KR20210042091A - Anti-L1CAM antibodies and uses thereof - Google Patents

Abstract

The present disclosure provides antibodies that specifically bind to L1CAM and compositions comprising such antibodies. Also provided herein is a method of preventing or treating a disease or condition, including a tumor, using an anti-L1CAM antibody.

Description

Anti-L1CAM antibodies and uses thereof

Cross-reference of related applications

This application claims priority to U.S. Provisional Application No. 62/865,871 (filing date: June 24, 2019) and Korean Application No. 10-2018-0075955 (filing date: June 29, 2018), each of which Their entirety is incorporated by reference.

Reference to electronically submitted sequence listings

The ASCII text file (file name: 4372_001PC02_SL_ST25.txt; size: 82,704 bytes; and creation date: June 27, 2019) submitted with this application is incorporated herein by reference in its entirety.

Technical field

The present disclosure relates to antibodies that specifically bind to the L1 Cell Adhesion Molecule (L1CAM), compositions comprising such antibodies, and tumors (e.g., biliary tract cancer, melanoma, pancreatic cancer, glioma, Breast cancer, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer and/or ovarian cancer).

L1 cell adhesion molecule (L1CAM) is one of the immunoglobulin superfamily cell adhesion molecules (CAM) that mediate cell to cell adhesion on the cell surface. It has a molecular weight of 200 to 220 kDa. L1CAM is first known as a protein that mediates neuron-neuron adhesion and is involved in neurite overgrowth 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).

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

An antibody that specifically binds to the L1CAM protein can be used for diagnosis and prevention or treatment of a disease (eg, cancer) in which L1CAM is overexpressed. Therefore, there is a need to develop an antibody capable of specifically binding to L1CAM and regulating L1CAM activity.

One aspect of the present disclosure is an isolated antibody or an isolated antibody 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). An antigen-binding fragment, wherein (a) the VH of the reference antibody comprises SEQ ID NO: 23, the VL of the reference antibody comprises SEQ ID NO: 24, or (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, the VL of the reference antibody comprises SEQ ID NO: 28, or (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.

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

The present disclosure further provides an isolated antibody or antigen-binding fragment thereof that cross competes with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL) for binding to the L1CAM epitope, wherein ( a) the VH of the reference antibody contains SEQ ID NO: 23, the VL of the reference antibody contains SEQ ID NO: 24, or (b) the VH of the reference antibody contains SEQ ID NO: 25, and the VL of the reference antibody contains SEQ ID NO: 26 Or (c) the VH of the reference antibody comprises SEQ ID NO: 27, the VL of the reference antibody comprises SEQ ID NO: 28, or (d) the VH of the reference antibody comprises SEQ ID NO: 29, and the VL of the reference antibody is 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 VH complementarity determining region 1 (CDR1), VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3, and VH CDR1, VH CDR2 of the antibody or antigen-binding fragment thereof, At least one amino acid in VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 is different from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 of mAb417 antibody, and VH CDR1 of mAb417 antibody Contains RFGMH (SEQ ID NO: 2); The VH CDR2 of the mAb417 antibody contains FISNDGSNKYYADSVKG (SEQ ID NO: 9); VH CDR3 of mAb417 antibody contains GRAYGSGSLFDP (SEQ ID NO: 10); The VL CDR1 of the mAb417 antibody contains RASRTISIYVN (SEQ ID NO: 6); The VL CDR2 of the mAb417 antibody comprises AASNLHS (SEQ ID NO: 7); The VH CDR3 of the mAb417 antibody contains QQSIGRGVVT (SEQ ID NO: 11).

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

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 is QQSIGRGPVT (SEQ ID NO: 13), QQAGFYTPWT (SEQ ID NO: Number 15), QQAGFYSPWT (SEQ ID NO: 17), QQSLHFYPWT (SEQ ID NO: 19) or QQSLVWYPWT (SEQ ID NO: 21).

The present disclosure further provides an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein having one or more characteristics selected from the group consisting of: (a) exhibits improved productivity compared to mAb417 antibody; (b) shows improved affinity compared to mAb417 antibody when measured by the equilibrium dissociation constant (K _{D );} (c) shows improved PI values compared to mAb417 antibody; (d) shows improved affinity compared to mAb417 antibody as measured by the association constant (K); Or (e) any combination thereof.

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein exhibits improved productivity compared to mAb417 antibody, wherein the improved productivity is at least 55 mg/L, at least 56 when expressed according to Example 3 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, or at least about 85 It is mg/ℓ.

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein exhibits improved affinity compared to mAb417 antibody as measured by the _{equilibrium dissociation constant (K D ), wherein the improved K D} is 2.6× less than 10 ^-10 M, 2.5 × 10 ^-10 M less, 2.0 × 10 ^-10 M less, 1.5 × 10 ^-10 M less, 1.0 × 10 ^-10 less than M, less than ^{9 × 10 -11 M, 8 ×} 10 - ^{Less than 11} M, less than 7×10 ^-11 M, less than 6×10 ^-11 M, less than 5×10 ^-11 M, less than 4×10 ^-11 M, less than 3×10 ^-11 M, 2×10 ^-11 M Less than 1×10 ^-11 M, less than 9×10 ^-12 M, less than 8×10 ^-12 M, less than 7×10 ^-12 M, less than 6×10 ^-12 M, less than 5×10 ^-12 M, Less than 4×10 ^-12 M, less than 3×10 ^-12 M, less than 2×10 ^-12 M, less than 1×10 ^-12 M, less than 9×10 ^-13 M, or less than 8×10 ^-13 M.

In another aspect, the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein exhibits improved affinity compared to mAb417 antibody as measured by the association constant (K), wherein the improved K is 5×10 ⁻¹⁰ Less than M, less than 4×10 ^-10 M, less than 3×10 ^-10 M, less than 2×10 ^-10 M, less than 1.0×10 ^-10 M, less than 9×10 ^-11 M, less than 8×10 ^-11 M , Less than 7×10 ^-11 M, less than 6×10 ^-11 M, less than 5×10 ^-11 M, less than 4×10 ^-11 M, less than 3×10 ^-11 M, less than 2×10 ^-11 M, 1 ×10 ^-11 M or less, 9×10 ^-12 M or less, 8×10 ^-12 M or less, 7×10 ^-12 M or less, 6×10 ^-12 M or less, 5×10 ^-12 M or less, 4×10 ^{Less than -12} M, less than 3×10 ^-12 M, less than 2×10 ^-12 M, less than 1×10 ^-12 M, less than 9×10 ^-13 M, or less than 8×10 ^-13 M. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein exhibits an improved PI value compared to mAb417 antibody and the improved PI value is less than 9.6, less than 9.5, less than 9.4, less than 9.3, less than 9.2, 9.1 Less than, 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.

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein comprises VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3; Wherein VH CDR1, CDR2 and CDR3 each include RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4); VL CDR1, CDR2 and CDR3 each comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSIGRGPVT (SEQ ID NO: 13).

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 each include RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4); VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQAGFYSPWT (SEQ ID NO: 17), respectively.

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 each include RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4); VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQAGFYTPWT (SEQ ID NO: 17), respectively.

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 each include RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4); VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSLHFYPWT (SEQ ID NO: 19), respectively.

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 each include RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4); VL CDR1, CDR2 and CDR3 comprise RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSLVWYPWT (SEQ ID NO: 19), respectively.

In some aspects, the VH of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is EVQLVESGGG WQPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAF ISNEGSNKYY ADSVKGRFTI SRDNSANTLY LQMNSLRAED TAVYYCARGR AYGSGSLF, at least about AYGSGSLF, and at least about 80 amino acid sequence shown in sequence number 23 (SEQ ID NO: 23) 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 amino acid sequences.

In another aspect, the VL of the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is DIQLTQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SIGRGPVTFGQGTKLEIK (at least about 85% with amino acid sequence set forth in SEQ ID NO: , 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 amino acid sequences.

In some aspects, the VH of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is EVQLVESGGG WQPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAF ISNEGSNKYY ADSVKGRFTI SRDNSANTLY LQMNSLRAED TAVYYCARGR AYGSGSLF, at least about 27 amino acid sequences set forth in TAVYYCARGR (SEQ ID NO: 27) and at least about AYGSGSLF SDP WGQGT 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 amino acid sequences.

In another aspect, the VL of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is DIQLTQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ AGFYSPWTFGQGTKLEIK (at least about 85% with amino acid sequence set forth in SEQ ID NO: , 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 amino acid sequences.

In some aspects, the VH of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is EVQLVESGGG WQPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAF ISNEGSNKYY ADSVKGRFTI SRDNSANTLY LQMNSLRAED TAVYYCARGR AYGSGSLF, at least about 80% amino acid sequence as set forth in AYGSGSLF, WGQGT 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 amino acid sequences.

In some aspects, the VL of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is DIQLTQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ AGFYTPWTFGQGTKLEIK (at least about 80% with amino acid sequence set forth in SEQ ID NO: , 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 amino acid sequences.

In another aspect, the VH of the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is EVQLVESGGG WQPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAF ISNEGSNKYY ADSVKGRFTI SRDNSANTLY LQMNSLRAED TAVYYCARGR AYGSGSLF, at least about 80 amino acid sequence shown in SEQ ID NO. 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 amino acid sequences.

In some aspects, the VL of the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is DIQLTQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQSLHFYPWT FGQGTKLEIK with at least about 80% amino acid sequence as set forth in SEQ ID NO: %, 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 amino acid sequences.

In another aspect, the VH of the anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is EVQLVESGGG WQPGGSLRL SCAASGFTFS RFGMHWVRQA PGKGLEWVAF ISNEGSNKYY ADSVKGRFTI SRDNSANTLY LQMNSLRAED TAVYYCARGR AYGSGSLF, at least about AYGSGSLF, and at least about 80 amino acid sequence shown in SEQ ID NO. 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 amino acid sequences.

In some aspects, the VL of an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is DIQLTQSPSS LSASVGDRVT ITCRASRTIS SYVNWYRQRP GKAPESLIYA ASNLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQSLVWYPWT FGQGTKLEIK with at least about 80% amino acid sequence as set forth in SEQ ID NO: %, 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 amino acid sequences.

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

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

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

Some aspects of the present disclosure relate to nucleic acids encoding anti-L1CAM antibodies, vectors comprising the nucleic acids, host cells comprising the vectors. In some aspects, the host cell is E. Coli (E. coli), Pseudomonas (Pseudomonas), Bacillus (Bacillus), Streptomyces (Streptomyces), yeasts, CHO, YB / 20, NS0 , PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2/0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells and human cells.

Some aspects of the disclosure relate to immunoconjugates comprising an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein linked to an agent.

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

Some aspects of the present disclosure relate to compositions comprising the anti-L1CAM antibodies, nucleic acids, vectors, host cells, immunoconjugates or bispecific or multispecific antibodies and carriers disclosed herein.

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

Some aspects of the present disclosure relate to methods of producing antibodies that specifically bind to human L1CAM protein, comprising culturing host cells under suitable conditions and isolating the antibody.

Also included herein is the treatment of a disease or condition comprising administering to a subject an anti-L1CAM antibody, nucleic acid, vector, host cell, immunoconjugate or bispecific or multispecific antibody disclosed herein. Methods of treating a disease or condition in a subject are provided. In some aspects, the disease or condition comprises a tumor. In some aspects, the tumor includes biliary tract cancer, melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer. In another aspect, the anti-L1CAM antibody, nucleic acid, vector, cell, immunoconjugate, bispecific or multispecific antibody inhibits tumor growth and/or enhances the invasion of immune cells into the tumor.

Some aspects of the disclosure relate to a method comprising a method of administering an additional therapeutic agent. In some aspects, the additional therapeutic agent includes chemotherapy, immunotherapy, radiotherapy, or a combination thereof. In some aspects, the additional therapeutic agent is an immune checkpoint inhibitor.

Other features of the present disclosure will become apparent from the following detailed description and examples, which should not be regarded as limiting. The contents of all cited references, including scientific articles, newspaper reports, Genbank entries, patents and patent applications cited throughout this application, are expressly incorporated herein by reference.

Figures 1A-1D show the analysis of the antigen-binding specificity of Ab417 variants for human LCAM1 in CHO-DG44 cells (Figure 1A), NCI-H522 (Figure 1B), SKOV3 (Figure 1C) and B16F1 (Figure 1D). One drawing.
Figures 2A-2F show purified Ab417 (Figure 2A) and Ab417 variants as measured by size exclusion high performance liquid chromatography (SEC-HPLC): Ab612 (Figure 2B), Ab4H5 (Figure 2C), Ab2C2 (Figure 2D), The quality of Ab4H6 (Figure 2E) and Ab5D12 (Figure 2F) is shown.
3A-3D depict analysis of tumor growth-inhibition of Ab417 variants. Figure 3A shows the tumor volume of 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). Figure showing the change (*p<0.05, significant difference from the isotype control group by Dunnett's t-test). Figure 3B is a diagram showing the weight change of the Choi-CK xenograft model after administration of Ab417, Ab612 and control hFc, and negative control (PBS). 3C shows Choi-CK xenograft 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). Figure showing the tumor weight of the model (*p<0.01, significant difference from the isotype control group by Dunnett's t-test). 3D is a diagram showing a tumor picture of each group of 8 mice sacrificed after the end of the experiment as described in FIG. 3C.

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

In order to facilitate an understanding of the present disclosure disclosed herein, a number of terms and phrases are defined. Further definitions are given in the detailed description below.

I. Definition

Throughout this specification, the singular expression refers to one or more of that entity; For example, “antibody” is understood to refer to one or more antibodies. As such, the singular expressions, “one or more” and “at least one” may be used interchangeably herein.

Additionally, when used herein, "and/or" should be taken as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term “and/or” as used herein, such as “A and/or B” herein, refers to “A and B”, “A or B”, “A” (alone) and “B” It is intended to include (alone). Likewise, the term “and/or” as used in the phrase, such as “A, B, and/or C”, is intended to include 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; B and C; A (alone); B (alone); And C (alone).

When an aspect is described herein with the term “comprising”, it is understood that other similar aspects described in connection with “consisting of” and/or “consisting essentially of” are also provided.

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 relates. See, eg, 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 the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press] give those skilled in the art a general dictionary meaning of many of the terms used in this disclosure.

Units, prefixes, and symbols are presented in the form accepted by the Systeme International de Unites (SI). Numerical ranges include numbers defining the range. Unless otherwise indicated, amino acid sequences are described in amino to carboxy orientation from left to right. The headings provided herein are not in general a limitation of the various aspects of the specification that may be referenced herein. Accordingly, the terms defined immediately below are more fully defined by reference in their entirety herein.

The term “about” is used herein to mean approximately, approximately, around or within a region thereof. When the term “about” is used in connection with a numerical range, it modifies this range by extending the boundary above and below the numerical value presented. In general, the term “about” can modify a numerical value above and below the value stated by the variable, eg, 10% upward or downward (higher or lower).

The term “L1 cell adhesion molecule” or “L1CAM” refers to one of the integrative membrane glycoproteins belonging to the immunoglobulin superfamily cell adhesion molecule (CAM).

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

Human L1CAM (UniProt ID No. P32004-1; SEQ ID NO: 1) is a type 1 integrated membrane glycoprotein composed of 1,257 amino acids and passes through the cell membrane once, and its amino terminal fragment exists outside the cell membrane, and its carboxyl end Fragments are present in the cytoplasm. The extracellular domain of L1CAM comprises 11 domains of 6 immunoglobulin type 2 domains (Ig1, Ig2, Ig3, Ig4, Ig5 and Ig6) and 5 fibronectin III-like domains (Fn1, Fn2, Fn3, Fn4 and Fn5) and It contains 20 N-glycosylation sites (US Pat. No. 9,777,060).

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 to 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 is deficient in amino acid residues 1177 to 1180, and has the following difference at amino acid residues 26 to 31 compared to the amino acid sequence of human L1CAM (YEGHHV → L).

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

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.

The terms “antibody” and “antibodies” are terms in the art, and can be used interchangeably herein, and refer to a molecule having an antigen binding site that specifically binds to an antigen. As used herein, this term includes whole antibodies and any antigen-binding fragment (ie, “antigen-binding fragment”) or single chain thereof. “Antibody” refers to a glycoprotein or antigen-binding fragment thereof comprising at least two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds in one aspect. In another aspect, “antibody” refers to a single chain antibody comprising a single variable domain, eg, a VHH domain. Each heavy chain is composed 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 is composed of three domains, CH1, CH2 and CH3. In certain naturally occurring antibodies, each light chain is composed 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.

The VH region and the VL region may be further subdivided into a more conserved region referred to as a framework region (FR) and a hypervariable region referred to as a complementarity determining region (CDR). Each VH and VL consists of 3 CDRs and 4 FRs and is 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 region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (eg, effector cells) and host tissues or factors, including the first component of the typical complement system (Clq).

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

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

The Kabat numbering of residues can be determined for a given antibody by alignment in the region of homology of the sequence of the antibody with the “standard” Kabat numbered sequence. Chothia refers to the location of the structural loop instead (Chothia and Lesk, J. Mol. Biol . 196:901-917 (1987)). The end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop when numbered using the Kabat numbering convention (this is because the Kabat numbering rules place inserts in H35A and H35B; 35A and 35B also exist) Otherwise, the loop ends at 32; if only 35A exists, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software.

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

For all heavy chain constant region amino acid positions discussed herein, the numbering is described in Edelman et al. , 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85]. The EU index of Edelman et al. is also described 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 old "EU index presented in Kabat" or "EU index of Kabat" and "position according to the EU index presented in Kabat" and their grammatical modifications are described in Edelman et al. human IgG1 EU antibody as presented in Kabat 1991. Refers to a residue numbering system based on.

The numbering system used for the variable domain (both heavy and light chain) and light chain constant region amino acid sequences is that set forth in Kabat 1991.

Antibodies can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgD, IgG2, IgG3, IgG4, IgA1 Or IgA2) or any subclass (eg, IgG1, IgG2, IgG3 and IgG4 in humans; and IgG1, IgG2a, IgG2b and IgG3 in mice). Immunoglobulins, such as IgG1, exist in several allotypes, and they differ from each other by at most several amino acids. The antibodies disclosed herein can be derived from any of the generally known isotypes, types, subtypes, or allotypes. In certain aspects, the antibody described herein is an IgG1, IgG2, IgG3, or IgG4 subtype or any hybrid thereof. In certain aspects, the antibody has a human IgG1 subtype or a human IgG2 or human IgG4 subtype.

“Antibody” refers to, for example, naturally occurring and non-naturally occurring antibodies; Monoclonal and polyclonal antibodies; Chimeric and humanized antibodies; Human and non-human antibodies, total synthetic coalescence; Single chain antibodies; Monospecific antibodies; Multispecific antibodies (including bispecific antibodies); A tetrameric antibody comprising two heavy and two light chain molecules; Antibody light chain monomer; Antibody heavy chain monomer; Antibody light chain dimers; Antibody heavy chain dimers; Antibody light chain-antibody heavy chain pair; Intrabody; Heteroconjugate antibodies; Monovalent antibody; Camelizing antibodies; Affibody; With anti-idiotype (anti-Id) antibodies (e.g., including anti-anti-Id antibodies), and a single monomeric variable antibody domain (e.g., VH domain or VL domain) capable of fully antigen binding And a single-domain antibody (sdAb) comprising a binding molecule consisting of (Harmen MM and Haard HJ Appl Microbiol Biotechnol . 77(1): 13-22 (2007)).

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 (eg, human L1CAM). Such “fragments” are, for example, about 8 to about 1500 amino acids long, suitably about 8 to about 745 amino acids long, suitably about 8 to about 300, for example about 8 to about 200 Dog amino acids, or about 10 to about 50 or 100 amino acids long. It has been found that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the term "antigen-binding portion" of an antibody, eg, an anti-L1CAM antibody described herein, include (i) Fab, a monovalent fragment consisting of VL, VH, CL, and CH1 domains. snippet; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of a VH domain and a CH1 domain; (iv) Fv fragments consisting of the VL domain and VH domain of a single arm of the antibody and Fvs (sdFv) linked by disulfide bonds; (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 that may be selectively linked by a synthetic linker. In addition, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but these can be linked by a synthetic linker, using a recombinant method, where the VL and VH regions form a pair to form a monovalent molecule. It can be one single protein chain (referred to as single chain Fv (scFv)) (see, for example, Bird et al. , (1988) Science 242:423-426; and Huston et al. , (1988) Proc). Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also included 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 intact antibodies. Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. The variable region typically refers to a fragment of an antibody, usually a fragment of a light or heavy chain, typically approximately 110 to 120 amino acids at the amino terminus in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which are between antibodies. The sequence differs significantly and is used for the binding and specificity of a specific antibody for its specific antigen. Sequence variability is concentrated in regions called complementarity determining regions (CDRs), while regions more highly conserved in variable domains are called framework regions (FR).

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 and antigen. In certain aspects, the variable region is a human variable region. In certain aspects, the variable region comprises a rodent or murine CDR and a human framework region (FR). In certain aspects, the variable region is a primate (eg, non-human primate) variable region. In certain aspects, the variable region comprises a rodent or murine CDR and a primate (eg, non-human primate) framework region (FR).

As used herein, the term "heavy chain" when used in connection with an antibody, based on the amino acid sequence of the constant domain, is of any different type, such as alpha (α), delta (δ), epsilon may refer to (ε), gamma (γ) and mu (μ), each of which is a subtype of IgG, e.g., IgA, IgD, IgE, IgG and IgM of antibodies, including IgG1, IgG2, IgG3 and IgG4. Generates type.

As used herein, the term “light chain” when used in connection with an antibody refers to any of the different types based on the amino acid sequence of the constant domain, such as kappa (κ) and lambda (λ). I can. Light chain amino acid sequences are well known in the art. In a specific aspect, the light chain is a human light chain.

The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.

As used herein, the terms “constant region” or “constant domain” are used interchangeably and have their usual meaning in the art. Constant domains are antibody fragments, e.g., carboxy-terminal portions of the light and/or heavy chains that are not directly involved in the binding of an antibody to an antigen, but may exhibit various effector functions, such as interaction with an Fc receptor. The constant region of the immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

The term “Fc region” (fragment crystallizable region) or “Fc domain” or “Fc” refers to an Fc receptor located on various cells of the immune system (eg, effector cells) or the first component of the orthodox complement system (C1q) and It refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of immunoglobulins to host tissues or factors, including the binding of. Thus, the Fc region comprises the constant region of the antibody except for the first constant region immunoglobulin domain (eg, CH1 or CL). In the IgG, IgA and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody; The IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region contains the immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. The boundaries of the Fc region of the immunoglobulin heavy chain may vary, but the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position C226 or P230 (or the amino acid between these two amino acids) to the carboxy-terminus of the heavy chain, The numbering here follows the same EU index as in Kabat. The CH2 domain of the human IgG Fc region extends from approximately amino acid 231 to approximately amino acid 340, while the CH3 domain is located on the C-terminal side of the CH2 domain in the Fc region, i.e., it is from approximately amino acid 341 to approximately amino acid 447 of IgG. Is extended. As used herein, the Fc region may be a native sequence Fc, including any allotype variant, or a variant Fc (eg, non-naturally-occurring Fc). Fc also refers to this region upon isolation, or an Fc-comprising protein poly, such as “binding protein comprising an Fc region”, also referred to as “Fc fusion protein” (eg, antibody or immunoadhesion). Refers to the region associated with the peptide.

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

"Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced forms of these receptors. The FcγR family consists of three activating (FcyRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIB) receptor. Human IgG1 binds to most human Fc receptors and elicits the strongest Fc effector function. It is considered equivalent to mouse IgG2a for the type of activating Fc receptor to which it binds. On the other hand, human IgG4 elicits minimal Fc effector function (see Vidarsson G. et al . Front Immunol . 5:520 (published online Oct. 20, 2014)).

The constant region can be manipulated to remove one or more effector functions, for example, by recombinant techniques. "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event 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 (eg, B cell receptor; BCR). These effector functions generally require that the Fc region is combined with a binding domain (eg, antibody variable domain). Thus, the term “constant region without Fc function” includes a constant region with reduced or no effector function mediated by the Fc region.

The effector function of an antibody can be reduced or avoided by different approaches. The effector function of the antibody can be reduced or avoided using antibody fragments lacking the Fc region (e.g., Fab, F(ab') ₂ , single chain Fv (scFv) or sdAb consisting of monomeric VH or VL domains). . Alternatively, in order to reduce the effector function of the antibody while retaining other valuable properties of the Fc region (e.g., prolonged half-life and heterodimerization), so-called aglycosylated antibodies are used in the Fc region. It can be produced by removing the sugar linked to the moiety. Non-glycosylated antibodies can glycosylate proteins or by producing antibodies in cells cultured in the presence of glycosylation inhibitors, e.g., by removing or enzymatically removing sugars by deleting or altering residues to which sugars are attached. It can be produced by expressing the antibody in cells that are absent (eg, bacterial host cells) (see, eg, US Patent Publication No. 20120100140). Another approach is to use an Fc region from an IgG subtype with reduced effector function. For example, IgG2 and IgG4 antibodies are characterized by having lower levels of Fc effector function than IgG1 and IgG3. The residue closest to the hinge region in the CH2 domain of the Fc portion is responsible for the effector function of the antibody, because it overlaps significantly for the C1q (complement) and IgG-Fc receptor (FcγR) on the effector cells of the innate immune system. This is because it contains a binding site (Vidarsson G. et al . Front Immunol . 5:520 (published online Oct. 20, 2014)). Thus, antibodies with reduced or no Fc effector function are chimeric Fc regions comprising, for example, 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 hinge region from IgG2. And a chimeric Fc region comprising a CH2 region from IgG4 (see, eg, Lau C. et al. J. Immunol . 191:4769-4777 (2013)), or altered Fc effector function, resulting in altered Fc effector function. For example, it can be prepared by generating an Fc region with a mutation with reduced or no Fc function. Such Fc regions with mutations are known in the art (e.g., U.S. Patent Publication No. 20120100140 and U.S. and PCT applications cited therein and in An et al. , mAbs 1:6, 572). See -579 (2009]; the disclosures of these are incorporated herein by reference in their entirety).

“Hinge”, “hinge domain” or “hinge region” or “antibody hinge region” are used interchangeably and bind the CH1 domain with the CH2 domain, and a heavy chain comprising the upper, middle and lower fragments of the hinge Refers to the domain of the constant region (Roux et al. , J. Immunol. 1998 161:4083). The hinge changes the level of flexibility between the binding and effector regions of the antibody, and also provides a site for intermolecular disulfide bonds between the two heavy chain constant regions. As used herein, the hinge starts at Glu216 and ends at Gly237 for all IgG isotypes (Roux et al. , 1998 J Immunol 161:4083). 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, US Department of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al . Front Immunol . 5:520 (published online Oct. 20, 2014)).

The term “CH1 domain” refers to a heavy chain constant region linking the hinge with the variable domain in the heavy chain constant domain. As used herein, the CH1 domain starts at A118 and ends at V215. The term “CH1 domain” includes the wild-type CH1 domain as well as naturally occurring variants (eg, allotypes) thereof. The CH1 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild type and allotype) are known in the art (eg, Kabat EA et al. , (1991), supra) and Vidarsson G. et al . Front Immunol . 5:520 (published online Oct. 20, 2014)). Exemplary CH1 domains include, for example, CH1 domains with mutations that modify the biological activity of the antibody, e.g., half-life, as described in US Patent Publication No. 20120100140 and US Patents and Publications and PCT Publications cited therein Includes.

The term “CH2 domain” refers to the heavy chain constant region linking the hinge to the CH3 domain in the heavy chain constant domain. As used herein, the CH2 domain starts at P238 and ends at K340. The term “CH2 domain” includes the wild-type CH2 domain as well as naturally occurring variants (eg, allotypes) thereof. The CH2 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild type and allotype) are known in the art (eg, Kabat EA et al. , (1991), supra) and Vidarsson G. et al . Front Immunol . 5:520 (published online Oct. 20, 2014)). Exemplary CH2 domains are described in, for example, U.S. Patent Publication No. 20120100140 and U.S. Patents and Publications and PCT Publications cited in this patent, the biological activity of the antibody, e.g., half-life and/or reduced Fc effector function. It includes a CH2 domain with a modifying mutation.

The term “CH3 domain” refers to a heavy chain constant region that is C-terminal to the CH2 domain in the heavy chain constant domain. As used herein, the CH3 domain starts at G341 and ends at K447. The term “CH3 domain” includes the wild-type CH3 domain as well as naturally occurring variants (eg, allotypes) thereof. The CH3 domain sequences of IgG1, IgG2, IgG3, and IgG4 (including wild type and allotype) are known in the art (eg, Kabat EA et al. , (1991), supra) and Vidarsson G. et al . Front Immunol . 5:520 (published online Oct. 20, 2014)). Exemplary CH3 domains include, for example, CH3 domains with mutations that modify the biological activity of the antibody, e.g., half-life, as described in US Patent Publication No. 20120100140 and US Patents and Publications and PCT Publications cited therein. Includes.

As used herein, “isotype” refers to the type of antibody encoded by the heavy chain constant region gene (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies). do.

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

The phrases "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the terms "antibody that specifically binds to an antigen".

“Isolated antibody” as used herein refers to an antibody that is substantially free of other antibodies with different antigen specificities (eg, an isolated antibody that specifically binds to L1CAM is an antigen other than L1CAM). There are substantially no antibodies that specifically bind to). However, isolated antibodies that specifically bind to the epitope of L1CAM may have cross-reactivity against other L1CAM proteins from different species.

“Binding affinity” generally refers to the total sum strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, “binding affinity” refers to an intrinsic binding affinity that reflects a 1:1 interaction between a member of a binding pair (eg, an antibody and an antigen). . The affinity of molecule X for partner Y can generally be expressed by the _{dissociation constant (K D ).} Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, the equilibrium dissociation constant (K _D ), and the equilibrium association constant (K _{A ).} K _D is calculated from the quotient of k _off /k _on and expressed as a molar concentration (M), while K _A is calculated from the quotient of _{k on} /k _off. k _on refers to, for example, the association rate constant of the antibody to the antigen, and k _off refers to, for example, the dissociation rate constant of the antibody to the antigen. k _on and k _off are immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), BIAcore ^® , BLI (Bio-layer interferometry) or epidemiologic exclusion assays. , it may be determined by techniques known to those skilled in the art, such as (KinExA ^®).

As used herein, the terms “specifically binds”, “specifically recognizes”, “specific binding”, “selective binding” and “selectively binds” are similar terms with respect to antibodies. , This binding refers to the binding of a molecule (e.g., an antibody) to an antigen (e.g., an epitope or immune complex), as understood by one of skill in the art. For example, molecules that specifically bind to an antigen are, for example, immunoassays, BIAcore ^® , KinExA ^® 3000 instruments (Sapidyne Instruments, Boise, Idaho, USA) or known in the art. When determined by other assays, it is generally possible to bind other peptides or polypeptides with a lower affinity. In a specific aspect, the molecule that specifically binds to the antigen is a molecule that binds to the antigen to K _A at least 2 log, 2.5 log, 3 log , 4 log or greater than the K _A than at the time of binding to other antigens.

Antibodies specifically bind to cognate antigens with high affinity, ^{typically reflected by 10 -5} to 10 ^{-11 M or lower dissociation constants.} Any K _D greater than about 10 ^-4 M is generally considered to exhibit non-specific binding. As used herein, an antibody that “specifically binds” to an antigen refers to an antibody that binds to the antigen and substantially the same antigen with high affinity, which is, for example, an immunoassay using a predetermined antigen. (E.g., ELISA) ^{, 10 -7} M or less, preferably 10 ^-8 M or less, as determined by surface plasmon resonance (SPR) technology or BLI (biolayer interferometry) in a ^{BIACORE TM 2000 instrument} , Even more preferably 10 ^-9 M or less, most preferably 10 ^-8 M to 10 ^-10 M or less, _{which means having a K D} of less than 10 -8 M, and this antibody does not bind with high affinity to unrelated antigens.

As used herein, “antigen” refers to any natural or synthetic immunogenic substance, such as a protein, peptide or hapten. The antigen can be L1CAM or a fragment thereof.

As used herein, “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, a contiguous amino acid (linear or contiguous epitope) of a polypeptide, or an epitope may be, for example, a polypeptide or a combination of two or more non-contiguous regions of a polypeptide (stereoconjunctival, Non-linear, discontinuous, or non-continuous epitopes). The epitopes formed are not always so, but are typically retained upon exposure to a denaturing solvent, and epitopes formed by tertiary folding are typically lost upon treatment with a denaturing solvent. Epitopes typically comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids in their own spatial conformation. Methods of determining that an epitope is bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, where (e.g., L1CAM (From) overlapping or contiguous peptides are tested for reactivity with a given antibody (eg, anti-L1CAM antibody). Methods of determining the spatial conformation of an epitope include techniques in the art and those disclosed herein, and include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (e.g., See Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

In certain aspects, the epitope to which the antibody binds is, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange combined with mass spectroscopy (e.g., liquid chromatography electron spray mass spectroscopy), Array-based oligo-peptide scanning assays and/or mutagenesis mapping (eg, site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any of the methods known in the art (see, for example, Gige R et al. , (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4 ): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques, and can be improved using computer software such as XPLOR (Ye distributed by Molecular Simulations, Inc.) University, 1992; see, for example, Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al. ,]; U.S. Patent No. 2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol. Crystallogr 49 (Pt 1): 37-60; Bricogne G (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 performed using any method known to those of skill in the art (see Champe M et al. , (1995) for a description of mutagenesis techniques, including, for example, alanine scanning mutagenesis techniques). J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085).

The term “epitope mapping” refers to the process of identification of molecular determinants for antibody-antigen recognition.

The term “binds to the same epitope” in the context of an 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 antibody described herein and "same epitope on L1CAM" include epitope mapping methods, such as x-ray analysis of the determination of an antigen:antibody complex that provides atomic resolution of the epitope. And hydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of an antibody to an antigenic fragment or mutated variant of an antigen, wherein the loss of binding due to modification of amino acid residues within the antigenic sequence is considered primarily as an indication of the epitope component. In addition to this, computer combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate certain short peptides from combinatorial phage display peptide libraries. Antibodies with identical VH and VL or identical CDR1, 2 and 3 sequences are expected to bind to the same epitope.

An antibody that “compets with another antibody for binding to a target” refers to an antibody that inhibits (partially or completely) the binding of the remaining antibody to the target. Whether two antibodies compete with each other for binding to the target, i.e., whether one antibody inhibits the binding of the other antibody to the target and the extent to which it is inhibited is determined by known competition experiments, e.g. BIACORE ^® surface. Plasmon resonance (SPR) analysis can be used to determine. In some aspects, the antibody competes with other antibodies for binding to the target and inhibits this binding by at least 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition may differ depending on whether the antibody is a “blocking antibody” (ie, a cold antibody first incubated with the target). Competition assays are described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101/pdb.prot4277] or “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999]. That is, regardless of whether one or the other antibodies are first contacted with the antigen in a competition experiment, two antibodies "cross-competit" if the antibodies block each other by at least 50%.

Competition binding assays to determine whether two antibodies compete for binding or cross compete for binding include competition for binding to cells expressing L1CAM, for example 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 (Stahli et al. , Methods in Enzymology 9:242 (1983)); Solid phase direct biotin-avidin EIA (see Kirkland et al. , J. Immunol . 137:3614 (1986)); Solid phase direct label assay, solid phase direct label sandwich assay (see, eg, 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)).

As used herein, the terms “specific binding”, “selective binding”, “selectively bind” and “specifically bind” refer to binding of an antibody to an epitope on a predetermined antigen that is not another antigen. do. Typically, the antibody (i), for example, a predetermined antigen, e.g., surface plasmon resonance in a BIACORE ^® 2000 instrument using recombinant human MICA or use MICB as the analyte, and an antibody as a ligand (SPR ^{) Less than about 10 -7} M, such as less than about 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or even more, as determined by technique, or by Scatchard analysis of the binding of the antibody to antigen-positive cells. Binds with a low equilibrium dissociation constant (K _D ), and (ii) at least its affinity for binding to a predetermined antigen or to a non-specific antigen other than a closely-related antigen (e.g., BSA, casein). It binds to a predetermined antigen with a 2-fold greater affinity. Thus, an antibody that "specifically binds to human L1CAM" is soluble or cellular with a _{K D} ^{less than 10 -7} M, such as about 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or even lower. Binding refers to an antibody that binds to human L1CAM. Antibodies that cross-react with cynomolgus L1CAM have a _{K D} ^{of 10 -7} M or less, such as approximately 10 ^-8 M, 10 ^-9 M or less or even lower than ^{10 -10 M or even lower cynomolgus L1CAM.} Refers to an antibody that binds In some aspects, such antibodies that do not cross-react with L1CAM from non-human species exhibit essentially undetectable binding to these proteins in standard binding assays.

The term “k _assoc ” or “k _a ” as used herein is intended to refer to the association rate constant of a particular antibody-antigen interaction, while the term “k _dis ” or “k dis” as used herein k _d "is intended to refer to the rate of dissociation of a particular antibody-antigen interaction. The term “K _D ”as used herein refers to the dissociation constant obtained from the ratio of k _d to k _a (ie k _d /k _a ), which is expressed in molar concentration (M). _{K D} values for antibodies can be determined using methods well established in the art. The available method for determining the K _D of an antibody comprises a surface plasmon resonance biosensor system, for example, BIACORE ^®, BLI (Bio layer interferometry) system or flow cytometry and Chad scatter analysis.

The term “high affinity” for an IgG antibody as used herein refers to a ^{K of 10 -8} M or less, more preferably 10 ^-9 M or less and even more preferably 10 ^-10 M or less for the target antigen. _Refers to an antibody with D. However, "high affinity" binding may differ for other antibody isotypes. For example, in the case of the IgM isotype, “high affinity” binding refers to an antibody having _{a K D} ^{of 10 -10} M or less, more preferably 10 ^-8 M or less.

_{The term "EC 50} " in reference to an in vitro or in vivo assay using an antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding thereof that induces a response that is 50% of the maximal response, ie, intermediate between the maximal response and the baseline. Refers to the concentration of fragments.

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 ligation of Fab' fragments (see, eg, Songsivilai & Lachmann, Clin. Exp. Immunol . 79:315-321 ( 1990); Kostelny et al. , J. Immunol . 148, 1547-1553 (1992)).

The term “monoclonal antibody” as used herein refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope, or a composition of antibodies in which all antibodies exhibit a single binding specificity and affinity for a particular epitope. Thus, the term “human monoclonal antibody” refers to an antibody or antibody composition that exhibits single binding specificity and has variable and selective constant regions derived from human germline immunoglobulin sequences. In one aspect, the human monoclonal antibody comprises a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse having a genome comprising a human heavy and light chain transgene fused to an immortalized cell. Produced by hybridomas.

The term “recombinant human antibody” refers to (a) an antibody isolated from an animal (eg, mouse) that is transgenic or transchromosomal to a human immunoglobulin gene, or a hybridoma prepared therefrom, (b) an antibody. Antibodies isolated from host cells transformed to express, for example, transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) human immunoglobulins against other DNA sequences. It includes all human antibodies produced, expressed, produced or isolated by recombinant means, such as antibodies made, expressed, produced or isolated by any other means involving splicing of the gene sequence. Such recombinant human antibodies are encoded by germline genes, but include variable and constant regions using specific human germline immunoglobulin sequences, including, for example, subsequent rearrangements and mutations that occur during antibody maturation. As is known in the art (see, for example, Lonberg (2005) Nature Biotech . 23(9): 1117-1125), the variable regions are various genes rearranged to form antibodies specific for foreign antigens. It contains an antigen binding domain, encoded by. In addition to rearrangements, 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 foreign antigens. The constant region will change in further response to the antigen (ie, isotype switch). Thus, rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to antigen cannot have sequence identity with the original nucleic acid molecule, but will instead be substantially identical or similar (i.e., at least 80 % Identity).

"Human antibody (HuMAb)" refers to an antibody in which both the framework and the CDR regions have variable regions derived from human germline immunoglobulin sequences. In addition, if this antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The antibodies described herein comprise amino acid residues that are 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). can do. However, "human antibodies" as used herein are not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species such as mice have been grafted onto human framework sequences. The terms “human antibody” and “fully human antibody” are used synonymously.

A “chimeric antibody” refers to an antibody from which the variable region is derived from one species and the constant region is derived from another species, such as an antibody from which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

The term “cross-react” refers to the ability of an antibody described herein to bind to L1CAM from different species. For example, antibodies described herein that bind to human L1CAM can also bind to another species of L1CAM (eg, mouse L1CAM). As used herein, cross-reactivity is functional by detecting specific reactivity with a purified antigen in a binding assay (e.g., SPR, ELISA) or by binding or otherwise physiologically expressing L1CAM with cells. It can be measured by detecting the interaction. Cross-method for determining the responsiveness standard combination described herein black, for example, Biacore ^® 2000 SPR instrument (Biacore AB (Uppsala, Sweden)) Biacore ^® surface plasmon resonance (SPR) black or flow cytometry using Includes technology.

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

“Polypeptide” refers to a chain comprising at least two consecutively linked amino acid residues, and there is no upper limit on the length of the chain. One or more amino acid residues in the protein may contain modifications such as, but not limited to, glycosylation, phosphorylation or disulfide bond formation. “Protein” may include one or more polypeptides.

The term “nucleic acid molecule” as used herein is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

The term “conservative amino acid substitution” refers to the substitution of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic Includes amino acids with side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). In some aspects, an expected non-essential amino acid residue in the anti-L1CAM antibody is substituted with another amino acid residue from the same side chain family. Methods of identifying nucleotide and amino acid conservative substitutions that do not eliminate antigen binding are well known in the art (see, for example, 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)] reference).

The percent identity between two sequences is a function of the number of identical positions shared by the sequence, taking into account the number of gaps and the length of each gap, which must be introduced for optimal alignment of the two sequences (i.e.% homology = # In the same location/# in total in the location × 100). Comparison of sequences and percent identity between two sequences can be achieved using a mathematical algorithm as described in the non-limiting examples below.

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

Nucleic acid and protein sequences described herein can be used as “query sequences” to perform searches in public databases, for example, to identify related sequences. This search is described in Altschul, et al. (1990) J. Mol. Biol . 215:403-10] of the NBLAST and XBLAST programs (version 2.0). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein search can be performed with the XBLAST program, score = 50, word length = 3, to obtain an amino acid sequence homologous to the protein molecules disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST is described by Altschul et al. , (1997) Nucleic Acids Res . 25(17):3389-3402. When using BLAST and Gapped BLAST programs, default parameters of each program (eg, XBLAST and NBLAST) may be used (refer to worldwideweb.ncbi.nlm.nih.gov).

Nucleic acids may be present in whole cells, in cell lysates or in partially purified or substantially pure form. Nucleic acids are prepared by standard techniques including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques known in the art, by other cellular components or other contaminants, e.g., other cellular nucleic acids ( For example, the rest of the chromosome) or when purified from a protein, "isolated" or "substantially pure" (F. Ausubel, et al. , ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley). Interscience, New York (1987)).

Nucleic acids, such as cDNA, can be mutated according to standard techniques to provide gene sequences. In the case of the coding sequence, these mutations can affect the amino acid sequence if desired. In particular, DNA sequences that are substantially homologous to or derived from native V, D, J, constants, switches and other such sequences described herein are contemplated (where "derived" means that the sequence is identical to or from other sequences. Means modified).

The term “vector” as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is 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, where additional DNA segments can be ligated to the viral genome. Certain vectors (eg, bacterial vectors and episomal mammalian vectors having a bacterial origin of replication) are capable of self-replicating in the host cells into which they have been introduced. Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating 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 that are useful in recombinant DNA technology are mainly in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably because plasmid is the most commonly used form of vector. However, other types of expression vectors are also included, such as viral (eg, replication defective retrovirus, adenovirus and adeno-associated virus) vectors, which perform equivalent functions.

The term “recombinant host cell” (or simply “host cell”) as used herein refers to a cell containing a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that these terms refer not only to the particular cell in question, but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term “host cell” as used herein.

As used herein, the term “linked” refers to the association of two or more molecules. Linkage can be covalent or non-covalent. Linkages can also be genetically linked (ie, recombinantly fused). Such linkage can be achieved using a variety of art-recognized techniques such as chemical conjugation and recombinant protein production.

“Immune response” is as understood in the art and generally refers to a biological response in vertebrates to foreign agents or abnormalities, eg, cancer cells, which reactions are these agents and diseases caused by them. Protects the organism from The immune response is 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 these cells or liver (antibodies, cytokines and complement). In the body of a vertebrate, produced by, and in the body of a vertebrate, an infected pathogen, a cell or tissue infected with a pathogen, a cancerous or other abnormal cell, or in the case of autoimmune or pathological inflammation, the selective targeting, binding, of normal human cells or tissues, It is mediated by the action of soluble macromolecules leading to damage, destruction and/or elimination. The immune response is, for example, activation or inhibition of T cells, e.g., effector T cells, Th cells, CD4 ⁺ cells, CD8 ⁺ T cells or Treg cells or any other cell of the immune system, such as NK cells. It includes the activation or inhibition of.

“Immune therapy” refers to treating a subject suffering from, at risk for, or recurring a disease by a method comprising inducing, enhancing, inhibiting or otherwise modifying the immune system or immune response.

As used herein, “administration” refers to physically introducing a therapeutic agent or a composition comprising a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. As used herein, the phrase "parenteral administration" generally refers to a mode of administration other than intestinal and local administration by injection, but is not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, Intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtraminal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular (intraventricle), intravitreal, epidural (epidural) and intrasternal injections and injections, as well as in vivo electroporation. Alternatively, the antibodies described herein can be administered via parenteral routes, such as topical, epidermal or mucosal routes of administration, for example intranasal, oral, vaginal, rectal, sublingual or topical administration. Administration can also be carried out, for example, once, several times and/or one or more times over an extended period of time.

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

"Cancer" as used herein 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 surrounding tissues and can metastasize to distal parts of the body through the lymphatic system or bloodstream.

As used herein, the terms “treat”, “treating” and “treatment” reverse or alleviate the progression, development, severity of symptoms, complications, conditions or biochemical markers associated with a disease, or Refers to any type of intervention or process performed on a subject or administering an active agent for the purpose of improving, inhibiting or slowing or preventing, or improving overall survival. Treatment may be for a subject with a disease or for a subject without a disease (eg, prevention).

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 other therapeutic agents, decreases the severity of disease symptoms and increases the frequency and duration of disease-free periods, and increases overall survival (cancer and It is a drug in any amount that promotes disease regression as evidenced by the diagnosis for the same disease, or the period of time the patient diagnosed with the disease is still alive from the date of initiation of treatment) or by preventing damage or disability due to the disease. A therapeutically effective amount or dosage of a drug includes a “prophylactically effective amount” or “prophylactically effective amount”, which is administered alone or in combination with other therapeutic agents to a subject at risk of developing or at risk of experiencing a recurrence of the disease or condition. When any amount of a drug that inhibits the onset or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit the onset or recurrence of a disease is determined in human subjects during clinical trials, using a variety of methods known to the skilled practitioner, such as in animal model systems for predicting efficacy in humans, or testing It can be assessed by analyzing the activity of the agent in an in vitro assay.

For example, an anticancer agent is a drug that promotes cancer regression in a subject. In some aspects, the therapeutically effective amount of the drug promotes cancer regression to the point where the cancer is eliminated. "Accelerating cancer regression" means reducing tumor growth or size, tumor necrosis, reducing the severity of at least one disease symptom, increasing the frequency and duration of periods without disease symptoms, by administering an effective amount of a drug alone or in combination with an anti-tumor agent, It means that the overall survival rate is increased, the damage or disability caused by the disease is prevented or the disease symptoms of the patient are improved. In addition, the terms "effective" and "effect" with respect to treatment include both pharmacological effects and physiological safety. Pharmacological effect refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity due to drug administration or other negative physiological effects of the cellular, organ and/or organism level (side effects).

For example, for the treatment of a tumor, a therapeutically effective amount or dosage of a drug can reduce cell growth or tumor growth by at least about 20%, at least about 40%, at least about 60%, at least about 80% compared to an untreated subject. Inhibit. In some aspects, a therapeutically effective amount or dosage of the 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 assay described below. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, and such inhibition can be measured in vitro by assays known to those of skill in the art. In some aspects described herein, tumor regression can be observed and can last for at least about 20 days, at least about 40 days, or at least about 60 days.

The term “subject” as used herein includes any human or non-human animal. The term “non-human animal” includes all vertebrate animals, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

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

The various aspects described herein are described in more detail in the following subsections.

II. Anti-L1CAM antibody

Disclosed herein are antibodies, e.g., monoclonal antibodies, that are characterized by a specific functional characteristic or characteristic. For example, antibodies specifically bind mammalian (e.g., human and mouse) L1CAM and exhibit one or more of the following functional properties:

(a) shows improved productivity compared to mAb417 antibody,

(b) shows improved affinity compared to mAb417 antibody when measured by the equilibrium dissociation constant (K _{D );}

(c) shows improved PI values compared to mAb417 antibody;

(d) shows improved affinity compared to mAb417 antibody when measured by the association constant (K);

(e) preventing and/or treating a disease or condition, including a tumor; or

(f) any combination thereof.

In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof exhibits improved productivity compared to mAb417 antibody, e.g., the improved productivity is at least 55 mg/L, at least 56 mg when expressed according to Example 3 /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, or at least about 85 mg /ℓ.

In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof, when measured by a biolayer interferometry (BLI) method (e.g., as described in the examples), has a high affinity, e.g., 2.6. ×10 ^-10 M or less, 2.5×10 ^-10 M or less, 2.0×10 ^-10 M or less, 1.5×10 ^-10 M or less, 1.0×10 ^-10 M or less, 9×10 ^-11 M or less, 8×10 ^{Less than -11} M, 7×10 ^-11 M or less, 6×10 ^-11 M or less, 5×10 ^-11 M or less, 4×10 ^-11 M or less, 3×10 ^-11 M or less, 2×10 ^-11 Less than M, less than 1×10 ^-11 M, less than 9×10 ^-12 M, less than 8×10 ^-12 M, less than 7×10 ^-12 M, less than 6×10 ^-12 M, less than 5×10 ^-12 M , K less than 4×10 ^-12 M, less than 3×10 ^-12 M, less than 2×10 ^-12 M, less than 1×10 ^-12 M, less than 9×10 ^-13 M, or less than 8×10 ^-13 M _{With D} , It specifically binds to human L1CAM. In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof is ^{less than 2×10 −10} M, less than 1.9×10 ⁻¹⁰ M, less than 1.8×10 ⁻¹⁰ M, less than 1.7×10 ⁻¹⁰ M, 1.6×10 Human L1CAM with a K _D ^{of less than -10} M, less than 1.5×10 ^-10 M, less than 1.4×10 ^-10 M, less than 1.3×10 ^-10 M, less than 1.2×10 ^-10 M or less than 1.1×10 ^{-10 M} Specifically binds to In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds human L1CAM with a K _D ^{of less than 1.1×10 −10 M.} In another aspect, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds human L1CAM with a K _D ^{of less than 9×10 −12 M.} In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds human L1CAM with a K _D ^{of less than 1×10 −12 M.} In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds human L1CAM with a K _D ^{of less than 8×10 −11 M.} In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds human L1CAM with a K _D ^{of less than 1×10 −12 M.} In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds human L1CAM with a K _D ^{of less than 1.05×10 −10 M.} In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds human L1CAM with a K _D ^{of less than 8.22×10 −12 M.} In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds human L1CAM with a K _D ^{of less than 7.4×10 −11 M.} In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof specifically binds human L1CAM with a K _D ^{of less than 9.6×10 −11 M.}

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

Standard assays are known in the art for assessing the ability of antibodies to bind to various species of L1CAM, including, for example, ELISA, Western blot and RIA. Suitable assays are described in detail in the Examples. Binding kinetics of an antibody (e.g., binding affinity) may also be assessed by standard assays known in the art, e.g., ELISA, Biacore analysis, or KinExA ^{® ®.} Assays evaluating the effect of antibodies on the functional properties of L1CAM (eg, ligand binding) are described in more detail below and in the Examples.

In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof is less than 9.6, less than 9.5, 9.4 as measured by a capillary isoelectricfocusing (cIEF) method (e.g., as described in the Examples). Less than, 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, It represents an improved isoelectric point (PI) value of less than 7.7 or less than 7.6.

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) of the reference antibody VH comprises SEQ ID NO: 23, the VL of the reference antibody comprises SEQ ID NO: 24, (b) the VH of the reference antibody comprises SEQ ID NO: 25, the VL of the reference antibody comprises SEQ ID NO: 26, or (c ) The VH of the reference antibody contains SEQ ID NO: 27, the VL of the reference antibody contains SEQ ID NO: 28, or (d) the VH of the reference antibody contains SEQ ID NO: 29, and the VL of the reference antibody contains SEQ ID NO: 30 Alternatively, 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.

In certain aspects, the anti-L1CAM antibody or antigen binding fragment thereof is specific for the same L1CAM epitope as a reference antibody comprising VH complementarity determining region 1 (CDR1), VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3. Wherein at least one amino acid in the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 of the antibody or antigen-binding fragment thereof is the VH of the reference antibody (e.g., mAb417 antibody) It is different from CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3, and the VH CDR1, VH CDR2, VH CDR3 of the reference antibody have the amino acid sequences of SEQ ID NO.2, SEQ ID NO. 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 may comprise (i) glutamine at residue 5 of VH CDR2 of the anti-L1CAM antibody or antigen-binding fragment thereof; (ii) serine at residue 8 of VL CDR1; And/or (iii) proline at residue 8 of VL CDR3. In certain aspects, the at least one amino acid difference comprises (i) alanine, glycine, phenylalanine, tyrosine, threonine, proline and tryptophan at residues 3 to 9 of each VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof; (ii) alanine, glycine, phenylalanine, tyrosine, serine, proline and tryptophan at residues 3 to 9 of VL CDR3, respectively, or (iii) leucine, valine or histidine, tryptophan or phenylalanine, tyrosine at residues 4 to 9 of VL CDR3, respectively , Proline and tryptophan.

In certain aspects, the anti-L1CAM antibody or antigen-binding fragment thereof is a reference antibody comprising VH complementarity determining region 1 (CDR1), VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3 for binding to the L1CAM epitope. Cross-competition for, wherein at least one amino acid in the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 of the antibody or antigen-binding fragment thereof is a reference antibody (e.g., mAb417 antibody) Is different from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3, wherein (i) VH CDR1, VH CDR2, VH CDR3 of the reference antibody are respectively SEQ ID NO: 2, SEQ ID NO: 9 and sequence It comprises the amino acid sequence of SEQ ID NO: 10, 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 may comprise (i) glutamine at residue 5 of VH CDR2 of the anti-L1CAM antibody or antigen-binding fragment thereof; (ii) serine at residue 8 of VL CDR1; And/or (iii) proline at residue 8 of VL CDR3. In certain aspects, the at least one amino acid difference comprises (i) alanine, glycine, phenylalanine, tyrosine, threonine, proline and tryptophan at residues 3 to 9 of each VL CDR3 of the anti-L1CAM antibody or antigen-binding fragment thereof; (ii) alanine, glycine, phenylalanine, tyrosine, serine, proline and tryptophan at residues 3 to 9 of VL CDR3, respectively, or (iii) leucine, valine or histidine, tryptophan or phenylalanine, tyrosine at residues 4 to 9 of VL CDR3, respectively , Proline and tryptophan.

In certain aspects, the anti-L1CAM antibody or antigen-binding fragment thereof cross-competes for binding to the L1CAM epitope with a reference antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein (a) a reference The VH of the antibody comprises SEQ ID NO: 23, 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, or (c) the VH of the reference antibody includes SEQ ID NO: 27, the VL of the reference antibody includes SEQ ID NO: 28, or (d) the VH of the reference antibody includes SEQ ID NO: 29, and the VL of the reference antibody includes 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.

Competitive antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (eg, 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.

Techniques for determining whether two antibodies bind to the same epitope include, for example, epitope mapping methods, such as x-ray analysis and hydrogen/deuterium exchange of crystals of antigen: antibody complexes that provide atomic resolution of the epitope. Mass spectroscopy (HDX-MS), a method of monitoring the binding of an antibody to an antigen fragment or mutated variant of an antigen, wherein the loss of binding due to modification of amino acid residues within the antigen sequence is mainly considered as an indication of an epitope component. , Methods, and computer combination methods for epitope mapping.

In certain aspects, the specification describes, for example, by immunoassays (e.g., ELISA), surface plasmon resonance, BLI (biolayer interferometry) or epidemiologic exclusion assays, compared to another protein of the L1CAM family. 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher Antibodies or antigen-binding fragments thereof that bind to L1CAM (e.g., human L1CAM) with affinity are provided.In a specific aspect, the present specification crosses with another protein of the L1CAM family when measured, e.g., by immunoassay. Antibodies or antigen-binding fragments thereof that bind to L1CAM (eg, human L1CAM) without reactivity are provided.

In certain aspects, the anti-L1CAM antibody is not a native antibody and is not a naturally occurring antibody. For example, anti-L1CAM antibodies have different post-translational modifications than those of naturally occurring antibodies, e.g., by having more, fewer and different types of post-translational modifications.

III. Exemplary anti-L1CAM antibody

Certain antibodies that can be used in the methods disclosed herein include antibodies with CDR and/or variable region sequences of antibodies Ab612, Ab4H5, Ab2C2, Ab4H6 and Ab5D12 constructed in Examples 1 and 2, e.g., monoclonal antibodies. As well as 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%) with a variable region or CDR sequence thereof. % Or about 100% identity), e.g., a monoclonal antibody. The VH amino acid sequences of Ab612, Ab4H5, Ab2C2, Ab4H6 and Ab5D12 are shown in SEQ ID NOs: 23, 25, 27, 29 and 31, respectively. The VL amino acid sequences of Ab612, Ab4H5, Ab2C2, Ab4H6 and Ab5D12 are shown in SEQ ID NOs: 24, 26, 28, 30 and 32, respectively.

[Table 4a]

[Table 4b]

Accordingly, provided herein is an isolated anti-L1CAM antibody or antigen-binding fragment thereof comprising a heavy chain 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 do. In another aspect, the isolated anti-L1CAM antibody or antigen binding fragment thereof comprises a CDR of a heavy chain variable region selected from the group consisting of SEQ ID NOs: 2, 10 or 4.

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

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

An isolated anti-L1CAM antibody or antigen-binding fragment thereof comprising a heavy and light chain variable region is also provided, (i) wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 23 and the light chain variable region is SEQ ID NO: 24 Contains the amino acid sequence of; (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.

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 set forth in SEQ ID NO: 23, 25, 27, 29 or 31 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 amino acid sequences.

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 is the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30 or 32. 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 .

Also provided is an isolated anti-L1CAM antibody or antigen-binding fragment thereof comprising a heavy and light chain variable region, wherein the heavy chain variable region is at least about 80% of the amino acid sequence set forth in SEQ ID NOs: 23, 25, 27, 29 and 31. , 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 amino acid sequences, wherein the light chain variable region 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% of the amino acid sequence set forth in SEQ ID NOs: 24, 26, 28, 30 or 32 , At least about 99% or about 100% identical amino acid sequences.

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

(a) a heavy and light chain variable region sequence comprising SEQ ID NOs: 23 and 24, respectively;

(b) a heavy and light chain variable region sequence comprising SEQ ID NOs: 25 and 26, respectively;

(c) a heavy and light chain variable region sequence comprising SEQ ID NOs: 27 and 28, respectively;

(d) a heavy and light chain variable region sequence comprising SEQ ID NOs: 29 and 30, respectively; or

(e) a heavy chain and light chain variable region sequence comprising SEQ ID NOs: 31 and 32, respectively.

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

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

(a) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2; And/or

(b) VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10; And/or

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

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds human L1CAM comprises:

(a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; And/or

(b) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; And/or

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

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds human L1CAM comprises:

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

(b) 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) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; And/or

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

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds human L1CAM comprises:

(a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; And/or

(b) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; And/or

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

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds human L1CAM comprises:

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

(b) 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) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; And/or

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

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds human L1CAM comprises:

(a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; And/or

(b) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; And/or

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

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds human L1CAM comprises:

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

(b) 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) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; And/or

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

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds human L1CAM comprises:

(a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; And/or

(b) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; And/or

(C) VL CDR3 comprising the amino acid sequence of SEQ ID NO: 19.

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds human L1CAM comprises:

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

(b) 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) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 7; And/or

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

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

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

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

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

In some aspects, the anti-L1CAM antibody or antigen-binding fragment thereof that specifically binds human L1CAM comprises a heavy chain (HC) and a light chain (LC), wherein the HC of the antibody or antigen-binding fragment thereof is SEQ ID NO: 46, and the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 47.

[Table 5a]

[Table 5b]

The VH domains described herein, or one or more CDRs thereof, may be linked to a constant domain to form a heavy chain, eg, a full-length heavy chain. Similarly, the VL domain, or one or more CDRs thereof, can be linked to a constant domain to form a light chain, e.g., a full-length light chain. The full-length heavy chain and the full-length light chain can be combined to form a full-length antibody.

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

With respect to the heavy chain, in some aspects, the heavy chain of an antibody described herein may be an alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In another specific aspect, the heavy chain of an antibody described herein may comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In one aspect, 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, wherein the constant region of the heavy chain is It contains the amino acid sequence of the human gamma (γ) heavy chain constant region. In another aspect, an antibody disclosed 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, wherein the constant region of the heavy chain Includes amino acids of the human heavy chain described herein or known in the art. Non-limiting examples of human constant region sequences are known in the art, see, for example, US Pat. No. 5,693,780 and Kabat EA et al, (1991), supra.

In some aspects, an antibody described herein that specifically binds to L1CAM (e.g., human L1CAM) comprises a VH CDR or VH CDR and a VL CDR or VL CDR described herein and a VL domain and a VH domain. Wherein the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule. In another specific aspect, an antibody described herein that specifically binds to L1CAM (e.g., human L1CAM) comprises a VL domain and a VH domain comprising any of the amino acid sequences described herein, wherein the constant The region comprises the amino acid sequence of the constant region of IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any subtype of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). . In some aspects, the constant region is naturally-occurring, including subtypes (e.g., IgG1, IgG2, IgG3 or IgG4) and allotypes (e.g., G1m, G2m, G3m and nG4m) and variants thereof. Contains the amino acid sequence of the constant region of human IgG (see, e.g., idarsson G. et al . Front Immunol . 5:520 (published online Oct. 20, 2014) and Jefferis R. and Lefranc MP, mAbs 1: 4, 1-7 (2009)]. In some aspects, the constant region comprises the amino acid sequence of the constant region of a phosphorus IgG1, IgG2, IgG3, or IgG4, or variant thereof.

In certain aspects, the anti-L1CAM antibody or antigen-binding portion thereof described herein does not have an Fc effector function, e.g., complement-dependent cytotoxicity (CDC) and/or antibody-dependent cell phagocytosis (ADCP). . Effector function is mediated by the Fc region, and the residue closest to the hinge region in the CH2 domain of the Fc region is responsible for the effector function of the antibody, because it is C1q (complement) and IgG- on effector cells of the innate immune system. This is because it contains quite overlapping binding sites for the Fc receptor (FcγR). In addition, IgG2 and IgG4 antibodies have a lower level of Fc effector function than IgG1 and IgG3 antibodies. Effector functions of antibodies can be reduced or avoided by different approaches known in the art, including: (1) antibody fragments lacking the Fc region (e.g., Fab, F(ab') ₂ , The use of single chain Fv (scFv), or sdAb) consisting of monomeric VH or VL domains; (2) Cells that cannot glycosylate proteins by deletion or alteration of residues to which sugars are attached, by removing sugars by enzymes, by producing antibodies in cells cultured in the presence of a glycosylation inhibitor, or by glycosylation of proteins (e.g., Production of glycosylated antibodies that may be generated by expressing the antibody in bacterial host cells, eg, see US Patent Publication No. 20120100140); (3) Use of an Fc region (e.g., an Fc region from an IgG2 or IgG4 antibody or a chimeric Fc region comprising a CH2 domain from an IgG2 or IgG4 antibody) from an IgG subtype with reduced effector function (e.g., U.S. See Patent Publication No. 20120100140 and Lau C. et al. J. Immunol . 191:4769-4777 (2013)); And (4) generation of an Fc region with a mutation with reduced or no Fc function (eg, US Patent Publication No. 20120100140 and US applications and PCT applications cited therein and in An et al. , mAbs 1: 6, 572-579 (2009)).

Thus, in some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein is a Fab, Fab', F(ab')2, Fv, single chain Fv(scFv) or sdAb consisting of a monomeric VH or VL domain. to be. Such antibody fragments are well known in the art and are described above.

In some aspects, an anti-L1CAM antibody or antigen-binding fragment thereof disclosed herein comprises an Fc region with reduced or no Fc effector function. In some aspects, the constant region comprises the amino acid sequence of the Fc region of human IgG2 or IgG4, and in some aspects, the anti-L1CAM antibody has an IgG2/IgG4 isotype. In some aspects, the anti-L1CAM antibody is 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 hinge region from IgG2 and a CH2 region from IgG4. It includes a chimeric Fc region or an Fc region having a mutation with 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 see US Patent Publication No. 20120100140 and US Patents and Publications and PCT Publications cited therein). In addition, an Fc region with reduced or no Fc effector function can be easily prepared by a person skilled in the art.

IV. Nucleic acid molecule

Another aspect of the invention relates to one or more nucleic acid molecules encoding any of the antibodies or antigen-binding fragments thereof described herein. Nucleic acids may be present in whole cells, in cell lysates or in partially purified or substantially pure form. Nucleic acids are prepared by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques known in the art, to other cellular components or other contaminants, e.g., other When purified from cellular nucleic acids (eg, chromosomal DNA linked to other chromosomal DNA, eg, DNA isolated in nature) or protein, “isolated” or “substantially pure” (F. Ausubel, et al. . , ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York]) The nucleic acids described herein may be, for example, DNA or RNA, and may contain intron sequences, or Or may not contain In certain aspects, the nucleic acid is a cDNA molecule.

The nucleic acids described herein can be obtained using standard molecular biology techniques. In the case of an antibody expressed by a hybridoma (e.g., a hybridoma made from a transgenic mouse carrying a human immunoglobulin gene as described further below), the light chain of the antibody produced by this hybridoma And cDNA encoding the heavy chain can be obtained by standard PCR amplification or cDNA cloning techniques. In the case of an antibody obtained from an immunoglobulin gene library (eg, using phage display technology), a nucleic acid encoding this antibody can be recovered from the library.

Certain nucleic acid molecules described herein are those encoding the VH and VL sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, Ab5D12 monoclonal antibodies. Exemplary DNA sequences encoding the VH sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, Ab5D12 are shown in SEQ ID NOs: 8, 14, 16, 18 or 22. Exemplary DNA sequences encoding the VL sequences of Ab612, Ab4H5, Ab2C2, Ab4H6, Ab5D12 are shown in SEQ ID NOs: 33, 34, 35, 36 and 37, respectively.

The method for producing an anti-L1CAM antibody described herein includes the step of expressing the heavy and light chains in a cell line comprising nucleotide sequences encoding heavy and light chains together with a signal peptide. Host cells comprising these nucleotide sequences are included in the present invention.

After the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments are further prepared by standard recombinant DNA techniques, e.g., to convert the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. Can be manipulated. 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 flexible linker. The term “operably linked” means that two DNA fragments are joined together such that the amino acid sequence encoded by the two DNA fragments remains in-frame.

The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (hinge, CH1, CH2 and/or CH3). Sequences of human heavy chain constant region genes are known in the art (e.g., Kabat, EA, et al. , (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242], DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, such as an IgG2 and/or IgG 4 constant region. In the case of the 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.

The isolated DNA encoding the VL region can be converted into a full-length light chain gene by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region CL. 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. 91-3242], DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

To generate the 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, so that the VL region And the VH region is linked by a flexible linker, so that the VH sequence and the VL sequence can be expressed as a contiguous single-stranded protein (see, eg, Bird et al. , (1988) Science 242:423-426; Huston et al. , (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al. , (1990) Nature 348:552-554)).

In some aspects, the invention provides a vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding portion thereof. In another aspect, the vector can be used in gene therapy.

Vectors suitable for the present invention include expression vectors, viral vectors, and plasmid vectors. In one aspect, the vector is a viral vector.

As used herein, expression vectors, when introduced into an appropriate host cell, contain essential elements for transcription and translation of the inserted coding sequence, or, in the case of RNA viral vectors, any essential elements for replication and translation. Refers to the nucleic acid construct of. Expression vectors may include plasmids, phagemids, viruses, and derivatives thereof.

The expression vector of the present invention may include a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein. In one aspect, the coding sequence for the antibody or antigen-binding fragment thereof is operably linked to an expression control sequence. 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. Coding sequences and gene expression control sequences are said to be operably linked when they are covalently linked in such a way that the expression or transcription and/or translation of the coding sequence is under the influence or control of the gene expression control sequence. In both DNA sequences, induction of the promoter in the 5'gene expression sequence causes transcription of the coding sequence, and the nature of the linkage between the two DNA sequences does not (1) cause the introduction of a frame-shift mutation, or (2) the coding sequence If it does not interfere with the ability of the promoter region to direct transcription or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein, it is said to be operably linked. Thus, the gene expression sequence is operably linked to the coding nucleic acid sequence if the gene expression sequence was able to achieve transcription of the coding nucleic acid sequence, whereby the transcript is translated into the desired antibody or antigen-binding fragment thereof.

Viral vectors include, but are not limited to, retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous' sarcoma virus; Lentivirus; Adenovirus; Adeno-associated virus; SV40-type virus; Polyomavirus; Epstein-Barr virus; Papilloma virus; Herpes virus; Vaccinia virus; Polio virus; And nucleic acid sequences from RNA viruses such as retroviruses. Other vectors well known in the art can readily be used. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with genes of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA and subsequently integration of the provirus into host cell DNA. Retrovirus has been approved for human gene therapy testing. Retroviruses with replication-defects (ie, can direct the synthesis of the desired protein but cannot produce infectious particles) are most useful. These genetically modified retroviral expression vectors have general utility in highly efficient transduction of genes in vivo. Standard protocols for producing replication-defective retroviruses (incorporation of foreign genetic material into plasmids, transfection of packaging cell lines into plasmids, production of recombinant retroviruses by packaging cell lines, collection of viral particles from tissue culture media, and target cells). Including the step of infection with viral particles), see Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, WH Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991)].

In one aspect, the virus is an adeno-associated virus, which is a double-stranded DNA virus. Adeno-associated viruses can be engineered to be replication defective and can infect a wide variety of cell types and species. This additionally results in thermal and lipid solvent stability; High frequency of transduction in various lineage cells, including hematopoietic cells; And a lack of inhibition of superinfection, thereby allowing multiple transductions. It has been reported that adeno-associated viruses can be integrated into human cellular DNA in a site-specific manner, thereby minimizing the probability of insertional mutagenesis and variability of the inserted gene expression characteristics of retroviral infection. In addition, wild-type adeno-associated virus infection was traced in tissue culture for more than 100 passages in the absence of selective pressure, suggesting that adeno-associated viral genome integration is a relatively stable event. In addition, adeno-associated viruses can function in an extrachromosomal manner.

In another aspect, the vector is derived from a lentivirus. In certain aspects, the vector is a vector of recombinant lentiviral capable of infecting non-dividing cells.

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

Adjacent to the 5'LTR is the sequence required for reverse transcription of the genome (tRNA primer binding site), which is effective for encapsidation of viral RNA into the particle (Psi site). When sequences essential for encapsidation (or packaging of retroviral RNA in infectious virions) are missing from the viral genome, a cis defect prevents encapsidation of genomic DNA.

However, the resulting mutation can still direct the synthesis of all virion proteins. The present disclosure relates to the production of recombinant lentiviruses capable of infecting non-dividing cells comprising transfecting suitable host cells with two or more vectors possessing packaging function, i.e. gag, pol and env, as well as rev and tat. Provides a way. As disclosed herein below, vectors lacking a functional tat gene are preferred for certain applications. Thus, for example, the first vector can provide a nucleic acid encoding viral gag and viral pol, and the other vector can provide a nucleic acid encoding viral env to produce a packaging cell. Introduction of a vector providing a heterologous gene, identified herein as a transfer vector, into a packaging cell provides a producer cell that releases an infectious viral particle carrying a foreign gene of interest.

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

Examples of retrovirus-derived env genes include, but are not limited to, Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcomavirus (HaMuSV or HSV), murine breast tumor virus (MuMTV or MMTV), gibbon monkey Leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV). Other env genes can also be used, for example, vesicular stomatitis virus (VSV) protein G (VSV G), hepatitis virus and influenza.

Vectors providing viral env nucleic acid sequences are operably associated with the regulatory sequences described herein.

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

Other vectors include plasmid vectors. Plasmid vectors are well known to those of skill in the art (eg, Sambrook et al ., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). Over the past few years, plasmid vectors have been shown to be particularly beneficial in delivering genes to cells in vivo due to their inability to replicate and integrate within the host genome. However, these plasmids having a promoter compatible with the host cell are capable of expressing peptides from genes operably encoded within the plasmid. Some commonly used plasmids available from commercial sources include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40, and pBlueScript. Further examples of specific plasmids are pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number V87020; pcDNA4/myc-His, catalog number V86320; And pBudCE4.1, catalog number V53220, all of which are products of Invitrogen (Invitrogen) (Carlsbad, CA). Other plasmids are also well 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.

V. Antibody production

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

In a specific aspect, an antibody described herein is an antibody (e.g., a monoclonal antibody) produced, expressed, produced or isolated by any means involving, for example, synthesis, generation through genetic manipulation of a DNA sequence. )to be. In certain aspects, such antibodies comprise sequences (eg, DNA sequences or amino acid sequences) that are not naturally present in the antibody germline repertoire of animals or mammals (eg, humans) in vivo.

In certain aspects, a method of making an antibody or antigen-binding fragment thereof that immunospecifically binds to L1CAM (e.g., human L1CAM), comprising the step of culturing the cell or host cell described herein. Is provided. In certain aspects, the present specification discloses an 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). A method of preparing an antibody or antigen-binding fragment thereof that immunospecifically binds to L1CAM (eg, human L1CAM) comprising the step of (eg, recombinantly expressing) is provided. In a particular aspect, the cell is an isolated cell. In a particular aspect, exogenous polynucleotides have been introduced into the cell. In a particular aspect, the method further comprises purifying the cell or antibody obtained from the host cell.

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

Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant and phage display techniques, or combinations thereof. For example, monoclonal antibodies are known in the art and are described, for example, in Arlow 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 produced recombinantly from host cells that exogenously express the antibodies or fragments thereof described herein, e.g., the light and/or heavy chains of such antibodies.

In certain aspects, a “monoclonal antibody” as used herein is an antibody produced by a single cell (eg, a hybridoma or host cell producing a recombinant antibody), wherein the antibody is, for example, For example, it specifically binds to L1CAM (eg, human L1CAM) when known in the art or as determined by ELISA or other antigen-binding or competitive binding assays of the examples provided herein. In a particular aspect, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In certain aspects, the monoclonal antibody is a monovalent antibody or a multivalent (eg, bivalent) antibody. In a particular aspect, the monoclonal antibody is a monospecific or multispecific antibody (eg, a bispecific antibody). Monoclonal antibodies described herein can be prepared, for example, by the hybridoma method described in Kohler G & Milstein C (1975) Nature 256: 495, which is incorporated herein by reference in its entirety, or , Or, for example, can be isolated from a phage library using the techniques described herein. Other methods for the production of clonal cell lines and monoclonal antibodies expressed thereby are known in the art (see, eg, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM, supra. et al. ]) .

Methods for producing and screening specific antibodies using hybridoma technology are routine and known in the art. For example, in the hybridoma method, a mouse or other suitable host animal, such as a mouse or sheep, goat, rabbit, rat, hamster or monkey, is specific for the protein used for immunization (e.g., human L1CAM). It is immunized to elicit lymphocytes that produce or are capable of producing antibodies to bind to 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)). In addition, the RIMMS (repetitive immunization multiple sites) technique can be used to immunize animals (Kilpatrick KE et al. , (1997) Hybridoma 16:381-9), which is incorporated by reference in its entirety.

In some aspects, a mouse (or other animal such as a rat, monkey, donkey, pig, sheep, hamster or dog) can be immunized with an antigen (e.g., L1CAM, such as human L1CAM), and an immune response is detected, For example, antigen-specific antibodies are detected in mouse serum, mouse spleens are harvested, and splenocytes are isolated. The splenocytes then form hybridomas in any suitable myeloma cells, e.g., cells from the cell line SP20 available from the American Type Culture Collection (ATCC) (Manassas, VA) by known methods. So that it is fused. Hybridomas are selected and cloned by limited dilution. In certain aspects, lymph nodes from immunized mice are harvested and fused with NSO myeloma cells.

The hybridoma cells thus prepared are preferably seeded and grown in an appropriate medium containing one or more substances that inhibit the growth and survival of unfused, maternal myeloma cells. For example, if maternal myeloma cells are deficient in the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas is hypoxanthine, aminopterin and thymidine, which prevent the growth of HGPRT-deficient cells. Will contain (HAT medium).

A specific aspect uses myeloma cells that fuse efficiently, support stable and high-level production of antibodies by selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these myeloma cell lines, murine myeloma cell lines such as NSO cell lines or those derived from MOPC-21 and MPC-11 mouse tumors available from the Sark Institute Cell Distribution Center (San Diego, CA), and SP-2 or X63-Ag8.653 cells available from ATCC (Rockville, MD). In addition, human myeloma and mouse-human heteromyeloma cell lines have 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)).

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

After hybridoma cells have been identified to produce antibodies of the desired specificity, affinity and/or activity, clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice]). Culture media suitable 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.

Monoclonal antibodies secreted by subclones are, for example, culture media by conventional immunoglobulin purification procedures such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, It is properly separated from ascites fluid or serum.

Antibodies described herein include antibody fragments that recognize specific L1CAM (eg, human L1CAM) and can be generated by any technique known to those of skill in the art. For example, the Fab and F(ab')2 fragments described herein are immunoglobulins using enzymes such as papain (to generate Fab fragments) or pepsin (to generate F(ab')2 fragments). It can be produced by proteolytic cleavage of a molecule. The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen-binding arms of an antibody molecule linked by disulfide bonds in the hinge region.

In one aspect, to generate a whole antibody, a PCR primer comprising a VH or VL nucleotide sequence, a restriction site and a flanking sequence to protect the restriction site is used to amplify the VH or VL sequence from a template, e.g., scFv clone. Can be used for Using cloning techniques known to those skilled in the art, the PCR amplified VH domain can be cloned into a vector expressing the VH constant region, and the PCR amplified VL domain contains a VL constant region, e.g., a human kappa or lambda constant region. It can be cloned into an expressing vector. In addition, the VH and VL domains can be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vector and light chain conversion vector can then be co-transduced into the cell line to generate stable or transient cell lines expressing full-length antibodies, such as IgG, using techniques known to those of skill in the art.

Chimeric antibodies are molecules in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain the variable region of a non-human animal (eg, 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 (see, for example, 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).

The humanized antibody is capable of binding to a predetermined antigen and has a framework region substantially having the amino acid sequence of a human immunoglobulin and an amino acid sequence of a non-human immunoglobulin (e.g., murine or chicken immunoglobulin). Includes CDRs. In certain aspects, the humanized antibody also comprises an immunoglobulin constant region (Fc), typically at least a fragment of a human immunoglobulin. Antibodies may also comprise the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4. Humanized antibodies are CDR-grafted (European Patent No. EP 239400; International Publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101 and 5,585,089), veneering or resurfacing. (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4/5): 489-498; Studnicka GM et al., (1994) Prot Engineering 7(6): 805 -814; And Roguska MA et al., (1994) PNAS 91: 969-973]), chain shuffling (US Pat. No. 5,565,332), and, for example, US Pat. No. 6,407,213, US Pat. No. 5,766,886 , International Publication No. WO 93/17105; Tan P et al., (2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng 9(10): 895 904; Couto JR et al., (1995) Cancer Res. 55 (23 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]. Can be produced using a variety of known methods (see also US Patent Publication No. 2005/0042664 A1 (February 24, 2005), incorporated herein by reference in its entirety).

Methods of making multispecific (e.g., bispecific antibodies) have been described (e.g., US Pat. Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620) No. 6,132,992 and 8,586,713).

Single domain antibodies, e.g., antibodies deficient in the light chain, can be prepared by methods well known in the art (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 No. WO 94/04678 , See WO 94/25591 and WO 01/44301).

Additionally, antibodies that specifically bind to the L1CAM antigen can, consequently, be used to generate anti-idiotypic antibodies that “mimic” the antigen using techniques known to those of skill in the art (eg, literature See Greenspan NS & Bona CA (1989) FASEB J 7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438).

In a particular aspect, an antibody described herein that binds to the same epitope of L1CAM (eg, human L1CAM) as an anti-L1CAM antibody described herein is a human antibody or antigen-binding fragment thereof. In a particular aspect, antibodies described herein (e.g., Ab612, Ab4H5, Ab2C2, Ab4H6 and Ab5D12) are competitively (e.g., in a dose dependent manner) from binding to L1CAM (e.g., human L1CAM). Blocking antibodies described herein are human antibodies or antigen-binding fragments thereof.

Human antibodies can be produced using any method known in the art. For example, transgenic mice, which cannot express functional endogenous immunoglobulins, but are capable of expressing human immunoglobulin genes, can be used. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy and light chain genes, the human variable region, constant region and diversity region can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be non-functional separately or simultaneously with introduction into human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region interferes with endogenous antibody production. The transformed embryonic stem cells are expanded and microinjected into the blastocyst to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in a normal manner with a selected antigen, eg, all or a fragment of an antigen (eg, L1CAM). Monoclonal antibodies directed against the antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques. Human immunoglobulin transgenes carried by transgenic mice rearrange during B cell differentiation, and then undergo class changeover and somatic mutation. Thus, using this technique, it is possible to produce IgG, IgA, IgM and IgE antibodies useful for treatment. For an overview of this technique for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93. A detailed discussion of such techniques for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies can be found in, for example, International Publication Nos. WO 98/24893, WO 96/34096 and WO 96/33735. ; And U.S. Patents 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 Xenomouse ^TM (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HuAb-Mouse ^TM (Mederex, Inc.)/ Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse ^™ (Kirin) and KM Mouse ^™ (Mederex/Kirin).

Human antibodies that specifically bind to L1CAM (e.g., human L1CAM) can be prepared by various methods known in the art, including the phage display method described above using an antibody library 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. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, See WO 96/34096, WO 96/33735 and WO 91/10741).

In some aspects, human antibodies can be produced using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas secreting human monoclonal antibodies, and these mouse-human high Bridoma can be screened to determine to secrete a human monoclonal antibody that specifically binds to a target antigen (eg, L1CAM, eg, human L1CAM). Such methods are known and described in the art and are described, for example, in Shinmoto H et al. , (2004) Cytotechnology 46: 19-23; Naganawa Y et al. , (2005) Human Antibodies 14: 27-31.

VI. Cells and vectors

In certain aspects, the present specification discloses a cell (e.g., a host cell) expressing (e.g., recombinantly) an antibody described herein that specifically binds to L1CAM (e.g., human L1CAM), and Related polynucleotides and expression vectors are provided. Provided herein is a vector (e.g., an expression vector) comprising a polynucleotide comprising 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 is a host cell comprising a vector for recombinantly expressing an anti-L1CAM antibody described herein (eg, a human or humanized antibody). In certain aspects, provided herein is a method of producing an antibody described herein comprising the step of expressing such antibody from a host cell.

Recombinant expression of an antibody described herein that specifically binds to L1CAM (e.g., human L1CAM) (e.g., a full-length antibody described herein, a heavy chain and/or light chain or single chain antibody of the antibody) is an antibody It includes the construction of an expression vector containing a polynucleotide encoding a. Once the polynucleotide encoding the antibody molecule described herein, the heavy chain and/or light chain of the antibody, or a fragment thereof (eg, heavy and/or light chain variable domain) is obtained, a vector for antibody molecule production is known in the art. It can be produced by recombinant DNA technology using conventional techniques. Thus, described herein is a method of preparing a protein by expressing a polynucleotide comprising an antibody or antibody fragment (eg, light or heavy chain) encoding a nucleotide sequence. Expression vectors comprising the coding sequence for an antibody or antibody fragment (eg, light or heavy chain) and appropriate transcriptional and translational control signals can be constructed using methods known to those of skill in the art. These methods include, for example, recombinant DNA technology in vitro, synthetic technology and gene recombination in vivo. In addition, the present specification includes an antibody molecule described herein, operably linked to a promoter, a heavy or light chain of an antibody, a heavy or light chain variable region of an antibody, or a fragment thereof, or a nucleotide sequence encoding a heavy or light chain CDR. A replicable vector is provided. Such vectors include, for example, a nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Patent Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Patent No. 5,122,464). And the variable domains of the antibody can be cloned into such vectors for expression of the entire heavy chain, all light chains, or all heavy and light chains.

Expression vectors can be delivered to cells (e.g., host cells) by conventional techniques, and the cells produced thereafter are cultured by conventional techniques to obtain antibodies (e.g., Ab612, Ab4H5, Ab2C2) described herein. , An antibody comprising at least one of the VH and/or VL, or VH and/or VL CDRs of Ab4H6 and Ab5D12) or fragments thereof. Accordingly, herein, the antibody described herein or a fragment thereof, or a heavy or light chain or fragment thereof, or a single chain antibody described herein, operably linked to a promoter for expression of such sequence in a host cell Host cells containing polynucleotides are provided. In certain aspects, for expression of double chain antibodies, vectors encoding both heavy and light chains separately can be co-expressed in host cells for expression of the entire immunoglobulin molecule, as described below. In certain aspects, the host cell comprises a vector comprising a polynucleotide encoding both the heavy and light chains or fragments thereof of an antibody described herein. In a specific aspect, the host cell is a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody or fragment thereof described herein, which is two different vectors, and a light chain of the antibody or fragment thereof described herein. Or a second vector comprising a polynucleotide encoding a light chain variable region. In another aspect, the first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody or fragment thereof described herein, and the second host cell is a light chain of an antibody described herein. Or a second vector comprising a polynucleotide encoding a light chain variable region. In a specific aspect, the heavy/heavy chain variable region expressed by the first cell is associated with the light/light chain variable region of the second cell to form the anti-L1CAM antibody or antigen-binding fragment thereof described herein. In certain aspects, provided herein are such first host cells and populations of host cells comprising such second host cells.

In a particular aspect, the present specification discloses a first vector comprising a polynucleotide encoding a light/light chain variable region of an anti-L1CAM antibody described herein and a heavy/heavy chain variable region of an anti-L1CAM antibody described herein. A population of vectors comprising a second vector comprising a polynucleotide is provided.

A variety of host-expression vector systems can be used to express the antibody molecules described herein. Such host-expression systems represent a vehicle from which the coding sequence of interest can be produced and then purified, but when transformed or transduced with the appropriate nucleotide coding sequence, the antibody molecule described herein is in situ. Represents a cell that can be expressed. These include bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences (eg, E. coli and B. subtilis ); Yeast transformed with a recombinant yeast expression vector containing the antibody coding sequence (eg, Saccharomyces Pichia ); Insect cell systems infected with a recombinant viral expression vector (eg, baculovirus) comprising an antibody coding sequence; Plant cells infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector containing an antibody coding sequence (e.g., Ti plasmid) Systems (eg, green algae such as Chlamydomonas reinhardtii); Alternatively, a recombinant containing a promoter derived from the genome of a mammalian cell (eg, metallothionein promoter) or derived from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter) Mammalian cell system (e.g., COS (e.g., COS1 or COS)), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, NIH 3T3, HEK-293T, HepG2, SP210, Rl.l, BW, LM, BSCl, BSC40, YB/20, SP2/0, Sf9, human lymphoblastoid, NS0, bow melanoma, HT-1080, PERC.6 and BMT10 cells)). In a specific aspect, the cells for expressing the antibodies described herein are CHO cells, eg, CHO cells of CHO GS System ^™ (Lonza). In certain aspects, a cell for expressing an antibody described herein is a human cell, eg, a human cell line. In a specific aspect, the mammalian expression vector is pOptiVEC ^™ or pcDNA3.3 In certain aspects, in particular bacterial cells, such as Escherichia coli or eukaryotic cells (e.g., for the expression of whole recombinant antibody molecules) Mammalian cells) are used for the expression of the recombinant antibody molecule. Mammalian cells, such as Chinese hamster ovary (CHO) cells, are effective expression systems for antibodies, for example, with vectors such as major intermediate early gene promoter elements from human cytomegalovirus (Foecking). MK & Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al. , (1990) Biotechnology 8(7): 662-7)). In certain aspects, the antibodies described herein are produced by CHO cells or NSO cells. In a specific aspect, expression of a nucleotide sequence encoding an antibody described herein that specifically binds to L1CAM (eg, human L1CAM) is regulated by a constitutive promoter, an inducible promoter or a tissue specific promoter.

In bacterial systems, multiple expression vectors can be advantageously selected depending on the intended use for the antibody molecule to be expressed. For example, when such antibodies are to be produced in large quantities for the production of pharmaceutical compositions of antibody molecules, a vector directing the expression of a high level of easily purified fusion protein product may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO, in which the antibody coding sequence is individually ligated to the vector within the frame of the lacZ coding region to produce a fusion protein). J 2: 1791-1794); 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 also be used to express foreign polypeptides, as fusion proteins with glutathione 5-transferase (GST). In general, these fusion proteins are soluble and can be readily purified from lysed cells by adsorbing and binding to matrix glutathione agarose beads and then eluting in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or factor Xa protease cleavage site, so that the cloned target gene product can be freed from the GST moiety.

In insect systems, AcNPV (Autographa californica nuclear polyhedrosis virus) can be used as a vector expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be individually cloned into a non-essential region of the virus (eg, polyhedrin gene) and located under the AcNPV promoter (eg, polyhedrin promoter).

In mammalian host cells, a number of virus-based expression systems can be used. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, such as a late promoter and tripartite leader sequence. These chimeric genes 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., the E1 or E3 region) will produce a recombinant virus that can survive in an infected host and express antibody molecules (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. Additionally, the start codon should be equivalent to the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can be of a variety of natural and synthetic origins. Expression efficiency can be improved by including appropriate transcription enhancer elements, transcription terminators, and the like (see, eg, Bitter G et al. , (1987) Methods Enzymol . 153:516-544).

In addition, the host cell can be selected to modulate the expression of the inserted sequence or to modify and process the gene product in the specific manner desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of the protein product may be important to the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells can be used that possess cellular machinery for the proper processing of the primary transcription, glycosylation and phosphorylation of the gene product. These mammalian host cells are CHO, SKOV-3, B16-F1, NCI-H522, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (any Murine myeloma cell line that does not inherently produce immunoglobulin chains), CRL7030, COS (e.g., COS 1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, Rl. l, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells. In certain aspects, the anti-L1CAM antibodies described herein are produced in mammalian cells, eg, CHO cells.

In a specific aspect, the antibody or antigen-binding fragment thereof described herein has a reduced or no fucose content. Such antibodies can be produced using techniques known to those of skill in the art. For example, antibodies can be expressed in cells that lack or lack the ability of fucosylate. In a specific example, a cell line in which both alleles of 1,6-fucosyltransferase are knocked out can be used to produce antibodies with reduced fucose content. The Potelligent ^® system (Lonza) is an example of a system that can be used to produce antibodies or antigen-binding fragments thereof with reduced fucose content.

For long-term, high-yield production of recombinant proteins, stable expressing cells can be generated. For example, cell lines stably expressing the anti-L1CAM antibody described herein can be engineered. In a specific aspect, the cells provided herein stably express a light/light chain variable region and a heavy/heavy chain variable region that are associated to form an antibody or antigen-binding fragment thereof described herein.

In certain aspects, rather than using an expression vector comprising a viral origin of replication, the host cell is DNA regulated by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and It can be transformed with a selectable marker. After introduction of the foreign DNA/polynucleotide, the engineered cells can be grown for 1-2 days in a concentrated medium and then converted to a selective medium. The selectable marker of the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into the chromosome, grow and form a foci that can be cloned in turn and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing the anti-L1CAM antibodies or fragments thereof described herein. Such engineered cell lines may be particularly useful for screening and evaluation of compositions that directly or indirectly interact with antibody molecules.

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. Multiple selection systems can be used that do not. In addition, antimetabolite resistance can be used as a selection basis for the following genes: dhfr conferring resistance to methotrexate (Wigler M et al. , (1980) PNAS 77(6): 3567-70; O 'Hare K et al. , (1981) PNAS 78: 1527-31); Gpt conferring resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); Neo conferring resistance to aminoglycoside G-418 (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 conferring resistance to hygromycin (Santerre RF et al. , (1984) Gene 30(1-3): 147-56). Methods generally known in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are incorporated herein by reference in their entirety, 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 Chapters 12 and 13, Dracopoli NC et al. , (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F et al. , (1981) J Mol Biol 150: 1-14.

In some aspects, an antibody or antigen-binding fragment thereof of the present disclosure can be expressed in immune cells, such as T cells and/or NK cells. In some aspects, the antibody or antigen-binding fragment thereof can be expressed as a chimeric antigen receptor (CAR). CAR-T cells are T cells that express chimeric antigen receptors. The old "chimeric antigen receptor (CAR)" as used herein refers to an antigen-specific extracellular (or Ectodomain) domain. Chimeric antigen receptors are distinguished from other antigen binding agents by their ability to bind MHC-independent antigens and transmit activation signals through the intracellular domain.

In some aspects, the antigen-specific extracellular domain of the chimeric antigen receptor recognizes and specifically binds to an antigen, ie, L1CAM. L1CAM-specific extracellular domains suitable for use in the CARs of the present disclosure can be any antigen-binding polypeptide, many of which are known in the art. In some examples, the antigen-binding domain is a single chain Fv (scFv) or Fab. In another aspect, antigen binding fragments useful for CARs of the present disclosure include antigen-binding fragments disclosed elsewhere herein.

In some aspects, the transmembrane domain useful for the CAR is linked to an extracellular domain and may comprise a naturally occurring transmembrane domain. In another aspect, the transmembrane domain useful for CAR is the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, CD8 or this It may be derived from the alpha chain, beta chain or zeta chain of any other known in the specification.

The term “intracellular domain” refers to the portion of a CAR that transmits effector function signals when an antigen binds to the extracellular domain and directs T cells to perform a particular function. In one aspect, the intracellular domain for the CAR comprises an immune receptor tyrosine-based activation motif activation motif (ITAM). In some aspects, the ITAM is CD3 zeta (ζ, zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d, 4-1 BB, DAP-10, OX40 or It is derived from Fc[epsilon] RI[gamma].

In some aspects, the CARs of the present disclosure further comprise a co-stimulatory domain that can be linked to an intracellular domain. The co-stimulatory domain in the CAR construct is capable of transducing signals and activating cells as part of the intracellular portion of the CAR. In some aspects, the co-stimulatory domain is CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD7 , LIGHT, NKG2C or B7-H3.

In another aspect, the CAR of the present disclosure further comprises a linker. Short oligopeptides or polypeptide linkers may exist between the transmembrane domain and the intracellular domain. In some aspects, the linker is not limited to a specific length as long as the intracellular domain of the CAR is capable of inducing T cell activation when the extracellular domain binds to the antigen, ie L1CAM. In some aspects, the linker comprises a (Gly4Ser)3 linker.

In another aspect, the disclosure includes a polynucleotide encoding a CAR of the disclosure or a vector comprising a polynucleotide.

As used herein, the term “T cell” is a lymphocyte derived from the thymus and contributes to the immune response of the cell. T cells include CD4 + T cells (helper T cells, TH cells), CD8 + T cells (cytotoxic T cells, CTL), memory T cells, regulatory T cells (Treg) or natural killer T cells. In some aspects, the T cell into which the CAR is introduced is a CD8 + T cell.

VII. Immunoconjugates, antibody derivatives and diagnostic agents

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 an appropriate detectable agent to form an immunoconjugate. . For diagnostic purposes, suitable agents are detectable labels, including radioisotopes for systemic imaging and radioisotopes for sample testing, enzymes, fluorescent labels and other suitable antibody tags.

Detectable labels that can be linked to any of the anti-L1CAM antibodies described herein are metal sols such as colloidal gold, isotopes such as N ₂ S ₂ , N ₃ S or N ₄ types of peptidic chelating ^{I 125} or Tc ⁹⁹ provided with an agent, a particulate label including a chromophore including a fluorescent marker, a luminescent marker, a phosphorescent marker, etc., as well as an enzymatic label that converts a given substrate into a detectable marker and, for example, by polymerase chain reaction. It can be of any of the various types currently used in the field of in vitro diagnostic agents, including polynucleotide tags that appear after amplification. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label is a 1,2-dioxetane substrate such as adamantyl methoxy phosphoryloxy phenyl dioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane- 3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decane}-4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-star ^® or other luminescence well known to those skilled in the art A substrate, for example an enzyme alkaline phosphatase, which is detected by measuring the presence or formation of chemiluminescence after conversion of a chelate of suitable lanthanides such as terbium (III) and europium (III). The appearance of a label or its reaction product can be achieved with the naked eye if the label is particulate and accumulated to an appropriate level, or by using instruments such as spectrophotometers, luminometers, fluorometers, etc. all in accordance with standard practice. I can.

In some aspects, the conjugation method is a substantially (or near) non-immunogenic linkage, e.g., a peptide- (i.e., amide-), sulfide-, (stereo-disorder), disulfide-, hydrazone- and ether. It generates linkage. These linkages are almost non-immunogenic and show reasonable stability in serum (eg, Sender, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244); International Patent Publication WO WO 2009/059278; see WO 95/17886).

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

In general, site-specific reactions and covalent couplings are based on the conversion of natural amino acids into amino acids with reactivity orthogonal to the reactivity of other functional groups present. For example, certain cysteines within a rare sequence context can be enzymatically converted in aldehydes (see Frese, MA, and Dierks, T., ChemBioChem . 10 (2009) 425-427). In addition, it is possible to obtain the desired amino acid modifications by using the specific enzymatic reactivity of a particular enzyme with natural amino acids in the context of a given sequence (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); Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403 uses protease-catalyzed formation of CN bonds. Site specific reactions and covalent coupling can also be achieved by selective reaction of terminal amino acids with appropriate modifying reagents.

Site-specific covalent couples using the reactivity of N-terminal cysteine and benzonitrile (see Ren, H. et al. , Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662). Ring can be achieved.

Native chemical ligation can also depend on C-terminal cysteine residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

US6437095 B1 describes a method of conjugation based on the faster reaction of cysteine located in a stretch of positively charged amino acids and cysteine in a stretch of negatively charged amino acids.

The moiety can also be a synthetic peptide or peptide mimetic. When the polypeptide is chemically synthesized, amino acids having orthogonal chemical reactivity can be incorporated during this synthesis (see, for example, de Graaf, AJ et al. , Bioconjug. Chem . 20 (2009) 1281-1295 ). Since a wide variety of orthogonal functional groups exist and can be introduced into synthetic peptides, conjugation of such peptides to linkers is standard chemistry.

To obtain single-labeled polypeptides, 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. Since the difference in charge and molecular weight can be used for separation, by using this kind of labeled and highly negatively charged binding pair member, a single conjugated polypeptide can contain a non-labeled polypeptide and more than one linker. It is easily separated from the retaining polypeptide. Fluorescent dyes can be useful for purifying complexes from non-binding components, such as labeled monovalent binders.

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

Anti-L1CAM antibodies described herein can also be conjugated to therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include metabolic antagonists, alkylating agents, DNA trough binding agents, DNA intercalating agents, DNA crosslinking agents, histone deacetylase inhibitors, nuclear transport 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. ADC is described in US Pat. No. 7,087,600; 6,989,452; And 7,129,261; PCT Publication No. WO 02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; And WO 08/103693; US Patent Publication No. 20060024317; US20060004081; And 20060247295.

In some aspects, 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-neoplastic agent, and any combination thereof.

In some aspects, the immunoconjugate comprises an anti-L1CAM antibody and a cytotoxin. The cytotoxin can be selected from any cytotoxin known in the art. In some aspects, the cytotoxin is dolastatin, monomethyl auristatin E (MMAE), maytansine, duocarmycin, calicheamicin, pyrrolobenzodia. Zepine, duokamycin, centanamycin, SN38, doxorubicin, derivatives thereof, synthetic analogues thereof, and any combination thereof. In certain aspects, the immunoconjugate comprises an anti-L1CAM antibody and cytotoxin A. In another aspect, the immunoconjugate comprises an anti-L1CAM antibody and a non-cytotoxic drug.

In some aspects, the immunoconjugate comprises an anti-L1CAM antibody and a radioactive agent. In some aspects, the radioactive agent is a radionucleotide. In certain aspects, the radioactive agent includes radioactive iodine. In certain aspects, the radioactive agent comprises 131-iodine. In another aspect, the radioactive agent comprises the radioactive isotope yttrium-90.

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

In certain aspects, the immunoconjugate comprises an anti-L1CAM antibody and an anti-neoplastic agent. The anti-neoplastic agent can be any such agent known in the art. In some aspects, the anti-neoplastic agent is epirubicin. In some aspects, the anti-neoplastic agent is a super antigen. In certain aspects, the super antigen is staphylococcal enterotoxin A (SEA/E-120; estafenatox).

Anti-L1CAM antibodies, such as those described herein, can be used to detect L1CAM on the surface of cells, such as human L1CAM, such as human L1CAM or soluble L1CAM in serum. Antibodies can be used, for example, in ELISA assays or flow cytometry. In some aspects, the anti-L1CAM antibody is contacted with the cells or serum for an appropriate time for specific binding to occur, followed by addition of a reagent, eg, an antibody detecting an anti-L1CAM antibody. An exemplary assay is provided in the Examples. L1CAM in the sample (serum), For example, an exemplary method for detecting stool expressed L1CAM or soluble L1CAM (sL1CAM) is (i) anti-L1CAM for a time sufficient to allow specific binding of the anti-L1CAM antibody to L1CAM in the sample. Contacting the antibody with the antibody, and (2) contacting the sample with an antibody that specifically binds to the Fc region of a detection reagent, e.g., an anti-L1CAM antibody, e.g., an anti-L1CAM antibody. And detecting the bound L1CAM. A washing step may be included after incubation with the antibody and/or detection reagent. Since individual detection reagents can be used, anti-L1CAM antibodies for use in this method need not be linked to a label or detection reagent.

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

VIII. Bispecific molecule

Anti-L1CAM antibodies described herein can be used to form bispecific molecules. The anti-L1CAM antibody or antigen-binding portion thereof is derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., a ligand for another antibody or receptor), so that at least 2 Bispecific molecules can be generated that bind to two different binding sites or target molecules. For example, an anti-L1CAM antibody can be linked to an antibody or scFv that specifically binds any protein that can be used as a potential target for combination therapy. Antibodies described herein can in fact be derivatized or linked to more than one other functional molecule, resulting in multispecific molecules that bind to more than two different binding sites and/or target molecules, such Multispecific molecules are also intended to be included in the term "bispecific molecule" as used herein. In order to generate the bispecific molecules described herein, the antibodies described herein are functionally linked to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic (e.g., By chemical coupling, gene fusion, non-covalent association or otherwise) bispecific molecules can be generated.

Accordingly, 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 aspects described herein where the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity.

In some aspects, the bispecific molecules described herein include at least one antibody, or e.g., Fab, Fab', F(ab')2, Fv, or single chain Fv (scFv) as binding specificity. , And antibody fragments thereof. The antibody may also be a light or heavy chain dimer or any minimal fragment thereof, such as an Fv or single chain construct, as described in US Pat. No. 4,946,778 (Ladner et al.).

Bispecific molecules described herein can be prepared by conjugating component binding specificities using methods known in the art. For example, each binding specificity of a bispecific molecule can be generated separately and then conjugated to each other. When the binding specificity is a protein or a peptide, various coupling or crosslinking agents can be used for covalent conjugation (see, for example, Karpovsky et al. (1984) J. Exp. Med . 160: 1686; Liu, MA et al. al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Another method is 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 bonding agents are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, IL).

IX. Composition

In the present specification, a composition formulated with a pharmaceutically acceptable carrier, e.g., one or a combination of anti-L1CAM antibodies or a combination with an antibody against another target, or an antigen-binding portion thereof S) containing, for example, pharmaceutical compositions are further provided. Such compositions may include (eg, two or more different) antibodies or one or a combination of the immunoconjugates or bispecific molecules described herein. For example, the pharmaceutical compositions described herein may comprise a combination of antibodies (or immunoconjugates or bispecific) that bind to different epitopes on a target antigen or have complementary activity.

The pharmaceutical compositions described herein can also be administered in combination therapy, ie in combination with other agents. For example, the combination therapy may comprise an anti-L1CAM antibody described herein in combination with at least one other anti-cancer and/or immunomodulatory, e.g., T-cell stimulating (e.g., activating) agent. . Examples of therapeutic agents that can be used in combination therapy are described in detail below in the section on the use of anti-L1CAM antibodies described herein.

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

In another aspect, the composition comprises a third antibody. In another aspect, the third antibody is any antibody disclosed herein.

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

As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In some aspects, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). The choice for subcutaneous injection is based on Halozyme Therapeutics' ENHANZE® drug-delivery technology and includes a co-formulation of Ab and recombinant human hyaluronidase enzyme (rHuPH20), which is an extracellular The substrate removes the traditional limitation on the volume of biological agents and drugs that can be delivered subcutaneously (US Pat. No. 7,767,429). Depending on the route of administration, active compounds, i.e. antibodies, immunoconjugates or bispecific molecules can be coated on the material to protect the compound from acids and other natural states that can inactivate the compound.

The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. “Pharmaceutically acceptable salt” means a salt that retains the desired biological activity of the parent compound and does not impart undesired toxic effects (see, eg, Berge, SM, et al. (1977) J Pharm. Sci. 66: 1-19A]. The pharmaceutical compositions described herein may also include pharmaceutically acceptable antioxidants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by the above sterilization procedures and by including various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid and the like. In addition, it may be desirable to include isotonic agents such as sugar, sodium chloride, and the like in the composition. In addition, absorption of injectable pharmaceutical forms can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except where any conventional medium or agent is incompatible with the active compound, its use in the pharmaceutical compositions described herein is contemplated. The pharmaceutical composition may or may not contain a preservative. Supplementary active compounds may be incorporated into the composition.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper flowability can be maintained, for example, by the use of coatings such as lecithin, maintenance of the required particle size in the case of dispersions and the use of surfactants. In many cases, the composition may include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride. Long-term absorption of the injectable composition can be caused by including in the composition an agent that delays absorption, such as monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the required amount of active compound in an appropriate solvent with one or a combination of the ingredients listed above as needed, followed by sterile microfiltration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those listed herein. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum drying and freeze-drying (lyophilization), which is the desired method from the active ingredient and previously sterile filtered solution. It produces a powder of additional ingredients.

For example, for administration of an anti-L1CAM antibody described herein, the dosage ranges from about 0.0001 to 100 mg/kg.

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 indicated ranges. Antibodies are generally administered several times.

Antibodies can be administered in sustained release formulations, in which case less frequent administration is required. Dosage and frequency depend on the patient's antibody half-life. In general, human antibodies exhibit the longest half-life, followed by humanized, chimeric and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic application, relatively low dosages are administered at relatively sparse 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 the patient shows partial or complete improvement of disease symptoms. Thereafter, the patient can be administered a prophylactic regime.

A "therapeutically effective dosage" 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 periods, or prevention of damage or disorder due to disease pain. have. With regard to cancer, a therapeutically effective dose may preferably result in increased survival, eg, overall survival and/or prevention of further exacerbation of the physical symptoms associated with cancer. Symptoms of cancer are well known in the art and include, for example, changes in the appearance of the nevus, including unusual nevus features, asymmetry, borders, color and/or diameter, newly pigmented skin areas, abnormal nevus, under the nail. Blackened areas, breast masses, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, worsening breathing difficulties, hemoptysis, hematuria, blood stool, nausea, vomiting, liver metastases , Lung metastasis, bone metastasis, abdominal satiety, abdominal swelling, peritoneal fluid, vaginal bleeding, constipation, abdominal distension, colon perforation, acute peritonitis (infection, fever, pain), pain, hematomes, hyperhidrosis, fever, high blood pressure, anemia, diarrhea , Jaundice, dizziness, chills, muscle spasms, colon metastasis, lung metastasis, bladder metastasis, liver metastasis, bone metastasis, kidney metastasis, and pancreatic metastasis, difficulty swallowing, and the like.

A therapeutically effective dose may prevent or delay the onset of cancer, which may be desirable if there are early or preliminary signs of disease. Laboratory tests used to diagnose cancer include chemistry (including measurement of L1CAM levels), hematology, serology and radiology. Thus, any clinical or biochemical assay monitoring any of the above can be used to determine whether a particular treatment is a therapeutically effective dose to treat cancer. One of skill in the art will be able to determine this amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.

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 one of skill in the art will understand, the route and/or mode of administration will depend on the desired outcome. Routes of administration of the anti-L1CAM antibodies described herein may include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. As used herein, the phrase "parenteral administration" generally refers to a mode of administration other than intestinal and topical administration by injection, but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intracystic, orbital Intracardiac, intradermal, intraperitoneal, transtraminal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intrathecal, epidural and intrasternal injections and infusions.

Alternatively, the antibodies described herein can potentially be administered via parenteral routes, such as topical, epidermal or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical.

X. Kit

Provided herein are kits comprising one or more antibodies or antigen-binding fragments thereof, bispecific molecules or immunoconjugates thereof described herein. In a specific aspect, the specification includes 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, one or more containers with optional instructions for use. Pharmaceutical packs or kits are provided. In some aspects, a kit comprises a pharmaceutical composition described herein and a prophylactic or therapeutic agent, such as those described herein.

XI. Use and method

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

Certain aspects of the present disclosure relate to a method of treating cancer in a subject in need thereof, which method provides an effective dose to the subject of a composition disclosed herein (e.g., antibody, polynucleotide, vector, Host cells, immunoconjugates or pharmaceutical compositions). In another aspect, the present disclosure relates to a method of inhibiting the shedding of L1CAM by tumor cells in a subject in need thereof, wherein the method provides an effective dose of the present disclosure to the subject. And administering a composition disclosed in the specification. In another aspect, the present disclosure relates to a method for inhibiting shedding of L1CAM in serum and/or inhibiting shedding of L1CAM in serum in a subject in need thereof and/or retaining L1CAM on the cell surface. And the method comprises administering to the subject an effective dose of a composition disclosed herein. In another aspect, the disclosure relates to a method of killing tumor cells in a subject in need thereof, the method comprising administering to the subject an effective dose of a composition disclosed herein. In another aspect, the present disclosure relates to a method of reducing the size of a tumor in a subject in need thereof, the method comprising administering to the subject an effective dose of a composition disclosed herein. In another aspect, the present disclosure relates to a method of inhibiting metastasis of a tumor in a subject in need thereof, the method comprising administering to the subject an effective dose of a composition disclosed herein. . In some aspects, the subject is a human.

The compositions of the present disclosure can be administered using any pharmaceutically acceptable route. In some aspects, the composition (e.g., antibody, polynucleotide, vector, host cell, immunoconjugate or pharmaceutical composition) is intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracystic , Intraorbital, intracardiac, intradermal, transtraminal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, intrasternal, topical, intradermal, intramucosal, or any combination thereof do. In some aspects, the composition is administered intravenously. In some aspects, the composition is administered subcutaneously.

In certain aspects, the method reduces the size of a cancer, eg, the size of a tumor, in the subject. In some aspects, the size of the cancer is 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 By 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

In some aspects, the method increases the overall survival of the subject. In some aspects, overall survival is increased relative to the average overall survival of subjects with the same cancer but treated with different therapies. In certain aspects, 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 times, at least about 3 times, at least about 5 times. In some aspects, overall survival is 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 It is increased by a year or at least about 10 years.

In some aspects, the method increases progression-free survival of the subject. In some aspects, overall survival is increased relative to the average progression-free survival of subjects with the same cancer but treated with different therapies. In certain aspects, 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 times, at least about 3 times, at least about 5 times. In some aspects, overall survival is 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 It is increased by a year or at least about 10 years.

In some aspects, the method increases the objective response rate in the subject. In certain aspects, the method elicits a complete response in the subject. In some aspects, the method induces a partial response in a subject.

In some aspects, the method comprises administering an anti-L1CAM antibody (or polynucleotide, vector, host cell or immunoconjugate) disclosed herein and a second therapy. In some aspects, the second therapy is administered prior to the anti-L1CAM antibody. In some aspects, the second therapy is administered after the anti-L1CAM antibody. In some aspects, the second therapy is administered concurrently with the anti-L1CAM antibody. In certain aspects, the anti-L1CAM antibody and the second therapy are administered separately. In another aspect, the anti-L1CAM antibody and the second therapy are administered in a single formulation.

The second therapy can be any other therapy known in the art. In some aspects, the second therapy comprises immunotherapy. In some aspects, the second therapy comprises chemotherapy. In some aspects, the second therapy comprises radiotherapy. In some aspects, the second therapy comprises surgery. In some aspects, the second therapy comprises administering a second therapeutic agent.

Anti-L1CAM antibodies can enhance the immune response against cancerous cells in patients with cancer. Accordingly, provided herein is a method of treating a subject having cancer, wherein the method is administered to the subject by administering the anti-L1CAM antibody described herein to treat the subject, e. Reducing and/or regressing the tumor and/or allowing prolonged survival to be achieved. Anti-L1CAM antibodies can be used alone to inhibit the growth of cancerous tumors. Alternatively, an anti-L1CAM antibody can be used in conjunction with another agent, e.g., another immunogenic agent, standard cancer treatment, or another antibody, as described below.

Accordingly, provided herein is a method of treating cancer comprising administering to the subject a therapeutically effective amount of an anti-L1CAM antibody described herein, eg, by inhibiting the growth of tumor cells in a replacement. Cancers whose growth can be inhibited using the antibodies of the present disclosure include cancers that are typically responsive to immunotherapy, and cancers that are typically not responsive to immunotherapy. Cancers that can be treated also include L1CAM positive cancers. The cancer can be a solid tumor or a cancer with hematologic malignancies (liquid tumors). Non-limiting examples of cancers for treatment include 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, kidney cancer ( For example, 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) Adenocarcinoma), thyroid cancer, neuroblastoma, pharyngeal cancer, laryngeal cancer, oral cancer, cancer of connective tissue, Hodgkin's lymphoma, lymphoma, multiple myeloma, pancreatic cancer, glioblastoma (glioblastoma polymorphic), cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer , Colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer, germ cell tumor, pediatric sarcoma, nasal sinus natural killer, melanoma (e.g., metastatic malignant melanoma, such as skin or intraocular malignant melanoma), bone cancer, Skin cancer, uterine cancer, cancer of the anus, testicular cancer, carcinoma of the fallopian tube, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, adrenal gland Cancer, soft tissue sarcoma, urethral cancer, penile cancer, rectal cancer, childhood solid tumor, ureteral cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal tumor, Environmentally induced cancer including brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal cancer, squamous cell carcinoma, cancer induced by asbestos, virus-related cancer or cancer of viral origin, e.g. human papilloma Viruses (HPV-associated or -originating tumors)), and any combination of these cancers.

In some aspects, the anti-L1CAM antibody is a patient with cancer that exhibits an inappropriate response to a previous treatment, e.g., a previous treatment with an immuno-oncology or immunotherapy drug, or is inherently refractory or resistant or in a resistant or refractory state. Is administered to patients with acquired, refractory or resistant cancer. For example, a subject that is not responsive or not sufficiently responsive to a first therapy or whose disease progression is observed after treatment can be treated by administration of an anti-L1CAM antibody alone or in combination with another therapy.

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

In some aspects, a method of treating cancer in a subject comprises the steps of first determining whether the subject is L1CAM positive, e.g., has tumor cells expressing L1CAM, and if the subject has L1CAM positive cancer, the subject is e.g. , Administering an anti-L1CAM antibody described herein. A method of treating a subject having cancer with an anti-L1CAM antibody may include administering a therapeutically effective amount of an L1CAM antibody to the subject having cancer cells expressing L1CAM. Also provided herein is a method of predicting whether a subject will respond to treatment with an anti-L1CAM antibody, wherein the method determines the level of L1CAM in the patient's cancer cells, and the subject's cancer cells are L1CAM positive. If so, determining that the subject is likely to respond to treatment with the L1CAM antibody.

Anti-L1CAM antibodies can be administered as a standard of care treatment. Anti-L1CAM antibodies can be administered as maintenance therapy, therapy intended to prevent the occurrence or recurrence of tumors.

Anti-L1CAM antibodies can be administered in conjunction with another treatment, eg radiation, surgery or chemotherapy. For example, anti-L1CAM antibody adjuvant therapy can be administered when there is a risk that micrometastasis may be present and/or to reduce the risk of recurrence.

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

Anti-L1CAM antibodies can also be combined with standards of care (eg, surgery, radiation, and chemotherapy). Administration of anti-L1CAM antibodies can be effectively combined with chemotherapy regimens. For example, the dose of chemotherapeutic agent administered can be reduced (Mokyr et al. (1998) Cancer Research 58: 5301-5304).

The practice of this disclosure will use conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology that are within the skill of the art, unless otherwise indicated. These techniques are fully described in the literature (see, for example, 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); DN Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis]; Mullis et al. 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, 2 ^nd Ed. CRC Press (2007) and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.)).

The following examples are presented by way of explanation and not by way of limitation.

Example

The following experimental methods and details are referred to in the following examples.

Example 1. Construction of Ab612 by site-directed mutagenesis

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. WO2014/077648, which are incorporated herein by reference. In order to improve the biophysical properties of the Ab417 antibody, the effect of each amino acid constituting the variable region of the Ab417 antibody on the binding ability of the Ab417 antibody was investigated.

DNA was synthesized to retain the mutations R16G, D54E, K76A, and P88A in the heavy chain variable region of the Ab417 antibody, and recombined with the gene encoding the CH1 domain of human Cγ1 by polymerase chain reaction (PCR). The resulting PCR product was electrophoresed on a 1.5% agarose gel. The band containing the DNA was cut out and purified using a PCR-purification kit (GeneAll®). Both ends of the purified DNA were digested with restriction enzymes Nco I and Apa I (New England Biolabs), and the human Fab phage display vector pKRIBB-FabD (ref) having the full length of Gene III was modified. Subcloned into the Nco I site and the Apa I site of the modified version (pKRIBB-full GIII). The resulting recombinant phagemid was named pKRIBB-full GIII-Fd612.

DNA was synthesized to retain the mutations I31S and V95P in the light chain variable region of the Ab417 antibody, and recombined with the gene encoding human Cκ by PCR. The resulting PCR product was electrophoresed on a 1.5% agarose gel. The band containing the DNA was cut out and purified using a PCR-purification kit (GeneAll®). Both ends of the purified DNA were digested with restriction enzyme Bst XI (New England Biolabs), and subcloned into the Bst XI site of pKRIBB-full GIII-Fd612. The resulting recombinant phagemid was named pKRIBB-full GIII-Fab612. To this end, an antibody containing the mutations R16G, D54E, K76A, and P88A in the heavy chain variable region, and the mutations I31S and V95P in the light chain variable region of the Ab417 antibody was named Ab612 antibody.

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

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

To isolate monoclonal antibodies that recognize human L1CAM, human L1CAM was used as an antigen in the first panning round, the second panning round and the third panning round.

After 3 rounds of panning, 470 colonies were randomly selected, individually inoculated into 96-well plates, and then incubated in 300 μl 2×YT/carbenicillin/glucose for 8 hours at 37°C. Thereafter, 30 μl of cells were seeded in each well containing 1 ml of 2×YT/carbenicillin/glucose medium, and cultured for 2 hours until OD600 reached 0.5. Cells were infected with KM13 helper phage at a multiplicity of infection (MOI) of 20 without shaking at 37° C. for 30 minutes, and then incubated for 30 minutes with shaking. The infected cells were centrifuged at 2900×g for 10 minutes, then resuspended in 2×YT/carbenicillin/kanamycin, and incubated at 30° C. for 12 hours. Supernatants containing Fab phage from 470 colonies were subjected to indirect and quantitative ELISA.

Among these, 41 clones that bind to highly active human L1CAM were selected, and plasmid DNA was isolated and sequenced. As a result, it was found that 32 clones differ from each other.

Indirect ELISA was performed to analyze the binding capacity of 32 unique clones to human L1CAM. As a result, it was found that four clones showed the highest antigen-binding ability (ie, Ab4H5, Ab2C2, Ab4H6 and Ab5D12).

Example 3. Conversion and production of Fab to IgG1

In order to convert the Fab to whole IgG1 using the codon-optimized sequence for mammalian cells, the heavy and light chain variable regions with the leader sequence were amplified by PCR, and in the mammalian IgG1 expression plasmid pdCMV-dhfr-Ab612 vector, respectively. It was subcloned into EcoRI and ApaI sites (New England Biolabs ) and Bsi WI and Hind III sites (New England Biolabs). HEK293F cells were cultured, and an IgG1 expression plasmid was transfected using ExpiFectamin (Thermo Fisher Scientific) according to the ExpiCHO protocol. 7 to 14 days after transfection, the cell culture supernatant was centrifuged and filtered using a bottle top filter (0.22 μm PES, Sartorius), as described in Example 5 below. Likewise, the productivity of each mutant was compared with the existing Ab417 antibody through ELISA.

As shown in Table 8, the mutant antibody showed 1.25 to 1.44 times higher productivity than Ab417. Ab612 improved 1.44 times, Ab4H5, Ab2C2, Ab4H6, and Ab5D12 improved by 1.25 times, 1.26 times, 1.36 times and 1.26 times, respectively.

Example 4. Purification of Ab417 variant antibody

Plasmid DNA of the expression vectors of these antibodies was obtained in large quantities, and expressed in ExpiCHO cells in the same manner as in Examples 1 to 3, respectively. 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 antibodies were eluted from Protein A using 0.1 M sodium citrate solution (pH 3.2). Then, 1.0M Tris solution (pH 8.0) was added immediately to the eluted antibody for neutralization. Purified IgG1 was dialyzed using a PD-10 column containing Sephadex G-25 (GE Healthcare) and then stored in a buffer solution (10 mM Napi 5% sorbitol 0.01% Tween 20). The concentration of the purified antibody was measured by Nanodrop (Thermo Fisher Scientific, Nanodrop 2000) based on the molar extinction coefficient. The purified antibody was stained with 10% SDS-PAGE and Coomassie to confirm that each heavy and light chain was expressed and assembled into whole IgG.

Example 5. Characterization of Ab417 variant antibodies

The affinity of the purified antibody for human L1 CAM was determined by competition ELISA. Human L1CAM was prepared at a density of 1×10-7M and serially diluted ^{to 1×10 −12} M using 0.1% PBA buffer (PBS containing 0.1% BSA) solution. Each antibody was prepared by dilution in a specific concentration of 0.1% PBA buffer solution according to its binding capacity. The diluted antigen and antibody were reacted with each other at 37° C. for 3 hours in the same volume ratio. A 96-well plate (MaxiSorp, Nunc) was diluted at a concentration of 100 ng/well in a buffer (15 mM Na ₂ CO ₃ , 34.84 mM NaHCO3, pH 9.6) using purified human L1 CAM at 4°C overnight Coated. The next day, Difco skim milk (BD) was dissolved in 0.05% PBS-T buffer at a concentration of 2%, 200 ul was added to each well, and incubated at 37° C. for 1 hour. Wells were washed twice with 0.05% PBS-T buffer solution. Then, 100ul of the antigen/antibody reactant reacted for 3 hours was added, followed by reaction at room temperature for 1 hour. The wells were washed 3 times with 0.05% PBS-T buffer to remove non-antigen binding antibodies. Goat anti-human IgG(Fc)-HRP (Invitrogen, 1/10000), which specifically recognizes the Fc region of a human antibody, was added as a secondary antibody and reacted at 37° C. for 1 hour. The wells were washed 4 times with 0.05% PBS-T buffer solution to remove the remaining secondary antibody. In order to investigate the affinity by color development, 100 ul of a solution (BD OptEIA, BD) containing TMB as a substrate for the enzyme HRP (covalently linked to the secondary antibody) was added to each well, and incubated at room temperature for 5 minutes. . Finally, 50ul of 2.5M HSO solution was added to each well to terminate the enzymatic reaction. After completion of the reaction, the absorbance was measured at 450 nm (VERSAmax microplate reader, Molecular Devices). The results are presented in Table 9. The affinity of Ab417 for human L1CAM was 5×10-10M, the affinity of Ab612 was 2.6×10-10M, the affinity of the Ab4H5 mutant was 6×10-11M, and the affinity of the Ab2C2 mutant was 5 ×10-11M, the affinity of the Ab4H6 mutant was 6×10-11M, and the affinity of the Ab5D12 mutant was 1.2×10-10M. The Ab417 variant antibody showed about 2-10 times higher binding affinity for human L1CAM than the Ab417 antibody.

Example 6. Analysis of antigen-binding specificity of Ab417 variants

To investigate whether the variant antibody selectively binds to human and mouse L1CAM, flow cytometry was performed using various types of cells. In this regard, as comparative antibodies, Ab417 antibodies that bind to human L1 CAM and mouse L1 CAM were used.

CHO-DG44 (ATCC No. PTA-3356) cells known not to express human L1CAM were cultured and used as a negative control. In addition, cells expressing human L1CAM, human ovarian carcinoma, SKOV3 (ATCC number HTB-77) and cells expressing human non-small cell lung carcinoma, NCI-H522 (AYCC NO. CRL-5810) were cultured. B16F1 (ATCC No. CRL-6323) was cultured as cells expressing mouse L1CAM and melanoma cell lines. Cultured cells were harvested using dissociation buffer or 0.05% trypsin for CHO-DG44 cells (GIBCO), resuspended in 1% PBA solution, and placed on ice for 20 minutes. The cells were then added to a round bottom polystyrene test tube (Falcon) at a density of ^{4×10 5 cells per tube.} The purified antibody was diluted in PBA solution at a concentration of 10 ug/ml, and 100 ul was added to each tube and mixed well. The tube was placed at 4° C. for 1 hour. A secondary antibody specifically binding to the Fc region of human IgG and covalently linked with FITC (Sigma) was added to each tube at a ratio of 1:2000. The plate was wrapped with foil to block light, and reacted at 4° C. for 1 hour. Staining reagent PI (propidium iodide, Sigma) was added at a ratio of 1:200 to evaluate cell viability. After all reactions were completed, FITC and PI fluorescence signals were detected in the cells. 1A shows that the variant antibody does not bind to L1CAM negative cells (ie, CHO-DG44). 1B-1D show that the variant antibody binds to human L1CAM-positive cells, NCI-H522 (FIG. 1B), SKOV3 (FIG. 1C) and mouse L1CAM-positive cells B16F1 (FIG. 1D). The variant antibody specifically binds to the mouse melanoma cell line B166F1, suggesting that the Ab417 variant of the present invention specifically binds to both human and mouse L1CAM.

Example 7. Quality measurement of purified Ab417 variants by SEC-HPLC

HPLC grade UPLC solvent and PBS were purchased from Fisher Scientific (Fair Lawn, NJ) and GIBCO (St. Louis, MO, USA), respectively. Size exclusion chromatography was used to separate antibody samples with a Biosuit High Resolution SEC column (7.5×300 mm, 250Å particle size). Samples were separated using PBS pH 7.4 with an isocratic flow. Waters® e2695 Separations Module was used for the experiment, and the 2489 UV/Vis Detector monitored the absorbance at 280nm. Size exclusion-high performance liquid chromatography (SEC-HPLC) analysis shows that the variant antibody exhibited monomeric peaks (Figs. 2A).

Example 8. Affinity of Ab417 variants for human L1CAM

The affinity of Ab417 and Ab417 variants for human L1CAM was investigated by BLI using Octet RED384 (ForteBio). For affinity analysis, the antibody was diluted with a PBA solution prepared by adding 0.1% BSA to PBS. Human L1CAM was serially diluted with PBA at concentrations of 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 or 0 nM, and then 200ul of diluted L1CAM was added to an opaque 96 well plate to prevent light transmission. By using an AHC (anti-human IgG Fc capture) sensor chip, PBA solution, antibody, PBA solution, antigen, and PBA solution are delivered to the sensor chip in this order, while investigating the change in refractive index that occurs during the association and dissociation of the antibody and antigen. The binding kinetics of antibodies to antigens were analyzed.

Table 10 shows that the affinity of Ab417 for human L1CAM (K _D ) is about 0.2 nM, the affinity of Ab612 is 0.1 nM, the affinity of Ab4H5 is 8 pM, the affinity of Ab2C2H is out of range, and that of Ab4H6. The affinity was 0.07nM, indicating that the affinity of Ab5D12 was 0.09nM. The increased affinity of the variant compared to Ab417 (eg, up to 25 times) was due to the slower dissociation rate.

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 equipped with a neutral coated capillary (Sciex PN 477441). All experiments were performed 3 times. 32 Karat software was used to determine the Pl value for each material. Table 11 shows that the pi value of Ab417 antibody is 9.62, the pi value of Ab612 is 9.25, the pi value of Ab4H5 is 8.96, the pi value of Ab2C2 is 8.96, the pi value of Ab4H6 is 9.03, and the pl value of Ab5D12 is 9.04. Indicates that it is.

Example 10. Analysis of tumor growth-inhibition of Ab417 variants

To investigate the anti-tumor effect of the Ab612 antibody, male Balb/c nude mice were transplanted with the human-derived cholangiocarcinoma cell line Choi-CK. The constructed Choi-Ck tumor tissue (3×3×3 mm 3) was inoculated on the back of mice. After tumor volume reached 100 mm 3 (n=8 per group), 10 mg/kg dose of Ab417 antibody, 10 mg/kg dose of Ab612 antibody, 3.3 mg/kg dose of control hFc antibody and negative control (PBS) or (Vehicle) was injected iv 3 times a week for 3 weeks. The tumor volume, the body weight of the mouse, and the tumor weight were measured and shown in FIGS. 1A to 1C, respectively.

3A and 3C show that the Ab612 treatment group showed an average tumor volume of 311 mm 3 and a tumor weight of 0.20 g, while the Ab417 treatment group showed an average tumor volume of 387 mm 3 and a tumor weight of 0.41 g. The sham treated control mice showed an average tumor volume of 1096 mm 3 and a tumor weight of 0.93 g. The results suggest that Ab612 (78.2% tumor growth inhibition rate (IR)) showed a 1.4-fold higher tumor growth inhibition compared to Ab417 (55.5% IR) based on the tumor weight (Fig. 3C). 3D shows tumor images of each group of 8 mice sacrificed after the end of the experiment as described in FIGS. 3A-3C. The size of cancer tissues extracted from 8 mice treated with Ab612 antibody was smaller than that treated with Ab417 antibody. The anticancer effect of the Ab612 antibody appears to work well in vivo, improving the tumor microenvironment (TME) through antibody penetration into cancerous tissues or immune cell-based attack environments.

Therefore, the group treated with the Ab612 antibody disclosed herein showed a significant inhibitory effect on tumor growth compared to the group administered with the negative control group. In addition, weight loss was not observed in mice during administration, and toxicity by antibody administration was not observed.

The above description of the specific aspect is that the reader can easily modify/modify this specific aspect for various applications by applying knowledge within the present technology without undue experimentation, without departing from the general concept of the present invention. The general character will be fully revealed. Accordingly, such adaptations and variations are intended to be within the meaning and scope of equivalents of the disclosed aspects, based on the teachings and guidelines presented herein. Since the phraseology or terminology herein is for the purpose of description and not limitation, it should be understood that the terminology or phraseology herein should be interpreted by those skilled in the art in light of the teachings and guidelines.

Other aspects of the invention will be apparent to those skilled in the art in view of the description and practice of the invention disclosed herein. The specification and examples are considered to be illustrative only, and the true scope and spirit of the invention are presented by the following claims.

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

SEQUENCE LISTING <110> Kangwon National University University-Industry Cooperation Foundation <120> ANTI-L1CAM ANTIBODY AND USES THEREOF <130> WO/2020/003210 <150> US 62/865,871 <151> 2019-06-24 <150> KR 10-2018-0075955 <151> 2018-06-29 <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 1257 <212> PRT <213> Artificial Sequence <220> <223> Human L1CAM <400> 1 Met Val Val Ala Leu Arg Tyr Val Trp Pro Leu Leu Leu Cys Ser Pro 1 5 10 15 Cys Leu Leu Ile Gln Ile Pro Glu Glu Tyr Glu Gly His His Val Met 20 25 30 Glu Pro Pro Val Ile Thr Glu Gln Ser Pro Arg Arg Leu Val Val Phe 35 40 45 Pro Thr Asp Asp Ile Ser Leu Lys Cys Glu Ala Ser Gly Lys Pro Glu 50 55 60 Val Gln Phe Arg Trp Thr Arg Asp Gly Val His Phe Lys Pro Lys Glu 65 70 75 80 Glu Leu Gly Val Thr Val Tyr Gln Ser Pro His Ser Gly Ser Phe Thr 85 90 95 Ile Thr Gly Asn Asn Ser Asn Phe Ala Gln Arg Phe Gln Gly Ile Tyr 100 105 110 Arg Cys Phe Ala Ser Asn Lys Leu Gly Thr Ala Met Ser His Glu Ile 115 120 125 Arg Leu Met Ala Glu Gly Ala Pro Lys Trp Pro Lys Glu Thr Val Lys 130 135 140 Pro Val Glu Val Glu Glu Gly Glu Ser Val Val Leu Pro Cys Asn Pro 145 150 155 160 Pro Pro Ser Ala Glu Pro Leu Arg Ile Tyr Trp Met Asn Ser Lys Ile 165 170 175 Leu His Ile Lys Gln Asp Glu Arg Val Thr Met Gly Gln Asn Gly Asn 180 185 190 Leu Tyr Phe Ala Asn Val Leu Thr Ser Asp Asn His Ser Asp Tyr Ile 195 200 205 Cys His Ala His Phe Pro Gly Thr Arg Thr Ile Ile Gln Lys Glu Pro 210 215 220 Ile Asp Leu Arg Val Lys Ala Thr Asn Ser Met Ile Asp Arg Lys Pro 225 230 235 240 Arg Leu Leu Phe Pro Thr Asn Ser Ser Ser His Leu Val Ala Leu Gln 245 250 255 Gly Gln Pro Leu Val Leu Glu Cys Ile Ala Glu Gly Phe Pro Thr Pro 260 265 270 Thr Ile Lys Trp Leu Arg Pro Ser Gly Pro Met Pro Ala Asp Arg Val 275 280 285 Thr Tyr Gln Asn His Asn Lys Thr Leu Gln Leu Leu Lys Val Gly Glu 290 295 300 Glu Asp Asp Gly Glu Tyr Arg Cys Leu Ala Glu Asn Ser Leu Gly Ser 305 310 315 320 Ala Arg His Ala Tyr Tyr Val Thr Val Glu Ala Ala Pro Tyr Trp Leu 325 330 335 His Lys Pro Gln Ser His Leu Tyr Gly Pro Gly Glu Thr Ala Arg Leu 340 345 350 Asp Cys Gln Val Gln Gly Arg Pro Gln Pro Glu Val Thr Trp Arg Ile 355 360 365 Asn Gly Ile Pro Val Glu Glu Leu Ala Lys Asp Gln Lys Tyr Arg Ile 370 375 380 Gln Arg Gly Ala Leu Ile Leu Ser Asn Val Gln Pro Ser Asp Thr Met 385 390 395 400 Val Thr Gln Cys Glu Ala Arg Asn Arg His Gly Leu Leu Leu Ala Asn 405 410 415 Ala Tyr Ile Tyr Val Val Gln Leu Pro Ala Lys Ile Leu Thr Ala Asp 420 425 430 Asn Gln Thr Tyr Met Ala Val Gln Gly Ser Thr Ala Tyr Leu Leu Cys 435 440 445 Lys Ala Phe Gly Ala Pro Val Pro Ser Val Gln Trp Leu Asp Glu Asp 450 455 460 Gly Thr Thr Val Leu Gln Asp Glu Arg Phe Phe Pro Tyr Ala Asn Gly 465 470 475 480 Thr Leu Gly Ile Arg Asp Leu Gln Ala Asn Asp Thr Gly Arg Tyr Phe 485 490 495 Cys Leu Ala Ala Asn Asp Gln Asn Asn Val Thr Ile Met Ala Asn Leu 500 505 510 Lys Val Lys Asp Ala Thr Gln Ile Thr Gln Gly Pro Arg Ser Thr Ile 515 520 525 Glu Lys Lys Gly Ser Arg Val Thr Phe Thr Cys Gln Ala Ser Phe Asp 530 535 540 Pro Ser Leu Gln Pro Ser Ile Thr Trp Arg Gly Asp Gly Arg Asp Leu 545 550 555 560 Gln Glu Leu Gly Asp Ser Asp Lys Tyr Phe Ile Glu Asp Gly Arg Leu 565 570 575 Val Ile His Ser Leu Asp Tyr Ser Asp Gln Gly Asn Tyr Ser Cys Val 580 585 590 Ala Ser Thr Glu Leu Asp Val Val Glu Ser Arg Ala Gln Leu Leu Val 595 600 605 Val Gly Ser Pro Gly Pro Val Pro Arg Leu Val Leu Ser Asp Leu His 610 615 620 Leu Leu Thr Gln Ser Gln Val Arg Val Ser Trp Ser Pro Ala Glu Asp 625 630 635 640 His Asn Ala Pro Ile Glu Lys Tyr Asp Ile Glu Phe Glu Asp Lys Glu 645 650 655 Met Ala Pro Glu Lys Trp Tyr Ser Leu Gly Lys Val Pro Gly Asn Gln 660 665 670 Thr Ser Thr Thr Leu Lys Leu Ser Pro Tyr Val His Tyr Thr Phe Arg 675 680 685 Val Thr Ala Ile Asn Lys Tyr Gly Pro Gly Glu Pro Ser Pro Val Ser 690 695 700 Glu Thr Val Val Thr Pro Glu Ala Ala Pro Glu Lys Asn Pro Val Asp 705 710 715 720 Val Lys Gly Glu Gly Asn Glu Thr Thr Asn Met Val Ile Thr Trp Lys 725 730 735 Pro Leu Arg Trp Met Asp Trp Asn Ala Pro Gln Val Gln Tyr Arg Val 740 745 750 Gln Trp Arg Pro Gln Gly Thr Arg Gly Pro Trp Gln Glu Gln Ile Val 755 760 765 Ser Asp Pro Phe Leu Val Val Ser Asn Thr Ser Thr Phe Val Pro Tyr 770 775 780 Glu Ile Lys Val Gln Ala Val Asn Ser Gln Gly Lys Gly Pro Glu Pro 785 790 795 800 Gln Val Thr Ile Gly Tyr Ser Gly Glu Asp Tyr Pro Gln Ala Ile Pro 805 810 815 Glu Leu Glu Gly Ile Glu Ile Leu Asn Ser Ser Ala Val Leu Val Lys 820 825 830 Trp Arg Pro Val Asp Leu Ala Gln Val Lys Gly His Leu Arg Gly Tyr 835 840 845 Asn Val Thr Tyr Trp Arg Glu Gly Ser Gln Arg Lys His Ser Lys Arg 850 855 860 His Ile His Lys Asp His Val Val Val Pro Ala Asn Thr Thr Ser Val 865 870 875 880 Ile Leu Ser Gly Leu Arg Pro Tyr Ser Ser Tyr His Leu Glu Val Gln 885 890 895 Ala Phe Asn Gly Arg Gly Ser Gly Pro Ala Ser Glu Phe Thr Phe Ser 900 905 910 Thr Pro Glu Gly Val Pro Gly His Pro Glu Ala Leu His Leu Glu Cys 915 920 925 Gln Ser Asn Thr Ser Leu Leu Leu Arg Trp Gln Pro Pro Leu Ser His 930 935 940 Asn Gly Val Leu Thr Gly Tyr Val Leu Ser Tyr His Pro Leu Asp Glu 945 950 955 960 Gly Gly Lys Gly Gln Leu Ser Phe Asn Leu Arg Asp Pro Glu Leu Arg 965 970 975 Thr His Asn Leu Thr Asp Leu Ser Pro His Leu Arg Tyr Arg Phe Gln 980 985 990 Leu Gln Ala Thr Thr Lys Glu Gly Pro Gly Glu Ala Ile Val Arg Glu 995 1000 1005 Gly Gly Thr Met Ala Leu Ser Gly Ile Ser Asp Phe Gly Asn Ile 1010 1015 1020 Ser Ala Thr Ala Gly Glu Asn Tyr Ser Val Val Ser Trp Val Pro 1025 1030 1035 Lys Glu Gly Gln Cys Asn Phe Arg Phe His Ile Leu Phe Lys Ala 1040 1045 1050 Leu Gly Glu Glu Lys Gly Gly Ala Ser Leu Ser Pro Gln Tyr Val 1055 1060 1065 Ser Tyr Asn Gln Ser Ser Tyr Thr Gln Trp Asp Leu Gln Pro Asp 1070 1075 1080 Thr Asp Tyr Glu Ile His Leu Phe Lys Glu Arg Met Phe Arg His 1085 1090 1095 Gln Met Ala Val Lys Thr Asn Gly Thr Gly Arg Val Arg Leu Pro 1100 1105 1110 Pro Ala Gly Phe Ala Thr Glu Gly Trp Phe Ile Gly Phe Val Ser 1115 1120 1125 Ala Ile Ile Leu Leu Leu Leu Val Leu Leu Ile Leu Cys Phe Ile 1130 1135 1140 Lys Arg Ser Lys Gly Gly Lys Tyr Ser Val Lys Asp Lys Glu Asp 1145 1150 1155 Thr Gln Val Asp Ser Glu Ala Arg Pro Met Lys Asp Glu Thr Phe 1160 1165 1170 Gly Glu Tyr Arg Ser Leu Glu Ser Asp Asn Glu Glu Lys Ala Phe 1175 1180 1185 Gly Ser Ser Gln Pro Ser Leu Asn Gly Asp Ile Lys Pro Leu Gly 1190 1195 1200 Ser Asp Asp Ser Leu Ala Asp Tyr Gly Gly Ser Val Asp Val Gln 1205 1210 1215 Phe Asn Glu Asp Gly Ser Phe Ile Gly Gln Tyr Ser Gly Lys Lys 1220 1225 1230 Glu Lys Glu Ala Ala Gly Gly Asn Asp Ser Ser Gly Ala Thr Ser 1235 1240 1245 Pro Ile Asn Pro Ala Val Ala Leu Glu 1250 1255 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Anti-L1CAM VH-CDR1 <400> 2 Arg Phe Gly Met His 1 5 <210> 3 <211> 1253 <212> PRT <213> Artificial Sequence <220> <223> Human L1CAM isoform 2 <400> 3 Met Val Val Ala Leu Arg Tyr Val Trp Pro Leu Leu Leu Cys Ser Pro 1 5 10 15 Cys Leu Leu Ile Gln Ile Pro Glu Glu Tyr Glu Gly His His Val Met 20 25 30 Glu Pro Pro Val Ile Thr Glu Gln Ser Pro Arg Arg Leu Val Val Phe 35 40 45 Pro Thr Asp Asp Ile Ser Leu Lys Cys Glu Ala Ser Gly Lys Pro Glu 50 55 60 Val Gln Phe Arg Trp Thr Arg Asp Gly Val His Phe Lys Pro Lys Glu 65 70 75 80 Glu Leu Gly Val Thr Val Tyr Gln Ser Pro His Ser Gly Ser Phe Thr 85 90 95 Ile Thr Gly Asn Asn Ser Asn Phe Ala Gln Arg Phe Gln Gly Ile Tyr 100 105 110 Arg Cys Phe Ala Ser Asn Lys Leu Gly Thr Ala Met Ser His Glu Ile 115 120 125 Arg Leu Met Ala Glu Gly Ala Pro Lys Trp Pro Lys Glu Thr Val Lys 130 135 140 Pro Val Glu Val Glu Glu Gly Glu Ser Val Val Leu Pro Cys Asn Pro 145 150 155 160 Pro Pro Ser Ala Glu Pro Leu Arg Ile Tyr Trp Met Asn Ser Lys Ile 165 170 175 Leu His Ile Lys Gln Asp Glu Arg Val Thr Met Gly Gln Asn Gly Asn 180 185 190 Leu Tyr Phe Ala Asn Val Leu Thr Ser Asp Asn His Ser Asp Tyr Ile 195 200 205 Cys His Ala His Phe Pro Gly Thr Arg Thr Ile Ile Gln Lys Glu Pro 210 215 220 Ile Asp Leu Arg Val Lys Ala Thr Asn Ser Met Ile Asp Arg Lys Pro 225 230 235 240 Arg Leu Leu Phe Pro Thr Asn Ser Ser Ser His Leu Val Ala Leu Gln 245 250 255 Gly Gln Pro Leu Val Leu Glu Cys Ile Ala Glu Gly Phe Pro Thr Pro 260 265 270 Thr Ile Lys Trp Leu Arg Pro Ser Gly Pro Met Pro Ala Asp Arg Val 275 280 285 Thr Tyr Gln Asn His Asn Lys Thr Leu Gln Leu Leu Lys Val Gly Glu 290 295 300 Glu Asp Asp Gly Glu Tyr Arg Cys Leu Ala Glu Asn Ser Leu Gly Ser 305 310 315 320 Ala Arg His Ala Tyr Tyr Val Thr Val Glu Ala Ala Pro Tyr Trp Leu 325 330 335 His Lys Pro Gln Ser His Leu Tyr Gly Pro Gly Glu Thr Ala Arg Leu 340 345 350 Asp Cys Gln Val Gln Gly Arg Pro Gln Pro Glu Val Thr Trp Arg Ile 355 360 365 Asn Gly Ile Pro Val Glu Glu Leu Ala Lys Asp Gln Lys Tyr Arg Ile 370 375 380 Gln Arg Gly Ala Leu Ile Leu Ser Asn Val Gln Pro Ser Asp Thr Met 385 390 395 400 Val Thr Gln Cys Glu Ala Arg Asn Arg His Gly Leu Leu Leu Ala Asn 405 410 415 Ala Tyr Ile Tyr Val Val Gln Leu Pro Ala Lys Ile Leu Thr Ala Asp 420 425 430 Asn Gln Thr Tyr Met Ala Val Gln Gly Ser Thr Ala Tyr Leu Leu Cys 435 440 445 Lys Ala Phe Gly Ala Pro Val Pro Ser Val Gln Trp Leu Asp Glu Asp 450 455 460 Gly Thr Thr Val Leu Gln Asp Glu Arg Phe Phe Pro Tyr Ala Asn Gly 465 470 475 480 Thr Leu Gly Ile Arg Asp Leu Gln Ala Asn Asp Thr Gly Arg Tyr Phe 485 490 495 Cys Leu Ala Ala Asn Asp Gln Asn Asn Val Thr Ile Met Ala Asn Leu 500 505 510 Lys Val Lys Asp Ala Thr Gln Ile Thr Gln Gly Pro Arg Ser Thr Ile 515 520 525 Glu Lys Lys Gly Ser Arg Val Thr Phe Thr Cys Gln Ala Ser Phe Asp 530 535 540 Pro Ser Leu Gln Pro Ser Ile Thr Trp Arg Gly Asp Gly Arg Asp Leu 545 550 555 560 Gln Glu Leu Gly Asp Ser Asp Lys Tyr Phe Ile Glu Asp Gly Arg Leu 565 570 575 Val Ile His Ser Leu Asp Tyr Ser Asp Gln Gly Asn Tyr Ser Cys Val 580 585 590 Ala Ser Thr Glu Leu Asp Val Val Glu Ser Arg Ala Gln Leu Leu Val 595 600 605 Val Gly Ser Pro Gly Pro Val Pro Arg Leu Val Leu Ser Asp Leu His 610 615 620 Leu Leu Thr Gln Ser Gln Val Arg Val Ser Trp Ser Pro Ala Glu Asp 625 630 635 640 His Asn Ala Pro Ile Glu Lys Tyr Asp Ile Glu Phe Glu Asp Lys Glu 645 650 655 Met Ala Pro Glu Lys Trp Tyr Ser Leu Gly Lys Val Pro Gly Asn Gln 660 665 670 Thr Ser Thr Thr Leu Lys Leu Ser Pro Tyr Val His Tyr Thr Phe Arg 675 680 685 Val Thr Ala Ile Asn Lys Tyr Gly Pro Gly Glu Pro Ser Pro Val Ser 690 695 700 Glu Thr Val Val Thr Pro Glu Ala Ala Pro Glu Lys Asn Pro Val Asp 705 710 715 720 Val Lys Gly Glu Gly Asn Glu Thr Thr Asn Met Val Ile Thr Trp Lys 725 730 735 Pro Leu Arg Trp Met Asp Trp Asn Ala Pro Gln Val Gln Tyr Arg Val 740 745 750 Gln Trp Arg Pro Gln Gly Thr Arg Gly Pro Trp Gln Glu Gln Ile Val 755 760 765 Ser Asp Pro Phe Leu Val Val Ser Asn Thr Ser Thr Phe Val Pro Tyr 770 775 780 Glu Ile Lys Val Gln Ala Val Asn Ser Gln Gly Lys Gly Pro Glu Pro 785 790 795 800 Gln Val Thr Ile Gly Tyr Ser Gly Glu Asp Tyr Pro Gln Ala Ile Pro 805 810 815 Glu Leu Glu Gly Ile Glu Ile Leu Asn Ser Ser Ala Val Leu Val Lys 820 825 830 Trp Arg Pro Val Asp Leu Ala Gln Val Lys Gly His Leu Arg Gly Tyr 835 840 845 Asn Val Thr Tyr Trp Arg Glu Gly Ser Gln Arg Lys His Ser Lys Arg 850 855 860 His Ile His Lys Asp His Val Val Val Pro Ala Asn Thr Thr Ser Val 865 870 875 880 Ile Leu Ser Gly Leu Arg Pro Tyr Ser Ser Tyr His Leu Glu Val Gln 885 890 895 Ala Phe Asn Gly Arg Gly Ser Gly Pro Ala Ser Glu Phe Thr Phe Ser 900 905 910 Thr Pro Glu Gly Val Pro Gly His Pro Glu Ala Leu His Leu Glu Cys 915 920 925 Gln Ser Asn Thr Ser Leu Leu Leu Arg Trp Gln Pro Pro Leu Ser His 930 935 940 Asn Gly Val Leu Thr Gly Tyr Val Leu Ser Tyr His Pro Leu Asp Glu 945 950 955 960 Gly Gly Lys Gly Gln Leu Ser Phe Asn Leu Arg Asp Pro Glu Leu Arg 965 970 975 Thr His Asn Leu Thr Asp Leu Ser Pro His Leu Arg Tyr Arg Phe Gln 980 985 990 Leu Gln Ala Thr Thr Lys Glu Gly Pro Gly Glu Ala Ile Val Arg Glu 995 1000 1005 Gly Gly Thr Met Ala Leu Ser Gly Ile Ser Asp Phe Gly Asn Ile 1010 1015 1020 Ser Ala Thr Ala Gly Glu Asn Tyr Ser Val Val Ser Trp Val Pro 1025 1030 1035 Lys Glu Gly Gln Cys Asn Phe Arg Phe His Ile Leu Phe Lys Ala 1040 1045 1050 Leu Gly Glu Glu Lys Gly Gly Ala Ser Leu Ser Pro Gln Tyr Val 1055 1060 1065 Ser Tyr Asn Gln Ser Ser Tyr Thr Gln Trp Asp Leu Gln Pro Asp 1070 1075 1080 Thr Asp Tyr Glu Ile His Leu Phe Lys Glu Arg Met Phe Arg His 1085 1090 1095 Gln Met Ala Val Lys Thr Asn Gly Thr Gly Arg Val Arg Leu Pro 1100 1105 1110 Pro Ala Gly Phe Ala Thr Glu Gly Trp Phe Ile Gly Phe Val Ser 1115 1120 1125 Ala Ile Ile Leu Leu Leu Leu Val Leu Leu Ile Leu Cys Phe Ile 1130 1135 1140 Lys Arg Ser Lys Gly Gly Lys Tyr Ser Val Lys Asp Lys Glu Asp 1145 1150 1155 Thr Gln Val Asp Ser Glu Ala Arg Pro Met Lys Asp Glu Thr Phe 1160 1165 1170 Gly Glu Tyr Ser Asp Asn Glu Glu Lys Ala Phe Gly Ser Ser Gln 1175 1180 1185 Pro Ser Leu Asn Gly Asp Ile Lys Pro Leu Gly Ser Asp Asp Ser 1190 1195 1200 Leu Ala Asp Tyr Gly Gly Ser Val Asp Val Gln Phe Asn Glu Asp 1205 1210 1215 Gly Ser Phe Ile Gly Gln Tyr Ser Gly Lys Lys Glu Lys Glu Ala 1220 1225 1230 Ala Gly Gly Asn Asp Ser Ser Gly Ala Thr Ser Pro Ile Asn Pro 1235 1240 1245 Ala Val Ala Leu Glu 1250 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Anti-L1CAM VH-CDR3 <400> 4 Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro 1 5 10 <210> 5 <211> 1248 <212> PRT <213> Artificial Sequence <220> <223> Human L1CAM isoform 3 <400> 5 Met Val Val Ala Leu Arg Tyr Val Trp Pro Leu Leu Leu Cys Ser Pro 1 5 10 15 Cys Leu Leu Ile Gln Ile Pro Glu Glu Leu Met Glu Pro Pro Val Ile 20 25 30 Thr Glu Gln Ser Pro Arg Arg Leu Val Val Phe Pro Thr Asp Asp Ile 35 40 45 Ser Leu Lys Cys Glu Ala Ser Gly Lys Pro Glu Val Gln Phe Arg Trp 50 55 60 Thr Arg Asp Gly Val His Phe Lys Pro Lys Glu Glu Leu Gly Val Thr 65 70 75 80 Val Tyr Gln Ser Pro His Ser Gly Ser Phe Thr Ile Thr Gly Asn Asn 85 90 95 Ser Asn Phe Ala Gln Arg Phe Gln Gly Ile Tyr Arg Cys Phe Ala Ser 100 105 110 Asn Lys Leu Gly Thr Ala Met Ser His Glu Ile Arg Leu Met Ala Glu 115 120 125 Gly Ala Pro Lys Trp Pro Lys Glu Thr Val Lys Pro Val Glu Val Glu 130 135 140 Glu Gly Glu Ser Val Val Leu Pro Cys Asn Pro Pro Pro Ser Ala Glu 145 150 155 160 Pro Leu Arg Ile Tyr Trp Met Asn Ser Lys Ile Leu His Ile Lys Gln 165 170 175 Asp Glu Arg Val Thr Met Gly Gln Asn Gly Asn Leu Tyr Phe Ala Asn 180 185 190 Val Leu Thr Ser Asp Asn His Ser Asp Tyr Ile Cys His Ala His Phe 195 200 205 Pro Gly Thr Arg Thr Ile Ile Gln Lys Glu Pro Ile Asp Leu Arg Val 210 215 220 Lys Ala Thr Asn Ser Met Ile Asp Arg Lys Pro Arg Leu Leu Phe Pro 225 230 235 240 Thr Asn Ser Ser Ser His Leu Val Ala Leu Gln Gly Gln Pro Leu Val 245 250 255 Leu Glu Cys Ile Ala Glu Gly Phe Pro Thr Pro Thr Ile Lys Trp Leu 260 265 270 Arg Pro Ser Gly Pro Met Pro Ala Asp Arg Val Thr Tyr Gln Asn His 275 280 285 Asn Lys Thr Leu Gln Leu Leu Lys Val Gly Glu Glu Asp Asp Gly Glu 290 295 300 Tyr Arg Cys Leu Ala Glu Asn Ser Leu Gly Ser Ala Arg His Ala Tyr 305 310 315 320 Tyr Val Thr Val Glu Ala Ala Pro Tyr Trp Leu His Lys Pro Gln Ser 325 330 335 His Leu Tyr Gly Pro Gly Glu Thr Ala Arg Leu Asp Cys Gln Val Gln 340 345 350 Gly Arg Pro Gln Pro Glu Val Thr Trp Arg Ile Asn Gly Ile Pro Val 355 360 365 Glu Glu Leu Ala Lys Asp Gln Lys Tyr Arg Ile Gln Arg Gly Ala Leu 370 375 380 Ile Leu Ser Asn Val Gln Pro Ser Asp Thr Met Val Thr Gln Cys Glu 385 390 395 400 Ala Arg Asn Arg His Gly Leu Leu Leu Ala Asn Ala Tyr Ile Tyr Val 405 410 415 Val Gln Leu Pro Ala Lys Ile Leu Thr Ala Asp Asn Gln Thr Tyr Met 420 425 430 Ala Val Gln Gly Ser Thr Ala Tyr Leu Leu Cys Lys Ala Phe Gly Ala 435 440 445 Pro Val Pro Ser Val Gln Trp Leu Asp Glu Asp Gly Thr Thr Val Leu 450 455 460 Gln Asp Glu Arg Phe Phe Pro Tyr Ala Asn Gly Thr Leu Gly Ile Arg 465 470 475 480 Asp Leu Gln Ala Asn Asp Thr Gly Arg Tyr Phe Cys Leu Ala Ala Asn 485 490 495 Asp Gln Asn Asn Val Thr Ile Met Ala Asn Leu Lys Val Lys Asp Ala 500 505 510 Thr Gln Ile Thr Gln Gly Pro Arg Ser Thr Ile Glu Lys Lys Gly Ser 515 520 525 Arg Val Thr Phe Thr Cys Gln Ala Ser Phe Asp Pro Ser Leu Gln Pro 530 535 540 Ser Ile Thr Trp Arg Gly Asp Gly Arg Asp Leu Gln Glu Leu Gly Asp 545 550 555 560 Ser Asp Lys Tyr Phe Ile Glu Asp Gly Arg Leu Val Ile His Ser Leu 565 570 575 Asp Tyr Ser Asp Gln Gly Asn Tyr Ser Cys Val Ala Ser Thr Glu Leu 580 585 590 Asp Val Val Glu Ser Arg Ala Gln Leu Leu Val Val Gly Ser Pro Gly 595 600 605 Pro Val Pro Arg Leu Val Leu Ser Asp Leu His Leu Leu Thr Gln Ser 610 615 620 Gln Val Arg Val Ser Trp Ser Pro Ala Glu Asp His Asn Ala Pro Ile 625 630 635 640 Glu Lys Tyr Asp Ile Glu Phe Glu Asp Lys Glu Met Ala Pro Glu Lys 645 650 655 Trp Tyr Ser Leu Gly Lys Val Pro Gly Asn Gln Thr Ser Thr Thr Leu 660 665 670 Lys Leu Ser Pro Tyr Val His Tyr Thr Phe Arg Val Thr Ala Ile Asn 675 680 685 Lys Tyr Gly Pro Gly Glu Pro Ser Pro Val Ser Glu Thr Val Val Thr 690 695 700 Pro Glu Ala Ala Pro Glu Lys Asn Pro Val Asp Val Lys Gly Glu Gly 705 710 715 720 Asn Glu Thr Thr Asn Met Val Ile Thr Trp Lys Pro Leu Arg Trp Met 725 730 735 Asp Trp Asn Ala Pro Gln Val Gln Tyr Arg Val Gln Trp Arg Pro Gln 740 745 750 Gly Thr Arg Gly Pro Trp Gln Glu Gln Ile Val Ser Asp Pro Phe Leu 755 760 765 Val Val Ser Asn Thr Ser Thr Phe Val Pro Tyr Glu Ile Lys Val Gln 770 775 780 Ala Val Asn Ser Gln Gly Lys Gly Pro Glu Pro Gln Val Thr Ile Gly 785 790 795 800 Tyr Ser Gly Glu Asp Tyr Pro Gln Ala Ile Pro Glu Leu Glu Gly Ile 805 810 815 Glu Ile Leu Asn Ser Ser Ala Val Leu Val Lys Trp Arg Pro Val Asp 820 825 830 Leu Ala Gln Val Lys Gly His Leu Arg Gly Tyr Asn Val Thr Tyr Trp 835 840 845 Arg Glu Gly Ser Gln Arg Lys His Ser Lys Arg His Ile His Lys Asp 850 855 860 His Val Val Val Pro Ala Asn Thr Thr Ser Val Ile Leu Ser Gly Leu 865 870 875 880 Arg Pro Tyr Ser Ser Tyr His Leu Glu Val Gln Ala Phe Asn Gly Arg 885 890 895 Gly Ser Gly Pro Ala Ser Glu Phe Thr Phe Ser Thr Pro Glu Gly Val 900 905 910 Pro Gly His Pro Glu Ala Leu His Leu Glu Cys Gln Ser Asn Thr Ser 915 920 925 Leu Leu Leu Arg Trp Gln Pro Pro Leu Ser His Asn Gly Val Leu Thr 930 935 940 Gly Tyr Val Leu Ser Tyr His Pro Leu Asp Glu Gly Gly Lys Gly Gln 945 950 955 960 Leu Ser Phe Asn Leu Arg Asp Pro Glu Leu Arg Thr His Asn Leu Thr 965 970 975 Asp Leu Ser Pro His Leu Arg Tyr Arg Phe Gln Leu Gln Ala Thr Thr 980 985 990 Lys Glu Gly Pro Gly Glu Ala Ile Val Arg Glu Gly Gly Thr Met Ala 995 1000 1005 Leu Ser Gly Ile Ser Asp Phe Gly Asn Ile Ser Ala Thr Ala Gly 1010 1015 1020 Glu Asn Tyr Ser Val Val Ser Trp Val Pro Lys Glu Gly Gln Cys 1025 1030 1035 Asn Phe Arg Phe His Ile Leu Phe Lys Ala Leu Gly Glu Glu Lys 1040 1045 1050 Gly Gly Ala Ser Leu Ser Pro Gln Tyr Val Ser Tyr Asn Gln Ser 1055 1060 1065 Ser Tyr Thr Gln Trp Asp Leu Gln Pro Asp Thr Asp Tyr Glu Ile 1070 1075 1080 His Leu Phe Lys Glu Arg Met Phe Arg His Gln Met Ala Val Lys 1085 1090 1095 Thr Asn Gly Thr Gly Arg Val Arg Leu Pro Pro Ala Gly Phe Ala 1100 1105 1110 Thr Glu Gly Trp Phe Ile Gly Phe Val Ser Ala Ile Ile Leu Leu 1115 1120 1125 Leu Leu Val Leu Leu Ile Leu Cys Phe Ile Lys Arg Ser Lys Gly 1130 1135 1140 Gly Lys Tyr Ser Val Lys Asp Lys Glu Asp Thr Gln Val Asp Ser 1145 1150 1155 Glu Ala Arg Pro Met Lys Asp Glu Thr Phe Gly Glu Tyr Ser Asp 1160 1165 1170 Asn Glu Glu Lys Ala Phe Gly Ser Ser Gln Pro Ser Leu Asn Gly 1175 1180 1185 Asp Ile Lys Pro Leu Gly Ser Asp Asp Ser Leu Ala Asp Tyr Gly 1190 1195 1200 Gly Ser Val Asp Val Gln Phe Asn Glu Asp Gly Ser Phe Ile Gly 1205 1210 1215 Gln Tyr Ser Gly Lys Lys Glu Lys Glu Ala Ala Gly Gly Asn Asp 1220 1225 1230 Ser Ser Gly Ala Thr Ser Pro Ile Asn Pro Ala Val Ala Leu Glu 1235 1240 1245 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 mAb417 <400> 6 Arg Ala Ser Arg Thr Ile Ser Ile Tyr Val Asn 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 mAb417 <400> 7 Ala Ala Ser Asn Leu His Ser 1 5 <210> 8 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Ab612 Variable Heavy Chain Polynucleotide Sequence <400> 8 gaggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggcggcag cctgagactg 60 agctgcgccg ccagcggctt caccttcagc agattcggca tgcactgggt gagacaggcc 120 cccggcaagg gcctggagtg ggtggccttc atcagcaacg agggcagcaa caagtactac 180 gccgacagcg tgaagggcag attcaccatc agcagagaca acagcgccaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cagaggcaga 300 gcctacggca gcggcagcct gttcgacccc tggggccagg gcaccctggt gaccgtgagc 360 agc 363 <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 mAb417 <400> 9 Phe Ile Ser Asn Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 mAb417 <400> 10 Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro 1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 mAb417 <400> 11 Gln Gln Ser Ile Gly Arg Gly Val Val Thr 1 5 10 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 12 Arg Ala Ser Arg Thr Ile Ser Ser Tyr Val Asn 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Ab612 VL CDR3 <400> 13 Gln Gln Ser Ile Gly Arg Gly Pro Val Thr 1 5 10 <210> 14 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Ab4H5 Variable Heavy Chain Polynucleotide Sequence <400> 14 gaggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggcggcag cctgagactg 60 agctgcgccg ccagcggctt caccttcagc agattcggca tgcactgggt gagacaggcc 120 cccggcaagg gcctggagtg ggtggccttc atcagcaacg agggcagcaa caagtactac 180 gccgacagcg tgaagggcag attcaccatc agcagagaca acagcgccaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cagaggcaga 300 gcctacggca gcggcagcct gttcgacccc tggggccagg gcaccctggt gaccgtgagc 360 agc 363 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Ab4H5 VL-CDR3 <400> 15 Gln Gln Ala Gly Phe Tyr Thr Pro Trp Thr 1 5 10 <210> 16 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Ab2C2 Variable Heavy Chain Polynucleotide Sequence <400> 16 gaggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggcggcag cctgagactg 60 agctgcgccg ccagcggctt caccttcagc agattcggca tgcactgggt gagacaggcc 120 cccggcaagg gcctggagtg ggtggccttc atcagcaacg agggcagcaa caagtactac 180 gccgacagcg tgaagggcag attcaccatc agcagagaca acagcgccaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cagaggcaga 300 gcctacggca gcggcagcct gttcgacccc tggggccagg gcaccctggt gaccgtgagc 360 agc 363 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Ab2C2 VL CDR3 <400> 17 Gln Gln Ala Gly Phe Tyr Ser Pro Trp Thr 1 5 10 <210> 18 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Ab4H6 Variable Heavy Chain Polynucleotide Sequence <400> 18 gaggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggcggcag cctgagactg 60 agctgcgccg ccagcggctt caccttcagc agattcggca tgcactgggt gagacaggcc 120 cccggcaagg gcctggagtg ggtggccttc atcagcaacg agggcagcaa caagtactac 180 gccgacagcg tgaagggcag attcaccatc agcagagaca acagcgccaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cagaggcaga 300 gcctacggca gcggcagcct gttcgacccc tggggccagg gcaccctggt gaccgtgagc 360 agc 363 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Ab4H6 VL-CDR3 <400> 19 Gln Gln Ser Leu His Phe Tyr Pro Trp Thr 1 5 10 <210> 20 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Ab5D12 Light Chain Amino Acid Sequence <400> 20 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr 20 25 30 Val Asn Trp Tyr Arg Gln Arg Pro Gly Lys Ala Pro Glu Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Val Trp Tyr Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Ab5D12 VL-CDR3 <400> 21 Gln Gln Ser Leu Val Trp Tyr Pro Trp Thr 1 5 10 <210> 22 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Ab5D12 Variable Heavy Chain Polynucleotide Sequence <400> 22 gaggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggcggcag cctgagactg 60 agctgcgccg ccagcggctt caccttcagc agattcggca tgcactgggt gagacaggcc 120 cccggcaagg gcctggagtg ggtggccttc atcagcaacg agggcagcaa caagtactac 180 gccgacagcg tgaagggcag attcaccatc agcagagaca acagcgccaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cagaggcaga 300 gcctacggca gcggcagcct gttcgacccc tggggccagg gcaccctggt gaccgtgagc 360 agc 363 <210> 23 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Ab612 VH Amino Acid Sequence <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Phe Ile Ser Asn Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ala Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 24 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Ab612 VL Amino Acid Sequence <400> 24 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr 20 25 30 Val Asn Trp Tyr Arg Gln Arg Pro Gly Lys Ala Pro Glu Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ile Gly Arg Gly Pro 85 90 95 Val Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 25 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Ab4H5 VH Amino Acid Sequence <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Phe Ile Ser Asn Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ala Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 26 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Ab4H5 VL Amino Acid Sequence <400> 26 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr 20 25 30 Val Asn Trp Tyr Arg Gln Arg Pro Gly Lys Ala Pro Glu Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Gly Phe Tyr Thr Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 27 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Ab2C2 VH Amino Acid Sequence <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Phe Ile Ser Asn Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ala Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 28 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Ab2C2 VL Amino Acid Sequence <400> 28 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr 20 25 30 Val Asn Trp Tyr Arg Gln Arg Pro Gly Lys Ala Pro Glu Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Gly Phe Tyr Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 29 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Ab4H6 VH Amino Acid Sequence <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Phe Ile Ser Asn Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ala Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 30 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Ab4H6 VL Amino Acid Sequence <400> 30 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr 20 25 30 Val Asn Trp Tyr Arg Gln Arg Pro Gly Lys Ala Pro Glu Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu His Phe Tyr Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 31 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Ab5D12 VH Amino Acid Sequence <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Phe Ile Ser Asn Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ala Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 32 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Ab5D12 VL Amino Acid Sequence <400> 32 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr 20 25 30 Val Asn Trp Tyr Arg Gln Arg Pro Gly Lys Ala Pro Glu Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Val Trp Tyr Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 33 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Ab612 Variable Light Chain Polynucleotide Sequence <400> 33 gacatccagc tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60 atcacctgca gagccagcag aaccatcagc agctacgtga actggtacag acagagaccc 120 ggcaaggccc ccgagagcct gatctacgcc gccagcaacc tgcacagcgg cgtgcccagc 180 agatttagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcag agcatcggca gaggccccgt gaccttcggc 300 cagggcacca agctggagat caag 324 <210> 34 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Ab4H5 Variable Light Chain Polynucleotide Sequence <400> 34 gacatccagc tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60 atcacctgca gagccagcag aaccatcagc agctacgtga actggtacag acagagaccc 120 ggcaaggccc ccgagagcct gatctacgcc gccagcaacc tgcacagcgg cgtgcccagc 180 agatttagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcag gccggcttct acaccccctg gaccttcggc 300 cagggcacca agctggagat caag 324 <210> 35 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Ab2C2 Variable Light Chain Polynucleotide Sequence <400> 35 gacatccagc tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60 atcacctgca gagccagcag aaccatcagc agctacgtga actggtacag acagagaccc 120 ggcaaggccc ccgagagcct gatctacgcc gccagcaacc tgcacagcgg cgtgcccagc 180 agatttagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcag gccggcttct actccccctg gaccttcggc 300 cagggcacca agctggagat caag 324 <210> 36 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Ab4H6 Variable Light Chain Polynucleotide Sequence <400> 36 gacatccagc tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60 atcacctgca gagccagcag aaccatcagc agctacgtga actggtacag acagagaccc 120 ggcaaggccc ccgagagcct gatctacgcc gccagcaacc tgcacagcgg cgtgcccagc 180 agatttagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcag tccctgcact tctacccctg gaccttcggc 300 cagggcacca agctggagat caag 324 <210> 37 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Ab5D12 Variable Light Chain Polynucleotide Sequence <400> 37 gacatccagc tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60 atcacctgca gagccagcag aaccatcagc agctacgtga actggtacag acagagaccc 120 ggcaaggccc ccgagagcct gatctacgcc gccagcaacc tgcacagcgg cgtgcccagc 180 agatttagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgccagcag tccctggtgt ggtacccctg gaccttcggc 300 cagggcacca agctggagat caag 324 <210> 38 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Ab612 Heavy Chain Amino Acid Sequence <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Phe Ile Ser Asn Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ala Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 39 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Ab612 Light Chain Amino Acid Sequence <400> 39 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr 20 25 30 Val Asn Trp Tyr Arg Gln Arg Pro Gly Lys Ala Pro Glu Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ile Gly Arg Gly Pro 85 90 95 Val Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 40 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Ab4H5 Heavy Chain Amino Acid Sequence <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Phe Ile Ser Asn Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ala Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 41 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Ab4H5 Light Chain Amino Acid Sequence <400> 41 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr 20 25 30 Val Asn Trp Tyr Arg Gln Arg Pro Gly Lys Ala Pro Glu Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Gly Phe Tyr Thr Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 42 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Ab2C2 Heavy Chain Amino Acid Sequence <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Phe Ile Ser Asn Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ala Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 43 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Ab2C2 Light Chain Amino Acid Sequence <400> 43 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr 20 25 30 Val Asn Trp Tyr Arg Gln Arg Pro Gly Lys Ala Pro Glu Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Gly Phe Tyr Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 44 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Ab4H6 Heavy Chain Amino Acid Sequence <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Phe Ile Ser Asn Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ala Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 45 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Ab4H6 Light Chain Amino Acid Sequence <400> 45 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr 20 25 30 Val Asn Trp Tyr Arg Gln Arg Pro Gly Lys Ala Pro Glu Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu His Phe Tyr Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 46 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Ab5D12 Heavy Chain Amino Acid Sequence <400> 46 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Phe Ile Ser Asn Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Ala Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Arg Ala Tyr Gly Ser Gly Ser Leu Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450

Claims (80)

An isolated antibody or antigen-binding fragment thereof, which 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, or
(b) the VH of the reference antibody comprises SEQ ID NO: 25, and the VL of the reference antibody comprises SEQ ID NO: 26, or
(c) the VH of the reference antibody comprises SEQ ID NO: 27, and the VL of the reference antibody comprises SEQ ID NO: 28, or
(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. An antibody or antigen-binding fragment thereof. The method of claim 1, wherein the antibody or antigen-binding fragment thereof comprises VH complementarity determining region 1 (CDR1), VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3, wherein the antibody Or at least one amino acid in the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL CDR3 of the antigen-binding fragment thereof is the VH CDR1, VH CDR2, VH CDR3, VL CDR1 of the mAb417 antibody, Different from VL CDR2 and/or VL CDR3,
The VH CDR1 of the mAb417 antibody contains RFGMH (SEQ ID NO: 2);
The VH CDR2 of the mAb417 antibody contains 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 contains 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), an antibody or an antigen-binding fragment thereof. An isolated antibody or antigen-binding fragment thereof, cross-compete 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, or
(b) the VH of the reference antibody comprises SEQ ID NO: 25, and the VL of the reference antibody comprises SEQ ID NO: 26, or
(c) the VH of the reference antibody comprises SEQ ID NO: 27, and the VL of the reference antibody comprises SEQ ID NO: 28, or
(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 VH complementarity determining region 1 (CDR1), VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1 of the antibody or antigen-binding fragment thereof, At least one amino acid in VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or VL 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 contains RFGMH (SEQ ID NO: 2);
The VH CDR2 of the mAb417 antibody contains 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 contains 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), an antibody or an antigen-binding fragment thereof. The method of claim 2 or 3, wherein the at least one amino acid difference is present in the VH CDR2 of the antibody or antigen-binding fragment thereof, and the VH CDR2 of the antibody or antigen-binding fragment thereof is at residue 5 An antibody or antigen-binding fragment thereof, comprising glutamine. The method of any one of claims 2 to 4, wherein the at least one amino acid difference is present in the VL CDR1 of the antibody or antigen-binding fragment thereof, and the VL CDR1 of the antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof, comprising serine at residue 8. The method of any one of claims 2 to 5, wherein the at least one amino acid difference is present in the VL CDR3 of the antibody or antigen-binding fragment thereof, and the VL CDR3 of the antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof, comprising proline at silver residue 8. The method of any one of claims 2 to 6, wherein the at least one amino acid difference is present in the VL CDR3 of the antibody or antigen-binding fragment thereof, and the VL CDR3 of the antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof, comprising alanine, glycine, phenylalanine, tyrosine, threonine, proline and tryptophan in each of the silver residues 3 to 9. The method of any one of claims 2 to 7, wherein the at least one amino acid difference is present in the VL CDR3 of the antibody or antigen-binding fragment thereof, and the VL CDR3 of the antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof, comprising alanine, glycine, phenylalanine, tyrosine, serine, proline and tryptophan at each of the silver residues 3 to 9. The method of any one of claims 2 to 8, wherein the at least one amino acid difference is present in the VL CDR3 of the antibody or antigen-binding fragment thereof, and the VL CDR3 of the antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof, comprising leucine, histidine, phenylalanine, tyrosine, proline and tryptophan at each of the silver residues 4 to 9. The method of any one of claims 2 to 9, wherein the at least one amino acid difference is present in the VL CDR3 of the antibody or antigen-binding fragment thereof, and the VL CDR3 of the antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof, comprising leucine, valine, tryptophan, tyrosine, proline and tryptophan at each of the silver residues 4 to 9. The method according to any one of claims 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. , An antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof according to any one of claims 2 to 11, wherein the VH CDR1 of the antibody or antigen-binding fragment thereof comprises RFGMH (SEQ ID NO: 2). The antibody or antigen-binding fragment thereof according to any one of claims 2 to 12, wherein the VH CDR2 of the antibody or antigen-binding fragment thereof comprises FISNEGSNKYYADSVKG (SEQ ID NO: 10). The antibody or antigen-binding fragment thereof according to any one of claims 2 to 13, wherein the VH CDR3 of the antibody or antigen-binding fragment thereof comprises GRAYGSGSLFDP (SEQ ID NO: 4). The antibody or antigen-binding fragment thereof according to any one of claims 2 to 14, wherein the VL CDR1 comprises RASRTISSYVN (SEQ ID NO: 12). The antibody or antigen-binding fragment thereof according to any one of claims 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 2 to 16, wherein the VL CDR3 comprises QQSIGRGPVT (SEQ ID NO: 13). The antibody or antigen thereof according to any one of claims 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)- Binding fragment. The antibody or antigen thereof according to any one of claims 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)- Binding fragment. The antibody or antigen thereof according to any one of claims 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)- Binding fragment. The antibody or antigen thereof according to any one of claims 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)- Binding fragment. The antibody or antigen thereof according to any one of claims 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)- Binding fragment. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein the antibody or antigen-binding fragment thereof has one or more characteristics selected from the group consisting of:
(a) the antibody or antigen-binding fragment thereof exhibits improved productivity;
(b) the antibody or antigen-binding fragment thereof exhibits improved affinity as measured by the _{equilibrium dissociation constant (K D );}
(c) the antibody or antigen-binding fragment thereof exhibits an improved PI value,
(d) the antibody or antigen-binding fragment thereof exhibits improved affinity as measured by the association constant (K); or
(e) any combination thereof. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment thereof exhibits improved productivity compared to the mAb417 antibody. 25. The method of claim 24, wherein the improved productivity when expressed according to Example 3 is at least 55 mg/l, at least 56 mg/l, at least 57 mg/l, at least 58 mg/l, at least 59 mg/l, at least About 60 mg/l, at least about 61 mg/l, at least about 62 mg/l, at least about 63 mg/l, at least about 64 mg/l, at least about 65 mg/l, at least about 66 mg/l, at least about 67 mg/L, at least about 68 mg/L, at least about 69 mg/L, at least about 70 mg/L, at least about 71 mg/L, at least about 72 mg/L, at least about 73 mg/L, at least about 74 Mg/l, at least about 75 mg/l, at least about 76 mg/l, at least about 77 mg/l, at least about 78 mg/l, at least about 79 mg/l, at least about 80 mg/l, at least about 81 mg /L, at least about 82 mg/L, at least about 83 mg/L, at least about 84 mg/L or at least about 85 mg/L. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, wherein the antibody or antigen-binding fragment thereof exhibits an improved K _D compared to the mAb417 antibody. The method of claim 26, wherein the improved K _D is less than 2.6 × 10 ^-10 M, less than 2.5 × 10 ^-10 M, less than 2.0 × 10 ^-10 M, less than 1.5 × 10 ^-10 M, 1.0 × 10 ^-10 M Less than, 9×10 ^-11 M or less, 8×10 ^-11 M or less, 7×10 ^-11 M or less, 6×10 ^-11 M or less, 5×10 ^-11 M or less, 4×10 ^-11 M or less, 3×10 ^-11 M or less, 2×10 ^-11 M or less, 1×10 ^-11 M or less, 9×10 ^-12 M or less, 8×10 ^-12 M or less, 7×10 ^-12 M or less, 6× 10 ^-12 M, less than 5 × 10 ^-12 M, less than 4 × 10 ^-12, less than ^{3 × 10 -12 M M, 2} × 10 less than ^-12 M, 1 × 10 ^-12 less than M, 9 × 10 ^{- An} antibody or antigen-binding fragment thereof, which is less than 13 M or less than 8×10 ^{-13 M.} The antibody or antigen-binding fragment thereof according to any one of claims 1 to 27, wherein the antibody or antigen-binding fragment thereof exhibits improved affinity (K) compared to the mAb417 antibody. The method of claim 28, wherein the improved affinity (K) is ^{less than 5×10 −10} M, less than 4×10 ⁻¹⁰ M, less than 3×10 ⁻¹⁰ M, less than 2×10 ⁻¹⁰ M, 1.0×10 ^{− Less than 10} M, less than 9×10 ^-11 M, less than 8×10 ^-11 M, less than 7×10 ^-11 M, less than 6×10 ^-11 M, less than 5×10 ^-11 M, 4×10 ^-11 M Less than 3×10 ^-11 M, 2×10 ^-11 M or less, 1×10 ^-11 M or less, 9×10 ^-12 M or less, 8×10 ^-12 M or less, 7×10 ^-12 M or less, Less than 6×10 ^-12 M, less than 5×10 ^-12 M, less than 4×10 ^-12 M, less than 3×10 ^-12 M, less than 2×10 ^-12 M, less than 1×10 ^-12 M, 9× 10 ^-13 M, or less than 8 × 10 ^-13 M is less than the antibody or an antigen-binding fragment. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, wherein the antibody or antigen-binding fragment thereof exhibits an improved PI value compared to the mAb417 antibody. The method of claim 30, wherein the improved PI value is less than 9.6, less than 9.5, less than 9.4, less than 9.3, less than 9.2, less than 9.1, less than 9.0, less than 8.9, less than 8.8, less than 8.7, less than 8.6, less than 8.5, less than 8.4 , Less than 8.3, less than 8.2, less than 8.1, less than 8.0, less than 7.9, less than 7.8, less than 7.7 or less than 7.6. An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3;
The VH CDR1, CDR2 and CDR3 each include RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4);
The VL CDR1, CDR2 and CDR3 are isolated antibodies or antigen-binding fragments thereof, each comprising RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSIGRGPVT (SEQ ID NO: 13). An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3;
The VH CDR1, CDR2 and CDR3 each include RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4);
The VL CDR1, CDR2 and CDR3 are isolated antibodies or antigen-binding fragments thereof, each comprising RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQAGFYSPWT (SEQ ID NO: 17). An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3;
The VH CDR1, CDR2 and CDR3 each include RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4);
The VL CDR1, CDR2 and CDR3 are isolated antibodies or antigen-binding fragments thereof, each comprising RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQAGFYTPWT (SEQ ID NO: 17). An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3;
The VH CDR1, CDR2 and CDR3 each include RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4);
The VL CDR1, CDR2 and CDR3 are isolated antibodies or antigen-binding fragments thereof, each comprising RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSLHFYPWT (SEQ ID NO: 19). An isolated antibody or antigen-binding fragment thereof that specifically binds to L1CAM, wherein the antibody or antigen-binding fragment thereof comprises VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3;
The VH CDR1, CDR2 and CDR3 each include RFGMH (SEQ ID NO: 2), FISNEGSNKYYADSVKG (SEQ ID NO: 10) and GRAYGSGSLFDP (SEQ ID NO: 4);
The VL CDR1, CDR2 and CDR3 are isolated antibodies or antigen-binding fragments thereof, each comprising RASRTISSYVN (SEQ ID NO: 12), AASNLHS (SEQ ID NO: 7) and QQSLVWYPWT (SEQ ID NO: 19). The method of any one of claims 1-36, wherein the VH is EVQLVESGGG WQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLF at least about 85%, at least about 90%, at least about 85%, at least about 90%, at least about 95%, at least about 90% of amino acid sequences An isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. The method of any one of claims 1-37, wherein the VL is DIQLTQSPSS LSASVGDRVTITCRASRTISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIGRGPVTFGQQSIGRGPVTFGQGTKLEIK (at least about 95%, at least about 80%, at least about 85%, at least about 90%, with amino acid sequence set forth in SEQ ID NO: 24). An isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. The method of any one of claims 1-38, wherein the VH is EVQLVESGGG WQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLF as set forth in about 85%, at least about 90%, at least about 85%, at least about 90%, at least about 85%, at least about 90%, amino acid sequence SEQ ID NO. An isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. The method of any one of claims 1-39, wherein the VL is set forth in DIQLTQSPSS LSASVGDRVTITCRASRTISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAGFYSPWTFGQAGFYSPWTFGQGTKLEIK (SEQ ID NO: 28) and at least about 85%, at least about 80%, at least about 85%, at least about 80%, amino acid sequence An isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. The method of any one of claims 1-40, wherein the VH is EVQLVESGGG WQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLF at least about 85%, at least about 90%, at least about 85%, at least about 90%, at least about 95%, at least about 90% of amino acid sequences An isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. The method of any one of claims 1-41, wherein the VL is set forth in DIQLTQSPSS LSASVGDRVTITCRASRTISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAGFYTPWTFGQAGFYTPWTFGQAGFYTPWTFGQQGTKLEIK (SEQ ID NO: 26 and at least about 85%, at least about 90%, at least about 85%, at least about 90%, amino acid sequence and at least about 90%, An isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. The method of any one of claims 1-42, wherein the VH is EVQLVESGGG WQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLF at least about 85%, at least about 90%, at least about 85%, at least about 90%, at least about 95%, at least about 90% of amino acid sequences, An isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. The method of any one of claims 1-43, wherein the VL is set forth in DIQLTQSPSSLSASVGDRVTITCRASRTISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLHFYPWTFGQSLHFYPWTFGQSLHFYPWTFGQSLHFYPWTFGQSLHFYPWTFGQSLHFYPWTFGQSLHFYPWTFGQGTKLEIK (SEQ ID NO: An isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. The method of any one of claims 1-44, wherein the VH is EVQLVESGGGWQPGGSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNEGSNKYYADSVKGRFTISRDNSANTLYLQMNSLRAEDTAVYYCARGRAYGSGSLF at least about 85%, at least about 80%, at least about 95%, at least about 90%, at least about 95%, at least about 90% of amino acid sequences An isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. The method of any one of claims 1-45, wherein the VL is set forth in DIQLTQSPSS LSASVGDRVTITCRASRTISSYVNWYRQRPGKAPESLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSLVWYPWTFGQSLVWYPWTFGQGTKLEIK (SEQ ID NO: 32 and at least about 80%, at least about 85%, at least about 90%, at least about 85%, at least about 90% amino acid sequence). An isolated antibody or antigen-binding fragment thereof comprising an amino acid sequence that is at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. The method of any one of claims 1-46, 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. An isolated antibody or antigen-binding fragment thereof comprising. The method of any one of claims 1-46, 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. An isolated antibody or antigen-binding fragment thereof comprising. The method of any one of claims 1-46, 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. An isolated antibody or antigen-binding fragment thereof comprising. The method of any one of claims 1-46, 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. An isolated antibody or antigen-binding fragment thereof comprising. The method of any one of claims 1-46, 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. An isolated antibody or antigen-binding fragment thereof comprising. 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 An isolated antibody or antigen-binding fragment thereof, wherein the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 39. 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 An isolated antibody or antigen-binding fragment thereof, wherein the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 41. 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 An isolated antibody or antigen-binding fragment thereof, wherein the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 43. 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 An isolated antibody or antigen-binding fragment thereof, wherein the LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 45. 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 The LC of the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 20, an isolated antibody or antigen-binding fragment thereof. The antibody of any one of claims 1-56, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, variants thereof and any combination thereof. 58. The antibody of any one of claims 1-57, which is a chimeric antibody, a human antibody or a humanized antibody. 57. The antibody of any one of claims 1-56, comprising a Fab, Fab', F(ab')2, Fv or single chain Fv (scFv). A nucleic acid encoding the antibody of any one of claims 1 to 59. A vector comprising the nucleic acid of claim 60. A host cell comprising the vector of claim 61. 64. The method of claim 63, wherein E. Coli (E. coli), Pseudomonas (Pseudomonas), Bacillus (Bacillus), Streptomyces (Streptomyces), yeasts, CHO, YB / 20, NS0 , PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, A host cell selected from the group consisting of Hep G2, SP2/0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, BMT10 cells, plant cells, insect cells and human cells. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 59, which is linked to an agent. A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 59 and the antibody or antigen-binding fragment thereof that binds to an antigen. The antibody of any one of claims 1-59, the nucleic acid of claim 60, the vector of claim 61, the cell of claim 62 or 63, the immunoconjugate of claim 64, or the bispecific or multispecific antibody of claim 65, and A composition comprising a carrier. The antibody of any one of claims 1-59, the nucleic acid of claim 60, the vector of claim 61, the cell of claim 62 or 63, the immunoconjugate of claim 64, or the bispecific or multispecific antibody of claim 65, and Kit, including instructions for use. An immune cell comprising the nucleic acid of claim 60 or the vector of claim 61. The immune cell of claim 68, which is a T cell or an NK cell. The immune cell of claim 68 or 69 comprising a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof of any one of claims 1-59. A method of producing an antibody or antigen-binding fragment thereof that specifically binds to a human L1CAM protein, comprising culturing the cells of claim 62 or 63 under suitable conditions and isolating the antibody. A method of treating a disease or condition in a subject in need thereof, comprising the antibody of any one of claims 1 to 59, the nucleic acid of claim 60, the vector of claim 61, or A treatment comprising administering the cell of claim 63, the immunoconjugate of claim 64, the bispecific or multispecific antibody of claim 65, the composition of claim 66, or the immune cell of any one of claims 68 to 70. Way. 73. The method of claim 72, wherein the disease or condition comprises a tumor. The method of any one of claims 72 or 73, wherein the tumor is biliary tract cancer, melanoma, pancreatic cancer, glioma, breast cancer, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer. Containing, treatment method. 75. The method of claim 74, wherein the tumor is biliary tract cancer. The method of any one of claims 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 is Inhibiting growth, a method of treatment. The treatment of any one of claims 72-76, wherein the antibody, the nucleic acid, the vector, the cell, the immunoconjugate, the composition or the immune cell enhances the invasion of immune cells into the tumor. Way. 78. The method of any one of claims 72-77, comprising administering an additional therapeutic agent. 79. The method of claim 78, wherein the additional therapeutic agent comprises chemotherapy, immunotherapy, radiotherapy, or a combination thereof. 80. The method of claim 79, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

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