Anti-L1CAM antibodies and uses thereof

Abstract

The present disclosure provides antibodies that specifically bind to L1CAM and compositions comprising such antibodies. Also provided herein are methods for using the anti-L1CAM antibodies to prevent or treat diseases or conditions, including tumors.

Description

[0001] This application is a divisional application of the invention patent application with application number 201980045545.5, application date June 27, 2019, applicant is Aibitai Biotechnology Co., Ltd., and invention name is "Anti-L1CAM antibodies and their uses".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application is a Chinese national phase application of International Application No. PCT / IB2019 / 055472 filed on June 27, 2019, which claims the benefit of U.S. Provisional Application No. 62 / 865,871 filed on June 24, 2019 and Korean Application No. 10-2018-0075955 filed on June 29, 2018, each of which is incorporated herein by reference in its entirety.

[0004] Reference to a sequence listing submitted electronically

[0005] The contents of the sequence listing in ASCII text file format submitted electronically with this application (name: 4372_001CN02_Seqlisting_ST25; size: 82,824 bytes; and creation date: October 7, 2021) are incorporated herein by reference in their entirety. Technical Field

[0006] The present disclosure provides antibodies that specifically bind to L1 cell adhesion molecule (L1CAM), compositions comprising such antibodies, and methods of using such antibodies to prevent or treat a disease or condition in a subject, including a tumor (e.g., cholangiocarcinoma, melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, and / or ovarian cancer). Background Art

[0007] L1 cell adhesion molecule (L1CAM) is one of the immunoglobulin superfamily cell adhesion molecules (CAM) on the cell surface that mediates cell-cell adhesion. Its molecular weight is 200 to 220 kDa. L1CAM was originally known as a protein that mediates neuron-neuron adhesion and is involved in neurite outgrowth and neuronal migration. Lee V. et al., Proc. Natl. Acad. Sci. 74: 5021-5025 (1997); McGuire JC. et al., Cell. 15 (2): 357-365 (1978).

[0008] L1CAM is mainly expressed in normal human brain. In addition, L1CAM expression is found in some hematopoietic cells and kidney cells, peripheral nerves, intestinal crypt cells and ganglia, but not in other normal cells. Huszar M. et al., Human Pathology 37: 1000-1008 (2006).

[0009] Antibodies that specifically bind to L1CAM protein can be used to diagnose and prevent or treat diseases in which L1CAM is overexpressed (eg, cancer). Therefore, it is necessary to develop antibodies that specifically bind to L1CAM and are capable of regulating L1CAM activity. Summary of the invention

[0010] One aspect of the present disclosure relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to the same L1 cell adhesion molecule (L1CAM) epitope as a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH of the reference antibody comprises SEQ ID NO:23, and the VL of the reference antibody comprises SEQ ID NO:24; (b) the VH of the reference antibody comprises SEQ ID NO:25, and the VL of the reference antibody comprises SEQ ID NO:26; (c) the VH of the reference antibody comprises SEQ ID NO:27, and the VL of the reference antibody comprises SEQ ID NO:28; (d) the VH of the reference antibody comprises SEQ ID NO:29, and the VL of the reference antibody comprises SEQ ID NO:30; or (e) the VH of the reference antibody comprises SEQ ID NO:31, and the VL of the reference antibody comprises SEQ ID NO:32.

[0011] Also provided herein are anti-L1CAM antibodies or antigen-binding fragments thereof, wherein the antibody or antigen-binding fragment thereof comprises a VH complementarity determining region 1 (CDR1), a VH CDR2 and a VH CDR3 and a VL CDR1, a VL CDR2 and a VL CDR3, wherein at least one amino acid in the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of the antibody or antigen-binding fragment thereof is different from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of the mAb417 antibody, and wherein the VH CDR1 of the mAb417 antibody comprises RFGMH (SEQ ID NO: 2); the VH CDR2 of the mAb417 antibody comprises FISNDGSNKYYADSVKG (SEQ ID NO: 9); the VH CDR3 of the mAb417 antibody comprises GRAYGSGSLFDP (SEQ ID NO: 10); ID NO:4); VL CDR1 of the mAb417 antibody comprises RASRTISIYVN (SEQ ID NO:6); VL CDR2 of the mAb417 antibody comprises AASNLHS (SEQ ID NO:7); and VL CDR3 of the mAb417 antibody comprises QQSIGRGVVT (SEQ ID NO:11).

[0012] The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that cross-competes with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL) for binding to the L1CAM epitope, wherein: (a) the VH of the reference antibody comprises SEQ ID NO:23, and the VL of the reference antibody comprises SEQ ID NO:24; (b) the VH of the reference antibody comprises SEQ ID NO:25, and the VL of the reference antibody comprises SEQ ID NO:26; (c) the VH of the reference antibody comprises SEQ ID NO:27, and the VL of the reference antibody comprises SEQ ID NO:28; (d) the VH of the reference antibody comprises SEQ ID NO:29, and the VL of the reference antibody comprises SEQ ID NO:30; or (e) the VH of the reference antibody comprises SEQ ID NO:31, and the VL of the reference antibody comprises SEQ ID NO:32, wherein the antibody or antigen-binding fragment thereof comprises a VH complementarity determining region 1 (CDR1), a VH CDR2, and a VH CDR3, and a VL CDR1, a VL CDR2, and a VL CDR3, wherein at least one amino acid in the VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2 and / or VL CDR3 of the antibody or antigen-binding fragment thereof is different from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of the mAb417 antibody, and wherein the VH CDR1 of the mAb417 antibody comprises RFGMH (SEQ ID NO: 2); the VH CDR2 of the mAb417 antibody comprises FISNDGSNKYYADSVKG (SEQ ID NO: 9); the VHCDR3 of the mAb417 antibody comprises GRAYGSGSLFDP (SEQ ID NO: 4); the VL CDR1 of the mAb417 antibody comprises RASRTISIYVN (SEQ ID NO: 6); the VL CDR2 of the mAb417 antibody comprises AASNLHS (SEQ ID NO: 7); and the VL CDR3 of the mAb417 antibody comprises GRAYGSGSLFDP (SEQ ID NO: 4). CDR3 comprises QQSIGRGVVT (SEQ ID NO: 11).

[0013] In certain aspects, the at least one amino acid difference comprises an anti-L1CAM antibody or antigen-binding fragment thereof: (i) glutamine at residue 5 in VH CDR2; (ii) serine at residue 8 in VL CDR1; and / or (iii) proline at residue 8 in VL CDR3. In certain aspects, the at least one amino acid difference comprises an anti-L1CAM antibody or antigen-binding fragment thereof: (i) alanine, glycine, phenylalanine, tyrosine, threonine, proline, and tryptophan at residues 3 to 9, respectively, in VL CDR3; (ii) alanine, glycine, phenylalanine, tyrosine, serine, proline, and tryptophan at residues 3 to 9, respectively, in VL CDR3; or (iii) leucine, valine or histidine, tryptophan or phenylalanine, tyrosine, proline, and tryptophan at residues 4 to 9, respectively, in VL CDR3. In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprise a VL CDR3 of the antibody or antigen-binding fragment thereof comprising SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 21. In some aspects, the VH CDR1 of the anti-L1CAM antibody comprises RFGMH (SEQ ID NO: 2). In some aspects, the VH CDR2 of the anti-L1CAM antibody comprises FISNEGSNKYYADSVKG (SEQ ID NO: 10). In some aspects, the VH CDR3 of the anti-L1CAM antibody comprises GRAYGSGSLFDP (SEQ ID NO: 4). In some aspects, the VL CDR1 of the anti-L1CAM antibody comprises RASRTISSYVN (SEQ ID NO: 12). In some aspects, the VL CDR2 of the anti-L1CAM antibody comprises AASNLHS (SEQ ID NO: 7). In some aspects, the VL CDR3 of the anti-L1CAM antibody comprises QQSIGRGPVT (SEQ ID NO: 13).

[0014] In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof of the present disclosure comprise heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the light chain CDR3 comprises QQSIGRGPVT (SEQ ID NO: 13), QQAGFYTPWT (SEQ ID NO: 15), QQAGFYSPWT (SEQ ID NO: 17), QQSLHFYPWT (SEQ ID NO: 19) or QQSLVWYPWT (SEQ ID NO: 21).

[0015] The present disclosure further provides an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein, which has one or more characteristics selected from the group consisting of: (a) exhibiting improved productivity compared to mAb417 antibody; (b) exhibiting improved yield as measured by equilibrium dissociation constant (K) compared to mAb417 antibody; D ) as measured by the association constant (K); (c) exhibiting an improved PI value compared to the mAb417 antibody; (d) exhibiting an improved affinity as measured by the association constant (K) as compared to the mAb417 antibody; or (e) any combination thereof.

[0016] In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein exhibit improved productivity compared to the mAb417 antibody, wherein the improved productivity when expressed according to Example 3 is at least 55 mg / L, at least 56 mg / L, at least 57 mg / L, at least 58 mg / L, at least 59 mg / L, at least about 60 mg / L, at least about 61 mg / L, at least about 62 mg / L, at least about 63 mg / L, at least about 64 mg / L, at least about 65 mg / L, at least about 66 mg / L, at least about 67 mg / L, at least about 68 mg / L, at least about 69 mg / L, at least about 70 mg / L, at least about 71 mg / L, at least about 72 mg / L, at least about 73 mg / L, at least about 74 mg / L, at least about 75 mg / L, at least about 76 mg / L, at least about 77 mg / L, at least about 78 mg / L, at least about 79 mg / L, at least about 80 mg / L, at least about 81 mg / L, at least about 82 mg / L, at least about 83 mg / L, at least about 84 mg / L, at least about 85 mg / L, at least about 86 mg / L, at least about 87 mg / L, at least about 88 mg / L At least about 67 mg / L, at least about 68 mg / L, at least about 69 mg / L, at least about 70 mg / L, at least about 71 mg / L, at least about 72 mg / L, at least about 73 mg / L, at least about 74 mg / L, at least about 75 mg / L, at least about 76 mg / L, at least about 77 mg / L, at least about 78 mg / L, at least about 79 mg / L, at least about 80 mg / L, at least about 81 mg / L, at least about 82 mg / L, at least about 83 mg / L, at least about 84 mg / L or at least about 85 mg / L.

[0017] In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein exhibit a relative affinity as measured by the equilibrium dissociation constant (K D ) measured by improved affinity, where the improved K D Less than 2.6×10 -10 M, less than 2.5×10 -10 M, less than 2.0×10 -10 M, less than 1.5×10 -10 M, less than 1.0×10 -10 M, less than 9×10 -11 M, less than 8×10 -11 M, less than 7×10 -11 M, less than 6×10 -11 M, less than 5×10 -11 M, less than 4×10 -11 M, less than 3×10 -11 M, less than 2×10-11 M, less than 1×10 -11 M, less than 9×10 -12 M, less than 8×10 -12 M, less than 7×10 -12 M, less than 6×10 -12 M, less than 5×10 -12 M, less than 4×10 -12 M, less than 3×10 -12 M, less than 2×10 -12 M, less than 1×10 -12 M, less than 9×10 -13 M or less than 8×10 -13 M.

[0018] In other aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein exhibit improved affinity as measured by the association constant (K) compared to the mAb417 antibody, wherein the improved K is less than 5×10 -10 M, less than 4×10 -10 M, less than 3×10 -10 M, less than 2×10 -10 M, less than 1.0×10 -10 M, less than 9×10 -11 M, less than 8×10 -11 M, less than 7×10 -11 M, less than 6×10 -11 M, less than 5×10 -11 M, less than 4×10 -11 M, less than 3×10 -11 M, less than 2×10 -11 M, less than 1×10 -11 M, less than 9×10 -12 M, less than 8×10 -12 M, less than 7×10 -12 M, less than 6×10 -12 M, less than 5×10 -12 M, less than 4×10 -12 M, less than 3×10 -12 M, less than 2×10 -12 M, less than 1×10 -12 M, less than 9×10 -13 M or less than 8×10 -13M. In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein exhibit an improved PI value compared to the mAb417 antibody, wherein the improved PI value is less than 9.6, less than 9.5, less than 9.4, less than 9.3, less than 9.2, less than 9.1, less than 9.0, less than 8.9, less than 8.8, less than 8.7, less than 8.6, less than 8.5, less than 8.4, less than 8.3, less than 8.2, less than 8.1, less than 8.0, less than 7.9, less than 7.8, less than 7.7 or less than 7.6.

[0019] In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprise VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3; wherein VH CDR1, CDR2 and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; and wherein VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSIGRGPVT (SEQ ID NO: 13), respectively.

[0020] In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprise VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3; wherein VH CDR1, CDR2 and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; and wherein VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQAGFYSPWT (SEQ ID NO: 17), respectively.

[0021] In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprise VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3; wherein VH CDR1, CDR2 and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; and wherein VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQAGFYTPWT (SEQ ID NO: 15), respectively.

[0022] In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprise VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3; wherein VH CDR1, CDR2 and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; and wherein VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSLHFYPWT (SEQ ID NO: 19), respectively.

[0023] In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprise VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3; wherein VH CDR1, CDR2 and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; and wherein VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSLVWYPWT (SEQ ID NO: 21), respectively.

[0024] In some aspects, the VH of the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as EVQLVESGGG VVQPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAFISNEGSNKYY ADSVKGRFTISRDNSANTL Y LQMNSLRAED TAVYYCARGR AYGSGSLFDP WGQGTLVTVS S (SEQ ID NO: 23).

[0025] In other aspects, the VL of the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as DIQLTQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCQQ SIGRGPVTFG QGTKLEIK (SEQ ID NO: 24).

[0026] In some aspects, the VH of the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as EVQLVESGGG VVQPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAFISNEGSNKYY ADSVKGRFTISRDNSANTL Y LQMNSLRAED TAVYYCARGR AYGSGSLFDP WGQGTLVTVS S (SEQ ID NO: 27).

[0027] In other aspects, the VL of the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as DIQLTQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCQQ AGFYSPWTFG QGTKLEIK (SEQ ID NO: 28).

[0028] In some aspects, the VH of the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as EVQLVESGGG VVQPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAFISNEGSNKYY ADSVKGRFTISRDNSANTL Y LQMNSLRAED TAVYYCARGR AYGSGSLFDP WGQGTLVTVSS (SEQ ID NO:25).

[0029] In some aspects, the VL of the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as DIQLTQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCQQ AGFYTPWTFG QGTKLEIK (SEQ ID NO: 26).

[0030] In other aspects, the VH of the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as EVQLVESGGG VVQPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAFISNEGSNKYY ADSVKGRFTISRDNSANTLY LQMNSLRAED TAVYYCARGR AYGSGSLFDP WGQGTLVTVS S (SEQ ID NO:29).

[0031] In some aspects, the VL of the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as DIQLTQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYC QQSLHFYPWT FG QGTKLEIK (SEQ ID NO: 30).

[0032] In other aspects, the VH of the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as EVQLVESGGG VVQPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAFISNEGSNKYY ADSVKGRFTISRDNSANTL Y LQMNSLRAED TAVYYCARGR AYGSGSLFDP WGQGTLVTVSS (SEQ ID NO:31).

[0033] In some aspects, the VL of the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence shown as DIQLTQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYC QQSLVWYPWT FG QGTKLEIK (SEQ ID NO: 32).

[0034] In some aspects, the VH of the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein comprises SEQ ID NO: 23, and the VL of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO: 24. In other aspects, the VH of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO: 25, and the VL of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO: 26. In some aspects, the VH of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO: 27, and the VL of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO: 28. In other aspects, the VH of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO: 29, and the VL of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO: 30. In some aspects, the VH of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO: 29, and the VL of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO: 30. In other aspects, the VH of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO:31, and the VL of the anti-L1CAM antibody or antigen-binding fragment thereof comprises SEQ ID NO:32.

[0035] Also provided herein is an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein, comprising a heavy chain (HC) and a light chain (LC), wherein the HC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 38, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 39. In some aspects, the HC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 40, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 41. In some aspects, the HC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 42, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 43. In some aspects, the HC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 44, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 45. In some aspects, the HC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 46, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 20.

[0036] In some aspects, the anti-L1CAM antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, variants thereof, and any combination thereof. In some aspects, the anti-L1CAM antibody is a chimeric antibody or a human antibody. In some aspects, the anti-L1CAM antibody comprises Fab, Fab′, F(ab′)2, Fv, or single-chain Fv (scFv).

[0037] Some aspects of the present disclosure relate to nucleic acids encoding anti-L1CAM antibodies, vectors comprising the nucleic acids, host cells comprising the vectors. In some aspects, the host cell is selected from the group consisting of Escherichia coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NSO, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture.

[0038] Some aspects of the present disclosure relate to an immunoconjugate comprising an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein linked to an agent.

[0039] Also provided herein is a bispecific or multispecific antibody comprising an anti-L1CAM antibody or antigen-binding fragment and an antibody or antigen-binding fragment thereof that binds to an antigen.

[0040] Some aspects of the present disclosure relate to a composition comprising an anti-L1CAM antibody disclosed herein, a nucleic acid, a vector, a host cell, an immunoconjugate, or a bispecific or multispecific antibody and a carrier.

[0041] Also provided herein is a kit comprising the anti-L1CAM antibody disclosed herein and instructions for use.

[0042] Some aspects of the present disclosure relate to a method for producing an antibody that specifically binds to human L1CAM protein, comprising culturing host cells under suitable conditions and isolating the antibody.

[0043] Also provided herein is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an anti-L1CAM antibody, nucleic acid, vector, host cell, immunoconjugate, or bispecific or multispecific antibody disclosed herein. In some aspects, the disease or condition comprises a tumor. In some aspects, the tumor comprises bile duct cancer, melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer. In other aspects, the anti-L1CAM antibody, the nucleic acid, the vector, the cell, the immunoconjugate, the bispecific or multispecific antibody inhibits tumor growth and / or enhances the infiltration of immune cells into the tumor.

[0044] Some aspects of the present disclosure relate to methods comprising administering an additional therapeutic agent. In some aspects, the additional therapeutic agent comprises chemotherapy, immunotherapy, radiotherapy, or a combination thereof. In some aspects, the additional therapeutic agent is an immune checkpoint inhibitor.

[0045] Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all cited references (including scientific articles, newspaper reports, GenBank entries, patents and patent applications cited throughout this application) are expressly incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figures 1A to 1D Flow cytometry was used to analyze the CHO-DG44 cells ( Figure 1A )、NCI-H522( Figure 1B )、SKOV3( Figure 1C ) and B16F1( Figure 1D ) Analysis of the antigen binding specificity of Ab417 variants to human LCAM1.

[0047] FIG. 2A to FIG. 2F The purified Ab417 ( Figure 2A ) and Ab417 variant Ab612 ( Figure 2B )、Ab4H5( Figure 2C )、Ab2C2( Figure 2D )、Ab4H6( Figure 2E ) and Ab5D12( Figure 2F )’s quality.

[0048] FIG. 3A to FIG. 3D Analysis of tumor growth inhibition effects of Ab417 variants is shown. Figure 3A Shown are changes in tumor volume in the Choi-CK xenograft model after administration of Ab417 (10 mg / kg), Ab612 (10 mg / kg), control hFc (human Fc) antibody (3.3 mg / kg) and negative control (PBS) (*p<0.05, significantly different from the isotype control group by Dunnett's t-test). Figure 3B Shown are body weight changes in the Choi-CK xenograft model after administration of Ab417, Ab612, control hFc, and negative control (PBS). Figure 3C Shown are the tumor weights of the Choi-CK xenograft model after administration of Ab417 (10 mg / kg), Ab612 (10 mg / kg), Ab612 (10 mg / kg), control hFc antibody (3.3 mg / kg) and negative control (vehicle). (*p<0.01, significantly different from the isotype control group by Dunnett's t-test). Figure 3D It shows that Figure 3C Tumor images of eight mice in each group sacrificed at the end of the experiment as described in . DETAILED DESCRIPTION

[0049] Disclosed herein is an isolated antibody or antigen-binding fragment thereof that: specifically binds to the same L1 cell adhesion molecule (L1CAM) epitope as a reference antibody; cross-competes with the reference antibody for binding to the L1CAM epitope and exhibits one or more properties disclosed herein; and / or prevents and / or treats a disease or condition including a tumor.

[0050] To facilitate understanding of the disclosure disclosed herein, various terms and phrases are defined. Additional definitions are presented throughout the detailed description.

[0051] I. Definitions

[0052] Throughout this disclosure, the term "a / an" entity refers to one or more of the entities; for example, "antibody" should be understood to represent one or more antibodies. Therefore, the terms "one (kind)", "one (kind) or more (kinds)" and "at least one (kind)" are used interchangeably herein.

[0053] Furthermore, when used herein, "and / or" should be considered to specifically disclose each of the two specified features or components, with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0054] It should be understood that whenever various aspects are described herein with the term "comprising," additional similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a comprehensive dictionary of many of the terms used in the present disclosure to the skilled person.

[0056] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in an amino to carboxyl orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be obtained by reference to the specification as a whole. Therefore, the terms defined immediately below are more fully explained by reference to the specification as a whole.

[0057] The term "about" is used herein to mean approximately, roughly, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by expanding the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variation up or down (higher or lower), for example, 10%.

[0058] The term "L1 cell adhesion molecule" or "L1 CAM" refers to one of the integral membrane glycoproteins belonging to the immunoglobulin superfamily of cell adhesion molecules (CAMs).

[0059] The term "L1CAM" includes any variant or isoform of L1CAM naturally expressed by cells. Thus, the antibodies described herein may cross-react with different isoforms in the same species (e.g., different isoforms of human L1CAM), or cross-react with L1CAM from species other than humans (e.g., mouse L1CAM). Alternatively, the antibodies may be specific for human L1CAM and may not exhibit any cross-reactivity with other species. L1CAM or any variant and isoform thereof may be isolated from cells or tissues naturally expressing them, or may be recombinantly produced using techniques well known in the art and / or the techniques described herein.

[0060] Human L1CAM (UniProt ID No. P32004-1; SEQ ID NO: 1) is a type 1 integral membrane glycoprotein consisting of 1,257 amino acids and spanning the cell membrane once, and its amino terminal fragment is present outside the cell membrane, while its carboxyl terminal fragment is present in the cytoplasm. The extracellular domain of L1CAM includes 11 domains, which have six immunoglobulin type 2 domains (Ig1, Ig2, Ig3, Ig4, Ig5 and Ig6), five fibronectin III-like domains (Fn1, Fn2, Fn3, Fn4 and Fn5) and twenty N-glycosylation sites. U.S. Patent No. 9,777,060.

[0061] At least two additional isoforms of human L1CAM have been identified. Isoform 2 (UniProt ID No. P32004-2; SEQ ID NO: 3) consists of 1,253 amino acids. Relative to the amino acid sequence of human L1CAM, isoform 2 lacks amino acid residues 1177-1180. Isoform 3 (UniProt ID No. P32004-3; SEQ ID NO: 5) consists of 1,248 amino acids. Relative to the amino acid sequence of human L1CAM, isoform 3 lacks amino acid residues 1177-1180 and has the following differences at amino acid residues 26-31 (YEGHHV→L).

[0062] Below are the amino acid sequences of the three known isoforms of human L1CAM.

[0063] (A) Human L1CAM (UniProt ID No. P32004-1; SEQ ID NO: 1)

[0064]

[0065]

[0066] (B) Human L1CAM isoform 2 (UniProt ID No. P32004-2; SEQ ID NO: 3)

[0067]

[0068] (C) Human L1CAM isoform 3 (UniProt ID No. P32004-3; SEQ ID NO: 5)

[0069]

[0070] The signal sequence of human L1CAM corresponds to amino acids 1-19 (underlined). Thus, the mature isoforms of human L1CAM isoform 1, human L1CAM isoform 2, and human L1CAM isoform 3 consist of amino acids 20 to 1,257, 1,253, or 1,248, respectively.

[0071] The terms "antibody" and "antibodies" are technical terms and are used interchangeably herein and refer to molecules having an antigen binding site that specifically binds to an antigen. The terms as used herein include complete antibodies and any antigen binding fragments thereof (i.e., "antigen binding fragments") or single chains. On the one hand, "antibody" refers to a glycoprotein or antigen binding fragment thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. On the other hand, "antibody" refers to a single-chain antibody comprising a single variable domain (e.g., a VHH domain). Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In some naturally occurring antibodies, the heavy chain constant region comprises three domains CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain CL.

[0072] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0073] The term "Kabat numbering" and similar terms are generally recognized in the art, and refer to a system in which the amino acid residues in the heavy chain variable region and light chain variable region of an antibody or its antigen-binding fragment are numbered. In some aspects, the CDR of an antibody can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDR in the heavy chain molecule of an antibody is usually present at amino acid positions 31 to 35 (which can be optionally included in one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2) and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are generally present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific aspect, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0074] The phrase "amino acid position numbering as in Kabat", "Kabat position" and grammatical variations thereof refer to the numbering system for the heavy chain variable domain or light chain variable domain used for the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of CDR2 (residue 52a according to Kabat) and an inserted residue after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). See Table 1.

[0075] Table 1

[0076]

[0077] The Kabat numbering of the residues of a given antibody can be determined by comparing the antibody sequence to the homology region of the "standard" Kabat numbering sequence. In contrast, Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). When numbering using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software.

[0078] IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions (including CDRs). See, for example, Lefranc, MP et al., Dev. Comp. Immunol. 27: 55-77 (2003), which is incorporated herein by reference. The IMGT numbering system is based on the alignment of more than 5,000 sequences, structural data, and characterization of hypervariable loops, and allows easy comparison of variable regions and CDR regions of all species. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0079] For all heavy chain constant region amino acid positions discussed in this disclosure, numbering is according to the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63 (1): 78-85, which describes the amino acid sequence of myeloma protein EU (which is the first sequenced human IgG1). The EU index of Edelman et al. is also shown in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, United States Public Health Service, National Institutes of Health, Bethesda. Therefore, the phrases "EU index as shown in Kabat" or "EU index of Kabat" and "positions according to the EU index as shown in Kabat ..." and grammatical variations thereof refer to the residue numbering system for human IgG1 EU antibodies based on Edelman et al. as shown in Kabat 1991.

[0080] The numbering system used for the variable domains (heavy and light chains) and light chain constant region amino acid sequences is that set forth in Kabat 1991.

[0081] The antibody can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgD, IgG2, IgG3, IgG4, IgA1 or IgA2) or any subclass (e.g., IgG1, IgG2, IgG3 and IgG4 of humans; and IgG1, IgG2a, IgG2b and IgG3 of mice). Immunoglobulins (e.g., IgG1) exist in the form of several allotypes, which differ from each other by at most a few amino acids. The antibodies disclosed herein may be from any of the commonly known isotypes, classes, subclasses or allotypes. In some aspects, the antibodies described herein are IgG1, IgG2, IgG3 or IgG4 subclasses or any hybrids thereof. In some aspects, the antibody is a human IgG1 subclass or a human IgG2 or human IgG4 subclass.

[0082] By way of example, "antibody" includes: naturally occurring antibodies and non-naturally occurring antibodies; monoclonal antibodies and polyclonal antibodies; chimeric antibodies and humanized antibodies; human antibodies and non-human antibodies; fully synthetic antibodies; single-chain antibodies; monospecific antibodies; multispecific antibodies (including bispecific antibodies); tetrameric antibodies comprising two heavy chain molecules and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers; antibody heavy chain dimers; antibody light chain-antibody heavy chain pairs; intrabodies; heteroconjugate antibodies; monovalent antibodies; camelized antibodies; affybodies; anti-idiotypic antibodies (including, e.g., anti-anti-Id antibodies) and single domain antibodies (sdAbs), which include binding molecules composed of a single monomeric variable antibody domain (e.g., a VH domain or a VL domain) that are fully capable of antigen binding. Harmen MM and Haard H J Appl Microbiol Biotechnol. 77(1):13-22 (2007)).

[0083] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human L1CAM). Such "fragments" are, for example, between about 8 and about 1500 amino acids in length, suitably between about 8 and about 745 amino acids in length, suitably between about 8 and about 300 amino acids in length, such as about 8 to about 200 amino acids or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody (e.g., the anti-L1CAM antibody described herein) include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody and a disulfide-linked Fv (sdFv); (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity determining regions (CDRs); or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected using recombinant methods by synthetic linkers that enable them to be made into a single protein chain that pairs the VL and VH regions to form a monovalent molecule, known as a single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as for intact antibodies. Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0084] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region generally refers to a fragment of an antibody, typically a fragment of a light chain or a heavy chain, typically the amino-terminal approximately 110 to 120 amino acids in a mature heavy chain, and the amino-terminal approximately 90 to 115 amino acids in a mature light chain, which differ widely in sequence between antibodies and are used for the binding and specificity of a particular antibody to its specific antigen. The variability of the sequence is concentrated in those regions known as complementary determining regions (CDRs), while the more highly conserved regions in the variable domains are known as framework regions (FRs).

[0085] Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light chain and heavy chain are primarily responsible for the interaction and specificity of the antibody with the antigen. In some aspects, the variable region is a human variable region. In some aspects, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In certain aspects, the variable region is a primate (e.g., non-human primate) variable region. In some aspects, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0086] As used herein, the term "heavy chain" when used in reference to antibodies can refer to any of the different types based on the amino acid sequence of the constant domains, such as α, δ, ε, γ, and μ, which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including the subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4.

[0087] As used herein, the term "light chain" when used with respect to an antibody can refer to any of the different types, such as κ or λ, based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific aspect, the light chain is a human light chain.

[0088] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0089] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0090] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meanings in the art. The constant region is an antibody fragment, such as a carboxyl terminal fragment of a light chain and / or a heavy chain, which is not directly involved in the binding of the antibody to an antigen but may exhibit various effector functions, such as interactions with Fc receptors. Relative to the immunoglobulin variable domain, the constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence.

[0091] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (C1q) of the classical complement system. Thus, the Fc region comprises the constant region of the antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody; in each polypeptide chain, the IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4). For IgG, the Fc region comprises the immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of immunoglobulin heavy chain Fc regions may be different, human IgG heavy chain Fc regions are generally defined as extending from the amino acid residue at position C226 or P230 (or the amino acid between these two amino acids) to the carboxyl terminus of the heavy chain, wherein numbering is according to the EU index as in Kabat. The CH2 domain in human IgG Fc region extends from about amino acid 231 to about amino acid 340, and the CH3 domain is positioned at the C-terminal side of the CH2 domain in the Fc region, i.e., it extends from about amino acid 341 of IgG to about amino acid 447. As used herein, the Fc region can be a native sequence Fc, including any allotype variant or variant Fc (e.g., non-naturally occurring Fc). Fc can also refer to this region individually or in the context of a protein polypeptide comprising Fc, such as "a binding protein comprising an Fc region" (also referred to as "Fc fusion protein" (e.g., antibody or immunoadhesion)).

[0092] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions, native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native sequence Fc includes various allotypes of Fc (see, e.g., Jefferis et al., (2009) mAbs 1:1; Vidarsson G. et al. Front Immunol. 5:520 (published online on October 20, 2014)).

[0093] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII and FcγRIV in mice; FcγRIA, FcγRIIA and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Human IgG1 binds to most human Fc receptors and elicits the strongest Fc effector functions. In terms of the type of activating Fc receptor bound, it is considered equivalent to mouse IgG2a. In contrast, human IgG4 elicits the least Fc effector function. Vidarsson G. et al. Front Immunol. 5:520 (published online on October 20, 2014).

[0094] The constant region can be manipulated, for example, by recombinant technology to eliminate one or more effector functions. "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand or the biochemical events resulting therefrom. Exemplary "effector functions" include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP) and down-regulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain). Therefore, the term "constant region without Fc function" includes a constant region with one or more effector functions mediated by the Fc region that are reduced or without the one or more effector functions.

[0095] The effector function of antibodies can be reduced or avoided by different methods. The effector function of antibodies can be reduced or avoided by using antibody fragments lacking the Fc region (e.g., Fab, F(ab') 2, single-chain Fv (scFv) or sdAb composed of monomeric VH or VL domains) to reduce or avoid. Alternatively, so-called aglycosylated antibodies can be generated by removing the sugar connected to specific residues in the Fc region to reduce the effector function of the antibody while retaining other valuable properties of the Fc region (such as extended half-life and heterodimerization). Aglycosylated antibodies can be generated by, for example, missing or changing the residues connected to the sugar, enzymatically removing the sugar, producing antibodies in cells cultured in the presence of glycosylation inhibitors, or expressing antibodies in cells (such as bacterial host cells) that cannot glycosylate proteins. See, for example, U.S. Publication No. 20120100140. Another method is to use the Fc region with reduced effector function from the IgG subclass. For example, IgG2 and IgG4 antibodies are characterized in that they have lower levels of Fc effector function compared to IgG1 and IgG3. The residues closest to the hinge region in the CH2 domain of the Fc portion are responsible for the effector function of the antibody because it contains a large number of overlapping binding sites for C1q (complement) and IgG-Fc receptors (FcγR) on effector cells of the innate immune system. Vidarsson G. et al. Front Immunol. 5: 520 (published online on October 20, 2014). Therefore, antibodies with reduced Fc effector function or without Fc effector function can be prepared by generating, for example, a chimeric Fc region comprising a CH2 domain of an IgG antibody from an IgG4 isotype and a CH3 domain of an IgG antibody from an IgG1 isotype, or a chimeric Fc region comprising a hinge region from IgG2 and a CH2 region from IgG4 (see, for example, Lau C. et al. J. Immunol. 191: 4769-4777 (2013)), or a Fc region with a mutation that causes a change in Fc effector function (e.g., reduced Fc function or no Fc function). Such Fc regions with mutations are known in the art. See, eg, US Pub. No. 20120100140 and the US applications and PCT applications cited therein, and An et al., mAbs 1:6, 572-579 (2009); the disclosures of which are incorporated by reference in their entireties.

[0096] "Hinge", "hinge domain", "hinge region" or "antibody hinge region" are used interchangeably and refer to the domain of the heavy chain constant region that connects the CH1 domain to the CH2 domain and includes the upper, middle and lower fragments of the hinge (Roux et al., J. Immunol. 1998 161: 4083). The hinge provides different levels of flexibility between the binding region and the effector region of the antibody, and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions. As used herein, for all IgG isotypes, the hinge starts at Glu216 and ends at Gly237 (Roux et al., 1998 J Immunol 161: 4083). The sequences of wild-type IgG1, IgG2, IgG3 and IgG4 hinges are known in the art. See, e.g., Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al. Front Immunol. 5:520 (published online Oct. 20, 2014).

[0097] The term "CH1 domain" refers to the heavy chain constant region that connects the variable domain to the hinge in the heavy chain constant domain. As used herein, the CH1 domain starts at A118 and ends at V215. The term "CH1 domain" includes wild-type CH1 domains and naturally occurring variants thereof (e.g., allotypes). The CH1 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, e.g., Kabat EA et al., (1991) supra and Vidarsson G. et al. Front Immunol. 5: 520 (published online on October 20, 2014). Exemplary CH1 domains include CH1 domains with mutations, and the mutations modify the biological activity of the antibody, such as half-life, such as described in U.S. Publication No. 20120100140 and the U.S. Patents and publications cited therein and PCT publications.

[0098] The term "CH2 domain" refers to the heavy chain constant region that connects the hinge to the CH3 domain in the heavy chain constant domain. As used herein, the CH2 domain starts at P238 and ends at K340. The term "CH2 domain" includes wild-type CH2 domains and naturally occurring variants thereof (e.g., allotypes). The CH2 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, e.g., Kabat EA et al., (1991) supra and Vidarsson G. et al. Front Immunol. 5: 520 (published online on October 20, 2014). Exemplary CH2 domains include CH2 domains with mutations that modify the biological activity of the antibody, such as half-life and / or reduced Fc effector function, such as described in U.S. Publication No. 20120100140 and the U.S. patents and publications cited therein and PCT publications.

[0099] The term "CH3 domain" refers to the heavy chain constant region at the C-terminus of the CH2 domain in the heavy chain constant domain. As used herein, the CH3 domain starts at G341 and ends at K447. The term "CH3 domain" includes wild-type CH3 domains and naturally occurring variants thereof (e.g., allotypes). The CH3 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, e.g., Kabat EA et al., (1991) supra and Vidarsson G. et al. Front Immunol. 5: 520 (published online on October 20, 2014). Exemplary CH3 domains include CH3 domains with mutations, and the mutations modify the biological activity of the antibody, such as half-life, such as described in U.S. Publication No. 20120100140 and the U.S. patents and publications cited therein and PCT publications.

[0100] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region genes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibodies).

[0101] "Allotype" refers to naturally occurring variants within a particular isotype group that differ by several amino acids (see, e.g., Jefferis et al., (2009) mAbs 1:1). The antibodies described herein can be of any allotype. Allotypes of IgG1, IgG2, IgG3, and IgG4 are known in the art. See, e.g., Kabat EA et al., (1991) supra; Vidarsson G. et al. Front Immunol. 5:520 (published online Oct. 20, 2014); and Lefranc MP, mAbs 1:4, 1-7 (2009).

[0102] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0103] As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to L1CAM is substantially free of antibodies that specifically bind to antigens other than L1CAM). However, an isolated antibody that specifically binds to a L1CAM epitope may have cross-reactivity to other L1CAM proteins from different species.

[0104] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be measured by the dissociation constant (K D ). Affinity can be expressed in a variety of ways known in the art, including but not limited to equilibrium dissociation constant (K D ) and equilibrium association constant (K A ) to measure and / or express. K D From k off / k on The quotient of is calculated and expressed as molar concentration (M), while K A From k on / k off k on refers to, for example, the rate constant for the association of an antibody with an antigen, and k off Refers to, for example, the dissociation of antibodies and antigens. on and k off The assay can be performed by techniques known to those of ordinary skill in the art such as immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), BLI (biolayer interferometry) or kinetic exclusion assay To determine.

[0105] As used herein, the terms "specifically binds", "specifically recognizes", "specific binding", "selective binding", and "selectively binds" are analogous terms in the context of antibodies and refer to the binding of a molecule (e.g., an antibody) to an antigen (e.g., an epitope or immune complex) as understood by those skilled in the art. For example, a molecule that specifically binds to an antigen can generally be detected, for example, by immunoassays, In certain aspects, a molecule that specifically binds to an antigen has a K value that is similar to that of the molecule when it binds to another antigen. A Compared to K of at least 2log, 2.5log, 3log, 4log or more A Binds to the antigen.

[0106] Antibodies usually bind specifically to their cognate antigens with high affinity. -5 Up to 10 -11 M or less dissociation constant (K D ) reflects. It is generally believed that any value greater than about 10 -4 M of K D As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen and substantially the same antigen with high affinity, but does not bind to unrelated antigens with high affinity, high affinity meaning that when measured by, for example, an immunoassay (e.g., ELISA), using a predetermined antigen in a BIACORE TM When measured by surface plasmon resonance (SPR) or BLI (biolayer interferometry) in the 2000 instrument, K D For 10 -7 M or less, preferably 10 -8 M or less, even more preferably 10 -9 M or less, and most preferably within 10 -8 M and 10 -10 M or smaller.

[0107] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide or hapten. The antigen may be L1CAM or a fragment thereof.

[0108] As used herein, "epitope" is a term in the art and refers to a local region of an antigen that can be specifically bound to an antibody. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or continuous epitope), or an epitope can be, for example, from two or more discontinuous regions of a polypeptide or polypeptide together (conformational, non-linear, discontinuous or discontinuous epitopes). Epitopes formed by continuous amino acids are usually, but not always, retained when exposed to a denaturing solvent, while epitopes formed by tertiary folding are usually lost when treated with a denaturing solvent. An epitope usually comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or continuous peptides from (e.g., from L1CAM) are tested for reactivity with a given antibody (e.g., anti-L1CAM antibody). Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, ed. (1996)).

[0109] In certain aspects, the epitope bound to the antibody can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assay, hydrogen / deuterium exchange coupled to a mass spectrometer (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any known method in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Part 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, issued by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) Vols. 114 and 115, Wyckoff HW et al., eds.; U.S. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Part 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, Carter CW eds.; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Part 10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-1085.

[0110] The term "epitope mapping" refers to the process of identifying molecular determinants of antibody-antigen recognition.

[0111] The term "binding to the same epitope" as a reference antibody means that the antibody binds to the amino acid residues of the same segment as determined by a given method. The techniques used to determine whether an antibody binds to the "same epitope on L1CAM" using the antibodies described herein include, for example, epitope mapping methods, such as X-ray analysis of antigen: antibody complex crystals (which provides atomic resolution of the epitope) and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of antibodies to antigen fragments or mutational variations of antigens, in which binding losses caused by modification of amino acid residues within the antigen sequence are often considered to be an indication of epitope components. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from a combinatorial phage display peptide library. Antibodies with identical VH and VL or identical CDR1, CDR2, and CDR3 sequences are expected to bind to the same epitope.

[0112] An antibody that "competes with another antibody for binding to a target" is an antibody that (partially or completely) inhibits the binding of another antibody to a target. Whether two antibodies compete with each other for binding to a target, that is, whether one antibody inhibits the binding of another antibody to a target and to what extent, can be determined using known competition assays such as The competition assay can be carried out as described in Chapter 11 of "Using Antibodies" by, for example, Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101 / pdb.prot4277 or Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. If two antibodies block each other in two ways by at least 50%, that is, whether one antibody or the other antibody contacts the antigen first in the competition experiment, the antibodies "cross-compete".

[0113] Competitive binding assays for determining whether two antibodies compete or cross-compete for binding include: competition for binding to cells expressing L1CAM, for example by flow cytometry, such as described in the Examples. Other methods include: SPR (e.g. ), BLI (biolayer interferometry), solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeling RIA using 1-125 labeling (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeling RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0114] As used herein, the terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, the antibody (i) is used when, for example, a predetermined antigen (e.g., recombinant human MICA and MICB) is used as an analyte and the antibody is used as a ligand in When measured by surface plasmon resonance (SPR) technology performed in the 2000 instrument or Scatchard analysis of antibody binding to antigen-positive cells, the binding rate is less than 10 -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even smaller equilibrium dissociation constant (K D ) and (ii) binds to the predetermined antigen with an affinity at least two times greater than its affinity for a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. -7 M or less such as less than about 10 -8 M, 10 -9 M or 10 -10 M or even smaller K DAntibodies that bind to soluble or cell-bound human L1CAM. Antibodies that "cross-react with cynomolgus monkey L1CAM" are antibodies that bind to soluble or cell-bound human L1CAM. -7 M or less such as less than about 10 -8 M, 10 -9 M or 10 -10 M or even smaller K D Antibodies that bind to cynomolgus monkey L1CAM. In some aspects, such antibodies that do not cross-react with L1CAM from non-human species exhibit essentially undetectable binding to these proteins in standard binding assays.

[0115] As used herein, the term “k assoc " or "k a " is intended to refer to the association rate of a particular antibody-antigen interaction, and as used herein, the term "k dis " or "k d " is intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "K D ” is intended to refer to the dissociation constant, which is derived from k d With k a The ratio (i.e. k d / k a ) and expressed as molar concentration (M). The K of an antibody can be determined using well-established methods in the art. D Used to determine the antibody K D Available methods include surface plasmon resonance, biosensor systems such as BLI (Bio-layer Interferometry) system) or flow cytometry and Scatchard analysis.

[0116] As used herein, the term "high affinity" of an IgG antibody refers to an antibody that has a 10 -8 M or smaller, 10 - 9 M or less or 10 -10 M or smaller K D However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is when the antibody has 10 -10 M or less or 10 -8 M or smaller K D。

[0117] In the context of in vitro or in vivo assays using antibodies or antigen-binding fragments thereof, the term “EC 50 ” refers to the concentration of an antibody or antigen-binding fragment thereof that induces a response that is 50% of the maximal response (ie, halfway between the maximal response and the baseline).

[0118] "Bispecific or bifunctional antibodies" are artificial hybrid antibodies with two different heavy chain / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0119] As used herein, the term "monoclonal antibody" refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope, or an antibody composition in which all antibodies exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody or antibody composition that exhibits a single binding specificity and has a variable region and optional constant region derived from human germline immunoglobulin sequences. On the one hand, human monoclonal antibodies are produced by hybridomas and / or by recombinant combinatorial human antibody libraries, the hybridomas comprising B cells obtained from transgenic non-human animals (e.g., transgenic mice), the B cells having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0120] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) or hybridomas prepared therefrom for human immunoglobulin genes, (b) antibodies isolated from host cells converted to express antibodies (e.g., from transfectomas), (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies include variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23 (9): 1117-1125), the variable region includes an antigen binding domain encoded by various genes that are rearranged to form antibodies specific to foreign antigens. In addition to rearrangement, the variable region may be further modified by multiple single amino acid changes (called somatic mutations or hypermutations) to increase the affinity of the antibody for foreign antigens. The constant region will change to further respond to the antigen (i.e., isotype switching). Therefore, rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides that respond to the antigen are unlikely to have sequence identity with the original nucleic acid molecule, but are substantially identical or similar (i.e., at least 80% identical).

[0121] "HuMAb" refers to an antibody having a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. The antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto human framework sequences. The terms "human" antibody and "fully human" antibody may be used synonymously.

[0122] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0123] As used herein, the term "cross-reactivity" refers to the ability of an antibody described herein to bind to L1CAM from a different species. For example, an antibody described herein that binds to human L1CAM may also bind to L1CAM of another species (e.g., mouse L1CAM). As used herein, cross-reactivity can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA) or binding or otherwise functionally interacting with cells that physiologically express L1CAM. Methods for determining cross-reactivity include standard binding assays as described herein, such as by using 2000SPR instrument (Biacore AB, Uppsala, Sweden) Surface plasmon resonance (SPR) analysis or flow cytometry techniques.

[0124] The term "naturally occurring" as applied to an object herein refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally occurring.

[0125] "Polypeptide" refers to a chain of at least two consecutively linked amino acid residues, wherein there is no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may contain one or more polypeptides.

[0126] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, and can be a cDNA.

[0127] "Conservative amino acid substitution" refers to the substitution of an amino acid residue by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some aspects, the predicted non-essential amino acid residue in the anti-L1CAM antibody is replaced by another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0128] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100). A mathematical algorithm can be used to achieve sequence comparison and determination of the percent identity between two sequences, as described in the non-limiting examples below.

[0129] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0130] The nucleic acid and protein sequences described herein can further be used as a "query sequence" for searching against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0131] Nucleic acids can be present in whole cells, cell lysates, or partially purified or substantially pure forms. Nucleic acids are "isolated" or "become substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art. See F. Ausubel et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0132] Nucleic acids, such as cDNAs, can be mutated to provide gene sequences according to standard techniques. For coding sequences, these mutations can affect the amino acid sequence as desired. Specifically, DNA sequences that are substantially homologous to or derived from the natural V, D, J, constant, switch and other such sequences described herein are contemplated (wherein "derived" means that the sequence is identical to or modified from another sequence).

[0133] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, certain vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors with practicality in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors providing equivalent functions are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0134] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains a nucleic acid that does not naturally occur in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular host cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0135] As used herein, the term "linked" refers to the association of two or more molecules. The linkage may be covalent or non-covalent. The linkage may also be genetic (i.e., recombinant fusion). Such linkages may be achieved using a variety of art-recognized techniques such as chemical conjugation and recombinant protein production.

[0136] "Immune response" is as understood in the art and generally refers to a biological response in a vertebrate to a foreign agent or abnormal cell (e.g., cancerous cell) that protects the organism from the effects of these agents and the diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which causes selective targeting, binding, damage, destruction, and / or elimination of normal human cells or tissues from the vertebrate body to invading pathogens, cells or tissues infected by pathogens, cancerous cells or other abnormal cells, or in the case of autoimmunity or pathological inflammation. An immune response includes, for example, activation or inhibition of T cells, such as effector T cells, Th cells, CD4 + Cells, CD8 + T cells or Treg cells; or any other cells that activate or suppress the immune system, such as NK cells.

[0137] "Immunotherapy" refers to the treatment of a subject afflicted with a disease or at risk of contracting a disease or suffering a recurrence of a disease by methods that include inducing, enhancing, suppressing or otherwise altering the immune system or immune response.

[0138] As used herein, "administering" refers to the use of various methods and delivery systems known to those skilled in the art to physically introduce therapeutic agents or compositions comprising therapeutic agents to a subject. Preferred routes of administration of antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means the mode of administration except enteral and topical administration, usually by injection, and includes but is not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, antibodies described herein can be administered by non-parenteral routes, such as topical, epidermal or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual or topical administration. It can also be administered, for example, once, repeatedly and / or in one or more extended periods.

[0139] As used herein, the phrase "inhibiting tumor growth" includes any measurable reduction in tumor growth, such as inhibiting tumor growth by at least about 10%, such as at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%. In some aspects, inhibition of tumor growth is measured as tumor growth inhibition percentage (TGI%). TGI% can be determined by calculating the TGI at dat "t" calculated from all treated animals according to the following formula: [1-((T t / T 0 ) / (C t / C 0 ))] / [(C t -C 0 ) / C t ]×100[Formula 1], where T t = individual tumor size of treated animal at time 't', T 0 = individual tumor size of treated animals at the first measurement, C t = median tumor size of control animals at time 't', C 0 = median tumor size of control animals at the first measurement.

[0140] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0141] As used herein, the term "treat / treating / treatment" refers to any type of intervention or process performed on a subject or the administration of an active agent to a subject, with the goal of reversing, alleviating, ameliorating, inhibiting or slowing down or preventing the progression, development, severity or recurrence of symptoms, complications, conditions or biochemical indicators associated with a disease, or enhancing overall survival. Treatment can be performed on subjects with the disease or subjects without the disease (e.g., for prevention).

[0142] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug, when used alone or in combination with another therapeutic agent, that promotes regression of a disease, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, an increase in overall survival (the length of time a patient diagnosed with a disease, such as cancer, is still alive from the date of diagnosis or initiation of treatment for the disease), or the prevention of impairment or disability due to affliction with the disease. A therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount" or "prophylactically effective dose," which is any amount of a drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or suffering a recurrence of the disease, inhibits the development or recurrence of the disease. The ability of a therapeutic agent to promote regression of a disease or inhibit the development or recurrence of a disease can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0143] For example, an anticancer agent is a drug that promotes the regression of cancer in a subject. In some aspects, a therapeutically effective amount of a drug promotes the regression of cancer to the extent of eliminating cancer. "Promoting cancer regression" means that an effective amount of a drug is administered alone or in combination with an anti-tumor agent, resulting in a reduction in tumor growth or tumor size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of a disease-free symptom period, an increase in overall survival, damage or disability caused by disease torture is prevented or otherwise improves the patient's disease symptoms. In addition, the terms "effective (effective)" and "effectiveness (effectiveness)" for treatment include pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote the regression of cancer in a patient. Physiological safety refers to the toxicity level or other adverse physiological effects (adverse reactions) at the cell, organ and / or organism level caused by the administration of a drug.

[0144] For example, for the treatment of tumors, a therapeutically effective amount or dose of a drug inhibits cell growth or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% relative to an untreated subject. In some aspects, a therapeutically effective amount or dose of a drug completely inhibits cell growth or tumor growth, i.e., 100% inhibition of cell growth or tumor growth. The ability of a compound to inhibit tumor growth can be assessed using the assay described below. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, and such inhibition can be measured in vitro by assays known to skilled practitioners. In some aspects described herein, tumor regression can be observed and lasts for at least about 20 days, at least about 40 days, or at least about 60 days.

[0145] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0146] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μΜ", respectively.

[0147] Various aspects described herein are described in further detail in the following subsections.

[0148] II. Anti-L1CAM Antibodies

[0149] Antibodies, such as monoclonal antibodies, characterized by specific functional properties or characteristics are disclosed herein. For example, the antibody specifically binds to mammalian (e.g., human and mouse) L1CAM and exhibits one or more of the following functional properties:

[0150] (a) Shows improved productivity compared to mAb417 antibody

[0151] (b) Compared with mAb417 antibody, the D ) measured improved affinity;

[0152] (c) showed improved PI values ​​compared to mAb417 antibody;

[0153] (d) exhibit improved affinity as measured by the association constant (K) compared to the mAb417 antibody;

[0154] (e) prevention and / or treatment of diseases or conditions including tumors; or

[0155] (f) any combination thereof.

[0156] In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof exhibit improved productivity compared to the mAb417 antibody, for example, when expressed according to Example 3, the improved productivity is at least 55 mg / L, at least 56 mg / L, at least 57 mg / L, at least 58 mg / L, at least 59 mg / L, at least about 60 mg / L, at least about 61 mg / L, at least about 62 mg / L, at least about 63 mg / L, at least about 64 mg / L, at least about 65 mg / L, at least about 66 mg / L, at least about 67 mg / L, at least about 68 mg / L, at least about 69 mg / L, at least about 70 mg / L, at least about 71 mg / L, at least about 72 mg / L, at least about 73 mg / L, at least about 74 mg / L, at least about 75 mg / L, at least about 76 mg / L, at least about 77 mg / L, at least about 78 mg / L, at least about 79 mg / L, at least about 80 mg / L, at least about 81 mg / L, at least about 82 mg / L, at least about 83 mg / L, at least about 84 mg / L, at least about 85 mg / L, at least about 86 mg / L, at least about 87 mg / L, at least about 88 mg / L In some embodiments, the present invention relates to at least about 80 mg / L, at least about 81 mg / L, at least about 82 mg / L, at least about 83 mg / L, at least about 84 mg / L, or at least about 85 mg / L.

[0157] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof binds with high affinity, for example, with less than 2.6×10 -10 M, less than 2.5×10 -10 M, less than 2.0×10 -10 M, less than 1.5×10 -10 M, less than 1.0×10 -10 M, less than 9×10 -11 M, less than 8×10 -11 M, less than 7×10 -11 M, less than 6×10 -11 M, less than 5×10 -11 M, less than 4×10 -11 M, less than 3×10 -11 M, less than 2×10 -11 M, less than 1×10 -11 M, less than 9×10 -12 M, less than 8×10 -12 M, less than 7×10 -12 M, less than 6×10 -12 M, less than 5×10 -12 M, less than 4×10 -12 M, less than 3×10 -12 M, less than 2×10 -12 M, less than 1×10 -12 M, less than 9×10 -13 M or less than 8×10 -13 M of KD , specifically binds to human L1CAM. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof has a specific binding affinity of less than 2×10 - 10 M, less than 1.9×10 -10 M, less than 1.8×10 -10 M, less than 1.7×10 -10 M, less than 1.6×10 -10 M, less than 1.5×10 - 10 M, less than 1.4×10 -10 M, less than 1.3×10 -10 M, less than 1.2×10 -10 M or less than 1.1×10 -10 M of K D Specifically binds to human L1CAM. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof has a specific binding affinity of less than 1.1×10 -10 M of K D Specifically binds to human L1CAM. In other aspects, the anti-L1CAM antibody or antigen-binding fragment thereof has a specific binding affinity of less than 9×10 -12 M of K D Specifically binds to human L1CAM. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof has a specific binding affinity of less than 1×10 -12 M of K D Specifically binds to human L1CAM. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof has a specific binding affinity of less than 8×10 -11 M of K D Specifically binds to human L1CAM. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof has a specific binding affinity of less than 1×10 -12 M of K D Specifically binds to human L1CAM. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof has a specific binding affinity of less than 1.05×10 -10 M of K D Specifically binds to human L1CAM. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof has a specific binding affinity of about 8.22×10 -12 M of K D Specifically binds to human L1CAM. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof has a specific binding affinity of about 7.4×10 -11 M of K D Specifically binds to human L1CAM. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof has a specific binding affinity of about 9.6×10 -11 M of K D Binds specifically to human L1CAM.

[0158] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof binds with high affinity, e.g., with less than 5×10 -10 M, less than 4×10 -10 M, less than 3×10 -10 M, less than 2×10 -10 M, less than 1.0×10 -10 M, less than 9×10 -11 M, less than 8×10 -11 M, less than 7×10 -11 M, less than 6×10 -11 M, less than 5×10 -11 M, less than 4×10 -11 M, less than 3×10 -11 M, less than 2×10 -11 M, less than 1×10 -11 M, less than 9×10 -12 M, less than 8×10 -12 M, less than 7×10 -12 M, less than 6×10 -12 M, less than 5×10 -12 M, less than 4×10 -12 M, less than 3×10 -12 M, less than 2×10 -12 M, less than 1×10 -12 M, less than 9×10 -13 M or less than 8×10 -13 K of M, specifically binds to human L1CAM.

[0159] Standard assays for assessing the ability of antibodies to bind to L1CAM of various species are known in the art, including, for example, ELISA, Western blot, and RIA. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibodies can also be determined by standard assays known in the art, such as by ELISA, Western blot, and RIA. Analysis or Assays for assessing the effects of antibodies on the functional properties of L1CAM (eg, ligand binding) are described in further detail below and in the Examples.

[0160] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof exhibits an improved isoelectric point (PI) value of less than 9.6, less than 9.5, less than 9.4, less than 9.3, less than 9.2, less than 9.1, less than 9.0, less than 8.9, less than 8.8, less than 8.7, less than 8.6, less than 8.5, less than 8.4, less than 8.3, less than 8.2, less than 8.1, less than 8.0, less than 7.9, less than 7.8, less than 7.7 or less than 7.6 as measured by capillary isoelectric focusing (cIEF) (e.g., as described in the Examples).

[0161] In certain aspects, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to the same L1CAM epitope as a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH of the reference antibody comprises SEQ ID NO:23, and the VL of the reference antibody comprises SEQ ID NO:24; (b) the VH of the reference antibody comprises SEQ ID NO:25, and the VL of the reference antibody comprises SEQ ID NO:26; (c) the VH of the reference antibody comprises SEQ ID NO:27, and the VL of the reference antibody comprises SEQ ID NO:28; (d) the VH of the reference antibody comprises SEQ ID NO:29, and the VL of the reference antibody comprises SEQ ID NO:30; or (e) the VH of the reference antibody comprises SEQ ID NO:31, and the VL of the reference antibody comprises SEQ ID NO:32.

[0162] In certain aspects, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to the same L1CAM epitope as a reference antibody comprising a VH complementarity determining region 1 (CDR1), a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR3, wherein at least one amino acid in the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of the antibody or antigen-binding fragment thereof is different from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of the reference antibody (e.g., mAb417 antibody), wherein the VH CDR1, VH CDR2, VH CDR3 of the reference antibody comprises the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 9 and SEQ ID NO: 4, respectively, and the VL CDR1, VLCDR2 and VL CDR3 of the reference antibody comprises the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. NO: 11. In certain aspects, the at least one amino acid difference comprises: (i) glutamine at the 5th residue in VH CDR2; (ii) serine at the 8th residue in VL CDR1; and / or (iii) proline at the 8th residue in VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof. In certain aspects, the at least one amino acid difference comprises: (i) alanine, glycine, phenylalanine, tyrosine, threonine, proline and tryptophan at the 3rd to 9th residues in VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof; (ii) alanine, glycine, phenylalanine, tyrosine, serine, proline and tryptophan at the 3rd to 9th residues in VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof; or (iii) leucine, valine or histidine, tryptophan or phenylalanine, tyrosine, proline and tryptophan at the 4th to 9th residues in VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof.

[0163] In certain aspects, the anti-L1CAM antibody or antigen-binding fragment thereof cross-competes for binding to the L1CAM epitope with a reference antibody comprising a VH complementarity determining region 1 (CDR1), a VH CDR2 and a VH CDR3, and a VL CDR1, a VL CDR2 and a VL CDR3, wherein at least one amino acid in the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of the antibody or antigen-binding fragment thereof differs from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of the reference antibody (e.g., mAb417 antibody), (i) wherein the VH CDR1, VH CDR2, VH CDR3 of the reference antibody comprises the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 9 and SEQ ID NO: 4, respectively, and the VL CDR1, VLCDR2 and VL CDR3 of the reference antibody comprises the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. ID NO: 11. In certain aspects, the at least one amino acid difference comprises: (i) glutamine at residue 5 in VH CDR2; (ii) serine at residue 8 in VL CDR1; and / or (iii) proline at residue 8 in VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof. In certain aspects, the at least one amino acid difference comprises: (i) alanine, glycine, phenylalanine, tyrosine, threonine, proline, and tryptophan at residues 3 to 9, respectively, in VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof; (ii) alanine, glycine, phenylalanine, tyrosine, serine, proline, and tryptophan at residues 3 to 9, respectively, in VL CDR3; or (iii) leucine, valine or histidine, tryptophan or phenylalanine, tyrosine, proline, and tryptophan at residues 4 to 9, respectively, in VL CDR3.

[0164] In certain aspects, the anti-L1CAM antibody or antigen-binding fragment thereof cross-competes with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL) for binding to the L1CAM epitope, wherein: (a) the VH of the reference antibody comprises SEQ ID NO:23, and the VL of the reference antibody comprises SEQ ID NO:24; (b) the VH of the reference antibody comprises SEQ ID NO:25, and the VL of the reference antibody comprises SEQ ID NO:26; (c) the VH of the reference antibody comprises SEQ ID NO:27, and the VL of the reference antibody comprises SEQ ID NO:28; (d) the VH of the reference antibody comprises SEQ ID NO:29, and the VL of the reference antibody comprises SEQ ID NO:30; or (e) the VH of the reference antibody comprises SEQ ID NO:31, and the VL of the reference antibody comprises SEQ ID NO:32.

[0165] Competing antibodies bind to the same epitope, overlapping epitope, or adjacent epitope (e.g., as demonstrated by steric hindrance). Competition assays known in the art such as RIA and EIA can be used to determine whether two antibodies compete with each other for binding to a target.

[0166] Techniques for determining whether two antibodies bind to the same epitope include, for example, epitope mapping methods such as X-ray analysis of crystals of the antigen:antibody complex (which provides atomic resolution of the epitope) and hydrogen / deuterium exchange mass spectrometry (HDX-MS)), methods for monitoring antibody binding to antigen fragments or mutational variations of the antigen (in which loss of binding due to modification of amino acid residues within the antigen sequence is often considered indicative of epitope composition), and computational combinatorial methods for epitope mapping.

[0167] In certain aspects, provided herein is an antibody or antigen-binding fragment thereof, which binds to L1CAM (e.g., human L1CAM) with 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more affinity compared to another protein in the L1CAM family, as measured by, e.g., immunoassay (e.g., ELISA), surface plasmon resonance, BLI (biolayer interferometry), or kinetic exclusion assay. In specific aspects, provided herein is an antibody or antigen-binding fragment thereof, which binds to L1CAM (e.g., human L1CAM) without cross-reactivity with another protein in the L1CAM family, as measured by, e.g., immunoassay.

[0168] In certain aspects, the anti-L1CAM antibody is not a natural antibody or is not a naturally occurring antibody. For example, the anti-L1CAM antibody has different post-translational modifications than naturally occurring antibodies, such as having more, fewer, or different types of post-translational modifications.

[0169] III. Exemplary Anti-L1CAM Antibodies

[0170] Specific antibodies that can be used in the methods disclosed herein are antibodies (e.g., monoclonal antibodies) having CDR and / or variable region sequences of antibodies Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 constructed in Examples 1 and 2, and antibodies having at least 80% identity (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity) to their variable region or CDR sequences. The VH amino acid sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 are shown in SEQ ID NOs: 23, 25, 27, 29, and 31, respectively. The VL amino acid sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 are shown in SEQ ID NOs: 24, 26, 28, 30, and 32, respectively.

[0171] Table 2. Variable heavy chain CDR amino acid sequences (according to the Kabat system)

[0172]

[0173] Table 3. Variable light chain CDR amino acid sequences (according to the Kabat system)

[0174]

[0175]

[0176] Table 4A. Variable heavy chain amino acid sequences

[0177]

[0178] Table 4B. Variable light chain amino acid sequences

[0179]

[0180] Thus, provided herein is an isolated anti-L1CAM antibody or antigen-binding fragment thereof, comprising heavy and light chain variable regions, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 23, 25, 27, 29 or 31. In other aspects, the isolated anti-L1CAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR selected from the group consisting of SEQ ID NO: 2, 10 or 4.

[0181] Also provided is an isolated anti-L1CAM antibody or antigen-binding fragment thereof, comprising heavy and light chain variable regions, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24, 26, 28, 30 or 32. In other aspects, the isolated anti-L1CAM antibody or antigen-binding fragment thereof comprises a light chain variable region CDR selected from the group consisting of SEQ ID NO: 12, 7, 13, 15, 17, 19 or 21.

[0182] In certain aspects, the isolated anti-L1CAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR selected from the group consisting of SEQ ID NO: 2, 10 or 4 and a light chain variable region CDR selected from the group consisting of SEQ ID NO: 12, 7, 13, 15, 17, 19 or 21.

[0183] Also provided is an isolated anti-L1CAM antibody or antigen-binding fragment thereof, comprising heavy and light chain variable regions, (i) wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:23, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:24; (ii) wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:25, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:26; (iii) wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:27, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:28; (iv) wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:29, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:30; or (v) wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:31, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:32.

[0184] Provided herein is an isolated anti-L1CAM antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identity to the amino acid sequence shown as SEQ ID NO: 23, 25, 27, 29 or 31.

[0185] Also provided herein is an isolated anti-L1CAM antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identity to the amino acid sequence shown as SEQ ID NO: 24, 26, 28, 30 or 32.

[0186] Also provided is an isolated anti-L1CAM antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identity to the amino acid sequence shown as SEQ ID NO: 23, 25, 27, 29 or 31, and wherein the light chain variable region comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identity to the amino acid sequence shown as SEQ ID NO: 24, 26, 28, 30 or 32.

[0187] In some aspects, the present disclosure provides an isolated anti-L1CAM antibody or antigen-binding fragment thereof comprising:

[0188] (a) comprising the heavy and light chain variable region sequences of SEQ ID NOs: 23 and 24, respectively;

[0189] (b) heavy chain and light chain variable region sequences comprising SEQ ID NOs: 25 and 26, respectively;

[0190] (c) heavy and light chain variable region sequences comprising SEQ ID NOs: 27 and 28, respectively;

[0191] (d) heavy chain and light chain variable region sequences comprising SEQ ID NOs: 29 and 30, respectively; or

[0192] (e) The heavy and light chain variable region sequences comprise SEQ ID NOs: 31 and 32, respectively.

[0193] The amino acid sequences of the VH CDR1, CDR2 and CDR3 of antibodies Ab612, Ab4H5, Ab2C2, Ab4H6 and Ab5D12 are shown in SEQ ID NOs: 2, 10 and 4, respectively. The amino acid sequences of the VL CDR1, CDR2 and CDR3 of Ab612 are shown in SEQ ID NOs: 12, 7 and 13, respectively. The amino acid sequences of the VL CDR1, CDR2 and CDR3 of Ab4H5 are shown in SEQ ID NOs: 12, 7 and 15, respectively. The amino acid sequences of the VL CDR1, CDR2 and CDR3 of Ab2C2 are shown in SEQ ID NOs: 12, 7 and 17, respectively. The amino acid sequences of the VL CDR1, CDR2 and CDR3 of Ab4H6 are shown in SEQ ID NOs: 12, 7 and 19, respectively. The amino acid sequences of the VL CDR1, CDR2 and CDR3 of Ab5D12 are shown in SEQ ID NOs: 12, 7 and 21, respectively.

[0194] In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof of the present disclosure that specifically binds to human L1CAM comprises:

[0195] (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2; and / or

[0196] (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and / or

[0197] (c) VH CDR3 comprising the amino acid sequence of SEQ ID NO:4.

[0198] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises:

[0199] (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; and / or

[0200] (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and / or

[0201] (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:13.

[0202] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises:

[0203] (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2;

[0204] (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0205] (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4;

[0206] (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12;

[0207] (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and / or

[0208] (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:13.

[0209] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises:

[0210] (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; and / or

[0211] (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and / or

[0212] (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:15.

[0213] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises:

[0214] (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2;

[0215] (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0216] (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4;

[0217] (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12;

[0218] (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and / or

[0219] (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:15.

[0220] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises:

[0221] (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; and / or

[0222] (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and / or

[0223] (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:17.

[0224] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises:

[0225] (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2;

[0226] (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0227] (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4;

[0228] (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12;

[0229] (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and / or

[0230] (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:17.

[0231] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises:

[0232] (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; and / or

[0233] (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and / or

[0234] (c) VL CDR3 comprising the amino acid sequence of SEQ ID NO:19.

[0235] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises:

[0236] (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2;

[0237] (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10;

[0238] (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4;

[0239] (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12;

[0240] (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and / or

[0241] (f) VL CDR3 comprising the amino acid sequence of SEQ ID NO:21.

[0242] In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises a heavy chain (HC) and a light chain (LC), wherein the HC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 38, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 39.

[0243] In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises a heavy chain (HC) and a light chain (LC), wherein the HC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 40, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 41.

[0244] In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises a heavy chain (HC) and a light chain (LC), wherein the HC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 42, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 43.

[0245] In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises a heavy chain (HC) and a light chain (LC), wherein the HC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 44, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 45.

[0246] In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM comprises a heavy chain (HC) and a light chain (LC), wherein the HC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 46, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 20.

[0247] Table 5A. Heavy chain amino acid sequence

[0248]

[0249]

[0250] Table 5B. Light chain amino acid sequences

[0251]

[0252]

[0253] The VH domains described herein or one or more CDRs thereof can be connected to a constant domain to form a heavy chain, such as a full-length heavy chain. Similarly, the VL domains described herein or one or more CDRs thereof can be connected to a constant domain to form a light chain, such as a full-length light chain. The full-length heavy chain and the full-length light chain are combined to form a full-length antibody.

[0254] Therefore, in a specific aspect, an antibody is provided herein, which comprises an antibody light chain and a heavy chain, such as a separated light chain and a heavy chain. With regard to the light chain, in a specific aspect, the light chain of the antibody described herein is a κ light chain. In another specific aspect, the light chain of the antibody described herein is a λ light chain. In another specific aspect, the light chain of the antibody described herein is a human κ light chain or a human λ light chain. In a specific aspect, the antibody described herein that specifically binds to a L1CAM polypeptide (e.g., human L1CAM) comprises a light chain, the light chain comprising any VL or VL CDR amino acid sequence described herein, and wherein the constant region of the light chain comprises the amino acid sequence of a human κ light chain constant region. In a specific aspect, the antibody described herein that specifically binds to a L1CAM polypeptide (e.g., human L1CAM) comprises a light chain, the light chain comprising a VL or VL CDR amino acid sequence described herein, and wherein the constant region of the light chain comprises the amino acid sequence of a human λ light chain constant region. Non-limiting examples of human constant region sequences have been described in the art, see, eg, US Pat. No. 5,693,780 and Kabat EA et al., (1991) supra.

[0255] With regard to the heavy chain, in some aspects, the heavy chain of the antibody described herein may be an α, δ, ε, γ or μ heavy chain. In another specific aspect, the heavy chain of the antibody may comprise a human α, δ, ε, γ or μ heavy chain. On the one hand, the antibody described herein that specifically binds to L1CAM (e.g., human L1CAM) comprises a heavy chain, the heavy chain comprises a VH or VH CDR amino acid sequence described herein, and wherein the constant region of the heavy chain comprises the amino acid sequence of a human γ heavy chain constant region. On the other hand, the antibody described herein that specifically binds to L1CAM (e.g., human L1CAM) comprises a heavy chain, the heavy chain comprises a VH or VHCDR amino acid sequence disclosed herein, and wherein the constant region of the heavy chain comprises the amino acids of a human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) supra.

[0256] In some aspects, the antibodies described herein that specifically bind to L1CAM (e.g., human L1CAM) comprise a VL domain and a VH domain, the VL domain and the VH domain comprising the VH or VH CDRs and the VL and VL CDRs described herein, and wherein the constant region comprises an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule or the amino acid sequence of the constant region of a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule. In another specific aspect, the antibodies described herein that specifically bind to L1CAM (e.g., human L1CAM) comprise a VL domain and a VH domain, the VL domain and the VH domain comprising any amino acid sequence described herein, and wherein the constant region comprises an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any subclass of an immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) amino acid sequence of the constant region. In some aspects, the constant region comprises the amino acid sequence of the constant region of a naturally occurring human IgG, including subclasses (e.g., IgG1, IgG2, IgG3, or IgG4) and allotypes (e.g., G1m, G2m, G3m, and nG4m) and variants thereof. See, e.g., Vidarsson G. et al. Front Immunol. 5:520 (published online on October 20, 2014) and Jefferis R. and Lefranc MP, mAbs 1:4, 1-7 (2009). In some aspects, the constant region comprises the amino acid sequence of the constant region of human IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0257] In certain aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein do not have Fc effector functions, such as complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP). The effector functions are mediated by the Fc region, and the residues closest to the hinge region in the CH2 domain of the Fc region are responsible for the effector functions of the antibody because it contains a large number of overlapping binding sites for C1q (complement) and IgG-Fc receptors (FcγR) on effector cells of the innate immune system. Similarly, IgG2 and IgG4 antibodies have lower levels of Fc effector functions than IgG1 and IgG3 antibodies. The effector functions of antibodies can be reduced or avoided by various methods known in the art, including (1) using antibody fragments lacking the Fc region (e.g., such as Fab, F(ab') 2 , single-chain Fv (scFv) or sdAb composed of monomeric VH or VL domains); (2) generating aglycosylated antibodies, which can be generated by, for example, deleting or changing the residues attached to the sugar, enzymatically removing the sugar, producing the antibody in cells cultured in the presence of a glycosylation inhibitor, or by expressing the antibody in cells that cannot glycosylate proteins (e.g., bacterial host cells) (see, e.g., U.S. Publication No. 20120100140); (3) using an Fc region with reduced effector function from an IgG subclass (e.g., an Fc region from an IgG2 or IgG4 antibody or a chimeric Fc region comprising a CH2 domain from an IgG2 or IgG4 antibody, see, e.g., U.S. Publication No. 20120100140 and Lau C. et al. J. Immunol. 191:4769-4777 (2013)); and (4) generating an Fc region with mutations that result in reduced or no Fc function. See, eg, US Publication No. 20120100140 and the US applications and PCT applications cited therein and An et al., mAbs 1:6, 572-579 (2009).

[0258] Thus, in some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein are Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), or sdAbs consisting of monomeric VH or VL domains. Such antibody fragments are well known in the art and are described, supra.

[0259] In some aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprise an Fc region with reduced or no Fc effector function. In some aspects, the constant region comprises the amino acid sequence of the Fc region of human IgG2 or IgG4, and in some aspects, the anti-L1CAM antibody is an IgG2 / IgG4 isotype. In some aspects, the anti-L1CAM antibody comprises a chimeric Fc region comprising the CH2 domain of an IgG antibody from an IgG4 isotype and the CH3 domain of an IgG antibody from an IgG1 isotype, or a chimeric Fc region comprising a hinge region from IgG2 and a CH2 region from IgG4, or an Fc region with a mutation that results in reduced or no Fc function. Fc regions with reduced or no Fc effector function include those known in the art. See, for example, Lau C. et al. J. Immunol. 191: 4769-4777 (2013); An et al., mAbs 1: 6, 572-579 (2009); and U.S. Publication No. 20120100140 and the U.S. patents and publications cited therein and PCT publications. Similarly, one of ordinary skill in the art can easily prepare an Fc region with reduced or no Fc effector function.

[0260] IV. Nucleic Acid Molecules

[0261] Another aspect described herein relates to one or more nucleic acid molecules encoding any one of the antibodies described herein or their antigen-binding fragments. Nucleic acid can exist in whole cell, cell lysate or partially purified or substantially pure form. When purified from other cell components or other contaminants, such as other cell nucleic acids (such as other chromosomal DNA (such as chromosomal DNA connected to the isolated DNA in nature)) or proteins by standard techniques, including alkaline / SDS treatment, CsCl bands, column chromatography, restriction enzymes, agarose gel electrophoresis and other methods well known in the art, nucleic acid is "isolated" or "becomes substantially pure". See F.Ausubel et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acid described herein can be, for example, DNA or RNA, and may or may not include intron sequences. In some aspects, nucleic acid is a cDNA molecule.

[0262] The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as further described below), cDNAs encoding the light and heavy chains of the antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the libraries.

[0263] Certain nucleic acid molecules described herein are nucleic acid molecules encoding the VH and VL sequences of the Ab612, Ab4H5, Ab2C2, Ab4H6, Ab5D12 monoclonal antibodies. Exemplary DNA sequences encoding the VH sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, Ab5D12 are shown in SEQ ID NO: 8, 14, 16, 18 or 22. Exemplary DNA sequences encoding the VL sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, Ab5D12 are shown in SEQ ID NO: 33, 34, 35, 36 and 37, respectively.

[0264] Table 6: Variable heavy chain polynucleotide sequences

[0265]

[0266]

[0267] Table 7: Variable light chain polynucleotide sequences

[0268]

[0269]

[0270] Methods for preparing anti-L1CAM antibodies as disclosed herein may include expressing heavy and light chains in a cell line comprising nucleotide sequences encoding heavy and light chains with a signal peptide. Host cells comprising these nucleotide sequences are contemplated herein.

[0271] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" is intended to mean connecting the two DNA fragments so that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0272] The DNA encoding VH can be operably linked to another DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2 and / or CH3), to convert the separated DNA encoding the VH region into a full-length heavy chain gene. The sequence of human heavy chain constant region genes is known in the art (see, e.g., Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, such as an IgG2 and / or IgG4 constant region. For a Fab fragment heavy chain gene, the DNA encoding VH can be operably linked to another DNA molecule encoding only a heavy chain CH1 constant region.

[0273] The DNA encoding VL can be operably linked to another DNA molecule encoding the light chain constant region CL to convert the separated DNA encoding the VL region into a full-length light chain gene (and a Fab light chain gene). The sequence of human light chain constant region genes is known in the art (see, for example, Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and the DNA fragments covering these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0274] To produce an scFv gene, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly4-Ser)3) so that the VH and VL sequences can be expressed as a continuous single-chain protein in which the VL region and the VH region are connected by a flexible linker (see, e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0275] In some aspects, the present disclosure provides a vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof. In other aspects, the vector can be used for gene therapy.

[0276] Suitable vectors for use in the present disclosure include expression vectors, viral vectors and plasmid vectors. In one aspect, the vector is a viral vector.

[0277] As used herein, expression vector refers to any nucleic acid construct that contains the necessary elements for transcription and translation of the inserted coding sequence, or for RNA viral vectors, the necessary elements for replication and translation when introduced into an appropriate host cell. Expression vectors may include plasmids, phagemids, viruses and their derivatives.

[0278] The expression vector of the present disclosure may include a polynucleotide encoding an antibody or its antigen-binding fragment as described herein. On the one hand, the coding sequence of the antibody or its antigen-binding fragment is operably connected to an expression control sequence. As used herein, when two nucleic acid sequences are covalently linked in a manner that allows each component nucleic acid sequence to retain its function, they are operably connected. When the coding sequence and the gene expression control sequence are covalently linked in a manner that allows the expression or transcription and / or translation of the coding sequence to be under the influence or control of the gene expression control sequence, they are referred to as being operably connected. If the promoter is induced in the 5' gene expression sequence to cause transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences does not (1) lead to the introduction of a frameshift mutation (frame-shift mutation), (2) interfere with the ability of the promoter region to guide the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into protein, then the two DNA sequences are referred to as being operably connected. Therefore, if the gene expression sequence can affect the transcription of the coding nucleic acid sequence so that the resulting transcript is translated into the desired antibody or its antigen-binding fragment, then the gene expression sequence is operably connected to the coding nucleic acid sequence.

[0279] Viral vectors include, but are not limited to, nucleic acid sequences of the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentivirus; adenovirus; adeno-associated virus; SV40 virus; polyoma virus; Epstein-Barr virus; papillomavirus; herpes virus; vaccinia virus; poliovirus; and RNA viruses, such as retroviruses. Other vectors well known in the art can be readily used. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced by genes of interest. Non-cytopathic viruses include retroviruses, whose life cycle includes reverse transcription of genomic viral RNA into DNA, followed by integration of the provirus into host cell DNA. Retroviruses have been approved for use in human gene therapy trials. The most useful are those that are replication-defective (i.e., capable of directing the synthesis of desired proteins, but unable to make infectious particles) retroviruses. Such genetically altered retroviral expression vectors have the general utility of efficiently transducing genes in vivo. Standard protocols for producing replication-defective retroviruses (comprising the following steps: incorporating exogenous genetic material into plasmids; transfecting packaging cell lines with plasmids; producing recombinant retroviruses by packaging cell lines; collecting viral particles from tissue culture medium; and infecting target cells with viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, WH Freeman Co., New York (1990) and Murry, EJ, Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0280] On the one hand, the virus is an adeno-associated virus, a double-stranded DNA virus. Adeno-associated virus can be engineered to be replication-defective and can infect a variety of cell types and species. It also has the following advantages, such as heat and lipid solvent stability; High transduction frequency in different lineage cells (including hematopoietic cells); And lack of repeated infection (superinfection) inhibition, thus allowing multiple series of transductions. It is reported that adeno-associated virus can be integrated into human cell DNA in a site-specific manner, thereby minimizing the possibility of insertion mutagenesis unique to retroviral infection and the variability of inserted gene expression. In addition, in the absence of selection pressure, wild-type adeno-associated virus infection has been passed down more than 100 times in tissue culture, meaning that adeno-associated virus genome integration is a relatively stable event. Adeno-associated virus can also work in an extrachromosomal manner.

[0281] In other aspects, the vector is derived from a lentivirus. In certain aspects, the vector is a recombinant lentiviral vector capable of infecting non-dividing cells.

[0282] The lentiviral genome and proviral DNA typically have three genes found in retroviruses: gag, pol, and env, flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes an RNA-directed DNA polymerase (reverse transcriptase), a protease, and an integrase; and the env gene encodes the viral envelope glycoprotein. The 5' and 3' LTRs function to facilitate transcription and polyadenylation of the virion RNA. The LTRs contain all other cis-acting sequences required for viral replication. Lentiviruses have additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0283] Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and efficient encapsidation of viral RNA into particles (Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of the genomic RNA.

[0284] However, the resulting mutant is still able to direct the synthesis of all virion proteins. The present disclosure provides a method for producing a recombinant lentivirus capable of infecting non-dividing cells, comprising transfecting a suitable host cell with two or more vectors carrying packaging functions (i.e., gag, pol and env as well as rev and tat). As will be disclosed below, a vector lacking a functional tat gene is desirable for certain applications. Thus, for example, a first vector can provide nucleic acids encoding viral gag and viral pol, and another vector can provide nucleic acids encoding viral env to produce packaging cells. A vector providing a heterologous gene (identified herein as a transfer vector) is introduced into the packaging cell to produce a producer cell, which releases infectious viral particles carrying the foreign gene of interest.

[0285] According to the configuration of the vector and foreign gene indicated above, the second vector can provide nucleic acid encoding the viral envelope (env) gene. The env gene can be derived from almost any suitable virus, including retroviruses. In some aspects, the env protein is an amphotropic envelope protein that allows transduction of cells of humans and other species.

[0286] Examples of retroviral derived env genes include, but are not limited to, Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), mouse mammary tumor virus (MuMTV or MMTV), Gibbon ape leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV), and Rous sarcoma virus (RSV). Other env genes, such as the vesicular stomatitis virus (VSV) protein G (VSV G) of hepatitis viruses and influenza, may also be used.

[0287] The vector providing the viral env nucleic acid sequence is operably associated with the regulatory sequences described elsewhere herein.

[0288] Examples of lentiviral vectors are disclosed in WO9931251, W09712622, W09817815, W09817816 and WO9818934, which are herein incorporated by reference in their entirety.

[0289] Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, 1989. In recent years, it has been found that plasmid vectors are particularly useful for delivering genes to cells in vivo because they cannot be replicated in the host genome and cannot be integrated into the host genome. However, these plasmids with promoters compatible with host cells can express peptides from genes operably encoded in the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40 and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, CA.). Other plasmids are well known to those of ordinary skill in the art. In addition, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific DNA fragments.

[0290] V. Antibody Production

[0291] Antibodies or fragments thereof that immunospecifically bind to L1CAM (e.g., human L1CAM) can be produced by any method known in the art for synthesizing antibodies, such as by chemical synthesis or by recombinant expression techniques. Unless otherwise indicated, the methods described herein use conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0292] In particular aspects, antibodies described herein are antibodies (e.g., monoclonal antibodies) prepared, expressed, produced, or isolated by any means involving production, such as synthesis of DNA sequences, genetic engineering. In certain aspects, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that are not naturally present in vivo in the antibody germline repertoire of an animal or mammal (e.g., a human).

[0293] In certain aspects, provided herein is a method for preparing an antibody or antigen-binding fragment thereof that immunospecifically binds to L1CAM (e.g., human L1CAM), comprising culturing a cell or host cell as described herein. In certain aspects, provided herein is a method for preparing an antibody or antigen-binding fragment thereof that immunospecifically binds to L1CAM (e.g., human L1CAM), comprising expressing (e.g., recombinantly expressing) an antibody or antigen-binding fragment thereof using a cell or host cell as described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody as described herein). In certain aspects, the cell is an isolated cell. In certain aspects, an exogenous polynucleotide has been introduced into the cell. In certain aspects, the method further comprises a step of purifying the antibody or antigen-binding fragment thereof obtained from the cell or host cell.

[0294] Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 in Short Protocols in Molecular Biology, (2002) 5th edition, Ausubel FM et al., eds., John Wiley and Sons, New York).

[0295] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display techniques or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and, for example, Harlow E and Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd Edition 1988); Hammerling GJ et al., Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981). As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be recombinantly produced from host cells that exogenously express antibodies described herein or fragments thereof (e.g., light chains and / or heavy chains of such antibodies).

[0296] In certain aspects, a "monoclonal antibody" as used herein is an antibody produced by a single cell (e.g., a hybridoma or host cell producing a recombinant antibody), wherein the antibody immunospecifically binds to L1CAM (e.g., human L1CAM), for example, as determined by ELISA or other antigen binding or competitive binding known in the art or in the Examples provided herein. In certain aspects, the monoclonal antibody can be a chimeric antibody or a humanized antibody. In certain aspects, the monoclonal antibody is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In certain aspects, the monoclonal antibody is a monospecific or multispecific antibody (e.g., a bispecific antibody). The monoclonal antibodies described herein can be prepared, for example, by the hybridoma method as described in Kohler G and Milstein C (1975) Nature 256:495, or can be isolated, for example, from a phage library using techniques such as those described herein. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art (see, e.g., Chapter 11 in Short Protocols in Molecular Biology, (2002) 5th ed., Ausubel FM et al., supra).

[0297] Methods for producing and screening specific antibodies using hybridoma technology are conventional and well known in the art. For example, in the hybridoma method, mice or other appropriate host animals, such as sheep, goats, rabbits, rats, hamsters or macaques, are immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization (e.g., human L1CAM). Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding JW (ed.), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). In addition, RIMMS (repetitive immunization multiple sites) technology can be used to immunize animals (Kilpatrick KE et al., (1997) Hybridoma 16: 381-9, which is incorporated by reference in its entirety).

[0298] In some aspects, mice (or other animals, such as chickens, rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (e.g., L1CAM, such as human L1CAM), and once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused with any suitable myeloma cells, such as cells from the cell line SP20 available from the American Type Culture Collection (ATCC) (Manassas, VA) by well-known techniques to form hybridomas. Hybridomas are selected and cloned by limiting dilution. In certain aspects, lymph nodes of immunized mice are harvested and fused with NSO myeloma cells.

[0299] The hybridoma cells thus prepared are inoculated and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0300] Particular aspects use myeloma cells that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these myeloma cell lines are murine myeloma lines, such as the NSO cell line, or cell lines derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor D (1984) J Immunol 133:3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0301] The culture medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies against L1CAM (e.g., human L1CAM). The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by methods known in the art, such as immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0302] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity and / or activity, clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (ed.), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium. In addition, hybridoma cells can be grown in vivo in animals as ascites tumors.

[0303] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0304] The antibodies described herein include antibody fragments that recognize specific L1CAM (e.g., human L1CAM) and can be generated by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen binding arms of the antibody molecule connected by a disulfide bond in the hinge region.

[0305] In one aspect, to generate a complete antibody, PCR primers comprising a VH or VL nucleotide sequence, a restriction site, and flanking sequences protecting the restriction site can be used to amplify the VH or VL sequence from a template (e.g., a scFv clone). Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a VL constant region (e.g., a human κ or λ constant region). The VH and VL domains can also be cloned into one vector expressing the necessary constant region. Then, the heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody, such as an IgG.

[0306] Chimeric antibodies are molecules in which different fragments of an antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may comprise a variable region of a non-human animal (e.g., mouse, rat, or chicken) monoclonal antibody fused to a constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison SL (1985) Science 229: 1202-7; Oi VT and Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

[0307] Humanized antibodies are capable of binding to a predetermined antigen and comprise a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin (e.g., mouse or chicken immunoglobulin). In particular aspects, humanized antibodies also comprise at least one fragment of an immunoglobulin constant region (Fc), typically a fragment of a human immunoglobulin. The antibody may also include CH1, hinge, CH2, CH3, and CH4 regions of a heavy chain. Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5):489-498; Studnicka GM et al., (1994) Prot Engineering 7(6):805-814; and Roguska MA et al., (1994) PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 93 / 17105; Tan P et al., (2002) J Immunol 169:1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3):267-79; Baca M et al., (1997) J Biol Chem 272(16):10678-84; Roguska MA et al., (1996) Protein Eng 9(10):895 904; Couto JR et al., (1995) Cancer Res. 55(23 Suppl):5973-5977; Couto JR et al., (1995) Cancer Res 55(8):1717-22; Sandhu JS (1994) Gene 150(2):409-10 and Pedersen JT et al., (1994) J Mol Biol 235(3):959-73.See also U.S. Application Publication No. US2005 / 0042664 Al (February 24, 2005), which is incorporated herein by reference in its entirety.

[0308] Methods for making multispecific (eg, bispecific) antibodies have been described. See, e.g., U.S. Pat. Nos. 7,951,917, 7,183,076, 8,227,577, 5,837,242, 5,989,830, 5,869,620, 6,132,992, and 8,586,713.

[0309] Single domain antibodies, e.g., antibodies lacking light chains, can be produced by methods well known in the art. See Riechmann L and Muyldermans S (1999) J Immunol 231:25-38; Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3):253-263; Muyldermans S, (2001) J Biotechnol 74(4):277-302; U.S. Pat. No. 6,005,079; and International Publication Nos. WO 94 / 04678, WO 94 / 25591, and WO 01 / 44301.

[0310] Furthermore, antibodies that immunospecifically bind to the L1CAM antigen can then be used to generate anti-idiotypic antibodies that "mimic" the antigen using techniques well known to those skilled in the art (see, e.g., Greenspan NS and Bona CA (1989) FASEB J 7(5):437-444; and Nissinoff A (1991) J Immunol 147(8):2429-2438).

[0311] In certain aspects, the antibodies described herein that bind to the same epitope of L1CAM (e.g., human L1CAM) as the anti-L1CAM antibodies described herein are human antibodies or antigen-binding fragments thereof. In certain aspects, the antibodies described herein that competitively block (e.g., in a dose-dependent manner) the binding of antibodies described herein (e.g., Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12) to L1CAM (e.g., human L1CAM) are human antibodies or antigen-binding fragments thereof.

[0312] Any method known in the art can be used to produce human antibodies. For example, transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. Specifically, human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions and diversity regions can also be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can be disabled separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. Specifically, homozygous deletions in the JH region prevent endogenous antibody production. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then bred to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized in a normal manner with selected antigens, such as all or fragments of antigens (e.g., L1CAM). Monoclonal antibodies against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgene carried by transgenic mice is rearranged during B cell differentiation, and then undergoes class switching and somatic mutation. Therefore, using this technology, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg N and Huszar D (1995) Int Rev Immunol 13: 65-93. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and the scheme for producing such antibodies, see, for example, International Publication Nos. WO98 / 24893, WO 96 / 34096 and WO 96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598. Examples of mice capable of producing human antibodies include the XENOMOUSE TM (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HUAB-MOUSE TM (Mederex, Inc. / Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569,825), TRANS CHROMO MOUSE TM (Kirin) and KM MOUSE TM (Medarex / Kirin).

[0313] Human antibodies that specifically bind to L1CAM (e.g., human L1CAM) can be prepared by a variety of methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741.

[0314] In some aspects, mouse-human hybridomas can be used to produce human antibodies. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine the mouse-human hybridomas that secrete human monoclonal antibodies that specifically bind to target antigens (e.g., L1CAM, such as human L1CAM). Such methods are known and described in the art, see, for example, Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31.

[0315] VI. Cells and Vectors

[0316] In certain aspects, cells (e.g., host cells) that express (e.g., recombinantly) an antibody (or antigen-binding fragment thereof) described herein, and related polynucleotides and expression vectors, the antibody specifically binds to L1CAM (e.g., human L1CAM). Vectors (e.g., expression vectors) comprising polynucleotides are provided herein, the polynucleotides comprising nucleotide sequences encoding anti-L1CAM antibodies or fragments for recombinant expression in host cells, such as in mammalian cells. Host cells comprising such vectors for recombinant expression of anti-L1CAM antibodies (e.g., human antibodies or humanized antibodies) described herein are also provided herein. In specific aspects, methods for producing antibodies described herein are provided herein, comprising expressing such antibodies from host cells.

[0317] Recombinant expression of an antibody described herein (e.g., a full-length antibody, an antibody heavy chain and / or light chain, or a single-chain antibody described herein) that specifically binds to L1CAM (e.g., human L1CAM) involves constructing an expression vector comprising a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule, an antibody heavy chain and / or light chain, or a fragment thereof (e.g., a heavy chain and / or light chain variable domain) described herein is obtained, a vector for producing the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Therefore, methods for preparing proteins by expressing a polynucleotide comprising an antibody or antibody fragment (e.g., a light chain or heavy chain) encoding nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct an expression vector comprising an antibody or antibody fragment (e.g., a light chain or heavy chain) encoding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided is a replicable vector comprising a nucleotide sequence encoding an antibody molecule described herein, an antibody heavy chain or light chain, a heavy chain or light chain variable domain, or a heavy chain or light chain CDR of an antibody or its fragment, operably linked to a promoter. Such vectors may, for example, include nucleotide sequences encoding the constant regions of antibody molecules (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Pat. No. 5,122,464), and the variable domains of an antibody may be cloned into such vectors to express the entire heavy chain, the entire light chain, or the entire heavy and light chains.

[0318] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce an antibody described herein (e.g., an antibody comprising one or more of the VH and / or VL or VH and / or VL CDRs of Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12) or a fragment thereof. Therefore, provided herein is a host cell comprising a polynucleotide encoding an antibody or fragment thereof described herein, or a heavy chain or light chain thereof, or a fragment thereof, or a single-chain antibody described herein, the polynucleotide being operably linked to a promoter to express such sequences in a host cell. In certain aspects, in order to express a double-chain antibody, as described in detail below, a vector encoding a heavy chain and a light chain can be co-expressed in a host cell alone to express the entire immunoglobulin molecule. In certain aspects, a host cell comprises a vector comprising a polynucleotide encoding a heavy chain and a light chain of an antibody or fragment thereof described herein. In a specific aspect, the host cell comprises two different vectors, the first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or fragment thereof described herein, and the second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody or fragment thereof described herein. In other aspects, the first host cell comprises a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or fragment thereof described herein, and the second host cell comprises a second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody described herein. In a specific aspect, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to form an anti-L1CAM antibody or antigen-binding fragment thereof described herein. In certain aspects, a host cell population is provided herein, comprising such a first host cell and such a second host cell.

[0319] In a specific aspect, provided herein is a vector population comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-L1CAM antibody described herein and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-L1CAM antibody described herein.

[0320] A variety of host-expression vector systems can be used to express the antibody molecules described herein. Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells that can in situ express the antibody molecules described herein when transformed or transfected with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Pichia pastoris) transformed with recombinant yeast expression vectors containing antibody coding sequences; Pichia); an insect cell system infected with a recombinant viral expression vector (e.g., baculovirus) comprising the antibody coding sequence; a plant cell system (e.g., green algae such as Chlamydomonas reinhardtii) infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) comprising the antibody coding sequence or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) comprising the antibody coding sequence; or a mammalian cell system (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, NIH) with a recombinant expression construct comprising a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, SP2 / 0, Sf9, human lymphoblastoid cells, NS0, bow melanoma, HT-1080, PERC.6 and BMT10 cells). In a specific aspect, the cell used to express the antibodies or antigen-binding fragments thereof described herein is a CHO cell, such as from a CHO GS SYSTEM TM In certain aspects, the cells used to express the antibodies described herein are human cells, such as human cell lines. In certain aspects, the mammalian expression vector is a POPTIVEC TMOr pcDNA3.3. In particular aspects, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells) that are particularly useful for expressing complete recombinant antibody molecules are used to express recombinant antibody molecules. For example, mammalian cells (such as Chinese hamster ovary (CHO) cells) combined with vectors (such as major intermediate early gene promoter elements from human cytomegalovirus) are effective expression systems for antibodies (Foecking MK and Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al., (1990) Biotechnology 8 (7): 662-7). In certain aspects, the antibodies described herein are produced by CHO cells or NSO cells. In particular aspects, the expression of the nucleotide sequence encoding the antibodies described herein that immunospecifically bind to L1CAM (e.g., human L1CAM) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0321] In bacterial systems, many expression vectors can be advantageously selected according to the intended use of the antibody molecule to be expressed. For example, when a large amount of such antibodies are to be produced, in order to generate a pharmaceutical composition of the antibody molecule, a vector that directs the expression of a high-level, easily purified fusion protein product may be required. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U and Mueller-Hill B (1983) EMBO J2: 1791-1794), in which the antibody coding sequence can be connected to the vector alone, in frame with the lac Z coding region, so that a fusion protein is produced; pIN vectors (Inouye S and Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G and Schuster SM (1989) J Biol Chem 24: 5503-5509) etc. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to the matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0322] In insect systems, for example, the California clover worm (Autographa californica) polyhedrosis virus (AcNPV) can be used as a vector for expressing foreign genes. The virus is grown in Spodoptera frugiperda cells. The antibody coding sequence can be cloned separately into a non-essential region (e.g., the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0323] In mammalian host cells, many virus-based expression systems can be used. In the case of using adenovirus as an expression vector, the antibody coding sequence of interest can be connected to an adenovirus transcription / translation control complex, such as a late promoter and a triplet leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in the non-essential region of the viral genome (e.g., region E1 or E3) will produce a recombinant virus that has vigor and can express antibody molecules in the infected host (e.g., see Logan J and Shenk T (1984) PNAS 81 (12): 3655-9). The antibody coding sequence inserted by efficient translation may also require a specific start signal. These signals include an ATG start codon and adjacent sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to ensure the translation of the entire insert. These exogenous translation control signals and start codons can be natural and synthetic in a variety of origins. The efficiency of expression can be increased by including appropriate transcription enhancer elements, transcription terminators, etc. (see, for example, Bitter G et al., (1987) Methods Enzymol. 153: 516-544).

[0324] In addition, host cell strains can be selected that modulate the expression of inserted sequences or modify and process gene products in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, SKOV-3, B16-F1, NCI-H522, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In certain aspects, the anti-L1CAM antibodies described herein are produced in mammalian cells such as CHO cells.

[0325] In certain aspects, the antibodies or antigen-binding fragments thereof described herein have reduced or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibodies can be expressed in cells lacking or deficient in fucosylation ability. In certain embodiments, a cell line with knockouts of both alleles of 1,6-fucosyltransferase can be used to produce antibodies or antigen-binding fragments thereof with reduced fucose content. The ANTIBODYNAMIC® system (Lonza) is an example of such a system that can be used to generate antibodies or antigen-binding fragments thereof with reduced fucose content.

[0326] In order to produce recombinant proteins in high yields over a long period of time, stable expressing cells can be generated. For example, cell lines that stably express the anti-L1CAM antibodies or antigen-binding fragments thereof described herein can be engineered. In a specific aspect, the cells provided herein stably express light chains / light chain variable domains and heavy chains / heavy chain variable domains, which associate to form antibodies or antigen-binding fragments thereof described herein.

[0327] In certain aspects, rather than using expression vectors containing viral origins of replication, host cells may be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selection markers. After the introduction of foreign DNA / polynucleotides, the engineered cells may be grown in an enriched medium for 1-2 days and then transferred to a selective medium. The selection marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the anti-L1CAM antibodies or antibody binding fragments thereof described herein. Such engineered cell lines can be particularly useful for screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0328] A number of selection systems can be used, including but not limited to the use of herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH and Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes in tk cells, hgprt cells, or aprt cells, respectively. Similarly, antimetabolite resistance can be used as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6):3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC and Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY and Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; and Morgan RA and Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ and Feigner PL (1993) Trends Biotechnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-56).Methods generally known in the field of recombinant DNA technology can be routinely applied to select desired recombinant clones, and such methods are described, for example, in Ausubel FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13 of Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F et al., (1981) J Mol Biol 150: 1-14, which are incorporated herein by reference in their entirety.

[0329] In some aspects, the antibodies or antigen binding fragments thereof disclosed herein may be expressed on immune cells such as T cells and / or NK cells. In some aspects, antibodies or antigen binding fragments thereof may be expressed as chimeric antigen receptors (CAR). CAR-T cells are T cells expressing chimeric antigen receptors. As used herein, the phrase "chimeric antigen receptor (CAR)" refers to the following recombinant fusion protein, which has an antigen-specific extracellular (or extracellular domain) domain coupled to an intracellular domain, and the intracellular domain instructs cells to perform specialized functions after the antigen binds to the extracellular domain. The difference between chimeric antigen receptors and other antigen binding agents is that they bind to MHC-independent antigens and the ability to transduce activation signals through their intracellular domains.

[0330] In some aspects, the antigen-specific extracellular domain of the chimeric antigen receptor recognizes and specifically binds to an antigen, i.e., L1CAM. The L1CAM-specific extracellular domain suitable for the CAR of the present disclosure can be any antigen-binding polypeptide, and a variety of such polypeptides are known in the art. In some cases, the antigen-binding domain is a single-chain Fv (scFv) or Fab. In other aspects, the antigen-binding fragments that can be used for the CAR of the present disclosure include antigen-binding fragments disclosed anywhere herein.

[0331] In some aspects, the transmembrane domain that can be used for CAR is connected to the extracellular domain, and can include a naturally occurring transmembrane domain. In other aspects, the transmembrane domain that can be used for CAR can be derived from the α chain, β chain or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, CD8 or any other substance known in the art.

[0332] The term "intracellular domain" refers to a part of CAR, which transduces effector function signals after antigen is combined with the extracellular domain and instructs T cells to perform specialized functions. On the one hand, the intracellular domain of CAR includes an activation motif (ITAM) based on immunoreceptor tyrosine. In some aspects, ITAM is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, CD66d, 4-1BB, DAP-1 0, OX40 or Fc[ε]RI[γ].

[0333] In some aspects, the CAR of the present disclosure also includes a costimulatory domain that can be connected to the intracellular domain. The costimulatory domain in the CAR construct can transmit signals and activate cells that are part of the intracellular portion of the CAR. In some aspects, the costimulatory domain is derived from CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD7, LIGHT, NKG2C or B7-H3.

[0334] In other aspects, the CAR of the present disclosure also includes a joint. A short oligopeptide or polypeptide joint may be present between the transmembrane domain and the intracellular domain. In some aspects, the joint is not limited to a specific length, as long as the intracellular domain of CAR can induce T cell activation when the extracellular domain binds to the antigen (i.e., L1CAM). In some aspects, the joint includes a (Gly4Ser)3 joint.

[0335] In other aspects, the present disclosure comprises a polynucleotide encoding a CAR of the present disclosure or a vector comprising the polynucleotide.

[0336] As used herein, the term "T cell" is a lymphocyte derived from the thymus and contributing to the cellular immune response. T cells include CD4+T cells (helper T cells, TH cells), CD8+T cells (cytotoxic T cells, CTL), memory T cells, regulatory T cells (Treg) or natural killer T cells. In some aspects, the T cells introduced into CAR are CD8+T cells.

[0337] VII. Immunoconjugates, Antibody Derivatives and Diagnostics

[0338] The anti-L1CAM antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, and for this purpose, the antibodies (or their binding fragments) can be conjugated to appropriate detectable agents to form immunoconjugates. For diagnostic purposes, appropriate agents are detectable labels, including radioisotopes for whole body imaging and radioisotopes, enzymes, fluorescent labels and other suitable antibody labels for sample testing.

[0339] The detectable label that can be attached to any of the anti-L1CAM antibodies described herein can be any of the various types of labels currently used in the field of in vitro diagnostics, including: particulate labels, including metal sols (such as colloidal gold); isotopes, such as N 2 S 2 、N 3 S or N 4 Types of Peptide Chelators Presented I 125 or Tc 99 ; chromophores, including fluorescent markers, luminescent markers, phosphorescent markers, etc.; and enzyme labels that convert a given substrate into a detectable label and polynucleotide tags that are displayed after amplification such as by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, etc. For example, the label can be alkaline phosphatase, which converts 1,2-dioxetane substrates (such as adamantyl methoxyphosphoryloxyphenyldioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}dec}-4-yl)phenyl phosphate disodium (CSPD) and CDP and CDP- ) or other luminescent substrates well known in the art (e.g., chelates of suitable lanthanides such as terbium (III) and europium (III))) are detected by measuring the presence or formation of chemiluminescence. The means of detection is determined by the selected label. The appearance of the label or its reaction product can be achieved using the naked eye, if the label is particulate and accumulates at an appropriate level, or using instruments such as spectrophotometers, luminometers, fluorometers, etc., all according to standard practice.

[0340] In some aspects, the conjugation method produces substantially (or almost) non-immunogenic linkages, such as peptide linkages (i.e., amide linkages), sulfide linkages (steric hindrance), disulfide linkages, hydrazone linkages, and ether linkages. These linkages are almost non-immunogenic and show reasonable stability in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886).

[0341] According to the biochemical nature of the part and the antibody, different conjugation strategies can be used. If the part is naturally occurring or recombined between 50 and 500 amino acids, there are standard procedures for describing the synthetic chemistry of protein conjugates in textbooks, and the procedures can be easily followed by technicians (see, for example, Hackenberger, CPR and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some aspects, the reaction of the maleimido moiety with the cysteine ​​residues in the antibody or the part is used. This is particularly suitable coupling chemistry, if, for example, the Fab fragment or Fab' fragment of an antibody is used. Alternatively, in some aspects, coupling with the C-terminal of the antibody or part is carried out. The C-terminal modification of a protein (e.g., Fab fragment) can be carried out as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

[0342] In general, site-specific reactions and covalent couplings are based on converting natural amino acids to amino acids with reactivity orthogonal to the reactivity of other functional groups present. For example, specific cysteines within a rare sequence context can be enzymatically converted in aldehydes (see Frese, MA and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain the desired amino acid modification in a given sequence context by exploiting the specific enzymatic reactivity of certain enzymes with natural amino acids (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; and Bordusa, F. Protease-catalyzed formation of C—N bonds used, Highlights in Bioorganic Chemistry (2004) 389-403). Site-specific reactions and covalent coupling can also be achieved by selective reaction of the terminal amino acids with appropriate modifying agents.

[0343] The reactivity of the N-terminal cysteine ​​with benzonitrile (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent coupling.

[0344] Native chemical ligation can also rely on a C-terminal cysteine ​​residue (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0345] US6437095 B1 describes a conjugation method based on the faster reaction of a cysteine ​​within a stretch of negatively charged amino acids with a cysteine ​​located in a stretch of positively charged amino acids.

[0346] The moiety may also be a synthetic peptide or peptide mimetic. If the polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity may be incorporated during such synthesis (see, e.g., de Graaf, AJ, et al., Bioconjug. Chem. 20 (2009) 1281-1295). Because a variety of orthogonal functional groups are at stake and can be introduced into synthetic peptides, conjugation of such peptides to linkers is standard chemistry.

[0347] To obtain a single labeled polypeptide, a conjugate with a 1:1 stoichiometry can be separated from other conjugated byproducts by chromatography. This process can be facilitated by using dye-labeled binding pair members and charged linkers. By using such labeled and negatively charged binding pair members, the single conjugated polypeptide is easily separated from non-labeled polypeptides and polypeptides carrying more than one linker, because differences in charge and molecular weight can be used for separation. Fluorescent dyes can be used to purify complexes from unbound components (such as labeled monovalent binders).

[0348] In some aspects, the moiety attached to the anti-L1CAM antibody is selected from the group consisting of: a binding moiety, a labeling moiety, and a biologically active moiety.

[0349] The anti-L1CAM antibodies described herein can also be conjugated with therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents. In ADCs, the antibody and therapeutic agent are preferably conjugated via a cleavable linker such as a peptidyl, disulfide or hydrazone linker. ADCs can be prepared as described in U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02 / 096910; WO 07 / 038658; WO 07 / 051081; WO 07 / 059404; WO 08 / 083312; and WO 08 / 103693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295.

[0350] In some aspects, the therapeutic agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a secondary antibody, an enzyme, an anti-tumor agent, and any combination thereof.

[0351] In some aspects, the immunoconjugate comprises an anti-L1CAM antibody and a cytotoxin. The cytotoxin can be selected from any cytotoxin known in the art. In some aspects, the cytotoxin is selected from the group consisting of: dolastatin, monomethyl auristatin E (MMAE), maytansine, duocarmycin, calicheamicin, pyrrolobenzodiazepine, dukamicin, centanamycin, SN38, doxorubicin, its derivatives, its synthetic analogs and any combination thereof. In certain aspects, the immunoconjugate comprises an anti-L1CAM antibody and cytotoxin A. In other aspects, the immunoconjugate comprises an anti-L1CAM antibody and a non-cytotoxic drug.

[0352] In some aspects, the immunoconjugate comprises an anti-L1CAM antibody and a radioactive agent. In some aspects, the radioactive agent is a radionucleotide. In certain aspects, the radioactive agent comprises radioactive iodine. In specific aspects, the radioactive agent comprises 131-iodine. In other aspects, the radioactive agent comprises the radioisotope yttrium-90.

[0353] In some aspects, the immunoconjugate comprises an anti-L1CAM antibody and a second antibody. In some aspects, the immunoconjugate comprises an anti-L1CAM antibody and an enzyme. In some aspects, the enzyme comprises glucose oxidase. In some aspects, the enzyme comprises peroxidase. In some aspects, the enzyme comprises myeloperoxidase. In some aspects, the enzyme comprises glucose oxidase. In some aspects, the enzyme comprises horseradish peroxidase.

[0354] In some aspects, the immunoconjugate comprises an anti-L1CAM antibody and an anti-tumor agent. The anti-tumor agent can be any such agent known in the art. In some aspects, the anti-tumor agent is epirubicin. In some aspects, the anti-tumor agent is a superantigen. In some aspects, the superantigen is Staphylococcal enterotoxin A (SEA / E-120; estafenatox).

[0355] Anti-L1CAM antibodies (such as those described herein) can also be used to detect L1CAM, such as human L1CAM, such as human L1CAM on the surface of cells or soluble L1CAM in serum. The antibodies can be used, for example, in ELISA assays or flow cytometry. In some aspects, the anti-L1CAM antibody is contacted with cells or serum for a period of time suitable for specific binding to occur, and then an agent, such as an antibody to detect the anti-L1CAM antibody, is added. Exemplary assays are provided in the Examples. An exemplary method for detecting L1CAM (such as surface-expressed L1CAM or soluble L1CAM (sL1CAM)) in a sample (serum) includes: (i) contacting the sample with an anti-L1CAM antibody for a time sufficient for the anti-L1CAM antibody to specifically bind to L1CAM in the sample; and (2) contacting the sample with a detection agent (such as an antibody) that specifically binds to the anti-L1CAM antibody (such as the Fc region of the anti-L1CAM antibody) to thereby detect L1CAM bound by the anti-L1CAM antibody. Incubation with the antibody and / or detection agent may include a washing step. The anti-L1CAM antibodies used in these methods do not have to be linked to a label or detection agent, as a separate detection agent can be used.

[0356] Other uses of anti-L1CAM antibodies (eg, as monotherapy or in combination therapy) are provided elsewhere herein (eg, in the subsections involving combination therapies).

[0357] VIII. Bispecific Molecules

[0358] The anti-L1CAM antibodies described herein can be used to form bispecific molecules. Anti-L1CAM antibodies or antigen-binding portions thereof can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand of a receptor), to generate bispecific molecules that bind to at least two different binding sites or target molecules. For example, anti-L1CAM antibodies can be linked to antibodies or scFvs that specifically bind to any protein that can be used as a potential target for combination therapy. In fact, the antibodies described herein can be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be covered by the term "bispecific molecule" as used herein. In order to produce the bispecific molecules described herein, the antibodies described herein can be functionally linked to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise), thereby obtaining bispecific molecules.

[0359] Thus, provided herein are bispecific molecules comprising at least a first binding specificity for L1CAM and a second binding specificity for a second target epitope. In some aspects described herein where the bispecific molecules are multispecific, the molecules can further include a third binding specificity.

[0360] In some aspects, the bispecific molecules described herein comprise at least one antibody or antibody fragment thereof as binding specificity, including, for example, Fab, Fab', F(ab')2, Fv or single-chain Fv (scFv). The antibody may also be a light chain or heavy chain dimer or any minimal fragment thereof, such as Fv or a single-chain construct as described in Ladner et al., U.S. Pat. No. 4,946,778.

[0361] The bispecific molecules described herein can be prepared by conjugating the component binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificity is a protein or peptide, a variety of coupling agents or cross-linking agents can be used for covalent conjugation. See, for example, Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648. Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83) and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Some conjugating agents are SATA and Sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0362] IX. Composition

[0363] Further provided herein are compositions, such as pharmaceutical compositions, comprising one or a combination of anti-L1CAM antibodies described herein, or a combination of antibodies directed to other targets, or one or more antigen-binding portions thereof, formulated with a pharmaceutically acceptable carrier. Such compositions may comprise one or a combination (e.g., two or more different substances) of antibodies, or immunoconjugates, or bispecific molecules described herein. For example, a pharmaceutical composition described herein may comprise a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on a target antigen or have complementary activities.

[0364] The pharmaceutical compositions described herein can also be administered in combination therapy, i.e., in combination with other agents. For example, a combination therapy can include an anti-L1CAM antibody described herein in combination with at least one other anticancer agent and / or immunomodulator, such as a T cell stimulator (e.g., activator). Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the subsection on uses of the anti-L1CAM antibodies described herein.

[0365] In some aspects, the composition of the present invention further comprises a bulking agent. The bulking agent may be selected from the group consisting of: NaCl, mannitol, glycine, alanine, and any combination thereof. In other aspects, the composition of the present invention comprises a stabilizer. The stabilizer may be selected from the group consisting of: sucrose, trehalose, raffinose, arginine, or any combination thereof. In other aspects, the composition of the present invention comprises a surfactant. The surfactant may be selected from the group consisting of: polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof. In certain aspects, the composition further comprises a chelating agent. The chelating agent may be selected from the group consisting of: diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid, nitrilotriacetic acid, and any combination thereof.

[0366] In other aspects, the composition comprises a third antibody. In some aspects, the third antibody is any antibody disclosed herein.

[0367] In one aspect, the composition further comprises NaCl, mannitol, valeric acid (DTPA), sucrose, PS80, and any combination thereof.

[0368] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some aspects, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The choice of subcutaneous injection is based on Halozyme Therapeutics' Drug delivery technology that involves co-formulating an Ab with recombinant human hyaluronidase (rHuPH20) to remove traditional limitations on the volume of biologics and drugs that can be delivered subcutaneously due to the extracellular matrix (U.S. Pat. No. 7,767,429). Depending on the route of administration, the active compound (i.e., antibody, immunoconjugate, or bispecific molecule) can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0369] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects (see, e.g., Berge, SM et al. (1977) J. Pharm. Sci. 66: 1-19). The pharmaceutical compositions described herein may also include a pharmaceutically acceptable antioxidant.

[0370] These compositions may also include adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms may be prevented by the same sterilization procedures and by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugar, sodium chloride, etc. in the compositions. In addition, by including agents that delay absorption, such as aluminum monostearate and gelatin, extended absorption of injectable drug forms may be caused.

[0371] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Such media and agents are known in the art for pharmaceutically active substances. Except for any conventional media or agents that are incompatible with the active compound, it is contemplated that they are used in the pharmaceutical compositions described herein. The pharmaceutical composition may or may not contain a preservative. Supplementary active compounds may be incorporated into the composition.

[0372] Under manufacturing and storage conditions, the therapeutic composition must generally be sterile and stable. The composition can be formulated into a solution, microemulsion, liposome or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersion and by using a surfactant. In many cases, the composition can include an isotonic agent, such as a sugar, a polyol (such as mannitol, sorbitol) or sodium chloride in the composition. By including an agent such as monostearate and gelatin that delays absorption in the composition, prolonged absorption of the injectable composition can be caused.

[0373] As required, a sterile injection solution can be prepared by incorporating the desired amount of the active compound into a suitable solvent with one or a combination of the ingredients listed above, followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle comprising a basic dispersion medium and other components required from those listed herein. In the case of sterile powders for preparing sterile injection solutions, some preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder of the active ingredient plus any additional required ingredients from its previously sterile filtered solution.

[0374] For administration of an anti-L1CAM antibody, such as described herein, the dosage range is about 0.0001 to 100 mg / kg.

[0375] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.The antibodies are typically administered on multiple occasions.

[0376] Antibodies can be administered as sustained release formulations, in which case less frequent administration is required. Dosage and frequency vary according to the half-life of the patient's antibody. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered at relatively infrequent time intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses at relatively short time intervals are sometimes required until the progression of the disease is reduced or terminated, and until the patient shows partial or complete improvement of symptoms of the disease. Thereafter, preventive regimens can be administered to the patient.

[0377] A "therapeutically effective dose" of an anti-L1CAM antibody described herein can result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of damage or disability due to disease affliction. In the case of cancer, a therapeutically effective dose can result in increased survival (e.g., overall survival) and / or prevent further worsening of physical symptoms associated with cancer. Symptoms of cancer are well known in the art and include, for example, unusual mole features, changes in the appearance of a mole (including asymmetry, borders, color and / or diameter), newly pigmented areas of skin, abnormal moles, darkened areas under the nails, breast lumps, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, worsening dyspnea, coughing up blood, blood in the urine, blood in the stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal fullness, bloating, abdominal effusion, vaginal bleeding, constipation, abdominal distension, colon perforation, acute peritonitis (infection, fever, pain), pain, vomiting blood, heavy sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, colon metastases, lung metastases, bladder metastases, liver metastases, bone metastases, kidney metastases and pancreatic metastases, difficulty swallowing, etc.

[0378] A therapeutically effective dose can prevent or delay the onset of cancer, such as may be required when there are early or preliminary signs of the disease. Laboratory tests used in diagnosing cancer include chemistry (including measurement of L1CAM levels), hematology, serology, and radiology. Therefore, any clinical or biochemical assay that monitors any of the foregoing can be used to determine whether a particular treatment is a therapeutically effective dose for treating cancer. One of ordinary skill in the art should be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0379] The compositions described herein can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending on the desired results. The routes of administration of the anti-L1CAM antibodies described herein may include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0380] Alternatively, the antibodies described herein can potentially be administered via non-parenteral routes such as topical, epidermal or mucosal administration routes, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0381] X.Medicine Box

[0382] Provided herein are kits comprising one or more antibodies or antigen-binding fragments thereof, bispecific molecules or immunoconjugates thereof as described herein. In particular aspects, provided herein are pharmaceutical packages or kits comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions described herein, such as one or more antibodies or antigen-binding fragments thereof provided herein, optionally with instructions for use. In some aspects, the kits comprise the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein.

[0383] XI. Use and Methods

[0384] Certain aspects of the present disclosure relate to methods of treating a subject, comprising administering to the subject an anti-L1CAM antibody disclosed herein, a polynucleotide encoding the anti-L1CAM antibody, a vector comprising the polynucleotide, a host cell comprising the polynucleotide, an immunoconjugate comprising the anti-L1CAM antibody, or any combination thereof.

[0385] Certain aspects of the present disclosure relate to methods for treating cancer in subjects in need, comprising administering to the subject an effective dose of a composition disclosed herein (e.g., an antibody, a polynucleotide, a vector, a host cell, an immunoconjugate, or a pharmaceutical composition). In other aspects, the present disclosure relates to methods for inhibiting the shedding of L1CAM by tumor cells in subjects in need, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the present disclosure relates to methods for reducing L1CAM shed in the serum of subjects in need and / or retaining L1CAM on the cell surface, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the present disclosure relates to a method for killing tumor cells in subjects in need, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the present disclosure relates to a method for reducing the size of a tumor in a subject in need, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the present disclosure relates to a method for inhibiting tumor metastasis in subjects in need, comprising administering to the subject an effective dose of a composition disclosed herein. In some aspects, the subject is human.

[0386] The compositions of the present disclosure can be used using any pharmaceutically acceptable approach. In some respects, compositions (such as antibodies, polynucleotides, carriers, host cells, immunoconjugates or pharmaceutical compositions) are intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, local, epidermal, mucosal or any combination thereof. In some respects, compositions are administered intravenously. In some respects, compositions are administered subcutaneously.

[0387] In some aspects, the methods reduce the size of a subject's cancer, such as the size of a tumor. In some aspects, the size of the cancer is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0388] In some aspects, the method increases the overall survival of the subject. In some aspects, relative to the average overall survival of the subject suffering from the same cancer but treated with different therapies, overall survival increases. In some aspects, overall survival increases by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 3 times, at least about 5 times. In some aspects, overall survival increases by at least about one month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 1 month, at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years or at least about 10 years.

[0389] In some aspects, the method increases the progression-free survival of the subject. In some aspects, relative to the average progression-free survival of the subject suffering from the same cancer but treated with different therapies, overall survival increases. In some aspects, progression-free survival increases by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 3 times, at least about 5 times. In some aspects, overall survival increases by at least about one month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 1 month, at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years or at least about 10 years.

[0390] In some aspects, the method increases the objective response rate of the subject. In some aspects, the method induces a complete response in the subject. In some aspects, the method induces a partial response in the subject.

[0391] In some aspects, the method comprises administering an anti-L1CAM antibody (or polynucleotide, vector, host cell or immunoconjugate) disclosed herein and a second therapy. In some aspects, the second therapy is administered before the anti-L1CAM antibody. In some aspects, the second therapy is administered after the anti-L1CAM antibody. In some aspects, the second therapy is administered simultaneously with the anti-L1CAM antibody. In certain aspects, the anti-L1CAM antibody and the second therapy are administered separately. In other aspects, the anti-L1CAM antibody and the second therapy are administered in the form of a single formulation.

[0392] The second therapy can be any other therapy known in the art. In some aspects, the second therapy includes immunotherapy. In some aspects, the second therapy includes chemotherapy. In some aspects, the second therapy includes radiation therapy. In some aspects, the second therapy includes surgery. In some aspects, the second therapy includes administering a second therapeutic agent.

[0393] Anti-L1CAM antibodies can enhance the immune response of patients with cancer to cancerous cells. Provided herein are methods for treating subjects with cancer, comprising administering to the subject an anti-L1CAM antibody described herein, such that the subject is treated, for example, such that the growth of a cancerous tumor is inhibited or reduced and / or the tumor regresses and / or prolonged survival is achieved. Anti-L1CAM antibodies can be used alone to inhibit the growth of cancerous tumors. Alternatively, anti-L1CAM antibodies can be used in combination with another agent (e.g., another immunogenic agent), a standard cancer treatment, or another antibody, as described below.

[0394] Thus, provided herein is a method for treating a subject's cancer, for example, by inhibiting the growth of tumor cells, comprising administering to the subject a therapeutically effective amount of an anti-L1CAM antibody described herein. Cancers whose growth can be inhibited using the antibodies disclosed herein include cancers that are typically responsive to immunotherapy and cancers that are typically unresponsive to immunotherapy. Treatable cancers also include L1CAM-positive cancers. Cancer can be a cancer with a solid tumor or a hematological malignancy (liquid tumor). Non-limiting examples of cancers for treatment include bile duct cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell epithelial carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, renal cancer (e.g., renal cell epithelial carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate epithelial carcinoma), thyroid cancer, neuroblastoma, pharyngeal cancer, laryngeal cancer, oral cancer, connective tissue cancer, Hodgkin's lymphoma (Hodgkin's lymphoma, multiple myeloma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon glandular cancer and head and neck cancer (or epithelial cancer), gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastatic malignant melanoma, such as skin or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, epithelial cancer of the fallopian tubes, epithelial cancer of the endometrium, epithelial cancer of the cervix, epithelial cancer of the vagina, epithelial cancer of the vulva, esophageal cancer, small intestine cancer, cancer of the endocrine system, parathyroid gland carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, rectal cancer, solid tumors of childhood, ureteral cancer, renal pelvic epithelial carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including asbestos-induced cancers), virus-related cancers or cancers of viral origin (such as human papillomavirus (HPV-related or HPV-derived tumors)); and any combination of said cancers.

[0395] In some aspects, anti-L1CAM antibodies are administered to patients with cancer who have shown an inadequate response or have progressed on a previous treatment (e.g., a previous treatment with an immuno-oncology or immunotherapy drug), or patients with refractory or resistant cancer (whether intrinsically refractory or resistant, or where the resistance or refractory state is acquired). For example, subjects who have not responded or have not responded adequately to a first therapy, or who have seen disease progression following treatment, can be treated by administering an anti-L1CAM antibody alone or in combination with another therapy.

[0396] In some aspects, an anti-L1CAM antibody is administered to a patient who has not previously received (ie, been treated with) an immuno-oncology agent.

[0397] In some aspects, a method of treating cancer in a subject comprises: first determining whether the subject is L1CAM positive (e.g., has tumor cells expressing L1CAM); and if the subject has L1CAM positive cancer, administering an anti-L1CAM antibody, such as an antibody described herein, to the subject. A method of treating a subject having cancer with an anti-L1CAM antibody may comprise administering a therapeutically effective amount of the L1CAM antibody to a subject having cancer cells expressing L1CAM. Also provided herein is a method for predicting whether a subject will respond to treatment with an anti-L1CAM antibody, wherein the method comprises determining the level of L1CAM in a patient's cancer cells, and if the subject's cancer cells are L1CAM positive, the subject may respond to treatment with the L1CAM antibody.

[0398] Anti-L1CAM antibodies can be administered with standard of care treatments.Anti-L1CAM antibodies can be administered as maintenance therapy, e.g., therapy intended to prevent tumor development or recurrence.

[0399] Anti-L1CAM antibodies can be administered together with another therapy such as radiation, surgery or chemotherapy. For example, anti-L1CAM antibodies can be administered as adjuvant therapy when there is a risk that micrometastasis may exist and / or to reduce the risk of recurrence.

[0400] Anti-L1CAM antibodies (e.g., anti-L1CAM antibodies described herein) can be combined with vaccination regimens. Many experimental strategies for vaccination against tumors have been designed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Chapter 61, pp. 3023-3043, in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition).

[0401] Administration of anti-L1CAM antibodies can also be combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). Administration of anti-L1CAM antibodies can be effectively combined with chemotherapeutic regimens. In these cases, it is possible to reduce the dose of the chemotherapeutic agent administered (Mokyr et al. (1998) Cancer Research 58:5301-5304).

[0402] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, Sambrook et al., eds. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., eds. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover, ed. (1985) DNA Cloning, Vols. I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al., U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the paper Methods In Enzymology (Academic Press, Inc., NY); Miller and Calos (1987) GeneTransfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., Methods In Enzymology, Volumes 154 and 155; Mayer and Walker (1987) ImmunochemicalMethods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the MouseEmbryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); Crooks, Antisense drug Technology: Principles, strategies and applications, 2nd edition CRC Press (2007) and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.). .

[0403] The following examples are offered by way of illustration and not by way of limitation.

[0404] Example

[0405] The following experimental methods and details are cited in the following examples.

[0406] Example 1. Construction of Ab612 by site-directed mutagenesis

[0407] Human antibody Ab417 that binds to mammalian (e.g., human and mouse) L1CAM and its preparation process are disclosed in U.S. Patent No. 9,777,060 and International Publication No. WO2014 / 077648, which are incorporated herein by reference. In order to improve the biophysical properties of Ab417 antibody, the effects of individual amino acids constituting the variable region of Ab417 antibody on the binding ability of Ab417 antibody were examined.

[0408] DNA was synthesized to carry the mutations R16G, D54E, K76A and P88A in the heavy chain variable region of Ab417 antibody and recombined with the gene encoding the CH1 domain of human Cγ1 by polymerase chain reaction (PCR). The resulting PCR product was electrophoresed on a 1.5% agarose gel. The band containing the DNA was cut and purified using a PCR purification kit. Purification was performed. Both ends of the purified DNA were digested with restriction enzymes NcoI and ApaI (New England Biolabs) and subcloned into the NcoI and ApaI sites of a modified version (pKRIBB-full GIII) of the human Fab phage display vector pKRIBB-FabD (ref), which carries the full length of gene III. The resulting recombinant phagemid was named pKRIBB-full GIII-Fd612.

[0409] DNA was synthesized to carry the mutations I31S and V95P in the variable region of the light chain of the Ab417 antibody and recombined with the gene encoding human Cκ by PCR. The resulting PCR product was electrophoresed on a 1.5% agarose gel. The band containing the DNA was cut out and purified using a PCR purification kit Purification was performed. The two ends of the purified DNA were digested with restriction enzyme BstXI (New England Biolabs) and subcloned into the BstXI site of pKRIBB-full GIII-Fd612. The resulting recombinant phagemid was named pKRIBB-full GIII-Fab612. For this reason, the antibody comprising the mutations R16G, D54E, K76A and P88A in the heavy chain variable region of Ab417 antibody and the mutations I31S and V95P in the light chain variable region was named Ab612 antibody.

[0410] Example 2. Construction of phage-displayed Ab612 variant Fab library

[0411] To improve the affinity and biophysical properties of the Ab612 antibody prepared in Example 1, five positions of the Ab612 LCDR3 were randomized by TRIM (trinucleotide mutagenesis, ELLA biotech, Germany). Fragment A containing the sequence from FR1 to FR3 of Ab612 was synthesized. Fragment B containing the sequence from Ab612 FR3 to FR4 with randomized LCDR3 was synthesized (5'-TTT AAT TTC CAC TTT AGT TCC CTG CCC GAA CGT CCA CGG X17 X15 X15X13 X15 TTG CTG ACA ATA ATA GGT GGC AAA ATC TTC-3'). X represents the number of amino acids; X17 is 17 amino acids, X15 and X13 are 15 amino acids and 13 amino acids, respectively. Fragments A and B were assembled by recombinant PCR using Phusion high-fidelity DNA polymerase (ThermoFisher Scientific). The obtained PCR product was purified using a PCR purification kit The Fab612 variant library was purified, digested with BstXI restriction enzyme (New England Biolabs), and ligated with BstXI-digested pKRIBB-full GIII-Fab612 expression vector at a vector-to-insert molar ratio of 1:3. The DNA was electroporated into competent E. coli TG1. The Ab612 variant Fab library generated 5.92 × 10 8 A colony.

[0412] To isolate monoclonal antibodies recognizing human L1CAM, human L1CAM was used as an antigen in the first, second, and third rounds of panning.

[0413] After three rounds of panning, 470 colonies were randomly selected and inoculated in 96-well plates, and then cultured for 8 hours at 37°C in 300 μl 2×YT / carbenicillin / glucose. Subsequently, 30 μl cells were inoculated into each well containing 1 ml 2×YT / carbenicillin / glucose culture medium, and cultured for 2 hours until OD600 reached 0.5. Cells were infected at 37°C with KM13 helper phage with a multiplicity of infection (MOI) 20 without shaking (shacking), and then incubated for 30 minutes under shaking. Infected cells were resuspended in 2×YT / carbenicillin / kanamycin after centrifugation for 10 minutes at 2900xg, and cultured for 12 hours at 30°C. Indirect and quantitative ELISA was performed to the supernatant containing the Fab phage from 470 colonies.

[0414] Among them, 41 clones that bind to human L1CAM with high activity were selected, and their plasmid DNAs were isolated and sequenced. As a result, 32 clones were found to be different from each other.

[0415] In order to analyze the binding ability of the 32 unique clones to human L1CAM, an indirect ELISA was performed. As a result, it was found that 4 clones showed the highest antigen binding ability (ie, Ab4H5, Ab2C2, Ab4H6, and Ab5D12).

[0416] Example 3. Conversion and production of Fab to IgG1

[0417] In order to convert Fab into a complete IgG1 with a codon optimized sequence for mammalian cells, the variable heavy and light chains with a leader sequence were amplified by PCR and subcloned into the EcoRI and ApaI sites (New England Biolabs) and BsiWI and HindIII sites (New England Biolabs) in the mammalian IgG1 expression plasmid pdCMV-dhfr-Ab612 vector, respectively. HEK293F cells were cultivated and transfected with the IgG1 expression plasmid using ExpiFectamin (Thermo Fisher Scientific) according to the ExpiCHO protocol. After 7-14 days from transfection, the cell culture supernatant was centrifuged and filtered using a bottle top filter (0.22 μm PES, Sartorius), and the productivity of the corresponding mutant was compared with the productivity of the existing Ab417 antibody by ELISA, as described in Example 5 below.

[0418] As shown in Table 8, the variant antibodies exhibited productivity that was 1.25-1.44 times that of Ab417. Ab612 was enhanced by 1.44 times, while Ab4H5, Ab2C2, Ab4H6, and Ab5D12 were enhanced by 1.25 times, 1.26 times, 1.36 times, and 1.26 times, respectively.

[0419] Table 8. Productivity of Ab612, Ab4H5, Ab2C2, Ab4H6 and Ab5D12 antibodies compared to Ab417

[0420]

[0421] Example 4. Purification of Ab417 variant antibody

[0422] The plasmid DNA of the expression vector of these antibodies was obtained in large quantities, and expressed in ExpiCHO cells in the same manner as in Example 1-3. The cell culture was centrifuged to collect the supernatant. The supernatant was filtered using a bottle top filter (0.22 μm PES, Sartorius) and purified by affinity chromatography. The corresponding supernatant was applied to a column packaged with protein A coupled beads (Amicogen), and the antibody was eluted from protein A using 0.1M sodium citrate solution (pH 3.2). Then, 1.0M Tris solution (pH 8.0) was immediately added to the eluted antibody for neutralization. After dialysis using a PD-10 column (GE Healthcare) with Sephadex G-25, the purified IgG1 was stored in a buffer (10mM Napi 5% sorbitol 0.01% tween 20). Based on the molar extinction coefficient, the concentration of the purified antibody was determined using Nanodrop (Thermo Fisher Scientific, Nanodrop2000). The purified antibodies were subjected to 10% SDS-PAGE and Coomassie staining to confirm that each of the heavy and light chains were expressed and assembled into complete IgG.

[0423] Example 5. Characterization of Ab417 variant antibodies

[0424] The affinity of the purified antibodies to human L1CAM was measured by competitive ELISA. Human L1CAM was prepared at a density of 1×10-7 M and serially diluted to 1×10-7 M using 0.1% PBA buffer solution (PBS containing 0.1% BSA). -12 M. The corresponding antibody was prepared by diluting it in a 0.1% PBA buffer solution at a specific concentration according to the binding capacity of the antibody. The diluted antigen and antibody were reacted with each other at the same volume ratio at 37°C for 3 hours. A 96-well plate (MaxiSorp, Nunc) was coated with purified human L1CAM diluted in a buffer solution (15 mM Na 2 CO 3, 34.84mMNaHCO3, pH 9.6), overnight at 4°C. The next day, Difco skim milk (BD) was dissolved in 0.05% PBS-T buffer solution at a concentration of 2%, and 200ul was added to each well, and then incubated at 37°C for 1 hour. The wells were washed twice with 0.05% PBS-T buffer solution. Then 100ul of antigen / antibody reactant that had reacted for 3 hours was added and allowed to react at room temperature for 1 hour. The wells were washed three times with 0.05% PBS-T buffer solution to remove non-antigen-bound antibodies. Goat anti-human IgG (Fc)-HRP (invitrogen, 1 / 10000) that specifically recognizes the Fc region of human antibodies was added as a secondary antibody and allowed to react at 37°C for 1 hour. The wells were washed four times with 0.05% PBS-T buffer solution to remove the remaining secondary antibody. In order to check affinity by color development, 100ul solution of TMB containing substrate as enzyme HRP (which is covalently linked to the secondary antibody) (BD OptEIA, BD) was added to each well and incubated at room temperature for 5 minutes. Finally, 50ul of 2.5M HSO solution was added to each well to terminate the enzymatic reaction. After the reaction was terminated, the absorbance was measured at 450nm (VERSAmax microplate reader, Molecular Devices). The results are shown in Table 9. The affinity of Ab417 to human L1CAM is 5×10-10M, the affinity of Ab612 is 2.6×10-10M, the affinity of Ab4H5 mutant is 6×10-11M, the affinity of Ab2C2 mutant is 5×10-11M, the affinity of Ab4H6 mutant is 6×10-11M, and the affinity of Ab5D12 mutant is 1.2×10-10M. The Ab417 variant antibodies exhibited approximately 2-10 fold higher binding affinity to human L1CAM than to the Ab417 antibody.

[0425] Table 9. Affinity (K) of Ab612, Ab4H5, Ab2C2, Ab4H6 and Ab5D12 antibodies for human L1CAM compared to Ab417

[0426]

[0427] Example 6. Antigen Binding Specificity Analysis of Ab417 Variants

[0428] In order to examine whether the variant antibodies selectively bind to human L1CAM and mouse L1CAM, flow cytometry was performed using various types of cells. In this regard, Ab417 antibody, which binds to human L1CAM and mouse L1CAM, was used as a comparative antibody.

[0429] CHO-DG44 (ATCC No. PTA-3356) cells known not to express human L1CAM were cultured and used as a negative control. Similarly, cells expressing human L1CAM, human ovarian epithelial carcinoma SKOV3 (ATCC No. HTB-77) and human non-small cell lung cancer NCI-H522 (AYCC No. CRL-5810) were cultured. As cells expressing mouse L1CAM, melanoma cell line B16F1 (ATCC No. CRL-6323) was cultured. Cultured cells of CHO-DG44 cells were harvested using dissociation buffer or 0.05% trypsin (GIBCO), resuspended in 1% PBA solution and placed on ice for 20 minutes. The cells were then plated at 4×10 5 The density of cells was added to a round-bottom polystyrene test tube (Falcon). The purified antibody was diluted in PBA solution at a concentration of 10ug / ml, and 100ul was added to each tube and mixed thoroughly. The tubes were placed at 4°C for 1 hour. The secondary antibody that specifically binds to the Fc region of human IgG and is covalently linked to FITC (Sigma) was added to each tube at a ratio of 1:2000. The plate was wrapped with foil to block light and allowed to react at 4°C for 1 hour. The staining reagent PI (propidium iodide, Sigma) was added at a ratio of 1:200 to assess cell viability. After all reactions were completed, FITC and PI fluorescence signals were detected in the cells. Figure 1A The variant antibody was shown not to bind to L1CAM negative cells (ie CHO-DG44). Figures 1B to 1D The variant antibodies are shown to bind to human L1CAM positive cells NCI-H522 ( Figure 1B )、SKOV3( Figure 1C ) and mouse L1CAM-positive cells B16F1 ( Figure 1D The variant antibodies specifically bound to the mouse melanoma cell line B166F1, indicating that the Ab417 variants of the present invention specifically bind to human L1CAM and mouse L1CAM.

[0430] Example 7. Properties of purified Ab417 variants measured by SEC-HPLC

[0431] HPLC grade UPLC solvents and PBS were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and GIBCO (St. Louis, MO, USA), respectively. Size exclusion chromatography was used to separate antibody samples using a Biosuit high resolution SEC column (7.5×300 mm, 250 grit). Samples were separated using an isocratic flow of PBS pH 7.4. The e2695 separation module was used for the experiment, and the 2489 UV / Vis detector monitored the absorbance at 280 nm. Size exclusion high performance liquid chromatography (SEC-HPLC) analysis showed that even though Ab417 had high molecular weight aggregates and low molecular weight fragments with broad peaks ( Figure 2A ), the variant antibody still showed a monomer peak ( FIG. 2B to FIG. 2F ).

[0432] Example 8. Affinity analysis of Ab417 variants for human L1CAM

[0433] Octet RED384 (ForteBio) was used to check the affinity of Ab417 and Ab417 variants to human L1CAM by BLI. For affinity analysis, the antibody was diluted with a PBA solution prepared by adding 0.1% BSA to PBS. Human L1CAM was serially diluted with PBA at a concentration of 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 or 0nM, and then 200ul of the diluted L1CAM was added to an opaque 96-well plate to prevent light transmission. The binding kinetics of antibodies and antigens were analyzed using an AHC (anti-human IgG Fc capture) sensor chip, which was carried out by checking the association of antibodies and antigens and the refractive index changes that occurred when the sensor chip was sequentially transferred to PBA solution, antibody, PBA solution, antigen and PBA solution.

[0434] Table 10 shows the affinity (K) of Ab417 for human L1CAM. D ) is about 0.2 nM, Ab612 has an affinity of 0.1 nM, Ab4H5 has an affinity of 8 pM, Ab2C2H has an affinity outside the range, Ab4H6 has an affinity of 0.07 nM, and Ab5D12 has an affinity of 0.09 nM. The increased affinity of the variants (e.g., up to 25-fold) compared to Ab417 is due to a slower off-rate.

[0435] Table 10. Affinity (K) of Ab612, Ab4H5, Ab2C2, Ab4H6 and Ab5D12 antibodies for human L1CAM compared to Ab417 D )

[0436]

[0437] Example 9. Isoelectric point (PI) analysis of Ab417 variants

[0438] Capillary isoelectric focusing (cIEF) of Ab417 and its mutants was performed using a Sciex PA800 plus instrument (Sciex PN 477441) with a neutral coating capillary. All experiments were performed in triplicate. The pI values ​​of the corresponding materials were determined using 32Karat software. Table 11 shows that the pI value of the Ab417 antibody is 9.62, the pI value of Ab612 is 9.25, the pI value of Ab4H5 is 8.96, the pI value of Ab2C2 is 8.96, the pI value of Ab4H6 is 9.03, and the pI value of Ab5D12 is 9.04.

[0439] Table 11. PI values ​​of Ab612, Ab4H5, Ab2C2, Ab4H6 and Ab5D12 compared to Ab417

[0440]

[0441] Example 10. Analysis of tumor growth inhibition effects of Ab417 variants

[0442] To study the anti-tumor effect of Ab612, male Balb / c nude mice were transplanted with human cholangiocarcinoma cell line Choi-CK. The Choi-CK tumor tissue (3×3×3mm 3 ) were inoculated into the back of mice. After the tumor volume reached 100 mm3 (n=8 per group), Ab417 antibody at a dose of 10 mg / kg, Ab612 antibody at a dose of 10 mg / kg, control hFc antibody at a dose of 3.3 mg / kg, and negative control (PBS) or (vehicle) were injected intravenously three times a week for 3 weeks. Tumor volume, mouse body weight, and tumor weight were measured and recorded on FIG. 3A to FIG. 3C Shown in.

[0443] Figure 3A and Figure 3C The Ab612 treatment group showed an average tumor volume of 311 mm 3 The tumor weight was 0.20 g, while the Ab417-treated group showed an average tumor volume of 387 mm 3 The tumor weight was 0.41 g. The mock-treated control mice showed an average tumor volume of 1096 mm 3 The results showed that based on tumor weight, Ab612 (78.2% tumor growth inhibition rate (IR)) showed 1.4 times higher tumor growth inhibition rate (IR) than Ab417 (55.5% IR). Figure 3C ). Figure 3D It shows that FIG. 3A to FIG. 3CImages of tumors from eight mice in each group that were sacrificed after the experiment described in Figure 1. The size of cancer tissue extracted from the eight mice treated with Ab612 antibody was smaller than that of cancer tissue treated with Ab417 antibody. The anti-cancer effect of Ab612 antibody seemed to work well in vivo, creating an improved tumor microenvironment (TME) through antibody infiltration of cancer tissue or immune cell-based attack environment.

[0444] Therefore, the group treated with the Ab612 antibody disclosed herein showed a significant inhibitory effect on tumor growth compared to the group administered with the negative control. In addition, no weight loss was observed in the mice during the administration period, and no toxicity caused by the administration of the antibody was observed.

[0445] The foregoing description of specific aspects will fully reveal the general nature of the invention so that others can easily modify and / or adapt such specific aspects for various applications without departing from the general concept of the invention by applying the knowledge in the art without undue experimentation. Therefore, based on the teachings and guidance presented herein, such adaptations and modifications are intended to be within the meaning and scope of the equivalent aspects of the disclosed aspects. It should be understood that the wording or terminology herein is for the purpose of description rather than limitation, so that the terms or wording of this specification will be interpreted by the skilled person based on the teachings and guidance.

[0446] Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein.It is intended that the specification and examples be considered exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0447] All publications, patents, and patent applications disclosed herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) complementarity determining region 1 (CDR1), CDR2 and CDR3 and a light chain variable region (VL) CDR1, CDR2 and CDR3; (i) wherein the VH CDR1, CDR2 and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; and wherein the VL CDR1, CDR2 and CDR3 respectively comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSIGRGPVT (SEQ ID NO: 13); (ii) wherein the VH CDR1, CDR2 and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; and wherein the VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQAGFYSPWT (SEQ ID NO: 17), respectively; (iii) wherein the VH CDR1, CDR2 and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; and wherein the VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQAGFYTPWT (SEQ ID NO: 15), respectively; (iv) wherein the VH CDR1, CDR2 and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; and wherein the VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSLHFYPWT (SEQ ID NO: 19), respectively; or (v) wherein the VH CDR1, CDR2 and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; and wherein the VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSLVWYPWT (SEQ ID NO: 21), respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence shown as EVQLVESGGGVVQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO:23), and wherein the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence shown as DIQLTQSPSSLSAVGDRVTITCRASRTISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIGRGPVTFGQGTKLEIK (SEQ ID NO:24). NO:24) has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical; wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence set forth as EVQLVESGGG VVQPGGSLRLSCAASGFTFS RFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYL QMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO:27), and wherein the VL ...90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence set forth as DIQLTQSPSS LSASVGDRVTITCRASR TISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQAGFYSPWTFGQGTKLEIK (SEQ ID NO:28) has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical; wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence set forth as EVQLVESGGG VVQPGGSLRLSCAASGFTFS RFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYL QMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO:25), and wherein the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence set forth as EVQLVESGGG VVQPGGSLRLSCAASGFTFS RFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYL QMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO:25), and NO:26) having an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical; wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence set forth as EVQLVESGGG VVQPGGSLRLSCAASGFTFS RFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYL QMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO:29), and wherein the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence set forth as EVQLVESGGG VVQPGGSLRLSCAASGFTFS RFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYL QMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO:29), and NO:30) has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity; or wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence set forth as EVQLVESGGGVVQPGGSLRLSCAASGFTFSR FGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQ MNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO:31), and wherein the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the amino acid sequence set forth as DIQLTQSPSS LSASVGDRVTITCRASRTISS YVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYC QQSLVWYPWTFGQGTKLEIK (SEQ ID NO:32). NO:32) has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical.

3. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2. A vector comprising the nucleic acid according to claim 3 . A host cell comprising the vector according to claim 4 .

6. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 linked to an agent. 7 . A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment of any one of claims 1 to 2 and an antibody or antigen-binding fragment thereof that binds to an antigen.

8. A composition comprising the antibody of any one of claims 1 to 2, the nucleic acid of claim 3, the vector of claim 4, the cell of claim 5, the immunoconjugate of claim 6 or the bispecific or multispecific antibody of claim 7 and a carrier.

9. A kit comprising the antibody of any one of claims 1 to 2, the nucleic acid of claim 3, the vector of claim 4, the cell of claim 5, the immunoconjugate of claim 6 or the bispecific or multispecific antibody of claim 7 and instructions for use.

10. Use of the antibody of any one of claims 1 to 2, the nucleic acid of claim 3, the vector of claim 4, the cell of claim 5, the immunoconjugate of claim 6, the bispecific or multispecific antibody of claim 7, or the composition of claim 8 for the preparation of a medicament for treating a disease or condition.

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