, wherein CDR-H1 comprises the amino acid sequence DYAMH (SEQ ID NO: 18), or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 18; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 81 or 82 or 19 or 72, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: 81 or to SEQ ID NO: 82 or to SEQ ID NO: 19 or to SEQ ID NO: 72; and / or CDR-H3 comprises the amino acid sequence of SEQ ID NO: 20 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO: NO:20 has a sequence that has at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.

[0014] In some embodiments, the antibody comprises: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 81 or 82, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 20.

[0015] In some embodiments, the antibody has a CDR-1 comprising the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X11 X 12 X 13 X 14 (SEQ ID NO: 110), wherein X1 is T, W, S, or R; X2 is G or A; X3 is I, T, D, or S; X4 is S, R, T, or Q; X5 is empty or S; X6 is empty, D, N, or G; X7 is empty, V, or L; X8 is X or empty; X9 is X or empty; X 10 It is X; X 11 It is X; X 12 is Y, F, D or W; X 13 is V, A or L, and X 14 is S, N or A. For example, in some embodiments, the antibody has a CDR-L1 comprising the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 (SEQ ID NO: 111), wherein X1 is T, Q, S or R; X2 is G or A; X3 is I, T, D or S; X4 is S, R, T or Q; X5 is empty or S; X6 is G, D, N or empty; X7 is empty, V or L; X8 is D, G, I, L, S or empty; X9 is S, G, A, I, R or empty; X 10 is H, Y, F, S or N; X 11 is R, N, D, H or Y; X 12 is Y, F, D or W; X 13 is V, A, or L; and X 14 is S, N or A; and / or

[0016] CDR-L2, the CDR-L2 comprising the following amino acid sequence: X1X2X3X4X5X6X7 (SEQ ID NO: 112), wherein X1 is D or S; X2 is F, V, N, K or A; X3 is S, T, D or N; X4 is K, V, N, Q or R; X5 is R, V or L; X6 is P, K, A or E; and X7 is S, P, A or T, and / or

[0017] CDR-L3, the CDR-L3 comprises the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 (SEQ ID NO: 115), wherein X1 is X; X2 is S, Q, A or T; X3 is Y, S, W, R; X4 is A, D, R, T or Y; X5 is X; X6 is X; X7 is S, P, L, Y, G; X8 is X or empty; X9 is X or empty; X10 L or empty; X 11 is X; and X 12 is V, T or L. For example, in some embodiments, the antibody has a CDR-L3 comprising the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 (SEQ ID NO: 114), wherein X1 is S, G, T, A, Q, C or N; X2 is S, Q, A or T; X3 is Y, S, W, R; X4 is A, D, R, T or Y; X5 is A, S, P, G, N or D; X6 is I, S, G, T, A, L, H, R, N; X7 is S, P, L, Y, G; X8 is P, T, S, Q, M, R, N or is empty; X9 is S, L, N, A, M or is empty; X 10 L or empty; X 11 is Y, W, F, V, A, or L; and X 12 It is V, T or L.

[0018] In some of the embodiments, in the CDR-L1, X3 is I, T, or S; X4 is S, T, or Q; X8 is D, G, I, S, or empty; X9 is S, G, I, or empty; X 10 is H, Y, S or N; X 11 is R, N, D or H; X 12 is Y or D; and X 13 is V or L; and / or, in said CDR-L2, X1 is D; X4 is K, V, N, Q or R; X6 is P, K or A; and X7 is S, A or T; and / or, in said CDR-L3, X1 is S, G, T, A, Q, C or N; X5 is A, S, P, G, N or D; X6 is I, S, G, T, A, L, H, R or N; X8 is P, T, S, Q, M, R, N or is empty; X9 is S, L, N, A, M or is empty; and X 11 is Y, W, F, V, A, or L. In some embodiments, in CDR-L3, X1 is S, G, Q, or N; X2 is S, Q, or T; X4 is A, D, T, or Y; X5 is A, S, or G; and X6 is I, S, N, R, A, H, or T.

[0019] In some embodiments, the CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 19 (GISWNSGRIGYADSVKG); or the CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 72 (GISWNSGSIGYADSVKG).

[0020] In some embodiments, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 80, 77, 74, 73, 75, 79, 78, 76, 21, 25, 28 or 31. In some embodiments, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 80, 77, 74, 73, 78, 21 or 28.

[0021] In some embodiments, the CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 100, 97, 94, 93, 95, 99, 98, 96, 22, 26, 29 or 32. In some embodiments, the CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 100, 97, 94, 93, 98, 22 or 29.

[0022] In some embodiments, the CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 109, 106, 103, 101, 104, 108, 107, 105, 102, 23, 24, 27, 30 or 33. In some embodiments, the CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 109, 106, 103, 101, 107, 24 or 30.

[0023] In some embodiments, the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 23, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 24, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; NO:25, 26 and 27, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO:28, 29 and 30, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO:29, 30, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; NO:31, 32 and 33, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO:80, 100 and 109, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO:80, 100 and 109, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; NO:77, 97 and 106, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO:74, 94 and 103, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively;CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 73, 93 and 101, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 75, 95 and 104, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 75, 95 and 104, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto; NO:79, 99 and 108, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO:78, 98 and 107, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO:78, 98 and 107, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; NO:76, 96 and 105, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequence of SEQ ID NO:73, 93 and 102, respectively, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; or CDR-L1, CDR-L2 and CDR-L3 comprise the sequence of SEQ ID NO:73, 93 and 102, respectively, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively. NO: 77, 97 and 106, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively.

[0024] In some embodiments, the CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 116, 117, 118, 119, 120 or 121, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively.

[0025] In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 81, and 20, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 19, and 20, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; NO:18, 82 and 20, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively; or, CDR-H1, CDR-H2 and CDR-H3 comprise the sequences of SEQ ID NO:18, 72 and 20, respectively, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively.

[0026] In some embodiments, the antibody has a CDR-L1 comprising the amino acid sequence X1GX3X4X5X6X7X8X9X 10 X 11 X 12 X 13 S (SEQ ID NO: 36), wherein X1 is T, S or Q, X3 is T, S or D, X4 is T or S, X5 is empty or S, X6 is empty, D or N, X7 is empty or V, X8 is empty, G or I, X9 is empty, G or R, X 10 Is S, Y or N, X 11 Is D or N, X 12 Is D or Y, X 13 is V or A; CDR-L2 comprises the amino acid sequence X1X2X3X4RPS (SEQ ID NO: 37), wherein X1 is D or S, X2 is V, N or K, X3 is S, N or D and X4 is K, Q or N; and / or, CDR-L3 comprises the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12(SEQ ID NO: 113), wherein X1 is C, S, A, G, or N; X2 is S, A, or T; X3 is Y, W, or R; X4 is A or D; X5 is G, D, or S; X6 is R, S, or N; X7 is Y, L, or G; X8 is N or S; X9 is S, N, or null; X 10 Empty; X 11 is V, A, or W; and X 12 It is L or V.

[0027] In some embodiments, the antibody has a CDR-L1 comprising the amino acid sequence X1GX3X4X5X6X7X8X9X 10 X 11 X 12 X 13 S (SEQ ID NO: 36), wherein X1 is T, S or Q, X3 is T, S or D, X4 is T or S, X5 is empty or S, X6 is empty, D or N, X7 is empty or V, X8 is empty, G or I, X9 is empty, G or R, X 10 Is S, Y or N, X 11 Is D or N, X 12 Is D or Y, X 13 is V or A; CDR-L2 comprises the amino acid sequence X1X2X3X4RPS (SEQ ID NO: 37), wherein X1 is D or S, X2 is V, N or K, X3 is S, N or D and X4 is K, Q or N; and / or CDR-L3 comprises the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 (SEQ ID NO: 38), wherein X1 is C, S, A, G, or N; X2 is S, A, or T; X3 is Y, W, or R; X4 is A or D; X5 is G, D, or S; X6 is R, S, or N; X7 is Y, L, or G; X8 is N or S; X9 is S or empty; X 10 is V, A or N; X 11 is W or empty; and X 12 It is L or V.

[0028] In some of the embodiments, in the CDR-L1, X1 is T or S, X3 is T or S, X 11 Is D or N and X 13 is V; and / or, in said CDR-L2, X2 is V or N and X4 is K or Q.

[0029] In some embodiments, the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:19 (GISWNSGRIGYADSVKG), or a sequence that has at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO:19.

[0030] In some embodiments, the CDR-L1 comprises the sequence of SEQ ID NO: 21, 25, 28 or 31, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto; and / or the CDR-L2 comprises the sequence of SEQ ID NO: 22, 26, 29 or 32, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto; and / or the CDR-L3 comprises the sequence of SEQ ID NO: 23, 24, 26 or 29, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto. The sequence shown in NO:23, 24, 27, 30 or 33, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0031] In some embodiments, the CDR-L1, CDR-L2 and / or CDR-L3 comprise the sequence of SEQ ID NO: 21, 22 and / or 23, respectively, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively.

[0032] In some embodiments, the CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22 and 24, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively.

[0033] In some embodiments, the CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 25, 26 and 27, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively.

[0034] In some embodiments, the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 28, 29, and 30, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively. In some embodiments, the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 31, 32, and 33, respectively, or sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively.

[0035] In some embodiments, the heavy and light chain CDRs are any combination of the aforementioned CDR-L and CDR-H sequences, including any combination of sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0036] In a specific embodiment, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 11, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0037] In a specific embodiment, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 12, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0038] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 13, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0039] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 14, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0040] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 15, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0041] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 16, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0042] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 17, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0043] In a specific embodiment, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 63, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0044] In a specific embodiment, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 60, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0045] In a specific embodiment, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO:61, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0046] In a specific embodiment, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 63, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0047] In a specific embodiment, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO:62, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0048] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO:71, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0049] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 90, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0050] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO:91, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0051] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 68, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0052] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 65, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0053] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 64, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0054] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO:66, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0055] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO:70, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0056] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO:69, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0057] In a specific embodiment, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 67, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0058] In a specific embodiment, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 11, 60, 63 or 62, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto; and / or the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 14, 16, 71, 90, 65, 64 or 69, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0059] In some embodiments, the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 17, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;

[0060] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 15, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto;

[0061] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 13, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto;

[0062] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 14, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto;

[0063] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 16, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto;

[0064] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 71, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;

[0065] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 68, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;

[0066] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto;

[0067] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 60 and 64, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto;

[0068] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 61 and 66, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto;

[0069] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 70, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;

[0070] the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 69, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto;

[0071] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 67, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;

[0072] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 91, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively; or

[0073] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 90, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, respectively.

[0074] In some embodiments, the VH region comprises SEQ ID NO: 11 and the VL region comprises SEQ ID NO: 13; in some embodiments, the VH region comprises SEQ ID NO: 11 and the VL region comprises SEQ ID NO: 14; in some embodiments, the VH region comprises SEQ ID NO: 11 and the VL region comprises SEQ ID NO: 15; in some embodiments, the VH region comprises SEQ ID NO: 11 and the VL region comprises SEQ ID NO: 16, or a sequence having at least or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; in some embodiments, the VH region comprises SEQ ID NO: 11 and the VL region comprises SEQ ID NO: 17, or a sequence having at least or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.

[0075] In some embodiments, the VH region comprises SEQ ID NO: 12 and the VL region comprises SEQ ID NO: 13; in some embodiments, the VH region comprises SEQ ID NO: 12 and the VL region comprises SEQ ID NO: 14; in some embodiments, the VH region comprises SEQ ID NO: 12 and the VL region comprises SEQ ID NO: 15; in some embodiments, the VH region comprises SEQ ID NO: 12 and the VL region comprises SEQ ID NO: 16, or a sequence having at least or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; in some embodiments, the VH region comprises SEQ ID NO: 12 and the VL region comprises SEQ ID NO: 16, or a sequence having at least or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.

[0076] In some embodiments, the antibody is a single-chain fragment, e.g., a single-chain fragment having two or more variable regions joined by one or more flexible immunoglobulin linkers. In some embodiments, the antibody is an scFv. In some embodiments, the scFv comprises a serine- and / or glycine-rich linker, e.g., a linker comprising GGGS (SEQ ID NO: 122) or GGGGS (SEQ ID NO: 123) repeats, e.g., a linker comprising the sequence set forth in SEQ ID NO: 34. In some embodiments, the linker comprises the sequence of SEQ ID NO: 43.

[0077] In some embodiments, the antibody fragment, e.g., scFv, comprises V H region or portion thereof, then the linker, then V L In some embodiments, the antibody fragment, e.g., scFv, comprises V L region or portion thereof, then the linker, then V H area or part thereof.

[0078] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0079] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87 or 89, or a sequence that is at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to said sequence.

[0080] In some embodiments, the antibody or fragment specifically binds to the same, similar, and / or overlapping epitope of CD19 as that specifically bound by the reference antibody, and / or the antibody competes with the reference antibody for binding to CD19. In some aspects, the reference antibody is a murine or chimeric or human or humanized anti-CD19 antibody, FMC63, SJ25C1, an antibody having a variable region sequence of SEQ ID NO: 39 and / or 40, or an antibody having a variable region sequence of SEQ ID NO: 41 and / or 42. In some aspects, the reference antibody comprises an antibody having a sequence as described herein, comprising one or more sequences as described in the preceding embodiments. For example, in some embodiments, the reference antibody can be a scFv comprising an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87, or 89. In some embodiments, the provided antibodies or fragments comprise one or more or all CDRs that are different from those in the reference antibody. For example, in some embodiments, provided antibodies or fragments comprise one or more or all CDRs that differ from the corresponding CDRs in the antibody designated FMC63 or SJ25C1.

[0081] For example, human antibodies and antigen-binding fragments are provided that specifically bind to the same or overlapping epitope of CD19 that is specifically bound by the reference antibody, e.g., FMC63, SJ25C1, an antibody having a variable region sequence of SEQ ID NO: 39 and / or 40, or an antibody having a variable region sequence of SEQ ID NO: 41 and / or 42, and comprising heavy and light chain CDRs that differ from the CDRs present in the reference antibody.

[0082] In some embodiments, the antibody competes for binding with a reference antibody to at least the same extent as the reference antibody competes with itself for binding to CD19, or competes to a degree that is no more than 1.5-fold, or 2-fold, or 3-fold, or 4-fold, or 5-fold, or 10-fold less than that competed by the reference antibody, and / or has a measured IC50 that is no greater than 1.5-fold, or 2-fold, or 3-fold, or 4-fold, or 5-fold, or 10-fold greater than the IC50 for the reference antibody competing with itself for binding, e.g., as measured in the same assay.

[0083] In some embodiments, the binding affinity of the antibody is at least or substantially as high as the binding affinity of the reference antibody to CD19. In some aspects, the EC50 of the binding affinity of the antibody is approximately equal to or lower than the EC50 of the reference antibody, or is no more than about 1.5 times, no more than about 2 times, no more than 3 times, and / or no more than 10 times greater than the EC50 of the reference antibody. In some embodiments, the binding affinity of the antibody is compared to the corresponding form of the reference antibody. Comparison is generally performed by the same or similar assay.

[0084] In some of any of the embodiments, CD 19 is human CD 19. In some of any of the embodiments, the antibody or fragment specifically binds to, exhibits binding affinity for, and / or competes for binding to, human CD 19.

[0085] In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is isolated.

[0086] In some embodiments, the antibody or fragment further comprises at least a portion of an immunoglobulin constant region. The constant region may comprise any one or more of CH1, CH2, CH3, and / or CH4, and / or CL of a human or other antibody and may be of any type, including IgG, IgM, IgA, IgE, and IgD, for example, including a human IgG, e.g., IgG1 or IgG4, constant region domain. In some embodiments, the constant region comprises or is an Fc region, e.g., a human IgG Fc region.

[0087] Also provided are molecules, such as chimeric and / or fusion molecules, comprising receptors, such as recombinant receptors, comprising an antibody of any embodiment (e.g., contained in or as part of an extracellular domain) and other domains, such as an intracellular signaling domain, a spacer, a linker, and / or a transmembrane domain. In some embodiments, the receptor is a chimeric antigen receptor comprising an extracellular portion comprising an antibody or fragment of any embodiment and an intracellular signaling domain.

[0088] In some embodiments, the antibody or fragment comprises scFv. In some embodiments, the intracellular signal transduction domain comprises ITAM and / or signal transduction domain, which can deliver a signal close to the natural connection of a molecule containing ITAM or a receptor complex (such as a TCR receptor complex). In some aspects, the intracellular signal transduction domain comprises the signal transduction domain of the ζ chain of the CD3-zeta (CD3 ζ) chain.

[0089] In some embodiments, the receptor further comprises one or more domains, such as a transmembrane domain, which connects an antibody transmembrane domain, which connects an extracellular domain and an intracellular signal transduction domain. In some aspects, the transmembrane domain comprises the transmembrane portion of a costimulatory molecule, such as a T cell costimulatory molecule, for example, CD28 and / or 41BB. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB and In some embodiments, the receptor includes a signal transduction domain of CD28 and 41BB.

[0090] Also provided are nucleic acids encoding the antibodies (including fragments) of any embodiment, or receptors, e.g., chimeric antigen receptors of any embodiment, vectors comprising the nucleic acids, and cells comprising the vectors and / or nucleic acids, e.g., for expression of the antibodies and / or molecules.

[0091] Therefore, cells and vectors are also provided for producing and expressing the molecules, including the antibodies and molecules, such as receptors, for example, chimeric antigen receptors (CARs). For example, engineered cells are provided that express the chimeric antigen receptors described in any embodiment. In some aspects, the cells are T cells. In some aspects, the cells are NK cells. In some aspects, the cells are stem cells.

[0092] Also provided are compositions comprising the antibodies, receptors, molecules and / or cells, including pharmaceutical compositions, eg, further comprising a pharmaceutically acceptable substance such as a carrier.

[0093] Also provided are methods of administration, including methods of treatment, by administering to a subject a cell, antibody, receptor, composition, or other molecule as described in any embodiment, e.g., in an effective amount, e.g., in a therapeutically effective amount. In some embodiments, the subject has or is suspected of having a disease or disorder associated with CD19, e.g., a B-cell malignancy, e.g., B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphocytic leukemia, non-acute lymphoblastic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, spleen, marginal zone lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, or Hodgkin's lymphoma, or an autoimmune or inflammatory disease in which B cells are involved.

[0094] In some embodiments, administration of the antibody or receptor is associated with a lower degree of immunogenicity compared to administration of a reference antibody (or a receptor comprising the reference antibody) that competes with the antibody for binding to an overlapping epitope. In some aspects, the reference antibody is a humanized, chimeric, or non-human antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1: Figure 1A and 1B Shown are results from binding assays comparing binding of exemplary human scFvs to HEK293 cells expressing CD 19 and to HEK293 cells not expressing CD 19. MFI = mean fluorescence intensity.

[0096] Figure 2 Shown is an SDS gel evaluating the purification of exemplary anti-CD 19 antibodies (scFv fragments).

[0097] Figure 3 : Figure 3 A, 3B, and 3C show the results of studies evaluating the binding affinity of various exemplary scFv antibodies (scFv fragments), including an anti-CD 19 antibody. MFI = mean fluorescence intensity.

[0098] Figure 4 Shown are the results of a study evaluating the binding affinity of various exemplary scFv antibodies, including anti-CD19 scFv antibody fragments. MFI = mean fluorescence intensity.

[0099] Figure 5 : Figure 5 A and 5B show results from competitive binding assays evaluating the binding of correspondingly labeled antibodies in the presence of varying concentrations of competing antibodies. MFI = mean fluorescence intensity.

[0100] Figure 6 Shown are results from a competitive binding assay evaluating the binding of a labeled reference scFv antibody in the presence of varying concentrations of a competing scFv antibody. MFI = mean fluorescence intensity.

[0101] Figure 7 : Figure 7 A shows the results from size exclusion chromatography; the column was calibrated, a standard protein was injected, and the fractions were collected to generate a reference. Figure 7 B shows the results obtained after injecting anti-CD19 scFv (clone 18B) into the same column and collecting the fractions under the same conditions.

[0102] Figure 8AResults from a binding assay evaluating exemplary human scFv clones for binding to cells expressing CD19 are shown, in order from left to right, as follows: cells only, simulated supernatant (Moc. Supe.), negative control antibody (Neg. Ctrl.), clone 18, clones 200 to 287, cells only, Moc. Supe, Neg. Ctrl., and clone 18. Exemplary hits showing specific binding to CD19 (shown with asterisks) are (in order from left to right): clone 213, clone 227, clone 241, clone 255, clone 272, clone 278, clone 283, and clone 285. MFI = mean fluorescence intensity.

[0103] Figure 8B Results from a binding assay evaluating exemplary human scFv clones for binding to cells expressing CD19 are shown, in order from left to right, as follows: cells only, Moc. Supe., negative control antibody (Neg. Ctrl.), clone 18B, clones 300 to 387, cells only, Moc. Supe., Neg. Ctrl., and clone 18B. Exemplary hits showing specific binding to CD19 (shown with asterisks) are (in order from left to right): clone 302, clone 305, clone 313, clone 314, clone 318, clone 324, clone 327, clone 328, clone 336, clone 339, clone 377, clone 379, and clone 382. MFI = mean fluorescence intensity.

[0104] Figure 8C Results from a binding assay evaluating the binding of exemplary human scFv clones to cells expressing CD19 are shown, in order from left to right, as follows: cells only, simulated supernatant (Moc. Supe.), negative control antibody (Neg. Ctrl.), clone 18B, clones 400 to 487, cells only, Moc. Supe., Neg. Ctrl., and clone 18B. Exemplary hits showing specific binding to CD19 (shown with asterisks) are (in order from left to right): clone 440 and clone 448.

[0105] Figure 8D Shown are results from binding assays comparing the binding of exemplary human scFvs to K562 cells expressing CD 19 compared to K562 cells not expressing CD 19. MFI = mean fluorescence intensity.

[0106] Figure 9 Shown is an SDS gel evaluating the purification of exemplary anti-CD 19 antibodies (scFv fragments).

[0107] Figure 10: Figure 10A-E shows results from separate binding assays evaluating the binding affinity of various exemplary scFv antibodies, including anti-CD19 scFv antibody fragments. MFI = mean fluorescence intensity.

[0108] Figure 11 Shown are results from a competitive binding assay evaluating the binding of a labeled reference scFv antibody in the presence of varying concentrations of a competing scFv antibody. MFI = mean fluorescence intensity.

[0109] Figure 12A Figure 2 shows cell surface expression of different CARs, in VH-VL (HL) orientation (HL; dark line) or VL-VH orientation (LH; gray line), in transduced CD8+ T cells, as determined before enrichment (pre) and after sorting with anti-EGFR antibody and using CD19 + The EGFRt expression of cells after B-LCL stimulation and expansion was detected.

[0110] Figure 12B Shown is an SDS gel evaluating the expression of an exemplary human anti-CD19 CAR in transduced primary human T cells.

[0111] Figure 13 A and 13B show primary human CD8 expressing different anti-CD19 specific CARs targeting CD19 expressing cells. + T cell cytolytic activity. C is EGFRt alone (negative control); FM is FMC63 scFv CAR, 18 is clone 18 scFv CAR, 17 is clone 17 scFv CAR, 76 is clone 76 scFv CAR, 5 is clone 5 scFv CAR, and 18B is clone 18B scFv CAR.

[0112] Figure 14 A and 14B show primary human CD8 expressing different anti-CD19 specific CARs after co-culture with CD19 expressing cells. + T cell cytokine secretion. C is EGFRt only (negative control); FM is FMC63 scFv CAR, 18 is clone 18 scFv CAR, 17 is clone 17 scFv CAR, 76 is clone 76 scFv CAR, 5 is clone 5 scFv CAR, and 18B is clone 18B scFv CAR.

[0113] Figure 15 Shows that primary human CD4 T cells expressing different anti-CD19 specific CARs after co-culture with CD19 expressing cells +T cell cytokine secretion. C is EGFRt only (negative control); FM is FMC63scFvCAR, 18 is clone 18scFvCAR, 17 is clone 17scFvCAR, 76 is clone 76scFvCAR, 5 is clone 5scFvCAR, and 18B is clone 18BscFvCAR.

[0114] Figure 16 A and 16B show the proliferation of primary human CD8+ T cells or CD4+ T cells, respectively, expressing different anti-CD19-specific CARs against CD19-expressing cells after co-culture with CD19-expressing cells.

[0115] Figure 17 : Figure 17 A shows the antitumor activity of primary human CD8+ T cells expressing different anti-CD19-specific CARs after administration to NSG mice transplanted with Raji cells expressing firefly luciferase. Figure 17 B shows the antitumor activity of primary human CD4+ and CD8+ T cells expressing different anti-CD19-specific CARs and administered at a 1:1 ratio to NSG mice transplanted with Raji cells.

[0116] Figure 18 : Figure 18 A shows the amino acid sequence of the 74-residue or 75-residue juxtamembrane region of three different chimeric CD19 molecules. Figure 18 Below all three sequences shown in A, positions in the regions where the human and rhesus macaque sequences contain identical amino acids are marked with an asterisk ("*"). Positions where the rhesus macaque sequence contains non-identical but conservative amino acid substitutions compared to the human sequence are marked with a ":". Positions where the rhesus macaque sequence contains non-identical but semi-conservative amino acid substitutions compared to the human sequence are marked with a ".". Positions where the rhesus macaque sequence contains insertions or non-identical, non-conservative / semi-conservative substitutions compared to the human sequence are not marked with a symbol. Figure 18 B shows the expression of primary human CD8 T cells expressing different anti-CD19 specific CARs after co-culture with cells expressing human CD19, rhesus CD19, or chimeric rhesus / human CD19 molecules (V1, V2, or V3). + T cell cytokine secretion. C is EGFRt only (negative control); FM is FMC63 scFv CAR, 18 is clone 18 scFv CAR, 17 is clone 17 scFv CAR, 76 is clone 76 scFv CAR, 5 is clone 5 scFv CAR, and 18B is clone 18B scFv CAR. Detailed Description of the Invention

[0117] Provided are CD19-binding molecules, including antibodies (including antigen-binding antibody fragments, such as single-chain fragments, including scFv) and recombinant receptors, including chimeric receptors containing the antibodies and fragments, nucleic acids encoding the antibodies and fragments, and cells, such as recombinant cells, that express and produce the antibodies and fragments. Also provided are methods of making and using the antibodies and fragments, and cells that express or contain the antibodies and fragments.

[0118] I. CD19 Binding Molecules

[0119] In some aspects, CD19 binding molecules, such as CD19-binding polypeptides, are provided. The binding molecules include antibodies that specifically bind to CD19, such as human CD19 molecules, including antigen-binding fragments thereof. The binding molecules include recombinant receptors, such as chimeric antigen receptors comprising the antibodies.

[0120] A. CD19 Antibody

[0121] Anti-CD19 antibodies are provided, including functional antibody fragments, including those containing variable heavy chains and variable light chains, such as scFv. Molecules comprising the antibodies are also provided, such as fusion proteins and / or recombinant receptors such as chimeric receptors, including antigen receptors. The anti-CD19 antibodies provided include human antibodies. In some embodiments, the antibodies, such as human antibodies, specifically bind to a specific epitope or region of CD19, generally an extracellular epitope or region. In some embodiments, the antibodies bind to the same or similar epitope or region of CD19, as bound by another antibody (such as one or more of a mouse antibody, FMC63 or SJ25C1). In some embodiments, the antibodies bind to overlapping epitopes of CD19, as bound by one of these known antibodies, and / or compete for binding with such antibodies. The antibodies include separated antibodies. The molecules include separated molecules.

[0122] The term "antibody" is used in the broadest sense herein and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv), and single-domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term includes genetically engineered and / or other modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, for example, bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" should be understood to include functional antibody fragments thereof. The term also includes intact or full-length antibodies, including antibodies of any type or subtype, including IgG and its subtypes, IgM, IgE, IgA, and IgD.

[0123] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR", are known in the art, and refer to non-contiguous amino acid sequences within the variable region of an antibody that confer specificity and / or binding affinity to the antibody. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known in the art and refer to the non-CDR portions of the variable region of the heavy and light chains. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.

[0124] The exact amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including the following: Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland ("Kabat" numbering scheme), Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography", J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme), Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme) and Honegger A and Plückthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70 (AHo numbering scheme).

[0125] The boundaries of a given CDR or FR may vary with the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence length, wherein insertion is represented by inserting letters, such as "30a", and deletion occurs in some antibodies. These two schemes place certain insertions and deletions ("insertion-deletion markers (indel)") at different sites, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0126] Table 1 below lists example position boundaries for CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by the Kabat, Chothia, and Contact schemes, respectively. For CDR-H1, the residue numbers are listed using the Kabat and Chothia numbering schemes. FRs are located between CDRs, e.g., FR-L1 is located between CDR-L1 and CDR-L2, etc. Note that because the shown Kabat numbering scheme places the insertion between H35A and H35B, the termini of the Chothi CDR-H1 loop, when numbered using the shown Kabat numbering convention, can differ between H32 and H34, depending on the length of the loop.

[0127] Table 1

[0128]

[0129] 1-Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition. Public Health Service, National Institutes of Health, Bethesda, Maryland

[0130] 2-Al-Lazikani et al., (1997) JMB 273,927-948

[0131] Therefore, unless otherwise indicated, it is understood that a reference to a "CDR" or "complementarity determining region" or an individual specific CDR (e.g., "CDR-H1, CDR-H2") of a given antibody or region thereof (e.g., a variable region thereof) encompasses a certain (or that specific) complementary determining region defined in any of the aforementioned schemes. For example, when a reference is made to a specific CDR (e.g., CDR-H3) comprising a given V H or V LWhen referring to an amino acid sequence corresponding to an amino acid sequence of a CDR, it is understood that the CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the variable region, as defined in any of the aforementioned schemes. In some embodiments, specific CDR sequences are indicated.

[0132] Similarly, unless otherwise indicated, it is understood that the FR or individual specific FR (e.g., FR-H1, FR-H2) of a given antibody or region thereof (e.g., its variable region) encompasses a certain (or that specific) framework region defined as any known scheme. In some cases, the scheme used to identify one or more specific CDRs or FRs is indicated, such as CDRs defined by Kabat, Chothia, or Contact methods. In other cases, the specific amino acid sequence of a CDR or FR is given.

[0133] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to the antigen. H and V L ) generally have a similar structure, wherein each domain comprises four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al., Kuby Immunology, 6th ed., WHF, p. 91 (2007)). A single V H or V L A domain may be sufficient to confer antigen binding specificity. In addition, a V domain from an antibody that binds a specific antigen may be used. H or V L Domain separation of antibodies that bind to the antigen to screen for complementary V L or V H Domain libraries. See, e.g., Portolano et al., J. immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0134] The antibodies provided include antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. In a specific embodiment, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as an scFv.

[0135] A single domain antibody is an antibody fragment that comprises all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody.

[0136] Antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment comprising a non-naturally occurring arrangement, such as an antibody region or chain having two or more connected by a synthetic linker, such as a peptide linker, and / or those that may not be produced by enzymatic hydrolytic digestion of naturally occurring intact antibodies. In some aspects, the antibody fragment is a scFv.

[0137] A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FRs. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has been humanized, generally to reduce immunogenicity against humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0138] The anti-CD19 antibodies provided include human antibodies. "Human antibodies" are antibodies having an amino acid sequence corresponding to an antibody produced by a human or human cell or using a non-human source (including human antibody libraries) of a human antibody repertoire or other human antibody encoding sequence. The term does not include humanized forms of non-human antibodies comprising non-human antigen binding regions, such as those wherein all or substantially all CDRs are non-human.

[0139] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. The animal typically contains all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or that is present extrachromosomally or randomly integrated into the chromosomes of the animal. In the transgenic animal, the endogenous immunoglobulin locus is generally inactivated. Human antibodies can also be derived from human antibody libraries, including phage display and cell-free libraries, which contain antibody coding sequences derived from a human repertoire.

[0140] The antibody provided comprises monoclonal antibody, comprises monoclonal antibody fragment.Term used herein " monoclonal antibody " refers to the antibody obtained from or present in basic homogeneous antibody group, that is, except comprising naturally occurring sudden change or the possible variant (described variant generally exists with a small amount) that occurs in the monoclonal antibody preparation production process, the individual antibody that described group comprises is identical.Be different from the polyclonal antibody preparation that usually comprises the different antibodies for different epi-positions, each monoclonal antibody in the monoclonal antibody preparation is all for the single epi-position on the antigen.This term is not interpreted as requiring to produce antibody by any ad hoc method.Monoclonal antibody can be prepared by multiple technology, includes but not limited to being produced by hybridoma, recombinant DNA method, phage display and other antibody display methods.

[0141] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, for example, linkers and CD19 binding peptides, may include amino acid residues containing natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, the polypeptide may include modifications to the original or native sequence, as long as the protein retains the desired activity. These modifications may be intentional, for example, through site-directed mutagenesis, or may be accidental, for example, through mutations in the host producing the protein or errors caused by PCR amplification.

[0142] Exemplary anti-CD19 antibodies

[0143] In some embodiments, the anti-CD19 antibody (e.g., antigen-binding antibody fragment) comprises specific heavy chain and / or light chain CDR sequences and / or heavy chain and / or light chain variable (V H or V L ) region sequence. The provided antibodies also include sequences that are at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the sequences.

[0144] In some embodiments, the antibody (e.g., an antigen-binding fragment thereof) comprises a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of a CDR-H3 present in a reference antibody, e.g., a reference antibody having a VH region comprising the amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63 62, 167, or 185, e.g., SEQ ID NO: 11, 12, 60, 61, 63, or 62. In some embodiments, the CDR-H3 comprises SEQ ID NO: 20. In some embodiments, the antibody (e.g., an antigen-binding fragment thereof) has a VH region that has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity (or 100% identity) to the VH region amino acid sequence of the reference antibody (e.g., to the VH region amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63 62, 167 or 185, e.g., the VH region amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63 or 62).

[0145] In some embodiments, the CDR-H1 comprises the amino acid sequence DYAMH (SEQ ID NO: 18), the CDR-H2 comprises the amino acid sequence GISWNSGRIG (SEQ ID NO: 81), GISWNSGSIG (SEQ ID NO: 82), the amino acid sequence shown in SEQ ID NO: 19 (GISWNSGRIGYADSVKG) or the amino acid sequence shown in SEQ ID NO: 72 (GISWNSGSIGYADSVKG), and / or the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 20.

[0146] In some embodiments, provided antibodies comprise a CDR-H3 having the amino acid sequence of SEQ ID NO:20.

[0147] In some embodiments, the antibody comprises a V having an amino acid sequence as set forth in SEQ ID NO: 11 or 12, or a sequence that is at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequence. HIn some embodiments, the antibody (e.g., an antigen-binding fragment thereof) comprises a VH region having an amino acid sequence as set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62, or a sequence that is at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequence. In some embodiments, the antibody (e.g., an antigen-binding fragment thereof) comprises a VH region having an amino acid sequence as set forth in SEQ ID NO: 11, 12, 60, 61, 63, 62, 167, or 185, or a sequence that is at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequence.

[0148] In some embodiments, the antibody comprises a sequence of residues 1-119 of SEQ ID NO: 11, 12, 60, 61, 63, 62, 167, or 185, or a sequence comprising a portion of SEQ ID NO: 11, 12, 60, 61, 63, 62, 167, or 185, including the first three framework regions and the three heavy chain CDRs. In some embodiments, the antibody comprises a sequence of residues 1-119 of SEQ ID NO: 11, 12, 60, 61, 63, or 62, or a sequence comprising a portion of SEQ ID NO: 11, 12, 60, 61, 63, or 62, including the first three framework regions and the three heavy chain CDRs.

[0149] In some embodiments, the anti-CD19 antibody comprises a light chain complementary determining region 1, 2, and / or 3 (CDR-L1, CDR-L2, and / or CDR-L3) having SEQ ID NO: 13, 14, 15, 16, 17, 71, 65, 64, 66, 70, 69, 67, 90, 91, or 187-205, respectively, such as a light chain variable region (V) set forth in SEQ ID NO: 13, 14, 15, 16, or 17 or SEQ ID NO: 13, 14, 15, 16, 17, 71, 90, 91, 68, 65, 64, 66, 70, 69, or 67. L ) region amino acid sequence.

[0150] In some embodiments, the anti-CD19 antibody comprises CDR-L1, CDR-L2 and / or CDR-L3, wherein:

[0151] In some embodiments, CDR-L1 comprises the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14(SEQ ID NO: 110), wherein X1 is T, W, S, or R; X2 is G or A; X3 is I, T, D, or S; X4 is S, R, T, or Q; X5 is empty or S; X6 is empty, D, N, or G; X7 is empty, V, or L; X8 is X or empty; X9 is X or empty; X 10 It is X; X 11 It is X; X 12 is Y, F, D or W; X 13 is V, A, or L, and X 14 is S, N or A. For example, in some embodiments, the CDR-L1 comprises the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 (SEQ ID NO: 226), wherein X1 is T, Q, S or R; X2 is G, A or E; X3 is I, T, A, D or S; X4 is S, R, T, Q, G or I; X5 is empty, S, R or T; X6 is G, D, N or empty; X7 is empty, V, L or I; X8 is D, G, I, L, S or empty; X9 is S, G, A, I, D, R or empty; X 10 is H, Y, F, S or N; X 11 is R, N, D, H, Y or T; X 12 is Y, F, D, W, H, T, or S; X 13 is V, A, or L; and X 14 is S, N or A. In some embodiments, the CDR-L1 comprises the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 (SEQ ID NO: 111), wherein X1 is T, Q, S or R; X2 is G or A; X3 is I, T, D or S; X4 is S, R, T or Q; X5 is empty or S; X6 is G, D, N or empty; X7 is empty, V or L; X8 is D, G, I, L, S or empty; X9 is S, G, A, I, R or empty; X 10 is H, Y, F, S or N; X 11 is R, N, D, H or Y; X 12 is Y, F, D or W; X 13 is V, A, or L; and X 14 It is S, N or A.

[0152] In some embodiments, the CDR-L2 comprises the following amino acid sequence: XXXXXXXXXXXXXXX (SEQ ID NO: 227), wherein X1 is D, S, or G; X2 is F, V, N, K, or A; X3 is S, T, D, or N; X4 is K, V, N, Q, or R; X5 is R, V, or L; X6 is P, K, A, or E; and X7 is S, P, A, or T. In some embodiments, the CDR-L2 comprises the following amino acid sequence: XXXXXXXXXXXXXXX (SEQ ID NO: 112), wherein X1 is D or S; X2 is F, V, N, K, or A; X3 is S, T, D, or N; X4 is K, V, N, Q, or R; X5 is R, V, or L; X6 is P, K, A, or E; and X7 is S, P, A, or T.

[0153] In some embodiments, the CDR-L3 comprises the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 (SEQ ID NO: 228), wherein X1 is S, G, T, A, Q, C, or N; X2 is S, Q, A, or T; X3 is Y, S, W, R; X4 is A, D, R, T, or Y; X5 is A, S, P, G, N, or D; X6 is I, S, G, T, A, L, H, R, or N; X7 is S, P, L, Y, G; X8 is P, T, S, Q, M, R, N, or is empty; X9 is S, L, N, A, M, R, or is empty; X 10 L, D or empty; X 11 is Y, W, F, V, A, or L; and X 12 is V, T, P or L. In some embodiments, the CDR-L3 comprises the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 (SEQ ID NO: 115), wherein X1 is X; X2 is S, Q, A or T; X3 is Y, S, W, R; X4 is A, D, R, T or Y; X5 is X; X6 is X; X7 is S, P, L, Y, G; X8 is X or empty; X9 is X or empty; X 10 L or empty; X 11 is X; and X 12 is V, T or L. For example, in some embodiments, the antibody has a CDR-L3 comprising the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12(SEQ ID NO: 114), wherein X1 is S, G, T, A, Q, C or N; X2 is S, Q, A or T; X3 is Y, S, W, R; X4 is A, D, R, T or Y; X5 is A, S, P, G, N or D; X6 is I, S, G, T, A, L, H, R, N; X7 is S, P, L, Y, G; X8 is P, T, S, Q, M, R, N or is empty; X9 is S, L, N, A, M or is empty; X 10 L or empty; X 11 is Y, W, F, V, A, or L; and X 12 It is V, T or L.

[0154] In some embodiments, in the CDR-L1, for example, in the CDR-L1 shown in SEQ ID NO: 110, 226 or 111, X3 is I, T or S; X4 is S, T or Q; X8 is D, G, I, S or empty; X9 is S, G, I or empty; X 10 is H, Y, S or N; X 11 is R, N, D or H; X 12 is Y or D; and X 13 is V or L; and / or in said CDR-L2, e.g., in the CDR-L2 of SEQ ID NO: 227 or 112, X1 is D; X4 is K, V, N, Q or R; X6 is P, K or A; and X7 is S, A or T; and / or in said CDR-L3, e.g., in the CDR-L3 of SEQ ID NO: 228, 114 or 115, X1 is S, G, T, A, Q, C or N; X5 is A, S, P, G, N or D; X6 is I, S, G, T, A, L, H, R or N; X8 is P, T, S, Q, M, R, N or is empty; X9 is S, L, N, A, M or is empty; and X 11 is Y, W, F, V, A, or L. In some embodiments, in CDR-L3, X1 is S, G, Q, or N; X2 is S, Q, or T; X4 is A, D, T, or Y; X5 is A, S, or G; and X6 is I, S, N, R, A, H, or T.

[0155] In some embodiments, the antibody comprises an amino acid sequence comprising CDR-L1 shown in SEQ ID NO:83, CDR-L2 shown in SEQ ID NO:84, and / or CDR-L3 shown in SEQ ID NO:85.

[0156] In some embodiments, the antibody, e.g., the antibody fragment comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 21, 25, 28, or 31. In some embodiments, the antibody or fragment comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 80, 77, 74, 73, 75, 79, 78, 76, 21, 25, 28, 31, or 146-152, e.g., a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 80, 77, 74, 73, 75, 79, 78, 76, 21, 25, 28, or 31. In some embodiments, the antibody or fragment comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 80, 77, 74, 73, 78, 21, or 28.

[0157] In some embodiments, the antibody or fragment comprises a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 22, 26, 29 or 32. In some embodiments, the antibody or fragment comprises a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 100, 97, 94, 93, 95, 99, 98, 96, 22, 26, 29, 32 or 153-157, e.g., a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 100, 97, 94, 93, 95, 99, 98, 96, 22, 26, 29 or 32. In some embodiments, the antibody or fragment comprises a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 100, 97, 94, 93, 98, 22 or 29.

[0158] In some embodiments, the antibody or fragment comprises a CDR-L3 comprising the sequence of SEQ ID NO: 23, 24, 27, 30, or 33. In some embodiments, the antibody or fragment comprises a CDR-L3 comprising the sequence of SEQ ID NO: 109, 106, 103, 101, 104, 108, 107, 105, 102, 23, 24, 27, 30, 33, 158, or 159, e.g., a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 109, 106, 103, 101, 104, 108, 107, 105, 102, 23, 24, 27, 30, or 33. In some embodiments, the antibody or fragment comprises a CDR-L3 comprising the sequence of SEQ ID NO: 109, 106, 103, 101, 107, 24, or 30.

[0159] In some embodiments, the CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NOs: 21, 22 and 23; CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NOs: 21, 22 and 24; CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NOs: 25, 26 and 27; CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NOs: 28, 29 and 30; or CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NOs: 31, 32 and 33.

[0160] In some embodiments, the CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22 and 23, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22 and 24, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 25, 26 and 27, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 28, 29 and 30, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 31, 32 and 33, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 80, 100 and 109, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 81, 82 and 83, respectively. NO: 77, 97 and 106; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO: 74, 94 and 103 respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO: 73, 93 and 101 respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO: 75, 95 and 104 respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO: 79, 99 and 108 respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO: 78, 98 and 107 respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO: 76, 96 and 105 respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO: 77, 97 and 106 respectively; NO:73, 93 and 102; CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NO:77, 97 and 106; CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NO:163, 164 and 165; CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NO:80, 100 and 109; CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NO:146, 97 and 106; CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NO:28, 153 and 158; CDR-L1, CDR-L2 and CDR-L3 respectively comprise the sequences of SEQ ID NO: NO:74, 94 and 103; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO:147, 154 and 121, respectively;CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 148, 94 and 103, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 75, 95 and 104, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 149, 155 and 119, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 150, 22 and 120, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22 and 159, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 151, 26 and 118, respectively; CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NOs: 152, 26 and 118, respectively. NO:28, 156 and 116; or CDR-L1, CDR-L2 and CDR-L3 comprise the sequences of SEQ ID NO:152, 157 and 117, respectively. ;

[0161] Also provided are antibodies having sequences that are at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the sequences.

[0162] In some embodiments, the CDR-H1, CDR-H2 and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 81 and 20, respectively; the CDR-H1, CDR-H2 and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 19 and 20, respectively; the CDR-H1, CDR-H2 and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 82 and 20, respectively; or the CDR-H1, CDR-H2 and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 72 and 20, respectively.

[0163] Also provided are antibodies having sequences that are at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the sequences.

[0164] In some embodiments, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 11, 12, 60, 61, 63, 62, 167 or 185, such as the amino acid sequence of SEQ ID NO: 11, 12, 60, 61, 63 or 62; and / or the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 71, 90, 91, 68, 65, 64, 66, 70, 69, 67 or 187-205, such as the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 71, 90, 91, 68, 65, 64, 66, 70, 69 or 67. In some embodiments, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 11, 60, 63 or 62; and / or the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 14, 16, 71, 90, 65, 64 or 69.

[0165] Also provided are antibodies having sequences that are at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the sequences.

[0166] In some embodiments, the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 17, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 15, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 13, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 14, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 16, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 71, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 68, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: NO:60 and 64; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO:61 and 66; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO:63 and 70; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO:62 and 69; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO:12 and 67; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO:12 and 91; or the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO:63 and 90. In some embodiments, the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 14, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 16, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 71, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 60 and 64, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 69, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 90, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 167 and 207, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 168 or 63 and 208, respectively;The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NO: 169 or 11 and 209, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NO: 170 or 61 and 210, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NO: 171 or 61 and 211, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NO: 172 and 212, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NO: 173 or 11 and 213, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NO: 174 or 11 and 214, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NO: 175 or 11 and 215, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NO: NO: 176 or 61 and 216 amino acid sequences; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO: 177 or 61 and 217; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO: 178 or 61 and 218; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO: 179 or 61 and 219; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO: 180 or 12 and 220; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO: 181 or 12 and 221; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO: 182 or 11 and 222; the VH and VL regions of the antibody or fragment respectively comprise the amino acid sequences of SEQ ID NO: NO: 183 or 60 and 223; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NO: 184 or 11 and 224, respectively; or the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NO: 185 and 225, respectively.

[0167] Also provided are antibodies having sequences that are at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the sequences.

[0168] In some embodiments, the antibody or fragment comprises V H A region comprising the amino acid sequence of SEQ ID NO: 11 or 12, or residues 1-119 of that sequence, or a sequence that is at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to that sequence.

[0169] In some embodiments, the antibody comprises or further comprises V L A region comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, or 17, or a sequence that is at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequence.

[0170] In some embodiments, the antibody is a single-chain antibody fragment, such as scFv, or a bispecific antibody. In some embodiments, the single-chain antibody comprises one or more linkers that engage two antibody domains or regions, such as a variable heavy chain (VH) region and a variable light chain (VL) region. L ). The linker is typically a peptide linker, for example, a flexible and / or soluble peptide linker. The linker includes those rich in glycine and serine, and / or in some cases, those rich in threonine. In some embodiments, the linker also includes charged residues, such as lysine and / or glutamic acid, which can increase solubility. In some embodiments, the linker also includes one or more proline residues.

[0171] Thus, single-chain antibody fragments, such as scFv and bispecific antibodies, are also provided, particularly human single-chain fragments, which generally comprise two antibody domains or regions (e.g., V H and V L The linker is typically a peptide linker, for example, a flexible and / or soluble peptide linker, such as a linker rich in glycine and serine.

[0172] In some aspects, the linker rich in glycine and serine (and / or threonine) includes at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% of such amino acids. In some embodiments, they include at least or at least about 50%, 55%, 60%, 70% or 75% glycine, serine and / or threonine. In some embodiments, the linker is substantially entirely composed of glycine, serine and / or threonine. The length of the linker is generally about 5 and about 50 amino acids, typically or about 10 and or about 30, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids, and in some examples, a length of 10-25 amino acids. Exemplary linkers include linkers with varying numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO: 123) or GGGS (3GS; SEQ ID NO: 122), such as 2, 3, 4, and 5 repeats of such sequences. Exemplary linkers include those having or consisting of the sequence shown in SEQ ID NO: 34 (GGGGSGGGGSGGGGS). Exemplary linkers also include those having or consisting of the sequence shown in SEQ ID NO: 43 (GSTSGSGKPGSGEGSTKG).

[0173] Therefore, in some embodiments, single-chain fragments, such as scFv, are also provided, which comprise one or more of the aforementioned linkers, such as glycine / serine-rich linkers, including linkers having repeats of GGGS (SEQ ID NO: 122) or GGGGS (SEQ ID NO: 123), such as the linker shown in SEQ ID NO: 34. In some embodiments, the linker has an amino acid sequence comprising the sequence shown in SEQ ID NO: 34.

[0174] The fragment, e.g., scFv, may include V H region or part thereof, followed by said linker, followed by V L or a portion thereof. The fragment, e.g., scFv, may include the V L , then the connector, then V H .

[0175] In some aspects, the scFv has the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10, or a sequence that is at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequence.

[0176] In some aspects, the scFv has the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87 or 89, or a sequence that is at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to said sequence.

[0177] In some aspects, the scFv has an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87, 89, or 207-225, or a sequence that is at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequence.

[0178] In some aspects, the scFv comprises a VH, a linker, and a VL as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87 89, or 207-225, or a sequence that is at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to said sequences, but wherein the VH and VL are arranged in an opposite orientation compared to said sequences, i.e., VL-VH.

[0179] The antibody, e.g., antibody fragment, may comprise at least a portion of an immunoglobulin constant region, e.g., one or more constant region domains. In some embodiments, the constant region comprises a light chain constant region and / or a heavy chain constant region 1 (CH1). In some embodiments, the antibody comprises a CH2 and / or CH3 domain, e.g., an Fc region. In some embodiments, the Fc region is the Fc region of a human IgG (e.g., IgG1 or IgG4).

[0180] In some embodiments, any of the above antibodies (e.g., antibody fragments) are human antibodies or antibody fragments. For example, provided herein are human anti-CD19 antibodies that specifically bind to CD19 (e.g., specifically bind to human CD19).

[0181] In some embodiments of the provided human anti-CD19 antibodies, the human antibody comprises a V Ha region comprising a portion that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and / or a portion that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence encoded by a germline nucleotide human heavy chain J segment; and / or comprising a V L A region comprising a portion that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment, and / or a portion that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment. In some embodiments, the V H The portion of the V region corresponds to CDR-H1, CDR-H2 and / or CDR-H3. H The portion of the region corresponds to framework region 1 (FR1), FR2, FR2 and / or FR4. In some embodiments, the V L The portion of the V region corresponds to CDR-L1, CDR-L2 and / or CDR-L3. L Portions of the region correspond to FR1, FR2, FR2 and / or FR4.

[0182] In some embodiments, the human antibody comprises a CDR-H1 that has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-H1 region in the sequence encoded by the germline nucleotide human heavy chain V segment. For example, the human antibody, in some embodiments, comprises a CDR-H1 that has a sequence that is 100% identical or has no more than one, two or three amino acid differences compared to the corresponding CDR-H1 region in the sequence encoded by the germline nucleotide human heavy chain V segment.

[0183] In some embodiments, the human antibody comprises a CDR-H2 that has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-H2 region in the sequence encoded by the germline nucleotide human heavy chain V segment. For example, the human antibody, in some embodiments, comprises a CDR-H2 that has a sequence that is 100% identical or has no more than one, two or three amino acid differences compared to the corresponding CDR-H2 region in the sequence encoded by the germline nucleotide human heavy chain V segment.

[0184] In some embodiments, the human antibody comprises a CDR-H3 that has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-H3 region in the sequence encoded by the germline nucleotide human heavy chain V segment, D segment and J segment. For example, the human antibody, in some embodiments, comprises a CDR-H3 that has a sequence that is 100% identical or has no more than one, two or three amino acid differences compared to the corresponding CDR-H3 region in the sequence encoded by the germline nucleotide human heavy chain V segment, D segment and J segment.

[0185] In some embodiments, the human antibody comprises a CDR-L1 that has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-L1 region in the sequence encoded by the germline nucleotide human light chain V segment. For example, the human antibody, in some embodiments, comprises a CDR-L1 that has a sequence that is 100% identical or has no more than one, two or three amino acid differences compared to the corresponding CDR-L1 region in the sequence encoded by the germline nucleotide human light chain V segment.

[0186] In some embodiments, the human antibody comprises a CDR-L2 that has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-L2 region in the sequence encoded by the germline nucleotide human light chain V segment. For example, the human antibody, in some embodiments, comprises a CDR-L2 that has a sequence that is 100% identical or has no more than one, two or three amino acid differences compared to the corresponding CDR-L2 region in the sequence encoded by the germline nucleotide human light chain V segment.

[0187] In some embodiments, the human antibody comprises a CDR-L3 that has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-L3 region in the sequence encoded by the germline nucleotide human light chain V segment and J segment. For example, the human antibody, in some embodiments, comprises a CDR-L3 that has a sequence that is 100% identical or has no more than one, two or three amino acid differences compared to the corresponding CDR-L3 region in the sequence encoded by the germline nucleotide human light chain V segment and J segment.

[0188] In some embodiments, the human antibody comprises a framework region comprising a human germline gene segment sequence. For example, in some embodiments, the human antibody comprises a V HRegions, wherein the framework regions, e.g., FR1, FR2, FR3, and FR4, have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with framework regions encoded by human germline antibody segments (e.g., V and / or J segments). In some embodiments, the human antibody comprises a V L Regions, wherein the framework regions, e.g., FR1, FR2, FR3, and FR4, have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the framework regions encoded by human germline antibody segments (e.g., V and / or V segments). For example, in some such embodiments, the V H and / or V L The framework sequence of the sequence differs by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, compared to the framework region encoded by the human germline antibody segment.

[0189] The antibody, e.g., antibody fragment, may comprise at least a portion of an immunoglobulin constant region, e.g., one or more constant region domains. In some embodiments, the constant region comprises a light chain constant region and / or a heavy chain constant region 1 (CH1). In some embodiments, the antibody comprises a CH2 and / or CH3 domain, e.g., an Fc region. In some embodiments, the Fc region is the Fc region of a human IgG (e.g., IgG1 or IgG4).

[0190] Also provided are nucleic acids encoding the antibodies and / or portions thereof (e.g., chains). The nucleic acids provided include those encoding the anti-CD19 antibodies described herein. Nucleic acids may include those encompassing natural and / or non-naturally occurring nucleotides and bases, for example, including those with backbone modifications. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably and refer to polymers of nucleotides. The polymers of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to a linear sequence of nucleotides comprising the nucleic acid molecule or polynucleotide. Exemplary nucleic acids and vectors are those having sequences shown in SEQ ID NO: 1, 3, 5, 7, 9, 44, 46, 48, 50, 52, 54, 56, 58, 86, and 88 and CDR encoding portions thereof, as well as sequences having at least or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto. The nucleic acid may encode a V sequence comprising the antibody (e.g., the light and / or heavy chains of the antibody). L The amino acid sequence of the antibody (eg, the light and / or heavy chain of the antibody) and / or the V H amino acid sequence.

[0191] Also provided are vectors comprising the nucleic acid, host cells comprising the vectors, for example, for producing the antibodies. Also provided are methods for producing the antibodies. In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acid are provided. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises the following (e.g., has been transformed with the following): (1) a vector comprising a V encoding the antibody; L The amino acid sequence of the antibody and the V H or (2) a first vector comprising a V encoding the antibody L A nucleic acid having an amino acid sequence of H In some embodiments, a method for preparing the anti-CD19 antibody is provided, wherein the method comprises: culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expressing the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0192] Also provided are methods for preparing the anti-CD19 antibodies (including Fabs). For the recombinant generation of the anti-CD19 antibodies, nucleic acids encoding the antibodies, e.g., those described above, can be separated and inserted into one or more vectors for further cloning and / or expression in a host cell. The nucleic acids can be easily separated and sequenced using conventional methods (e.g., using oligonucleotide probes that specifically bind to the heavy and light chain genes encoding the antibodies).

[0193] In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are also suitable as cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been modified to mimic or approximate those in human cells, thereby producing antibodies with partial or complete human glycosylation characteristics. See Gerngros S, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0194] Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells; cells; and, NSO cells. In some embodiments, the antibody heavy and / or light chains can be expressed in yeast. See, for example, U.S. Publication No. US 2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to produce the desired post-translational modifications to the heavy and / or light chains. For example, in some embodiments, a polypeptide produced in CHO cells has a higher level of sialylation than the same polypeptide produced in 293 cells.

[0195] In some embodiments, the antibody is produced in a cell-free system. Exemplary cell-free systems are described in, for example, Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0196] Provided embodiments also include vectors and host cells and other expression systems for expressing and producing the antibodies and other binding proteins, including eukaryotic and prokaryotic host cells, including bacteria, filamentous fungi and yeast, and mammalian cells, such as human cells, as well as cell-free expression systems.

[0197] Exemplary features

[0198] In some aspects, antibodies, including antigen-binding fragments, are provided that have one or more specific functional characteristics, such as binding properties, including binding to a specific epitope, such as an epitope that is similar or overlapping with an epitope of another antibody, the ability to compete with other antibodies for binding, and / or a specific binding affinity.

[0199] In some embodiments, the antibody specifically binds to the CD19 protein. In some aspects of the embodiments described herein, CD19 refers to human CD19. In general, observations about an antibody or other binding molecule binding to CD19 or specifically binding to CD19 do not necessarily indicate that it binds to CD19 of all species. For example, in some embodiments, a feature of binding to CD19, such as the ability to specifically bind to it and / or the ability to compete with a reference antibody for binding to it, and / or the ability to bind with a specific affinity or to compete to a specific degree, in some embodiments, refers to the ability with respect to human CD19 protein, and the antibody may not have this feature for CD19 of other species (e.g., monkey or mouse).

[0200] In some embodiments, provided antibodies, including antigen binding fragments, bind to human CD19, for example, bind to an epitope or region of human CD19 (e.g., bind to human CD19 shown in 92 (Accession No. P15391) or an allelic variant or splice variant thereof). In certain embodiments, the anti-CD19 antibodies bind to a CD19 epitope that is conserved between CD19 of different species. In some embodiments, the anti-CD19 antibodies bind to a CD19 epitope that is not conserved or not fully conserved between CD19 of different species, for example, between humans and macaque (Macaca mulatta) (rhesus) CD19.

[0201] In some embodiments, the antibody binds to an epitope comprising one or more amino acids located within (or entirely within) the extracellular domain of CD19 and / or located within (or entirely within) the membrane-proximal region of the extracellular portion of CD19. In some embodiments, the antibody binds to an epitope comprising one or more amino acids located within or entirely within the following: Ig-like domain 2 of CD19, a portion encoded by the fourth exon of CD19, a portion corresponding to positions 176-277 of the human CD19 sequence shown in SEQ ID NO: 92, and / or a portion of the 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 44, 43, 43, 41, or 40 amino acids closest to the membrane of the extracellular portion of CD19. In some embodiments, the portion or domain is required for the antibody to bind to CD19. In some embodiments, the epitope comprises (or further comprises) one or more amino acids located within or entirely within the following portion: Ig-like domain 1 of CD19, the portion encoded by the second exon of CD19, and / or the portion corresponding to positions 20-117 of the human CD19 sequence set forth in SEQ ID NO: 92. In some embodiments, the portion or domain is required for the antibody to bind to CD19. In some embodiments, the antibody specifically binds to a peptide that comprises, consists of, or essentially consists of the sequence of the portion and does not comprise the entire sequence of full-length CD19.

[0202] In some embodiments, the epitope comprises one or more amino acids located in a portion of CD19 corresponding to residues 218-249 of the human CD19 sequence set forth in SEQ ID NO:92 (e.g., a portion having the sequence set forth in SEQ ID NO:143), or comprises such a portion of CD19.

[0203] In some embodiments, the epitope includes one or more amino acids at positions corresponding to the following amino acids at one or more positions of the human CD19 sequence shown in SEQ ID NO: 92: histidine (H) at position 218, alanine (A) at position 236, methionine (M) at position 242, glutamic acid (E) at position 243, proline (P) at position 249, and / or lysine (K) and / or serine (S) at positions 223 and 224. In some embodiments, the amino acids at the one or more positions are important for the binding of the antibody to CD19. In some embodiments, the amino acids in the epitope at the one or more positions correspond to the amino acids at the corresponding positions in the human CD19 sequence shown in SEQ ID NO: 92.

[0204] In some embodiments, the epitope includes an amino acid (e.g., histidine) at a position on CD19 corresponding to the histidine at position 218 of the human CD19 sequence set forth in SEQ ID NO:92; in some embodiments, the amino acid is important for binding of the antibody to CD19.

[0205] In some embodiments, the epitope includes an amino acid (e.g., alanine) at a position on CD19 corresponding to the alanine at position 236 of the human CD19 sequence set forth in SEQ ID NO:92; in some embodiments, the amino acid is important for binding of the antibody to CD19.

[0206] In some embodiments, the epitope includes an amino acid (e.g., methionine) at a position on CD19 corresponding to the methionine at position 242 of the human CD19 sequence set forth in SEQ ID NO:92; in some embodiments, the amino acid is important for binding of the antibody to CD19.

[0207] In some embodiments, the epitope includes an amino acid (e.g., glutamic acid) at a position on CD19 corresponding to glutamic acid at position 243 of the human CD19 sequence set forth in SEQ ID NO:92; in some embodiments, the amino acid is important for binding of the antibody to CD19.

[0208] In some embodiments, the epitope includes an amino acid (e.g., proline) at a position on CD19 corresponding to the proline at position 249 of the human CD19 sequence set forth in SEQ ID NO:92; in some embodiments, the amino acid is important for binding of the antibody to CD19.

[0209] In some embodiments, the epitope comprises amino acids (e.g., lysine and / or serine) at one or both positions corresponding to the lysine and / or serine at positions 223 and 224 of the human CD19 sequence set forth in SEQ ID NO:92; in some embodiments, the amino acids are important for binding of the antibody to CD19.

[0210] In some embodiments, the epitope is equal to, similar to, overlapping with, or comprises one or more of the same amino acids as the epitope that specifically binds to a reference antibody (e.g., FMC63 or SJ25C1). In some embodiments, the same one or more amino acids are important for the binding of the provided antibody to the reference antibody.

[0211] In some embodiments, the extent of binding of an anti-CD19 antibody to an unrelated, non-CD19 protein (e.g., non-human CD19 or other non-CD19 protein) is less than about 40% of the binding of the antibody to human CD19 (e.g., as measured by radioimmunoassay (RIA)). In some embodiments, provided antibodies include antibodies wherein binding to non-human CD19 or other non-CD19 proteins is less than about 30%, less than about 20%, or less than about 10% of the binding of the antibody to human CD19.

[0212] In some embodiments, the properties of the antibodies (including antigen-binding fragments) provided are described with respect to the properties observed for another antibody (e.g., a reference antibody). In some embodiments, the reference antibody is a non-human anti-CD19 antibody, such as a mouse or chimeric or humanized anti-CD19 antibody. In some aspects, the reference antibody is an antibody called FMC63 or an antibody called SJ25C1 (see, e.g., Zola H et al., Immunol Cell Biol. 1991 Dec; 69(Pt 6): 411-22; U.S. Patent 7,446,190), and / or a fragment thereof, such as an scFv fragment thereof, and / or an antibody comprising the following portions: a V H and V L sequences, and / or, the heavy and light chain CDRs of said antibodies.

[0213] For example, in some embodiments, the reference antibody has a V H A region comprising the sequence shown in SEQ ID NO: 39 or 41, or comprising CDR1, CDR2 and / or CDR3 in said sequence, and / or having such V L, which comprises the sequence shown in SEQ ID NO: 40 or 42, or comprises CDR1, CDR2 and / or CDR3 of said sequence. Therefore, in some embodiments, the antibody competes for binding with FMC63 or SJ25C1 or an antigen-binding fragment thereof and / or binds to the same or overlapping epitope of CD19.

[0214] In some embodiments, the reference antibody has a sequence present in an antibody described herein or a portion thereof. For example, in some embodiments, the reference antibody has a light chain variable (V) sequence shown in SEQ ID NO: 13, 14, 15, 16, or 17 and / or a light chain variable (V) sequence shown in SEQ ID NO: 13, 14, 15, 16, 17, 71, 90, 91, 68, 65, 64, 66, 70, 69, or 67. L ) region amino acid sequence, and / or has a heavy chain variable (VH) region shown in SEQ ID NO: 11, 12, 60, 61, 63 or 62. In some embodiments, the antibody has a heavy chain and / or light chain CDR 1, 2 and / or 3 present in the antibody. In some embodiments, the reference antibody can be a scFv comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87 or 89.

[0215] In some embodiments, the antibody further comprises heavy and light chain CDRs that are different from the CDRs present in one or more reference antibodies (e.g., FMC63 and SJ25C1). For example, the antibodies provided include competing for binding and / or binding to those identical or overlapping CD19 epitopes bound to one or more reference antibodies, but comprising different CDRs, e.g., different heavy and / or light chain CDR1s, CDR2s, and CDR3s. In some embodiments, the antibodies provided include heavy and light chain CDRs that are different from the CDRs present in the antibody referred to as FMC63 (e.g., present in the VH region shown in SEQ ID NO: 39 and / or in the VL region shown in SEQ ID NO: 40). In some embodiments, the antibodies provided include heavy and light chain CDRs that are different from the CDRs present in the antibody referred to as SJ25C1 (e.g., present in the VH region shown in SEQ ID NO: 41 and / or in the VL region shown in SEQ ID NO: 42).

[0216] For example, in some embodiments, the antibody specifically binds to an epitope that overlaps with the CD19 epitope bound by the reference antibody, such as an antibody that binds to the same or similar epitope as the reference antibody. In some embodiments, the antibody competes with the reference antibody for binding to CD19.

[0217] In some embodiments, the antibody shows a binding preference for cells expressing CD19 compared to CD19 negative cells (e.g., specific cells known in the art and / or described herein). In some embodiments, the binding preference is observed in the following situations: the degree of binding to cells expressing CD19 is detected to be significantly greater than that of non-CD19 expressing cells. In some embodiments, for example, the fold change of the degree of binding detected by the mean fluorescence intensity in a flow cytometry-based assay and / or the dissociation constant or EC50 for cells expressing CD19 compared to cells not expressing CD19 is at least or about 1.5, 2, 3, 4, 5, 6 or more, and / or is roughly the same, roughly the same, at least the same or at least about the same or greater than the fold change observed for the reference antibody (e.g., the corresponding form of the reference antibody). In some cases, the total degree of binding to CD19 or to cells expressing CD19 is roughly the same, at least the same or greater than that observed for the reference antibody. In any of the provided embodiments, comparison of binding properties (eg, affinity or competition) can be performed by testing in the same or similar assays.

[0218] An antibody "competes for binding" to CD 19 with a reference antibody if it competitively inhibits binding of the reference antibody to CD 19 and / or if the reference antibody competitively inhibits binding of the antibody to CD 19. An antibody competitively inhibits binding of a reference antibody to an antigen if, when present in excess, it detectably inhibits (blocks) the binding of the other antibody to its antigen. The specific degree of inhibition can be determined.

[0219] In some embodiments, an excess of the provided antibody, e.g., 1, 2, 5, 10, 50, or 100-fold, compared to the amount or concentration of the reference antibody, inhibits binding of the antigen to the reference antibody (or vice versa). In some embodiments, the inhibition of binding is at least 50%, and in some embodiments, at least 75%, 90%, or 99%. In some aspects, competitive inhibition is measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990: 50: 1495-1502).

[0220] In some embodiments, if the reference antibody is present at a concentration of 10 nM, the provided antibody inhibits binding of the reference antibody with an IC50 of less than about or less than 100, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nM or less than about or less than 9, 8, 7, 6 or 5 nM. In some embodiments, if the provided antibody is present at a concentration of 10 nM, the reference antibody inhibits binding of the provided antibody with an IC50 of less than about or less than 100, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nM or less than about or less than 9, 8, 7, 6 or 5 nM.

[0221] In some embodiments, the competitive inhibition of binding of the provided antibody to the reference antibody (or vice versa) is or is about or is at least or is about the same degree as the competitive inhibition of binding of the reference antibody itself (e.g., an unlabeled reference antibody) to the reference antibody. In some embodiments, the provided antibody inhibits the binding of the reference antibody (e.g., FMC63 or SJ25C1) to human CD19 by at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Competitive inhibition assays are known and include ELISA-based, flow cytometry-based, and RIA-based assays. In some aspects, competitive inhibition assays are performed by incorporating an excess of one of the unlabeled forms of the antibody and assessing its ability to suppress the binding of the other antibody, which is labeled with a detectable marker so that its binding and reduction can be assessed by detecting the marker or marker.

[0222] In some embodiments, if all or substantially all amino acid mutations in an antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody, then the two antibodies specifically bind to the same epitope. In some embodiments, if at least some of the amino acid mutations in the antigen that reduce or eliminate binding of the antigen to one antibody also reduce or eliminate binding of the antigen to the other antibody, then the two antibodies specifically bind to overlapping epitopes.

[0223] In some embodiments, provided antibodies are capable of binding to CD19, such as human CD19, with at least a certain affinity (as measured by any of a variety of known methods). In some embodiments, the affinity is represented by a dissociation constant (Kd); in some embodiments, the affinity is represented by an EC50. In certain embodiments, the binding affinity (EC50) and / or dissociation constant of the antibody to CD19 is or is about or is less than about or is less than 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM, such as from or about 1 nM to or about 15 nM, such as from or about 5 to or about 10 nM. In one embodiment, the extent of binding of an anti-CD 19 antibody to an unrelated, non-CD 19 protein is less than about or less than 10% of the binding of the antibody to CD 19, e.g., as detected by radioimmunoassay (RIA).

[0224] In some aspects, the affinity is or is about the same or substantially the same as the affinity of a reference antibody (e.g., a murine CD19 antibody, such as FMC63 or SJ25C1). In some aspects, the affinity is at least 80%, 85%, 90%, 95%, or 99% the same as the affinity of the reference antibody. In some embodiments, the binding affinity is compared to the corresponding form of the reference antibody.

[0225] In some embodiments, the affinity, e.g., EC50 or Kd, of the antibody is approximately equal to or lower than the affinity of a reference antibody (e.g., a corresponding form of the reference antibody), e.g., no more than about 1.5-fold or no more than about 2-fold greater, no more than 3-fold greater, and / or no more than 10-fold greater than the EC50 of the reference antibody, e.g., as measured in the same or similar assay.

[0226] Anti-CD19 antibodies provided herein can be identified, screened or characterized with respect to their physical / chemical properties and / or biological activity by different known tests. In one aspect, the antigen binding activity of the antibody is tested, for example, by known methods, such as ELISA, Western blot and / or flow cytometry, including cell-based binding assays, for example, assessing the combination of the antibody (for example, coupled to a fluorescent marker or with a label) and a cell expressing a target antigen (for example, CD19), in some cases, compared with the result of a cell not expressing the target antigen (for example, CD19). Binding affinity is detected in the form of Kd or EC50.

[0227] Competition assays can be used to identify antibodies that compete with any of the antibodies described herein.Assays for mapping the epitopes bound by the antibodies and reference antibodies can also be used and are known.

[0228] Immunoconjugates

[0229] In some embodiments, the antibody is an immunoconjugate or portion thereof, wherein the antibody is conjugated to one or more heterologous molecules, such as, but not limited to, a cytotoxic agent, an imaging agent, a detectable moiety, a multimerization domain, or other heterologous molecules. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu); chemotherapeutic agents (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunomycin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins. In some embodiments, the antibody is conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatic toxins, or fragments thereof of bacterial, fungal, plant, or animal origin), or radioactive isotopes.

[0230] Such immunoconjugates include antibody-drug conjugates (ADCs) in which an antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatins such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines, such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Biocon J. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, leronate, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0231] The immunoconjugates also include those in which the antibody is conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0232] The immunoconjugates also include those in which the antibody is conjugated to a radioactive atom to form a radioconjugate. Exemplary radioisotopes include At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 , Pb 212 and radioactive isotopes of Lu.

[0233] Conjugates of antibodies and cytotoxic agents can be prepared using any of a variety of known protein coupling agents, for example, linkers (see Vitetta et al., Science 238:1098 (1987)), WO94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell, such as an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, and a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020).

[0234] Conjugates may also include fusion proteins, such as Fc-fusions and chimeric molecules.

[0235] Multispecific antibodies

[0236] In certain embodiments, the CD19-binding molecule, e.g., an antibody, is multispecific. The multispecific binding molecule includes a multispecific antibody, including, for example, a bispecific. A multispecific binding partner, e.g., an antibody, has binding specificity for at least two different sites, which may be located in the same or different antigens. In certain embodiments, one of the binding specificities is for CD19, and the other binding specificity is for another antigen. In certain embodiments, a bispecific antibody can bind to two different CD19 epitopes. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CD19. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. The bispecific antibodies include multispecific single-chain antibodies, e.g., bispecific antibodies, trispecific antibodies, and tetraspecific antibodies, tandem bi-scFv, and tandem tri-scFv. Also provided are multispecific chimeric receptors, e.g., multispecific CARs, comprising the antibodies.

[0237] Exemplary other antigens include other B cell specific antigens and antigens expressed on T cells. Exemplary antigens include CD4, CD5, CD8, CD14, CD15, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, angiogenic factors, VEGF, PIGF, ED-B fibronectin, oncogenes, oncogene products, CD66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5).

[0238] Variants

[0239] In certain embodiments, the antibody comprises one or more amino acid variations, such as substitutions, deletions, insertions, and / or mutations, compared to the antigen sequences described herein. Exemplary variants include those designed to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. The modifications include, for example, deletions from the amino acid sequence of the antibody, and / or insertions into the amino acid sequence of the antibody and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be formed to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0240] In certain embodiments, for example, compared to the antibody sequences described herein and / or compared to the natural spectrum (e.g., human spectrum) sequence, the antibody includes one or more amino acid substitutions. Sites of interest for substitution mutagenesis include CDRs and FRs. Amino acid substitutions can be introduced into the antibody of interest, and the desired activity of the product can be screened, for example, retained / improved antigen binding, reduced immunogenicity, improved half-life, and / or improved effector function, for example, the ability to promote antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cellular cytotoxicity (CDC). In some embodiments, the variant antibody shows retained or improved CD19 binding.

[0241] In some embodiments, one or more residues in the CDR of a parent antibody (e.g., a humanized or human antibody) can be substituted. In some embodiments, the substitution is performed to restore the sequence or position in the sequence to a germline sequence, such as an antibody sequence present in a germline (e.g., a human germline), e.g., to reduce the likelihood of immunogenicity after administration to a human subject.

[0242] In some embodiments, changes are made in CDR "hot spots": residues encoded by codons that undergo mutation at high frequency during maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and / or residues that contact antigen, wherein the resulting variants V are tested. H or V L Binding affinity. Affinity maturation by constructing and reselecting from a secondary library has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-guided approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. Specifically, CDR-H3 and CDR-L3 are often targeted.

[0243] In certain embodiments, substitutions, insertions, or deletions may occur in one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may, for example, be outside of the antigen contacting residues in the CDRs. In the variants V provided above, H and V L In certain embodiments of the sequences, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0244] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusing the N- or C-terminus of the antibody to an enzyme or polypeptide, which increases the serum half-life of the antibody.

[0245] Modification

[0246] In certain embodiments, the antibody is altered to increase or decrease the extent of glycosylation of the antibody, for example, by removing or inserting one or more glycosylation sites by altering the amino acid sequence and / or by modifying the oligosaccharide attached to the glycosylation site (e.g., using certain cell lines). The glycosylation site includes asparagine 297 (by Kabat numbering) of the heavy chain.

[0247] Exemplary modifications, variants, and cell lines are described, for example, in Patent Publication Nos. US2003 / 0157108, US2004 / 0093621, US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US 2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1, Presta, L; and WO 2004 / 056312A1, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al. Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107); WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.); WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0248] Such modified antibodies include those with one or more amino acid modifications in the Fc region, e.g., those having a human Fc region sequence or other portion of a constant region (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0249] Such modifications can be made, for example, to improve half-life, alter binding to one or more types of Fc receptors, and / or alter effector function.

[0250] Such variants also include cysteine ​​engineered antibodies, such as "sulfur MAbs" and other cysteine ​​engineered variants, in which one or more residues of the antibody are substituted with cysteine ​​residues to generate reactive sulfhydryl groups at accessible sites, e.g., for conjugation with reagents and linker-reagents to produce immunoconjugates. Cysteine ​​engineered antibodies are described, e.g., in U.S. Patent Nos. 7,855,275 and 7,521,541.

[0251] In some embodiments, the antibody is modified to include other non-protein moieties, including water-soluble polymers. Exemplary polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymer or random copolymer), and, dextran or poly (vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageously manufactured due to its stability in water. The polymer may have any molecular weight and may be branched or non-branched. The number of polymers connected to the antibody may be different, and if more than one polymer is connected, they may be identical or different molecules. Generally speaking, the amount and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used for therapy for a particular condition, etc.

[0252] B. Recombinant receptors

[0253] The CD19 binding molecules provided include recombinant receptors, such as antigen receptors and other chimeric receptors, which specifically bind to CD19, such as receptors comprising provided anti-CD19 antibodies (e.g., antibody fragments). Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CAR). Cells expressing the recombinant receptors are also provided, and their applications in adoptive cell therapy, such as treating diseases and disorders associated with CD19 expression.

[0254] Exemplary antigen receptors, including CARs, and methods for engineering and introducing receptors into cells include, for example, those disclosed in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US2013 0149337, those described in U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP2537416, and / or Sadelain et al., Cancer Discov. 2013 Apr; 3(4): 388–398; Davila et al., (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 Oct; 24(5): 633-39; Wu et al., Cancer, 2012 Mar 18(2): 160-75. In some aspects, the antigen receptor includes the CAR described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO / 2014055668A1. Exemplary CARs include those described in any of the foregoing disclosures, e.g., WO2014031687, US 8,339,645, US 7,446,179, US2013 / 0149337, U.S. Pat. No. 7,446,190, U.S. Pat. No. 8,389,282, for example, and wherein the antigen binding portion (e.g., scFv) is replaced by an antibody provided herein.

[0255] The chimeric receptor includes a chimeric antigen receptor (CAR). The chimeric receptor, such as CAR, generally includes an extracellular antigen binding domain, which includes, is or is contained in one of the anti-CD19 antibodies provided. Therefore, the chimeric receptor, such as CAR, generally includes one or more CD19-binding molecules, such as one or more antigen binding fragments, domains or parts or one or more antibody variable domains, and / or antibody molecules, such as those described herein, in their extracellular part. In some embodiments, the CAR includes one or more CD19-binding parts of the antibody molecule, such as the variable heavy (V) domains of the antibody. H) chain region and / or variable light (V L ) chain region, for example, a scFv antibody fragment.

[0256] CD19-targeted CARs are described in, for example, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US2013 / 0149337, U.S. Patent No. 7,446,190 and U.S. Patent No. 8,389,282.

[0257] In some embodiments, the recombinant receptor, such as a CAR, such as an antibody portion thereof, further comprises a spacer, which may be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region, for example, an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In some aspects, the portion of the constant region serves as an antigen-recognition portion, such as a scFv, and a spacer region between the transmembrane domain. The spacer may have a length that provides increased cell responsiveness after antigen binding compared to when the spacer is not present. In some examples, the length of the spacer is or is approximately 12 amino acids, or no more than 12 amino acids. Exemplary spacers include those having a length of at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, and including integers between any endpoints of the above-listed ranges. In some embodiments, the spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge connected to the CH2 and CH3 domains, or an IgG4 hinge connected to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, International Patent Application Publication No. WO2014031687, US Patent No. 8,822,647, or Published Application No. US2014 / 0271635.

[0258] In some embodiments, the constant region or portion is human IgG, such as IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (shown in SEQ ID NO: 124) and is encoded by the sequence shown in SEQ ID NO: 125. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 126. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 127. In some embodiments, the constant region or portion is IgD. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 128. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 124, 126, 127, or 128.

[0259] Antigen recognition domain is generally connected to one or more intracellular signal transduction moieties, for example, in the case of CAR, by antigen receptor complex (such as TCR complex) simulation activation signal transduction moiety, and / or by the signal of another cell surface receptor.Therefore, in some embodiments, the CD19 specific binding moiety (for example, antibody) is connected to one or more transmembrane and intracellular signal transduction domains.In some embodiments, the transmembrane domain is fused to the extracellular domain.In one embodiment, the transmembrane domain of one of the domains in natural associated receptor (such as CAR) is used.In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid the domain from being bound to the transmembrane domain of the same or different surface membrane proteins, so as to minimize the interaction with other members of the receptor complex.

[0260] In some embodiments, the membrane-spanning domain is derived from a natural or synthetic source. When the source is a natural source, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. The membrane-spanning region includes α, β or ζ chains derived from T-cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, and / or the membrane-spanning region includes those (i.e., including at least their membrane-spanning regions) of its functional variants (such as those that substantially retain its structural portion (e.g., membrane-spanning portion), properties). In some embodiments, the membrane-spanning domain is a membrane-spanning domain derived from CD4, CD28 or CD8, for example, CD8α or its functional variant. In some embodiments, the membrane-spanning domain is synthetic in some embodiments. In some aspects, the synthetic membrane-spanning domain mainly includes hydrophobic residues such as leucine and valine. In some aspects, a trimer of phenylalanine, tryptophan, and valine will be present at each end of the synthetic transmembrane domain.In some embodiments, the linkage occurs via a linker, spacer, and / or transmembrane domain.

[0261] The intracellular signal transduction domain includes a signal that simulates or approximates a natural antigen receptor, a signal that is combined with a costimulatory receptor by such a receptor, and / or a signal that is passed through a separate costimulatory receptor. In some embodiments, there is a short oligopeptide or polypeptide linker, for example, a linker having a length of 2-10 amino acids, for example, comprising glycine and serine, for example, a linker of a glycine-serine doublet, and forms a connection between the cytoplasmic signal transduction domain and the transmembrane domain of the CAR.

[0262] The receptor, e.g., CAR, generally includes at least one or more intracellular signal transduction moieties. In some embodiments, the receptor includes an intracellular component of the TCR complex, e.g., TCR CD3 that mediates T-cell activation and cytotoxicity. + Chain, for example, CD3 ζ chain. Therefore, in some aspects, the CD19 binding antibody is connected to one or more cell signal transduction modules. In some embodiments, the cell signal transduction module includes a CD3 transmembrane domain, a CD3 intracellular signal transduction domain, and / or other CD transmembrane domains. In some embodiments, the receptor, for example, CAR, also includes part of one or more other molecules, such as Fc receptor γ, CD8, CD4, CD25 or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-ζ (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.

[0263] In some embodiments, after the connection of CAR, the cytoplasmic domain of the CAR or the intracellular signal transduction domain activates the normal effector function of immune cells or at least one of the responses, for example, engineered T cells are to express the CAR.For example, in some cases, CAR induces the function of T cells, such as cytolytic activity or helper T cell activity, such as the secretion of cytokines or other factors.In some embodiments, the truncated portion of the intracellular signal transduction domain of the antigen receptor portion or the costimulatory molecule is used to replace the complete immunostimulatory chain, for example, if its transduction effector function signal.In some embodiments, the one or more intracellular signal transduction domains include the cytoplasmic sequence of T cell receptor (TCR), and in some aspects, act together with such receptors in natural conditions to initiate signal transduction after antigen receptor engagement.Those of the common receptors, and / or any derivatives or variants of such molecules, and / or any synthetic sequences with the same functional capabilities.

[0264] In the case of natural TCR, full activation generally requires not only signal transduction by TCR, but also costimulatory signals. Therefore, in some embodiments, in order to promote full activation, components for generating a second or costimulatory signal are also included in the CAR. In other embodiments, CAR does not include components for generating costimulatory signals. In some aspects, other CARs are expressed in the same cell and are provided for generating a second or costimulatory signal component.

[0265] In some aspects, T cell activation is described as being mediated by two types of cytoplasmic signal transduction sequences: those (first cytoplasmic signal transduction sequences) that initiate antigen-dependent first activation by TCR, and those (second cytoplasmic signal transduction sequences) that act in an antigen-independent manner to provide a second or costimulatory signal. In some aspects, the CAR includes one or both of such signal transduction components.

[0266] In some aspects, the CAR includes a primary cytoplasmic signal transduction sequence that regulates the initial activation of the TCR complex. The first cytoplasmic signal transduction sequence acting in a stimulatory manner may include a signal transduction motif, which is known to be an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM include the first cytoplasmic signal transduction sequence, which includes those derived from the following: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD8, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signal transduction molecule in CAR includes a cytoplasmic signal transduction domain, which is partially or partially derived from a sequence of CD3ζ.

[0267] In some embodiments, the CAR includes a transmembrane portion and / or a signal transduction domain of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, or ICOS or CD27. In some aspects, the same CAR includes both activation and costimulatory moieties.

[0268] In some embodiments, the activation domain (such as CD3ζ) is included in a CAR, and the costimulatory portion (such as CD28 or 4-1BB) is provided by another CAR that recognizes another antigen. In some embodiments, the CAR includes activation or stimulation CAR, costimulatory CAR, which are all expressed on the same cell (see WO2014 / 055668). In some aspects, CD19-targeted CAR is stimulatory or activating CAR; in other aspects, it is costimulatory CAR. In some embodiments, the cell also includes inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5 (215) (December 2013), such as CAR that recognizes non-CD19 antigens, whereby the activation signal delivered by CD19-targeted CAR is weakened or inhibited by the combination of inhibitory CAR and its ligand, for example, to reduce off-target effects.

[0269] In some embodiments, the intracellular signal transduction portion of the recombinant receptor, such as CAR, includes a CD3 ζ intracellular domain and a costimulatory signal transduction region. In certain embodiments, the intracellular signal transduction domain includes a CD28 transmembrane and signal transduction domain, which is connected to a CD3 (e.g., CD3-ζ) intracellular domain. In some embodiments, the intracellular signal transduction domain includes a chimeric CD28 and / or CD137 (4-1BB, TNFRSF9) costimulatory domain, which is connected to a CD3 ζ intracellular domain.

[0270] In some embodiments, the CAR encompasses one or more, for example, two or more, costimulatory domains and activation domains, for example, the initial activation domain in the cytoplasmic portion. Exemplary CARs include the intracellular portion of CD3-ζ, CD28, and 4-1BB.

[0271] In some embodiments, the CAR or other antigen receptors further include a marker, such as a cell surface marker, which can be used to determine that the cell is used to express the transduction or engineering of the receptor, such as a truncated form of a cell surface receptor, such as a truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, NGFR or epidermal growth factor receptor (e.g., tEGFR) or its functional variant. In some embodiments, the nucleic acid encoding the marker is operably connected to a polynucleotide encoding a joint sequence (e.g., a cleavable joint sequence, e.g., T2A). For example, a marker, and optionally a joint sequence, can be any of the species described in disclosed patent application number WO2014031687. For example, the marker can be a truncated EGFR (tEGFR), i.e., it is optionally connected to a joint sequence, such as a T2A cleavable joint sequence. Exemplary polypeptides for truncated EGFR (e.g., tEGFR) include the amino acid sequence set forth in SEQ ID NO: 138, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 138. Exemplary T2A linker sequences include the amino acid sequence set forth in SEQ ID NO: 137, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 137.

[0272] In some embodiments, the marker is a molecule, e.g., a cell surface protein, that is not naturally found on a T cell or is not naturally found on the surface of a T cell or a portion thereof.

[0273] In some embodiments, the molecule is a non-self molecule, eg, a non-self protein, ie, a protein that is not recognized as "self" by the immune system of the host into which the cell is to be adoptively transferred.

[0274] In some embodiments, the marker has no therapeutic function and / or has no effect except as a marker for genetic engineering (e.g., for selecting successfully engineered cells). In other embodiments, the marker can be a therapeutic molecule or a molecule that plays some desired effects in other cases, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule, to enhance and / or inhibit the response of the cell after adoptive transfer and encountering a ligand.

[0275] In some cases, CAR is referred to as the first, second and / or third generation CAR. In some aspects, the first generation CAR is a CAR that provides a signal of CD3-chain induction only after antigen binding; In some aspects, the second generation CAR is a CAR that provides a signal and a costimulatory signal, such as a CAR comprising an intracellular signal transduction domain of a costimulatory receptor such as CD28 or CD137 from a costimulatory receptor; In some aspects, the third generation CAR is a CAR comprising a variety of costimulatory domains of different costimulatory receptors in some aspects.

[0276] In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment described herein. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv, and the intracellular domain comprises an ITAM. In some aspects, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain connecting the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain comprises the transmembrane portion of CD28. The extracellular domain and the transmembrane domain may be directly or indirectly connected. In some embodiments, the extracellular domain and the transmembrane domain are connected by a spacer, such as any spacer described herein. In some embodiments, the receptor comprises the extracellular portion of a molecule from which the transmembrane domain is derived, such as the extracellular portion of CD28. In some embodiments, the chimeric antigen receptor comprises an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between a transmembrane domain and an intracellular signal transduction domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.

[0277] For example, in some embodiments, the CAR includes an antibody provided herein (e.g., an antibody fragment), is or includes the transmembrane domain of the transmembrane portion of CD28 or its functional variant, and the intracellular signal transduction domain of the signal transduction portion comprising CD28 or its functional variant and CD3 ζ or its functional variant. In some embodiments, the CAR includes an antibody provided herein (e.g., an antibody fragment), is or includes the transmembrane domain of the transmembrane portion of CD28 or its functional variant, and, the intracellular signal transduction domain of the signal transduction portion comprising 4-1BB or its functional variant and CD3 ζ or its functional variant. In some of the embodiments, the receptor further includes a spacer, which includes a portion of an Ig molecule (e.g., human Ig molecule), such as an Ig hinge, such as an IgG4 hinge, such as only a hinge spacer.

[0278] In some embodiments, the transmembrane domain of the recombinant receptor (e.g., CAR) is or includes the transmembrane domain of human CD28 (e.g., Accession No. P01747.1) or a variant thereof, such as a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 129, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 129; in some embodiments, the transmembrane domain comprising the portion of the recombinant receptor comprises the amino acid sequence set forth in SEQ ID NO: 130, or an amino acid sequence that has at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0279] In some embodiments, the intracellular signal transduction portion of the recombinant receptor (eg, CAR) comprises an intracellular co-stimulatory signal transduction domain of human CD28 or a functional variant or portion thereof, such as a domain having an LL-GG substitution at position 186-187 of the original CD28 protein. For example, the intracellular signal transduction domain may comprise an amino acid sequence as shown in SEQ ID NO: 131 or 132, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 131 or 132. In some embodiments, the intracellular domain comprises an intracellular co-stimulatory signal transduction domain of 4-1BB (e.g., (Accession No. Q07011.1) or a functional variant or a portion thereof, such as the amino acid sequence shown in SEQ ID NO: 133, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 133.

[0280] In some embodiments, the intracellular signal transduction domain of the recombinant receptor (eg, CAR) comprises a human CD3 zeta stimulatory signal transduction domain or a functional variant thereof, such as a 112AA cytoplasmic domain of human CD3 zeta (Accession No.: P20963.2) isoform 3, or a CD3 zeta signal transduction domain, which is described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993. For example, in some embodiments, the intracellular signal transduction domain comprises amino acid sequence 134, 135 or 136 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 134, 135 or 136.

[0281] In some aspects, the spacer comprises only the hinge region of IgG, such as only the hinge of IgG4 or IgG1, such as only the spacer hinge shown in SEQ ID NO: 124. In other embodiments, the spacer is or comprises an Ig hinge, such as an IgG4-derived hinge, which is optionally connected to the CH2 and / or CH3 domains. In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge, which is connected to the CH2 and CH3 domains, such as shown in SEQ ID NO: 127. In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge, which is connected to the CH3 domain only, such as shown in SEQ ID NO: 126. In some embodiments, the spacer is or comprises a glycine-serine-rich sequence or other flexible linker, such as a known flexible linker.

[0282] For example, in some embodiments, the CAR includes an anti-CD19 antibody, such as an anti-CD19 antibody fragment, such as any human anti-CD19 antibody provided, for example, a single-chain antibody including scFv as described herein, a spacer, for example, a spacer comprising a portion of an immunoglobulin molecule, such as a hinge region and / or one or more heavy chain molecule constant regions, such as an Ig-hinge comprising a spacer, a transmembrane domain comprising all or part of a transmembrane domain derived from a CD28, an intracellular signaling domain derived from CD28, and a CD3 ζ signaling domain. In some embodiments, the CAR includes an anti-CD19 antibody or fragment, such as any of the human anti-CD19 antibodies, including scFv as described herein, a spacer, for example, any of the Ig-hinge comprising a spacer, a transmembrane domain derived from CD28, an intracellular signaling domain derived from 4-1BB, and a CD3 ζ signaling domain.

[0283] In some embodiments, the CAR construct further comprises a T2A ribosomal skipping element and / or a tEGFR sequence, e.g., downstream of the CAR, e.g., as shown in SEQ ID NO: 137 and / or 138, respectively, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 137 or 138.

[0284] C. Engineered cells

[0285] Also provided are cells, cell populations, and compositions comprising the cells, e.g., engineered cells, e.g., comprising an engineered antigen receptor, e.g., comprising an extracellular domain comprising the anti-CD19 antibody or fragment, as described herein. Compositions include pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are methods of treatment, e.g., administering the cells and compositions to a subject (e.g., a patient).

[0286] Therefore, there is also provided a genetically engineered cell that expresses a recombinant receptor comprising the antibody, for example, a cell comprising the CAR. The cell is generally a eukaryotic cell, such as a mammalian cell, and is typically a human cell. In some embodiments, the cell is derived from blood, bone marrow, lymph or lymphoid organs, and is a cell of the immune system, such as a cell of innate or adaptive immunity, for example, bone marrow or lymphoid cells, including lymphocytes, generally T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSC). Cells are generally primary cells, such as those separated directly from an object and / or separated and frozen from an object. In some embodiments, cell includes one or more subsets of T cells or other cell types, such as full T cell populations, CD4+ cells, CD8+ cells and subgroups thereof, such as those defined by function, activation state, maturation, differentiation potential, amplification, recirculation, positioning, and / or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With regard to object to be treated, cell can be allogenic and / or autologous. Method includes existing methods. In some respects, such as for existing technology, cell is multipotent and / or specialized, such as stem cell, such as induced pluripotent stem cell (iPSC). In some embodiments, the method includes separating cells from object, preparing, processing, culturing and / or engineering it, as described herein, and re-importing it into the same patient before or after freezing preservation.

[0287] T cells and / or CD4+ and / or CD8 + T cell subtypes and subsets include: naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM), central memory T (T CM ), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated non-variant T (MAIT) cells, naturally occurring and adoptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells.

[0288] In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a monocyte or a granulocyte, e.g., a bone marrow cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil.

[0289] In some embodiments, the cell comprises one or more nucleic acids introduced by genetic engineering and thereby expresses a recombinant or genetically engineered product of the nucleic acid. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or a sample obtained from the cell, such as a sample obtained from another organism or cell, e.g., not normally found in the cell being engineered and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, e.g., the nucleic acid is not found in nature, including chimeric combinations comprising nucleic acids encoding various domains from a variety of different cell types.

[0290] Methods and vectors for genetic engineering

[0291] Also provided are methods, nucleic acids, compositions, and kits for expressing the binding molecules, including receptors containing the antibodies, and for producing genetically engineered cells that express the binding molecules. Genetic engineering generally involves introducing a nucleic acid encoding the recombinant or engineered moiety into a cell, for example, by retroviral transduction, transfection, or transformation.

[0292] In some embodiments, gene transfer is performed by first stimulating the cells, e.g., by combining them with a stimulator that induces a response, e.g., proliferation, survival, and / or activation, e.g., as detected by expression of a cytokine or activation marker, and then transducing the activated cells and expanding them in culture to a number sufficient for clinical use.

[0293] In some cases, overexpression of stimulatory factors (e.g., lymphokines or cytokines) may be toxic to the subject. Therefore, in some cases, engineered cells include, for example, after administration in adoptive immunotherapy, gene segments that make the cells susceptible to negative selection in vivo. For example, in some aspects, the cells are engineered so that they can be eliminated due to changes in the in vivo conditions of the patient to whom they are administered. Negatively selectable phenotypes can be produced by providing genes that confer sensitivity to the administered reagent (e.g., compound). Negatively selectable genes include: herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II: 223, 1977), which provides ganciclovir sensitivity; cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89: 33 (1992)).

[0294] In some aspects, cells are also engineered to promote the expression of cytokines or other factors. Various methods for introducing genetically engineered components, for example, antigen receptors (e.g., CAR) are well known, and methods and compositions provided herein can be used. Exemplary methods include those for transferring nucleic acids encoding receptors, including by viruses, for example, retroviruses or slow viruses, transduction, transposons, and electroporation.

[0295] In some embodiments, the recombinant nucleic acid is transferred into cells using recombinant infectious viral particles, e.g., vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is transferred into T cells using recombinant lentiviral vectors or retroviral vectors, e.g., gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi:10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10):1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al. Trends Biotechnol. 2011 Nov;29(11):550–557).

[0296] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), for example, a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen focus forming virus (SFFV) or adeno-associated virus (AAV). Most retroviral vectors are derived from mouse retroviruses. In some embodiments, the retrovirus includes those derived from any avian or mammalian cell source. Retroviruses are typically amphibious, which means that they are able to infect host cells of several species (including humans). In one embodiment, the retroviral gag, pol and / or env sequences are substituted with the gene to be expressed. Many exemplary retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0297] Lentiviral transduction methods are known. Exemplary methods are described in, for example, Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101: 1637–1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

[0298] In some embodiments, the recombinant nucleic acid is transferred into T cells by electroporation (see, e.g., Chicaybam et al. (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, the recombinant nucleic acid is transferred into T cells by transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4):427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microprojectile bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0299] Other methods and vectors for transferring nucleic acids encoding recombinant products are those described in, for example, International Patent Application Publication No. WO2014055668 and US Patent No. 7,446,190.

[0300] Other nucleic acids (e.g., genes) for introduction include those for improving therapeutic efficacy, for example, by promoting the viability and / or function of transferred cells; genes for providing genetic markers for selection and / or evaluation of cells, for example, to assess survival or location in vivo; genes for improving safety, for example, by making cells susceptible to negative selection in vivo, as described in Lupton SD et al., Mol. and Cell Biol., 11: 6 (1991); and Riddell et al., Human Gene Therapy 3: 319-338 (1992); see also Lupton's publications PCT / US91 / 08442 and PCT / US94 / 05601, etc., which describe the application of bifunctional selectable fusion genes derived from the fusion of dominant positive selectable markers and negative selectable markers. See, for example, Riddell et al., U.S. Patent No. 6,040,177, columns 14-17.

[0301] Preparation of cells for engineering

[0302] In some embodiments, the preparation of engineered cells includes one or more cultures and / or one or more preparation steps. Cells for importing CD19- binding molecules (e.g., CAR) can be separated from samples (e.g., biological samples, such as samples obtained from or derived from objects). In some embodiments, the object from which cells are separated is an object suffering from a certain disease or illness or requiring cell therapy or to be given a cell therapy. In some embodiments, the object is a person who needs specific therapeutic intervention, for example, a person who needs adoptive cell therapy, and the cells for the therapy are separated, processed, and / or engineered.

[0303] Thus, in some embodiments, the cells are primary cells, e.g., primary human cells. The samples include tissues, body fluids, and other samples taken directly from the subject, as well as samples obtained from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction using a viral vector), washing, and / or incubation. The biological sample can be a sample directly obtained from a biological source or a processed sample. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples, including processed samples derived therefrom.

[0304] In some aspects, the sample from which cells are derived or separated is a blood or blood-derived sample or is or is derived from a single collection or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, intestinal associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix uteri, testis, ovary, tonsil or other organs, and / or cells derived therefrom. In the case of cell therapy (e.g., adoptive cell therapy), sample includes samples from autologous and allogeneic sources.

[0305] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from a xenogeneic source, e.g., from a mouse, rat, non-human primate, or pig.

[0306] In some embodiments, the separation of cells comprises one or more preparation and / or non-affinity-based cell separation steps. In some instances, cells are washed, centrifuged, and / or incubated in the presence of one or more substances, e.g., to remove unwanted components, enrich for desired components, lyse, or remove cells sensitive to a particular substance. In some instances, cells are separated based on one or more properties, e.g., density, adhesion properties, size, sensitivity, and / or resistance to a particular component.

[0307] In some examples, cells are obtained from the subject's circulating blood, e.g., by apheresis or leukapheresis. In some aspects, the sample comprises lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and, in some aspects, comprises cells other than red blood cells and platelets.

[0308] In some embodiments, the blood cells collected from the subject are washed, for example, to remove the plasma portion, and the cells are placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the cleaning solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the cleaning step is completed by semi-automated "flow-through" centrifugation (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the cleaning step is completed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, after cleaning, the cells are resuspended in a variety of biocompatible buffers, for example, without Ca. ++ / Mg ++ PBS. In certain embodiments, the blood cell sample components are removed and the cells are directly resuspended in culture medium.

[0309] In some embodiments, the method comprises a density-based cell separation method, for example, preparing white blood cells from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

[0310] In some embodiments, the separation method includes, based on one or more specific molecules in the cell, such as surface markers, for example, expression or presence of surface proteins, intracellular markers or nucleic acids to separate different cell types. In some embodiments, any known method for separating based on such markers can be adopted. In some embodiments, the separation is based on affinity or separation based on immunoaffinity. For example, in some aspects, the separation includes expression or expression level of one or more markers (typically cell surface markers) based on the cell to separate cells and cell populations, for example, by hatching with an antibody or binding partner that is specifically bound to such markers, followed by typically a washing step, and separating the cells in conjunction with the antibody or binding partner from those cells that have not yet been bound to the antibody or binding partner.

[0311] Such separation steps can be based on positive selection, wherein cells that have bound the reagent are retained for further use, and / or, negative selection, wherein cells that have not bound to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when there are no antibodies available to specifically identify cell types in a heterogeneous population, and thus separation is best performed based on markers expressed by cells that differ from the desired population.

[0312] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as those expressing a marker, refers to an increase in the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as those expressing a marker, refers to a decrease in the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0313] In some examples, multiple rounds of separation steps are performed, wherein the fraction from the positive or negative selection in one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step simultaneously depletes cells expressing multiple markers, such as by incubating the cells with multiple antibodies or binding partners each specific for a marker targeted by the negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on the various cell types.

[0314] For example, in some aspects, a specific subpopulation of T cells, such as cells positive for one or more surface markers or cells expressing high levels of one or more surface markers, e.g., CD28 + 、CD62L + 、CCR7 + 、CD27 + 、CD127 + 、CD4 + 、CD8 + 、CD45RA + and / or CD45RO + T cells are isolated by positive or negative selection techniques.

[0315] For example, CD3 + 、CD28 + T cells can be treated with CD3 / CD28-linked magnetic beads (e.g., DYNA M-450 CD3 / CD28 T cell expander) for positive selection.

[0316] In some embodiments, separation is performed by enriching a particular cell population by positive selection or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers expressed on the positively or negatively selected cells, respectively (markers). + ) or expressed at relatively high levels (marker high).

[0317] In some embodiments, T cells are isolated from PBMC samples by negative selection for markers expressed on non-T cells (e.g., B cells, monocytes, or other blood leukocytes), such as CD14. + or CD8 + Select steps to isolate helper CD4 + and CD8 + Cytotoxic T cells. These CD4 + and CD8 + The group was further sorted into subgroups.

[0318] In some embodiments, CD8 + The cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with the corresponding subpopulations. In some embodiments, the cells are enriched or depleted for central memory T (T CM ) cells to increase efficacy, for example to improve long-term survival, expansion and / or engraftment after administration, and in some aspects, they are particularly robust in such subpopulations. See Terakura et al. (2012) Blood. 1:72–82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, T CM -Enriched CD8 + T cells and CD4 + T cells were pooled to further enhance efficacy.

[0319] In an embodiment, memory T cells are CD8 + CD62L on peripheral blood lymphocytes + PBMC can be directed against CD62L-CD8 + and / or CD62L + CD8 + The fraction can be enriched or depleted, for example, using anti-CD8 and anti-CD62L antibodies.

[0320] In some embodiments, for the central memory T(T CM ) cells based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, negative selection is performed based on cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, the enrichment of T cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, negative selection is performed based on cells expressing or highly expressing CD45RA and / or granzyme B. CM CD8 cell enrichment + The population is isolated by depleting cells expressing CD4, CD14, CD45RA, and positively selecting or enriching for cells expressing CD62L. CM ) cells are enriched by starting with a negative fraction of cells selected based on CD4 expression, negatively selecting based on expression of CD14 and CD45RA, and positively selecting based on CD62L. In some aspects, such selections are performed simultaneously, and in other aspects, such selections are performed sequentially in a certain order. In some aspects, the same selection steps based on CD4 expression are used to prepare CD8 + Cell populations or subpopulations, also used to produce CD4 + The cell populations or subpopulations, thereby retaining the positive and negative fractions from the CD4-based separation, are used in the subsequent steps of the method, optionally after one or more further positive or negative selection steps.

[0321] In a specific example, a PBMC sample or other blood leukocyte sample is subjected to CD4 + Cell selection, wherein the negative and positive fractions are retained. The negative fraction is then negatively selected based on the expression of CD14 and CD45RA or CD19, and positively selected based on markers characteristic of central memory T cells, such as CD62L or CCR7, wherein the positive and negative selections are performed in a certain order.

[0322] By identifying cell populations with cell surface antigens, helper CD4 + T cells are sorted into naive, central memory and effector cells. CD4 + Lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO - 、CD45RA + 、CD62L + 、CD4 + In some embodiments, central memory CD4 + cells are CD62L + and CD45RO + In some embodiments, effector CD4+ cells are CD62L - and CD45RO - .

[0323] In one example, to negatively select CD4 + Cells are enriched, and the monoclonal antibody mixture generally includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as magnetic beads or paramagnetic beads, to allow separation of cells for positive and / or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (for review, see Methods in Molecular Medicine, Vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, pp. 17-25, eds. SA Brooks and U. Schumacher). Humana Press Inc., Totowa, New Jersey.

[0324] In some aspects, the sample or cell composition to be separated is incubated with a smaller magnetizable or magnetically responsive material, such as a magnetically responsive particle or microparticle, for example, a paramagnetic bead (e.g., Dynalbeads beads or MACS beads). The magnetically responsive material (e.g., particle) is generally directly or indirectly linked to a binding partner (e.g., an antibody) that specifically binds to a molecule, for example, a surface marker present on one or more cells or cell populations that require separation (e.g., negative or positive selection).

[0325] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material that is bound to a specific binding moiety, such as an antibody or other binding partner. A variety of well-known magnetically responsive materials are used for magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Patent No. 4,452,773 and European Patent Specification EP 452342B, which are incorporated herein by reference. Colloidally shaped particles, such as those described in Owen U.S. Patent No. 4,795,698, and Liberti et al., U.S. Patent No. 5,200,084 are other examples.

[0326] The incubation is generally performed under conditions whereby the antibody or binding partner or molecule, such as a secondary antibody or other reagent, which specifically binds to the antibody or binding partner, which is attached to the magnetic particles or beads, specifically binds to the cell surface molecule, if present, on the cells in the sample.

[0327] In some aspects, the sample is placed in a magnetic field, and those cells with magnetically responsive or magnetizable particles attached thereto are attracted by the magnet and separated from the unlabeled cells. For positive selection, cells attracted by the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selections is performed in the same selection step, wherein both the positive and negative fractions are retained and further processed or subjected to a further separation step.

[0328] In some embodiments, the magnetic responsive particles are coated in a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme or streptavidin. In some embodiments, the magnetic particles are connected to cells by a coating having a specific primary antibody for one or more markers. In some embodiments, the cells, rather than beads, are labeled with a primary antibody or a binding partner, and then, the magnetic particles coated with a secondary antibody or other binding partner (e.g., streptavidin) specific for the cell type are added. In some embodiments, the streptavidin-coated magnetic particles are used in conjunction with a biotinylated primary antibody or a secondary antibody.

[0329] In some embodiments, the magnetically responsive particles remain attached to cells to be subsequently incubated, cultured, and / or engineered; in some aspects, the particles remain attached to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, magnetizable particles, or antibodies attached to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0330] In some embodiments, the affinity-based selection is carried out by magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, California). Magnetic activated cell sorting (MACS) systems can select cells with magnetized particles attached thereto in high purity. In some embodiments, MACS operates in the following mode, wherein after applying an external magnetic field, non-target and target substances are eluted sequentially. That is, the cells attached to the magnetized particles are maintained in place, and the unattached substances are eluted. Then, after the first elution step is completed, the substances that are retained in the magnetic field and prevented from being eluted are liberated in a certain way, so that they can be eluted and recovered. In some embodiments, the non-target cells are labeled and consumed from a heterogeneous cell population.

[0331] In certain embodiments, the isolation or separation is performed using a system, device, or apparatus that performs one or more of the separation, cell preparation, separation, treatment, incubation, cultivation, and / or formulation steps of the method. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, e.g., to minimize errors, user handling, and / or contamination. In one example, the system is a system described in International Patent Application Publication No. WO2009 / 072003 or US 20110003380A1.

[0332] In some embodiments, the system or device performs one or more, e.g., all, of the separation, processing, engineering, and formulation steps in an integrated or standalone system, and / or in an automated or programmable manner. In some aspects, the system or device comprises a computer and / or computer program in information communication with the system or device that allows a user to program, control, evaluate the results of the processing, separation, engineering, and formulation steps, and / or adjust various aspects of the processing, separation, engineering, and formulation steps.

[0333] In some aspects, the separation and / or other steps are performed using a CliniMACS system (Miltenyi Biotic), for example, for automated separation of cells at clinical grade levels in a closed and sterile system. Components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some aspects, an integrated computer controls all components of the instrument and instructs the system to perform repeated steps in a standardized order. In some aspects, the magnetic separation unit includes a removable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate through the tubing set and, together with the pinch valves, ensures a controlled flow of buffer through the system and a continuous suspension of cells.

[0334] In some aspects, the CliniMACS system uses the antibody-coupled magnetizable particles provided in the form of sterile, pyrogen-free solution. In some embodiments, after labeling cells with magnetic particles, the cells are cleaned to remove excessive particles. Then, the cell preparation bag is connected to a tube set, which is further connected to a bag and a cell collection bag comprising a buffer solution. The tube set is composed of preassembled sterile tubing, includes a precolumn and a separation column, and is only used for single use. After the separation procedure is initiated, the system automatically applies the cell sample to the separation column. Labeled cells are retained in the column, and unlabeled cells are removed by a series of cleaning steps. In some embodiments, the cell mass used for methods described herein is unlabeled, and is not retained in the column. In some embodiments, the cell mass used for methods described herein is labeled, and is retained in the column. In some embodiments, after removing the magnetic field, the cell mass used for methods described herein is eluted from the column, and is collected in the cell collection bag.

[0335] In some embodiments, separation and / or other steps adopt CliniMACS Prodigy system (Miltenyi Biotech) to carry out.In some aspects, CliniMACS Prodigy system is equipped with cell treatment unit, and described cell treatment unit allows automation cleaning and by centrifugal fractionation cell.CliniMACS Prodigy system can also include built-in camera and image recognition software, and it determines the most preferred cell fractionation terminal point by the visible layer of identification source cell product.For example, peripheral blood is automatically separated into red blood cell, white blood cell and plasma layer.CliniMACS Prodigy system can also include the cell cultivation chamber of integration, and it completes cell culture scheme, for example, cell differentiation and amplification, antigen loading and long-term cell culture.Input port can allow aseptic removal and supplementary culture medium, and cell can adopt integrated microscope to monitor. See, eg, Klebanoff et al. (2012) J Immunother. 35(9):651–660, Terakura et al. (2012) Blood. 1:72–82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0336] In some embodiments, cell populations as described herein are collected and enriched (or consumed) by flow cytometry, wherein cells stained for multiple cell surface markers are carried in a fluid. In some embodiments, cell populations as described herein are collected and enriched (or consumed) by preparative scale (FACS)-sorting. In certain embodiments, cell populations as described herein are collected and enriched (or consumed) by using a microelectromechanical system (MEMS) chip in conjunction with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1 (5): 355–376. In both cases, cells can be labeled with multiple markers to allow for the separation of well-defined T cell subsets with high purity.

[0337] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation can be based on binding to a fluorescently labeled antibody. In some instances, cell separation based on binding of an antibody or other binding partner specific for one or more cell surface markers is carried in a fluid stream, such as by fluorescence activated cell sorting (FACS) (which includes preparation scale (FACS) and / or microelectromechanical system (MEMS) chips), for example, in combination with a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0338] In some embodiments, the preparation method includes: a freezing step, for example, before or after separation, incubation and / or engineering, freezing the cells. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, to some extent, remove monocytes in the cell population. In some embodiments, for example, after a washing step to remove plasma and platelets, the cells are suspended in a freezing solution. In some aspects, any of various known freezing solutions and parameters can be used. One example involves using PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HAS). It is then diluted with culture medium 1: 1 so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to -80°C at a rate of 1° / minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0339] In some embodiments, provided methods include cultivating, incubating, culturing, and / or genetic engineering steps. For example, in some embodiments, provided methods are for incubating and / or engineering spent cell populations and culture initiation compositions.

[0340] Thus, in some embodiments, the cell population is incubated in a culture initiation composition. Incubation and / or engineering can be performed in a culture vessel, such as a cell, chamber, well, column, tube, tube set, valve, vial, culture dish, bag, or other container for culturing or growing cells.

[0341] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic engineering. The incubation step may include cultivating, nurturing, stimulating, activating, and / or propagating. In some embodiments, cells or compositions are incubated in the presence of stimulating conditions or irritating agents. Such conditions include being designed to induce cell proliferation, propagation, activation, and / or survival in a population to simulate antigen exposure, and / or to initiate cells for genetic engineering, such as those for importing recombinant antigen receptors.

[0342] The conditions may include one or more of the following: a specific culture medium, temperature, oxygen content, carbon dioxide content, time, reagents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other substances designed to activate cells.

[0343] In some embodiments, stimulation conditions or reagents include one or more substances, for example, ligands, which can activate the intracellular signal transduction domain of the TCR complex. In some aspects, the substance opens or starts the TCR / CD3 intracellular signal transduction cascade in T cells. Such substances may include antibodies, such as those specific to TCR components and / or costimulatory receptors, for example, anti-CD3, anti-CD28, for example, which are bound to a solid support, such as a bead, and / or one or more cytokines. Optionally, the amplification method may also include the steps of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (for example, with a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agent includes IL-2 and / or IL-15, for example, at least about 10 units / mL of IL-2.

[0344] In some aspects, incubation is performed according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9):651–660, Terakura et al. (2012) Blood. 1:72–82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0345] In some embodiments, the T cell population is expanded by adding to a culture starting composition feeder layer cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), (e.g., so that for each T lymphocyte in the initial population to be expanded, the resulting cell population comprises at least about 5, 10, 20, or 40 or more PBMC feeder layer cells); and incubating the culture (e.g., a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder layer cells may include gamma-irradiated PBMC feeder layer cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000-3600 rads to prevent cell division. In some aspects, the feeder layer cells are added to the culture medium before adding the population of T cells.

[0346] In some embodiments, the stimulation conditions include a temperature suitable for growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally or approximately 37 degrees Celsius. Optionally, incubation may also include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays within the range of about 6000-10000 rad. In some aspects, LCL feeder cells are provided in any suitable amount, for example, the ratio of LCL feeder cells to naive T lymphocytes is at least about 10:1.

[0347] In an embodiment, antigen-specific T cells, such as antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating natural or antigen-specific T lymphocytes with an antigen. For example, an antigen-specific T cell line or clone can be generated against a cytomegalovirus antigen by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0348] II. Compositions, Methods, and Uses

[0349] Also provided are compositions comprising the CD19 binding molecules and engineered cells, including pharmaceutical compositions and formulations, and methods of using the molecules and compositions, and uses of the molecules and compositions, e.g., for treating diseases, conditions and disorders in which CD19 is expressed, and / or for detecting, diagnostic and prognostic methods.

[0350] A. Pharmaceutical Compositions and Formulations

[0351] Pharmaceutical formulations are provided that include the CD19-binding molecules, e.g., antibodies or chimeric receptors, and / or engineered cells expressing the molecules. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the compositions include at least one other therapeutic agent.

[0352] The term "pharmaceutical formulation" refers to a preparation in such form that it permits the biological activity of the active ingredient contained therein to be effective, and does not contain additional ingredients that are unacceptably toxic to a subject to which the formulation would be administered.

[0353] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is not an active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0354] In some aspects, the selection of carriers is determined in part by specific cells, binding molecules and / or antibodies, and / or by the method of administration. Therefore, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The amount of preservative or its mixture is typically about 0.0001% to about 2% (based on the total weight of the composition). Carriers are described in, for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate, and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0355] In some aspects, the composition comprises a buffer. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and a variety of other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or its mixture is typically present in an amount of about 0.001% to about 4% (based on the total weight of the composition). Methods for preparing a pharmaceutical composition that can be administered are known. Exemplary methods are specifically described in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (May 1, 2005).

[0356] Formulations of the antibodies may include lyophilized formulations and aqueous solutions.

[0357] The formulation or composition may also comprise more than one active ingredient that is beneficial for the specific indication, disease or condition to be treated with the binding molecule or cell, preferably those that have complementary activities to the binding molecule or cell, wherein the respective active agents do not negatively affect each other. Such active ingredients are suitable for being present in combination in an amount effective for the desired purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active substances or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cell or antibody is administered in the form of a salt, for example, a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid, and organic acids, such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid and arylsulfonic acids, e.g., p-toluenesulfonic acid.

[0358] Active ingredients can be encapsulated in microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as a cyclodextrin inclusion complex or as a liposome. Liposomes can be used to target host cells (e.g., T- cells or NK cells) to specific tissues. Many methods can be used to prepare liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980) and U.S. Patents 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0359] In some aspects, the pharmaceutical composition employs a time-release, delayed-release, and sustained-release delivery system so that delivery of the composition occurs before the site to be treated becomes sensitized and has sufficient time to cause sensitization of the site. Many types of release delivery systems are available and known. Such systems can avoid repeated administration of the composition, thereby increasing convenience for both the subject and the physician.

[0360] In some embodiments, the pharmaceutical composition comprises the amount of an effective treatment or prevention disease or condition, such as a binding molecule and / or cell for the treatment of an effective or preventive amount. In some embodiments, the treatment or preventive efficacy is monitored by regularly evaluating the object of treatment. For repeated administration over several days or longer, treatment can be repeated until symptoms of the disease are required to be suppressed according to the condition. However, other dosage regimens may be useful and can be determined. The desired dose can be given by a single pill, by multiple pills, or by continuous infusion to deliver the composition.

[0361] In certain embodiments, in the case of genetically engineered cells comprising the binding molecule, the subject is administered in a range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 50 billion cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined between any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, or a range defined between any two of the foregoing values). In some cases, the number of cells is about 100 million to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined between any two of the foregoing values), and in some cases, about 100 million to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges, and / or this number of cells per kilogram of body weight of the subject.

[0362] They can adopt standard to give technology, preparation and / or device to give.Preparation and device, such as syringe and bottle, for composition storage and giving are provided.The giving of described cell can be autologous or heterologous.For example, immunosuppressive cell or progenitor cell can be obtained from a subject, and give same subject or different compatible subjects.Immunosuppressive cell derived from peripheral blood or its offspring (for example, in vivo, in vitro or in vitro derived) can be given by local injection, comprise catheter administration, systemic injection, local injection, intravenous injection or parenteral administration.When giving therapeutic composition (for example, pharmaceutical composition containing genetically modified immunosuppressive cell), it is usually formulated into the injectable form (solution, suspension, emulsion) of unit dosage form.

[0363] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, mucosal, sublingual or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the cell population is administered to a subject using peripheral systemic delivery via intravenous, intraperitoneal or subcutaneous injection.

[0364] In some embodiments, the composition is provided in the form of a sterile liquid preparation, for example, an isotonic aqueous solution, suspension, emulsion, dispersion or viscous composition, which can be buffered to the pH of selection in some aspects. Liquid preparations are generally easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are more or less convenient for administration, especially by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact time with a specific tissue. Liquid or viscous compositions can include a vehicle, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0365] Sterile injectable solutions can be prepared by incorporating the binding molecule into a solvent, such as with a suitable carrier, diluent, or excipient such as sterile water, normal saline, glucose, dextrose, etc. The composition can also be lyophilized. The composition can contain auxiliary substances, such as wetting, dispersion, or emulsifiers (e.g., methylcellulose), pH buffers, gelling or viscosity enhancing additives, preservatives, flavorings, pigments, etc., depending on the desired administration and preparation route. In some aspects, standard textbooks can be consulted to prepare suitable preparations.

[0366] Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms may also be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Absorption of injectable pharmaceutical forms may be enhanced by the use of substances that delay absorption, such as aluminum monostearate and gelatin.

[0367] Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0368] Preparations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0369] B. Treatment and prevention methods and applications

[0370] Also provided are methods and uses of the CD19 binding molecules, including anti-CD19 antibodies, e.g., antibody fragments, and / or engineered cells expressing the recombinant receptor. The methods and uses include therapeutic methods and uses, e.g., involving administering the molecules, cells or compositions comprising the molecules to a subject having a disease, condition or disorder that expresses or is associated with CD19 expression, and / or wherein the cells or tissue express CD19. In some embodiments, the molecules, cells and / or compositions are administered in an amount effective to treat the disease or disorder. Uses include the use of the antibodies and cells in the methods and treatments, as well as in the preparation of a medicament for performing the therapeutic methods. In some embodiments, the methods are performed by administering the antibodies or cells or compositions comprising them to a subject having or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0371] As used herein, the term "treat" (and grammatical variations thereof such as "therapeutic" and "therapeutic") refers to the complete or partial alleviation or reduction of a disease or condition or disorder or a symptom, adverse effect, consequence, or phenotype associated therewith. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or reducing the disease state, and alleviating or improving prognosis. The term does not imply a complete cure of the disease or complete elimination of any symptom or having an effect on all symptoms or consequences.

[0372] As used herein, "delaying the progression of a disease" means postponing, hindering, slowing, slowing, stabilizing, curbing, and / or stalling the progression of the disease (e.g., cancer). The delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. One skilled in the art will appreciate that, in practice, a sufficient or significant delay can encompass prevention (for individuals who do not develop the disease). For example, the progression of advanced cancer, such as metastasis, can be delayed.

[0373] As used herein, "prevention" includes providing methods for preventing the occurrence or recurrence of a disease in a subject predisposed to developing the disease but not yet diagnosed with the disease. In some embodiments, provided molecules and compositions are used to delay disease development or slow disease progression.

[0374] As used herein, "suppressing" a function or activity means a decrease in the function or activity when compared to an otherwise identical condition (except for the condition or parameter of interest) or compared to another condition. For example, an antibody, composition, or cell that suppresses tumor growth reduces the growth rate of the tumor compared to the growth rate of the tumor in the absence of the antibody, composition, or cell.

[0375] An "effective amount" of an agent, e.g., a pharmaceutical agent, binding molecule, antibody, or cell or composition, in the context of administration, refers to an amount that, at dosages / amounts and for periods of time necessary, is effective to achieve a desired result, e.g., a therapeutic or prophylactic result.

[0376] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation, antibody, or cell, refers to an amount that is effective, at dosages and over the necessary time course, to achieve a desired therapeutic outcome, e.g., treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effect of that treatment. A therapeutically effective amount can vary according to a variety of factors, e.g., disease state, age, sex, and weight of the subject, as well as the cell population being administered. In some embodiments, provided methods involve administering an effective amount (e.g., a therapeutically effective amount) of the described molecules, cells, and / or compositions.

[0377] A "prophylactically effective amount" refers to an amount that, at a given dosage and for a given duration, is effective to achieve the desired preventive result. Typically (but not necessarily), since a prophylactic dose is used in a subject at an early stage or before the onset of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0378] As used herein, " subject " is a mammal, such as a human or other animal, and is typically a human. The disease and disorder include: B cell malignancies, such as B cell leukemia and lymphoma, including B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphocytic leukemia, non-acute lymphoblastic leukemia, non-Hodgkin's lymphoma, diffuse large B cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, spleen, marginal zone lymphoma, mantle cell lymphoma, inert B cell lymphoma, Hodgkin's lymphoma. The disease and disorder also include autoimmune and inflammatory diseases, including those associated with inappropriate or enhanced B cell quantity and / or activation. Exemplary diseases and disorders include multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus (SLE).

[0379] In some embodiments, the subject has persistent or recurrent disease, for example, after treatment with another CD19-specific antibody and / or cells expressing a CD19-targeting chimeric receptor and / or other therapies (including chemotherapy, radiotherapy and / or hematopoietic stem cell transplantation (HSCT), for example, allogeneic HSCT). In some embodiments, the administration effectively treats the subject regardless of whether the subject has been resistant to another CD19-targeted therapy. In some embodiments, the subject has not yet relapsed but has been determined to be at risk of relapse, for example, at high risk of relapse, whereby the compound or composition is administered prophylactically, for example, to reduce the likelihood of relapse or prevent relapse.

[0380] In some embodiments, the treatment does not induce the immune response of the subject for the therapy, and / or does not induce such responses to the extent that effectively treat the disease or illness. In some aspects, the degree of immunogenicity and / or the targeted host response of the graft are lower than the observed situation using different but suitable treatments. For example, in the case of adoptive cell therapy using cells expressing CAR (including provided anti-CD19 antibodies), the degree of immunogenicity is compared to including being bound to similar (for example, overlapping epitopes) and / or competing with provided antibodies to be bound to different antibodies (such as mouse antibodies) of CD19 for reduced CAR.

[0381] In some embodiments, the method includes adoptive cell therapy, whereby genetically engineered cells expressing a receptor comprising provided anti-CD19 (e.g., a CAR targeted by CD19) are administered to a subject. Such administration can promote activation (e.g., T cell activation) of the cells in a CD19-targeted manner, thereby targeting the disease or disordered cells and destroying them.

[0382] Therefore, provided method and application include the method and application for adoptive cell therapy.In some embodiments, the method includes giving cells or compositions containing the cells to an object, tissue or cell, such as having a disease, illness or disorder risk or suspected object, tissue or cell with a disease, illness or disorder.In some embodiments, the cell, group and composition are given to the object with a specific disease to be treated (for example, by adoptive cell therapy, such as adoptive T cell therapy) or illness.In some embodiments, to an object, such as having a disease or illness or being in a disease or illness risk, cell or composition are given.In some aspects, the method is thus treated, for example, to alleviate one or more symptoms of the disease or illness, for example, by reducing the tumor load in the cancer expressing CD19.

[0383] Methods for administering cells for adoptive cell therapy are known and can be used in conjunction with the methods and compositions provided herein. For example, adoptive T cell therapy methods are described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

[0384] In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is performed by autologous transfer, wherein the cells are isolated and / or otherwise prepared from a subject receiving the cell therapy or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject (e.g., a patient) in need of treatment and the cells are administered to the same subject after isolation and processing.

[0385] In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is performed by allogeneic transfer, wherein the cells are isolated and / or otherwise prepared from a subject that is different from the subject to be or ultimately receive the cell therapy (e.g., a first subject). In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0386] In some embodiments, the subject to be administered the cells, cell populations, or compositions is a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and can be of any suitable age, including infants, teenagers, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is an animal model for disease, adoptive cell therapy, and / or evaluation of toxicity results such as cytokine release syndrome (CRS).

[0387] The CD19-binding molecules, such as antibodies and chimeric receptors comprising the antibodies, and cells expressing them, can be administered in any suitable manner, for example, by injection, for example, intravenous or subcutaneous injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, reverse septal injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjunctival injection, subconjunctival injection, suprascleral injection, retrobulbar injection, periocular injection or peribulbar delivery. In some embodiments, they are administered parenterally, intrapulmonary and intranasally, and if desired for local treatment, intralesional administration. Extraperitoneal infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration and administration may depend in part on whether the administration is short-term or long-term. Different dosing regimens include, but are not limited to, single or multiple administration at different time points, bolus administration and pulse infusion.

[0388] For the prevention or treatment of disease, the appropriate dosage of the binding molecule or cell may depend on the type of disease to be treated, the type of binding molecule, the severity and course of the disease, whether the binding molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the binding molecule, and the discretion of the attending physician. In some embodiments, the compositions, molecules, and cells are suitable for administration to the patient at one time or over a series of treatments.

[0389] Depending on the type and severity of the disease, antibody dosages may include about 1 μg / kg-15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg), about 1 μg / kg-100 mg / kg or more, about 0.05 mg / kg-about 10 mg / kg, 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg. Multiple doses may be given intermittently, for example, weekly or every three weeks. A higher loading dose may be given first, followed by one or more lower doses.

[0390] In certain embodiments, in the case of genetically engineered cells comprising the binding molecule, the subject is administered in a range of about 1 million to about 100 billion cells, and / or such number of cells / kg body weight, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 50 billion cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined between any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, or a range defined between any two of the foregoing values). In some cases, the dosage may be from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range determined between any two of the foregoing values), and in some cases, from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 90 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges, and / or this number of cells / kg body weight. Likewise, the dosage may depend on the specific attributes of the disease or disorder and / or patient and / or other treatments.

[0391] In some embodiments, the cells or antibodies are administered as part of a combination therapy, e.g., simultaneously or sequentially, in any order, with another therapeutic intervention, e.g., another antibody or engineered cell or receptor or agent, e.g., a cytotoxic or therapeutic agent.

[0392] In some embodiments, the cells or antibodies are co-administered with one or more other therapeutic agents or in combination with another therapeutic intervention, either simultaneously or sequentially in any order. In some cases, the cells are co-administered with another treatment close enough in time so that the cell population enhances the effect of one or more other therapeutic agents, or vice versa. In some embodiments, the cells or antibodies are administered before one or more other therapeutic agents. In some embodiments, the cells or antibodies are administered after one or more other therapeutic agents.

[0393] Once cells are administered to a mammal (e.g., a human), in some aspects, the bioactivity of the engineered cell population and / or antibody is measured by any of a variety of known methods. Parameters for evaluation include: specific binding of engineered or natural T cells or other immune cells to antigens, in vivo (e.g., by imaging) or in vitro (e.g., by ELISA or flow cytometry). In certain embodiments, the ability of engineered cells to destroy target cells can be detected using any suitable method known in the art, such as the cytotoxicity assays described in the following literature, e.g., Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the bioactivity of cells can also be measured by measuring the expression and / or secretion of certain cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is detected by assessing a clinical outcome, such as a reduction in tumor burden or load.

[0394] In certain embodiments, the engineered cells are modified in any number of ways to increase their therapeutic or preventive efficacy. For example, the engineered CAR or TCR expressed by the group can be directly or indirectly connected to a targeting moiety via a joint. Connecting compounds, for example, connecting CAR or TCR to a targeting moiety is known in the art. See, for example, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Patent No. 5,087,616.

[0395] C. Diagnostic and Detection Methods

[0396] Also provided are methods for use of the provided binding molecules, such as antibodies, including antibody fragments, and molecules comprising one or more of the antibodies (e.g., conjugates and complexes), for detecting, prognosing, diagnosing, stratifying, determining the binding of a specific treatment to one or more tissues or cell types, and / or understanding treatment decisions in a subject (e.g., by detecting the presence of CD19 and / or its epitope recognized by the antibody). In some embodiments, the method is a diagnostic and / or prognostic method associated with a disease or condition that expresses CD19. In some embodiments, the method comprises incubating and / or probing a biological sample with the antibody, and / or administering the antibody to a subject. In certain embodiments, the biological sample comprises cells or tissues or portions thereof, such as tumor or cancer tissue or biopsies or sections thereof. In certain embodiments, the contacting is performed under conditions that allow the anti-CD19 antibody to bind to CD19 present in the sample. In some embodiments, the method further comprises detecting whether a complex is formed between the anti-CD19 antibody and CD19 in the sample, e.g., detecting the presence or absence or level of the binding. The method can be an in vitro or in vivo method. In one embodiment, anti-CD 19 antibodies are used to select subjects suitable for treatment with anti-CD 19 antibodies or engineered antigen receptors, e.g., where CD 19 is a biomarker used to select patients.

[0397] In some embodiments, a sample, such as a cell, tissue sample, lysate, composition or other sample thereof, is contacted with an anti-CD19 antibody, and the binding or complex formation of the antibody and the sample (e.g., CD19 in the sample) is measured or detected. If binding in the test sample is observed or detected compared to a reference cell of the same tissue type, the presence of a related disease or disorder is indicated, and / or the therapeutic comprising the antibody (e.g., antibody fragment) will specifically bind to the tissue or cell or other biological substance identical or belonging to the same type of tissue or cell from which the sample is derived. In some embodiments, the sample is from human tissue and can be from diseased and / or normal tissue, for example, from a subject with a disease or disorder to be treated and / or from the same species as the subject but not suffering from the disease to be treated or disorder. In some cases, normal tissue or cells are from a subject suffering from a disease or disorder to be treated but are not themselves diseased cells or tissues, for example, from normal tissue of the same or different organs that are not the cancer present in a given subject.

[0398] Different methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), inhibition of immune scattering turbidimetric assay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). Indicator moieties or label groups can be attached to the subject antibody and can be selected to meet the needs of different method applications often dictated by test equipment and compatible immunoassay procedures. Exemplary labels include radionuclides (e.g., 125 I. 131 I. 35 S. 3 H or 32 P and / or chromium ( 51 Cr), cobalt ( 57 Co), fluorine ( 18 F), gadolinium ( 153 Gd, 159 Gd), Germanium ( 68 Ge), holmium ( 166 Ho), indium ( 115 In, 113 In, 112 In, 111 In), iodine ( 125 I. 123 I. 121 I), lanthanum ( 140 La), Lutetium ( 177 Lu), manganese ( 54 Mn), molybdenum ( 99 Mo), palladium ( 103 Pd), phosphorus ( 32 P), praseodymium ( 142 Pr), promethium ( 149 Pm), rhenium (186Re, 188Re), rhodium (105Rh), ruthenium (97Ru), samarium ( 153 Sm), Scandium ( 47 Sc), selenium ( 75 Se)、( 85 Sr), sulfur ( 35 S), technetium ( 99 Tc), thallium ( 201 Ti), tin ( 113 Sn, 117 Sn), tritium (3H), xenon ( 133 Xe), Ytterbium ( 169 Yb, 175 Yb), yttrium ( 90Y)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or β-galactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot® supplied by Quantum Dot Corporation, Palo Alto, CA). TM Nanoparticles). The above-mentioned different general techniques for allowing different immunoassays are known.

[0399] For diagnostic purposes, antibodies can be labeled with a detectable moiety, including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels known in the art. Methods for coupling labels to antibodies are known in the art.

[0400] In some embodiments, the antibodies need not be labeled, but their presence can be detected using a labeled antibody that binds to any of the antibodies.

[0401] The antibodies provided herein can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0402] The antibodies and polypeptides can also be used in in vivo diagnostic assays, such as in vivo imaging. Generally, the antibodies are treated with radionuclides (e.g. 111 In, 99 Tc, 14 C. 131 I. 125 I or 3 H) Labeling so that cells or tissues of interest can be localized in vivo following administration to a subject.

[0403] The antibodies can also be used as pathology staining reagents, for example, using known techniques.

[0404] III. Products

[0405] Also provided are articles comprising the provided binding molecules, e.g., antibodies and CARs, and / or genetically engineered cells, and / or compositions. The articles may include a container and a label or a package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be made of various materials such as glass or plastic. In some embodiments, the container holds the composition itself or a combination thereof with another composition effective for treating, preventing, and / or diagnosing the condition. In some embodiments, the container has a sterile inlet. Exemplary containers include intravenous solution bags and vials, including those with stoppers that can be pierced by an injection needle. The label or package insert may indicate that the composition is used to treat a disease or condition that expresses CD19 or is associated with CD19. The article may include (a) a first container comprising a composition, wherein the composition includes the antibody or engineered antigen receptor; and (b) a second container comprising a composition, wherein the composition includes other agents, such as a cytotoxic agent or other therapeutic agent. The article may also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or in addition, the article of manufacture may further comprise another or the same container comprising a pharmaceutically acceptable buffer. It may also comprise other materials, such as other buffers, diluents, filters, needles, and / or syringes.

[0406] As used herein, reference to a "corresponding form" of an antibody means that when comparing a property or activity of two antibodies, the properties being compared are those of the same form of the antibody. For example, reference to an antibody having greater activity than a corresponding form of a first antibody means that the activity of that particular form of the antibody, such as an scFv, is greater than that of the scFv form of the first antibody.

[0407] As used herein, reference to a nucleotide or amino acid position "corresponding to" a nucleotide or amino acid position in a disclosed sequence (e.g., shown in the sequence listing) refers to a nucleotide or amino acid position identified when aligned to maximum identity with the disclosed sequence using a standard alignment algorithm (e.g., the GAP algorithm). For example, in some embodiments, exemplary corresponding residues of a CD19 protein (e.g., a human CD19 protein) can be identified by aligning the sequence with the exemplary VpX sequence shown in SEQ ID NO: 92. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as a guide. In general, to identify corresponding positions, amino acid sequences are aligned so that the highest degree of match is given (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., New York: Oxford University Press, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Publishing Co., NJ, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48:1073).

[0408] "Effector functions" refer to those biological activities attributed to the Fc region of an antibody, which vary with the antibody isotype. Exemplary "effector functions" include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0409] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise defined herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index, see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Services, National Institutes of Health, Bethesda, Maryland, 1991).

[0410] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure, or has heavy chains that comprise an Fc region as defined herein.

[0411] An "isolated" antibody is one that has been partially separated from its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). Methods for assessing antibody purity are reviewed in, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0412] An "isolated" nucleic acid refers to a nucleic acid molecule that has been partially separated from its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0413] "Isolated nucleic acid encoding an anti-CD19 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including said nucleic acid molecules in a single vector or separate vectors, and said nucleic acid molecules are present at one or more locations in a host cell.

[0414] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and its derived progeny, regardless of the number of passages. The nucleic acid content of the progeny may not be identical to that of the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that screened or selected for in the originally transformed cell are included herein.

[0415] As used herein, "percent (%) amino acid sequence identity" and "percent identity" when applied to amino acid sequences (reference polypeptide sequences) are defined as the percentage of amino acid residues in a candidate sequence (e.g., a subject antibody or fragment) that are identical to the amino acid residues in the reference polypeptide sequence, after alignment of the sequences and introduction of gaps (if necessary) to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment of the compared sequences over their entire lengths.

[0416] Amino acid substitutions can include replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1: Amino acid substitutions can be introduced into binding molecules, e.g., antibodies of interest, and the resulting products screened for a desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0417] Amino acids can generally be classified according to the following general side chain properties:

[0418] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0419] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0420] (3) Acidic: Asp, Glu;

[0421] (4) Basic: His, Lys, Arg;

[0422] (5) Residues that affect chain orientation: Gly, Pro;

[0423] (6) Aromatic: Trp, Tyr, Phe.

[0424] Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.

[0425] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are introduced into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0426] The term "package insert" refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the use, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning such therapeutic products.

[0427] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. For example, "a" or "an" means "at least one or one" or "one or more." It should be understood that the aspects and variations described herein include "consisting of" and / or "consisting essentially of."

[0428] In this disclosure, various aspects of the claimed subject matter are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be interpreted as a hard limit to the scope of the claimed subject matter. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, where a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other stated or intermediate values ​​within the range are included in the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller range, and they also fall within the scope of the claimed subject matter unless the upper and lower limits of the range are explicitly excluded. When a range is set to include one or two limits, the claimed subject matter also includes a range that excludes one or both of the limits. This applies regardless of the width of the range.

[0429] As used herein, the term "about" refers to the typical error range of each value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter itself. For example, a description of "about X" includes a description of "X."

[0430] As used herein, a composition refers to any mixture of two or more products, substances or compounds, including cells, which can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.

[0431] As used herein, referring to a cell or cell population as "positive" for a particular marker refers to the detectable presence of the particular marker (typically a surface marker) on or in the cell. When referring to a surface marker, the term refers to the presence of surface expression detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry at a level that is significantly higher than the level of staining detected when the same procedure is performed using an isotype-matched control under otherwise identical conditions, and / or is substantially similar to the level of cells known to be positive for the marker, and / or is significantly higher than the level of cells known to be negative for the marker.

[0432] As used herein, referring to a cell or cell population as "negative" for a particular marker means that the cell or cells have a substantial detectable absence of the particular marker, typically a surface marker, on or in the cell. When referring to a surface marker, the term refers to the presence of surface expression detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry at a level that is significantly higher than the level of staining detected when the same procedure is performed using an isotype-matched control under otherwise identical conditions, and / or significantly lower than the level in cells known to be positive for the marker, and / or substantially similar to the level in cells known to be negative for the marker.

[0433] Unless otherwise defined, all technical and scientific terms or proprietary words used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with commonly understood meanings are defined herein for the purpose of clarification and / or ease of reference, and the inclusion of such definitions herein should not be construed as indicating a significant difference from commonly understood meanings in the art.

[0434] All publications, including patent documents, academic papers, and databases, mentioned herein are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If definitions set forth herein differ from or are otherwise inconsistent with definitions set forth in patents, published applications, and other publications incorporated herein by reference, the definitions set forth herein control over definitions in the documents incorporated herein by reference.

[0435] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0436] IV. Exemplary Embodiments

[0437] Embodiments provided herein include:

[0438] 1. An anti-CD19 antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:

[0439] The VH region comprises a heavy chain complementary determining region 3 (CDR-H3), which comprises the amino acid sequence shown in SEQ ID NO: 20, or

[0440] The VH region comprises at least 90% sequence identity to the VH region amino acid sequence shown in SEQ ID NO: 11, 12, 60, 61, 63 or 62.

[0441] 2. An antibody or antigen-binding fragment thereof, comprising:

[0442] CDR-H1, CDR-H2 and CDR-H3 respectively comprise the amino acid sequences of the CDR-H1, CDR-H2 and CDR-H3 sequences contained in the VH region amino acid sequence shown in SEQ ID NO: 11, 12, 60, 61, 63 or 62; and / or

[0443] Light chain complementary determining regions 1, 2 and 3 (CDR-L1, CDR-L2 and CDR-L3), which respectively comprise the amino acid sequences of the CDR-L1, CDR-L2 and CDR-L3 sequences contained in the light chain variable (VL) region amino acid sequence shown in SEQ ID NO: 13, 14, 15, 16, 17, 71, 65, 64, 66, 70, 69, 67, 90 or 91.

[0444] 3. An antibody or antigen-binding fragment thereof, comprising:

[0445] CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 81 or 82, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 20; and / or

[0446] CDR-L1, the CDR-L1 comprises the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 (SEQ ID NO: 111), wherein X1 is T, Q, S or R; X2 is G or A; X3 is I, T, D or S; X4 is S, R, T or Q; X5 is empty or S; X6 is G, D, N or empty; X7 is empty, V or L; X8 is D, G, I, L, S or empty; X9 is S, G, A, I, R or empty; X 10is H, Y, F, S or N; X 11 is R, N, D, H or Y; X 12 is Y, F, D or W; X 13 is V, A, or L; and X 14 is S, N or A;

[0447] CDR-L2, comprising the following amino acid sequence: X1X2X3X4X5X6X7 (SEQ ID NO: 112), wherein X1 is D or S; X2 is F, V, N, K or A; X3 is S, T, D or N; X4 is K, V, N, Q or R; X5 is R, V or L; X6 is P, K, A or E; and X7 is S, P, A or T, and

[0448] CDR-L3, the CDR-L3 comprises the following amino acid sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 (SEQ ID NO: 115), wherein X1 is X; X2 is S, Q, A or T; X3 is Y, S, W, R; X4 is A, D, R, T or Y; X5 is X; X6 is X; X7 is S, P, L, Y, G; X8 is X or empty; X9 is X or empty; X 10 L or empty; X 11 is X; and X 12 It is V, T or L.

[0449] 4. The antibody or antigen-binding fragment thereof according to embodiment 3, wherein:

[0450] In the CDR-L1, X3 is I, T or S; X4 is S, T or Q; X8 is D, G, I, S or empty; X9 is S, G, I or empty; X 10 is H, Y, S or N; X 11 is R, N, D or H; X 12 is Y or D; and X 13 is V or L; and / or

[0451] In said CDR-L2, X1 is D; X4 is K, V, N, Q or R; X6 is P, K or A; and X7 is S, A or T; and / or

[0452] In said CDR-L3, X1 is S, G, T, A, Q, C or N; X5 is A, S, P, G, N or D; X6 is I, S, G, T, A, L, H, R or N; X8 is P, T, S, Q, M, R, N or empty; X9 is S, L, N, A, M or empty; and X 11 It is Y, W, F, V, A or L.

[0453] 5. The antibody or antigen-binding fragment of embodiment 3 or embodiment 4, wherein, in the CDR-L3, X1 is S, G, Q, or N; X2 is S, Q, or T; X4 is A, D, T, or Y; X5 is A, S, or G; and X6 is I, S, N, R, A, H, or T.

[0454] 6. The antibody or fragment of any one of embodiments 1-5, wherein:

[0455] The CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 19 (GISWNSGRIGYADSVKG); or

[0456] The CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 72 (GISWNSGSIGYADSVKG).

[0457] 7. The antibody or fragment of any one of embodiments 1-6, wherein the CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 80, 77, 74, 73, 75, 79, 78, 76, 21, 25, 28 or 31.

[0458] 8. The antibody or fragment of embodiment 7, wherein the CDR-L1 comprises the amino acid sequence shown in SEQ ID NO: 80, 77, 74, 73, 78, 21 or 28.

[0459] 9. The antibody or fragment of any one of embodiments 1-8, wherein the CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 100, 97, 94, 93, 95, 99, 98, 96, 22, 26, 29 or 32.

[0460] 10. The antibody or fragment of embodiment 9, wherein the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 100, 97, 94, 93, 98, 22 or 29.

[0461] 11. The antibody or fragment of any one of embodiments 1-10, wherein the CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 109, 106, 103, 101, 104, 108, 107, 105, 102, 23, 24, 27, 30 or 33.

[0462] 12. The antibody or fragment of embodiment 11, wherein the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 109, 106, 103, 101, 107, 24 or 30.

[0463] 13. The antibody or fragment of any one of embodiments 1-12, wherein:

[0464] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 21, 22 and 23, respectively;

[0465] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 21, 22 and 24, respectively;

[0466] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 25, 26 and 27, respectively;

[0467] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 28, 29 and 30, respectively;

[0468] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 31, 32 and 33, respectively;

[0469] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 80, 100 and 109, respectively;

[0470] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 77, 97 and 106, respectively;

[0471] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 74, 94 and 103, respectively;

[0472] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 73, 93 and 101, respectively;

[0473] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 75, 95 and 104, respectively;

[0474] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 79, 99 and 108, respectively;

[0475] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 78, 98 and 107, respectively;

[0476] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 76, 96 and 105, respectively;

[0477] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 73, 93 and 102, respectively; or

[0478] The CDR-L1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 77, 97 and 106, respectively.

[0479] 14. The antibody or antigen-binding fragment thereof of any one of embodiments 1-13, wherein:

[0480] The CDR-H1, CDR-H2 and CDR-H3 comprise SEQ ID NOs: 18, 81 and 20, respectively;

[0481] The CDR-H1, CDR-H2 and CDR-H3 comprise SEQ ID NOs: 18, 19 and 20, respectively;

[0482] The CDR-H1, CDR-H2 and CDR-H3 comprise SEQ ID NOs: 18, 82 and 20, respectively; or

[0483] The CDR-H1, CDR-H2 and CDR-H3 comprise SEQ ID NOs: 18, 72 and 20, respectively.

[0484] 15. The antibody or fragment of any one of embodiments 1-14, wherein:

[0485] The VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 11, 12, 60, 61, 63 or 62; and / or

[0486] The VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 71, 90, 91, 68, 65, 64, 66, 70, 69 or 67.

[0487] 16. The antibody or fragment of embodiment 15, wherein:

[0488] The VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 11, 60, 63 or 62; and / or

[0489] The VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 14, 16, 71, 90, 65, 64 or 69.

[0490] 17. The antibody or fragment of any one of embodiments 1-16, wherein:

[0491] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 17, respectively;

[0492] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 15, respectively;

[0493] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 13, respectively;

[0494] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 14, respectively;

[0495] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 16, respectively;

[0496] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 71, respectively;

[0497] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 68, respectively;

[0498] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively;

[0499] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 60 and 64, respectively;

[0500] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 61 and 66, respectively;

[0501] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 70, respectively;

[0502] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 69, respectively;

[0503] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 67, respectively;

[0504] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 91, respectively; or

[0505] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 90, respectively.

[0506] 18. The antibody or fragment of embodiment 17, wherein:

[0507] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 14, respectively;

[0508] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 16, respectively;

[0509] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 71, respectively;

[0510] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively;

[0511] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 60 and 64, respectively;

[0512] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 69, respectively; or

[0513] The VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 90, respectively.

[0514] 19. The antibody or fragment of any one of embodiments 1-18, wherein the antibody specifically binds to CD19.

[0515] 20. The antibody or fragment of embodiment 19, wherein the antibody specifically binds to a CD19 epitope that is identical to or overlaps with an epitope specifically bound by a reference anti-CD19 antibody selected from FMC63 and SJ25C1.

[0516] 21. The antibody or fragment of embodiment 19, wherein the antibody competes with an anti-CD19 antibody selected from FMC63 and SJ25C1 for binding to CD19.

[0517] 22. A human antibody fragment that specifically binds to a CD19 epitope that is identical to or overlaps with the epitope specifically bound by a reference antibody, which is the antibody or fragment thereof as described in any one of embodiments 1-21, or an anti-CD19 antibody selected from FMC63 and SJ25C1, wherein the human antibody fragment comprises heavy chain and light chain CDRs that are different from the CDRs present in FMC63 and SJ25C1.

[0518] 23. A human antibody fragment that specifically binds to CD19 and competes with a reference antibody for binding to CD19, which is the antibody or fragment of any one of embodiments 1-21, or an anti-CD19 antibody selected from FMC63 and SJ25C1, wherein the antibody fragment comprises heavy chain and light chain CDRs that are different from the CDRs present in FMC63 and SJ25C1.

[0519] 24. The antibody or fragment of embodiment 21 or 23, which competes with a reference antibody for binding to CD19 to at least the same extent as the reference antibody competes with itself for binding to CD19, or competes to a degree no more than 1.5-fold or 2-fold less than the degree of competition by the reference antibody.

[0520] 25. The antibody or fragment of any one of embodiments 1-24, wherein the binding affinity of the antibody is at least the same as, or at least substantially the same as, the binding affinity of a reference antibody for CD19, the reference antibody being selected from FMC63 and SJ25C1.

[0521] 26. The antibody or fragment of embodiment 25, wherein the EC50 of the binding affinity is approximately equal to or lower than the EC50 of the reference antibody, or is no more than about 1.5 times, or no more than about 2 times, no more than 3 times, and / or no more than 10 times greater than the EC50 of the reference antibody.

[0522] 27. The antibody or fragment of any one of embodiments 1-26, wherein the antibody or fragment is a human antibody or fragment.

[0523] 28. The antibody or fragment of any one of embodiments 1-27, wherein the antibody or fragment is a recombinant antibody or fragment.

[0524] 29. The antibody or fragment of any one of embodiments 1-28, which is monoclonal.

[0525] 30. The antibody or fragment of any one of embodiments 1-29, which is a single-chain fragment.

[0526] 31. The antibody or fragment of any one of embodiments 1-30, which is a fragment comprising an antibody variable region joined by a flexible immunoglobulin linker.

[0527] 32. The antibody or fragment of embodiment 30 or 31, wherein the fragment comprises an scFv.

[0528] 33. The antibody or fragment of embodiment 32, wherein the scFv comprises a linker comprising the sequence shown in SEQ ID NO:34.

[0529] 34. The antibody or fragment of embodiment 32, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87 or 89.

[0530] 35. The antibody or fragment of any one of embodiments 1-34, further comprising at least a portion of an immunoglobulin constant region.

[0531] 36. The antibody or fragment of embodiment 35, wherein at least a portion of the immunoglobulin constant region comprises an Fc region.

[0532] 37. The antibody or fragment of embodiment 36, wherein the Fc region is the Fc region of human IgG.

[0533] 38. The antibody or fragment of any one of embodiments 1-37, wherein the CD19 is human CD19.

[0534] 39. A chimeric antigen receptor (CAR) comprising an extracellular portion comprising the antibody or fragment of any one of embodiments 1-38, and an intracellular signaling domain.

[0535] 40. The chimeric antigen receptor of embodiment 38, wherein the antibody or fragment comprises an scFv and the intracellular signaling domain comprises an ITAM.

[0536] 41. The chimeric antigen receptor of embodiment 39 or 40, wherein the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3ζ) chain.

[0537] 42. The chimeric antigen receptor of any one of embodiments 39-41, further comprising a transmembrane domain connecting the extracellular domain and the intracellular signaling domain.

[0538] 43. The chimeric antigen receptor of embodiment 42, wherein the transmembrane domain comprises the transmembrane portion of CD28.

[0539] 44. The chimeric antigen receptor of any one of embodiments 39-43, further comprising an intracellular signal transduction domain of a T cell co-stimulatory molecule.

[0540] 45. The chimeric antigen receptor of embodiment 44, wherein the T cell co-stimulatory molecule is selected from the group consisting of CD28 and 41BB.

[0541] 46. ​​An engineered cell expressing a chimeric antigen receptor as described in any of embodiments 39-45.

[0542] 47. The engineered cell of embodiment 46, which is a T cell.

[0543] 48. A method of treatment comprising administering the cells of embodiment 46 or 47 to a subject having a disease or disorder associated with CD19.

[0544] 49. A method of treatment comprising administering the antibody of any one of embodiments 1-38 to a subject having a disease or disorder associated with CD19.

[0545] 50. The method of embodiment 48 or 49, wherein the disease or disorder is a B-cell malignancy.

[0546] 51. The method of embodiment 50, wherein the B cell malignancy is selected from the group consisting of B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphocytic leukemia, non-acute lymphoblastic leukemia, non-Hodgkin's lymphoma, diffuse large B cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, spleen, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, and Hodgkin's lymphoma.

[0547] 52. A nucleic acid encoding the antibody or fragment thereof of any one of embodiments 1-38 or the chimeric antigen receptor of any one of embodiments 39-45.

[0548] 53. A composition comprising the antibody or fragment thereof of any one of embodiments 1-38, the CAR of any one of embodiments 39-45, or the cell of embodiment 46 or 47.

[0549] 54. A method of treatment comprising administering the composition of embodiment 53 to a subject having a disease or disorder associated with CD19.

[0550] 55. An antibody or antigen-binding fragment thereof, comprising:

[0551] Heavy chain complementarity determining regions 1, 2, and 3 (CDR-H1, CDR-H2, and CDR-H3), which respectively comprise the amino acid sequences of the CDR 1, 2, and 3 sequences contained in the heavy chain variable (VH) region amino acid sequence shown in SEQ ID NO: 11 or 12; and

[0552] Light chain complementary determining regions 1, 2 and 3 (CDR-L1, CDR-L2 and CDR-L3), which respectively comprise the amino acid sequences of CDR 1, 2 and 3 sequences contained in the light chain variable (VL) region amino acid sequence shown in SEQ ID NO: 13, 14, 15, 16 or 17.

[0553] 56. The antibody or fragment of embodiment 55, wherein:

[0554] The CDR-H1 comprises the amino acid sequence DYAMH (SEQ ID NO: 18);

[0555] The CDR-H2 comprises the amino acid sequence GISWNSGRIGY (SEQ ID NO: 35);

[0556] The CDR-H3 comprises the amino acid sequence of SEQ ID NO: 20;

[0557] The CDR-L1 comprises the amino acid sequence X1GX3X4X5X6X7X8X9X 10 X 11 X 12 X 13 S (SEQ ID NO: 36), wherein X1 is T, S or Q, X3 is T, S or D, X4 is T or S, X5 is empty or S, X6 is empty, D or N, X7 is empty or V, X8 is empty, G or I, X9 is empty, G or R, X 10 Is S, Y or N, X 11 Is D or N, X 12 Is D or Y, X 13 It is V or A;

[0558] The CDR-L2 comprises the amino acid sequence XXRPS (SEQ ID NO: 37), wherein X1 is D or S, X2 is V, N or K, X3 is S, N or D, and X4 is K, Q or N; and

[0559] The CDR-L3 comprises the amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 (SEQ ID NO: 38), wherein X1 is C, S, A, G or N, X2 is S, A or T, X3 is Y, W or R, X4 is A or D, X5 is G, D or S, X6 is R, S or N, X7 is Y, L or G, X8 is N or S, X9 is S or empty, X 10 is V, A or N, X 11 is W or empty, and X 12 It is L or V.

[0560] 57. The antibody or fragment of embodiment 56, wherein: in the CDR-L1, X1 is T or S, X3 is T or S, X 11 Is D or N and X 13 is V; in said CDR-L2, X2 is V or N and X4 is K or Q; and / or in said CDR-L3, X1 is C, S, A or G, X3 is Y or W, X5 is G or D, X7 is Y or L, X 10 is V or A, and X 11 Empty.

[0561] 58. The antibody or fragment of any one of embodiments 55-57, wherein the CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 19 (GISWNSGRIGYADSVKG).

[0562] 59. The antibody or fragment of any one of embodiments 55-58, wherein the CDR-L1 comprises the sequence shown in SEQ ID NO: 21, 25, 28 or 31.

[0563] 60. The antibody or fragment of any one of embodiments 55-59, wherein the CDR-L2 comprises the sequence shown in SEQ ID NO: 22, 26, 29 or 32.

[0564] 61. The antibody or fragment of any one of embodiments 55-60, wherein the CDR-L3 comprises the sequence shown in SEQ ID NO: 23, 24, 27, 30 or 33.

[0565] 62. The antibody or fragment of any one of embodiments 55-61, wherein:

[0566] The CDRL1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 21, 22 and 23, respectively;

[0567] The CDRL1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 21, 22 and 24, respectively;

[0568] The CDRL1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 25, 26 and 27, respectively;

[0569] The CDRL1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 28, 29 and 30, respectively; or

[0570] The CDRL1, CDR-L2 and CDR-L3 comprise SEQ ID NOs: 31, 32 and 33, respectively.

[0571] 63. The antibody or fragment of any one of embodiments 55-62, wherein the antibody or fragment comprises:

[0572] a VH region comprising the amino acid sequence of SEQ ID NO: 11 or 12; and

[0573] A VL region comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16 or 17.

[0574] 64. The antibody or fragment of embodiment 63, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 11.

[0575] 65. The antibody or fragment of embodiment 63, wherein the VH region comprises the amino acid sequence of SEQ ID NO:12.

[0576] 66. The antibody or fragment of any one of embodiments 55-65, wherein the antibody specifically binds to CD19.

[0577] 67. The antibody or fragment of embodiment 66, wherein the antibody specifically binds to a CD19 epitope that is identical to or overlaps with an epitope specifically bound by a reference anti-CD19 antibody selected from FMC63 and SJ25C1.

[0578] 68. The antibody or fragment of embodiment 66, wherein the antibody competes for binding to CD19 with an anti-CD19 antibody selected from FMC63 and SJ25C1.

[0579] 69. A human antibody fragment that specifically binds to a CD19 epitope that is identical to or overlaps with the epitope specifically bound by a reference antibody, which is an antibody or fragment thereof as described in any one of embodiments 55-68, or an anti-CD19 antibody selected from FMC63 and SJ25C1, wherein the human antibody fragment comprises heavy chain and light chain CDRs that are different from the CDRs present in FMC63 and SJ25C1.

[0580] 70. A human antibody fragment that specifically binds to CD19 and competes with a reference antibody for binding to CD19, which is the antibody or fragment of any one of embodiments 55-68, or an anti-CD19 antibody selected from FMC63 and SJ25C1, wherein the antibody fragment comprises heavy chain and light chain CDRs that are different from the CDRs present in FMC63 and SJ25C1.

[0581] 71. The antibody of embodiment 68 or 70, which competes with a reference antibody for binding to CD19 to at least the same extent as the reference antibody competes with itself for binding to CD19, or competes to a degree no more than 1.5-fold or 2-fold less than the degree of competition by the reference antibody.

[0582] 72. The antibody of any one of embodiments 55-71, wherein the binding affinity of the antibody is at least the same as, or at least substantially the same as, the binding affinity of a reference antibody for CD19, the reference antibody being selected from FMC63 and SJ25C1.

[0583] 73. The antibody of embodiment 72, wherein the EC50 of the binding affinity is approximately equal to or lower than the EC50 of the reference antibody, or is no more than about 1.5 times, or no more than about 2 times, no more than 3 times, and / or no more than 10 times greater than the EC50 of the reference antibody.

[0584] 74. The antibody or fragment of any one of embodiments 55-73, wherein the antibody is a human antibody.

[0585] 75. The antibody or fragment of any one of embodiments 55-74, wherein the antibody is a recombinant antibody.

[0586] 76. The antibody or fragment of any one of embodiments 55-75, which is a monoclonal antibody or fragment.

[0587] 77. The antibody or fragment of any one of embodiments 55-76, which is a single-chain fragment.

[0588] 78. The antibody or fragment of any one of embodiments 55-77, which is a fragment comprising an antibody variable region joined by a flexible immunoglobulin linker.

[0589] 79. The antibody or fragment of embodiment 77 or 78, wherein the fragment comprises an scFv.

[0590] 80. The antibody or fragment of embodiment 79, wherein the scFv comprises a linker comprising the sequence shown in SEQ ID NO:34.

[0591] 81. The antibody or fragment of embodiment 80, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10.

[0592] 82. The antibody or fragment of any of embodiments 55-81, further comprising at least a portion of an immunoglobulin constant region.

[0593] 83. The antibody or fragment of embodiment 82, wherein at least a portion of the immunoglobulin constant region comprises an Fc region.

[0594] 84. The antibody or fragment of embodiment 83, wherein the Fc region is the Fc region of human IgG.

[0595] 85. The antibody or fragment of any one of embodiments 55-84, wherein the CD19 is human CD19.

[0596] 86. A chimeric antigen receptor comprising an extracellular portion comprising the antibody or fragment of any one of embodiments 55-85, and an intracellular signaling domain.

[0597] 87. The chimeric antigen receptor of embodiment 86, wherein the antibody or fragment comprises an scFv and the intracellular signaling domain comprises an ITAM.

[0598] 88. The chimeric antigen receptor of embodiment 87, wherein the intracellular signaling domain comprises the signaling domain of the ζ chain of the CD3-ζ (CD3ζ) chain.

[0599] 89. The chimeric antigen receptor of any one of embodiments 86-88, further comprising a transmembrane domain connecting the extracellular domain and the intracellular signaling domain.

[0600] 90. The chimeric antigen receptor of embodiment 89, wherein the transmembrane domain comprises the transmembrane portion of CD28.

[0601] 91. The chimeric antigen receptor of any one of embodiments 86-90, further comprising an intracellular signal transduction domain of a T cell co-stimulatory molecule.

[0602] 92. The chimeric antigen receptor of embodiment 91, wherein the T cell co-stimulatory molecule is selected from the group consisting of CD28 and 41BB.

[0603] 93. An engineered cell expressing a chimeric antigen receptor as described in any of embodiments 86-92.

[0604] 94. The engineered cell of embodiment 93, which is a T cell.

[0605] 95. A method of treatment comprising administering the cells of embodiment 93 or 94 to a subject having a disease or disorder associated with CD19.

[0606] 96. A method of treatment comprising administering the cells of any one of embodiments 55-85 to a subject having a disease or disorder associated with CD19.

[0607] 97. The method of embodiment 95 or 96, wherein the disease or disorder is a B cell malignancy.

[0608] 98. The method of embodiment 97, wherein the B cell malignancy is selected from the group consisting of B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphocytic leukemia, non-acute lymphoblastic leukemia, non-Hodgkin lymphoma, diffuse large B cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, spleen, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, and Hodgkin lymphoma.

[0609] 99. A nucleic acid encoding an antibody as described in any of embodiments 55-85 or a chimeric antigen receptor as described in any of embodiments 86-92.

[0610] 100. A composition comprising the antibody of any one of embodiments 55-85, the CAR of any one of embodiments 86-92, or the cell of embodiment 93 or 94.

[0611] 101. A method of treatment comprising administering the composition of embodiment 100 to a subject having a disease or disorder associated with CD19.

[0612] 102. The antibody or fragment of any one of embodiments 1-38 or 55-85, the CAR of any one of embodiments 39-45 or 86-92, the cell of any one of embodiments 46, 47, 93 and 94, the method of any one of embodiments 48-51, 54, 95-98 and 101, the nucleic acid of any one of embodiments 52 or 99, or the composition of any one of embodiments 53 and 100, wherein the antibody or fragment specifically binds to an epitope of one or more amino acids in a region comprising the extracellular portion of CD19.

[0613] 103. The antibody or fragment of any one of embodiments 1-38 or 55-85, the CAR of any one of embodiments 39-45 or 86-92, the cell of any one of embodiments 46, 47, 93 and 94, the method of any one of embodiments 48-51, 54, 95-98 and 101, the nucleic acid of any one of embodiments 52 or 99, or the composition of any one of embodiments 53 and 100, wherein the antibody or fragment specifically binds to an epitope located within a region of the extracellular portion of CD19.

[0614] 103. The antibody or fragment of any one of embodiments 1-38 or 55-85, the CAR of any one of embodiments 39-45 or 86-92, the cell of any one of embodiments 46, 47, 93 and 94, the method of any one of embodiments 48-51, 54, 95-98 and 101, the nucleic acid of any one of embodiments 52 or 99, or the composition of any one of embodiments 53 and 100, wherein the antibody or fragment specifically binds to a polypeptide consisting of or consisting essentially of a region of the extracellular portion of CD19, or a polypeptide comprising a region of the extracellular portion of CD19 but not comprising or essentially comprising other portions of CD19.

[0615] 104. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 102-103, wherein the region of the extracellular portion of CD19 is a juxtamembrane region.

[0616] 105. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 102-104, wherein the region of the extracellular portion of CD19 is the portion encoded by the fourth exon of CD19 or the portion corresponding to positions 176-277 of the human CD19 sequence shown in SEQ ID NO:92.

[0617] 106. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 102-105, wherein the region of the extracellular portion of CD19 comprises or consists of the membrane-most 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 44, 43, 43, 41 or 40 amino acid portion of the extracellular portion of CD19.

[0618] 107. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 102-106, wherein the region of the extracellular portion comprises, or consists of, the Ig-like domain of CD19, the portion encoded by the second exon of CD19 and / or the portion corresponding to positions 20-117 of the human CD19 sequence shown in SEQ ID NO:92.

[0619] 108. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 102-106, which is optionally any of the portions of the extracellular region described in any one of embodiments 105-107.

[0620] 109. The antibody or fragment of any one of embodiments 1-38 or 55-85, the CAR of any one of embodiments 39-45 or 86-92, the cell of any one of embodiments 46, 47, 93 and 94, the method of any one of embodiments 48-51, 54, 95-98 and 101, the nucleic acid of any one of embodiments 52 or 99, or the composition of any one of embodiments 53 and 100, wherein the antibody or fragment specifically binds to an epitope comprising CD19 comprising one or more amino acids located at, present in or including the portion of CD19 corresponding to residues 218-249 of the human CD19 sequence shown in SEQ ID NO:92.

[0621] 110. The antibody, fragment, cell, method, nucleic acid or composition of embodiment 109, wherein the portion comprises the sequence shown in SEQ ID NO:143.

[0622] 111. The antibody or fragment of any one of embodiments 1-38 or 55-85, the CAR of any one of embodiments 39-45 or 86-92, the cell of any one of embodiments 46, 47, 93 and 94, the method of any one of embodiments 48-51, 54, 95-98 and 101, the nucleic acid of embodiment 52 or 99, or the composition of any one of embodiments 53 and 100, wherein the antibody or fragment specifically binds to a CD19 epitope comprising amino acids at positions corresponding to positions of the human CD19 sequence shown in SEQ ID NO: 92 selected from the group consisting of histidine (H) at position 218, alanine (A) at position 236, methionine (M) at position 242, glutamic acid (E) at position 243, proline (P) at position 249, and / or lysine (K) and / or serine (S) at positions 223 and 224, and combinations thereof.

[0623] 112. The antibody or fragment of any one of embodiments 1-38 or 55-85, the CAR of any one of embodiments 39-45 or 86-92, the cell of any one of embodiments 46, 47, 93 and 94, the method of any one of embodiments 48-51, 54, 95-98 and 101, the nucleic acid of embodiment 52 or 99, or the composition of any one of embodiments 53 and 100, wherein the amino acids at positions corresponding to positions of the human CD19 sequence shown in SEQ ID NO: 92 selected from the group consisting of histidine (H) at position 218, alanine (A) at position 236, methionine (M) at position 242, glutamic acid (E) at position 243, proline (P) at position 249, and / or lysine (K) and / or serine (S) at positions 223 and 224, and combinations thereof, are important for binding of the antibody to human CD19.

[0624] 113. The antibody, fragment, cell, method, nucleic acid or composition of embodiment 111 or 112, wherein the amino acid is the same as the amino acid present at the corresponding position in SEQ ID NO:92.

[0625] 114. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 111-113, wherein the amino acid is or comprises an amino acid at a position corresponding to histidine at position 218 of human CD19, optionally wherein the amino acid at said position is histidine.

[0626] 115. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 111-114, wherein the amino acid is or comprises an amino acid at a position corresponding to the alanine at position 236 of the human CD19 sequence, optionally wherein the amino acid at said position is alanine.

[0627] 116. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 111-115, wherein the amino acid is or comprises an amino acid at a position corresponding to methionine at position 242 of human CD19, optionally wherein the amino acid at said position is methionine.

[0628] 117. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 111-116, wherein the amino acid is or comprises an amino acid at a position corresponding to glutamic acid at position 243 of human CD19, optionally wherein the amino acid at said position is glutamic acid.

[0629] 118. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 111-117, wherein the amino acid is or comprises an amino acid at a position corresponding to proline at position 249 of human CD19, optionally wherein the amino acid at said position is proline.

[0630] 119. The antibody, fragment, cell, method, nucleic acid or composition of any one of embodiments 111-118, wherein the amino acid is or comprises one or more amino acids at one or both of the positions corresponding to lysine and / or serine at positions 223 and 224 of human CD19.

[0631] 120. The antibody or fragment of any one of embodiments 1-38 or 55-85, the CAR of any one of embodiments 39-45 or 86-92, the cell of any one of embodiments 46, 47, 93 and 94, the method of any one of embodiments 48-51, 54, 95-98 and 101, the nucleic acid of any one of embodiments 52 or 99, or the composition of any one of embodiments 53 and 100, wherein the antibody or fragment specifically binds to an epitope that overlaps with, is equal to, or comprises the epitope specifically bound by the reference antibody, wherein the overlapping portion comprises or is located within the portion of CD19 that (a) comprises SEQ ID NO: 143, (b) corresponds to residues 218-249 of the human CD19 sequence set forth in SEQ ID NO: 92, or (c) corresponds to the region of CD19 encoded by exon 4 of human CD19, or (d) is within the portion corresponding to the 75th membrane-proximal residue or the 80th membrane-proximal residue of human CD19.

[0632] 121. The antibody, fragment, cell, method, nucleic acid or composition of embodiment 120, wherein the reference antibody is FMC63, or wherein the reference antibody is SJ25C1. Example

[0633] The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0634] Example 1: Preparation and evaluation of anti-CD19 antibodies

[0635] Exemplary anti-CD19 antibodies that specifically bind to cells expressing CD19, have similar binding properties to, and / or compete for binding with, a murine anti-CD19 reference antibody were generated and evaluated.

[0636] 1A. Library selection and antibody preparation

[0637] An exemplary anti-CD19 antibody (scFv) was generated by a series of selection steps performed on a human primary antibody library encoding dsDNA displayed in a cell-free system. V was selected for binding to living cells through three consecutive rounds of selection. H Library members were enriched for those that specifically bound to stably transfected HEK293 cells expressing CD19 but not to parental HEK293 cells and / or to CHOK1 cells that do not express CD19. At the end of each round of selection, three separate elution pools were generated by: (a) surface stripping to recover binders from target cells, (b) competitive elution using a murine anti-CD19 antibody, FMC63 IgG, and (c) competitive elution using another murine anti-CD19 antibody, SJ25C1 (both (b) and (c) were performed to enrich for binders that compete with FMC63 and / or SJ25C1 for binding to CD19).

[0638] At the end of three rounds of selection, these enriched V H The library was converted to a scFv library by combining the V H Members and V H -(G4S)3-V L The original man of form V L The resulting scFv library was subjected to a fourth round of enrichment for members that specifically bound to CD19-expressing HEK293 cells but not to parental cells, followed by surface stripping.

[0639] The fifth round was carried out to further enrich for members bound to another cell expressing CD19 (CD19 / K562). Separate elution pools were generated after selection, using (a) surface stripping, (b) FMC63 competitive elution, or (c) SJ25C1 competitive elution. In the sixth round, each of these three pools was further enriched for members not bound to parental cells (HEK293, twice, K562) through negative selection, and then the members of the HEK293 cells expressing CD19 were enriched through positive selection, followed by immunoprecipitation with anti-Myc antibodies that recognized the C-terminal tag on CD19 expressed on HEK293 cells.

[0640] In one study, forty-eight (48) clones from each of three R6 scFv pools were sequenced using forward and reverse primers to determine the amino acid sequence. 130 of the scFv sequences determined showed full-length reads. Convergence was observed in the sequences. Eighteen (18) duplicates were identified in these 130 scFv sequences (representing forty-six (46) of the 130 clones). In this study, a V sequence comprising CDRs 1-3 and FRs 1-3 was detected in two of the different pools. HPartial sequence fourteen (14) times (10 copies from one and 4 copies from the other), with 5 different V L Pairing. Other copies were identified 2-5 times in different pools; others were single copy sequences. In another study, other CD19-binding clones were identified and sequenced. They showed the same V H parts, with different V L sequence.

[0641] 1B. Specific binding to cells expressing CD19

[0642] The binding of sequenced clones to HEK293 cells expressing CD19 and control HEK293 cells was assessed by flow cytometry using crude cell lysates or bacterially produced supernatants from in vitro translation compared to cells that did not express CD19. Briefly, the RNA of each clone was standardized and in vitro translated as a crude scFv with a C-terminal FLAG tag. HEK293 expressing CD19 and control (intended transfected) HEK293 cells were used for the assay. The binding of individual scFv to CD19 and control cells was detected using a secondary anti-FLAG-AleXa647 conjugate. Alternatively, the scFv binding pool was cloned into an E. coli expression vector and produced as a scFv with a HIS tag, which was detected in a flow cytometric assay using an anti-HIS-AleXa647 conjugate. Mouse anti-CD19 antibodies (FMC63 scFv and FMC63 IgG) were used as positive controls; control scFvs were also used. The mean fluorescence intensity (MFI) was assessed by flow cytometry. Figure 1A and 1B , showing binding of identified clones to cells expressing CD 19. The clones evaluated included scFvs, including clones 5, 17, 18 (identified using in vitro translated lysates) and 76 (identified using bacterial supernatants), which clearly showed preferential binding to cells expressing CD 19 compared to CD 19 negative cells.

[0643] like Figure 1A and 1B As shown, for some clones, the fold change in the extent of binding to cells expressing CD19 as measured by mean fluorescence intensity in this example was approximately equal to, at least equal to, or greater than the fold change observed for the positive control reference antibodies, murine anti-CD19 antibody FMC63 scFv and / or FMC63 IgG. In some cases, the observed overall extent of binding to CD19 or to cells expressing CD19 was approximately the same as, at least equal to, or greater than that observed for one or more positive control reference antibodies.

[0644] Four (4) scFv clones ("Clone 18," "Clone 17," "Clone 5," and "Clone 76") that clearly showed binding preference for cells expressing CD19 relative to cells not expressing CD19 were further analyzed. Sequencing showed that these clones shared their V H The CDR sequences in the sequence and have different V L Sequences and different CDR-L. Multiple sequences corresponding to these four clones, including exemplary scFv, V H 、V L The sequence identifiers of the CDR (Kabat) amino acid sequences and scFv sequence encoding nucleotide sequences are listed in Table 2. A germline variant of clone 18 (designated "clone 18B") was generated by a cysteine ​​(C) → serine (S) substitution at Kabat position 89; the sequence of this clone is also listed in Table 2. Each of these clones has a V H 3 chain sequence. Clone 18 included a light chain framework derived from a Vλ2 sequence (of which clone 18B had a Vλ2 germline framework sequence); clones 17 and 76 had a Vλ1 sequence, and clone 5 included a Vλ3 sequence. Clones 18 and 17 were derived from multiple batches and libraries, including V H -V L Shuffled and scFv. Clone 76 was derived from V H -V L SJ25C1 competitive elution (6th round); Clone 5 was derived from V H -V L FMC63 competitive elution (round 6).

[0645]

[0646] 1C. Binding affinity, competition with reference antibodies

[0647] Clone 5, 17, 18, 18B and 76 were purified in a single step and evaluated by SDS gel. An exemplary gel of the study is shown in Figure 2 (Lanes 1 and 2 = clone 5, non-reduced, reduced; Lanes 3 and 4 = clone 17, non-reduced, reduced; Lanes 5 and 6 = clone 18, non-reduced and reduced; Lanes 7 and 8 = clone 76, non-reduced and reduced.) In this study, the isoelectric points were measured to be 5.36, 5.32, 7.11, and 5.32 for clones 5, 17, 18, and 76, respectively.

[0648] Melting temperature (T m ) measurements were performed using a BioTad CFX96 instrument to analyze the incorporation of Sypro Orange protein at increasing temperatures, showing T values ​​similar to those observed for the reference antibody FMC63 scFv m The results are shown in Table 3.

[0649]

[0650] The clones were titered and their binding affinity to CD19-expressing K562 cells was assessed by flow cytometry (EC 50 ), using a reference mouse CD19 antibody, FMC63 scFv, as a positive control. The results of three separate experiments, each including and comparing other binding affinities with the results of clone 18, are shown in Figure 3 A-3C.

[0651] The results are shown in Figure 3 In the experiment of A, the EC values ​​of clone 18, clone 17, another clone identified by this study (called clone 192; sequence is shown in Table 6), and the reference antibody (FMC63 scFv) were 50 The values ​​were measured to be 3.79nM, 14.86nM, 12.80nM and 7.37nM respectively. Figure 3 In the experiment of B, the EC of clone 18, clone 18B and clone 76 50 The values ​​were measured to be 7.1 nM, 9.3 nM and 7.9 nM respectively. Figure 3 In the experiment of C, the EC of clone 18 and clone 76 50 The values ​​were measured to be 4.1 nM and 8.8 nM, respectively.

[0652] Thus, each clone tested specifically binds to cells expressing CD19 with similar affinity as the reference antibody, e.g., with an EC50 that is about equal to or lower than the reference antibody, or no more than about 1.5-fold, or no more than about 2-fold, or no more than about 3-fold greater than the EC50 of the reference antibody. 50 .

[0653] In another experiment, clones 18, 5, 17, other clones identified (161, 170, 1 (see sequence information in Table 6)), and the positive control reference antibody FMC63 scFv (one plate) and clone 18, other clones identified (177, 184, 192, 198), and the positive control reference antibody FMC63 scFv (another plate) were evaluated by the same experiment. The results are shown in Figure 4 The EC observed on both plates 50 The values ​​are shown in Tables 4A and 4B. As shown, the clones were observed to have comparable binding affinities to the reference antibody.

[0654]

[0655] Competition binding assays were performed to evaluate competition for binding of different antibodies to cells expressing CD19. In one assay, 0.5 nM (~EC 50 ) Binding of FITC-labeled SJ25C1 to Ramos cells; Binding was assessed by flow cytometry (mean fluorescence intensity). The results are shown in Figure 5 A, indicates that in this study, FMC63 IgG competed with SJ25C1 IgG1 for binding to CD19, indicating that SJ25C1 and FMC63 bind to overlapping epitopes of CD19, e.g., shared epitopes. In another experiment, cells expressing CD19 were incubated with labeled FMC63 IgG in the presence (or absence) of different concentrations of clone 18 scFv, FMC63 scFv (positive control), and control cFv (negative control). The results are shown in Figure 5 B. As shown, both clone 18 scFv and FMC63 scFv (but not negative control cFv) competed with FMC63 IgG for binding to CD19-expressing cells with comparable IC 50 values ​​(24.0 nM and 19.8 nM, respectively), indicating that clone 18 binds to an epitope of CD19 that overlaps with the epitope recognized by FMC63 and competes for binding to a similar extent with the reference antibody.

[0656] In another experiment, 10 nM (EC 50 ) AleXa647-labeled FMC63 scFv was incubated with CD19-expressing K562 cells in the presence or absence of different concentrations of clone 18scFv, clone 18BscFv, clone 17 scFv, clone 76scFv, reference antibody (FMC63 scFv) and negative control antibody (R12). The results are shown in Figure 6 The clones and the reference antibody, but not the negative control antibody, showed competition with the FMC63 scFv for binding to CD19, and competition by the reference antibody with itself was similar to that observed for the clones tested.

[0657] In summary, the following ECs were observed for different clones in multiple studies: 50 (binding affinity) and IC 50 (Competition) values ​​are listed in Table 5. As shown therein, the identified human CD19 antibodies include those having similar degrees of binding affinity for CD19 and similar degrees of competitive inhibition of a murine anti-CD19 reference antibody (compared to the reference antibody itself), e.g., about the same, less than or no more than 1.5-fold, 2-fold, or 3-fold greater EC 50 and / or IC 50 .

[0658]

[0659] 1D. Size Exclusion Chromatography

[0660] The biophysical properties of clone 18B were evaluated by size exclusion chromatography. A HiLoad 16 / 600 SuperdeX200 column was calibrated and injected with a Bio-Rad gel filtration standard of 150-1901 kDa protein, and fractions were collected at 1.5 mL / min to generate a reference. 770 μg of clone 18B scFv was injected into the column and fractions were collected under the same conditions. The results are shown in Figure 7 ( Figure 7 A = Standard; Figure 7 B = Clone 18B). The results for clone 18B scFv showed a single peak with minimal large aggregates observed.

[0661] Example 2: Preparation and evaluation of other anti-CD19 antibodies

[0662] Additional exemplary anti-CD 19 antibodies (scFv fragments) that have similar binding properties to (and / or compete for binding with) the murine anti-CD 19 reference antibody were generated and evaluated.

[0663] 2A. Library selection and antibody preparation

[0664] Other exemplary anti-CD19 scFvs were generated by two different selection schemes, each involving a series of selection steps performed on human antibody libraries encoding dsDNA displayed in a cell-free system.

[0665] In one approach (referred to as "Clone 18 CDR3 grafting"), the heavy chain CDR3 (CDR-H3) sequence (SEQ ID NO: 20, DQGYHYYDSAEHAFDI) present in the clone identified in Example 1 was grafted into the original human V H Library framework. The resulting CDR3-transferred V H The members of the library have been compared with the original human V L Library members were shuffled to generate V H -(G4S)3-V L The resulting scFv library underwent three rounds of selection to enrich for members that specifically bound to CD19-expressing HEK293 cells and did not bind to parental cells, followed by surface stripping for round 1 (R1) and immunoprecipitation and dissociation for round 2 (R2).

[0666] In another approach (termed "FMC63-directed selection"), two initial scFv libraries were generated, each by (a) shuffling the original V HThe members of the library are related to the V L region, and (b) reorganization of the original V L The members of the library are related to the V H After two and three rounds of selection, the parent FMC63 V H or V L Under the guidance of , the library members from (a) and (b) were enriched for CD19-binding. The binding molecules were eluted from CD19 / HEK293 cells (R1) by surface stripping, and FMC63 was eluted from CD19 / K562 cells (R2 and R3). The third scFv library was generated by shuffling the selected V from (a) H Sequence and V selected from (b) L Sequence. CD19 / HEK293 cells were subjected to three additional rounds of selection using surface stripping (R1), followed by elution of CD19 / K562 cells with FMC63 (R2), and immunoprecipitation of CD19 / HEK293 cells with immunoprecipitation (R3). Binding of the selected scFv clones to cells expressing CD19 was confirmed by flow cytometry using bacterially produced supernatants. The selected scFv pool was cloned into an E. coli expression vector and produced as a HIS-tagged scFv. Binding of individual clones to CD19-transfected HEK293 cells was detected by flow cytometry using an anti-HIS-AleXa647 conjugate. Clone 18 or clone 18B was used as a positive control, along with various negative controls. The results are shown in Figure 8A -C (MFI = mean fluorescence intensity).

[0667] Shown in Figure 8D The results confirmed that the exemplary twenty-three (23) hits specifically bound CD19 ( Figure 8A (Asterisks in -C represent 4 matches identified by CDR3 grafting and 19 matches identified by FMC63-guided selection). Binding of in vitro translated FLAG-tagged scFv to CD19-expressing K562 cells was assessed by flow cytometry as described in Example 1 compared to control (to-be-transfected) K562 cells. As shown, these clones specifically bind to cells expressing CD19.

[0668] These and other CD19-specific scFv clones generated by the selection schemes of Examples 1 and 2 were further evaluated. Sequencing revealed several CD19-specific binding antibodies (scFvs) with various light chain sequences and a common CDR-H3 sequence (SEQ ID NO: 20) that is also present in the scFv described in Example 1. Sequence identifiers corresponding to the various sequences of other CD19-binding scFvs are listed in Table 6, including scFv, V H 、VL and CDR (Kabat) amino acid sequences (and encoding scFv nucleotide sequences). The CD19-specific scFv clones include those with several different light chain variable and CDR sequences, some of which have CDR-H1, CDR-H2, and / or CDR-H3 shown in SEQ ID NO: 11, CDR-H1, CDR-H2, and / or CDR-H3 with sequences of SEQ ID NO: 18, 19, and / or 20, and / or CDR-H1, CDR-H2, and / or CDR-H3 with sequences of 18, 72, and 20. Each clone listed in Table 6 is derived from a human V H 3 frames (indicating the kappa and lambda gene V segments of the origin of the clone).

[0669]

[0670] 2B. Purification and Evaluation

[0671] The above clones, including those listed in Table 6 and / or described in Example 1, were purified by a single-step purification method and evaluated by SDS gel. The results are shown in Figure 9 (Lane 1 = MW marker; Lanes 2, 9, and 10 = clone 5 (1530, 2880, 1130 μg / mL); Lane 3 = clone 18B (660 μg / mL); Lanes 4, 11, 12, and 13 = clone 17 (300, 1060, 180, 1440 μg / mL); Lane 5 = clone 192B (1580 μg / mL); Lanes 6 and 14 = clone 76 (1340, 3220 μg / mL); Lane 7 = clone 835 (470 μg / mL); Lane 8 = clone 488 (340 μg / mL)). Melting temperature (T m ) measurements were performed as described in Example 1, revealing T values ​​similar to those observed for the reference antibodies and clones in Example 1. m Values ​​(Table 7).

[0672]

[0673] Different clones were titrated and their binding affinity (EC50) to different CD19 expressing cells was assessed by flow cytometry. FMC63 scFv reference antibody was used as a positive control. Results from five separate experiments evaluating the binding affinity of different CD19 specific scFv clones are shown in Figures 10A-10E As shown, selection resulted in several CD19-specific sc...

Claims

1. An anti-CD19 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: (a) the VH region comprises a heavy chain complementary determining region 1 (CDR-H1), CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 consist of the amino acid sequences shown in SEQ ID NOs: 18, 19 and 20, respectively; and The VL region comprises a light chain complementary determining region 1 (CDR-L1), CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 are composed of the amino acid sequences shown in SEQ ID NOs: 74, 94 and 103, respectively; are composed of the amino acid sequences shown in SEQ ID NOs: 73, 93 and 101, respectively; or are composed of the amino acid sequences shown in SEQ ID NOs: 75, 95 and 104, respectively; or (b) the VH region comprises CDR-H1, CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 consist of the amino acid sequences shown in SEQ ID NOs: 18, 72 and 20, respectively; and The VL region comprises CDR-L1, CDR-L2 and CDR-L3, and the CDR-L1, CDR-L2 and CDR-L3 are respectively composed of the amino acid sequences as shown in SEQ ID NO:80, 100 and 109; are respectively composed of the amino acid sequences as shown in SEQ ID NO:77, 97 and 106; or are respectively composed of the amino acid sequences as shown in SEQ ID NO:78, 98 and 107.

2. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH and VL regions of the antibody or antigen-binding fragment thereof comprise amino acid sequences that are at least 90% identical to the sequences shown in SEQ ID NOs: 63 and 71, respectively; The VH and VL regions of the antibody or antigen-binding fragment thereof comprise amino acid sequences that are at least 90% identical to the sequences shown in SEQ ID NOs: 11 and 65, respectively; The VH and VL regions of the antibody or antigen-binding fragment thereof comprise amino acid sequences that are at least 90% identical to the sequences shown in SEQ ID NOs: 60 and 64, respectively; The VH and VL regions of the antibody or antigen-binding fragment thereof comprise amino acid sequences that are at least 90% identical to the sequences shown in SEQ ID NOs: 61 and 66, respectively; The VH and VL regions of the antibody or antigen-binding fragment thereof comprise amino acid sequences that are at least 90% identical to the sequences shown in SEQ ID NOs: 62 and 69, respectively; or The VH and VL regions of the antibody or antigen-binding fragment thereof comprise amino acid sequences that are at least 90% identical to the sequences shown in SEQ ID NOs: 63 and 90, respectively.

3. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 63 and 71, respectively; The VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively; The VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 60 and 64, respectively; The VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 61 and 66, respectively; The VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 62 and 69, respectively; or The VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 63 and 90, respectively.

4. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof can specifically bind to human CD19.

5. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The binding affinity of the antibody or antigen-binding fragment thereof is as high as the binding affinity of a reference anti-CD19 antibody to CD19, wherein the reference anti-CD19 antibody is selected from the group consisting of FMC63 and SJ25C1.

6. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 100 nM.

7. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 50 nM.

8. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 40 nM.

9. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 30 nM.

10. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 25 nM.

11. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 20 nM.

12. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 19 nM.

13. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 18 nM.

14. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 17 nM.

15. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 16 nM.

16. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 15 nM.

17. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 14 nM.

18. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 13 nM.

19. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 12 nM.

20. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 11 nM.

21. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 10 nM.

22. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 9 nM.

23. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 8 nM.

24. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 7 nM.

25. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 6 nM.

26. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 5 nM.

27. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 4 nM.

28. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 3 nM.

29. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 2 nM.

30. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein: The antibody or antigen-binding fragment thereof has a binding affinity EC50 and / or a dissociation constant for human CD19 that is lower than 1 nM.

31. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a human antibody or fragment.

32. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 3, wherein: The antibody or antigen-binding fragment thereof is a human antibody or fragment.

33. The anti-CD19 antibody or antigen-binding fragment thereof of claim 1, which is a single-chain fragment.

34. The anti-CD19 antibody or antigen-binding fragment thereof of claim 3, which is a single-chain fragment.

35. The anti-CD19 antibody or antigen-binding fragment thereof of claim 1, which is an antigen-binding fragment comprising an antibody variable region joined by a flexible linker.

36. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 33, wherein The antigen binding fragment comprises a scFv.

37. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 34, wherein The antigen binding fragment comprises a scFv.

38. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 36, wherein The scFv is in VH-VL orientation.

39. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 37, wherein The scFv is in VH-VL orientation.

40. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 36, wherein The scFv comprises a linker, and the linker comprises the sequence shown in SEQ ID NO:

34.

41. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 37, wherein The scFv comprises a linker, and the linker comprises the sequence shown in SEQ ID NO:

34.

42. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 36, wherein The scFv comprises a sequence having at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 45, 49, 51, 53, 57, or 89.

43. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 36, wherein The scFv comprises the amino acid sequence shown in any one of SEQ ID NO: 45, 49, 51, 53, 57, or 89.

44. A conjugate comprising a) an anti-CD19 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: (i) the VH region comprises a heavy chain complementary determining region 1 (CDR-H1), CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 have the amino acid sequences shown in SEQ ID NOs: 18, 19 and 20, respectively, and The VL region comprises a light chain complementary determining region 1 (CDR-L1), CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 have the amino acid sequences shown in SEQ ID NOs: 21, 22 and 23, respectively; have the amino acid sequences shown in SEQ ID NOs: 21, 22 and 24, respectively; have the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27, respectively; have the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30, respectively; have the amino acid sequences shown in SEQ ID NOs: 31, 32 and 33, respectively; have the amino acid sequences shown in SEQ ID NOs: 74, 94 and 103, respectively; have the amino acid sequences shown in SEQ ID NOs: 73, 93 and 101, respectively; or have the amino acid sequences shown in SEQ ID NOs: 75, 95 and 104, respectively; or (ii) the VH region comprises CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1, CDR-H2 and CDR-H3 consist of the amino acid sequences shown in SEQ ID NOs: 18, 72 and 20, respectively; and The VL region comprises CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 are respectively composed of the amino acid sequences shown in SEQ ID NOs: 80, 100 and 109; respectively composed of the amino acid sequences shown in SEQ ID NOs: 77, 97 and 106; or respectively composed of the amino acid sequences shown in SEQ ID NOs: 78, 98 and 107; and b) a cytotoxic agent, an imaging agent, an enzyme or a fragment thereof, or a multimerization domain.

45. The conjugate of claim 44, wherein The conjugate comprises the anti-CD19 antibody or the antigen-binding fragment thereof, and a cytotoxic agent.

46. ​​The conjugate of claim 45, wherein The cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, a growth inhibitory agent, an enzyme, a toxin, and a radioisotope.

47. The conjugate of claim 45, wherein The cytotoxic agent is a chemotherapeutic agent selected from the group consisting of maytansinoids, auristatins, dolastatins, calicheamicins, anthracyclines, methotrexate, taxanes, trichothecenes, CC1065, and vinca alkaloids.

48. The conjugate of claim 45, wherein The cytotoxic agent is vindesine.

49. The conjugate of claim 47, wherein: (a) the auristatin is monomethyl auristatin E or monomethyl auristatin F; and / or (b) the anthracycline antibiotic is daunomycin or doxorubicin; and / or (c) the taxane is selected from the group consisting of docetaxel, paclitaxel, levofloxacin, tadalafil and octataxel; and / or (d) The vinca alkaloid is selected from the group consisting of vincristine and vinblastine.

50. The conjugate of claim 45, wherein The cytotoxic agent is etoposide.

51. The conjugate of claim 45, wherein The cytotoxic agent is a chemotherapeutic agent selected from the group consisting of methotrexate, vincristine, vinblastine, etoposide, doxorubicin, melphalan, mitomycin C, chlorambucil and daunomycin.

52. The conjugate of claim 45, wherein The cytotoxic agent is doxorubicin.

53. The conjugate of claim 45, wherein The cytotoxic agent is a toxin or a fragment thereof selected from the group consisting of diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein, momordica charantia inhibitor, curcin, crotin, saponin, gelonin, schistosomiasis toxin, restrictocin, phenomycin, enomycin and trichothecenes.

54. The conjugate of claim 45, wherein The cytotoxic agent is a radioactive isotope selected from the group consisting of: At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 ,Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu.

55. A conjugate as described in claim 44, wherein the conjugate comprises the anti-CD19 antibody or antigen-binding fragment thereof and an imaging agent.

56. The conjugate of claim 55, wherein The imaging agent is a radionuclide.

57. The conjugate of claim 56, wherein The radionuclide is selected from the group consisting of: 111 In, 99 Tc, 14 C, 131 I, 125 I and 3 H.

58. The conjugate of claim 44, wherein the conjugate comprises the anti-CD 19 antibody or antigen-binding fragment thereof and an enzyme or fragment thereof.

59. The conjugate of claim 58, wherein The enzyme or fragment thereof is a nucleolytic enzyme.

60. A conjugate comprising: a) an anti-CD19 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: (i) the VH region comprises a heavy chain complementary determining region 1 (CDR-H1), CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 have the amino acid sequences shown in SEQ ID NOs: 18, 19 and 20, respectively, and The VL region comprises a light chain complementary determining region 1 (CDR-L1), CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 have the amino acid sequences shown in SEQ ID NOs: 21, 22 and 23, respectively; have the amino acid sequences shown in SEQ ID NOs: 21, 22 and 24, respectively; have the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27, respectively; have the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30, respectively; have the amino acid sequences shown in SEQ ID NOs: 31, 32 and 33, respectively; have the amino acid sequences shown in SEQ ID NOs: 74, 94 and 103, respectively; have the amino acid sequences shown in SEQ ID NOs: 73, 93 and 101, respectively; or have the amino acid sequences shown in SEQ ID NOs: 75, 95 and 104, respectively; or (ii) the VH region comprises CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1, CDR-H2 and CDR-H3 consist of the amino acid sequences shown in SEQ ID NOs: 18, 72 and 20, respectively; and The VL region comprises CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 are respectively composed of the amino acid sequences shown in SEQ ID NOs: 80, 100 and 109; respectively composed of the amino acid sequences shown in SEQ ID NOs: 77, 97 and 106; or respectively composed of the amino acid sequences shown in SEQ ID NOs: 78, 98 and 107; and b) a detectable moiety selected from the group consisting of a radionuclide, an enzyme, a fluorescent moiety and a luminescent moiety.

61. The conjugate of claim 60, wherein The detectable moiety is a fluorescent moiety.

62. The conjugate of claim 61, wherein The fluorescent moiety is a fluorescent protein.

63. The conjugate of claim 60, wherein The detectable moiety is a radioactive isotope of chromium, cobalt, fluorine, gadolinium, germanium, holmium, indium, iodine, lanthanum, lutetium, manganese, platinum, palladium, phosphorus, praseodymium, promethium, rhenium, rhodium, ruthenium, samarium, scandium, selenium, strontium, sulfur, technetium, thallium, tin, tritium, xenon, ytterbium, and yttrium.

64. The conjugate of claim 60, wherein: The detectable moiety is a radionuclide selected from the group consisting of: 51 Cr, 57 Co, 18 F, 153 Gd, 159 Gd, 68 Ge, 166 Ho, 115 In, 113 In, 112 In, 111 In, 125 I, 131 I, 123 I, 121 I, 140 La, 177 Lu, 54 Mn, 99 Mo, 103 Pd, 32 P, 142 Pr, 149 Pm, 186 Re, 188 Re, 105 Rh, 97 Ru, 153 Sm, 47 Sc, 75 Se, 85 Sr. 35 S, 99 Tc, 201 Ti, 113 Sn, 117 Sn, 3 H, 133 Xe, 169 Yb, 175 Yb, and 90 Y.

65. The conjugate of claim 60, wherein the enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase, luciferase, and beta-galactosidase.

66. The conjugate of claim 60, wherein the detectable moiety is a fluorescent moiety or a protein selected from the group consisting of fluorescein, rhodamine, phycoerythrin, green fluorescent protein, and blue fluorescent protein.

67. The conjugate of claim 60, wherein the detectable moiety is a luminescent moiety and the luminescent moiety is a Qdot nanoparticle.

68. A chimeric antigen receptor comprising: An extracellular portion comprising an anti-CD19 antibody or an antigen-binding portion thereof as described in any one of claims 1 to 43; and Intracellular signaling domain.

69. The chimeric antigen receptor of claim 68, wherein The antibody or antigen-binding fragment thereof comprises a scFv, and the intracellular signaling domain comprises an ITAM.

70. The chimeric antigen receptor of claim 68, wherein The intracellular signal transduction domain comprises the signal transduction domain of the CD3-ζ chain.

71. The chimeric antigen receptor of claim 68, further comprising a transmembrane domain connecting the extracellular portion and the intracellular signaling domain.

72. The chimeric antigen receptor of claim 71, wherein The transmembrane domain comprises the transmembrane portion of CD28.

73. The chimeric antigen receptor of any one of claims 68-72, further comprising an intracellular signaling domain of a T cell co-stimulatory molecule.

74. The chimeric antigen receptor of claim 73, wherein The T cell co-stimulatory molecule is selected from the group consisting of CD28 and 41BB.

75. A nucleic acid encoding the anti-CD19 antibody or antigen-binding fragment thereof of any one of claims 1-43 or the chimeric antigen receptor of any one of claims 68-74.

76. An engineered cell expressing a receptor comprising an anti-CD 19 antibody or antigen-binding fragment thereof as described in any one of claims 1-43, a conjugate as described in any one of claims 44-67, or a chimeric antigen receptor as described in any one of claims 68-74.

77. The engineered cell of claim 76, wherein the engineered cell is a T cell.

78. A composition comprising an anti-CD19 antibody or antigen-binding fragment thereof as described in any one of claims 1-43, a conjugate as described in any one of claims 44-67, or a chimeric antigen receptor as described in any one of claims 68-74.

79. The composition of claim 78, further comprising a pharmaceutically acceptable excipient.

80. Use of an anti-CD19 antibody or antigen-binding fragment thereof as described in any one of claims 1-43, a conjugate as described in any one of claims 44-67, or a chimeric antigen receptor as described in any one of claims 68-74 in the preparation of a medicament for treating B-cell malignancies.

81. The use of claim 80, wherein The B-cell malignancy is a leukemia or a lymphoma.

82. The use of claim 80, wherein The B cell malignancy is selected from the group consisting of B cell chronic lymphocytic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, hairy cell leukemia, non-acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, indolent B cell lymphoma and Hodgkin's lymphoma.

83. The use of claim 80, wherein The B-cell malignancy is acute lymphoblastic leukemia.

84. The use of claim 80, wherein The B-cell malignancy is selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, splenic marginal zone lymphoma and mantle cell lymphoma.

85. Use of a composition as claimed in claim 78 or claim 79 in the preparation of a medicament for the treatment of a B-cell malignancy.

86. The use according to claim 85, wherein The B-cell malignancy is a leukemia or a lymphoma.

87. The use of claim 85, wherein The B cell malignancy is selected from the group consisting of B cell chronic lymphocytic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, hairy cell leukemia, non-acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, indolent B cell lymphoma and Hodgkin's lymphoma.

88. The use of claim 85, wherein The B-cell malignancy is commonly acute lymphoblastic leukemia.

89. The use of claim 85, wherein The B-cell malignancy is selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, splenic marginal zone lymphoma and mantle cell lymphoma.

Images