Anti-L1CAM antibodies and uses thereof

Abstract

To provide anti-L1CAM antibodies and uses thereof.SOLUTION: The present disclosure provides antibodies that specifically bind to L1CAM and compositions comprising such antibodies. Also provided herein are methods for preventing or treating diseases or conditions which include a tumor using the anti-L1CAM antibodies. An antibody that specifically binds to L1CAM protein may be used for diagnosis and prevention or treatment of diseases in which L1CAM is overexpressed (e.g., cancer). Accordingly, there is a need to develop antibodies that specifically bind to L1CAM and that are capable of modulating L1CAM activity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 865,871, filed June 24, 2019, and Korean Patent Application No. 10-2018-0075955, filed June 29, 2018. The international application PCT / IB2019 / 055472 filed on June 27, 2019, has entered the Japanese national phase. and each of which is incorporated herein by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing The contents of the sequence listing submitted electronically in an ASCII text file submitted with this application (name: 4372_001 JP 02_S eqlisting _ST2 5、 Size:82, 889 Byte and creation date: 20 22 June 2 1 (Dates) are incorporated herein by reference in their entirety.

[0003] 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 diseases or conditions, including tumors (e.g., cholangiocarcinoma, melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, renal cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, and / or ovarian cancer) in a subject. [Background technology]

[0004] L1 cell adhesion molecule (L1CAM) is a member of the immunoglobulin superfamily of cell adhesion molecules (CAMs) that mediates cell-cell adhesion on the cell surface. It has a molecular weight of 200-220 kDa. L1CAM was first identified as a protein mediating 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))

[0005] L1CAM is primarily expressed in the normal human brain. Furthermore, L1CAM expression is found in some hematopoietic and renal cells, peripheral nerves, intestinal crypt cells, and ganglia, but not in other normal cells. Huszar M. et al., Human Pathology 37:1000-1008 (2006). Antibodies that specifically bind to the L1CAM protein can be used for the diagnosis and prevention or treatment of diseases in which L1CAM is overexpressed (e.g., cancer). Thus, there is a need to develop antibodies that specifically bind to L1CAM and are capable of regulating L1CAM activity. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lee V. et al.,Proc.Natl.Acad.Sci.74:5021-5025(1997) [Non-patent document 2] McGuire JC.et al.,Cell.15(2):357-365(1978) [Non-patent document 3] Huszar M. et al., Human Pathology 37:1000-1008(2006) Summary of the Invention [Means for solving the problem]

[0007] One aspect of the present disclosure is directed 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.

[0008] Also provided herein is an anti-L1CAM antibody or antigen-binding fragment 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, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and / or the VL CDR3 of the antibody or antigen-binding fragment differs from the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and / or the VL CDR3 of the mAb417 antibody, 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), and the VH CDR3 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 QQSIGRGVVT (SEQ ID NO: 11).

[0009] 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 an 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; and 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. and 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 mAb417 antibody, 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), and the VH CDR3 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 VH CDR3 of the mAb417 antibody comprises QQSIGRGVVT (SEQ ID NO: 11).

[0010] In certain embodiments, the at least one amino acid difference comprises (i) a glutamine at residue 5 of VH CDR2, (ii) a serine at residue 8 of VL CDR1, and / or (iii) a proline at residue 8 of VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof. In certain embodiments, the at least one amino acid difference comprises (i) an alanine, glycine, phenylalanine, tyrosine, threonine, proline, and tryptophan at residues 3-9, respectively, in the VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof; (ii) an alanine, glycine, phenylalanine, tyrosine, serine, proline, and tryptophan at residues 3-9, respectively, in the VL CDR3; or (iii) a leucine, valine or histidine, tryptophan or phenylalanine, tyrosine, proline, and tryptophan at residues 4-9, respectively, in the VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof. In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein comprises 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 embodiments, the VH CDR1 of the anti-L1CAM antibody comprises RFGMH (SEQ ID NO: 2). In some embodiments, the VH CDR2 of the anti-L1CAM antibody comprises FISNEGSNKYYADSVKG (SEQ ID NO: 10). In some embodiments, the VH CDR3 of the anti-L1CAM antibody comprises GRAYGSGSLFDP (SEQ ID NO: 4). In some embodiments, the VL CDR1 of the anti-L1CAM antibody comprises RASRTISSYVN (SEQ ID NO: 12). In some embodiments, the VL CDR2 of the anti-L1CAM antibody comprises AASNLHS (SEQ ID NO: 7). In some embodiments, the VL CDR3 of the anti-L1CAM antibody comprises QQSIGRGPVT (SEQ ID NO: 13).

[0011] In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof of the present disclosure comprises 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).

[0012] The present disclosure provides a method for the production of mAb-1-containing mAb-1-based ...containing mAb-1-containing mAb-1-containing mAb-1 D ), (c) compared to the mAb417 antibody. Improved Further provided is 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 a PI value; (b) exhibiting improved affinity as measured by the association constant (K) compared to the mAb417 antibody; or (c) any combination thereof.

[0013] In some embodiments, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein exhibit improved productivity compared to the mAb417 antibody, the improved productivity being 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, or at least about 67 mg / L when expressed according to Example 3. 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.

[0014] In some embodiments, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein have a lower equilibrium dissociation constant (K D ) and showed improved affinity as measured by improved K D is 2.6 x 10 -10 Less than M, 2.5 x 10 -10 Less than M, 2.0 x 10 -10 Less than M, 1.5 x 10 -10 Less than M, 1.0 x 10 -10 Less than M, 9 x 10 -11 Less than M, 8 x 10 -11 Less than M, 7 x 10 -11 Less than M, 6 x 10 -11 Less than M, 5 x 10 -11 Less than M, 4 x 10 -11 Less than M, 3 x 10 -11 Less than M, 2 x 10 -11 Less than M, 1 x 10 -11 Less than M, 9 x 10 -12 Less than M, 8 x 10 -12Less than M, 7 x 10 -12 Less than M, 6 x 10 -12 Less than M, 5 x 10 -12 Less than M, 4 x 10 -12 Less than M, 3 x 10 -12 Less than M, 2 x 10 -12 Less than M, 1 x 10 -12 Less than M, 9 x 10 -13 Less than M or 8 x 10 -13 It is less than M.

[0015] In other aspects, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein exhibit improved affinity as measured by association constant (K) compared to the mAb417 antibody, wherein the improved K is greater than or equal to 5×10 -10 Less than M, 4 x 10 -10 Less than M, 3 x 10 -10 Less than M, 2 x 10 -10 Less than M, 1.0 x 10 -10 Less than M, 9 x 10 -11 Less than M, 8 x 10 -11 Less than M, 7 x 10 -11 Less than M, 6 x 10 -11 Less than M, 5 x 10 -11 Less than M, 4 x 10 -11 Less than M, 3 x 10 -11 Less than M, 2 x 10 -11 Less than M, 1 x 10 -11 Less than M, 9 x 10 -12 Less than M, 8 x 10 -12 Less than M, 7 x 10 -12 Less than M, 6 x 10 -12 Less than M, 5 x 10 -12 Less than M, 4 x 10 -12 Less than M, 3 x 10 -12 Less than M, 2 x 10 -12 Less than M, 1 x 10 -12 Less than M, 9 x 10 -13 Less than M or 8 x 10 -13 In some embodiments, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein have a mAb binding activity of less than 1 M, as compared to the mAb417 antibody. Improved Shows the PI value, ImprovedThe 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.

[0016] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, 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 the VL CDR1, CDR2, and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7), and QQSIGRGPVT (SEQ ID NO: 13), respectively.

[0017] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein comprises VH CDR1, CDR2, CDR3, and VL CDR1, CDR2, CDR3, 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 the VL CDR1, CDR2, and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7), and QQAGFYSPWT (SEQ ID NO: 17), respectively.

[0018] In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein comprises 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 VL CDR1, CDR2, and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7), and QQAGFYTPWT (SEQ ID NO: 1 5 ) is included.

[0019] In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein comprises VH CDR1, CDR2, CDR3, and VL CDR1, CDR2, and CDR3, 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 the VL CDR1, CDR2, and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7), and QQSLHFYPWT (SEQ ID NO: 19), respectively.

[0020] In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein comprises a VH CDR1, CDR2, CDR3, and a VL CDR1, CDR2, CDR3, 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 the VL CDR1, CDR2, and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7), and QQSLVWYPWT (SEQ ID NO: 16), respectively. 21 ) is included.

[0021] In some embodiments, the VH of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is VV The amino acid sequence of the present invention includes 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 set forth as QPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAF ISNEGSNKYY ADSVKGRFTI SRDNSANTL Y LQMNSLRAED TAVYYCARGR AYGSGSLFDP WGQGTLVTVS S (SEQ ID NO: 23).

[0022] In other aspects, the VL of an anti-L1CAM antibody or antigen-binding fragment 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 set forth as DIQL TQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SIGRGPVTFG QGTKLEIK (SEQ ID NO: 24).

[0023] In some embodiments, the VH of an anti-L1CAM antibody or antigen-binding fragment disclosed herein is VVThe amino acid sequence of the present invention includes 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 set forth as QPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAF ISNEGSNKYY ADSVKGRFTI SRDNSANTL Y LQMNSLRAED TAVYYCARGR AYGSGSLFDP WGQGTLVTVS S (SEQ ID NO: 27).

[0024] In other aspects, the VL of an anti-L1CAM antibody or antigen-binding fragment 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 set forth as DIQL TQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ AGFYSPWTFG QGTKLEIK (SEQ ID NO: 28).

[0025] In some embodiments, the VH of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is VV QPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAF ISNEGSNKYY ADSVKGRFTI SRDNSANTL Y LQMNSLRAED TAVYYCARGR AYGSGSLFDP WGQGTLVTVS S (SEQ ID NO: 25), and includes 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 set forth as

[0026] In some embodiments, the VL of an anti-L1CAM antibody or antigen-binding fragment 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 set forth as DIQL TQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ AGFYTPWTFG QGTKLEIK (SEQ ID NO: 26).

[0027] In other aspects, the VH of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is VV QPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAF ISNEGSNKYY ADSVKGRFTI SRDNSANTLY LQMNSLRAED TAVYYCARGR AYGSGSLFDP WGQGTLVTVS S (SEQ ID NO: 29), and includes 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 set forth as

[0028] In some embodiments, the VL of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is selected from the group consisting of DIQL TQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQSLHFYPWT FG It includes 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 set forth as QGTKLEIK (SEQ ID NO: 30).

[0029] In other embodiments, the VH of an anti-L1CAM antibody or antigen-binding fragment disclosed herein is VV QPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAF ISNEGSNKYY ADSVKGRFTI The amino acid sequence includes 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 set forth as SRDNSANTL Y LQMNSLRAED TAVYYCARGR AYGSGSLFDP WGQGTLVTVS S (SEQ ID NO: 31).

[0030] In some embodiments, the VL of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is selected from the group consisting of DIQL TQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQSLVWYPWT FG It includes 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 set forth as QGTKLEIK (SEQ ID NO: 32).

[0031] In some embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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.

[0032] 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 embodiments, 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 embodiments, 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 embodiments, 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 embodiments, 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.

[0033] In some embodiments, the anti-L1CAM antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, variants thereof, and any combination thereof. In some embodiments, the anti-L1CAM antibody is a chimeric antibody or a human antibody. In some embodiments, the anti-L1CAM antibody comprises a Fab, a Fab', a F(ab')2, an Fv, or a single-chain Fv (scFv).

[0034] Some aspects of the present disclosure are directed to nucleic acids encoding anti-L1CAM antibodies, vectors containing the nucleic acids, and host cells containing the vectors. In some aspects, the host cells are selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, and BMT10 cells in tissue culture, plant cells, insect cells, and human cells.

[0035] Some aspects of the present disclosure are directed to immunoconjugates comprising an anti-L1CAM antibody, or antigen-binding fragment thereof, disclosed herein, conjugated to an agent.

[0036] Also provided herein are bispecific or multispecific antibodies comprising an anti-L1CAM antibody or antigen-binding fragment and an antibody or antigen-binding fragment thereof that binds to an antigen.

[0037] Some aspects of the present disclosure are directed to compositions comprising an anti-L1CAM antibody, nucleic acid, vector, host cell, immunoconjugate, or bispecific or multispecific antibody disclosed herein and a carrier.

[0038] Also provided herein are kits containing the anti-L1CAM antibodies disclosed herein and instructions for use.

[0039] Some aspects of the present disclosure are directed to methods for producing antibodies that specifically bind to human L1CAM protein, comprising culturing host cells under suitable conditions and isolating the antibodies.

[0040] Also provided herein are methods for 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 embodiments, the disease or condition comprises a tumor. In some embodiments, the tumor comprises cholangiocarcinoma, melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, renal cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer. In other embodiments, the anti-L1CAM antibody, nucleic acid, vector, cell, immunoconjugate, bispecific or multispecific antibody inhibits tumor growth and / or enhances immune cell infiltration into the tumor.

[0041] Some aspects of the present disclosure are directed to methods comprising administering an additional therapeutic agent. In some aspects, the additional therapeutic agent comprises chemotherapy, immunotherapy, radiation therapy, or a combination thereof. In some aspects, the additional therapeutic agent is an immune checkpoint inhibitor.

[0042] 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, press reports, GenBank entries, patents, and patent applications cited throughout this application, are expressly incorporated herein by reference. [Brief explanation of the drawings]

[0043] [Figure 1A] Analysis of the antigen-binding specificity of Ab417 variants against human LCAM1 in CHO-DG44 cells (Figure 1A), NCI-H522 (Figure 1B), SKOV3 (Figure 1C), and B16F1 (Figure 1D) using flow cytometry is shown. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 2A] The quality of purified Ab417 (Figure 2A) and Ab417 variants: Ab612 (Figure 2B), Ab4H5 (Figure 2C), Ab2C2 (Figure 2D), Ab4H6 (Figure 2E), and Ab5D12 (Figure 2F) as determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 3A] Figure 3A shows the tumor growth inhibitory effects of Ab417 mutants. Figure 3A shows the 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, significant difference from the isotype control group by Dunnett's t-test). Figure 3B shows the changes in body weight in the Choi-CK xenograft model after administration of Ab417, Ab612, control hFc, and negative control (PBS). Figure 3C shows the tumor weight in 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, significant difference from isotype control group by Dunnett's t-test.) Figure 3D shows tumor images from each group of eight mice sacrificed at the end of the experiment, as described in Figure 3C. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0044] 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, exhibits one or more of the properties disclosed herein, and / or prevents and / or treats a disease or condition, including a tumor.

[0045] To facilitate understanding of the present disclosure set forth herein, certain terms and phrases are defined. Additional definitions are set forth throughout the detailed description.

[0046] I. Definition Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity, for example, "an antibody" is understood to refer to one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0047] Furthermore, as used herein, "and / or" should be considered a specific disclosure of each of two particular features or components, with or without the other features or components. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and; B and C; A (alone); B (alone); and C (alone).

[0048] As used herein, where an embodiment is described in terms of "comprising," it is understood that analogous embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0050] Units, prefixes, and symbols are represented in their Systeme International de Unites (SI) accepted format. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations on the various aspects of this disclosure, which may be had by reference to the specification in its entirety. Accordingly, the terms defined below are more fully defined by reference to the specification in its entirety.

[0051] The term "about" is used herein to mean approximately, roughly, roughly, or within the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify numerical values ​​above and below the stated value by a variance of, for example, 10 percent, upward or downward (higher or lower).

[0052] The term "L1 cell adhesion molecule" or "L1CAM" refers to one of the integral membrane glycoproteins belonging to the immunoglobulin superfamily of cell adhesion molecules (CAMs).

[0053] The term "L1CAM" includes any variant or isoform of L1CAM that is naturally expressed by a cell. Thus, the antibodies described herein may cross-react with different isoforms in the same species (e.g., different isoforms of human L1CAM) or with L1CAM from species other than human (e.g., mouse L1CAM). Alternatively, the antibody may be specific for human L1CAM and not exhibit any cross-reactivity with other species. L1CAM, or any variants and isoforms thereof, may be isolated from cells or tissues that naturally express them, or may be recombinantly produced using techniques well known in the art and / or described herein.

[0054] Human L1CAM (UniProt ID No. P32004-1, SEQ ID NO: 1) is a 1,257 amino acid type 1 integral membrane glycoprotein that spans the plasma membrane once, with its amino-terminal fragment present on the outside of the plasma membrane and its carboxyl-terminal fragment present in the cytoplasm. The extracellular domain of L1CAM contains six immunoglobulin type 2 domains (Ig1, Ig2, Ig3, Ig4, Ig5, and Ig6), five fibronectin III-like domains (Fn1, Fn2, Fn3, Fn4, and Fn5), and 20 N-glycosylation sites. U.S. Patent No. 9,777,060.

[0055] 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. Isoform 2 lacks amino acid residues 1177-1180 compared to the amino acid sequence of human L1CAM. Isoform 3 (UniProt ID No. P32004-3, SEQ ID NO: 5) consists of 1,248 amino acids. Isoform 3 lacks amino acid residues 1177-1180 and has the following difference compared to the amino acid sequence of human L1CAM: amino acid residues 26-31 (YEGHHV → L).

[0056] Below are the amino acid sequences of the three known human L1CAM isoforms. (A) Human L1CAM (UniProt ID number P32004-1, SEQ ID NO: 1) [ka] (B) Human L1CAM isoform 2 (UniProt ID number P32004-2, SEQ ID NO: 3) [ka] (C) Human L1CAM isoform 3 (UniProt ID number P32004-3, SEQ ID NO: 5) [ka]

[0057] The signal sequence of human L1CAM corresponds to amino acids 1 to 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.

[0058] The terms "antibody" and "antibodies" are terms of the art and may be used interchangeably herein to refer to a molecule having an antigen-binding site that specifically binds to an antigen. As used herein, the terms include whole antibodies and any antigen-binding fragment (i.e., "antigen-binding fragment") or single chains thereof. In one embodiment, "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. In another embodiment, "antibody" refers to a single-chain antibody comprising a single variable domain, e.g., a VHH domain. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region consists of three domains: CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL.

[0059] The VH and VL regions can be further subdivided into regions of hypervariability 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, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins 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.

[0060] The term "Kabat numbering" and similar terms are art-recognized and refer to a system for numbering amino acid residues within the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In certain embodiments, the CDRs of an antibody can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat (See EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically located at amino acid positions 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3), which may optionally include one or two additional amino acids following 35 (designated 35A and 35B in the Kabat numbering scheme). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). In certain aspects, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0061] The phrases "Kabat amino acid position numbering," "Kabat position," and grammatical variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the antibody compilation in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. 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 a shortening of, or insertion into, the FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of CDR2 (residue 52a according to Kabat) and inserted residues after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). See Table 1. [Table 1]

[0062] The Kabat numbering of residues can be determined for a given antibody by aligning the antibody sequence with the "standard" Kabat numbering sequence in the homologous region. Instead, Chothia refers to the location of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDRs and the Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software.

[0063] IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, e.g., Lefranc, MP et al., Dev. Comp. Immunol. 27:55-77 (2003), incorporated herein by reference. The IMGT numbering system is based on the alignment of over 5,000 sequences, structural data, and characterization of hypervariable loops, allowing for easy comparison of variable and CDR regions across all species. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26-35, VH-CDR2 is located at positions 51-57, VH-CDR3 is located at positions 93-102, VL-CDR1 is located at positions 27-32, VL-CDR2 is located at positions 50-52, and VL-CDR3 is located at positions 89-97.

[0064] For all heavy chain constant region amino acid positions discussed in this disclosure, numbering follows 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, the first human IgG1 to be sequenced. The EU index of Edelman et al. is also incorporated herein by reference in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. Thus, the phrases "EU index as described in Kabat" or "EU index of Kabat" and "position... according to the EU index as described in Kabat," and grammatical variations thereof, refer to the residue numbering system based on the human IgG1 EU antibody of Edelman et al., as described in Kabat 1991.

[0065] The numbering system used for the variable domains (both heavy and light chain) and light chain constant region amino acid sequences is that described in Kabat 1991.

[0066] An antibody can be an immunoglobulin molecule of any type (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 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice). Immunoglobulins, such as IgG1, exist in several allotypes, which differ from each other in at most a few amino acids. The antibodies disclosed herein can be derived from any of the commonly known isotypes, classes, subclasses, or allotypes. In certain aspects, the antibodies described herein are of the IgG1, IgG2, IgG3, or IgG4 subclass, or any hybrid thereof. In certain aspects, the antibodies are of the human IgG1 subclass, or of the human IgG2 or human IgG4 subclass.

[0067] "Antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, fully synthetic antibodies, single-chain antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), tetrameric antibodies comprising two heavy chain 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, affinity antibodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and single domain antibodies (sdAbs), which include binding molecules consisting of a single monomeric variable antibody domain entirely capable of antigen binding (e.g., a VH domain or a VL domain). Harmen M M and Haard H J Appl Microbiol Biotechnol. 77(1):13-22 (2007)).

[0068] The term "antigen-binding fragment" of an antibody, as used herein, 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, about 8 to about 1500 amino acids in length, preferably about 8 to about 745 amino acids in length, and preferably about 8 to about 300 amino acids in length, e.g., about 8 to about 200 amino acids in length, 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 fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody, for example, an 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 in the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a 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 the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR), or (vii) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker, which allows them to be produced as a single protein chain using recombinant methods, in which the VL and VH regions pair to form a monovalent molecule (also known as a single-chain Fv (scFv)); see, e.g., Bird et al., (1988) Science 242:423-426, and Huston (See, e.g., W. 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 of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0069] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a fragment of an antibody, generally a light or heavy chain fragment, typically the amino-terminal 110-120 amino acids in the mature heavy chain and approximately 90-115 amino acids in the mature light chain, which vary significantly in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions within variable domains are called framework regions (FRs).

[0070] Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0071] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively (including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4), based on the amino acid sequence of the constant domain.

[0072] As used herein, the term "light chain," when used in reference to an antibody, can refer to any distinct type based on the amino acid sequence of the constant domain, e.g., kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In certain aspects, the light chain is a human light chain.

[0073] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0074] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0075] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is an antibody fragment, e.g., the carboxyl-terminal fragment of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen but can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0076] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates immunoglobulin binding to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or the first component (C1q) of the classical complement system. Thus, the Fc region includes the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region contains two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains, while IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) on each polypeptide chain. In IgG, the Fc region includes immunoglobulin domains Cγ2 and Cγ3, as well as the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is usually defined as extending from the amino acid residue at position C226 or P230 (or the amino acids between these two amino acids) to the carboxy terminus of the heavy chain, where numbering is according to the EU index as in Kabat. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, while the CH3 domain is located C-terminal to the CH2 domain within the Fc region, i.e., from about amino acid 341 to about amino acid 447 of IgG. As used herein, an Fc region can be a native-sequence Fc, including any allotypic variants, or a variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to this region alone or in the context of an Fc-containing protein polypeptide, such as an "Fc region-containing binding protein," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0077] 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 the various allotypes of Fc (see, e.g., Jefferis et al., (2009) mAbs 1:1; Vidarsson G. et al., Front Immunol. 5:520 (Published online October 20, 2014)).

[0078] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include the FcγR family of receptors, 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 induces the strongest Fc effector functions. It is considered equivalent to mouse IgG2a in terms of the type of activating Fc receptor it binds. Conversely, human IgG4 induces the least Fc effector functions. Vidarsson G. et al. Front Immunol. 5:520 (Published online October 20, 2014).

[0079] The constant region can be engineered, for example, by recombinant techniques, 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 receptors; BCRs). Such effector functions generally require the combination of the Fc region with a binding domain (e.g., an antibody variable domain). Thus, the term "constant region without Fc functions" includes constant regions in which one or more effector functions mediated by the Fc region are reduced or have no effector function.

[0080] The effector function of antibodies can be reduced or avoided by different approaches. Antibody effector function can be reduced or avoided by using antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, single-chain Fv (scFv), or sdAbs consisting of monomeric VH or VL domains). Alternatively, so-called aglycosylated antibodies can be generated by removing sugars attached to specific residues in the Fc region to reduce antibody effector function while retaining other valuable attributes of the Fc region (e.g., long half-life and heterodimerization). Aglycosylated antibodies can be generated, for example, by deleting or modifying the residue to which the sugar is attached, enzymatically removing the sugar, producing the antibody in cells cultured in the presence of a glycosylation inhibitor, or expressing the antibody in cells (e.g., bacterial host cells) that cannot glycosylate proteins. See, e.g., U.S. Publication No. 20120100140. Another approach is to employ Fc regions from IgG subclasses with reduced effector function. For example, IgG2 and IgG4 antibodies are characterized by lower levels of Fc effector function than IgG1 and IgG3. Residues closest to the hinge region within the CH2 domain of the Fc portion are responsible for antibody effector function because they contain largely overlapping binding sites for C1q (complement) and IgG-Fc receptors (FcγRs) on effector cells of the innate immune system. Vidarsson G. et al. Front Immunol. 5:520 (Published online October 20, 2014).Thus, antibodies with reduced or no Fc effector function can be prepared, for example, by generating a chimeric Fc region comprising a CH2 domain from an IgG antibody of the IgG4 isotype and a CH3 domain from an IgG antibody of the IgG1 isotype, or a chimeric Fc region comprising a hinge region from an IgG2 and a CH2 region from an IgG4 (see, e.g., Lau C. et al. J. Immunol. 191:4769-4777 (2013)), or an Fc region with mutations that result in altered Fc effector function (e.g., reduced or no Fc function). Such Fc regions with mutations are known in the art. See, e.g., U.S. Publication No. 20120100140 and the U.S. 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.

[0081] The terms "hinge," "hinge domain," "hinge region," or "antibody hinge region" are used interchangeably and refer to the domain of the heavy chain constant region that joins 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 varying levels of flexibility between the binding and effector regions of an antibody and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions. As used herein, the hinge begins at Glu216 and ends at Gly237 for all IgG isotypes (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, for example, Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014).

[0082] 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 begins at A118 and ends at V215. The term "CH1 domain" includes wild-type CH1 domains as well as naturally occurring variants (e.g., allotypes) thereof. CH1 domain sequences for IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, for example, Kabat EA et al., (1991) supra, and Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014). Exemplary CH1 domains include CH1 domains with mutations that modify the biological activity (e.g., half-life) of the antibody, and are described, for example, in U.S. Publication No. 20120100140 and the U.S. patents and publications and PCT publications cited therein.

[0083] 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 begins at P238 and ends at K340. The term "CH2 domain" includes wild-type CH2 domains and naturally occurring variants (e.g., allotypes) thereof. CH2 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, for example, Kabat EA et al., (1991) supra, and Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014). Exemplary CH2 domains include CH2 domains with mutations that modify the biological activity (e.g., half-life) of the antibody and / or have reduced Fc effector function, and are described, for example, in U.S. Publication No. 20120100140, and the U.S. patents and publications and PCT publications cited therein.

[0084] The term "CH3 domain" refers to the heavy chain constant region C-terminal to the CH2 domain within the heavy chain constant domain. As used herein, the CH3 domain begins at G341 and ends at K447. The term "CH3 domain" includes wild-type CH3 domains as well as naturally occurring variants (e.g., allotypes) thereof. CH3 domain sequences for IgG1, IgG2, IgG3, and IgG4 (including wild-type and allotypes) are known in the art. See, for example, Kabat EA et al., (1991) supra, and Vidarsson G. et al. Front Immunol. 5:520 (published online October 20, 2014). Exemplary CH3 domains include CH3 domains with mutations that modify the biological activity (e.g., half-life) of the antibody, and are described, for example, in U.S. Publication No. 20120100140 and the U.S. patents and publications and PCT publications cited therein.

[0085] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by heavy chain constant region genes.

[0086] "Allotype" refers to naturally occurring variants within a particular isotype group, which variants differ in 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 October 20, 2014); and Lefranc MP, mAbs 1:4, 1-7 (2009).

[0087] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0088] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigen 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 an epitope of L1CAM may have cross-reactivity with other L1CAM proteins from different species.

[0089] "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 molecule X for partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A K D is k off / k on It is calculated from the quotient of K and expressed as molar concentration (M). A is k on / k off It is calculated from the quotient of k on refers to the association rate constant of, for example, an antibody to an antigen, and k off refers to, for example, the dissociation of an antibody against an antigen. on and k off can be determined by techniques known to those skilled in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assays (ELISAs)), BIACORE®, BLI (biolayer interferometry), or kinetic exclusion assays (KINEXA®).

[0090] As used herein, the terms "specifically bind," "specifically recognize," "specific binding," "selective binding," and "selectively bind" are similar terms in the context of antibodies and refer to a molecule (e.g., an antibody) that binds to an antigen (e.g., an epitope or immune complex) as such binding is understood by those of skill in the art. For example, a molecule that specifically binds to an antigen is generally capable of binding other peptides or polypeptides with lower affinity, as determined, for example, by immunoassay, BIACORE®, KINEXA® 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain aspects, a molecule that specifically binds to an antigen has a K A At least 2 logs, 2.5 logs, 3 logs, 4 logs, or more than A binds to the antigen.

[0091] Antibodies are typically -5 ~10 -11 The dissociation constant (K D ) specifically binds to its cognate antigen with high affinity, reflected by a .about.10 -4 Any K greater than M D As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds with high affinity to the antigen and a substantially identical antigen, as determined, for example, by immunoassay (e.g., ELISA) surface plasmon resonance (SPR) technology on a BIACORE™ 2000 instrument using a given antigen or BLI (biolayer interferometry). -7 M or less, preferably 10 -8 M or less, and even more preferably 10 -9 M or less, most preferably 10 -8 M~10 -10 K below M D This means that the antibody has a high affinity for the antigen but does not bind with high affinity to unrelated antigens.

[0092] 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.

[0093] As used herein, "epitope" is a term used in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, composed of two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or discontinuous epitope). Epitopes formed from consecutive amino acids are typically, but not necessarily, retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically 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 epitope is bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblot and immunoprecipitation assays, in which overlapping or contiguous peptides (e.g., derived from L1CAM) are tested for reactivity with a given antibody (e.g., an anti-L1CAM antibody). Methods for determining the spatial conformation of an epitope include techniques in the art and 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)).

[0094] In certain embodiments, the epitope to which an antibody binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al., US2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne (See Carter CW, Roversi P et al., (1997) Meth Enzymol 276A:361-423, ed. Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, for example, Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085.

[0095] The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition.

[0096] The term "binds to the same epitope" as a reference antibody means that the antibody binds to the same segment of amino acid residues as determined by a given method. Techniques for determining whether an antibody binds to the "same epitope on L1CAM" with the antibodies described herein include epitope mapping methods, such as X-ray analysis of crystals of antigen:antibody complexes, which provide atomic resolution and hydrogen / deuterium exchange mass spectrometry (HDX-MS) of the epitope. Other methods monitor antibody binding to antigen fragments or mutated variants of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is often considered an indication of epitope content. 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 combinatorial phage display peptide libraries. Antibodies with the same VH and VL or the same CDR1, 2, and 3 sequences are expected to bind to the same epitope.

[0097] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to the target, can be determined using known competition experiments, e.g., BIACORE® surface plasmon resonance (SPR) analysis. In some embodiments, an antibody competes with another antibody and inhibits the binding of the other antibody to the target by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). Competition assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277 or Chapter 11 of "Using Antibodies″by Ed Harlow and David Lane,Cold Spring Harbor Laboratory Press,Cold This can be performed as described in Spring Harbor, NY, USA 1999. Two antibodies "cross-compete" if they block each other by at least 50% in both directions, i.e., regardless of whether one or the other antibody comes into contact with the antigen first in the competition experiment.

[0098] Competitive binding assays to determine whether two antibodies compete or cross-compete for binding include, for example, competition for binding to cells expressing L1CAM by flow cytometry as described in the Examples. Other methods include SPR (e.g., BIACORE®), BLI (BioLayer Interferometry), solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahl 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 label assay, solid-phase direct label sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)), solid-phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)), solid-phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546 (1990)) and direct label RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0099] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to antibody binding to an epitope on a predetermined antigen. Typically, an antibody (i) binds to an epitope of approximately 10, as determined, for example, by surface plasmon resonance (SPR) technology on a BIACORE® 2000 instrument using a predetermined antigen, e.g., recombinant human MICA or MICB, as the analyte and the antibody as the ligand, or by Scatchard analysis of antibody binding to antigen-positive cells. -7 Less than M, e.g., about 10 -8 Under M, 10 -9 Less than M or 10 -10 M or even lower equilibrium dissociation constant (KD ) and (ii) bind to a predetermined antigen with an affinity at least two-fold higher than the affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). Thus, an antibody that "specifically binds to human L1CAM" binds to soluble or cell-bound human L1CAM with an affinity of at least 10 -7 M or less, e.g., about 10 -8 Under M, 10 -9 Less than M or 10 -10 K less than or equal to M D An antibody that "cross-reacts with cynomolgus monkey L1CAM" refers to an antibody that binds to cynomolgus monkey L1CAM at a concentration of 10 -7 M or less, e.g., about 10 -8 Under M, 10 -9 Less than M or 10 -10 K less than or equal to M D In some embodiments, such antibodies that do not cross-react with L1CAM from non-human species exhibit essentially undetectable binding to these proteins in standard binding assays.

[0100] "k assoc " or "k a The term "k" as used herein is intended to refer to the association rate of a particular antibody-antigen interaction, while "k dis " or "k d The term "K", as used herein, is intended to refer to the off-rate of a particular antibody-antigen interaction. D " as used herein means k d vs. k a The ratio of (i.e., k d / k a ) and expressed as a molar concentration (M). D The K value can be determined using methods well established in the art. DAvailable methods for determining include surface plasmon resonance, biosensor systems such as BIACORE®, BLI (biolayer interferometry) systems, or flow cytometry, and Scatchard analysis.

[0101] As used herein, the term "high affinity" for an IgG antibody refers to an antibody that has an affinity of 10 or more for a target antigen. -8 M or less, 10 -9 M or less, or 10 -10 K below M D However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is defined as 10 -10 M or less, or 10 -8 K below M D It refers to an antibody having the following structure:

[0102] "EC" in the context of an in vitro or in vivo assay using an antibody or antigen-binding fragment thereof 50 The term "antibody or antigen-binding fragment" refers to the concentration of an antibody or antigen-binding fragment that induces 50% of the maximal response, i.e., a response that is halfway between the maximal response and the baseline.

[0103] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy / 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 & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0104] The term "monoclonal antibody," as used herein, refers to an antibody displaying a single binding specificity and affinity for a particular epitope, or a composition of antibodies in which all of the antibodies display a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody or antibody composition displaying a single binding specificity and having variable and optional constant regions derived from human germline immunoglobulin sequences. In one aspect, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell, and / or by recombinant combinatorial human antibody libraries.

[0105] The term "recombinant human antibody," as used herein, includes all human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes, or hybridomas prepared therefrom, (b) antibodies isolated from host cells, e.g., transfectomas, transformed to express the antibody, (c) antibodies isolated from a recombinant combinatorial human antibody library, and (d) antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain 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), variable regions contain antigen-binding domains, which are encoded by various genes that rearrange to form antibodies specific to foreign antigens. In addition to rearrangement, variable regions can be further modified by multiple single amino acid changes (referred to as somatic mutations or hypermutations) to increase the affinity of the antibody for the foreign antigen. The constant regions will also change in response to antigen (i.e., isotype switching). Thus, rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to antigen may not have sequence identity to the original nucleic acid molecule, but instead are substantially identical or similar (i.e., have at least 80% identity).

[0106] A "human antibody" (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is 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 mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0107] A "chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, for example, an antibody whose variable region is derived from a murine antibody and whose constant region is derived from a human antibody.

[0108] The term "cross-reactivity," as used herein, 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 can also bind to another species of L1CAM (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 by binding to or otherwise functionally interacting with cells that physiologically express L1CAM. Methods for determining cross-reactivity include standard binding assays described herein, for example, by BIACORE® surface plasmon resonance (SPR) analysis using a BIACORE® 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or flow cytometry techniques.

[0109] The term "naturally occurring" as applied to an object herein refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and that has not been intentionally modified by humans in a laboratory is naturally occurring.

[0110] A "polypeptide" refers to a chain containing at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in a protein can contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" can include one or more polypeptides.

[0111] The term "nucleic acid molecule," as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.

[0112] A "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having 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), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted non-essential amino acid residue in an anti-L1CAM antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions 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)).

[0113] 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 must be introduced to optimally align the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0114] 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 gap weights of 40, 50, 60, 70, or 80, and length weights 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)) 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. Additionally, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0115] The nucleic acid and protein sequences described herein can further be used as "query sequences" to search public databases, for example, to 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 using 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 using 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 utilized 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.

[0116] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially purified" 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 alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0117] Nucleic acids, e.g., cDNA, can be mutated according to standard techniques to provide gene sequences. For coding sequences, these mutations can affect the desired amino acid sequence. In particular, DNA sequences that are substantially homologous to or derived from the naturally occurring V, D, J, constant, switch, and other such sequences described herein are contemplated ("derived" indicates that the sequence is identical or modified from another sequence).

[0118] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions are also included.

[0119] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell that contains a nucleic acid that does not naturally occur within 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 subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0120] 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 linkage may be achieved using a wide range of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0121] "Immune response," as understood in the art, generally refers to a biological response within a vertebrate to foreign substances or abnormalities, e.g., cancer cells, which protects the organism from these foreign substances and the diseases they cause. The 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 produced by these cells or the liver (including antibodies, cytokines, and complement), which result in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues from the vertebrate body. Immune responses include, for example, T cells, e.g., effector T cells, Th cells, CD4 + cells, CD8 +This includes the activation or inhibition of T cells, or Treg cells, or any other cells of the immune system, such as NK cells.

[0122] "Immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or experiencing a recurrence of a disease by methods involving inducing, enhancing, suppressing, or otherwise modifying the immune system or immune response.

[0123] As used herein, "administering" refers to physically introducing a therapeutic agent or a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration of the antibodies described herein include, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration by injection or infusion. The phrase "parenteral administration," as used herein, generally refers to modes of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered via a non-parental route, e.g., a topical, epidermal, or mucosal administration route, e.g., an intranasal, oral, intravaginal, rectal, sublingual, or topical route. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0124] As used herein, the phrase "inhibiting tumor growth" includes any measurable decrease in tumor growth, e.g., at least about 10%, e.g., 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% inhibition of tumor growth. In some embodiments, tumor growth inhibition is measured as percent tumor growth inhibition (TGI%). TGI% can be determined by calculating the TGI at dat "t", where TGI is calculated using the formula: [1-((T t / T0) / (C t / C0))] / [(C t -C0) / C t ]*100 [Equation 1] calculated from all treated animals, where T t = individual tumor size of treated animals at time "t", TO = individual tumor size of treated animals at first measurement, C t = median tumor size in control animals at time "t", C0 = median tumor size in control animals at the time of the first measurement.

[0125] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled 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 via the lymphatic system or bloodstream.

[0126] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on a subject or administering an active agent with the intent to reverse, alleviate, ameliorate, inhibit, or slow or prevent the progression, onset, severity, or recurrence of symptoms, complications, pathology, or biochemical manifestations associated with a disease, or to enhance overall survival. Treatment can be of subjects with disease or subjects not having the disease (e.g., for prophylaxis).

[0127] 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 dosage" of a drug or therapeutic agent, when used alone or in combination with another therapeutic agent, is any amount of drug that promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, an increase in overall survival (the period from either the date of diagnosis of a disease, such as cancer, or the date treatment begins, during which a patient diagnosed with the disease is still alive), or the prevention of functional impairment or disability due to disease affliction. A therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount" or "prophylactically effective dosage," which is any amount of drug that inhibits the onset or recurrence of disease when administered alone or in combination with another therapeutic agent to a subject at risk of developing or experiencing disease recurrence. The ability of a therapeutic agent to promote disease regression or inhibit the onset or recurrence of disease can be assessed using a variety of methods known to those skilled in the art, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0128] For example, an anti-cancer drug is a drug that promotes cancer regression in a subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administering an effective amount of a drug, alone or in combination with an anti-tumor drug, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free symptom intervals, an increase in overall survival, prevention of functional impairment or disability due to disease affliction, or otherwise improvement of disease symptoms in a patient. Additionally, the terms "effective" and "efficacy" with respect to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level resulting from the administration of a drug.

[0129] For example, for the treatment of tumors, a therapeutically effective amount or dosage 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% compared to untreated subjects. In some embodiments, a therapeutically effective amount or dosage of a drug completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be assessed using the assays described below. Alternatively, this property of a composition can be assessed by examining the ability of a compound to inhibit cell growth, which can be measured in vitro by assays known to those skilled in the art. In some embodiments described herein, tumor regression can be observed and can last for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

[0130] 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.

[0131] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM," respectively.

[0132] The various aspects described herein are described in further detail in the following subsections.

[0133] II. Anti-L1CAM antibody Disclosed herein are antibodies, e.g., monoclonal antibodies, characterized by particular functional features or properties. For example, the antibodies specifically bind to mammalian (e.g., human and mouse) L1CAM and exhibit one or more of the following functional properties: (a) Showing improved productivity compared to mAb417 antibody, (b) The equilibrium dissociation constant (KD ) exhibiting improved affinity, as measured by (c) Compared to mAb417 antibody Improved Showing the PI value, (d) exhibiting improved affinity, as measured by the association constant (K), compared to the mAb417 antibody; (e) preventing and / or treating a disease or condition, including a tumor; or (f) Any combination of these.

[0134] In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof exhibits improved productivity compared to the mAb417 antibody, for example, 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, at least about 89 mg / L, at least about 90 mg / L, at least about 91 mg / L, at least about 92 mg / L, at least about 93 mg / L, at least about 94 mg / L, at least about 95 mg / L, at least about 96 mg / L, at least about 97 mg / L, at least about 98 mg / L, at least about 99 mg / L, at least about 100 mg / L, at least about 101 mg / L, at least about 1 7 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.

[0135] In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof has high affinity, e.g., a cytotoxicity of 2.6×10, e.g., as measured by biolayer interferometry (BLI) (e.g., as described in the Examples). -10 Less than M, 2.5 x 10 -10 Less than M, 2.0 x 10 -10 Less than M, 1.5 x 10 -10 Less than M, 1.0 x 10 -10Less than M, 9 x 10 -11 Less than M, 8 x 10 -11 Less than M, 7 x 10 -11 Less than M, 6 x 10 -11 Less than M, 5 x 10 -11 Less than M, 4 x 10 -11 Less than M, 3 x 10 -11 Less than M, 2 x 10 -11 Less than M, 1 x 10 -11 Less than M, 9 x 10 -12 Less than M, 8 x 10 -12 Less than M, 7 x 10 -12 Less than M, 6 x 10 -12 Less than M, 5 x 10 -12 Less than M, 4 x 10 -12 Less than M, 3 x 10 -12 Less than M, 2 x 10 -12 Less than M, 1 x 10 -12 Less than M, 9 x 10 -13 Less than M or 8 x 10 -13 K less than M D In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to human L1CAM at 2×10 -10 Less than M, 1.9 x 10 -10 Less than M, 1.8 x 10 -10 Less than M, 1.7 x 10 -10 Less than M, 1.6 x 10 -10 Less than M, 1.5 x 10 -10 Less than M, 1.4 x 10 -10 Less than M, 1.3 x 10 -10 Less than M, 1.2 x 10 -10 Less than M, or 1.1 x 10 -10 K less than M D In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to human L1CAM at a concentration of 1.1 x 10 -10 K less than M D In other embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to human L1CAM at 9×10 -12 K less than M D In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to human L1CAM at 1×10 -12 K less than M DIn some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to human L1CAM at 8×10 -11 K less than M D In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to human L1CAM at 1×10 -12 K less than M D In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to human L1CAM at a concentration of 1.05 x 10 -10 K less than M D In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to human L1CAM at a concentration of about 8.22 x 10 -12 K of M D In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to human L1CAM at a concentration of about 7.4 x 10 -11 K of M D In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds to human L1CAM at a concentration of about 9.6 x 10 -11 K of M D It specifically binds to human L1CAM.

[0136] In some embodiments, the anti-L1CAM antibody or antigen-binding fragment thereof has high affinity, e.g., as measured by ELISA (e.g., as described in the Examples), e.g., 5×10 -10 Less than M, 4 x 10 -10 Less than M, 3 x 10 -10 Less than M, 2 x 10 -10 Less than M, 1.0 x 10 -10 Less than M, 9 x 10 -11 Less than M, 8 x 10 -11 Less than M, 7 x 10 -11 Less than M, 6 x 10 -11 Less than M, 5 x 10 -11 Less than M, 4 x 10 -11 Less than M, 3 x 10 -11 Less than M, 2 x 10 -11 Less than M, 1 x 10 -11 Less than M, 9 x 10 -12 Less than M, 8 x 10-12 Less than M, 7 x 10 -12 Less than M, 6 x 10 -12 Less than M, 5 x 10 -12 Less than M, 4 x 10 -12 Less than M, 3 x 10 -12 Less than M, 2 x 10 -12 Less than M, 1 x 10 -12 Less than M, 9 x 10 -13 Less than M or 8 x 10 -13 It specifically binds to human L1CAM with a K less than M

[0137] Standard assays for assessing the binding ability of antibodies to various species of L1CAM 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 antibodies can also be assessed by standard assays known in the art, such as ELISA, BIACORE® analysis, or KINEXA®. Assays for assessing the effect of antibodies on the functional properties (e.g., ligand binding) of L1CAM are described in further detail below and in the Examples.

[0138] In some embodiments, 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).

[0139] 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.

[0140] 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, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and / or a VL CDR3 of the antibody or antigen-binding fragment is different from the VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and / or a VL CDR3 of the reference antibody (e.g., mAb417 antibody), and the VH CDR1, VH CDR2, and VH CDR3 of the reference antibody are set forth in SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. 4 and the VL CDR1, VL CDR2, and VL CDR3 of the reference antibody comprise the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 11, respectively. In certain aspects, the at least one amino acid difference is (i) a glutamine at residue 5 of VH CDR2; (ii) a VL CDR3 at residue 1 of VL CDR3; and / or (iii) a proline at residue 8 of the VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof. In certain embodiments, the at least one amino acid difference comprises (i) alanine, glycine, phenylalanine, tyrosine, threonine, proline, and tryptophan at residues 3-9, respectively, in the VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof; (ii) alanine, glycine, phenylalanine, tyrosine, serine, proline, and tryptophan at residues 3-9, respectively, in the VL CDR3; or (iii) leucine, valine or histidine, tryptophan or phenylalanine, tyrosine, proline, and tryptophan at residues 4-9, respectively, in the VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof.

[0141] In certain aspects, the anti-L1CAM antibody or antigen-binding fragment thereof cross-competes for binding to an 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, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and / or a VL CDR3 of the antibody or antigen-binding fragment differs from the VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and / or a VL CDR3 of the reference antibody (e.g., mAb417 antibody), and wherein (i) the VH CDR1, the VH CDR2, and the VH CDR3 of the reference antibody are set forth in SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. 4and the VL CDR1, VL CDR2, and VL CDR3 of the reference antibody comprise the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 11, respectively. In certain aspects, the at least one amino acid difference comprises (i) a glutamine at residue 5 of VH CDR2, (ii) a serine at residue 8 of VL CDR1, and / or (iii) a proline at residue 8 of 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 3-9, respectively, in the VL CDR3 of an anti-L1CAM antibody or antigen-binding fragment thereof; (ii) alanine, glycine, phenylalanine, tyrosine, serine, proline, and tryptophan at residues 3-9, respectively, in the VL CDR3; or (iii) leucine, valine or histidine, tryptophan or phenylalanine, tyrosine, proline, and tryptophan at residues 4-9, respectively, in the VL CDR3.

[0142] In certain aspects, the anti-L1CAM antibody or antigen-binding fragment thereof cross-competes for binding to an L1CAM epitope with 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.

[0143] Competing antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as evidenced by steric hindrance). Whether two antibodies compete with each other for binding to a target can be determined using competition experiments known in the art, such as RIA and EIA.

[0144] Techniques for determining whether two antibodies bind to the same epitope include, for example, X-ray analysis of crystals of antigen:antibody complexes that provide atomic resolution and hydrogen / deuterium exchange mass spectrometry (HDX-MS) of the epitope, methods that monitor antibody binding to antigen fragments or mutated variants of the antigen (loss of binding due to modification of amino acid residues within the antigen sequence is often taken as an indication of epitope composition), and computational combinatorial methods for epitope mapping.

[0145] In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that bind 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 higher affinity than another protein in the L1CAM family, as measured, e.g., by immunoassay (e.g., ELISA), surface plasmon resonance, BLI (biolayer interferometry), or kinetic exclusion assay. In certain embodiments, provided herein are antibodies or antigen-binding fragments thereof that bind to L1CAM (e.g., human L1CAM) without cross-reactivity with another protein in the L1CAM family, as measured, e.g., by immunoassay.

[0146] In certain embodiments, the anti-L1CAM antibody is not a native or naturally occurring antibody, e.g., the anti-L1CAM antibody has post-translational modifications that differ from those of naturally occurring antibodies, e.g., by having more, fewer, or different types of post-translational modifications.

[0147] III. Exemplary Anti-L1CAM Antibodies Particular antibodies that can be used in the methods disclosed herein are antibodies (e.g., monoclonal antibodies) having the CDR and / or variable region sequences of antibodies Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 constructed in Examples 1 and 2, as well as 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 those variable region or CDR sequences. The VH amino acid sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 are set forth in SEQ ID NOs: 23, 25, 27, 29, and 31, respectively. The VL amino acid sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 are set forth in SEQ ID NOs: 24, 26, 28, 30, and 32, respectively. [Table 2] [Table 3] [Table 4A] [Table 4B-1] [Table 4B-2]

[0148] Thus, 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 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 the CDRs of the heavy chain variable region selected from the group consisting of SEQ ID NO: 2, 10, or 4.

[0149] 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 the CDRs of the light chain variable region selected from the group consisting of SEQ ID NO: 12, 7, 13, 15, 17, 19, or 21.

[0150] 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.

[0151] 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 (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 23 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24; (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 25 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26; (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 28; (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 29 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30; or (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32.

[0152] 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 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 set forth as SEQ ID NO: 23, 25, 27, 29, or 31.

[0153] 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 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 set forth as SEQ ID NO: 24, 26, 28, 30, or 32.

[0154] 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 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 set forth as SEQ ID NO: 23, 25, 27, 29, and 31, and the light chain variable region 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 set forth as SEQ ID NO: 24, 26, 28, 30, or 32.

[0155] In some aspects, the disclosure provides an isolated anti-L1CAM antibody, or antigen-binding fragment thereof, (a) heavy and light chain variable region sequences comprising SEQ ID NOs: 23 and 24, respectively; (b) heavy and light chain variable region sequences comprising SEQ ID NOs: 25 and 26, respectively; (c) heavy and light chain variable region sequences comprising SEQ ID NOs: 27 and 28, respectively; (d) heavy and light chain variable region sequences comprising SEQ ID NOs: 29 and 30, respectively; or (e) comprising heavy and light chain variable region sequences comprising SEQ ID NOs: 31 and 32, respectively.

[0156] The amino acid sequences of VH CDR1, CDR2, and CDR3 for antibodies Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 are set forth in SEQ ID NOs: 2, 10, and 4, respectively. The amino acid sequences of VL CDR1, CDR2, and CDR3 for Ab612 are set forth in SEQ ID NOs: 12, 7, and 13, respectively. The amino acid sequences of VL CDR1, CDR2, and CDR3 for Ab4H5 are set forth in SEQ ID NOs: 12, 7, and 15, respectively. The amino acid sequences of VL CDR1, CDR2, and CDR3 for Ab2C2 are set forth in SEQ ID NOs: 12, 7, and 17, respectively. The amino acid sequences of VL CDR1, CDR2, and CDR3 for Ab4H6 are set forth in SEQ ID NOs: 12, 7, and 19, respectively. The amino acid sequences of the VL CDR1, CDR2, and CDR3 for Ab5D12 are set forth in SEQ ID NOs: 12, 7, and 21, respectively.

[0157] In some embodiments, an anti-L1CAM antibody or antigen-binding fragment thereof of the present disclosure that specifically binds to human L1CAM is (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2, and / or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and / or (c) comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO:4.

[0158] In some embodiments, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM is (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and / or (c) comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0159] In some embodiments, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM is (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and / or (f) comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0160] In some embodiments, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM is (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and / or (c) comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 15.

[0161] In some embodiments, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM is (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and / or (f) comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 15.

[0162] In some embodiments, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM is (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and / or (c) comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0163] In some embodiments, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM is (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and / or (f) comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0164] In some embodiments, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM is (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12, and / or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and / or (c) comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 19.

[0165] In some embodiments, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds to human L1CAM is (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and / or (f) comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21.

[0166] In some aspects, the 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.

[0167] In some aspects, the 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.

[0168] In some aspects, the 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.

[0169] In some aspects, the 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.

[0170] In some embodiments, the 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 Includes. [Table 5A] [Table 5B-1] [Table 5B-2]

[0171] A VH domain described herein, or one or more CDRs thereof, can be combined with a constant domain to form a heavy chain, e.g., a full-length heavy chain. Similarly, a VL domain described herein, or one or more CDRs thereof, can be combined with a constant domain to form a light chain, e.g., a full-length light chain. The full-length heavy chain and full-length light chain combine to form a full-length antibody.

[0172] Thus, in certain aspects, provided herein are antibodies comprising an antibody light chain and a heavy chain, e.g., separate light and heavy chains. With respect to the light chain, in certain aspects, the light chain of an antibody described herein is a kappa light chain. In another specific aspect, the light chain of an antibody described herein is a lambda light chain. In yet another specific aspect, the light chain of an antibody described herein is a human kappa light chain or a human lambda light chain. In certain aspects, an antibody described herein that specifically binds to an L1CAM polypeptide (e.g., human L1CAM) comprises a light chain comprising any of the VL or VL CDR amino acid sequences described herein, and the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region. In certain aspects, an antibody described herein that specifically binds to an L1CAM polypeptide (e.g., human L1CAM) comprises a light chain comprising any of the VL or VL CDR amino acid sequences described herein, and the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region. Non-limiting examples of human constant region sequences are described in the art, see, eg, US Pat. No. 5,693,780 and Kabat EA et al., (1991) supra.

[0173] With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In another specific embodiment, the heavy chain of a described antibody can comprise a human α (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In one embodiment, an antibody described herein that specifically binds to L1CAM (e.g., human L1CAM) comprises a heavy chain comprising a VH or VH CDR amino acid sequence described herein, and the constant region of the heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region. In another embodiment, an antibody described herein that specifically binds to L1CAM (e.g., human L1CAM) comprises a heavy chain comprising a VH or VH CDR amino acid sequence disclosed herein, and 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 are described in the art, see, eg, US Pat. No. 5,693,780 and Kabat EA et al., (1991) supra.

[0174] In some aspects, antibodies described herein that specifically bind to L1CAM (e.g., human L1CAM) comprise a VL domain and a VH domain comprising the VH or VH CDRs and the VL and VL CDRs described herein, and the constant region comprises the amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or the constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In another specific aspect, antibodies described herein that specifically bind to L1CAM (e.g., human L1CAM) comprise a VL domain and a VH domain comprising any amino acid sequence described herein, and the constant region comprises the amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any subclass of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In some embodiments, the constant region comprises the amino acid sequence of the constant region of 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 October 20, 2014) and Jefferis R. and Lefranc MP, mAbs 1:4, 1-7 (2009). In some embodiments, the constant region comprises the amino acid sequence of the constant region of human IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0175] In certain embodiments, 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). Effector functions are mediated by the Fc region, and residues closest to the hinge region in the CH2 domain of the Fc region are responsible for antibody effector functions because they contain largely overlapping binding sites for C1q (complement) and IgG-Fc receptors (FcγRs) on effector cells of the innate immune system. Furthermore, IgG2 and IgG4 antibodies have lower levels of Fc effector functions than IgG1 and IgG3 antibodies. Antibody effector functions can be reduced or avoided by different approaches known in the art, including: (1) using antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, single-chain Fv (scFv), or sdAbs consisting of monomeric VH or VL domains); (2) generating aglycosylated antibodies (e.g., by deleting or modifying sugar-binding residues, enzymatically removing the sugars, and producing the antibody in cells cultured in the presence of glycosylation inhibitors, or by expressing the antibody in cells unable to glycosylate proteins (e.g., bacterial host cells; see, e.g., U.S. Publication No. 20120100140); and (3) employing Fc regions from IgG subclasses with reduced effector function (e.g., Fc regions from IgG2 or IgG4 antibodies, or chimeric Fc regions comprising CH2 domains from IgG2 or IgG4 antibodies; see, e.g., U.S. Publication No. 20120100140 and Lau C. et al.). (4) generating Fc regions with mutations that reduce or eliminate Fc function. See, e.g., U.S. Publication No. 20120100140 and the U.S. and PCT applications cited therein, and An et al. See, e.g., et al., mAbs 1:6, 572-579 (2009).

[0176] Thus, in some embodiments, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein are Fab, Fab', F(ab'), Fv, single-chain Fv (scFv), or sdAb consisting of monomeric VH or VL domains. Such antibody fragments are well known in the art and are described above.

[0177] In some embodiments, the anti-L1CAM antibodies or antigen-binding fragments thereof disclosed herein comprise an Fc region with reduced or no Fc effector function. In some embodiments, the constant region comprises the amino acid sequence of an Fc region of human IgG2 or IgG4, and in some embodiments, the anti-L1CAM antibody is of the IgG2 / IgG4 isotype. In some embodiments, the anti-L1CAM antibody comprises a chimeric Fc region comprising a CH2 domain from an IgG antibody of the IgG4 isotype and a CH3 domain from an IgG antibody of the IgG1 isotype, or a chimeric Fc region comprising a hinge region from IgG2 and a CH2 region from IgG4, or an Fc region with mutations that result 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 and PCT publications cited therein. In addition, Fc regions with reduced or no Fc effector function can be readily produced by one skilled in the art.

[0178] IV. Nucleic acid molecules Another aspect described herein relates to one or more nucleic acid molecules encoding any one of the antibodies or antigen-binding fragments thereof described herein. The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA associated with the DNA from which it was isolated in nature) or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein can be, for example, DNA or RNA and may or may not contain intronic sequences. In certain aspects, the nucleic acid is a cDNA molecule.

[0179] 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 described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acids encoding the antibodies can be recovered from the library.

[0180] Certain nucleic acid molecules described herein encode the VH and VL sequences of the Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 monoclonal antibodies. Exemplary DNA sequences encoding the VH sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 are set forth in SEQ ID NOs: 8, 14, 16, 18, or 22. Exemplary DNA sequences encoding the VL sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 are set forth in SEQ ID NOs: 33, 34, 35, 36, and 37, respectively. [Table 6-1] [Table 6-2] [Table 6-3] [Table 7-1] [Table 7-2]

[0181] Methods for producing the anti-L1CAM antibodies disclosed herein can include expressing the heavy and light chains in a cell line containing nucleotide sequences encoding the heavy and light chains along with their signal peptides. Host cells containing these nucleotide sequences are encompassed herein.

[0182] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked," as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0183] The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA, et al.). al.,(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,USDepartment of Health and Human Services,NIH (See Publication No. 91-3242), and DNA fragments encompassing 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, e.g., an IgG2 and / or IgG4 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0184] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are 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. 10-14022). (See, e.g., J. Immunol. 2004, 10:111-1132, 2004; see ...

[0185] To generate an scFv gene, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a flexible linker, e.g., the amino acid sequence (Gly4-Ser)3, such that the VH and VL strings can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the 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 ... McCafferty et al., (1988) Science 242:423-426; McCafferty et al., (1988) Science 242:423-426; McCafferty et al., (1988) al., (1990) Nature 348:552-554).

[0186] In some aspects, the present disclosure provides a vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof. In other aspects, the vector can be used in gene therapy.

[0187] Vectors suitable for the present disclosure include expression vectors, viral vectors, and plasmid vectors. In one aspect, the vector is a viral vector.

[0188] As used herein, an expression vector refers to any nucleic acid construct that contains the necessary elements for the transcription and translation of an inserted coding sequence, or, in the case of an RNA viral vector, for replication and translation when introduced into an appropriate host cell. Expression vectors include plasmids, phagemids, viruses, and their derivatives.

[0189] The expression vector of the present disclosure can include a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein. In one embodiment, the coding sequence for the antibody or antigen-binding fragment thereof is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in a manner that allows each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in a manner that places the expression, transcription, and / or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked if induction of the promoter in the 5' gene expression sequence results in transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to induce transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence is capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired antibody or antigen-binding fragment thereof.

[0190] Viral vectors include, but are not limited to, 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; polyomavirus; Epstein-Barr virus; papilloma virus; herpesvirus; vaccinia virus; poliovirus; and nucleic acid sequences derived from RNA viruses, such as retroviruses. Other vectors known in the art can be readily adapted. Certain viral vectors are based on noncytopathic eukaryotic viruses in which nonessential genes have been replaced with a gene of interest. Noncytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA and subsequent proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy trials. The most useful are replication-deficient retroviruses (i.e., capable of directing synthesis of desired proteins but unable to manufacture infectious particles). Such genetically modified retroviral expression vectors have general utility for highly efficient gene transduction in vivo. Standard protocols for producing replication-defective retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retrovirus by the packaging cell line, collecting viral particles from tissue culture medium, and infecting target cells with the viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E.J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0191] In one embodiment, the virus is an adeno-associated virus, a double-stranded DNA virus. Adeno-associated viruses can be engineered to be replication-deficient and can infect a wide range of cell types and species. Furthermore, they have advantages such as heat and lipid solvent stability, high transduction frequencies in cells of various lineages, including hematopoietic cells, and the lack of superinfection inhibition, thus enabling multiple rounds of transduction. Reportedly, adeno-associated viruses can integrate into human cellular DNA in a site-specific manner, thereby minimizing the potential for insertional mutagenesis and variability in inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been tracked in tissue culture for over 100 passages in the absence of selective pressure, suggesting that adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also function in an extrachromosomal manner.

[0192] 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.

[0193] Lentiviral genomes and proviral DNA typically contain 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 RNA-directed DNA polymerase (reverse transcriptase), protease, and integrase; and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs facilitate transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses contain additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0194] Adjacent to the 5' LTR are sequences required for reverse transcription of the genome (tRNA primer binding site) and efficient packaging of viral RNA into particles (Psi site). If sequences required for packaging from the viral genome (or packaging of retroviral RNA into infectious virions) are missing, the cis defect prevents packaging of genomic RNA.

[0195] However, the resulting mutants are still capable of directing 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 herein below, vectors lacking a functional tat gene are desirable for certain applications. Thus, for example, one vector can provide nucleic acid encoding viral gag and viral pol, and another vector can provide nucleic acid encoding viral env to produce packaging cells. Introducing a vector providing a heterologous gene, identified herein as a transcription vector, into the packaging cells results in producer cells that release infectious viral particles carrying the foreign gene of interest.

[0196] According to the above-described configuration of vector and foreign gene, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from almost any suitable virus, including retroviruses. In some embodiments, the env protein is an amphipathic envelope protein that allows for transduction of cells of human and other species.

[0197] Examples of env genes from retroviruses 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 vesicular stomatitis virus (VSV) protein G (VSV G) from hepatitis virus and influenza, can also be used.

[0198] The vector providing the viral env nucleic acid sequence is operably associated with the regulatory sequences described elsewhere herein.

[0199] Examples of lentiviral vectors are disclosed in WO9931251, WO9712622, WO9817815, WO9817816, and WO9818934, which are incorporated by reference in their entirety.

[0200] 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, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because they cannot replicate or integrate into the host genome. However, these plasmids with a promoter compatible with the host cell can express peptides from genes operably encoded within the plasmid. 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 will be known to those of skill in the art. Additionally, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific fragments of DNA.

[0201] V. Antibody production Antibodies or fragments thereof that immunospecifically bind to L1CAM (e.g., human L1CAM) can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields that are within the skill of the art. These techniques are described, for example, in the references cited herein and are explained fully in the literature. See, for example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J ... al.,(1989),Molecular Cloning:A Laboratory Manual,Second Edition,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 See Manual, Cold Spring Harbor Laboratory Press.

[0202] In certain embodiments, the antibodies described herein are antibodies (e.g., monoclonal antibodies) that are prepared, expressed, generated, or isolated by any means involving, for example, synthesis of DNA sequences, creation through genetic engineering, etc. In certain embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that do not naturally occur within the antibody germline repertoire of an animal or mammal (e.g., a human) in vivo.

[0203] In certain aspects, provided herein are methods for producing an antibody or antigen-binding fragment thereof that immunospecifically binds to L1CAM (e.g., human L1CAM), comprising culturing a cell or host cell described herein. In certain aspects, provided herein are methods for producing an antibody or antigen-binding fragment thereof that immunospecifically binds to L1CAM (e.g., human L1CAM), comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment thereof using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody 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 purifying the antibody or antigen-binding fragment thereof obtained from the cell or host cell.

[0204] Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0205] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques known in the art and including those taught in, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981). The term "monoclonal antibody," as used herein, is not limited to antibodies produced through hybridoma technology. For example, monoclonal antibodies can be recombinantly produced from host cells exogenously expressing an antibody described herein or a fragment thereof, e.g., the light chain and / or heavy chain of such an antibody.

[0206] In certain embodiments, 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), which antibody immunospecifically binds to L1CAM (e.g., human L1CAM) as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or in the Examples provided herein. In certain embodiments, a monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent or polyvalent (e.g., bivalent) antibody. In certain embodiments, a monoclonal antibody is a monospecific or multispecific antibody (e.g., a bispecific antibody). The monoclonal antibodies described herein can be made, for example, by hybridoma methods described in Kohler G & Milstein C (1975) Nature 256:495, or can be isolated from phage libraries, for example, using the techniques described herein. Other methods for the preparation of clonal cell lines and the 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).

[0207] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters, or macaques, are immunized to induce lymphocytes that produce or can produce antibodies that specifically bind to the protein used for immunization (e.g., human L1CAM). Alternatively, lymphocytes can be immunized in vitro. The 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)). Additionally, animals can be immunized using the RIMMS (repeated immunization multiple site) technique (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, incorporated by reference in its entirety).

[0208] In some embodiments, a mouse (or other animal such as a chicken, rat, monkey, donkey, pig, sheep, hamster, or dog) can be immunized with an antigen (e.g., an L1CAM, such as human L1CAM), and once an immune response is detected, e.g., once antibodies specific to the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes are isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, e.g., cells from cell line SP20 available from the American Type Culture Collection (ATCC) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes from the immunized mouse are harvested and fused with NSO myeloma cells.

[0209] The hybridoma cells thus prepared are seeded 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 the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas typically contains the substances hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0210] Certain embodiments employ myeloma cells that fuse efficiently, support stable high-level production of antibody 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 Salk These include those derived from the MOPC-21 and MPC-11 mouse tumors available from the 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)).

[0211] The culture medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies directed 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 in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0212] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and expanded 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 as ascites tumors in animals.

[0213] 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.

[0214] The antibodies described herein include antibody fragments that recognize a specific L1CAM (e.g., human L1CAM) and can be produced 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 an antibody molecule and contains an intact light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains the two antigen-binding arms of an antibody molecule linked by disulfide bonds in the hinge region.

[0215] In one embodiment, to produce whole antibodies, VH or VL sequences can be amplified from a template, e.g., an scFv clone, using PCR primers containing the VH or VL nucleotide sequence, restriction sites, and flanking sequences to protect the restriction sites. 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 kappa or lambda constant region. The VH and VL domains can also be cloned into a single vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to generate stable or transient cell lines expressing full-length antibodies, e.g., IgG.

[0216] A chimeric antibody is a molecule in which different fragments of an antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain the variable region of a non-human animal (e.g., mouse, rat, or chicken) monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. For example, see Morrison SL (1985) Science 229:1202-7, Oi VT & Morrison SL (1986) BioTechniques 4:214-221, Gillies SD et al., (1989) J Immunol Methods 125:191-202, and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

[0217] A humanized antibody is capable of binding to a predetermined antigen and comprises framework regions having substantially the amino acid sequence of a human immunoglobulin and CDRs having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a mouse or chicken immunoglobulin). In certain embodiments, the humanized antibody also comprises at least a fragment of an immunoglobulin constant region (Fc), typically a fragment of a human immunoglobulin. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody may be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.Humanized antibodies can be produced by a variety of techniques, including CDR grafting (EP 239400, WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (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), as well as e.g. U.S. Pat. No. 6,407,213, U.S. Pat. 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 Supp):5973s-5977s, 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. Patent Application Publication No. US2005 / 0042664A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

[0218] Methods for making multispecific (e.g., bispecific) antibodies have been described. See, e.g., U.S. Patent 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.

[0219] Single domain antibodies, e.g., antibodies lacking light chains, can be produced by methods well known in the art (see Riechmann L & 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. Patent No. 6,005,079, and International Publication Nos. WO94 / 04678, WO94 / 25591, and WO01 / 44301).

[0220] Furthermore, antibodies that immunospecifically bind to the L1CAM antigen can 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 & Bona CA (1989) FASEB J 7(5):437-444, and Nissinoff A (1991) J Immunol 147(8):2429-2438).

[0221] In certain aspects, an antibody described herein that binds to the same epitope of L1CAM (e.g., human L1CAM) as the anti-L1CAM antibodies described herein is a human antibody or antigen-binding fragment thereof. In certain aspects, an antibody described herein that competitively blocks (e.g., in a dose-dependent manner) an antibody described herein (e.g., Ab612, Ab4H5, Ab2C2, Ab4H6, and Ab5D12) from binding to L1CAM (e.g., human L1CAM) is a human antibody or antigen-binding fragment thereof.

[0222] Human antibodies can be produced using any method known in the art. For example, transgenic mice that are incapable of expressing functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are normally immunized with a selected antigen, such as all or a fragment of the antigen (e.g., L1CAM). Monoclonal antibodies directed against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such techniques, 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 & Huszar D (1995) Int Rev Immunol 13:65-93. For a detailed discussion of this technology and protocols for producing human antibodies and human monoclonal antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735, and U.S. Pat. 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™ (Abgenix, Inc., U.S. Pat. Nos. 6,075,181 and 6,150,184), the HUAB-MOUSE™ (Mederex, Inc. / Gen Pharm, U.S. Pat. Nos. 5,545,806 and 5,569,825), the TRANS CHROMO MOUSE™ (Kirin), and the KM MOUSE™ (Medarex / Kirin).

[0223] Human antibodies that specifically bind to L1CAM (e.g., human L1CAM) can be produced by various methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences (see also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793, and International Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741).

[0224] In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, Epstein-Barr virus (EBV)-transformed human peripheral blood lymphocytes can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, which can then be screened to determine those that secrete human monoclonal antibodies that immunospecifically bind to a target antigen (e.g., L1CAM, such as human L1CAM). Such methods are known and described in the art. See, e.g., Shinmoto H et al., (2004) Cytotechnology 46:19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31.

[0225] VI. Cells and Vectors In certain aspects, provided herein are cells (e.g., host cells) that (e.g., recombinantly) express an antibody (or antigen-binding fragment thereof) described herein that specifically binds to L1CAM (e.g., human L1CAM) and related polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) comprising a polynucleotide that comprises a nucleotide sequence encoding an anti-L1CAM antibody or fragment for recombinant expression in a host cell, e.g., a mammalian cell. Also provided herein are host cells comprising such vectors for recombinantly expressing an anti-L1CAM antibody (e.g., a human or humanized antibody) described herein. In certain aspects, provided herein are methods for producing the antibodies described herein, comprising expressing such an antibody from a host cell.

[0226] Recombinant expression of an antibody described herein (e.g., a full-length antibody, the antibody heavy and / or light chain, or a single-chain antibody described herein) that specifically binds to L1CAM (e.g., human L1CAM) involves construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule described herein, the antibody heavy and / or light chain, or a fragment thereof (e.g., a heavy and / or light chain variable domain) is obtained, vectors for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Accordingly, described herein are methods for preparing proteins by expressing polynucleotides containing antibody or antibody fragment (e.g., light or heavy chain) encoding nucleotide sequences. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (e.g., light or heavy chain) coding sequences 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 are replicable vectors containing a nucleotide sequence encoding an antibody molecule described herein, an antibody heavy or light chain, a heavy or light chain variable domain of an antibody or fragment thereof, or a heavy or light chain CDR operably linked to a promoter. Such vectors can contain, for example, a nucleotide sequence encoding the constant region of an antibody molecule (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036 and U.S. Patent No. 5,122,464), and an antibody variable domain can be cloned into such a vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0227] The expression vector can be transferred into a cell (e.g., a host cell) by conventional techniques, and the resulting cell 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 fragment thereof. Accordingly, provided herein are host cells containing a polynucleotide encoding an antibody or fragment thereof described herein, or a heavy or light chain thereof, or a fragment thereof, or a single-chain antibody described herein, operably linked to a promoter for expression of such sequences in the host cell. In certain aspects, for expression of a double-chain antibody, vectors encoding both the heavy and light chains can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as described in more detail below. In certain aspects, the host cell contains a vector comprising a polynucleotide encoding both the heavy and light chains of an antibody or fragment thereof described herein. In certain embodiments, a host cell comprises two different vectors: a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or fragment thereof described herein, and a second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody or fragment thereof described herein. In other embodiments, a 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 a second host cell comprises a second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody or fragment thereof described herein. In certain embodiments, 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 embodiments, a population of host cells comprising such a first host cell and such a second host cell is provided herein.

[0228] In a particular aspect, provided herein is a population of vectors 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.

[0229] A variety of host-expression vector systems can be utilized to express the antibody molecules described herein. Such host-expression systems represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the antibody molecules described herein in situ. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequence; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequence; and plant cell systems (e.g., Chlamydomonas spp.) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequence. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSCl, BSC40, YB / 20, SP2 / 0, Sf9, human lymphoblastoid, NS0, bow melanoma, HT-1080, PERC.6, and BMT10 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter). In certain embodiments, cells for expressing the antibodies or antigen-binding fragments thereof described herein are CHO cells, for example, CHO cells derived from the CHO GS SYSTEM™ (Lonza).In certain embodiments, cells for expressing the antibodies described herein are human cells, e.g., human cell lines. In certain embodiments, the mammalian expression vector is POPTIVEC™ or pcDNA3.3. In certain embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), particularly for the expression of whole recombinant antibody molecules, are used for the expression of recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in conjunction with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45:101-5, and Cockett MI et al., (1990) Biotechnology 8(7):662-7). In certain embodiments, the antibodies described herein are produced by CHO cells or NSO cells. In certain embodiments, expression of nucleotide sequences 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.

[0230] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, when large quantities of such antibodies are to be produced, a vector that directs the expression of high levels of fusion protein products that are easily purified may be desirable for generating pharmaceutical compositions of the antibody molecule. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794) (the antibody coding sequence can be ligated separately into the vector in frame with the lac Z coding region to produce a fusion protein), pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24:5503-5509), and the like. For example, pGEX vectors can be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to 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.

[0231] In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0232] Several virus-based expression systems are available for mammalian host cells. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated into an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or region E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655-9). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, for example, Bitter G et al., (1987) Methods Enzymol. 153:516-544).

[0233] In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen 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, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, NSO (a mouse myeloma cell line that does not endogenously produce 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 embodiments, the anti-L1CAM antibodies described herein are produced in mammalian cells, such as CHO cells.

[0234] In certain embodiments, 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 that have insufficient or no fucosylation ability. In certain instances, a cell line with knockout of both alleles of l,6-fucosyltransferase can be used to produce antibodies or antigen-binding fragments thereof with reduced fucose content. The POTELLIGENT® system (Lonza) is an example of such a system that can be used to produce antibodies or antigen-binding fragments thereof with reduced fucose content.

[0235] For long-term, high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines that stably express the anti-L1CAM antibodies described herein, or antigen-binding fragments thereof, can be engineered. In certain aspects, the cells provided herein stably express the light chain / light chain variable domain and heavy chain / heavy chain variable domain that associate to form the antibodies or antigen-binding fragments thereof described herein.

[0236] In certain embodiments, rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and a selectable marker. After introduction of the foreign DNA / polynucleotide, engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing the anti-L1CAM antibodies or antibody-binding fragments thereof described herein. Such engineered cell lines can be particularly useful in screening and evaluating compositions that interact directly or indirectly with the antibody molecule.

[0237] Several selection systems can be employed in tk cells, hgprt cells, or aprt cells, respectively, including, but not limited to, herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes. Additionally, antimetabolite resistance can be used as the basis for selection of 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 & Berg P (1981) PNAS 78(4):2072-6), neo (Wu GY & 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 & Anderson WF (1993) Ann Rev Biochem 62:191-217, Nabel GJ & 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 commonly known in the field of recombinant DNA technology can be routinely applied to select the 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 Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Chapters 12 and 13; Colbere-Garapin F et al., (1981) J Mol Biol 150:1-14, which are incorporated herein by reference in their entireties.

[0238] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure can be expressed on immune cells, such as T cells and / or NK cells. In some embodiments, the antibodies or antigen-binding fragments thereof can be expressed as chimeric antigen receptors (CARs). CAR-T cells are T cells that express chimeric antigen receptors. The term "chimeric antigen receptor (CAR)," as used herein, refers to a recombinant fusion protein having an antigen-specific extracellular (or ectodomain) domain linked to an intracellular domain that enables the cell to perform a specialized function upon binding of an antigen to the extracellular domain. Chimeric antigen receptors are distinguished from other antigen-binding agents by their ability to bind MHC-independent antigens and transduce activation signals via the intracellular domain.

[0239] In some embodiments, 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 use in the CAR of the present disclosure can be any antigen-binding polypeptide, a wide variety of which are known in the art. In some cases, the antigen-binding domain is a single-chain Fv (scFv) or Fab. In other embodiments, the antigen-binding fragment useful in the CAR of the present disclosure includes any antigen-binding fragment disclosed herein.

[0240] In some embodiments, a transmembrane domain useful in a CAR is connected to the extracellular domain and can comprise a naturally occurring transmembrane domain, hi other embodiments, a transmembrane domain useful in a CAR can be derived from the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, CD8, or any other known in the art.

[0241] The term "intracellular domain" refers to the portion of the CAR that transduces an effector function signal upon binding to the extracellular domain of an antigen, instructing the T cell to perform a specialized function. In one embodiment, the intracellular domain of the CAR comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the ITAM is derived from CD3 zeta (ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, CD66d, 4-1 BB, DAP-10, OX40, or Fc[ε]RI[γ].

[0242] In some embodiments, the CAR of the present disclosure further comprises a costimulatory domain capable of binding to the intracellular domain. The costimulatory domain in the CAR construct can transmit a signal and activate the cell as part of the intracellular portion of the CAR. In some embodiments, 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.

[0243] In other embodiments, the CAR of the present disclosure further comprises a linker.A short oligopeptide or polypeptide linker can be present between the transmembrane domain and the intracellular domain.In some embodiments, the linker is not limited to a specific length, as long as the intracellular domain of the CAR can induce T cell activation when the extracellular domain binds to an antigen, i.e., L1CAM.In some embodiments, the linker comprises a (Gly4Ser)3 linker.

[0244] In other aspects, the disclosure includes a polynucleotide encoding a CAR of the disclosure, or a vector comprising the polynucleotide.

[0245] As used herein, the term "T cell" refers to a lymphocyte derived from the thymus and is a contributor to cellular immune responses. T cells include CD4+ T cells (helper T cells, TH cells), CD8+ T cells (cytotoxic T cells, CTL), memory T cells, regulatory T cells (Tregs), or natural killer T cells. In some embodiments, the T cells into which the CAR is introduced are CD8+ T cells.

[0246] VII. Immunoconjugates, Antibody Derivatives, and Diagnostics The anti-L1CAM antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging. For this purpose, the antibody (or binding fragment thereof) can be conjugated to a suitable detectable agent to form an immunoconjugate. For diagnostic purposes, suitable agents are detectable labels, including radioisotopes for whole-body imaging, and radioisotopes, enzymes, fluorescent labels, and other suitable antibody tags for sample testing.

[0247] Detectable labels that can be attached to any of the anti-L1CAM antibodies described herein include particle labels that include metal sols, such as colloidal gold, and are presented with peptide chelators of the N2S2, N3S, or N4 type. 125 or Tc 99The label may be any of a variety of types currently used in the field of in vitro diagnostics, including isotopes such as , chromophores including fluorescent markers, luminescent markers, phosphorescent markers, etc., as well as enzyme labels that convert a given substrate into a detectable marker, and polynucleotide tags that are revealed after amplification, such as by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, etc. For example, the label can be the enzyme alkaline phosphatase, which is detected by measuring the presence or formation of chemiluminescence following conversion of a 1,2 dioxetane substrate such as adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}decan}-4-yl)phenyl phosphate (CSPD), and CDP and CDP-STAR®, or other luminescent substrates well known to those of skill in the art, e.g., chelates of suitable lanthanides such as terbium(III) and europium(III). The means of detection will be determined by the label chosen. The appearance of the label or its reaction products can be achieved using the naked eye, if the label is particulate and accumulates at appropriate levels, or using instruments such as a spectrophotometer, luminometer, or fluorometer, all according to standard practices.

[0248] In some embodiments, the conjugation methods result in substantially (or nearly) non-immunogenic linkages, such as peptide (i.e., amide), sulfide, (sterically hindered), disulfide, hydrazone, and ether linkages, which are largely non-immunogenic and exhibit reasonable stability in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO2009 / 059278; WO95 / 17886).

[0249] Depending on the biochemical properties of the moiety and antibody, different conjugation strategies can be employed. Whether the moiety is naturally occurring or recombinant, consisting of 50-500 amino acids, standard procedures describing the synthesis of protein conjugates are available in textbooks and can be easily followed by those skilled in the art (see, e.g., Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some embodiments, reaction of a maleimide moiety with a cysteine ​​residue within an antibody or moiety is used. This is a particularly suitable coupling chemistry, for example, when Fab or Fab' fragments of an antibody are used. Alternatively, in some embodiments, coupling to the C-terminal end of the antibody or moiety is performed. C-terminal modification of proteins, e.g., C-terminal modification of Fab fragments, can be performed as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

[0250] Generally, site-specific reactions and covalent bonds are based on converting natural amino acids into amino acids with reactivity orthogonal to that of other functional groups present. For example, specific cysteines in rare sequence configurations can be enzymatically converted into aldehydes (see Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain desired amino acid modifications by utilizing the specific enzymatic reactivity of a particular enzyme with natural amino acids in a given sequence configuration (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 protease-catalyzed formation of CN bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403). Site-specific reaction and covalent attachment can also be achieved by selectively reacting the terminal amino acid with an appropriate modifying reagent.

[0251] The reactivity of N-terminal cysteines with benzonitrile (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent conjugation.

[0252] 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).

[0253] US6437095B1 describes a conjugation method based on the faster reaction of cysteines within a stretch of negatively charged amino acids with cysteines located within a stretch of positively charged amino acids.

[0254] The portion can also be a synthetic peptide or peptidomimetic.When a polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity can be incorporated during such synthesis (see, for example, de Graaf, AJ et al., Bioconjug.Chem.20(2009)1281-1295).Since various orthogonal functional groups are of interest and can be introduced into synthetic peptides, the conjugation of such peptides to linkers is standard chemistry.

[0255] To obtain a mono-labeled polypeptide, conjugates with a 1:1 stoichiometry can be separated from other conjugation by-products by chromatography. This procedure can be facilitated by using dye-labeled binding pair members and charged linkers. By using this type of label and highly negatively charged binding pair members, mono-conjugated polypeptides are easily separated from unlabeled polypeptides and polypeptides bearing two or more linkers, since differences in charge and molecular weight can be used for separation. Fluorescent dyes can be useful for purifying conjugates from unbound components, such as labeled monovalent binding agents.

[0256] In some embodiments, the moiety that binds to the anti-L1CAM antibody is selected from the group consisting of a binding moiety, a labeling moiety, and a biologically active moiety.

[0257] The anti-L1CAM antibodies described herein can also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linking agents, 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. Pat. Nos. 7,087,600, 6,989,452, and 7,129,261; PCT Publication Nos. WO02 / 096910, WO07 / 038658, WO07 / 051081, WO07 / 059404, WO08 / 083312, and WO08 / 103693; U.S. Patent Publication Nos. 20060024317, 20060004081, and 20060247295.

[0258] In some embodiments, the therapeutic agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

[0259] In some embodiments, the immunoconjugate comprises an anti-L1CAM antibody and a cytotoxin. The cytotoxin can be selected from any cytotoxin known in the art. In some embodiments, the cytotoxin is selected from the group consisting of dolastatin, monomethylauristatin E (MMAE), maytansine, duocarmycin, calicheamicin, pyrrolobenzodiazepine, duocarmycin, centanamycin, SN38, doxorubicin, derivatives thereof, synthetic analogs thereof, and any combination thereof. In certain embodiments, the immunoconjugate comprises an anti-L1CAM antibody and cytotoxin A. In other embodiments, the immunoconjugate comprises an anti-L1CAM antibody and a non-cytotoxic drug.

[0260] In some embodiments, the immunoconjugate comprises an anti-L1CAM antibody and a radiopharmaceutical. In some embodiments, the radiopharmaceutical is a radionucleotide. In certain embodiments, the radiopharmaceutical comprises radioactive iodine. In certain embodiments, the radiopharmaceutical comprises 131-iodine. In other embodiments, the radiopharmaceutical comprises the radioisotope yttrium-90.

[0261] In some embodiments, the immune complex comprises an anti-L1CAM antibody and a second antibody. In certain embodiments, the immune complex comprises an anti-L1CAM antibody and an enzyme. In some embodiments, the enzyme comprises glucose oxidase. In some embodiments, the enzyme comprises peroxidase. In some embodiments, the enzyme comprises myeloperoxidase. In some embodiments, the enzyme comprises glucose oxidase. In some embodiments, the enzyme comprises horseradish peroxidase.

[0262] In certain embodiments, 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 embodiments, the anti-tumor agent is epirubicin. In some embodiments, the anti-tumor agent is a superantigen. In certain embodiments, the superantigen is staphylococcal enterotoxin A (SEA / E-120, estafenatox).

[0263] Anti-L1CAM antibodies, such as those described herein, can also be used to detect human L1CAM, such as human L1CAM on the cell surface or soluble L1CAM in serum. The antibodies can be used, for example, in ELISA assays or flow cytometry. In some embodiments, the anti-L1CAM antibody is contacted with cells or serum for a time appropriate for specific binding to occur, and then a reagent, such as an antibody that detects the anti-L1CAM antibody, is added. An exemplary assay is provided in the Examples. An exemplary method for detecting L1CAM, e.g., surface-expressed L1CAM or soluble L1CAM (sL1CAM) in a sample (serum), includes (i) contacting the sample with the anti-L1CAM antibody for a time sufficient to allow specific binding of the anti-L1CAM antibody to L1CAM in the sample, and (2) detecting L1CAM bound by the anti-L1CAM antibody by contacting the sample with a detection reagent, e.g., an antibody, that specifically binds to the anti-L1CAM antibody, e.g., the Fc region of the anti-L1CAM antibody. A washing step can be included after incubation with the antibody and / or detection reagent. Anti-L1CAM antibodies for use in these methods do not need to be labeled or conjugated to a detection agent, as a separate detection agent can be used.

[0264] Other uses of anti-L1CAM antibodies, eg, as monotherapy or combination therapy, are provided elsewhere herein, eg, in the section on combination therapy.

[0265] VIII. Bispecific molecules The anti-L1CAM antibodies described herein can be used to form bispecific molecules. An anti-L1CAM antibody, or an antigen-binding portion thereof, can be derivatized or conjugated to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. For example, an anti-L1CAM antibody can be conjugated to an antibody or scFv that specifically binds to any protein that can be used as a potential target for combination therapy. The antibodies described herein can actually be derivatized or conjugated to two or more other functional molecules to generate multispecific molecules that bind to three or more different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create the bispecific molecules described herein, the antibodies described herein can be functionally conjugated (e.g., by chemical coupling, genetic fusion, noncovalent binding, or other methods) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, to obtain a bispecific molecule.

[0266] Thus, provided herein are bispecific molecules comprising at least one first binding specificity for L1CAM and a second binding specificity for a second target epitope. In some embodiments described herein where the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity.

[0267] In some embodiments, the bispecific molecules described herein comprise as binding specificities at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab'), Fv, or single-chain Fv (scFv). The antibody can also be a light or heavy chain dimer, or any minimal fragment thereof, such as the Fv or single-chain constructs described in U.S. Patent No. 4,946,778 to Ladner et al.

[0268] 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 specificities are proteins or peptides, various coupling 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).

[0269] IX. Composition Further provided herein are compositions, e.g., pharmaceutical compositions, comprising one or a combination of the anti-L1CAM antibodies described herein, or a combination with antibodies against other targets, or antigen-binding portion(s) thereof, formulated with a pharmaceutically acceptable carrier. Such compositions can comprise one or a combination of (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules, described herein. For example, the pharmaceutical compositions described herein can comprise a combination of antibodies (or immunoconjugates or bispecific antibodies) that bind to different epitopes on the target antigen or have complementary activities.

[0270] The pharmaceutical compositions described herein can also be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy can include an anti-L1CAM antibody described herein in combination with at least one other anti-cancer and / or immunomodulatory agent, such as a T cell stimulating (e.g., activating) agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on uses of the anti-L1CAM antibodies described herein.

[0271] In some embodiments, the compositions of the present invention further comprise a bulking agent. The bulking agent may be selected from the group consisting of NaCl, mannitol, glycine, alanine, and any combination thereof. In other embodiments, the compositions of the present invention comprise a stabilizer. The stabilizer may be selected from the group consisting of sucrose, trehalose, raffinose, arginine, or any combination thereof. In other embodiments, the compositions of the present invention comprise a surfactant. The surfactant may be selected from the group consisting of polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof. In certain embodiments, the compositions further comprise 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.

[0272] In other embodiments, the composition comprises a third antibody. In some embodiments, the third antibody is any antibody disclosed herein.

[0273] In one embodiment, the composition further comprises NaCl, mannitol, pentetic acid (DTPA), sucrose, PS80, and any combination thereof.

[0274] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). An option for subcutaneous injection is based on Halozyme Therapeutics' ENHANZE® drug delivery technology, which involves co-formulation of an Ab with a recombinant human hyaluronidase enzyme (rHuPH20), eliminating traditional limitations on the volume of biologics and drugs that can be delivered subcutaneously due to the extracellular matrix (U.S. Patent 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.

[0275] The pharmaceutical compounds described herein may contain one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19A). The pharmaceutical compositions described herein may also contain a pharmaceutically acceptable antioxidant.

[0276] These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by both the above-mentioned sterilization procedures and the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0277] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injections or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Unless any conventional media or agents are compatible with the active compound, their use in the pharmaceutical compositions described herein is contemplated.Pharmaceutical compositions can contain or lack preservatives.Auxiliary active compounds can be incorporated into the composition.

[0278] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. In many cases, the composition can include isotonic agents, for example, sugars, polyalcohols (e.g., mannitol, sorbitol, or sodium chloride), in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0279] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent containing one or a combination of the ingredients listed above, as needed, followed by sterile microfiltration.Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed herein.For sterile powders for preparing sterile injectable solutions, some preparation methods are vacuum drying and freeze-drying (lyophilization), which obtain a powder of the active ingredient and any additional desired ingredients from the solution that has previously been sterile-filtered.

[0280] For example, for administration of the anti-L1CAM antibodies described herein, dosages range from about 0.0001 to 100 mg / kg.

[0281] 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 usually administered multiple times.

[0282] The antibody can be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages are sometimes required at relatively short intervals until the progression of the disease is reduced or terminated, and until the patient shows partial or complete improvement in the symptoms of the disease. Thereafter, the patient can undergo a preventive regime.

[0283] A "therapeutically effective dose" of an anti-L1CAM antibody described herein may result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptom intervals, or prevention of functional impairment or disability due to disease affliction. In the context of cancer, a therapeutically effective dose may result in an increase in survival, e.g., overall survival, and / or prevention of further deterioration of physical symptoms associated with cancer. Symptoms of cancer are well known in the art and include, for example, abnormal mole characteristics, asymmetry, changes in the appearance of a mole including border, color and / or diameter, newly pigmented skin areas, abnormal moles, dark areas under the fingernails, breast lumps, changes in the nipple, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, worsening shortness of breath, hemoptysis, blood in the urine, blood in the stool, nausea, vomiting, liver metastasis, lung metastasis, bone metastasis, abdominal fullness, bloating, fluid in the abdominal cavity, 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 metastasis, lung metastasis, bladder metastasis, liver metastasis, bone metastasis, kidney metastasis, and pancreatic metastasis, difficulty swallowing, etc.

[0284] A therapeutically effective dose can prevent or delay the onset of cancer, as may be desired when there are early or preliminary signs of disease. Laboratory tests used to diagnose cancer include chemistry (including measuring L1CAM levels), hematology, serology, and radiology. Therefore, any clinical or biochemical assay that monitors any of the above can be used to determine whether a particular treatment is a therapeutically effective dose for treating cancer. Those skilled in the art will 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 administration route selected.

[0285] The compositions described herein can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. The route of administration of the anti-L1CAM antibodies described herein can include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. The term "parenteral administration" as used herein refers to forms of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intradermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0286] Alternatively, the antibodies described herein can potentially be administered via a non-parental route, e.g., a topical, epidermal, or mucosal route of administration, e.g., intranasal, oral, intravaginal, rectal, sublingual, or topical route.

[0287] X.Kit Provided herein are kits comprising one or more antibodies, or antigen-binding fragments thereof, bispecific molecules, or immunoconjugates thereof described herein. In certain aspects, provided herein are pharmaceutical packs or kits comprising one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more antibodies or antigen-binding fragments thereof provided herein, and optionally instructions for use. In some aspects, the kits contain the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein.

[0288] XI. Uses and Methods Certain aspects of the present disclosure are directed 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 an anti-L1CAM antibody, or any combination thereof.

[0289] Certain aspects of the present disclosure are directed to a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective dose of a composition (e.g., an antibody, polynucleotide, vector, host cell, immunoconjugate, or pharmaceutical composition) disclosed herein. In other aspects, the present disclosure is directed to a method of inhibiting L1CAM shedding by tumor cells in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the present disclosure is directed to a method of reducing shed L1CAM in serum and / or retaining L1CAM on the cell surface in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the present disclosure is directed to a method of killing tumor cells in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the present disclosure is directed to a method of reducing tumor size in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the present disclosure is directed to inhibiting tumor metastasis in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein. In some embodiments, the subject is a human.

[0290] The compositions of the present disclosure can be administered using any pharmaceutically acceptable route. In some embodiments, the compositions (e.g., antibodies, polynucleotides, vectors, host cells, immunoconjugates, or pharmaceutical compositions) are administered intravenously, intraperitoneally, intramuscularly, intraarterially, intrathecally, intralymphatically, intralesionally, intracapsularly, intraorbitally, intracardially, intradermally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidally, intraspinally, epidurally, intrasternally, topically, epidermally, mucosally, or any combination thereof. In some embodiments, the compositions are administered intravenously. In some embodiments, the compositions are administered subcutaneously.

[0291] In certain embodiments, the method reduces the size of a cancer, e.g., tumor size, in a subject. In some embodiments, 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%.

[0292] In some embodiments, the method increases the overall survival of the subject. In some embodiments, the overall survival is increased compared to the average overall survival of subjects with the same cancer but treated with a different therapy. In certain embodiments, the overall survival is increased by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 3-fold, or at least about 5-fold. In some embodiments, the overall survival is increased by at least about 1 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.

[0293] In some embodiments, the method increases the progression-free survival of a subject. In some embodiments, overall survival is increased compared to the progression-free survival of a subject with the same cancer but treated with a different therapy. In certain embodiments, progression-free survival is increased by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 3-fold, or at least about 5-fold. In some embodiments, overall survival is increased by at least about 1 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.

[0294] In some embodiments, the method increases the objective response rate in the subject. In certain embodiments, the method induces a complete response in the subject. In some embodiments, the method induces a partial response in the subject.

[0295] In some embodiments, the method comprises administering an anti-L1CAM antibody (or polynucleotide, vector, host cell, or immunoconjugate) disclosed herein and a second therapy. In some embodiments, the second therapy is administered before the anti-L1CAM antibody. In some embodiments, the second therapy is administered after the anti-L1CAM antibody. In some embodiments, the second therapy is administered simultaneously with the anti-L1CAM antibody. In certain embodiments, the anti-L1CAM antibody and the second therapy are administered separately. In other embodiments, the anti-L1CAM antibody and the second therapy are administered in a single formulation.

[0296] The second therapy can be any other therapy known in the art. In some embodiments, the second therapy comprises immunotherapy. In some embodiments, the second therapy comprises chemotherapy. In some embodiments, the second therapy comprises radiation therapy. In some embodiments, the second therapy comprises surgery. In some embodiments, the second therapy comprises administering a second therapeutic agent.

[0297] Anti-L1CAM antibodies can enhance the immune response against cancerous cells in patients with cancer. Provided herein are methods for treating a subject with cancer, comprising administering an anti-L1CAM antibody described herein to the subject, such that the subject is treated, for example, so that cancerous tumor growth is inhibited or reduced, and / or so that tumor regression occurs, and / or so that long-term survival is achieved. Anti-L1CAM antibodies can be used alone to inhibit cancerous tumor growth. Alternatively, anti-L1CAM antibodies can be used in combination with another agent, such as another immunogenic agent, standard cancer therapy, or another antibody, as described below.

[0298] Thus, for example, provided herein is a method for treating cancer by inhibiting tumor cell growth in a subject, 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 of the present disclosure include cancers that typically respond to immunotherapy and cancers that typically do not respond to immunotherapy.Cancers that can be treated also include L1CAM-positive cancers.Cancers can be cancers with solid tumors or hematological malignancies (liquid tumors). Non-limiting examples of cancers for treatment include cholangiocarcinoma, 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 carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, neuroblastoma, pharyngeal cancer, laryngeal cancer, oral cancer, cancer of connective tissue, Hodgkin's lymphoma, lymphoma, multiple myeloma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon adenocarcinoma, as well as head and neck cancer (or carcinoma), stomach cancer, germ cell tumors, pediatric sarcoma, sinus natural killer, melanoma (e.g., skin or intraocular malignancies), and tumors of the brain, including thyroid cancer, ... cancers of the ovaries, rectum, urethra, rectum, rectum, pelvis, rectum, rectum, uterine, and anal regions, cancers of the endocrine system, cancers of the parathyroid glands, cancers of the adrenal glands, sarcomas of soft tissue, cancers of the urethra, penile, rectal, pediatric solid tumors, cancers of the ureter, cancers of the renal pelvis, tumors of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including those induced by asbestos, virus-associated cancers, or virus-derived cancers (e.g., human papillary neoplasia (HPV-related or derived tumors)), and any combination of the foregoing cancers.

[0299] In some embodiments, the anti-L1CAM antibody is administered to patients with cancers that have responded poorly to previous treatments, such as immuno-oncology or immunotherapy drugs, or that have progressed, or that are either inherently refractory or resistant, or that have acquired a resistant or refractory state. For example, subjects who do not respond or do not respond adequately to a first therapy, or who experience disease progression after treatment, can be treated by administering an anti-L1CAM antibody alone or in combination with another therapy.

[0300] In some embodiments, the anti-L1CAM antibody is administered to a patient who has not previously received (ie, has not been treated with) an immuno-oncology agent.

[0301] In some embodiments, a method for treating cancer in a subject includes first determining whether the subject is L1CAM-positive, e.g., whether the subject has tumor cells that express L1CAM, and if the subject has an L1CAM-positive cancer, then administering to the subject, e.g., an anti-L1CAM antibody described herein. A method for treating a subject with cancer with an anti-L1CAM antibody can include administering to the subject cancer cells that express L1CAM, a therapeutically effective amount of an L1CAM antibody. Also provided herein is a method for predicting whether a subject will respond to treatment with an anti-L1CAM antibody, comprising determining the level of L1CAM in the patient's cancer cells, wherein if the subject's cancer cells are L1CAM-positive, the subject is likely to respond to treatment with the L1CAM antibody.

[0302] The anti-L1CAM antibody can be administered in conjunction with standard of care treatment. The anti-L1CAM antibody can be administered as a maintenance therapy, for example, a therapy intended to prevent the development or recurrence of a tumor.

[0303] The anti-L1CAM antibody can be administered in conjunction with another treatment, such as radiation, surgery, or chemotherapy. For example, adjuvant anti-L1CAM antibody therapy can be administered when there is a risk of micrometastases and / or to reduce the risk of recurrence.

[0304] Anti-L1CAM antibody, for example, the anti-L1CAM antibody described herein, can be combined with vaccination protocol.Many experimental strategies have been devised for tumor vaccination (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; also see Restifo, N. and Sznol, M., Cancer Vaccines, Ch.61, pp.3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition).

[0305] The administration of anti-L1CAM antibodies can also be combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). The administration of anti-L1CAM antibodies can be effectively combined with chemotherapy regimens. In these cases, the dose of the administered chemotherapy agent can be reduced (Mokyr et al. (1998) Cancer Research 58:5301-5304).

[0306] The practice of the present disclosure will employ, unless otherwise indicated, 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 explained fully in the literature, see, e.g., Sambrook et al., ed. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press), Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY), DNGlover ed., (1985) DNA Cloning, Volumes I and II, Gait, ed. (1984) Oligonucleotide Synthesis, Mullis et al. U.S. Patent 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 treatise,Methods In Enzymology(Academic Press,Inc.,NY), Miller and Calos eds.(1987)Gene Transfer Vectors For Mammalian Cells,(Cold Spring Harbor Laboratory),Wu et al.,eds.,Methods In Enzymology,Vols.154 and 155,Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London), Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986), Crooks, Antisense drug Technology:Principles, strategies and applications,2nd Ed.CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0307] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0308] The following experimental methods and details are referenced in the Examples below.

[0309] Example 1. Construction of Ab612 by site-directed mutagenesis The mammalian (e.g., human and mouse) L1CAM-binding human antibody Ab417 and procedures for its preparation are disclosed in U.S. Patent No. 9,777,060 and International Publication No. WO 2014 / 077648, which are incorporated herein by reference. To improve the biophysical properties of Ab417 antibody, the effects of each amino acid constituting the variable region of Ab417 antibody on the binding ability of Ab417 antibody were examined.

[0310] DNA carrying the mutations R16G, D54E, K76A, and P88A in the heavy chain variable region of the Ab417 antibody was synthesized and recombined with a 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 excised and purified using a PCR purification kit (GeneAll®). 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-allGIII) of the human Fab phage display vector pKRIBB-FabD (reference) carrying the full-length gene III. The resulting recombinant phagemid was designated pKRIBB-allGIII-Fd612.

[0311] DNA carrying the I31S and V95P mutations in the light chain variable region of the Ab417 antibody was synthesized and recombined with a gene encoding human Cκ by PCR. The resulting PCR product was electrophoresed on a 1.5% agarose gel. The band containing the DNA was excised and purified using a PCR purification kit (GeneAll®). Both ends of the purified DNA were digested with the restriction enzyme BstXI (New England Biolabs) and subcloned into the BstXI site of pKRIBB-allGIII-Fd612. The resulting recombinant phagemid was designated pKRIBB-allGIII-Fab612. For this purpose, an antibody containing the R16G, D54E, K76A, and P88A mutations in the heavy chain variable region and the I31S and V95P mutations in the light chain variable region of the Ab417 antibody was designated the Ab612 antibody.

[0312] Example 2. Construction of a phage-displayed Ab612 mutant Fab library 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 of FR1 to FR3 of Ab612 was synthesized. Fragment B (5'-TTT AAT TTC) containing the sequence of FR3 to FR4 of Ab612 was synthesized using the randomized LCDR3. CAC TTT AGT TCC CTG CCC GAA CGT CCA CGG X17 X15 X15 X13 X15 TTG CTG ACA ATA ATA The following fragments were synthesized: GGT GGC AAA ATC TTC-3'. X represents the number of amino acids; X17 is 17 amino acids, and X15 and X13 are 15 and 13 amino acids, respectively. Fragment A and Fragment B were assembled by recombinant PCR with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific). The resulting PCR products were purified using a PCR purification kit (GeneAll®), digested with BstXI restriction enzyme (New England Biolabs), and ligated with BstXI-digested pKRIBB-all GIII-Fab612 expression vector at a vector:insert molar ratio of 1:3. DNA was electroporated into competent E. coli TG1. The Ab612 mutant Fab library consisted of 5.92 × 10 8 Colonies were produced.

[0313] To isolate monoclonal antibodies that recognize human L1CAM, human L1CAM was used as an antigen in the first, second, and third rounds of panning.

[0314] After three rounds of panning, 470 colonies were randomly selected and separately inoculated into 96-well plates. They were then cultured in 300 μL of 2xYT / carbenicillin / glucose at 37°C for 8 hours. Then, 30 μL of cells were seeded into each well containing 1 mL of 2xYT / carbenicillin / glucose medium and cultured for 2 hours until the OD600 reached 0.5. The cells were infected with KM13 helper phage at a multiplicity of infection (MOI) of 20 for 30 minutes at 37°C without shaking, and then incubated for 30 minutes with shaking. The infected cells were centrifuged at 2900 × g for 10 minutes, resuspended in 2xYT / carbenicillin / kanamycin, and cultured at 30°C for 12 hours. The supernatants containing Fab phage from the 470 colonies were subjected to indirect and quantitative ELISA.

[0315] Among these, 41 clones with high activity binding to human L1CAM were selected, and their plasmid DNA was isolated and sequenced, revealing that 32 clones were distinct from each other.

[0316] Indirect ELISA was performed to analyze the binding ability of 32 unique clones to human L1CAM, and four clones were found to exhibit the highest antigen-binding ability (i.e., Ab4H5, Ab2C2, Ab4H6, and Ab5D12).

[0317] Example 3. Conversion and production of Fab to IgG1 To convert the Fab into a full IgG1 with a codon-optimized sequence for mammalian cells, the heavy and light chain variable regions with leader sequences were amplified by PCR and subcloned into the mammalian IgG1 expression plasmid pdCMV-dhfr-Ab612 vector at the EcoRI and ApaI sites (New England Biolabs) and the BsiWI and HindIII sites (New England Biolabs), respectively. HEK293F cells were cultured and transfected with the IgG1 expression plasmid using ExpiFectamin (Thermo Fisher Scientific) according to the ExpiCHO protocol. Seven to 14 days after transfection, the cell culture supernatant was centrifuged and filtered using a bottle-top filter (0.22 μm PES, Sartorius). The productivity of each mutant was compared with that of the existing Ab417 antibody by ELISA, as described in Example 5 below.

[0318] As shown in Table 8, the mutant antibodies exhibited 1.25- to 1.44-fold higher productivity than Ab417. Ab612 was enhanced by 1.44-fold, while Ab4H5, Ab2C2, Ab4H6, and Ab5D12 were enhanced by 1.25-fold, 1.26-fold, and 1.36-fold, and 1.26-fold, respectively. [Table 8]

[0319] Example 4. Purification of Ab417 mutant antibodies Plasmid DNA of the expression vectors for these antibodies was obtained in large quantities and expressed in ExpiCHO cells in the same manner as in Examples 1 to 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. Each supernatant was applied to a column packed with protein A-binding beads (Amicogen), and the antibody was eluted from the protein A using 0.1 M sodium citrate solution (pH 3.2). 1.0 M Tris solution (pH 8.0) was then immediately added to the eluted antibody for neutralization. The purified IgG1 was dialyzed using a PD-10 column with Sephadex G-25 (GE Healthcare) and stored in buffer (10 mM NaPi, 5% sorbitol, 0.01% Tween 20). The concentration of the purified antibody was determined based on the molar extinction coefficient using a Nanodrop (Thermo Fisher Scientific, Nanodrop 2000). The purified antibody was subjected to 10% SDS-PAGE and Coomassie staining to confirm that each of the heavy and light chains was expressed and assembled as whole IgG.

[0320] Example 5. Characterization of Ab417 variant antibodies The affinity of the purified antibodies to human L1CAM was measured by competitive ELISA. Human L1CAM was prepared at a density of 1 × 10 M and diluted to 1 × 10 using 0.1% PBA buffer (PBS containing 0.1% BSA). -12The antibodies were serially diluted to 100 μM. Each antibody was prepared by diluting it in 0.1% PBA buffer at a specific concentration according to its binding capacity. The diluted antigen and antibody were reacted with each other in the same volume ratio for 3 hours at 37°C. A 96-well plate (MaxiSorp, Nunc) was coated overnight at 4°C with purified human L1 CAM diluted in buffer (15 mM Na2CO3, 34.84 mM NaHCO3, pH 9.6) at a concentration of 100 ng / well. The next day, Difco nonfat milk (BD) was dissolved in 0.05% PBS-T buffer at a concentration of 2%, and 200 μL was added to each well, followed by incubation at 37°C for 1 hour. The wells were washed twice with 0.05% PBS-T buffer. Next, 100 μL of the antigen / antibody reaction mixture that had been reacted for 3 hours was added and reacted at room temperature for 1 hour. The wells were washed three times with 0.05% PBS-T buffer to remove non-antigen-bound antibodies. Goat anti-human IgG (Fc)-HRP (Invitrogen, 1 / 10000), which specifically recognizes the Fc region of human antibodies, was added as a secondary antibody and incubated at 37°C for 1 hour. The wells were washed four times with 0.05% PBS-T buffer to remove residual secondary antibody. To examine affinity by color development, 100 μl of a solution containing TMB as a substrate for the enzyme HRP (covalently bound to the secondary antibody) (BD OptEIA, BD) was added to each well and incubated at room temperature for 5 minutes. Finally, 50 μl of 2.5 M HSO solution was added to each well to terminate the enzyme reaction. After the reaction was terminated, absorbance was measured at 450 nm (VERSAmax microplate reader, Molecular Devices). The results are shown in Table 9. The affinity of Ab417 for human L1CAM was 5 x 10-10 M, that of Ab612 was 2.6 x 10-10 M, that of the Ab4H5 mutant was 6 x 10-11 M, that of the Ab2C2 mutant was 5 x 10-11 M, that of the Ab4H6 mutant was 6 x 10-11 M, and that of the Ab5D12 mutant was 1.2 x 10-10 M. The Ab417 mutant antibodies showed approximately 2- to 10-fold higher binding affinity for human L1CAM than the Ab417 antibody. [Table 9]

[0321] Example 6. Analysis of antigen binding specificity of Ab417 variants To examine whether the mutant antibodies selectively bind to human and mouse L1CAM, we performed flow cytometry using various cell types, using Ab417, which binds to both human and mouse L1CAM, as a comparative antibody.

[0322] CHO-DG44 cells (ATCC No. PTA-3356), known not to express human L1CAM, were cultured and used as a negative control. Cells expressing human L1CAM, human ovarian cancer, SKOV3 (ATCC No. HTB-77), and human non-small cell lung cancer, NCI-H522 (ATCC No. CRL-5810), were also cultured. The melanoma cell line B16F1 (ATCC No. CRL-6323) was cultured as a cell expressing mouse L1CAM. Cultured 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 collected at 4 × 10 per tube. 5The cells were added to round-bottom polystyrene test tubes (Falcon) at a density of 10 μg / mL. Purified antibodies were diluted in PBA solution at a concentration of 10 μg / mL, and 100 μl was added to each tube and mixed thoroughly. The tubes were then incubated at 4°C for 1 hour. A 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 in foil to protect from light, and the incubation was continued at 4°C for 1 hour. Cell viability was assessed by adding the staining reagent PI (propidium iodide, Sigma) at a ratio of 1:200. After the entire incubation period, intracellular FITC and PI fluorescent signals were detected. Figure 1A shows that the mutant antibodies do not bind to L1CAM-negative cells (i.e., CHO-DG44). Figures 1B-1D show that the mutant antibodies bind to human L1CAM-positive cells, NCI-H522 (Figure 1B), SKOV3 (Figure 1C), and mouse L1CAM-positive cells, B16F1 (Figure 1D). The mutant antibodies specifically bind to the mouse melanoma cell line B166F1, suggesting that the Ab417 variants of the present invention specifically bind to both human and mouse L1CAM.

[0323] Example 7. Determining the quality of purified Ab417 variants by SEC-HPLC 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 on a Biosuit high-resolution SEC column (7.5 × 300 mm, 250 Å particle size). Samples were separated using PBS pH 7.4 with isocratic flow. A Waters® e2695 Separation Module was used for the experiment, and a 2489 UV / Vis detector monitored absorbance at 280 nm. Size-exclusion high-performance liquid chromatography (SEC-HPLC) analysis showed that the mutant antibodies exhibited a monomer peak (Figures 2B–F), whereas Ab417 contained broad peaks of high-molecular-weight aggregates and low-molecular-weight fragments (Figure 2A).

[0324] Example 8. Analysis of the affinity of Ab417 variants for human L1CAM The affinity of Ab417 and Ab417 variants for human L1CAM was examined by BLI using Octet RED384 (ForteBio). For affinity analysis, the antibody was diluted in PBA solution prepared by adding 0.1% BSA to PBS. Human L1CAM was serially diluted with PBA at concentrations of 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, or 0 nM. 200 μl of diluted L1CAM was then added to an opaque 96-well plate to prevent light transmission. Using an AHC (anti-human IgG Fc capture) sensor chip, the binding kinetics of the antibody to the antigen were analyzed by examining the changes in refractive index that occur upon association and dissociation of the antibody and antigen while transferring the sensor chip to PBA solution, antibody, PBA solution, antigen, and PBA solution in that order.

[0325] Table 10 shows the affinity (K D ) was approximately 0.2 nM, the affinity of Ab612 was 0.1 nM, the affinity of Ab4H5 was 8 pM, the affinity of Ab2C2H was outside the range, the affinity of Ab4H6 was 0.07 nM, and the affinity of Ab5D12 was 0.09 nM. The increased affinity of the mutants compared to Ab417 (e.g., up to 25-fold) was attributed to slower dissociation rates. [Table 10]

[0326] Example 9. Isoelectric Point (PI) Analysis of Ab417 Variants Capillary isoelectric focusing (cIEF) of Ab417 and its mutants was performed using a Sciex PA800 Plus instrument (Sciex PN477441) with neutral-coated capillaries. All experiments were performed in triplicate. The pI values ​​of each material were determined using 32Karat software. Table 11 shows that the pI values ​​of Ab417 antibody were 9.62, Ab612 were 9.25, Ab4H5 were 8.96, Ab2C2 were 8.96, Ab4H6 were 9.03, and Ab5D12 were 9.04. [Table 11-1] [Table 11-2]

[0327] Example 10. Analysis of the tumor growth inhibitory effects of Ab417 mutants To investigate the antitumor effect of Ab612 antibody, the human cholangiocarcinoma cell line Choi-CK was transplanted into male Balb / c nude mice. 3 ) was inoculated into the back of mice. After 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 intravenously injected three times a week for three weeks. Tumor volume, mouse body weight, and tumor weight were measured and are shown in Figure 1A-1C, respectively.

[0328] Figures 3A and 3C show that the Ab612 treatment group had a mean of 311 mm 3 The Ab417-treated group showed a mean tumor volume of 0.20 g and tumor weight of 387 mm 3 The figures show that the mice had a mean tumor volume of 1096 mm and a tumor weight of 0.41 g. Mock-treated control mice had a mean tumor volume of 1096 mm 3The mean tumor volume was 0.93 g and tumor weight was 0.93 g. Based on tumor weight, the results show that Ab612 (78.2% tumor growth inhibition rate (IR)) exhibited 1.4-fold higher tumor growth inhibition compared to Ab417 (55.5% IR) (Figure 3C). Figure 3D shows tumor images from each group of eight mice sacrificed after the completion of the experiment, as described in Figures 3A-3C. The size of cancer tissue extracted from the eight mice treated with Ab612 antibody was smaller than that of those treated with Ab417 antibody. The anti-cancer effect of Ab612 antibody appears to be well-functioning in vivo, resulting in an improvement of the tumor microenvironment (TME) through antibody infiltration into cancer tissue or an immune cell-based aggressive environment.

[0329] Therefore, the group treated with the Ab612 antibody disclosed herein showed a significant inhibitory effect on tumor growth compared to the group treated with the negative control. In addition, no weight loss was observed in the mice during administration, and no toxicity due to antibody administration was observed.

[0330] The foregoing description of specific embodiments will sufficiently clarify the general nature of the invention so that others may readily modify and / or adapt such specific embodiments for various uses without departing from the general concept thereof and without undue experimentation by applying knowledge within the skill of the art. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology used herein is for the purpose of description and not of limitation, as would be interpreted by one of skill in the art in light of the teaching and guidance.

[0331] 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 as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0332] All publications, patents, and patent applications disclosed in this specification 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. In particular embodiments, for example, the following items are provided: (Item 1) 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), (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 isolated antibody or antigen-binding fragment thereof, wherein the VH of the reference antibody comprises SEQ ID NO: 31 and the VL of the reference antibody comprises SEQ ID NO: 32. (Item 2) 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, and the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of the antibody or antigen-binding fragment thereof At least one amino acid in CDR3 is selected from the group consisting of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of the mAb417 antibody. Unlike CDR3, 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); 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); 2. The antibody or antigen-binding fragment thereof of item 1, wherein the VL CDR3 of the mAb417 antibody comprises QQSIGRGVVT (SEQ ID NO: 11). (Item 3) 1. 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 an L1 cell adhesion molecule (L1CAM) epitope, (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; 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, and the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of the antibody or antigen-binding fragment thereof at least one amino acid in CDR3 is different from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of the mAb417 antibody; 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); 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); The isolated antibody or antigen-binding fragment thereof, wherein the VH CDR3 of the mAb417 antibody comprises QQSIGRGVVT (SEQ ID NO: 11). (Item 4) 4. The antibody or antigen-binding fragment thereof of item 2 or 3, wherein the at least one amino acid difference is in the VH CDR2 of the antibody or antigen-binding fragment thereof, and the VH CDR2 of the antibody or antigen-binding fragment thereof comprises a glutamine at residue 5. (Item 5) 5. The antibody or antigen-binding fragment thereof of any one of items 2 to 4, wherein the at least one amino acid difference is in the VL CDR1 of the antibody or antigen-binding fragment thereof, and the VL CDR1 of the antibody or antigen-binding fragment thereof comprises a serine at residue 8. (Item 6) 6. The antibody or antigen-binding fragment thereof of any one of items 2 to 5, wherein the at least one amino acid difference is in the VL CDR3 of the antibody or antigen-binding fragment thereof, and the VL CDR3 of the antibody or antigen-binding fragment thereof comprises a proline at residue 8. (Item 7) 7. The antibody or antigen-binding fragment thereof according to any one of items 2 to 6, wherein the at least one amino acid difference is in the VL CDR3 of the antibody or antigen-binding fragment thereof, and the VL CDR3 of the antibody or antigen-binding fragment thereof comprises alanine, glycine, phenylalanine, tyrosine, threonine, proline, and tryptophan at residues 3 to 9, respectively. (Item 8) 8. The antibody or antigen-binding fragment thereof according to any one of items 2 to 7, wherein the at least one amino acid difference is in the VL CDR3 of the antibody or antigen-binding fragment thereof, and the VL CDR3 of the antibody or antigen-binding fragment thereof comprises alanine, glycine, phenylalanine, tyrosine, serine, proline, and tryptophan at residues 3 to 9, respectively. (Item 9) 9. The antibody or antigen-binding fragment thereof according to any one of items 2 to 8, wherein the at least one amino acid difference is in the VL CDR3 of the antibody or antigen-binding fragment thereof, and the VL CDR3 of the antibody or antigen-binding fragment thereof comprises leucine, histidine, phenylalanine, tyrosine, proline, and tryptophan at residues 4 to 9, respectively. (Item 10) 10. The antibody or antigen-binding fragment thereof according to any one of items 2 to 9, wherein the at least one amino acid difference is in the VL CDR3 of the antibody or antigen-binding fragment thereof, and the VL CDR3 of the antibody or antigen-binding fragment thereof comprises leucine, valine, tryptophan, tyrosine, proline, and tryptophan at residues 4 to 9, respectively. (Item 11) 11. The antibody or antigen-binding fragment thereof according to any one of items 2 to 10, wherein the VL CDR3 of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21. (Item 12) 12. The antibody or antigen-binding fragment thereof according to any one of items 2 to 11, wherein the VH CDR1 of the antibody or antigen-binding fragment thereof comprises RFGMH (SEQ ID NO: 2). (Item 13) 13. The antibody or antigen-binding fragment thereof according to any one of items 2 to 12, wherein the VH CDR2 of the antibody or antigen-binding fragment thereof comprises FISNEGSNKYYADSVKG (SEQ ID NO: 10). (Item 14) 14. The antibody or antigen-binding fragment thereof according to any one of items 2 to 13, wherein the VH CDR3 of the antibody or antigen-binding fragment thereof comprises GRAYGSGSLFDP (SEQ ID NO: 4). (Item 15) 15. The antibody or antigen-binding fragment thereof according to any one of items 2 to 14, wherein the VL CDR1 comprises RASRTISSYVN (SEQ ID NO: 12). (Item 16) 16. Any of items 2 to 15, wherein the VL CDR2 comprises AASNLHS (SEQ ID NO: 7). The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14. (Item 17) 17. The antibody or antigen-binding fragment thereof according to any one of items 2 to 16, wherein the VL CDR3 comprises QQSIGRGPVT SEQ ID NO: 13. (Item 18) 18. The antibody or antigen-binding fragment thereof according to any one of items 1 to 17, comprising heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, wherein the light chain CDR3 comprises QQSIGRGPVT (SEQ ID NO: 13). (Item 19) 18. The antibody or antigen-binding fragment thereof according to any one of items 1 to 17, comprising heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, wherein the light chain CDR3 comprises QQAGFYTPWT (SEQ ID NO: 15). (Item 20) 18. The antibody or antigen-binding fragment thereof according to any one of items 1 to 17, comprising heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, wherein the light chain CDR3 comprises QQAGFYSPWT (SEQ ID NO: 17). (Item 21) 18. The antibody or antigen-binding fragment thereof according to any one of items 1 to 17, comprising heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, wherein the light chain CDR3 comprises QQSLHFYPWT (SEQ ID NO: 19). (Item 22) 18. The antibody or antigen-binding fragment thereof according to any one of items 1 to 17, comprising heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, wherein the light chain CDR3 comprises QQSLVWYPWT (SEQ ID NO: 21). (Item 23) the antibody or antigen-binding fragment thereof (a) the antibody or antigen-binding fragment thereof exhibits improved productivity; (b) the antibody or antigen-binding fragment thereof has an equilibrium dissociation constant (K D ) exhibiting improved affinity as measured by (c) the antibody or antigen-binding fragment thereof is Improved Showing the PI value, (d) the antibody or antigen-binding fragment thereof exhibits improved affinity as measured by the association constant (K), or (e) The antibody or antigen-binding fragment thereof according to any one of items 1 to 22, having one or more features selected from the group consisting of any combination thereof. (Item 24) 24. The antibody or antigen-binding fragment thereof according to any one of items 1 to 23, wherein the antibody or antigen-binding fragment thereof exhibits improved productivity compared to the mAb417 antibody. (Item 25) The increased 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, or at least about 25. The method according to claim 24, wherein the HCl concentration is 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. An antibody or antigen-binding fragment thereof. (Item 26) The antibody or antigen-binding fragment thereof has an improved K D 26. The antibody or antigen-binding fragment thereof according to any one of items 1 to 25, wherein (Item 27) The improved K D But 2.6 x 10 -10 Less than M, 2.5 x 10 -10 Less than M, 2.0 x 10 -10 Less than M, 1.5 x 10 -10 Less than M, 1.0 x 10 -10 Less than M, 9 x 10 -11 Less than M, 8 x 10 -11 Less than M, 7 x 10 -11 Less than M, 6 x 10 -11 Less than M, 5 x 10 -11 Less than M, 4 x 10 -11 Less than M, 3 x 10 -11 Less than M, 2 x 10 -11 Less than M, 1 x 10 -11 Less than M, 9 x 10 -12 Less than M, 8 x 10 -12 Less than M, 7 x 10-12 Less than M, 6 x 10 -12 Less than M, 5 x 10 -12 Less than M, 4 x 10 -12 Less than M, 3 x 10 -12 Less than M, 2 x 10 -12 Less than M, 1 x 10 -12 Less than M, 9 x 10 -13 Less than M or 8 x 10 -13 27. The antibody or antigen-binding fragment thereof according to item 26, wherein the antibody or antigen-binding fragment thereof has a molecular weight of less than M. (Item 28) 28. The antibody or antigen-binding fragment thereof according to any one of items 1 to 27, wherein the antibody or antigen-binding fragment thereof exhibits improved affinity (K) compared to the mAb417 antibody. (Item 29) The improved affinity (K) is 5×10 -10 Less than M, 4 x 10 -10 Less than M, 3 x 10 -10 Less than M, 2 x 10 -10 Less than M, 1.0 x 10 -10 Less than M, 9 x 10 -11 Less than M, 8 x 10 -11 Less than M, 7 x 10 -11 Less than M, 6 x 10 -11 Less than M, 5 x 10 -11 Less than M, 4 x 10 -11 Less than M, 3 x 10 -11 Less than M, 2 x 10 -11 Less than M, 1 x 10 -11 Less than M, 9 x 10 -12 Less than M, 8 x 10 -12 Less than M, 7 x 10 -12 Less than M, 6 x 10 -12 Less than M, 5 x 10 -12 Less than M, 4 x 10 -12 Less than M, 3 x 10 -12 Less than M, 2 x 10 -12 Less than M, 1 x 10 -12 Less than M, 9 x 10 -13 Less than M or 8 x 10 -13 29. The antibody or antigen-binding fragment thereof according to item 28, wherein the antibody or antigen-binding fragment thereof is less than M. (Item 30) The antibody or antigen-binding fragment thereof is Improved 30. The antibody or antigen-binding fragment thereof according to any one of items 1 to 29, which exhibits a PI value. (Item 31) The aforementioned Improved 31. The antibody or antigen-binding fragment thereof of item 30, having a 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. (Item 32) 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises a VH CDR1, CDR2, and CDR3, and a VL CDR1, CDR2, and CDR3; the VH CDR1, CDR2, and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10), and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; The isolated antibody or antigen-binding fragment thereof, wherein the VL CDR1, CDR2, and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7), and QQSIGRGPVT (SEQ ID NO: 13), respectively. (Item 33) 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises a VH CDR1, CDR2, CDR3, and a VL CDR1, CDR2, CDR3; the VH CDR1, CDR2, and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10), and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; The isolated antibody or antigen-binding fragment thereof, 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. (Item 34) 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises a VH CDR1, CDR2, CDR3, and a VL CDR1, CDR2, CDR3; the VH CDR1, CDR2, and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10), and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; The isolated antibody or antigen-binding fragment thereof, wherein the VL CDR1, CDR2, and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7), and QQAGFYTPWT (SEQ ID NO: 17), respectively. (Item 35) 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises a VH CDR1, CDR2, CDR3, and a VL CDR1, CDR2, CDR3; the VH CDR1, CDR2, and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10), and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; The isolated antibody or antigen-binding fragment thereof, 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. (Item 36) 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises a VH CDR1, CDR2, CDR3, and a VL CDR1, CDR2, CDR3; the VH CDR1, CDR2, and CDR3 comprise RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10), and GRAYGSGSLFDP (SEQ ID NO: 4), respectively; The isolated antibody or antigen-binding fragment thereof, wherein the VL CDR1, CDR2, and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7), and QQSLVWYPWT (SEQ ID NO: 19), respectively. (Item 37) 37. The antibody or antigen-binding fragment thereof of any one of items 1 to 36, wherein the VH 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 set forth as EVQLVESGGGWQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO: 23). (Item 38) The VL is DIQLTQSPSSLSASVGDRVTITCRASRTISSYV 38. The antibody or antigen-binding fragment thereof of any one of items 1 to 37, comprising 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 set forth as NWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIGRGPVTFGQGTKLEIK (SEQ ID NO: 24). (Item 39) 39. The antibody or antigen-binding fragment thereof of any one of items 1 to 38, wherein the VH 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 set forth as EVQLVESGGGWQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO: 27). (Item 40) 39. The antibody or antigen-binding fragment thereof of any one of items 1 to 39, wherein the VL 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 set forth as DIQLTQSPSSLSASVGDRVTITCRASRTISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAGFYSPWTFGQGTKLEIK (SEQ ID NO: 28). (Item 41) 41. The antibody or antigen-binding fragment thereof of any one of items 1 to 40, wherein the VH 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 set forth as EVQLVESGGGWQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO: 25). (Item 42) 42. The antibody or antigen-binding fragment thereof of any one of items 1 to 41, wherein the VL 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 set forth as DIQLTQSPSSLSASVGDRVTITCRASRTISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAGFYTPWTFGQGTKLEIK (SEQ ID NO: 26). (Item 43) 43. The antibody or antigen-binding fragment thereof of any one of items 1 to 42, wherein the VH 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 set forth as EVQLVESGGGWQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO: 29). (Item 44) 44. The antibody or antigen-binding fragment thereof of any one of items 1 to 43, wherein the VL 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 set forth as DIQLTQSPSSLSASVGDRVTITCRASRTISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLHFYPWTFGQGTKLEIK (SEQ ID NO: 30). (Item 45) 45. The antibody or antigen-binding fragment thereof of any one of items 1 to 44, wherein the VH 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 set forth as EVQLVESGGGWQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS (SEQ ID NO: 31). (Item 46) 46. ​​The antibody or antigen-binding fragment thereof of any one of items 1 to 45, wherein the VL 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 set forth as DIQLTQSPSSLSASVGDRVTITCRASRTISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLVWYPWTFGQGTKLEIK (SEQ ID NO: 32). (Item 47) 47. The antibody or antigen-binding fragment thereof according to any one of items 1 to 46, wherein the VH of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 23 and the VL of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 24. (Item 48) 47. The antibody or antigen-binding fragment thereof according to any one of items 1 to 46, wherein the VH of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 25 and the VL of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 26. (Item 49) 47. The antibody or antigen-binding fragment thereof according to any one of items 1 to 46, wherein the VH of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 27 and the VL of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 28. (Item 50) 47. The antibody or antigen-binding fragment thereof according to any one of items 1 to 46, wherein the VH of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 29 and the VL of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 30. (Item 51) 47. The antibody or antigen-binding fragment thereof according to any one of items 1 to 46, wherein the VH of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 31 and the VL of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 32. (Item 52) An antibody or antigen-binding fragment thereof that specifically binds to L1CAM, 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. (Item 53) An antibody or antigen-binding fragment thereof that specifically binds to L1CAM, 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: 40 and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 41. (Item 54) An antibody or antigen-binding fragment thereof that specifically binds to L1CAM, 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: 42 and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 43. (Item 55) An antibody or antigen-binding fragment thereof that specifically binds to L1CAM, 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: 44 and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 45. (Item 56) An antibody or antigen-binding fragment thereof that specifically binds to L1CAM, 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: 46 and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 20. (Item 57) 57. The antibody according to any one of items 1 to 56, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, variants thereof, and any combination thereof. (Item 58) 58. The antibody according to any one of items 1 to 57, which is a chimeric antibody, a human antibody, or a humanized antibody. (Item 59) 57. The antibody of any one of items 1 to 56, comprising a Fab, a Fab', a F(ab')2, an Fv, or a single-chain Fv (scFv). (Item 60) A nucleic acid encoding the antibody according to any one of items 1 to 59. (Item 61) A vector comprising the nucleic acid according to Item 60. (Item 62) A host cell comprising the vector described in Item 61. (Item 63) 64. The host cell of item 63, selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells in tissue culture, plant cells, insect cells, and human cells. (Item 64) 60. An immune complex comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 59, conjugated to a drug. (Item 65) 60. A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 59, and an antibody or antigen-binding fragment thereof that binds to an antigen. (Item 66) The antibody according to any one of Items 1 to 59, the nucleic acid according to Item 60, or the nucleic acid according to Item 61 66. A composition comprising a vector, a cell according to item 62 or 63, an immunoconjugate according to item 64, or a bispecific or multispecific antibody according to item 65, and a carrier. (Item 67) A kit comprising the antibody according to any one of Items 1 to 59, the nucleic acid according to Item 60, the vector according to Item 61, the cell according to Item 62 or 63, the immunoconjugate according to Item 64, or the bispecific or multispecific antibody according to Item 65, and instructions for use. (Item 68) 62. An immune cell comprising the nucleic acid of item 60 or the vector of item 61. (Item 69) 69. The immune cell of item 68, which is a T cell or an NK cell. (Item 70) 70. The immune cell according to item 68 or 69, comprising a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 59. (Item 71) A method for producing an antibody or antigen-binding fragment thereof that specifically binds to human L1CAM protein, the method comprising culturing the cell described in item 62 or 63 under suitable conditions and isolating the antibody. (Item 72) 60. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the antibody according to any one of items 1 to 59, the nucleic acid according to item 60, the vector according to item 61, the cell according to item 62 or 63, the immune complex according to item 64, the bispecific or multispecific antibody according to item 65, the composition according to item 66, or the immune cell according to any one of items 68 to 70. (Item 73) 73. The method of claim 72, wherein the disease or condition comprises a tumor. (Item 74) 74. The method of item 72 or 73, wherein the tumor comprises cholangiocarcinoma, melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, renal cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer. (Item 75) Item 75. The method of item 74, wherein the tumor is cholangiocarcinoma. (Item 76) 76. The method of any one of items 72 to 75, wherein the antibody, the nucleic acid, the vector, the cell, the immunoconjugate, the bispecific or multispecific antibody, the composition, or the immune cell suppresses growth of the tumor. (Item 77) 77. The method according to any one of items 72 to 76, wherein the antibody, the nucleic acid, the vector, the cell, the immunoconjugate, the composition, or the immune cell enhances immune cell infiltration into the tumor. (Item 78) 78. The method of any one of items 72 to 77, comprising administering an additional therapeutic agent. (Item 79) 79. The method of item 78, wherein the additional therapeutic agent comprises chemotherapy, immunotherapy, radiation therapy, or a combination thereof. (Item 80) 80. The method of claim 79, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

Claims

1. 1. An immunoconjugate comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to L1 cell adhesion molecule (L1CAM) and is conjugated to a drug, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) complementarity determining region 1 (CDR1), a VL CDR2, and a VL CDR3, and a heavy chain variable region (VH) CDR1, a VH CDR2, and a VH CDR3, wherein the VL CDR1 comprises RASRTISSYVN (SEQ ID NO: 12), the VL CDR2 comprises AASNLHS (SEQ ID NO: 7), the VL CDR3 comprises QQSIGRGPVT (SEQ ID NO: 13), the VH CDR1 comprises RFGMH (SEQ ID NO: 2), and the VH CDR2 comprises FISNEGSNKYYADSVKG (SEQ ID NO: 10), and the VH 1. An immunoconjugate wherein CDR3 comprises GRAYGSGSLFDP (SEQ ID NO: 4), and the agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

2. 1. An immunoconjugate comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to L1 cell adhesion molecule (L1CAM) and is conjugated to a drug, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) complementarity determining region 1 (CDR1), a VL CDR2, and a VL CDR3, and a heavy chain variable region (VH) CDR1, a VH CDR2, and a VH CDR3, wherein the VL CDR1 comprises RASRTISSYVN (SEQ ID NO: 12), the VL CDR2 comprises AASNLHS (SEQ ID NO: 7), the VL CDR3 comprises QQSLHFYPWT (SEQ ID NO: 19), the VH CDR1 comprises RFGMH (SEQ ID NO: 2), and the VH CDR2 comprises FISNEGSNKYYADSVKG (SEQ ID NO: 10), and the VH 1. An immunoconjugate wherein CDR3 comprises GRAYGSGSLFDP (SEQ ID NO: 4), and the agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

3. 1. An immunoconjugate comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to L1 cell adhesion molecule (L1CAM) and is conjugated to a drug, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) complementarity determining region 1 (CDR1), a VL CDR2, and a VL CDR3, and a heavy chain variable region (VH) CDR1, a VH CDR2, and a VH CDR3, wherein the VL CDR1 comprises RASRTISSYVN (SEQ ID NO: 12), the VL CDR2 comprises AASNLHS (SEQ ID NO: 7), and the VL CDR3 comprises QQAGFYSPWT (SEQ ID NO: 17), the VH CDR1 comprises RFGMH (SEQ ID NO: 2), and the VH CDR2 comprises FISNEGSNKYYADSVKG (SEQ ID NO: 10), and the VH 1. An immunoconjugate wherein CDR3 comprises GRAYGSGSLFDP (SEQ ID NO: 4), and the agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

4. 1. An immunoconjugate comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to L1 cell adhesion molecule (L1CAM) and is conjugated to a drug, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) complementarity determining region 1 (CDR1), a VL CDR2, and a VL CDR3, and a heavy chain variable region (VH) CDR1, a VH CDR2, and a VH CDR3, wherein the VL CDR1 comprises RASRTISSYVN (SEQ ID NO: 12), the VL CDR2 comprises AASNLHS (SEQ ID NO: 7), the VL CDR3 comprises QQAGFYTPWT (SEQ ID NO: 15), the VH CDR1 comprises RFGMH (SEQ ID NO: 2), and the VH CDR2 comprises FISNEGSNKYYADSVKG (SEQ ID NO: 10), and the VH 1. An immunoconjugate wherein CDR3 comprises GRAYGSGSLFDP (SEQ ID NO: 4), and the agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

5. An immunoconjugate comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to L1 cell adhesion molecule (L1CAM) and is conjugated to a drug, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) complementarity determining region 1 (CDR1), a VL CDR2, and a VL CDR3, and a heavy chain variable region (VH) CDR1, a VH CDR2, and a VH CDR3, wherein the VL CDR1 comprises RASRTISSYVN (SEQ ID NO: 12), the VL CDR2 comprises AASNLHS (SEQ ID NO: 7), the VL CDR3 comprises QQSLVWYPWT (SEQ ID NO: 21), the VH CDR1 comprises RFGMH (SEQ ID NO: 2), and the VH CDR2 comprises FISNEGSNKYYADSVKG (SEQ ID NO: 10), and the VH 1. An immunoconjugate wherein CDR3 comprises GRAYGSGSLFDP (SEQ ID NO: 4), and the agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

6. An immunoconjugate comprising an antibody or antigen-binding fragment thereof that specifically binds to L1CAM and is bound to a drug, wherein the antibody or antigen-binding fragment thereof comprises a VH and a VL, the VH of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 23, and the VL of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 24, and the drug is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive drug, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

7. An immunoconjugate comprising an antibody or antigen-binding fragment thereof that specifically binds to L1CAM and is bound to a drug, wherein the antibody or antigen-binding fragment thereof comprises a VH and a VL, the VH of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 29 and the VL of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 30, and the drug is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive drug, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

8. An immunoconjugate comprising an antibody or antigen-binding fragment thereof that specifically binds to L1CAM and is bound to a drug, wherein the antibody or antigen-binding fragment thereof comprises a VH and a VL, the VH of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 27, and the VL of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 28, and the drug is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive drug, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

9. An immunoconjugate comprising an antibody or antigen-binding fragment thereof that specifically binds to L1CAM and is bound to a drug, wherein the antibody or antigen-binding fragment thereof comprises a VH and a VL, the VH of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 25 and the VL of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 26, and the drug is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive drug, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

10. An immunoconjugate comprising an antibody or antigen-binding fragment thereof that specifically binds to L1CAM and is bound to a drug, wherein the antibody or antigen-binding fragment thereof comprises a VH and a VL, the VH of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 31, and the VL of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 32, and the drug is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive drug, a second antibody, an enzyme, an anti-tumor agent, and any combination thereof.

11. The immune complex of claim 6, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain (HC) constant region and a light chain (LC) constant region, wherein the HC constant region comprises amino acids 122 to 451 of SEQ ID NO: 38, and the LC constant region comprises amino acids 109 to 215 of SEQ ID NO:

39.

12. The immune complex of claim 7, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain (HC) constant region and a light chain (LC) constant region, wherein the HC constant region comprises amino acids 122 to 451 of SEQ ID NO: 44, and the LC constant region comprises amino acids 109 to 215 of SEQ ID NO:

45.

13. The immune complex of claim 8, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain (HC) constant region and a light chain (LC) constant region, wherein the HC constant region comprises amino acids 122 to 451 of SEQ ID NO: 42 and the LC constant region comprises amino acids 109 to 215 of SEQ ID NO:

43.

14. The immune complex of claim 9, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain (HC) constant region and a light chain (LC) constant region, wherein the HC constant region comprises amino acids 122 to 451 of SEQ ID NO: 40, and the LC constant region comprises amino acids 109 to 214 of SEQ ID NO:

41.

15. The immune complex of claim 10, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain (HC) constant region and a light chain (LC) constant region, wherein the HC constant region comprises amino acids 122 to 451 of SEQ ID NO: 46 and the LC constant region comprises amino acids 109 to 215 of SEQ ID NO:

20.

16. The immune complex of any one of claims 1 to 15, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, variants thereof, and any combination thereof.

17. The immune complex of any one of claims 1 to 16, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

18. The immune complex of any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof comprises Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv).

19. The immune conjugate of any one of claims 1 to 18, wherein the immune conjugate is an antibody-drug conjugate (ADC).

20. A composition comprising the immunoconjugate of any one of claims 1 to 19 and a carrier.

21. A kit comprising the immunoconjugate of any one of claims 1 to 19 and instructions for use.

22. A composition for treating a disease or condition in a subject in need thereof, the composition comprising an immunoconjugate according to any one of claims 1 to 19.

23. 23. The composition of claim 22, wherein the disease or condition comprises a tumor.

24. 24. The composition of claim 23, wherein the tumor comprises cholangiocarcinoma, melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, renal cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer.

25. 25. The composition of claim 24, wherein the tumor is cholangiocarcinoma.

26. The composition of any one of claims 23 to 25, wherein the immunoconjugate inhibits the growth of the tumor and / or enhances the infiltration of immune cells into the tumor.

27. The composition of any one of claims 23 to 26, wherein the immunoconjugate enhances immune cell infiltration into the tumor.

28. The composition of any one of claims 23 to 27, wherein the composition is for use in combination with an additional therapeutic agent.

29. 30. The composition of claim 28, wherein the additional therapeutic agent comprises chemotherapy, immunotherapy, radiation therapy, or a combination thereof.

30. 30. The composition of claim 29, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

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