Anti-L1-CAM antibodies and uses thereof

Abstract

The present application generally relates to immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that can bind to and neutralize the activity of the L1-CAM protein. The antibodies of the present application are useful in methods of detecting and treating L1-CAM positive cancer in a subject in need thereof.

Description

[0001] This invention application is a divisional application based on an invention patent application with an application date of June 14, 2018, application number 201880052266.7 (international application number PCT / US2018 / 037645) and name “Anti-L1-CAM antibodies and uses thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 520,382, filed on June 15, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on June 14, 2018 is named 115872-0375_SL.txt and is 147,283 bytes in size. Technical Field

[0006] The present technology generally relates to the preparation of immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to L1-CAM protein and the use of the immunoglobulin-related compositions. Specifically, the present technology relates to the preparation of L1-CAM neutralizing antibodies and the use of the antibodies in the detection and treatment of L1-CAM-related cancers. Background Art

[0007] The following description of the background of the present technology is provided only to assist in understanding the present technology, and it is not admitted that the description describes or constitutes prior art against the present technology.

[0008] The advent of chimeric antigen receptor (CAR) technology (Sadelain M et al., Cancer Discov 3:388-98 (2013)) is rapidly expanding therapeutic research of genetically modified T cells redirected against L1-CAM, with several clinical trials using T cells modified with L1-CAM-CAR: NCT00889954 (all L1-CAM (+) cancers), NCT01935843 (L1-CAM (+) solid tumors). See Hong H et al., J Immunother 37:93-104 (2014). Although T cells can effectively target tumors with low levels of L1-CAM, there are concerns about potential bystander toxicity in normal tissues with low levels of L1-CAM expression.

[0009] In addition to toxicity, cell harvesting, processing, storage, shipping, and product release regulation for lymphocyte therapies can also be challenging, especially when the cells must be genetically modified. Despite the infusion of billions of these cells, T cell consumption, survival, and homing are suboptimal. In addition, CAR-modified T cells are no exception to the immunosuppressive tumor microenvironment, in which Tregs, tumor-associated macrophages, and myeloid suppressor cells work together to circumvent the anti-tumor properties of CAR-modified T cells. In addition, CAR-modified T cells are subject to the same immunosuppressive limitations faced by classical T cells, including anergy after CTLA4 is engaged by B7 or PD-1 is engaged by PD-L1 (B7-H1) on tumor cells. Therefore, clinically effective alternatives to CAR-modified T cell therapies for the treatment of cancer are necessary. Summary of the invention

[0010] In one aspect, the disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) or its antigen-binding fragment, wherein (a) said V H Contains V of GYTFTSYWMQ H -CDR1 sequence (SEQ ID NO: 53), V H -CDR2 sequence (SEQ ID NO: 54) and V of YDGYYAMDY H -CDR3 sequence (SEQ ID NO: 55); and / or (b) the V L Contains V of KSSQSLLYSSNQKNYLA L -CDR1 sequence (SEQ ID NO: 56), V L -CDR2 sequence (SEQ ID NO: 57) and V of QQYHSYPFT L -CDR3 sequence (SEQ ID NO: 58). In some embodiments of the antibody or antigen-binding fragment, the V H Contains the amino acid sequence of SEQ ID NO: 1, and the V LComprising the amino acid sequence of SEQ ID NO:3. Additionally or alternatively, the antibody may further comprise an Fc domain of an isotype selected from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. In some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from N297A and K322A. Additionally or alternatively, in some embodiments, the antibody comprises an IgG4 constant region comprising an S228P mutation. In certain embodiments, the antigen binding fragment is selected from Fab, F(ab') 2 , Fab', scF v and F v In some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In certain embodiments, the antibody or antigen-binding fragment binds to an epitope of the L1-CAM protein, the epitope comprising at least five to eight consecutive amino acid residues of the second Ig-like domain of L1-CAM (SEQ ID NO: 74). In some embodiments, the epitope is a conformational epitope.

[0011] In another aspect, the present disclosure provides an antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:51, or a variant thereof having one or more conservative amino acid substitutions, and / or a light chain (LC) amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:52, or a variant thereof having one or more conservative amino acid substitutions. In certain embodiments, the antibody comprises a HC amino acid sequence and a LC amino acid sequence selected from the group consisting of: SEQ ID NO: 9 and SEQ ID NO: 13 (huE71 H1 / L1); SEQ ID NO: 9 and SEQ ID NO: 14 (huE71 H1 / L2); SEQ ID NO: 10 and SEQ ID NO: 13 (huE71 H2 / L1); SEQ ID NO: 10 and SEQ ID NO: 14 (huE71 H2 / L2); SEQ ID NO: 17 and SEQ ID NO: 21 (huE72 H1 / L1); SEQ ID NO: 17 and SEQ ID NO: 22 (huE72 H1 / L2); SEQ ID NO: 18 and SEQ ID NO: 21 (huE72 H2 / L1); SEQ ID NO: 18 and SEQ ID NO: 22 (huE72 H2 / L2); SEQ ID NO: 26 and SEQ ID NO: 25 (chE71 IgG1); SEQ ID NO: 30 and SEQ ID NO: SEQ ID NO:29 (chE72 IgG1); SEQ ID NO:42 and SEQ ID NO:41 (chE71 IgG4); SEQ ID NO:46 and SEQ ID NO:45 (huE71 IgG4); SEQ ID NO:49 and SEQ ID NO:50 (huE71 BsAb); and SEQ ID NO:51 and SEQ ID NO:52 (huE72 BsAb).

[0012] In one aspect, the disclosure provides an antibody comprising (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 13, 14, 21, 22, 25, 29, 41, 45, 50 or 52; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 9, 10, 17, 18, 26, 30, 42, 46, 49 and 51.

[0013] In another aspect, the present disclosure provides an antibody comprising (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the LC sequence present in any one of SEQ ID NOs: 13, 14, 21, 22, 25, 29, 41, 45, 50 or 52; and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the HC sequence present in any one of SEQ ID NOs: 9, 10, 17, 18, 26, 30, 42, 46, 49 and 51.

[0014] In any one of the above embodiments, the antibody is a chimeric antibody, a humanized antibody or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody comprises an IgG1 constant region containing one or more amino acid substitutions selected from N297A and K322A. In certain embodiments, the antibody of the present technology comprises an IgG4 constant region containing an S228P mutation. In any one of the above embodiments, the antibody binds to an epitope of the L1-CAM protein, the epitope comprising at least five to eight consecutive amino acid residues of the 2nd Ig-like domain (SEQ ID NO: 74) of L1-CAM or the 6th Ig-like domain (SEQ ID NO: 75) of L1-CAM. In some embodiments, the epitope is a conformational epitope. Additionally or alternatively, in some embodiments, the antibody of the present technology lacks α-1,6-fucose modification.

[0015] In one aspect, the disclosure provides a recombinant nucleic acid sequence encoding any of the antibodies described herein. In some embodiments, the recombinant nucleic acid sequence is selected from: SEQ ID NO: 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 43, 44, 47 and 48.

[0016] In another aspect, the present disclosure provides a host cell or a vector comprising any of the recombinant nucleic acid sequences disclosed herein.

[0017] In one aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment of the present technology and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the following: an isotope, a dye, a chromagen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA or any combination thereof.

[0018] In some embodiments of the bispecific antibodies of the present technology, the bispecific antibodies bind to T cells, B cells, bone marrow cells, plasma cells or mast cells. Additionally or alternatively, in some embodiments, the bispecific antibodies bind to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, KIR or small molecule DOTA hapten. The small molecule DOTA hapten can be selected from DOTA, DOTA-Bn, DOTA-deferoxamine, DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH 2 ,Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH 2 , DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH 2 ;DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 , DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 , DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 , DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH 2 , Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH 2 , Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2, Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH 2 , Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 , DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 , (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH 2 , Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 , (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 , Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH 2 , Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2 , Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH 2 and Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH 2 .

[0019] In another aspect, the disclosure provides a method of treating L1-CAM-related cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any one of the antibodies disclosed herein. In certain embodiments, the antibody comprises a HC amino acid sequence and a LC amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 9 and SEQ ID NO: 13 (huE71 H1 / L1); (ii) SEQ ID NO: 9 and SEQ ID NO: 14 (huE71 H1 / L2); (iii) SEQ ID NO: 10 and SEQ ID NO: 13 (huE71 H2 / L1); (iv) SEQ ID NO: 10 and SEQ ID NO: 14 (huE71

[0020] : (i) SEQ ID NO:17 and SEQ ID NO:21 (huE72 H1 / L2); (ii) SEQ ID NO:17 and SEQ ID NO:22 (huE72 H1 / L2); (iii) SEQ ID NO:18 and SEQ ID NO:21 (huE72 H2 / L1); (ix) SEQ ID NO:26 and SEQ ID NO:25 (chE71 IgG1); (x) SEQ ID NO:30 and SEQ ID NO:29 (chE72 IgG1); (xi) SEQ ID NO:42 and SEQ ID NO:41 (chE71 IgG4); (xii) SEQ ID NO:46 and SEQ ID NO:45 (huE71 IgG4); (xiii) SEQ ID NO:49 and SEQ ID NO:50 (huE71 IgG1); BsAb); and (xiv) SEQ ID NO:51 and SEQ ID NO:52 (huE72 BsAb), wherein the antibodies specifically bind to L1-CAM and neutralize L1-CAM activity.

[0021] In some embodiments, the L1-CAM-associated cancer is leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, bile duct cancer, carcinoid, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), Pheochromocytoma, glioma, pancreatic neuroectodermal cancer, pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma, tumor vascular, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine cancer, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, nasal glioma, medulloblastoma, capillary hemangioma, Kaposi sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, or head and neck squamous cell carcinoma.

[0022] Additionally or alternatively, in some embodiments of the method, the antibody is administered to the subject separately, sequentially or simultaneously with an additional therapeutic agent. Examples of additional therapeutic agents include one or more of the following: alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutics.

[0023] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising (a) administering to the subject an effective amount of an antibody of the present technology, wherein the antibody is configured to localize to a tumor expressing L1-CAM and is labeled with a radioactive isotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody that is higher than a reference value. In some embodiments, the subject is diagnosed with or suspected of having cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0024] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle emitting isotopes include 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu and 67 Cu. In some embodiments of the methods, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (eg, via a N297A mutation in the Fc region, which results in deglycosylation).

[0025] Also disclosed herein is a kit for detecting and / or treating L1-CAM-related cancers, the kit comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein) or a functional variant thereof (e.g., a substitution variant). In certain embodiments, the immunoglobulin-related composition is coupled to one or more detectable labels. In one embodiment, the one or more detectable labels include a radioactive label, a fluorescent label, or a chromogenic label.

[0026] Additionally or alternatively, in some embodiments, the kit further comprises a second antibody that specifically binds to the anti-L1-CAM immunoglobulin-related composition described herein. In some embodiments, the second antibody is coupled to at least one detectable label selected from a radioactive label, a fluorescent label, or a chromogenic label. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The molecular structure of human L1-CAM is shown.

[0028] Figure 2 The interaction of L1-CAM with integrins and cell signaling is shown (adapted from Gavert et al., Expert opinion Biol Therapy 8: 1749, 2008).

[0029] Figure 3 Expression of L1-CAM in normal human tissues is shown (adapted from Weidle et al., Anticancer Research 29:4919-4932, 2009).

[0030] Figure 4 The expression of L1-CAM in human tumors is shown (adapted from Rawnaq T et al., J Surg Res 173:314-9 (2012)).

[0031] Figure 5 Different murine and chimeric antibodies against L1-CAM are shown.

[0032] Figure 6 The heavy chain variable domain (V H ) and the light chain variable domain (V L ) and its homologous human sequences (SEQ ID NO: 1-4). The CDRs of the heavy and light chains of murine E71 and their homologous human sequences are underlined.

[0033] Figure 7 The V of mouse antibody E72 is shown H and V L The amino acid sequences of murine E72 and their homologous human sequences (SEQ ID NOs: 5-8). The CDRs of the heavy and light chains of murine E72 and their homologous human sequences are underlined. H CDR1, V H CDR2 and V HThe amino acid sequences of CDR3 are GYWMH (SEQ ID NO: 68), EINPSNGRTNYNERFKS (SEQ ID NO: 69) and DYYGTSYNFDY (SEQ ID NO: 70), respectively, and V L CDR1, V L CDR2 and V L The CDR3 amino acid sequences are KANEDINNRLA (SEQ ID NO: 71), GATNLVT (SEQ ID NO: 72) and QQYWSTPFT (SEQ ID NO: 73), respectively.

[0034] Figure 8 The amino acid sequences of the humanized heavy chains of E71 are shown (SEQ ID NOs: 9-10).

[0035] Fig. 9 The cDNA sequence of the humanized heavy chain of E71 is shown (SEQ ID NOs: 11-12).

[0036] Fig.10 The amino acid sequence of the humanized light chain of E71 is shown (SEQ ID NOs: 13-14).

[0037] Fig.11 The cDNA sequence of the humanized light chain of E71 is shown (SEQ ID NOs: 15-16).

[0038] Fig.12 The amino acid sequence of the humanized heavy chain of E72 is shown (SEQ ID NOs: 17-18).

[0039] Fig.13 The cDNA sequences of the humanized heavy chains of E72 are shown (SEQ ID NOs: 19-20).

[0040] Fig.14 The amino acid sequence of the humanized light chain of E72 is shown (SEQ ID NOs: 21-22).

[0041] Fig.15 The cDNA sequence of the humanized light chain of E72 is shown (SEQ ID NOs: 23-24).

[0042] Fig.16 The amino acid sequences of the heavy and light chains of chimeric chE71 are shown (SEQ ID NOs: 25-26).

[0043] Fig.17 The cDNA sequences of the heavy and light chains of chimeric chE71 are shown (SEQ ID NOs: 27-28).

[0044] Fig.18 The amino acid sequences of the heavy and light chains of chimeric chE72 are shown (SEQ ID NOs: 29-30).

[0045] Fig.19 The cDNA sequences of the heavy and light chains of chimeric chE72 are shown (SEQ ID NOs: 31-32).

[0046] Fig. 20 The amino acid and cDNA sequences of the heavy chain of huE71 are shown (SEQ ID NOs: 33-34).

[0047] Fig.21 The amino acid and cDNA sequences of the light chain of huE71 are shown (SEQ ID NOs: 35-36).

[0048] Fig. 22 The amino acid and cDNA sequences of the heavy chain of huE72 are shown (SEQ ID NOs: 37-38).

[0049] Fig.23 The amino acid and cDNA sequences of the light chain of huE72 are shown (SEQ ID NOs: 39-40).

[0050] Fig.24 The amino acid sequences of the heavy and light chains of the chimeric IgG4, chE71-IgG4, are shown (SEQ ID NOs: 41-42).

[0051] Fig.25 The cDNA sequences of the heavy and light chains of the chimeric IgG4, chE71-IgG4, are shown (SEQ ID NOs: 43-44).

[0052] Fig.26 The amino acid sequences of the heavy and light chains of the humanized IgG4, huE71-IgG4, are shown (SEQ ID NOs: 45-46).

[0053] Fig. 27 The cDNA sequences of the heavy and light chains of humanized IgG4, huE71-IgG4, are shown (SEQ ID NOs: 47-48).

[0054] Fig.28 The kinetics of binding of humanized E71 or E72 IgG1 and BsAb are shown.

[0055] Fig.29 Shown are the binding kinetics of humanized IgG variants of huE71 measured on L1-CAM-Fc using SPR (Biacore T100).

[0056] Fig.30 Shown are the binding kinetics of humanized IgG variants of huE72 determined on L1-CAM-Fc using SPR (Biacore T100).

[0057] Fig.31 Shown are the binding kinetics of humanized IgG variants of huE72 determined using SPR (Biacore T100) on L1-CAM-His.

[0058] Fig.32 Shown are cell surface staining of mesothelioma cell lines by flow cytometry using mouse E71 and MOPC21 (as a control).

[0059] Fig.33 Shown are cell surface staining of leukemia, breast cancer, and Ewing's sarcoma cell lines by flow cytometry using humanized huE71-IgGn and Rituxan (as a control).

[0060] Fig.34 Shown are cell surface staining of melanoma, neuroblastoma, osteosarcoma, rhabdomyosarcoma, small cell lung cancer (SCLC), and head and neck cancer by flow cytometry using humanized huE71-IgGn and Rituxan (as a control).

[0061] Fig.35 Shown is the construction of T cell engaging bispecific antibodies (BsAbs) against both huE71 and huE72.

[0062] Fig.36 The amino acid sequences of T cell engaging bispecific antibodies (BsAb) huE71-BsAb and huE72-BsAb are shown (V H and V L )(SEQ ID NO:49-52).

[0063] Fig.37 Shown are the binding kinetics of humanized huE71 or huE72 IgG and their BsAb on L1-CAM-Fc measured using SPR (Biacore T100).

[0064] Figure 38 (A) shows the binding of humanized E71-BsAb and humanized E72-BsAb to L1-CAM(+) tumor cells. Figure 38 (B) shows the binding of humanized E71-BsAb and humanized E72-BsAb to T cells.

[0065] Figure 39 (A) shows the results of IL-6 cytokine release assays for antibodies of the present technology in the presence or absence of L1-CAM (+) tumor cell lines. Figure 39 (B) shows the results of IL-10 cytokine release assays for antibodies of the present technology in the presence or absence of L1-CAM (+) tumor cell lines. Figure 39 (C) shows the results of IFN-γ cytokine release assays for antibodies of the present technology in the presence or absence of L1-CAM (+) tumor cell lines. Figure 39 (D) shows the results of IL-2 cytokine release assays for antibodies of the present technology in the presence or absence of L1-CAM (+) tumor cell lines. Figure 39 (E) shows the results of TNF-α cytokine release assays for antibodies of the present technology in the presence or absence of L1-CAM (+) tumor cell lines.

[0066] Fig.40 Shows 51 Results of chromium release assay. Humanized E71-BsAb or humanized E72-BsAb were incubated with T cells and IMR-32 luciferase cell line (E:T ratio = 10:1) for 4 hours.

[0067] Fig.41 The cytotoxicity (EC50) of humanized E71-BsAb or humanized E72-BsAb as well as the L1-CAM expression (mean fluorescence index or MFI) of tumor cell lines are shown.

[0068] Figure 42 (A) shows that the cytotoxicity (EC50) of humanized E71-BsAb is inversely correlated with the L1-CAM expression (MFI) of tumor cell lines. Figure 42 (B) shows that the cytotoxicity (EC50) of humanized E72-BsAb is inversely correlated with the L1-CAM expression (MFI) of tumor cell lines.

[0069] Fig.43 The effects of huE71-BsAb and huE72-BsAb on neuroblastoma tumors in a humanized mouse model are shown. L1-CAM(+) neuroblastoma patient-derived xenografts (PDX) were subcutaneously transplanted into DKO mice. HuE71-BsAb or huE72-BsAb treatment was initiated on day 15 when tumors were measurable, using a dosing schedule of twice weekly injections for five weeks. PBMCs (7.5×10 6 ), and then injected once a week for 3 weeks. Tumor size was measured once a week. Tumor growth in mice treated with huE72-BsAb and PBMC was equivalent to that of controls. HuE71-BsAb and PBMC significantly inhibited tumor growth.

[0070] Fig.44 The effects of huE71-BsAb and huE72-BsAb on neuroblastoma tumors in a humanized mouse model are shown. The L1-CAM (+) neuroblastoma cell line IMR32 was mixed with human PBMCs and implanted subcutaneously into DKO mice. HuE71-BsAb or huE72-BsAb treatment was started on day 5 when the tumor was measurable, using a dosing schedule of twice weekly injections for five weeks. The size of the tumor was measured once a week. Mice treated with huE72-BsAb or control (deglycosylated huE71-IgG1 + deglycosylated huOKT3) had equal tumor growth. HuE71-BsAb or huE71-IgGn significantly inhibited tumor growth.

[0071] Fig.45 Shows 89 Continuous PET imaging of Zr-HuE71-IgGn(MAGE). Small animal PET imaging in a subcutaneous ovarian cancer tumor model reveals high tumor and lymph node activity concentrations. In athymic nude mice bearing subcutaneous SKOV-3 tumors xenografted on the right shoulder 89 Representative maximum intensity projection (MIP) PET images of Zr-HuE71-1 MAGE (192-197 μCi in 150 μL PBS injected via tail vein). Selective tumor uptake began as early as 24 hours and continued to increase until 96 hours.

[0072] Fig.46 Shows 89 Continuous PET imaging of Zr-HuE72-IgGn(MAGE). Small animal PET imaging in a subcutaneous ovarian cancer tumor model reveals high tumor and lymph node activity concentrations. In athymic nude mice bearing subcutaneous SKOV-3 tumors xenografted on the right shoulder 89 Representative maximum intensity projection (MIP) PET images of Zr-HuE72-IgGn(MAGE) (approximately 200 μCi in 150 μL PBS injected via tail vein).Selective tumor uptake was suboptimal at 24 hours; it did increase over time.

[0073] Fig.47 Shown is a table summarizing the characteristics of humanized anti-L1-CAM antibodies of the present technology.

[0074] Figure 48(A) to Figure 48(E) This technology shows 89Immunoreactivity of Zr radiolabeled L1-CAM antibodies. The radioimmunoconjugates were functionally characterized for binding to antigen-expressing cells (SKOV-3) and the immunoreactivity scores were determined via the Lindmo assay (Ben QW et al., Ann Surg Oncol 17:2213-21 (2010)). An aliquot of each radioimmunoconjugate, 50 μL of a 1 μCi / mL stock solution, was added to 500 μL of PBS, 1% BSA (pH 7.4 to a final volume of 550 μL) containing 5.0×10 5 -5.0×10 6 Cells / mL (triplicate) were added to the tubes. Cell binding activity was counted and the data were plotted on a double reciprocal graph. Immunoreactivity scores were expressed as the mean percentage ± SEM of triplicate samples.

[0075] Fig.49 Shows 89 Serial PET imaging of Zr-HuE71-1 WT antibody. Small animal PET imaging in a subcutaneous ovarian cancer tumor model reveals high tumor, liver, and lymph node activity concentrations. In athymic nude mice bearing subcutaneous L1-CAM-positive SKOV-3 tumors xenografted on the right shoulder 89 Representative maximum intensity projection (MIP) PET images of Zr-HuE71-1 WT (194-201 μCi in 150 μL PBS injected via tail vein).

[0076] Fig.50 Shows 89 Continuous PET imaging of Zr-deglycosylated HuE71-1 antibody. Small animal PET imaging in a subcutaneous ovarian cancer tumor model reveals high tumor and very low background activity concentrations. In athymic nude mice bearing subcutaneous L1-CAM-SKOV-3 tumors xenografted on the right shoulder 89 Representative maximum intensity projection (MIP) PET images of Zr-deglycosylated HuE71-1 (207-214 μCi in 150 μL PBS injected via tail vein).

[0077] Fig.51 Shows 89 Serial PET imaging of Zr-HuE71-4 WT antibody. Small animal PET imaging in a subcutaneous ovarian cancer tumor model reveals high tumor and kidney activity concentrations. In athymic nude mice bearing subcutaneous L1-CAM-positive SKOV-3 tumors xenografted on the right shoulder 89 Representative maximum intensity projection (MIP) PET images of Zr-HuE71-4 WT (198-204 μCi in 150 μL PBS injected via tail vein).

[0078] Fig.52 Shows 89 Serial PET imaging of the Zr-HuE71-4 mutant antibody. Small animal PET imaging in a subcutaneous ovarian cancer tumor model reveals high tumor and background tissue activity, with minimal kidney activity. In athymic nude mice bearing subcutaneous L1-CAM-positive SKOV-3 tumors xenografted on the right shoulder 89 Representative maximum intensity projection (MIP) PET images of Zr-HuE71-4 mutant (206-212 μCi in 150 μL PBS injected via tail vein).

[0079] Fig.53 Shows 89 Serial PET imaging of Zr-HuCtrl-4 WT antibody. Small animal PET imaging in a subcutaneous ovarian cancer tumor model reveals low tumor activity but high kidney activity. In athymic nude mice bearing subcutaneous L1-CAM-positive SKOV-3 tumors xenografted on the right shoulder 89 Representative maximum intensity projection (MIP) PET images of Zr-HuCtrl-4WT (200-211 μCi in 150 μL PBS injected via tail vein).

[0080] Fig.54 The ex vivo biodistribution of IgG1 antibody 96 hours after tracer injection is shown. Acute biodistribution of radioimmunoconjugates in SKOV-3 tumor-bearing mice. Biodistribution data from athymic nude mice (n=4 per group) following administration of IgG1 radioimmunoconjugates (17-24 μCi, 3-6 μg) via tail vein injection. A 50-fold excess was injected for the blocking group. %ID / g values ​​were Fig.55 Asterisks indicate P < 0.05.

[0081] Fig.55 Ex vivo biodistribution of IgG1 antibody 96 hours after injection is shown.

[0082] Fig.56 The ex vivo biodistribution of IgG4 antibody 96 hours after tracer injection is shown. Acute biodistribution of radioimmunoconjugates in SKOV-3 tumor-bearing mice. Biodistribution data obtained from athymic nude mice (n=4 per group) following administration of IgG4 radioimmunoconjugates (21-26 μCi, 3-6 μg) via tail vein injection. A 50-fold excess was injected for the blockade group. %ID / g values ​​were Fig.57 Asterisks indicate P < 0.05.

[0083] Fig.57Ex vivo biodistribution values ​​for IgG4 antibodies 96 hours after injection are shown.

[0084] Fig.58 Ex vivo histological analysis of tumor and lymph node tissues is shown. Tumor and lymph node tissues from mice injected with the indicated radioimmunoconjugates were collected, embedded in paraffin, and subjected to routine H&E staining and analysis.

[0085] Figure 59(A) to Figure 59(C) Shows 177 Results of Lu-deglycosylated HuE71-1 imaging and ex vivo biodistribution. Figure 59 (A): 177 SPECT-CT images of athymic nude mice bearing SKOV-3 tumors 168 hours after injection of Lu-deglycosylated HuE71-1 (858 μCi in 150 μL PBS). FIG59(B): 177 Cerenkov images of athymic nude mice bearing SKOV-3 tumors 168 hours after injection of Lu-deglycosylated HuE71-1 (858 μCi in 150 μL PBS). FIG59(C): 177 Ex vivo biodistribution of Lu-deglycosylated HuE71-1 (18-26 μCi in 150 μL PBS, n=4) in athymic nude mice bearing SKOV-3 tumors 168 hours after injection.

[0086] Fig.60 Shows the progress 177 Mean tumor volume in animals treated with Lu-deglycosylated HuE71-1 radioimmunotherapy. A graph showing the mean tumor volume for each mouse cohort during the first 80 days of the PRIT study is shown, with error bars representing standard deviation. Black arrows indicate the first injection of a given treatment, and gray arrows indicate the first injection of a given treatment in a split-dose group. 177 Second dose of Lu-deglycosylated HuE71-1. Tumor volumes were obtained using a handheld TM900 scanner (Peira, Brussels, Belgium). After initial tumor measurements, mice were randomized into groups (n=8-9 per group) to ensure that all groups had approximately equal mean tumor volumes at the start.

[0087] Fig.61 It was shown that huE71-IgG1n mediates ADCC in the LAN-1 cell line.

[0088] Fig.62 It was shown that huE71-IgG1n mediates ADCC in the NB1691 cell line. DETAILED DESCRIPTION

[0089] It should be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail to provide a substantial understanding of the present technology.

[0090] The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that can specifically bind to and neutralize the biological activity of L1-CAM polypeptides. The immunoglobulin-related compositions of the present technology can be used in methods for detecting or treating L1-CAM-related cancers in subjects in need thereof. Therefore, various aspects of the present method relate to the preparation, characterization and manipulation of anti-L1-CAM antibodies. The immunoglobulin-related compositions of the present technology can be used alone or in combination with additional therapeutic agents for treating cancer. In some embodiments, the immunoglobulin-related compositions are humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0091] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; Ausubel et al., eds. (2007) Current Protocols in Molecular Biology Series; Methods in Enzymology Series (Academic Press, Inc., New York); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press of Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane, eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th ed.; Gait, ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization. Acid Hybridization; Hames and Higgins, eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos, eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides et al. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker, eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al., eds. (1996) Weir's Handbook of Experimental Immunology.Methods for detecting and measuring the level of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantitative techniques. (See also Strachan & Read, Human Molecular Genetics, 2nd Edition (John Wiley & Sons, New York, 1999)).

[0092] definition

[0093] Unless otherwise limited, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which the technology belongs. Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural indicators. For example, reference to "a cell" includes combinations of two or more cells, and so on. In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below are those well known and commonly used in the art.

[0094] Unless otherwise indicated or apparent from the context, as used herein, the term "about" with respect to a number is generally considered to include numbers within 1%, 5%, or 10% in either direction (greater than or less than) of the stated number (except where such a number is below 0% or above 100% of the possible value).

[0095] As used herein, "administering" an agent or drug to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration can be by any suitable route, including, but not limited to, oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, intratumoral, or topical. Administration includes self-administration and administration by another person.

[0096] "Adjuvant" refers to one or more substances that cause immune system stimulation. In this article, adjuvants are used to enhance the immune response to one or more vaccine antigens or antibodies. The adjuvant can be administered to the subject before, together with or after the administration of the vaccine. Examples of compounds used as adjuvants include aluminum compounds, oils, block polymers, immunostimulatory complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium and vitamin B12), Quil A (saponin), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins and glycoproteins), hormones, cytokines and costimulatory factors.

[0097] As used herein, the term "antibody" collectively refers to immunoglobulins or immunoglobulin-like molecules, including, for example, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, such as mammals (such as humans, goats, rabbits, and mice), and non-mammalian species (such as shark immunoglobulins). As used herein, "antibodies" (including complete immunoglobulins) and "antigen-binding fragments" specifically bind to a target molecule (or a group of highly similar target molecules) while substantially excluding binding to other molecules (e.g., the binding constant for the target molecule is at least 10 greater than the binding constant for other molecules in the biological sample). 3 M -1 , at least 10 4 M -1 Or at least 10 5 M -1 The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Edition, WH Freeman & Co., New York, 1997.

[0098] More specifically, an antibody refers to a polypeptide ligand that contains at least a light chain immunoglobulin variable region or a heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen epitope. Antibodies are composed of heavy chains and light chains, each of which has a variable region, called a heavy chain variable (V H ) region and light chain variable (V L ) area. H Area and V LThe regions are jointly responsible for binding to the antigen recognized by the antibody. Typically, immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains, namely λ (lambda) and κ (kappa). There are five main heavy chain categories (or isotypes): IgM, IgD, IgG, IgA and IgE, which determine the functional activity of the antibody molecule. Each heavy chain and light chain contains a constant region and a variable region (the region is also referred to as a "domain"). The combined heavy chain variable region and light chain variable region specifically bind to the antigen. The light chain variable region and the heavy chain variable region contain a "framework" region interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). The scope of the framework region and CDR has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is currently maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody, i.e., the combined framework region of the constituent light and heavy chains, mainly adopts a β-sheet conformation, and the CDR forms a loop connecting the β-sheet structure and forming a part of the β-sheet structure in some cases. Therefore, the framework region plays a role in forming a scaffold that provides for positioning the CDR in the correct orientation through non-covalent interactions between the chains.

[0099] The CDRs are primarily responsible for binding to the antigen epitope. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are usually also identified by the chain in which the particular CDR is located. H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while V L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds to the L1-CAM protein will have a specific V H Area and V L Region sequence, thus having a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs are different between different antibodies, only a limited number of amino acid positions in the CDR are directly involved in antigen binding. These positions in the CDR are called specificity determining residues (SDRs). As used herein, "immunoglobulin-related compositions" refer to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) and antibody fragments. Antibodies or their antigen-binding fragments specifically bind to antigens.

[0100] As used herein, the term "antibody-related polypeptide" refers to an antigen-binding antibody fragment, including a single-chain antibody, which may contain one or more variable regions alone or in combination with all or part of the following polypeptide elements: the hinge region of an antibody molecule, a CH 1 , CH 2 and CH 3 The technology also includes one or more variable regions and hinge regions, CH 1 , CH 2 and CH 3 Any combination of domains. Antibody-related molecules that can be used in this method include, but are not limited to, Fab, Fab' and F(ab') 2 , Fd, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (sdFv) and V L or V H Examples include: (i) Fab fragments, i.e., fragments of V L 、V H , C L and CH 1 Monovalent fragment composed of structural domains; (ii) F(ab') 2 A fragment comprising two Fab fragments connected by a disulfide bond in the hinge region; (iii) a fragment comprising V H and CH 1 (iv) an Fd fragment consisting of a single-arm V L and V H (v) dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of V H domains; and (vi) isolated complementarity determining regions (CDRs). Thus, an "antibody fragment" or "antigen binding fragment" may comprise a portion of a full-length antibody, typically its antigen binding or variable region. Examples of antibody fragments or antigen binding fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0101] As used herein, "bispecific antibody" or "BsAb" refers to an antibody that can simultaneously bind to two targets with different structures (e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen). A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding portion in the bispecific antibody includes a V H and / or V L region; in some such embodiments, VH and / or V L The V regions are those found in a particular monoclonal antibody. In some embodiments, a bispecific antibody contains two antigen-binding portions, each comprising a V region from a different monoclonal antibody. H and / or V L In some embodiments, the bispecific antibody contains two antigen-binding moieties, wherein one of the two antigen-binding moieties comprises a V H and / or V L region of the immunoglobulin molecule, the V H and / or V L The antigen binding portion comprises a CDR region from the first monoclonal antibody; and the other antigen binding portion comprises a V H and / or V L Antibody fragments (e.g., Fab, F(ab'), F(ab') 2 , Fd, Fv, dAB, scFv, etc.), the V H and / or V L The region contains the CDRs from the second monoclonal antibody.

[0102] As used herein, the term "conjugated" refers to the association of two molecules by any method known to those skilled in the art. Suitable types of association include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordination bonds. Physical bonds include, for example, hydrogen bonds, dipole interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic stacking.

[0103] As used herein, the term "diabodies" refers to small antibody fragments with two antigen-binding sites, said fragments comprising two antigen-binding sites bound to the same polypeptide chain (V H V L ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and 30 Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0104] As used herein, the term "single-chain antibody" or "single-chain Fv (scFv)" refers to the two domains V and Fv fragments. L and V H The single-chain antibody molecule may include a polymer having multiple individual molecules, such as a dimer, trimer, or other polymer.v The two domains of the fragment V L and V H The two proteins are encoded by separate genes, but they can be joined by synthetic linkers using recombinant methods to make them into a single protein chain in which V L and V H The domains are paired to form a monovalent molecule called a single-chain Fv (scFv). Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85: 5879-5883. Such single-chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0105] Fragments of any of the foregoing antibodies are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for binding specificity and neutralization activity in the same manner as are intact antibodies.

[0106] As used herein, "antigen" refers to a molecule to which an antibody (or its antigen-binding fragment) can selectively bind. The target antigen can be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen can be a polypeptide (e.g., L1-CAM polypeptide). Antigens can also be administered to animals to generate an immune response in the animal.

[0107] The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that has a portion of the polypeptide responsible for binding to the antigen. Examples of antigen-binding fragments that can be used in the present technology include scFv, (scFv) 2 , scFvFc, Fab, Fab′ and F(ab′) 2 , but not limited to this.

[0108] "Binding affinity" refers to the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K d ) is represented by. Affinity can be measured by standard methods known in the art (including those described herein). Low affinity complexes contain antibodies that generally tend to dissociate easily from the antigen, while high affinity complexes contain antibodies that generally tend to remain bound to the antigen for a longer period of time.

[0109] As used herein, the term "biological sample" means a sample material derived from living cells. Biological samples can include tissues, cells, proteins or membrane extracts of cells separated from a subject and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)), as well as tissues, cells and fluids present in a subject. The biological samples of this technology include but are not limited to samples taken from: breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsil, thymus, blood, hair, cheek, skin, serum, plasma, CSF, sperm, prostatic fluid, semen, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph and tears. Biological samples can also be obtained from a biopsy of an internal organ or from cancer. Biological samples can be obtained from subjects for diagnosis or research; or can be obtained from individuals who are not ill, as controls or for basic research. Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a breast, lung, colon or prostate tissue sample obtained by needle biopsy.

[0110] As used herein, the term "CDR-grafted antibody" means an antibody in which at least one CDR of a "recipient" antibody is replaced by a CDR "graft" from a "donor" antibody having the desired antigen specificity.

[0111] As used herein, the term "chimeric antibody" refers to an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse Fc constant region) is replaced with the Fc constant region of an antibody from another species (e.g., a human Fc constant region) using recombinant DNA technology. See generally Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 0125,023; Better et al., Science 240:1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443, 1987; Liu et al., J. Immunol 139:3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218, 1987; Nishimura et al., Cancer Res 47:999-1005, 1987; Wood et al., Nature 314:446-449, 1885; and Shaw et al., J. Natl. Cancer Inst. 80:1553-1559, 1988.

[0112] As used herein, the term "consensus FR" means the framework (FR) antibody region in the consensus immunoglobulin sequence. The FR region of an antibody does not contact the antigen.

[0113] As used herein, a "control" is an alternative sample used in an experiment for comparison purposes. A control can be "positive" or "negative." For example, if the purpose of an experiment is to determine the relevance of a therapeutic agent's efficacy in the treatment of a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive therapy or receives a placebo) are typically used.

[0114] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic and / or preventive effect, such as an amount that can prevent or reduce a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or preventive applications, the amount of the composition administered to the subject will vary according to the composition, the extent, type and severity of the disease, and according to individual characteristics (such as general health, age, sex, body weight and drug tolerance). The technician will be able to determine the appropriate dose based on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition can be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a composition level in which the physiological effects of a disease or condition are improved or eliminated. The therapeutically effective amount can be administered in one or more administrations.

[0115] As used herein, the term "effector cell" means an immune cell involved in the effector phase of an immune response that is distinct from the cognitive phase and activation phase of an immune response. Exemplary immune cells include cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear leukocytes, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and perform specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as neutrophils capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcαR participate in the specific killing of target cells and present antigens to other components of the immune system, or bind to cells presenting antigens.

[0116] As used herein, the term "epitope" means a protein determinant that can specifically bind to an antibody. An epitope is usually composed of chemically active surface groups of a molecule (such as amino acids or sugar side chains) and usually has specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between a conformational epitope and a non-conformational epitope is that the binding to a conformational epitope (rather than a non-conformational epitope) is lost in the presence of a denaturing solvent. In some embodiments, the "epitope" of the L1-CAM protein is a region of the protein to which the anti-L1-CAM antibodies of the present technology specifically bind. In some embodiments, the epitope is a conformational epitope. In order to screen for anti-L1-CAM antibodies that bind to an epitope, a conventional cross-blocking assay can be performed, such as the assay described in the following document: Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Harlow and David Lane (1988). This assay can be used to determine whether an anti-L1-CAM antibody binds to the same site or epitope as the anti-L1-CAM antibodies of the present technology. Alternatively or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized, such as by alanine scanning, to identify contact residues. In a different approach, peptides corresponding to different regions of the L1-CAM protein can be used in competition assays with multiple test antibodies, or with a test antibody and an antibody with a characterized or known epitope.

[0117] As used herein, "expression" includes one or more of the following: transcription of a gene into a pre-mRNA; splicing and other processing of the pre-mRNA to produce a mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product (if required for proper expression and function).

[0118] As used herein, the term "gene" means a segment of DNA that contains all the information regulating the biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0119] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be used for comparison purposes. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (polypeptide or polypeptide region) has a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence means that, when compared, the percentage of bases (or amino acids) in the comparison of the two sequences is the same. This comparison and homology or sequence identity percentage can be determined using software programs known in the art. In some embodiments, default parameters are used for comparison. One comparison program is BLAST, using default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; chain = two; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting mode = high score (HIGH SCORE); database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Detailed information on these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are polynucleotides with a specified homology percentage and encoding polypeptides with the same or similar biological activity. If two sequences have less than 40% identity or less than 25% identity to each other, they are considered "unrelated" or "non-homologous".

[0120] As used herein, the term "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody that contains minimal sequence derived from a non-human immunoglobulin. For the most part, a humanized antibody is a human immunoglobulin in which the hypervariable region residues of the receptor are replaced by hypervariable region residues (donor antibody) from a non-human species (e.g., mouse, rat, rabbit, or non-human primate) with the desired specificity, affinity, and ability. In some embodiments, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may include residues not found in the receptor antibody or the donor antibody. These modifications are performed to further improve antibody performance such as binding affinity. Typically, a humanized antibody will include at least one, usually two, variable domains (e.g., Fab, Fab', F(ab') 2or Fv), wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins and all or substantially all of the FR regions are regions of human immunoglobulin consensus FR sequences, but the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in FR is generally no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody may also optionally include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For further details, see Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). See, for example, Ahmed & Cheung, FEBS Letters 588 (2): 288-297 (2014).

[0121] As used herein, the term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. A hypervariable region generally includes amino acid residues from a "complementarity determining region" or "CDR" (e.g., V L Approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in V H 31-35B (H1), 50-65 (H2), and 95-102 (H3)) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991))) and / or those residues from the "hypervariable loops" (e.g., V L Residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in V H 26-32 (H1), 52A-55 (H2) and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0122] As used herein, when used in the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity" percentage refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence of an antibody described herein) when compared and aligned for maximum correspondence over a comparison window or a specific region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm using the default parameters described below or by manual alignment and visual inspection (e.g., NCBI website). Such sequences are then referred to as "substantially identical". This term also refers to or may be applicable to the complement of a test sequence. The term also includes sequences with deletions and / or additions, as well as sequences with substitutions. In some embodiments, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or 50-100 amino acids or nucleotides in length.

[0123] As used herein, the term "intact antibody" or "intact immunoglobulin" means an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or V H ) and the heavy chain constant region. The heavy chain constant region consists of three domains CH 1 , CH 2 and CH 3 Each light chain is composed of a light chain variable region (abbreviated as LCVR or V L ) and the light chain constant region. The light chain constant region consists of a domain C L Composition. H and V L The V region can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR 1 , CDR 1 , FR 2 , CDR 2 , FR 3 , CDR 3 , FR 4The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0124] As used herein, the term "individual," "patient," or "subject" can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human.

[0125] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially uniform antibody population, that is, except for possible naturally occurring mutations that may be present in small amounts, the individual antibodies constituting the population are identical. For example, a monoclonal antibody may be an antibody derived from a single clone (including any eukaryotic, prokaryotic or phage clone) rather than a method for producing it. A monoclonal antibody composition exhibits a single binding specificity and affinity to a specific epitope. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, compared to conventional (polyclonal) antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on the antigen. The modifier "monoclonal" indicates that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibodies to be produced by any ad hoc method. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including but not limited to hybridoma, recombinant and phage display techniques. For example, the monoclonal antibodies used according to the present method can be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). For example, "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described by Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0126] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, etc., that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, A. Gennaro, 2000, Lippincott ed., Williams & Wilkins, Philadelphia, Pennsylvania).

[0127] As used herein, the term "polyclonal antibody" means a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. The use of this term includes preparations of at least two (2) antibodies containing antibodies that specifically bind to different epitopes or regions of an antigen.

[0128] As used herein, the term "polynucleotide" or "nucleic acid" means any RNA or DNA, which can be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA, which can be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, polynucleotides refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA to which the backbone is modified for stability or other reasons.

[0129] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres). Polypeptides refer to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and longer chains, commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified by natural processes (such as post-translational processing) or by chemical modification techniques well known in the art. Such modifications are fully described in basic texts and more detailed monographs as well as a large research literature.

[0130] As used herein, the term "recombinant" when used with respect to, for example, a cell or a nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found within the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

[0131] As used herein, the term "separate" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by different routes.

[0132] As used herein, the term "sequential" therapeutic use refers to administration of at least two active ingredients at different times, with the same or different routes of administration. More specifically, sequential use refers to the entire administration of one of the active ingredients before the administration of the other or other active ingredients begins. Thus, administration of one of the active ingredients may be followed by administration of the other active ingredient or ingredients within minutes, hours or days. In this case there is no simultaneous treatment.

[0133] As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize and bind to other molecules. As used herein, the terms "specifically bind," "specifically binds," or "specific for" a particular molecule (e.g., a polypeptide or an epitope on a polypeptide) can be, for example, characterized by a molecule having about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M of K d The term "specifically binds" may also refer to binding in which a molecule (e.g., an antibody or antigen-binding fragment thereof) binds to a specific polypeptide (e.g., L1-CAM polypeptide) or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0134] As used herein, the term "simultaneous" therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or substantially the same time.

[0135] As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0136] As used herein, "treating" or "treatment" encompasses treating a disease or disorder described herein in a subject, such as a human, and includes: (i) inhibiting the disease or disorder, i.e., preventing its development; (ii) alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease, for example, have been alleviated, mitigated, cured, or are in a state of remission.

[0137] It is also understood that the various treatment modalities for disorders as described herein are intended to mean "substantially," which includes complete treatment but also less than complete treatment, and in which some biologically or medically relevant result is achieved. The treatment can be continuous, extended treatment for chronic conditions, or a single or several administrations for acute condition treatment.

[0138] One or more amino acid sequence modifications of the anti-L1-CAM antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the anti-L1-CAM antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions and substitutions may be performed to obtain the desired antibody, as long as the antibody obtained has the desired properties. The modifications also include changes in the glycosylation pattern of the protein. The sites of greatest interest for substitution mutagenesis include the hypervariable regions, but FR changes are also contemplated. "Conservative substitutions" are shown in the table below.

[0139]

[0140] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody. A convenient method for generating such substitution variants involves affinity maturation using phage display. In particular, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody variants thus produced are displayed as fusions of the gene III product of M13 packaged in each particle in a monovalent form from filamentous phage particles. The variants displayed by phage are then screened for their biological activity (e.g., binding affinity) as disclosed herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, analyzing the crystal structure of the antigen-antibody complex may be beneficial to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution according to the technology described in detail herein. Once such variants are produced, this group of variants is screened as described herein, and antibodies with similar or better properties in one or more related assays can be selected for further development.

[0141] L1-CAM

[0142] L1-CAM is a member of the L1 family of adhesion molecules, which is part of the immunoglobulin superfamily (IgSF CAM) and is involved in axon guidance, neuronal migration and differentiation. The L1 family includes L1-CAM (CD171), a near homologue of L1-CAM (CHL1), neurofascin and NgCAM-related cell adhesion molecules (NR-CAM). These proteins are expressed in neurons, especially on their axons and glial cells (such as Schwann cells). L1-CAM is a neural cell adhesion molecule involved in the development of the central nervous system. L1-CAM is composed of 28 exons and 27 introns, and the molecular weight of its gene product ranges between 200 and 220 kDa (Coutelle O et al., Gene 208: 7-15 (1998)). The extracellular domain (ECM, exon 1 to exon 24) has six Ig-like domains ( Figure 1 ) and five fibronectin-like domains, of which N-glycosylation is in the first fibronectin domain. The RGD motif is present in the sixth Ig-like domain. ECM is involved in homophilic binding ( Figure 2 ), and shares homology with FGFR. The cytoplasmic domain contains five potential phosphoserine residues and can interact with the cytoskeleton, second messenger pathways and kinases. Two exons (2 and 27) are spliced ​​alternately (Coutelle O et al., Gene 208:7-15 (1998)).

[0143] L1-CAM mutations cause an X-linked neurological disorder called CRASH (genesis of the corpus callosum, developmental delay, adducted thumbs, spastic paraplegia, and hydrocephalus). The clinical syndrome caused by L1-CAM mutations is variable: more than 70 L1-CAM mutations in all parts of the L1-CAM molecule have been described in patients with CRASH. L1-CAM knockout mice show hyperplasia of the corticospinal tracts and abnormalities of the ventricular system. L1-CAM mediates adhesion to different substrates in a context-dependent manner. Through hemophilic binding, L1-CAM interacts with other adhesion molecules such as axonin-1 / TAX-1, contactin, neurocan, neuropilin 1, and integrins such as αvβ3, α5β1, αvβ1, and αvβ5. Both cis (between molecules in the same cell membrane) and trans (between molecules on opposite membranes) interactions have been described. N-linked carbohydrates in the ECM account for 25% of the molecular weight of L1-CAM. The ECM has two proteolytic cleavage sites: a distal site that is cleaved by the metalloproteinase ADAM 10, resulting in fragments of 200 and 32 kDa. L1-CAM has been shown to be involved in multiple proliferation pathways, anti-apoptosis pathways, and angiogenesis-related pathways.

[0144] L1-CAM is commonly found in neural tissue ( Figure 3 ), while non-neuronal cells, including cancer cells, predominantly express variants lacking exons 2 and 27. Exon 2 (YEGHHV, encoding the N-terminal Ig1 domain) is important for homophilic L1-L1 binding in vitro and is required for optimal binding to heterophilic ligands. RSLE sequences (cytoplasmic tails) containing L1-CAM encoded by exon 27 are internalized 2-3 times faster than L1Δ (RSLE). RSLE-dependent endocytosis is a mechanism that regulates L1-CAM surface density, which in turn controls neurite branching and cell adhesion. Ovarian cancer cell lines predominantly express L1-CAMΔ in vitro. Activation of ERK by L1-CAM requires endocytosis of L1-CAM mediated by exon 27. These different splice variants may have different or even opposing functions in cancer biology.

[0145] L1-CAM signaling in human cancer. L1-CAM and its cleavage enzyme ADAM10 are associated with the metastatic potential of colorectal cancer when found at the invasion front. β-Catenin-Wnt is known to confer cell motility, invasion, and tumorigenesis in fibroblasts and colon cancer cells by signaling through L1-CAM. L1-CAM promotes cell motility and maintains an invasive phenotype, whether through homophilic or heterophilic binding. In the presence of serum or platelet-derived growth factor, L1-CAM stimulates the extracellular signal-related kinase (ERK) pathway, leading to the expression of gene products associated with motility and invasion, such as β3 integrin subunits, small GTPases, and cysteine ​​proteases, cathepsin-L and cathepsin-B. L1-CAM, together with IGF2R and SCL31A1, has been identified as a survival factor that protects tumor cells from apoptosis.

[0146] L1-CAM on ovarian cancer combines with neuropilin-1 on mesothelial cells to form the inner layer of the peritoneum, thereby inducing tumor growth through mutual signaling between mesothelial cells and tumors. Expression of non-neuronal isoforms of L1-CAM was found in 16 of 17 tumor cell lines derived from different tumor types (Shtutman M et al., Cancer Res66:11370-80 (2006)). The knockdown of non-neuronal L1-CAM destroys adherens junctions and increases the transcriptional activity of β-catenin in breast cancer cell line MCF-7. Full-length L1-CAM is subjected to sequential cutting of ADAM 10 and presenilin / γ-secretase, and then the C-terminal fragment of L1-CAM is transferred to the nucleus for gene regulation. The RGD binding site located in the 6th Ig domain of L1-CAM shows that it is important for nuclear signaling.

[0147] Metalloproteinase-mediated shedding of the L1-CAM ectodomain to its soluble form (sL1-CAM) has physiological impacts. sL1-CAM may mediate angiogenesis through ligation of integrins via the RGD motif. sL1-CAM induces proliferation, matrigel invasion, tube formation and pro-angiogenic activity of bovine aortic endothelial cells. sL1-CAM is a ligand for several integrins and can be deposited in the extracellular matrix. The cytoplasmic domain of L1-CAM regulates basal shedding and association with the cytoskeleton through ankyrin binding sites. Exosomes carrying constitutive cleavage products of L1-CAM from ovarian cancer cell lines can be found in the ascites and serum of ovarian cancer patients. L1-CAM can also mediate vascular co-option and metastatic growth in a brain metastasis model. Tissue plasmin destroys L1-CAM, thereby stopping the metastatic process. Tumor-derived antiplasminogens, neuroserpins, can prevent plasmin-mediated destruction of L1-CAM or the release of membrane-bound astrocytic FasL, paracrine death signals for cancer cells.

[0148] L1-CAM is also involved in chemoresistance. For example, ovarian cancer cells expressing L1-CAM are more resistant to apoptosis, in part through the anti-apoptotic molecule Bcl-2. In L1-CAM(+) HEK-293 cells, L1-CAM mediates ERK, FAK, and PAK phosphorylation. Cell lines selected for cisplatin resistance upregulate L1-CAM expression, and its knockdown restores sensitivity.

[0149] L1-CAM in human tissues and tumors. In normal human tissues, L1-CAM is detected in neural tissue and peripheral nerves, basal cells of the skin and small blood vessels, especially in the mature placenta ( Figure 3 Tumors expressing L1-CAM include those of neuroectodermal and neural crest origin (including neuroblastoma, GIST, pheochromocytoma, paraganglioma, glioma, pancreatic neuroectodermal cancer, etc.), pancreatic adenocarcinoma, ovarian and uterine cancer, colorectal cancer, small cell lung cancer and non-small cell lung cancer, renal cell carcinoma, triple-negative breast cancer, and tumor vascularization ( Figure 4 IHC performed on tissue microarrays of 128 different tumor types (approximately 5500 different samples) using a mouse monoclonal IgG antibody (UJ127) revealed L1-CAM expression in tumors of neural and neural crest origin, but not so much in tumors of epithelial origin (see Figure 4Negative (no staining detected when compared to nonspecific background staining), weak (1+ staining or 2+ staining in <30% of tumor cells), or strong (3+ staining or 2+ staining in >30% of tumor cells)). More than 25% positivity (weak enhancement) is found in neuroblastoma, ovarian granulosa cell tumor, schwannoma, pheochromocytoma, GIST, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, nasal glioma, medulloblastoma, paraganglioma, capillary hemangioma, and Kaposi's sarcoma. It is found between 10% and 25% for malignant melanoma, chondrosarcoma, esophageal adenocarcinoma, colorectal carcinoma, and oligodendroglioma. It is found between 5%-10% for ependymoma, pancreatic neuroendocrine carcinoma, small cell lung cancer, adrenal adenoma, leiomyosarcoma, liposarcoma, astrocytoma, and endometrial carcinoma. Less than 5% were found for benign nevi, malignant mesothelioma, cervical cancer, esophageal squamous cell carcinoma, meningioma, mucosa-associated lymphoid tissue, neurofibroma, and pancreatic adenocarcinoma and prostate cancer. In general, L1-CAM is associated with poorly differentiated advanced disease stages often accompanied by metastasis. Anti-L1-CAM antibodies such as CE7 (E72) fail to react with monocytes expressing L1-CAM transcripts. This suggests that L1-CAM may undergo different post-translational modifications in tumors compared to normal tissues that allow preferential binding of anti-L1-CAM antibodies.

[0150] Existing antibodies against L1-CAM. chCE7 binds to the 6th Ig-like domain (with RGD motif) of human L1-CAM on renal cancer cells ( Figure 1 ), and is internalized by human neuroblastoma cells (Meli ML et al., Int J Cancer 83:401-8 (1999); Novak-Hofer I et al., Int J Cancer 57:427-432 (1994)). Other monoclonal antibodies have also been described: 5G3, A10-A3, UJ127, L1-14.10, MAB777 (R&D systems Inc., Minneapolis, MN) and O.N.378 (US Biological Life Sciences, Salem, MA), which represent a variety of epitopes and affinities ( Figure 5 ). However, each of these L1-CAM antibodies directed against different epitopes has not been systematically evaluated for in vitro and in vivo properties, and the clinical impact of the antibodies remains uncertain. Many anti-L1-CAM antibodies (e.g., chCE7) are inefficient in eliciting antibody-dependent cell-mediated cytotoxicity (ADCC). Some anti-L1-CAM antibodies may induce unwanted immunogenic responses in patients.

[0151] Immunoglobulin-related compositions of the present technology

[0152] The present technology describes methods and compositions for producing and using anti-L1-CAM immunoglobulin-related compositions (e.g., anti-L1-CAM antibodies or antigen-binding fragments thereof). The anti-L1-CAM immunoglobulin-related compositions of the present disclosure can be used to diagnose or treat L1-CAM positive cancers. Anti-L1-CAM immunoglobulin-related compositions within the scope of the present technology include, for example, but not limited to, monoclonal antibodies, chimeric antibodies, humanized antibodies, and diabodies that specifically bind to a target polypeptide, its homologs, derivatives, or fragments. The present disclosure also provides an antigen-binding fragment of any anti-L1-CAM antibody disclosed herein, wherein the antigen-binding fragment is selected from Fab, F(ab)'2, Fab', scF v and F v .

[0153] In one aspect, the present technology provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) or its antigen-binding fragment, wherein (a) said V H Contains V of GYTFTSYWMQ H -CDR1 sequence (SEQ ID NO: 53), V H -CDR2 sequence (SEQ ID NO: 54) and V of YDGYYAMDY H -CDR3 sequence (SEQ ID NO: 55); and / or (b) the V L Contains V of KSSQSLLYSSNQKNYLA L -CDR1 sequence (SEQ ID NO: 56), V L -CDR2 sequence (SEQ ID NO: 57) and V of QQYHSYPFT L -CDR3 sequence (SEQ ID NO: 58). In some embodiments of the antibody or antigen-binding fragment, the V H Contains the amino acid sequence of SEQ ID NO: 1, and the V L The antibody further comprises an amino acid sequence of SEQ ID NO: 3. Additionally or alternatively, in some embodiments, the antibody further comprises an Fc domain of any isotype, such as but not limited to IgG (including IgG1, IgG2, IgG3 and IgG4), IgA (including IgA 1 and IgA 2 ), IgD, IgE or IgM and IgY. Non-limiting examples of constant region sequences include:

[0154] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 59)

[0155]

[0156] Human IgG1 constant region, Uniprot: P01857 (SEQ ID NO: 60)

[0157]

[0158] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO: 61)

[0159]

[0160] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO: 62)

[0161]

[0162]

[0163] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 63)

[0164]

[0165] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO: 64)

[0166]

[0167] Human IgA1 constant region, Uniprot: P01876 (SEQ ID NO: 65)

[0168]

[0169]

[0170] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 66)

[0171]

[0172] Human Igκ constant region, Uniprot: P01834 (SEQ ID NO: 67)

[0173]

[0174] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 59-66. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 67. In some embodiments, the immunoglobulin-related compositions of the present technology bind to an epitope of a L1-CAM polypeptide that comprises at least five to eight consecutive amino acid residues of the second Ig-like domain of L1-CAM (SEQ ID NO: 74). In some embodiments, the epitope is a conformational epitope.

[0175] In another aspect, the present disclosure provides an isolated immunoglobulin-related composition (e.g., an antibody or antigen-binding fragment thereof) comprising the following heavy chain (HC) amino acid sequence:

[0176]

[0177]

[0178]

[0179]

[0180] or variants thereof having one or more conservative amino acid substitutions.

[0181] Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise the following light chain (LC) amino acid sequence:

[0182]

[0183]

[0184] GDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSG SGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLQITR (SEQ ID NO:52) (huE72-BsAb light chain-huOKT3scFv), or a variant thereof having one or more conservative amino acid substitutions.

[0186] In some embodiments, the immunoglobulin-related compositions of the present technology comprise an HC amino acid sequence and an LC amino acid sequence, respectively, selected from the group consisting of: SEQ ID NO: 9 and SEQ ID NO: 13 (huE71 H1 / L1); SEQ ID NO: 9 and SEQ ID NO: 14 (huE71 H1 / L2); SEQ ID NO: 10 and SEQ ID NO: 13 (huE71 H2 / L1); SEQ ID NO: 10 and SEQ ID NO: 14 (huE71

[0187] SEQ ID NO: 17 and SEQ ID NO: 21 (huE72 H1 / L1); SEQ ID NO: 17 and SEQ ID NO: 22 (huE72 H1 / L2); SEQ ID NO: 18 and SEQ ID NO: 21 (huE72 H2 / L1); SEQ ID NO: 18 and SEQ ID NO: 22 (huE72 H2 / L2); SEQ ID NO: 26 and SEQ ID NO: 25 (chE71 IgG1); SEQ ID NO: 30 and SEQ ID NO: 29 (chE72 IgG1); SEQ ID NO: 42 and SEQ ID NO: 41 (chE71 IgG4); SEQ ID NO: 46 and SEQ ID NO: 45 (huE71 IgG4); SEQ ID NO: 49 and SEQ ID NO: 50 (huE71 BsAb); and SEQ ID NO: 51 and SEQ ID NO: 52 (huE72 BsAb).

[0188] In any of the above embodiments of the immunoglobulin-related compositions, the HC and LC immunoglobulin variable domain sequences form an antigen binding site that binds to an epitope of the L1-CAM polypeptide, the epitope comprising at least five to eight consecutive amino acid residues of the 2nd Ig-like domain of L1-CAM (SEQ ID NO: 74) or the 6th Ig-like domain of L1-CAM (SEQ ID NO: 75). In some embodiments, the epitope is a conformational epitope.

[0189] In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0190] In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one L1-CAM polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology are present in an amount of about 10 -3 M, 10 -4 M, 10 -5 M, 10 - 6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 The dissociation constant (K d ) binds to at least one L1-CAM polypeptide. In certain embodiments, the immunoglobulin-related composition is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In some embodiments, the antibody comprises a human antibody framework region.

[0191] In certain embodiments, the immunoglobulin-related compositions include one or more of the following features: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 13, 14, 21, 22, 25, 29, 41, 45, 50, or 52; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 9, 10, 17, 18, 26, 30, 42, 46, 49, and 51. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted with another amino acid. The substitution may be a "conservative substitution" as defined herein.

[0192] In some embodiments, the immunoglobulin-related composition comprises (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the LC sequence present in any one of SEQ ID NOs: 13, 14, 21, 22, 25, 29, 41, 45, 50 or 52; and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the HC sequence present in any one of SEQ ID NOs: 9, 10, 17, 18, 26, 30, 42, 46, 49 and 51.

[0193] In certain embodiments, the immunoglobulin-related composition comprises an IgG1 constant region comprising one or more amino acid substitutions selected from N297A and K322A. Additionally or alternatively, in some embodiments, the immunoglobulin-related composition comprises an IgG4 constant region comprising an S228P mutation.

[0194] In some aspects, the anti-L1-CAM immunoglobulin-related compositions described herein contain structural modifications to promote rapid binding and cellular uptake and / or slow release. In some aspects, the anti-L1-CAM immunoglobulin-related compositions (e.g., antibodies) of the present technology may contain deletions in the CH2 constant heavy chain region to promote rapid binding and cellular uptake and / or slow release. In some aspects, Fab fragments are used to promote rapid binding and cellular uptake and / or slow release. In some aspects, F(ab)' 2 Fragments are used to promote rapid binding and cellular uptake and / or slow release.

[0195] In one aspect, the present technology provides a nucleic acid sequence encoding a heavy chain or light chain of an immunoglobulin-related composition described herein. Also disclosed herein is a recombinant nucleic acid sequence encoding any antibody described herein. In some embodiments, the nucleic acid sequence is selected from SEQ ID NO: 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 43, 44, 47 and 48. In another aspect, the present technology provides a host cell that expresses any nucleic acid sequence encoding a heavy chain or light chain of an immunoglobulin-related composition described herein.

[0196] The immunoglobulin-related compositions (e.g., anti-L1-CAM antibodies) of the present technology can be monospecific, bispecific, trispecific, or have greater multispecificity. Multispecific antibodies can be specific for different epitopes of one or more L1-CAM polypeptides, or can be specific for both one or more L1-CAM polypeptides and heterologous compositions (e.g., heterologous polypeptides or solid support materials). See, e.g., WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt et al., J. Immunol. 147: 60-69 (1991); U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; 6,106,835; Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related compositions are chimeric. In certain embodiments, the immunoglobulin-related compositions are humanized.

[0197] The immunoglobulin-related compositions of the present technology can be further recombinantly fused to a heterologous polypeptide at the N-terminus or C-terminus, or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated to molecules and effector molecules (such as heterologous polypeptides, drugs or toxins) that can be used as markers in detection assays. See, for example, WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; U.S. Pat. No. 5,314,995; and EP 0 396 387.

[0198] In any of the above embodiments of the immunoglobulin-related compositions of the present technology, the antibody or antigen-binding fragment can be optionally conjugated to an agent selected from the following: an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. For chemical or physical bonds, the functional groups on the immunoglobulin-related compositions are typically associated with the functional groups on the agent. Alternatively, the functional groups on the agent are associated with the functional groups on the immunoglobulin-related compositions.

[0199] Functional groups on the medicament and immunoglobulin-related compositions can be directly associated. For example, functional groups (such as sulfhydryl groups) on the medicament can be associated with functional groups (such as sulfhydryl groups) on the immunoglobulin-related compositions to form disulfide bonds. Alternatively, the functional groups can be associated by cross-linking agents (i.e., joints). Some examples of cross-linking agents are described below. The cross-linking agent can be attached to the medicament or immunoglobulin-related compositions. The number of medicaments or immunoglobulin-related compositions in the conjugate is also limited by the number of functional groups present on each other. For example, the maximum number of medicaments associated with the conjugate depends on the number of functional groups present on the immunoglobulin-related compositions. Alternatively, the maximum number of immunoglobulin-related compositions associated with the medicament depends on the number of functional groups present on the medicament.

[0200] In another embodiment, the conjugate comprises an immunoglobulin-related composition associated with an agent. In one embodiment, the conjugate comprises at least one agent chemically bonded (e.g., conjugated) to at least one immunoglobulin-related composition. The agent can be chemically bonded to the immunoglobulin-related composition by any method known to those skilled in the art. For example, the functional group on the agent can be directly attached to the functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate, and hydroxyl.

[0201] The agent may also be chemically bonded to the immunoglobulin-related composition by a cross-linking agent such as a dialdehyde, carbodiimide, dimaleimide, etc. Cross-linking agents may be obtained, for example, from Pierce Biotechnology, Inc. of Rockford, IL. The Pierce Biotechnology, Inc. website may provide assistance. Additional cross-linking agents include the platinum cross-linking agents described in U.S. Pat. Nos. 5,580,990; 5,985,566; and 6,133,038 to Kreatech Biotechnology, BV, Amsterdam, The Netherlands.

[0202] Alternatively, the functional groups on the agent and the immunoglobulin-related composition can be the same. Homobifunctional crosslinkers are generally used to crosslink the same functional groups. Examples of homobifunctional crosslinkers include EGS (i.e., ethylene glycol bis[succinimidyl succinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate. 2HCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (i.e., 1,4-bis-[3'-(2'-pyridyldithio)-propionamido] butane) and BMH (i.e., bismaleimidohexane). Such homobifunctional crosslinkers can also be obtained from Pierce Biotechnology, Inc.

[0203] In other cases, it may be beneficial to cleave the agent from the immunoglobulin-related composition. The above-mentioned Pierce Biotechnology, Inc. website can also provide assistance to those skilled in the art in selecting a suitable cross-linking agent, which can be cleaved by, for example, an enzyme in the cell. Thus, the agent can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-α-[2-pyridyldithio]toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (e.g., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl).

[0204] In another embodiment, the conjugate comprises at least one agent physically bonded to at least one immunoglobulin-related composition. The agent can be physically bonded to the immunoglobulin-related composition by any method known to those skilled in the art. For example, the immunoglobulin-related composition and the agent can be mixed by any method known to those skilled in the art. The order of mixing is not important. For example, the agent can be physically mixed with the immunoglobulin-related composition by any method known to those skilled in the art. For example, the immunoglobulin-related composition and the agent can be placed in a container and stirred by, for example, shaking the container to mix the immunoglobulin-related composition and the agent.

[0205] The immunoglobulin-related compositions can be modified by any method known to those skilled in the art. For example, as described above, the immunoglobulin-related compositions can be modified by cross-linking agents or functional groups.

[0206] A. Methods for preparing anti-L1-CAM antibodies of the present technology

[0207] General Overview. First, a target polypeptide against which antibodies of the present technology can be generated is selected. For example, antibodies can be generated against the full-length L1-CAM protein or against a portion of the extracellular domain of the L1-CAM protein comprising six Ig-like domains and five fibronectin-like domains (see Figure 1 ). Techniques for generating antibodies against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, techniques involving display libraries, heterologous or human mice, hybridomas, and the like. Target polypeptides within the scope of the present technology include any polypeptide derived from the L1-CAM protein that contains an extracellular domain capable of eliciting an immune response. The preparation of antibodies specific for the L1-CAM protein is described in Examples 1, 2, and 5.

[0208] It will be appreciated that recombinantly engineered antibodies and antibody fragments (eg, antibody-related polypeptides) directed against L1-CAM protein and fragments thereof are suitable for use in accordance with the present disclosure.

[0209] Anti-L1-CAM antibodies that can be subjected to the techniques described herein include monoclonal antibodies and polyclonal antibodies, as well as antibody fragments (e.g., Fab, Fab′, F(ab′) 2 , Fd, scFv), diabodies, antibody light chains, antibody heavy chains and / or antibody fragments. Polypeptides such as Fab′ and F(ab′) containing antibody Fv have been described for high yield production. 2 Methods for antibody fragments. See U.S. Patent No. 5,648,237.

[0210] Typically, antibodies are obtained from a species of origin. More specifically, nucleic acid or amino acid sequences of the variable portions of the light chain, heavy chain, or both of an antibody of the species of origin that is specific for a target polypeptide antigen are obtained. The species of origin is any species that can be used to generate antibodies or antibody libraries of the present technology, such as rats, mice, rabbits, chickens, monkeys, humans, etc.

[0211] Phage or phagemid display technology is a technology that can be used to derive antibodies of the present technology. The technology used to produce and clone monoclonal antibodies is well known to those skilled in the art. The expression of sequences encoding antibodies of the present technology can be carried out in Escherichia coli.

[0212] Due to the degeneracy of the sequence encoding nucleic acid, other sequences encoding amino acid sequences substantially identical to the amino acid sequence of naturally occurring proteins can be used in the practice of the present technology. These sequences include, but are not limited to, nucleic acid sequences including all or part of the nucleic acid sequence encoding the above-mentioned polypeptides, which are altered by substitution of different codons for functionally equivalent amino acid residues in the coding sequence, thereby producing silent changes. It should be understood that the nucleotide sequence of the immunoglobulin according to the present technology allows up to 25% sequence homology changes as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pages 127-149, 1998, Alan R. Liss, Inc.), as long as such variants form effective antibodies that recognize L1-CAM protein. For example, one or more amino acid residues in a polypeptide sequence can be substituted by another amino acid of similar polarity, which acts as a functional equivalent, thereby resulting in silent changes. Substitutes for amino acids in the sequence can be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins or fragments or derivatives thereof, which are modified differently during or after translation, such as by glycosylation, proteolytic cleavage, connection to antibody molecules or other cellular ligands, etc. In addition, the nucleic acid sequence encoding the immunoglobulin can be mutated in vitro or in vivo to produce and / or destroy translation, start and / or stop sequences, or to produce changes in the coding region and / or form new restriction endonuclease sites or destroy pre-existing sites to facilitate further in vitro modification. Any mutagenesis technique known in the art may be used, including but not limited to in vitro site-directed mutagenesis (J. Biol. Chem. 253:6551, use of Tab linkers (Pharmacia)), etc.

[0213] Preparation of polyclonal antisera and immunogens. Methods for producing antibodies or antibody fragments of the present technology generally include immunizing a subject (usually a non-human subject, such as a mouse or rabbit) with a purified L1-CAM protein or a fragment thereof or with a cell expressing the L1-CAM protein or a fragment thereof. A suitable immunogenic preparation may contain, for example, a recombinantly expressed L1-CAM protein or a chemically synthesized L1-CAM peptide. Using standard techniques for polyclonal and monoclonal antibody preparation, the ECM of the L1-CAM protein or a portion or fragment thereof may be used as an immunogen to produce anti-L1-CAM antibodies that bind to the L1-CAM protein or a portion or fragment thereof.

[0214] The full-length L1-CAM protein or its fragments can be used as fragments, as immunogens. In some embodiments, the L1-CAM fragment comprises the amino acid sequence

[0215] (Ig-like C2 type 2 domain of L1-CAM) or

[0216] The peptide comprises at least five to eight consecutive amino acid residues of (Ig-like C2 type 6 domain of L1-CAM) and covers the epitope of the L1-CAM protein, so that antibodies against the peptide form a specific immune complex with the L1-CAM protein.

[0217] In some embodiments, the antigenic L1-CAM peptide that overlaps the 2nd or 6th Ig-like domain comprises at least 5, 8, 10, 15, 20 or 30 amino acid residues. Depending on the application and according to methods well known to those skilled in the art, longer antigenic peptides are sometimes required rather than shorter antigenic peptides. Multimers of a given epitope are sometimes more effective than monomers.

[0218] If desired, the immunogenicity of the L1-CAM protein (or a fragment thereof) can be increased by fusion or conjugation with a hapten, such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such haptens are known in the art. The L1-CAM protein can also be used in combination with conventional adjuvants, such as Freund's complete or incomplete adjuvant, to enhance the subject's immune response to the polypeptide. Various adjuvants used to enhance the immune response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants (e.g., Bacille Calmette-Guerin and Corynebacterium parvum), or similar immunostimulatory compounds. These techniques are standard in the art.

[0219] In describing the present technology, immune responses may be described as "primary" or "secondary" immune responses. A primary immune response, also referred to as a "protective" immune response, refers to an immune response generated in an individual as a result of some initial exposure (e.g., an initial "immunization") to a particular antigen (e.g., a L1-CAM protein). In some embodiments, immunization can be performed by vaccinating an individual with a vaccine containing the antigen. For example, the vaccine can be an L1-CAM vaccine comprising one or more antigens derived from L1-CAM proteins. Over time, the primary immune response may weaken or diminish, and may even disappear or at least be so diminished that it cannot be detected. Therefore, the present technology also relates to a "secondary" immune response, also referred to herein as a "memory immune response." The term secondary immune response refers to an immune response elicited in an individual after a primary immune response has been generated.

[0220] Thus, a secondary immune response may be elicited, for example to enhance an existing immune response that has weakened or diminished, or to regenerate a previous immune response that has disappeared or is no longer detected. The stimulation or memory immune response may be a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs following stimulation of memory B cells produced when an antigen is first presented. Delayed-type hypersensitivity (DTH) is a CD4 + Type of cellular secondary or memory immune response mediated by T cells. The first exposure to an antigen primes the immune system, and one or more additional exposures result in DTH.

[0221] After appropriate immunization, anti-L1-CAM antibodies can be prepared from the subject's serum. If necessary, antibody molecules against L1-CAM protein can be isolated from mammals (eg, from blood) and further purified by well-known techniques such as polypeptide A chromatography to obtain IgG fractions.

[0222] Monoclonal antibodies. In one embodiment of the present technology, the antibody is an anti-L1-CAM monoclonal antibody. For example, in some embodiments, the anti-L1-CAM monoclonal antibody can be a human or mouse anti-L1-CAM monoclonal antibody. In order to prepare monoclonal antibodies or derivatives, fragments, analogs or homologs thereof against L1-CAM protein, any technique for producing antibody molecules by continuous cell line culture can be used. Such techniques include, but are not limited to, hybridoma technology (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); tri-source hybridoma technology; human B cell hybridoma technology (see, e.g., Kozbor et al., 1983. Immunol. Today 4: 72) and EBV hybridoma technology to produce human monoclonal antibodies (see, e.g., Cole et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used in the practice of the present technology and can be produced by using human hybridomas (see, for example, Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see, for example, Cole et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a nucleic acid population encoding an antibody region can be isolated. PCR using primers derived from sequences encoding conserved regions of antibodies is used to amplify sequences encoding partial antibodies from the population, and then DNA encoding antibodies or fragments thereof (such as variable domains) is reconstructed from the amplified sequences. Such amplified sequences can also be fused to DNA encoding other proteins, such as phage coats or bacterial cell surface proteins, to express and display fusion polypeptides on phages or bacteria. The amplified sequences can then be expressed and further selected or isolated based on, for example, the affinity of the expressed antibodies or fragments thereof for an antigen or epitope present on the L1-CAM protein. Alternatively, hybridomas expressing anti-L1-CAM monoclonal antibodies can be prepared by immunizing a subject and then isolating the hybridomas from the spleen of the subject using conventional methods. See, for example, Milstein et al., (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening of hybridomas using standard methods will produce monoclonal antibodies with different specificities (i.e., against different epitopes) and affinities.A selected monoclonal antibody having a desired property (e.g., L1-CAM binding) as expressed by a hybridoma may be used, it may be conjugated to a molecule such as polyethylene glycol (PEG) to alter its properties, or the cDNA encoding the monoclonal antibody may be isolated, sequenced, and manipulated in a variety of ways. Synthetic dendromeric trees may be added to reactive amino acid side chains such as lysine to enhance the immunogenic properties of the L1-CAM protein. Likewise, CPG-dinucleotide technology may be used to enhance the immunogenic properties of the L1-CAM protein. Other manipulations include substitution or deletion of specific aminoacyl residues that contribute to antibody instability during storage or after administration to a subject, and affinity maturation techniques for improving the affinity of antibodies to the L1-CAM protein.

[0223] Hybridoma technology. In some embodiments, the antibody of the present technology is an anti-L1-CAM monoclonal antibody produced by a hybridoma, wherein the hybridoma includes a B cell obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma technology includes those known in the art and taught in the following documents: Harlow et al., Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0224] Phage display technology. As described above, antibodies of the present technology can be produced by applying recombinant DNA and phage display technology. For example, various phage display methods known in the art can be used to prepare anti-L1-CAM antibodies. In the phage display method, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. By directly selecting with antigens (usually antigens bound or captured on solid surfaces or beads), phages with desired binding properties are selected from libraries or combinatorial antibody libraries (e.g., humans or mice). The phages used in these methods are typically filamentous phages, which contain fd and M13 with Fab, Fv or disulfide-stabilized Fv antibody domains, and the domains are recombinantly fused with phage gene III or gene VIII proteins. In addition, the method is suitable for the construction of Fab expression libraries (see, e.g., Huse et al., Science 246: 1275-1281, 1989) to quickly and efficiently identify monoclonal Fab fragments having the desired specificity for L1-CAM polypeptide, e.g., polypeptide or derivatives, fragments, analogs or homologs thereof.Other examples of phage display methods that can be used to prepare antibodies of the present technology include those disclosed in the following literature: Huston et al., Proc. Natl. Acad. Sci USA, 85:5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci U.S.A, 87:1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182:41-50, 1995; Ames et al., J. Immunol. Methods 184:177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24:952-958, 1994; Persic et al., Gene 187:9-18, 1997; Burton et al., Advances in Immunology 57:191-280,1994; PCT / GB91 / 01134; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; WO 96 / 06213; WO 92 / 01047 (Medical Research Council et al.); WO 97 / 08320 (Morphosys); WO 92 / 01047 (CAT / MRC); WO 91 / 17271 (Affymax); and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743. U.S. Pat. No. 6,753,136 to Lohning has described a method for displaying polypeptides on the surface of phage particles by attaching the polypeptides via disulfide bonds. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to produce whole antibodies (including human antibodies) or any other desired antigen-binding fragments and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast and bacteria).For example, Fab, Fab' and F(ab') can also be produced recombinantly using methods known in the art, such as those disclosed in WO 92 / 22324; Mullinax et al., BioTechniques 12:864-869, 1992; and Sawai et al., AJRI 34:26-34, 1995; and Better et al., Science 240:1041-1043, 1988. 2 Fragment technology.

[0225] Typically, hybrid antibodies or hybrid antibody fragments cloned into display vectors can be selected for appropriate antigens to identify variants that retain good binding activity because the antibodies or antibody fragments will be present on the surface of phage or phagemid particles. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector formats can be used for this process, such as cloning antibody fragment libraries into lytic phage vectors (modified T7 or Lambda Zap systems) for selection and / or screening.

[0226] Expression of recombinant anti-L1-CAM antibodies. As described above, antibodies of the present technology can be produced by applying recombinant DNA technology. Recombinant polynucleotide constructs encoding anti-L1-CAM antibodies of the present technology generally include expression control sequences operably linked to the coding sequence of the anti-L1-CAM antibody chain, including naturally associated or heterologous promoter regions. Therefore, another aspect of the present technology includes vectors containing one or more nucleic acid sequences encoding anti-L1-CAM antibodies of the present technology. In order to recombinantly express one or more polypeptides of the present technology, nucleic acids containing all or part of the nucleotide sequence encoding the anti-L1-CAM antibody are inserted into suitable cloning vectors or expression vectors (i.e., vectors containing the necessary elements for transcribing and translating the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and described in detail below. Methods for generating a variety of vector populations have been described in U.S. Patent Nos. 6,291,160 and 6,680,192 to Lerner et al.

[0227] In general, expression vectors that can be used in recombinant DNA technology are usually in the form of plasmids. In this disclosure, "plasmid" and "vector" are sometimes used interchangeably because plasmids are the most commonly used form of vectors. However, the present technology is intended to include such other forms of expression vectors that are not plasmids technically and perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). Such viral vectors allow infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of nucleotide sequences encoding anti-L1-CAM antibodies and collection and purification of anti-L1-CAM antibodies (e.g., cross-reactive anti-L1-CAM antibodies). See generally US2002 / 0199213. These expression vectors are typically replicated in a host organism as an episome or as an integral part of the host chromosomal DNA. Typically, the expression vector contains a selection marker, such as ampicillin resistance or hygromycin resistance, to allow detection of cells transformed with the desired DNA sequence. The vector may also encode a signal peptide that can be used to direct secretion of the extracellular antibody fragment, such as pectin lyase. See U.S. Patent No. 5,576,195.

[0228] The recombinant expression vector of the present technology comprises a nucleic acid encoding a protein having L1-CAM binding properties, and the nucleic acid is in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences selected according to the host cell for expression, and the one or more regulatory sequences are operably connected to the nucleic acid sequence to be expressed. In the recombinant expression vector, "operably connected" is intended to mean that the target nucleotide sequence is connected to one or more regulatory sequences in a manner that allows (for example, in an in vitro transcription / translation system or in a host cell when the vector is introduced into a host cell) the expression of the nucleotide sequence. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (for example, polyadenylation signals). Such regulatory sequences are described in, for example, Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). Regulatory sequences include those that direct constitutive expression of nucleotide sequences in many types of host cells and those that direct expression of nucleotide sequences only in certain host cells (for example, tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the expression level of the desired polypeptide, and the like. Typical regulatory sequences that can be used as promoters for expression of recombinant polypeptides (e.g., anti-L1-CAM antibodies) include, for example, but not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, in particular, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. In one embodiment, the polynucleotide encoding the anti-L1-CAM antibody of the present technology is operably linked to the ara B promoter and is expressible in the host cell. See U.S. Patent No. 5,028,530. The expression vector of the present technology can be introduced into a host cell to produce a polypeptide or peptide encoded by the nucleic acid described herein, including a fusion polypeptide (e.g., anti-L1-CAM antibody, etc.).

[0229] Another aspect of the present technology relates to host cells expressing anti-L1-CAM antibodies, which contain nucleic acids encoding one or more anti-L1-CAM antibodies. The recombinant expression vectors of the present technology can be designed to express anti-L1-CAM antibodies in prokaryotic or eukaryotic cells. For example, anti-L1-CAM antibodies can be expressed in bacterial cells (such as E. coli), insect cells (using baculovirus expression vectors), fungal cells (such as yeast, yeast cells) or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods that can be used to prepare and screen polypeptides (such as anti-L1-CAM antibodies) with predetermined properties via expression of randomly generated polynucleotide sequences have been previously described. See U.S. Patent Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; 6,569,641.

[0230] The expression of polypeptides in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters that direct the expression of fusion or non-fusion polypeptides. Fusion vectors add many amino acids to the polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors usually have three purposes: (i) increase the expression of the recombinant polypeptide; (ii) increase the solubility of the recombinant polypeptide; and (iii) assist in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Typically, in a fusion expression vector, a proteolytic cleavage site is introduced at the junction of the fusion part and the recombinant polypeptide so that the recombinant polypeptide can be separated from the fusion part after the fusion polypeptide is purified. Such enzymes and their homologous recognition sequences include factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, New Jersey), which fuse glutathione S-transferase (GST), maltose E binding polypeptide or polypeptide A to the target recombinant polypeptide, respectively.

[0231] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, CA (1990) 60-89). Pack et al., U.S. Pat. Nos. 6,294,353; 6,692,935, have described methods for targeted assembly of different active peptides or protein domains via polypeptide fusion to produce multifunctional polypeptides. One strategy for maximizing expression of recombinant polypeptides (e.g., anti-L1-CAM antibodies) in E. coli is to express the polypeptide in a host bacterium that is impaired in its ability to proteolytically cleave the recombinant polypeptide. See, for example, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990) 119-128. Another strategy is to change the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that the individual codons for each amino acid are codons that are preferentially used in the expression host (e.g., E. coli) (see, for example, Wada et al., 1992. Nucl. Acids Res. 20: 2111-2118). This change in the nucleic acid sequence of the present technology can be performed by standard DNA synthesis techniques.

[0232] In another embodiment, the anti-L1-CAM antibody expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, California) and picZ (Invitrogen Corp, San Diego, California). Alternatively, baculovirus expression vectors can be used to express anti-L1-CAM antibodies in insect cells. Baculovirus vectors that can be used to express polypeptides (eg, anti-L1-CAM antibodies) in cultured insect cells (eg, SF9 cells) include the pAc series (Smith et al., Mol. Cell. Biol. 3:2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

[0233] In yet another embodiment, a nucleic acid encoding an anti-L1-CAM antibody of the present technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, for example, but not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman et al., EMBO J. 6: 187-195, 1987). When used in mammalian cells, the control functions of the expression vector are generally provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that can be used to express the anti-L1-CAM antibodies of the present technology, see, for example, Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL. 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Chapters 16 and 17.

[0234] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a specific cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., Genes Dev. 1: 268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), promoters of T cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g., neurofilament promoters; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86:5473-5477, 1989), pancreas-specific promoters (Edlund et al., 1985. Science 230:912-916) and mammary gland-specific promoters (e.g., whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also contemplated, such as the mouse hox promoter (Kessel and Gruss, Science 249:374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546, 1989).

[0235] Another aspect of the present method relates to a host cell into which the recombinant expression vector of the present technology has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to a specific subject cell, but also to the progeny or potential progeny of such a cell. Because some changes may occur in the offspring due to mutations or environmental influences, such progeny may in fact be different from the parental cell, but are still included in the scope of the term as used herein.

[0236] The host cell can be any prokaryotic or eukaryotic cell. For example, anti-L1-CAM antibodies can be expressed in bacterial cells such as Escherichia coli, insect cells, yeast or mammalian cells. Mammalian cells are hosts suitable for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987). Many suitable host cell lines capable of secreting complete heterologous proteins have been developed in the art, and the suitable host cell lines include Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. In some embodiments, the cells are non-human. The expression vectors of these cells can include expression control sequences, such as replication origins, promoters, enhancers, and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. Co et al., J Immunol. 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

[0237] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional conversion or transfection techniques. As used herein, the terms "conversion" and "transfection" are intended to refer to various art-recognized techniques for introducing exogenous nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, gene guns, or transfection based on viruses. Other methods for transforming mammalian cells include using polybrene, protoplast fusion, liposomes, electroporation, and microinjection (generally referring to Sambrook et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (MOLECULAR CLONING:A LABORATORY MANUAL. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. Depending on the type of cell host, the vector containing the target DNA segment can be transferred to the host cell by a well-known method.

[0238] For stable transfection of mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. In order to identify and select these components, genes encoding selectable markers (e.g., resistance to antibiotics) are usually introduced into host cells together with the gene of interest. Various selectable markers include selectable markers that confer resistance to drugs such as G418, hygromycin, and methotrexate. Nucleic acids encoding selectable markers can be introduced into host cells on the same vector as the vector encoding the anti-L1-CAM antibody, or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while other cells die).

[0239] Host cells (e.g., prokaryotic or eukaryotic host cells in culture) comprising an anti-L1-CAM antibody of the present technology can be used to produce (i.e., express) a recombinant anti-L1-CAM antibody. In one embodiment, the method comprises culturing a host cell (into which a recombinant expression vector encoding an anti-L1-CAM antibody has been introduced) in a suitable culture medium, thereby producing an anti-L1-CAM antibody. In another embodiment, the method further comprises the step of isolating the anti-L1-CAM antibody from the culture medium or the host cell. Once expressed, the anti-L1-CAM antibody, such as a collection of anti-L1-CAM antibodies or anti-L1-CAM antibody-related polypeptides, is purified from the culture medium and host cells. The anti-L1-CAM antibody can be purified according to standard procedures in the art (including HPLC purification, column chromatography, gel electrophoresis, etc.). In one embodiment, the anti-L1-CAM antibody is produced in a host organism by the method of U.S. Pat. No. 4,816,397 of Boss et al. Typically, the anti-L1-CAM antibody chain is expressed together with a signal sequence and is thus released into the culture medium. However, if the anti-L1-CAM antibody chains are not naturally secreted by the host cells, the anti-L1-CAM antibody chains can be released by treatment with a mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, etc. (see generally Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0240] Polynucleotides encoding anti-L1-CAM antibodies, for example, sequences encoding anti-L1-CAM antibodies, can be incorporated into transgenes for introduction into the genome of transgenic animals and subsequently expressed in the milk of transgenic animals. See, for example, U.S. Pat. Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding sequences for light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes, such as casein or beta-lactoglobulin. To produce transgenic animals, the transgene can be microinjected into fertilized oocytes, or it can be incorporated into the genome of embryonic stem cells and the nuclei of such cells transferred into enucleated oocytes.

[0241] Single-chain antibodies. In one embodiment, the anti-L1-CAM antibody of the present technology is a single-chain anti-L1-CAM antibody. According to the present technology, the technology can be applied to produce single-chain antibodies specific for L1-CAM protein (see, e.g., U.S. Patent No. 4,946,778). Examples of technologies that can be used to produce single-chain Fvs and antibodies of the present technology include those described in the following references: U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999, 1993; and Skerra et al., Science 240:1038-1040, 1988.

[0242] Chimeric and humanized antibodies. In one embodiment, the anti-L1-CAM antibody of the present technology is a chimeric anti-L1-CAM antibody. In one embodiment, the anti-L1-CAM antibody of the present technology is a humanized anti-L1-CAM antibody. In one embodiment of the present technology, the donor antibody and the recipient antibody are monoclonal antibodies from different species. For example, the recipient antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody.

[0243] Recombinant anti-L1-CAM antibodies (such as chimeric monoclonal antibodies and humanized monoclonal antibodies) comprising human and non-human parts can be prepared using standard recombinant DNA techniques and are within the scope of the present technology. For certain uses, including in vivo uses of the anti-L1-CAM antibodies of the present technology and uses of these agents in in vitro detection assays, chimeric or humanized anti-L1-CAM antibodies can be used. Such chimeric monoclonal antibodies and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, for example, but are not limited to, those described in the following references: International Application No. PCT / US86 / 02269; U.S. Pat. No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Patent No. WO 86 / 01533; U.S. Pat. Nos. 4,816,567; 5,225,539; European Patent No. 125023; Better et al., 1988. Science 240:1041-1043; Liu et al., 1987. Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987. J. Immunol. 139:3521-3526; Sun et al., 1987. Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987. Cancer Res. 47:999-1005; Wood et al., 1985. Nature 314:446-449; Shaw et al., 1988. J. Natl. Cancer Inst. 80:1553-1559; Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Jones et al., 1986. Nature 321:552-525; Verhoeyan et al., 1988. Science 239:1534; Morrison, Science 229:1202, 1985; Oi et al., BioTechniques 4:214, 1986; Gillies et al., J. Immunol. Methods, 125:191-202, 1989; U.S. Patent No. 5,807,715; and Beidler et al., 1988. J. Immunol. 141:4053-4060.For example, antibodies can be humanized using a variety of techniques including CDR grafting (EP 0 239 400; WO 91 / 09967; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP 460 167), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E.A., Molecular Immunology, 28:489-498, 1991; Studnicka et al., Protein Engineering 7:805-814, 1994; Roguska et al., PNAS 91:969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332). In one embodiment, the cDNA encoding the murine anti-L1-CAM monoclonal antibody is digested with restriction enzymes specifically selected to remove sequences encoding the Fc constant region, and the same portion of the cDNA encoding the human Fc constant region is substituted (see Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J Immunol 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cancer Res 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559; U.S. Pat. No. 6,180,370; U.S. Pat. Nos. 6,300,064; 6,696,248; 6,706,484; 6,828,422.

[0244] In one embodiment, the present technology provides the construction of humanized anti-L1-CAM antibodies that are less likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response while still having effective antibody effector functions. As used herein, the terms "human" and "humanized" with respect to antibodies refer to any antibody that is expected to elicit a weak immunogenic response that is therapeutically tolerable in a human subject. In one embodiment, the present technology provides humanized anti-L1-CAM antibodies, heavy chain and light chain immunoglobulins.

[0245] CDR antibodies. In some embodiments, the anti-L1-CAM antibodies of the present technology are anti-L1-CAM CDR antibodies. Typically, the donor and recipient antibodies used to generate anti-L1-CAM CDR antibodies are monoclonal antibodies from different species; typically, the recipient antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody. The graft may have a single V H or V L A single CDR (or even a portion of a single CDR) within a H and V L Multiple CDRs (or portions thereof) within one or both of the variable domains of the recipient antibody will be replaced by the corresponding donor CDRs, but only as many replacements as necessary are required to allow the resulting CDR-grafted antibody to fully bind to the L1-CAM protein. Methods for producing CDR-grafted and humanized antibodies are taught by the following references: Queen et al., U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; and Winter US5,225,539; and EP 0682040. Can be used to prepare V H and V L Polypeptide methods are taught by the following references: Winter et al., US Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP 0451216; and EP0120694.

[0246] After selecting suitable framework region candidates from the same family and / or same family member, one or both of the heavy chain and light chain variable regions are produced by transplanting the CDR from the species of origin into the hybrid framework region. Conventional methods known to those skilled in the art can be used to complete the assembly of hybrid antibodies or hybrid antibody fragments with hybrid variable chain regions in any of the above aspects. For example, DNA sequences encoding hybrid variable domains described herein (i.e., frameworks based on target species and CDRs from species of origin) can be produced by oligonucleotide synthesis and / or PCR. Suitable restriction enzymes can also be used to separate the nucleic acids encoding the CDR regions from the species of origin antibodies, and connected to the target species framework by connecting with a suitable ligase. Alternatively, the framework regions of the variable chains of the species of origin antibodies can be changed by site-directed mutagenesis.

[0247] Since hybrids are constructed from selections between multiple candidates corresponding to each framework region, there are many sequence combinations suitable for construction according to the principles described herein. Thus, libraries of hybrids can be assembled whose members have different combinations of various framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

[0248] This process usually does not change the FR of the recipient antibody flanking the transplanted CDR. However, those skilled in the art can sometimes improve the antigen binding affinity of the resulting anti-L1-CAM CDR-grafted antibody by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable substitution positions include amino acid residues adjacent to the CDR, or amino acid residues that can interact with the CDR (see, for example, US 5,585,089, especially columns 12-16). Or those skilled in the art can start with the donor FR and modify it to make it more similar to the recipient FR or human shared FR. The techniques for making these modifications are known in the art. In particular, if the resulting FR conforms to the human shared FR at the position or is at least 90% or more identical to this shared FR, then compared with the same antibody with fully human FR, doing so may not significantly increase the antigenicity of the resulting modified anti-L1-CAM CDR-grafted antibody.

[0249] Bispecific antibodies (BsAb). Bispecific antibodies are antibodies that can simultaneously bind to two targets with different structures (e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen). BsAb can be prepared, for example, by combining heavy chains and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, by molecular function, a bispecific binder binds to an antigen (or epitope) on one of its two binding arms (a VH / VL pair), and binds to different antigens (or epitopes) on its second arm (different VH / VL pairs). By this definition, a bispecific binder has two different antigen binding arms (both specificity and CDR sequences are different), and is monovalent for each antigen it binds to.

[0250] The bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) of the present technology have at least one arm that specifically binds to, for example, L1-CAM and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of a B cell, a T cell, a bone marrow cell, a plasma cell, or a mast cell. Additionally or alternatively, in certain embodiments, the second target antigen is selected from CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, and KIR. In certain embodiments, BsAb is capable of binding to tumor cells expressing L1-CAM antigens on the cell surface. In some embodiments, BsAb has been engineered to promote killing of tumor cells by guiding (or recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include those having a first antigen binding site specific for L1-CAM and a second antigen binding site specific for a small molecule hapten (e.g., DTP A, IMP288, DOTA, DOTA-Bn, DOTA-deferoxamine, other DOTA chelates described herein, biotin, fluorescein, or those disclosed in Goodwin, D A. et al., 1994, Cancer Res. 54(22):5937-5946).

[0251] A variety of bispecific fusion proteins can be produced using molecular engineering. For example, BsAb has been constructed, which utilizes a complete immunoglobulin framework (e.g., IgG), a single-chain variable fragment (scFv), or a combination thereof. In some embodiments, the bispecific fusion protein is divalent, comprising, for example, an scFv with a single binding site for an antigen and a Fab fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, comprising, for example, an immunoglobulin (e.g., IgG) with two binding sites for an antigen and two identical scFvs for a second antigen. The BsAb consisting of two scFv units in series has been shown to be a clinically successful bispecific antibody form. In some embodiments, BsAb comprises two single-chain variable fragments (scFv) in series, which are designed to connect the scFv of a tumor antigen (e.g., L1-CAM) to the scFv of a T cell (e.g., by combining CD3). In this way, T cells are recruited to the tumor site so that they can mediate the cytotoxic killing of tumor cells. See, e.g., Dreier et al., J. Immunol. 170:4397-4402 (2003); Bargou et al., Science 321:974-977 (2008)).

[0252] The latest methods for producing BsAbs include engineered recombinant monoclonal antibodies with additional cysteine ​​residues that make them more firmly cross-linked than more common immunoglobulin isotypes. See, for example, FitzGerald et al., Protein Eng. 10 (10): 1221-1225 (1997). Another method is an engineered recombinant fusion protein that connects two or more different single-chain antibodies or antibody fragment segments with the desired dual specificity. See, for example, Coloma et al., Nature Biotech. 15: 159-163 (1997). A variety of bispecific fusion proteins can be produced using molecular engineering.

[0253] Bispecific fusion proteins connecting two or more different single-chain antibodies or antibody fragments are produced in a similar manner. Recombinant methods can be used to produce a variety of fusion proteins. In some specific embodiments, the BsAb according to the present technology comprises an immunoglobulin containing a heavy chain and a light chain and an scFv. In some specific embodiments, the scFv is connected to the C-terminus of the heavy chain of any L1-CAM immunoglobulin disclosed herein. In some specific embodiments, the scFv is connected to the C-terminus of the light chain of any L1-CAM immunoglobulin disclosed herein. In various embodiments, the scFv is connected to the heavy chain or light chain via a linker sequence. By PCR reaction, the appropriate linker sequence necessary for in-frame connection of the heavy chain Fd with the scFv is introduced into the VL and V κ The DNA fragment encoding the scFv was then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct was excised and ligated to a V vector containing a DNA sequence encoding the L1-CAM antibody. H The obtained vector can be used to transfect suitable host cells, such as mammalian cells, to express the bispecific fusion protein.

[0254] In some embodiments, the length of the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but to provide flexibility (e.g., the first and / or second antigen binding site) for the polypeptide. In some embodiments, based on the specific characteristics of the BsAb, such as increased stability, a linker is used in the BsAb described herein. In some embodiments, the BsAb of the present technology comprises G 4 S linker (SEQ ID NO: 76). In some specific embodiments, the BsAb of the present technology comprises (G 4 S) n A linker wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more (SEQ ID NO: 78).

[0255] Fc modification. In some embodiments, the anti-L1-CAM antibody of the present technology comprises a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to the wild-type Fc region (or parent Fc region), such that the affinity of the molecule for an Fc receptor (e.g., FcγR) is altered, provided that the variant Fc region is not substituted at a position in direct contact with the Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions (such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000)). Examples of positions within the Fc region that are in direct contact with an Fc receptor (such as FcγR) include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G) loop.

[0256] In some embodiments, the anti-L1-CAM antibodies of the present technology have altered affinity for activating and / or inhibitory receptors, wherein the variant Fc region has one or more amino acid modifications, wherein the one or more amino acid modifications are N297 to alanine or K322 to alanine.

[0257] Glycosylation modifications. In some embodiments, the anti-L1-CAM antibodies of the present technology have an Fc region containing variant glycosylation compared to a parent Fc region. In some embodiments, the variant glycosylation includes the absence of fucose; in some embodiments, expression in GnT1-deficient CHO cells results in variant glycosylation.

[0258] In some embodiments, the antibodies of the present technology may have modified glycosylation sites relative to an appropriate reference antibody that binds to an antigen of interest (e.g., L1-CAM) without altering the functionality of the antibody, such as binding activity to the antigen. As used herein, "glycosylation site" includes any specific amino acid sequence in an antibody that is specifically and covalently attached to an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together).

[0259] Oligosaccharide side chains are usually connected to the backbone of the antibody via N or O. N-linked glycosylation refers to the attachment of the oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of the oligosaccharide moiety to a hydroxyamino acid, such as serine, threonine. For example, an Fc-glycoform (huL1-CAM-IgGln) lacking certain oligosaccharides (including fucose and terminal N-acetylglucosamine) can be produced in special CHO cells and exhibits enhanced ADCC effector function.

[0260] In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included in the present technology, see, for example, U.S. Patent No. 6,218,149; EP 0359096B1; U.S. Patent Publication No. US2002 / 0028486; International Patent Application Publication WO03 / 035835; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent No. 6,218,149; U.S. Patent No. 6,472,511; All of these references are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content (or its related parts or components) of an antibody is modified by deleting one or more endogenous carbohydrate parts of the antibody. In some specific embodiments, the present technology includes deleting the glycosylation site of the Fc region of an antibody by modifying the asparagine at position 297 to alanine.

[0261] Engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms can be produced by any method known to those skilled in the art, such as by using engineered or variant expression strains, by coexpression with one or more enzymes (e.g., DI N-acetylglucosamine transferase III (GnTIII)), by expressing molecules comprising the Fc region in various organisms or cell lines from various organisms, or by modifying one or more carbohydrates after having expressed a molecule comprising the Fc region. Methods for generating engineered glycoforms are known in the art and include, but are not limited to, those described in the following references: Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application Serial No. 10 / 277,370; U.S. Patent Application Serial No. 10 / 113,929; International Patent Application Publication Nos. WO 00 / 61739A1; WO 01 / 292246A1; WO 02 / 311140A1; WO 02 / 30954A1; POTILLEGENT TM Technology (Biowa, Inc. Princeton, NJ); GLYCOMAB TM Glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of these references is incorporated herein by reference in its entirety. See, e.g., International Patent Application Publication No. WO 00 / 061739; U.S. Patent Application Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[0262] Fusion protein. In one embodiment, the anti-L1-CAM antibody of the present technology is a fusion protein. When fused with a second protein, the anti-L1-CAM antibody of the present technology can be used as an antigen tag. Examples of domains that can be fused to a polypeptide include not only heterologous signal sequences, but also other heterologous functional regions. The fusion is not necessarily direct, but can be performed through a linker sequence. Moreover, the fusion protein of the present technology can also be engineered to improve the characteristics of the anti-L1-CAM antibody. For example, a region of additional amino acids (particularly charged amino acids) can be added to the N-terminus of the anti-L1-CAM antibody to improve stability and durability during purification from a host cell or subsequent processing and storage. Similarly, a peptide portion can be added to the anti-L1-CAM antibody to facilitate purification. Such regions can be removed before the final preparation of the anti-L1-CAM antibody. Adding a peptide portion to facilitate the processing of a polypeptide is a well-known and conventional technique in the art. The anti-L1-CAM antibody of the present technology can be fused to a marker sequence (such as a peptide that promotes the purification of the fusion polypeptide). In selected embodiments, the marker amino acid sequence is a hexa-histidine peptide (SEQ ID NO: 79), such as the tag provided in the pQE vector (QIAGEN, Inc., Chatsworth, Calif.) and others, many of which are commercially available. For example, hexa-histidine (SEQ ID NO: 79) provides convenient purification of the fusion protein, as described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989. Another peptide tag that can be used for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

[0263] Thus, any of these above-described fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology. Likewise, in some embodiments, the fusion proteins described herein exhibit increased half-life in vivo.

[0264] Fusion proteins with a disulfide-linked dimeric structure (due to IgG) can bind and neutralize other molecules more efficiently than individual monomeric secreted proteins or protein fragments. Fountoulakis et al., J. Biochem. 270:3958-3964, 1995.

[0265] Similarly, EP-AO 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various parts of the constant region of an immunoglobulin molecule and another human protein or a fragment thereof. In many cases, the Fc portion in the fusion protein is beneficial in therapy and diagnosis, and can therefore lead to, for example, improved pharmacokinetic properties. See EP-A 0232262. Alternatively, it may be necessary to delete or modify the Fc portion after expression, detection and purification of the fusion protein. For example, if the fusion protein is used as an antigen for immunization, the Fc portion may hinder therapy and diagnosis. In drug discovery, for example, human proteins (such as hIL-5) have been fused to the Fc portion for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al., J.Molecular Recognition 8:52-58, 1995; Johanson et al., J.Biol.Chem., 270:9459-9471, 1995.

[0266] Labeled anti-L1-CAM antibodies. In one embodiment, the anti-L1-CAM antibodies of the present technology are coupled to a labeling moiety, i.e., a detectable group. The specific label or detectable group conjugated to the anti-L1-CAM antibody is not a critical aspect of the present technology, as long as it does not significantly interfere with the specific binding of the anti-L1-CAM antibody of the present technology to the L1-CAM protein. The detectable group can be any material with detectable physical or chemical properties. Such detectable labels have been well developed in the fields of immunoassays and imaging. In general, almost any label that can be used in such methods can be applied to the present technology. Therefore, a label is any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels that can be used in the practice of the present technology include magnetic beads (e.g., Dynabeads TM ), fluorescent dyes (e.g. fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radioactive labels (e.g. 3 H. 14 C. 35 S. 125 I. 121 I. 131 I. 112 In, 99 mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18 F. 11 C. 15 O (for positron emission tomography), 99m TC, 111In (for single photon emission tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each of which is incorporated herein by reference in its entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6th Edition, Molecular Probes, Inc., Eugene OR.).

[0267] The label can be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As described above, a variety of labels can be used, with the label being selected based on factors such as desired sensitivity, ease of conjugation to the compound, stability requirements, available instrumentation, and disposal regulations.

[0268] Non-radioactive labels are usually attached by indirect means. Typically, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand is then bound to an anti-ligand (e.g., streptavidin) molecule, which has inherent detectability or is covalently bound to a signaling system (e.g., a detectable enzyme, a fluorescent compound, or a chemiluminescent compound). Many ligands and anti-ligands can be used. When a ligand (e.g., biotin, thyroxine, and cortisol) has a natural anti-ligand, the ligand can be used in combination with a labeled naturally occurring anti-ligand. Alternatively, any hapten or antigenic compound can be used in combination with an antibody, such as an anti-L1-CAM antibody.

[0269] The molecule can also be directly conjugated with the signal generating compound, for example by conjugating with an enzyme or a fluorophore. The target enzyme as a label will mainly be a hydrolase, particularly a phosphatase, an esterase and a glycosidase, or an oxidoreductase, particularly a peroxidase. Fluorescent compounds that can be used as labeling parts include, but are not limited to, for example, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds that can be used as labeling parts include, but are not limited to, for example, fluorescein and 2,3-dihydrophthalazinediones, such as luminol. For a review of various labels or signal production systems that can be used, see U.S. Patent No. 4,391,904.

[0270] The means of detecting the label is well known to those skilled in the art. Therefore, for example, when the label is a radioactive label, the detection means includes a scintillation counter or a photographic film, such as in an autoradiography. If the label is a fluorescent label, it can be detected by exciting the fluorescent dye with light of an appropriate wavelength and detecting the fluorescence produced. Fluorescence can be detected visually by film using an electronic detector such as a charge coupled device (CCD) or a photomultiplier tube. Similarly, enzyme labels can be detected by providing an appropriate substrate for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels can be simply detected by observing the color associated with the label. Therefore, in various dipstick assays, the conjugated gold often presents a pink color, and the various conjugated beads present the color of the beads.

[0271] Some assay formats do not require the use of labeled components. For example, an agglutination assay can be used to detect the presence of a target antibody, such as an anti-L1-CAM antibody. In this case, antigen-coated particles are agglutinated by a sample containing the target antibody. In this format, no components need to be labeled, and the presence of the target antibody can be detected by simple visual inspection.

[0272] B. Identification and Characterization of Anti-L1-CAM Antibodies of the Present Technology

[0273] Methods for identifying and / or screening anti-L1-CAM antibodies of the present technology. Methods for identifying and screening antibodies against L1-CAM polypeptides that have the desired specificity for L1-CAM protein include any immune-mediated techniques known in the art. Components of the immune response can be detected in vitro by various methods well known to those of ordinary skill in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radiolabeled target cells and the lysis of these target cells detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigen and antigen-presenting cells, and cytokine synthesis and secretion measured by standard methods (Windhagen A et al., Immunity, 2:373-80, 1995); (3) antigen-presenting cells can be incubated with whole protein antigen and the presentation of the antigen on MHC detected by T lymphocyte activation assay or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86:4230-4, 1989); (4) mast cells can be incubated with agents (cross-linking their Fc-ε receptors) and histamine release measured by enzyme immunoassay (Siraganian et al., TIPS, 4:432-437, 1983); (5) enzyme-linked immunosorbent assay (ELISA).

[0274] Similarly, the products of immune responses in model organisms (e.g., mice) or human subjects can also be detected by various methods well known to those of ordinary skill in the art. For example, (1) the production of antibodies in response to vaccination can be easily detected by standard methods currently used in clinical laboratories, such as ELISA; (2) the migration of immune cells to the site of inflammation can be detected by scratching the skin surface and placing a sterile container to capture the migrating cells at the scratch site (Peters et al., Blood, 72:1310-5, 1988); (3) the use of 3 H-thymidine measures the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions; (4) the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells with labeled particles (Peters et al., Blood, 72:1310-5, 1988); and (5) the differentiation of immune system cells can be measured by labeling PBMCs with antibodies to CD molecules (such as CD4 and CD8) and measuring the fraction of PBMCs that express these markers.

[0275] In one embodiment, the display of L1-CAM peptides on the surface of replicable genetic packages is used to select anti-L1-CAM antibodies of the present technology. See, e.g., U.S. Patent Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585 287; EP 605522; EP 616640; EP1024191; EP 589 877; EP 774 511; EP 844 306. Methods useful for producing / selecting filamentous phage particles containing a phagemid genome encoding a binding molecule with a desired specificity have been described. See, for example, EP 774 511; US ​​5871907; US 5969108; US 6225447; US 6291650; US 6492160.

[0276] In some embodiments, the anti-L1-CAM antibodies of the present technology are selected using display of L1-CAM peptides on the surface of yeast host cells. Methods that can be used to isolate scFv polypeptides by display on the surface of yeast have been described by Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10.

[0277] In some embodiments, anti-L1-CAM antibodies of the present technology are selected using ribosome display. Methods that can be used to identify ligands in peptides using ribosome display have been described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91:9022-26, 1994; and Hanes et al., Proc. Natl. Acad. Sci. USA 94:4937-42, 1997.

[0278] In certain embodiments, tRNA display of L1-CAM peptides is used to select anti-L1-CAM antibodies of the present technology. Methods that can be used for in vitro selection of ligands using tRNA display have been described by Merryman et al., Chem. Biol., 9:741-46, 2002.

[0279] In one embodiment, anti-L1-CAM antibodies of the present technology are selected using RNA display. Methods that can be used to select peptides and proteins using RNA display libraries have been described by Roberts et al., Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al., FEBS Lett., 414: 405-8, 1997. Methods that can be used to select peptides and proteins using non-natural RNA display libraries have been described by Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

[0280] In some embodiments, the anti-L1-CAM antibodies of the present technology are expressed in the periplasm of Gram-negative bacteria and mixed with labeled L1-CAM protein. See WO 02 / 34886. In clones expressing recombinant polypeptides with affinity for L1-CAM protein, the concentration of labeled L1-CAM protein bound to the anti-L1-CAM antibody is increased, and the cells are allowed to separate from the rest of the library, as described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Patent Publication No. 2004 / 0058403.

[0281] After the desired anti-L1-CAM antibody is selected, it is expected that the antibody can be produced in large quantities by any technique known to those skilled in the art (e.g., prokaryotic or eukaryotic cell expression, etc.). Anti-L1-CAM antibodies (which are, for example, but not limited to, anti-L1-CAM hybrid antibodies or fragments) can be produced by constructing expression vectors using conventional techniques, encoding antibody heavy chains in which the CDRs required for the binding specificity of the original species antibody are retained and, if necessary, a minimal portion of the variable region framework (such as engineered according to the techniques described herein) is derived from the origin species antibody, while the remainder of the antibody is derived from a target species immunoglobulin, which can be manipulated as described herein to produce a vector for expressing the hybrid antibody heavy chain.

[0282] Measurement of L1-CAM binding. In some embodiments, the L1-CAM binding assay refers to an assay format in which L1-CAM protein and anti-L1-CAM antibodies are mixed under conditions suitable for binding between L1-CAM protein and anti-L1-CAM antibody and assessing the amount of binding between L1-CAM protein and anti-L1-CAM antibody. The amount of binding is compared with a suitable control, which can be the amount of binding in the absence of L1-CAM protein, the amount of binding in the presence of a non-specific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assays include, for example, ELISA, radioimmunoassay, proximity scintillation assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for directly measuring L1-CAM protein bound to anti-L1-CAM antibodies are, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), etc. Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. If the specific binding of a candidate anti-L1-CAM antibody is at least 1% greater than the binding observed in the absence of the candidate anti-L1-CAM antibody, the candidate anti-L1-CAM antibody can be used as an anti-L1-CAM antibody according to the present technology.

[0283] Measurement of L1-CAM neutralization. As used herein, "L1-CAM neutralization" refers to the reduction of activity and / or expression of L1-CAM protein by binding of an anti-L1-CAM antibody. The ability of the anti-L1-CAM antibodies of the present technology to neutralize L1-CAM activity / expression can be assessed in vitro or in vivo using methods known in the art. Uses of the anti-L1-CAM antibodies of the present technology

[0284] Overview. The anti-L1-CAM antibodies of the present technology can be used in methods known in the art related to the localization and / or quantification of L1-CAM proteins (e.g., for measuring the level of L1-CAM proteins in appropriate physiological samples, for diagnostic methods, for polypeptide imaging, etc.). The antibodies of the present technology can be used to separate L1-CAM proteins by standard techniques such as affinity chromatography or immunoprecipitation. The anti-L1-CAM antibodies of the present technology can facilitate the purification of naturally immunoreactive L1-CAM proteins from biological samples such as mammalian serum or cells, as well as the purification of recombinantly produced immunoreactive L1-CAM proteins expressed in host systems. In addition, the anti-L1-CAM antibodies can be used to detect immunoreactive L1-CAM proteins (e.g., in plasma, cell lysates, or cell supernatants) to evaluate the expression abundance and pattern of immunoreactive polypeptides. The anti-L1-CAM antibodies of the present technology can be used diagnostically to monitor the level of immunoreactive L1-CAM proteins in tissues as part of a clinical testing program, for example, to determine the efficacy of a given treatment regimen. As described above, detection can be facilitated by coupling (i.e., physically connecting) the anti-L1-CAM antibodies of the present technology to a detectable substance.

[0285] Detection of L1-CAM protein. An exemplary method for detecting the presence of an immunoreactive L1-CAM protein in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with an anti-L1-CAM antibody of the present technology capable of detecting an immunoreactive L1-CAM protein, thereby detecting the presence of an immunoreactive L1-CAM protein in the biological sample. Detection can be accomplished by a detectable label attached to the antibody.

[0286] The term "labeled" with respect to anti-L1-CAM antibodies is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound (e.g., a secondary antibody) that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0287] In some embodiments, the anti-L1-CAM antibodies disclosed herein are conjugated to one or more detectable labels. For such uses, the anti-L1-CAM antibodies can be detectably labeled by covalent or non-covalent attachment of chromogenic agents, enzymatic agents, radioisotopic agents, isotopic agents, fluorescent agents, toxic agents, chemiluminescent agents, nuclear magnetic resonance contrast agents, or other labels.

[0288] Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0289] Examples of suitable radioisotope labels include 3 H. 111 In, 125 I. 131 I. 32 P. 35 S. 14 C. 51 Cr, 57 To, 58 Co. 59 Fe, 75 Se, 152 Eu, 90 Y. 67 Cu, 217 Ci, 211 At 212 Pb, 47 Sc, 109 Pd etc. 111 In is an exemplary isotope when using in vivo imaging because it avoids 125 I or 131 The problem of I-labeled L1-CAM binding antibodies being dehalogenated by the liver. In addition, this isotope has a more favorable gamma emission energy for imaging (Perkins et al., Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, a monoclonal antibody conjugated with 1-(p-isothiocyanatobenzyl)-DPTA 111 In exhibits little uptake in non-tumor tissues (particularly the liver) and enhances the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioactive isotope labels include 157 Gd, 55 Mn, 162 Dy, 52 Tr and 56 Fe.

[0290] Examples of suitable fluorescent labels include 152Eu labeling, fluorescein labeling, isothiocyanate labeling, rhodamine labeling, phycoerythrin labeling, phycocyanin labeling, allophycocyanin labeling, green fluorescent protein (GFP) labeling, o-phthalaldehyde labeling and fluorescamine labeling. Examples of suitable toxin labels include diphtheria toxin, ricin toxin and cholera toxin.

[0291] Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate labels, luciferin labels, luciferase labels, and aequorin labels. Examples of nuclear magnetic resonance contrast agents include heavy metal nuclei such as Gd, Mn, and iron.

[0292] The detection methods of the present technology can be used to detect immunoreactive L1-CAM proteins in biological samples in vitro and in vivo. In vitro techniques for detecting immunoreactive L1-CAM proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, radioimmunoassay and immunofluorescence. In addition, in vivo techniques for detecting immunoreactive L1-CAM proteins include introducing labeled anti-L1-CAM antibodies into a subject. For example, the anti-L1-CAM antibody can be labeled with a radioactive marker, the presence and location of which in the subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains L1-CAM protein molecules from the test subject.

[0293] Immunoassays and imaging. Using antibody-based techniques, the anti-L1-CAM antibodies of the present technology can be used to determine the level of immunoreactive L1-CAM protein in biological samples (e.g., human plasma). For example, protein expression in tissues can be studied using classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101:976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105:3087-3096, 1987. Other antibody-based methods that can be used to detect protein gene expression include immunoassays such as enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA). Suitable antibody assay labels are known in the art and include enzyme labels (such as glucose oxidase) and radioisotopes or other radioactive agents (such as iodine ( 125 I. 121 I. 131 I), carbon ( 14 C) Sulfur ( 35 S), tritium ( 3 H), Indium ( 112 In) and technetium ( 99mTc)) and fluorescent labels such as fluorescein, rhodamine, and green fluorescent protein (GFP), as well as biotin.

[0294] In addition to determining the level of immunoreactive L1-CAM protein in a biological sample, the anti-L1-CAM antibodies of the present technology can also be used for in vivo imaging of L1-CAM. Antibodies that can be used in this method include antibodies that can be detected by X-ray radiography, NMR or ESR. For X-ray radiography, suitable labels include radioisotopes, such as barium or cesium, which emit detectable radiation but are not significantly harmful to the subject. Labels suitable for NMR and ESR include labels with detectable characteristic spins, such as deuterium, which can be incorporated into the anti-L1-CAM antibody by labeling nutrients for the relevant scFv clones.

[0295] The cells have been treated with an appropriate detectable imaging moiety such as a radioisotope (e.g. 131 I. 112 In, 99 An anti-L1-CAM antibody labeled with a radiopaque substance (mTc), a radiopaque substance, or a material detectable by nuclear magnetic resonance) is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into a subject. It will be understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is generally between about 5 and 20 millicuries. 99 The labeled anti-L1-CAM antibody will then accumulate in the cell location containing the specific target polypeptide. For example, the labeled anti-L1-CAM antibody of the present technology will accumulate in the cells and tissues in the subject where the L1-CAM protein has been located.

[0296] Therefore, the present technology provides a method for diagnosing a medical condition, which involves: (a) determining the expression of an immunoreactive L1-CAM protein by measuring the binding of an anti-L1-CAM antibody of the present technology in cells or body fluids of an individual; (b) comparing the amount of immunoreactive L1-CAM protein present in the sample with a standard reference, wherein an increase or decrease in the level of the immunoreactive L1-CAM protein compared to the standard indicates a medical condition.

[0297] Affinity purification. The anti-L1-CAM antibodies of the present technology can be used to purify immunoreactive L1-CAM proteins from a sample. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics (such as polycarbonates), complex carbohydrates (such as agarose and sepharose), acrylic resins, and beads such as polyacrylamide and latex. The techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., "Handbook of Experimental Immunology" 4th Edition, Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, NY (1974)).

[0298] The simplest method of combining antigen with antibody support matrix is ​​to collect beads in a column and transfer the antigen solution downward along the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, and the contact time can be extended by using a low flow rate. The immobilized antibody captures the antigen when the antigen flows through. Alternatively, the antigen can be contacted with the antibody support matrix in the following manner: the antigen solution is mixed with a support (e.g., beads) and rotated or shaken to maximize the contact between the antigen and the immobilized antibody. After the binding reaction is completed, the slurry is transferred to the column to collect beads. The beads are washed with suitable washing buffer, and then the pure or substantially pure antigen is eluted.

[0299] The antibody or polypeptide of interest may be conjugated to a solid support such as a bead. In addition, if desired, a first solid support such as a bead may be conjugated to a second solid support by any suitable means, including means disclosed herein for conjugating a polypeptide to a support, wherein the second solid support may be a second bead or other support. Therefore, any conjugation methods and means disclosed herein for conjugating a polypeptide to a solid support may also be used to conjugate a first support to a second support, wherein the first and second solid supports may be the same or different.

[0300] Suitable linkers (which may be cross-linkers) for conjugating polypeptides to solid supports include a variety of agents that can react with functional groups present on the support surface or with polypeptides or both. Reagents that can be used as cross-linkers include homobifunctional agents, and in particular heterobifunctional agents. Useful bifunctional cross-linkers include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. Cross-linkers can be selected to provide selectively cleavable bonds between polypeptides and solid supports. For example, photolabile cross-linkers such as 3-amino-(2-nitrophenyl) propionic acid can be used as a means of cleaving polypeptides from solid supports. (Brown et al., Mol. Divers, pp. 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24: 351-66 (1996); and U.S. Pat. No. 5,643,722). Other cross-linking agents are well known in the art. (See, e.g., Wong (1991), supra; and Hermanson (1996), supra).

[0301] The antibody or polypeptide can be fixed on a solid support (such as beads) by a covalent amide bond formed between the carboxyl-functionalized beads and the amino terminus of the polypeptide, or conversely by a covalent amide bond formed between the amino-functionalized beads and the carboxyl terminus of the polypeptide. In addition, a difunctional trityl linker can be attached to the support via an amino resin through an amino group or a carboxyl group on the resin, such as a 4-nitrophenyl active ester attached to a resin (such as Wang resin). When using the difunctional trityl method, the solid support may need to be treated with a volatile acid (such as formic acid or trifluoroacetic acid) to ensure that the polypeptide is cut and can be removed. In this case, the polypeptide can be deposited on the bottom of the hole of the solid support or on the flat surface of the solid support as a fragment without beads. After adding the matrix solution, the polypeptide can be desorbed into the MS.

[0302] Hydrophobic trityl linkers can also be used as acid-labile linkers by cleaving the amino-linked trityl group from the polypeptide using a volatile acid or an appropriate matrix solution (e.g., a matrix solution containing 3-HPA). Acid instability can also be changed. For example, a trityl group, a monomethoxytrityl group, a dimethoxytrityl group, or a trimethoxytrityl group can be changed to a suitable para-substituted or more acid-labile tritylamine derivative of the polypeptide, i.e., trityl ether and tritylamine can be bonded to the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker, for example, by destroying the hydrophobic attraction or by cleaving the trityl ether or tritylamine bond under acidic conditions where a matrix such as 3-HPA is used as an acid (including, if necessary, under typical MS conditions).

[0303] Orthogonal cleavable linkers can also be used to bind a first solid support (e.g., beads) to a second solid support, or can be used to bind a polypeptide of interest to a solid support. Using such linkers, a first solid support (e.g., beads) can be selectively cleaved from a second solid support without cleaving the polypeptide from the support; the polypeptide can then be cleaved from the beads later. For example, a disulfide linker that can be cleaved with a reducing agent such as DTT can be used to bind the beads to a second solid support, and an acid-cleavable bifunctional trityl group can be used to fix the polypeptide to the support. As desired, the connection between the polypeptide and the solid support can be cleaved first, for example, to keep the connection between the first and second supports intact. Trityl linkers can provide covalent or hydrophobic conjugation, and regardless of the nature of the conjugation, the trityl group is easily cleaved under acidic conditions.

[0304] For example, the beads can be bound to the second support via a linking group, which can be selected to have a length and chemical nature that promotes high density binding of the beads to the solid support or high density binding of the polypeptide to the beads. Such linking groups can have, for example, a "tree-like" structure, thereby providing a variety of functional groups at each attachment site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, penta-erythrole, and trihydroxyaminomethane.

[0305] Non-covalent binding association. Through non-covalent interactions, antibodies or polypeptides can be conjugated to a solid support, or a first solid support can also be conjugated to a second solid support. For example, magnetic beads made of ferromagnetic materials that can be magnetized can be attracted to a magnetic solid support and can be released from the support by removing the magnetic field. Alternatively, the solid support can have ionic or hydrophobic moieties, which can allow the ionic or hydrophobic moieties to interact with a polypeptide (e.g., a polypeptide containing an attached trityl group) or with a second solid support having a hydrophobic character, respectively.

[0306] The solid support may also be provided with a member of a specific binding pair and thus may be conjugated to a polypeptide containing a complementary binding moiety or to a second solid support. For example, beads coated with avidin or with streptavidin may be bound to a polypeptide into which a biotin moiety has been incorporated, or to a second solid support coated with biotin or a biotin derivative such as iminobiotin.

[0307] It should be appreciated that any binding member disclosed herein or known in the art can be reversed. Thus, correspondingly, for example, biotin can be incorporated into a polypeptide or solid support, and conversely, avidin or other biotin binding moiety can be incorporated into a support or polypeptide. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and their specifically interacting antigens, and other such pairs known to those skilled in the art.

[0308] A. Diagnostic Uses of Anti-L1-CAM Antibodies of the Present Technology

[0309] Overview. The anti-L1-CAM antibodies of the present technology can be used in diagnostic methods. Therefore, the present technology provides methods of diagnosing L1-CAM activity in a subject using the antibodies. The anti-L1-CAM antibodies of the present technology can be selected so that they have any level of epitope binding specificity and very high binding affinity to the L1-CAM protein. In general, the higher the binding affinity of the antibody, the more stringent washing conditions can be performed in the immunoassay to remove non-specifically bound material without removing the target polypeptide. Therefore, the anti-L1-CAM antibodies of the present technology that can be used in diagnostic assays generally have about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 or 10 12 M -1 In addition, it is desirable that anti-L1-CAM antibodies for use as diagnostic reagents have sufficient kinetic association rates to reach equilibrium under standard conditions within at least 12 h, at least five (5) h, or at least one (1) hour.

[0310] Anti-L1-CAM antibodies can be used to detect immunoreactive L1-CAM proteins in a variety of standard assay formats. Such formats include immunoprecipitation, Western blot, ELISA, radioimmunoassay, and immunometric assays. See Harlow & Lane, Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, New York, 1988); U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. The biological sample can be obtained from any tissue or fluid of the subject. In certain embodiments, the subject is in an early stage of cancer. In one embodiment, the early stage of cancer is determined by the level or expression pattern of L1-CAM protein in a sample obtained from the subject. In certain embodiments, the sample is selected from urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsy body tissue.

[0311] Immunoassay or sandwich assay is a form of diagnostic method of the present technology. See U.S. Patent Nos. 4,376,110, 4,486,530, 5,914,241 and 5,965,375. Such assays use an antibody (e.g., an anti-L1-CAM antibody or an anti-L1-CAM antibody population) fixed to a solid phase and another anti-L1-CAM antibody or an anti-L1-CAM antibody population in a solution. Typically, the solution anti-L1-CAM antibody or anti-L1-CAM antibody population is labeled. If an antibody population is used, the population may contain antibodies that specifically bind to different epitopes within the target polypeptide. Therefore, the same population can be used for both solid phase antibodies and solution antibodies. If an anti-L1-CAM monoclonal antibody is used, the first and second L1-CAM monoclonal antibodies with different binding specificities are used for the solid phase and the solution phase. The solid phase (also referred to as "capture") and solution (also referred to as "detection") antibodies can be contacted with the target antigen in any order or simultaneously. If the solid phase antibody is contacted first, the assay is called a forward assay. In contrast, if the solution antibody is contacted first, the assay is called a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is called a simultaneous assay. After contacting the L1-CAM protein with the anti-L1-CAM antibody, the sample is incubated for a period of time, typically ranging from about 10 minutes to about 24 hours, and typically about 1 hour. A washing step is then performed to remove components in the sample that are not specifically bound to the anti-L1-CAM antibody used as a diagnostic reagent. When the solid phase antibody and the solution antibody are combined in separate steps, washing can be performed after either or both of the binding steps. After washing, the binding is quantified, typically by detecting a label attached to the solid phase via the binding of a labeled solution antibody. Typically, for a given antibody pair or antibody population and given reaction conditions, a calibration curve is prepared from a sample containing a known concentration of the target antigen. The concentration of the immunoreactive L1-CAM protein in the sample being tested is then read by interpolation from the calibration curve (i.e., standard curve). The analyte can be measured from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of the bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of this curve is a measure of the L1-CAM protein concentration in the sample.

[0312] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes and derivatized nylon membranes, and also particles such as agarose, dextran-based gels, dipsticks, microparticles, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX TM(Amersham Pharmacia Biotech, Piscataway, NJ) etc. Immobilization can be by absorption or by covalent attachment. Optionally, the anti-L1-CAM antibody can be linked to a linker molecule (such as biotin) to attach to a surface-bound linker (such as avidin).

[0313] In some embodiments, the present disclosure provides an anti-L1-CAM antibody of the present technology conjugated to a diagnostic agent. The diagnostic agent can include a radioactive or non-radioactive label, a contrast agent (such as for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label can be a gamma, beta, alpha, Auger electron or positron emitting isotope. The diagnostic agent is a molecule that is administered conjugated to an antibody portion, i.e., an antibody or antibody fragment or subfragment, and can be used to diagnose or detect disease by locating cells containing the antigen.

[0314] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancers (such as paramagnetic ions) for magnetic resonance imaging (MRI). U.S. Patent No. 6,331,175 describes MRI technology and the preparation of antibodies conjugated to MRI enhancers, and is incorporated by reference in its entirety. In some embodiments, the diagnostic agent is selected from radioisotopes, enhancers for magnetic resonance imaging, and fluorescent compounds. In order to load radioactive metals or paramagnetic ions on the antibody component, it may be necessary to react it with a reagent having a long tail, to which a variety of chelating groups for binding ions are attached. This tail can be a polymer, such as polylysine, polysaccharides or other derivatized or derivatizable chains, and the polymer has a side group that can be combined with a chelating group, and the chelating group is, for example, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrin, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and similar groups known to be useful for this purpose. Chelates can be coupled to the antibodies of the present technology using standard chemical methods. Chelates are typically linked to antibodies via a group that is capable of forming a bond with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal crosslinking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Patent No. 4,824,659. Particularly useful metal chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs used with diagnostic isotopes for radiography. The same chelate when combined with non-radioactive metals (such as manganese, iron, and gadolinium) can be used for MRI when used with the L1-CAM antibody of the present technology. Macrocyclic chelates such as NOTA (1,4,7-triaza-cyclononane-N,N′,N″-triacetic acid), DOTA and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) can be used with a variety of metals and radioactive metals (radionuclides such as gallium, yttrium and copper, respectively). Such metal chelate complexes can be stabilized by tailoring the ring size to the metal of interest. Other examples of DOTA chelates include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH 2 ; (ii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH 2 ; (iii)DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH 2 ; (iv)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ; (v)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2 ; (vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ; (vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH 2 ; (viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH 2 ; (ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2 ; (x) Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH 2 ; (xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 ; (xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH 2 ; (xiii) (Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH 2 ; (xiv) Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2 ; (xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH 2 ; (xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH 2 ; (xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH 2 ; and (xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH 2 .

[0315] Also considered are the stable binding nuclides (such as those used in RAIT). 223 Other cyclic chelates of interest include macrocyclic polyethers.

[0316] B. Therapeutic Uses of Anti-L1-CAM Antibodies of the Present Technology

[0317] The immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) of the present technology can be used to treat L1-CAM-related cancers. Such treatment can be used for patients identified as having pathologically high levels of L1-CAM (e.g., patients diagnosed by the methods described herein) or patients diagnosed with a disease known to be associated with such pathological levels. In one aspect, the present disclosure provides a method for treating L1-CAM-related cancers in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology. Examples of cancers that can be treated by the antibodies of the present technology include, but are not limited to, leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, bile duct cancer, carcinoid, neuroendocrine tumors, gastrointestinal stromal tumors (GIST) , pheochromocytoma, glioma, pancreatic neuroectodermal cancer, pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma, tumor vascular, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine cancer, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, nasal glioma, medulloblastoma, capillary hemangioma, Kaposi sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, and head and neck squamous cell carcinoma.

[0318] The composition of the present technology can be used in combination with other therapeutic agents that can be used to treat L1-CAM-related cancers. For example, the antibody of the present technology can be administered separately, sequentially or simultaneously with at least one additional therapeutic agent selected from the following: alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cell-inhibiting alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutic agents and targeted biological therapeutic agents (e.g., therapeutic peptides described in US 6306832, WO2012007137, WO2005000889, WO 2010096603, etc.). In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapeutic agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolomide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, a Nastrazole, exemestane, letrozole, leuprolide, abarelix, buserelin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, lapatinib, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, nitrogen mustard, bleomycin, microtubule poison, annonaceous acetone, or a combination thereof.

[0319] The compositions of the present technology can optionally be administered to a subject in need thereof in the form of a single bolus injection. Alternatively, the dosing regimen can include multiple administrations at different times after the appearance of a tumor.

[0320] Administration can be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), rectal, intracranial, intrathecal or topical. Administration includes self-administration and administration by another person. It is also understood that the various treatments of medical conditions as described herein are intended to mean "substantially," which includes complete treatment but also less than complete treatment, and in which some biologically or medically relevant results are obtained.

[0321] In some embodiments, the antibodies of the present technology comprise a pharmaceutical formulation that can be administered to a subject in need thereof in one or more doses. The dosage regimen can be adjusted to provide a desired response (e.g., a therapeutic response).

[0322] Typically, the effective amount of the antibody composition of the present technology sufficient to achieve a therapeutic effect is within the range of about 0.000001 mg per kilogram of body weight per day to about 10,000 mg per kilogram of body weight per day. Typically, the dosage range is about 0.0001 mg per kilogram of body weight per day to about 100 mg per kilogram of body weight per day. For administration of anti-L1-CAM antibodies, the dosage range is 0.0001 to 100 mg / kg of subject body weight per week, every two weeks or every three weeks, and more typically 0.01 to 5 mg / kg of subject body weight. For example, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight per week, every two weeks or every three weeks, or within the range of 1-10 mg / kg per week, every two weeks or every three weeks. In one embodiment, the single dose range of the antibody is 0.1-10,000 micrograms per kilogram of body weight. In one embodiment, the antibody concentration range in the carrier is 0.2 to 2000 micrograms / delivered milliliter. Exemplary treatment regimens require administration once every two weeks or once a month or once every 3 to 6 months. Anti-L1-CAM antibodies can be administered at multiple times. The intervals between individual doses can be hourly, daily, weekly, monthly or annual. Intervals can also be irregular as indicated by measuring the blood level of the antibody in the subject. In some methods, the dose is adjusted so that the serum antibody concentration in the subject reaches about 75 μg / mL to about 125 μg / mL, 100 μg / mL to about 150 μg / mL, about 125 μg / mL to about 175 μg / mL or about 150 μg / mL to about 200 μg / mL. Alternatively, anti-L1-CAM antibodies can be administered as sustained release formulations, in which case less frequent administration is required. The dose and frequency vary according to the half-life of the antibody in the subject. The dose and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered for a long time at relatively infrequent intervals. In therapeutic applications, relatively high doses with relatively short intervals are sometimes required until the progression of the disease is reduced or terminated, or until the subject shows partial or complete improvement in symptoms of the disease. Thereafter, the patient may be given a preventive regimen.

[0323] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising (a) administering to the subject an effective amount of an antibody (or an antigen-binding fragment thereof) of the present technology, wherein the antibody is configured to localize to a tumor expressing L1-CAM and is labeled with a radioactive isotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as injected dose per gram (%ID / g). The reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissues and calculating the average radioactivity level ± standard deviation present in non-tumor (normal) tissues. In some embodiments, the ratio of radioactivity levels between tumor and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0324] In some embodiments, the subject is diagnosed with or suspected of having cancer.The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0325] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle emitting isotopes include 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu and 67 Cu. Examples of alpha-emitting isotopes include 213 Bi, 211 At 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr. 217 At and 255Fm. Examples of Auger emitters include 111 In, 67 Ga, 51 Cr, 58 Co. 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl and 203 Pb. In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via the N297A mutation in the Fc region, which results in deglycosylation). The therapeutic effect of this immunoconjugate can be determined by calculating the area under the curve (AUC) tumor: AUC normal tissue ratio. In some embodiments, the AUC tumor: AUC normal tissue ratio of the immunoconjugate is about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1, 85: 1, 90: 1, 95: 1 or 100: 1.

[0326] Toxicity. Optimally, an effective amount (e.g., dose) of an anti-L1-CAM antibody described herein will provide therapeutic benefit without causing substantial toxicity to the subject. The toxicity of the anti-L1-CAM antibodies described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as by measuring LD 50 (dose lethal to 50% of the population) or LD 100 The dose ratio between toxicity and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is non-toxic to humans. The dosage of the anti-L1-CAM antibody described herein is within a range of circulating concentrations that include an effective dose with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route and dosage of administration can be selected by each physician based on the condition of the subject. See, for example, Fingl et al., In: The Pharmacological Basis of Therapeutics, Chapter 1 (1975).

[0327] Formulation of pharmaceutical compositions. According to the methods of the present technology, anti-L1-CAM antibodies can be incorporated into pharmaceutical compositions suitable for administration. Pharmaceutical compositions typically comprise recombinant or substantially purified antibodies and a pharmaceutically acceptable carrier in a form suitable for administration to a subject. The pharmaceutically acceptable carrier depends in part on the specific composition being administered, as well as on the specific method for administering the composition. Thus, there are a variety of suitable formulations of pharmaceutical compositions for administering antibody compositions (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18th edition, 1990). Pharmaceutical compositions are typically formulated to be sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0328] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variants thereof, when they refer to compositions, carriers, diluents and agents, are used interchangeably and indicate that the material can be administered to or onto a subject without producing an undesirable physiological effect to a degree that would prohibit administration of the composition. For example, a "pharmaceutically acceptable excipient" means an excipient that can be used to prepare a pharmaceutical composition that is generally safe, non-toxic and desirable, and includes excipients that are acceptable for veterinary as well as human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, a gas. "Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the composition are capable of reacting with an inorganic or organic base. Suitable inorganic salts include salts formed with alkali metals such as sodium and potassium, magnesium, calcium and aluminum. Suitable organic salts include organic salts formed with organic bases, such as amine bases, for example, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkanesulfonic acids and aromatic hydrocarbon sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed with carboxyl, sulfonyloxy, and phosphonooxy groups present in the anti-L1-CAM antibody, such as C 1-6Alkyl ester. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a monoacid monosalt or ester or a disalt or ester; and similarly, when more than two acidic groups are present, some or all of such groups may be salified or esterified. The anti-L1-CAM antibodies named in this technology may exist in an unsalted or unesterified form, or in a salified and / or esterified form, and the naming of such anti-L1-CAM antibodies is intended to include the original (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. In addition, certain embodiments of the present technology may exist in more than one stereoisomer form, and the naming of such anti-L1-CAM antibodies is intended to include all single stereoisomers and all mixtures of such stereoisomers (whether racemic or otherwise). It is not difficult for a person of ordinary skill in the art to determine the appropriate timing, sequence, and dosage for the administration of specific drugs and compositions of the present technology.

[0329] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Unless any conventional media or compounds are incompatible with the anti-L1-CAM antibody, their use in the composition is contemplated. Supplementary active compounds may also be incorporated into the composition.

[0330] The pharmaceutical compositions of the present technology are formulated to be compatible with their intended route of administration. The anti-L1-CAM antibody compositions of the present technology can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intrathecally, intraperitoneally, intranasally; or intramuscularly, or as an inhalant. The anti-L1-CAM antibodies can be administered optionally in combination with other drugs that are at least partially effective in treating various L1-CAM-related cancers.

[0331] Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial compounds, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating compounds, such as ethylenediaminetetraacetic acid (EDTA); buffers (such as acetates, citrates or phosphates), and compounds for adjusting tonicity (such as sodium chloride or dextrose). pH can be adjusted with acids or bases (such as hydrochloric acid or sodium hydroxide). Parenteral preparations can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0332] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under manufacturing and storage conditions and must be preserved against the contaminating effects of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, appropriate fluidity can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Preventing the effects of microorganisms can be achieved by a variety of antibacterial and antifungal compounds (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is desirable to include isotonic compounds in the composition, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride. Extended absorption of injectable compositions can be achieved by including compounds that delay absorption, such as aluminum monostearate and gelatin in the composition.

[0333] Sterile injectable solutions can be prepared in the following manner: the anti-L1-CAM antibody of the present technology is incorporated in the required amount into an appropriate solvent with one or a combination of the ingredients listed above as needed, followed by filtration sterilization. Typically, the dispersion is prepared by incorporating the anti-L1-CAM antibody into a sterile vehicle containing an alkaline dispersion medium and other required ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient and any additional required ingredients from a previously sterile filtered solution. The antibodies of the present technology can be administered in the form of a depot injection or implant preparation, which can be prepared in such a way that the injection or implant preparation is depotted to allow for sustained or pulsed release of the active ingredient.

[0334] Oral compositions generally contain an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, anti-L1-CAM antibodies can be mixed with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is orally administered and rinsed, and spit out or swallowed. Pharmaceutically compatible binding compounds and / or auxiliary materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders, such as microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch or lactose, disintegrating compounds, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silica; sweetening compounds, such as sucrose or saccharin; or flavoring compounds, such as mint, methyl salicylate or orange flavor.

[0335] For administration by inhalation, the anti-L1-CAM antibody can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0336] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, the anti-L1-CAM antibody is formulated into an ointment, salves, gel, or cream as generally known in the art.

[0337] Anti-L1-CAM antibodies can also be prepared as pharmaceutical compositions for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

[0338] In one embodiment, the anti-L1-CAM antibody is prepared with a carrier that will protect the anti-L1-CAM antibody from rapid clearance from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are clear to those skilled in the art. The materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Pat. No. 4,522,811.

[0339] C. Kit

[0340] The present technology provides a kit for detecting and / or treating L1-CAM-related cancers, the kit comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein) or a functional variant thereof (e.g., a substitution variant). Optionally, the above components of the kit of the present technology are packaged in a suitable container and labeled for diagnosis and / or treatment of L1-CAM-related cancers. The above components can be stored in unit dose or multi-dose containers, such as sealed ampoules, vials, bottles, syringes and test tubes, as an aqueous solution (preferably a sterile solution) or as a lyophilized (preferably sterile) preparation for reconstitution. The kit may also include a second container containing a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients and saline solutions of the pharmaceutical composition. In addition, the kit may include instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The container can be made of a variety of materials (such as glass or plastic) and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The test kit can further include more containers containing pharmaceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. From a commercial and user perspective, it can also include other materials, including other buffers, diluents, filters, needles, syringes, culture media for one or more suitable hosts. The test kit can optionally include instructions, which are typically included in the commercial packaging of the treatment or diagnosis product, and the instructions include information about, for example, the indications, usage, dosage, manufacture, administration, contraindications, and / or warnings for using such treatment or diagnosis products.

[0341] The kit can be used to detect the presence of immunoreactive L1-CAM protein in a biological sample, such as any body fluid, including but not limited to, for example, serum, plasma, lymph, cystic fluid, urine, feces, cerebrospinal fluid, ascites or blood, and includes a biopsy sample of body tissue. For example, the kit can include: one or more humanized, chimeric or bispecific anti-L1-CAM antibodies (or antigen-binding fragments thereof) of the present technology that can bind to L1-CAM protein in a biological sample; a device for determining the amount of L1-CAM protein in a sample; and a device for comparing the amount of immunoreactive L1-CAM protein in a sample with a standard. One or more anti-L1-CAM antibodies can be labeled. Kit components (e.g., reagents) can be packaged in a suitable container. The kit can further include instructions for using the kit to detect immunoreactive L1-CAM protein.

[0342] For antibody-based kits, the kit can include, for example, 1) a first antibody, such as a humanized, chimeric or bispecific L1-CAM antibody (or antigen-binding fragment thereof) of the present technology, attached to a solid support, which first antibody binds to the L1-CAM protein; and, optionally; 2) a second, different antibody that binds to the L1-CAM protein or the first antibody and is conjugated to a detectable label.

[0343] The kit may also contain, for example, a buffer, a preservative, or a protein stabilizer. The kit may further contain other components required for detecting the detectable label, such as an enzyme or a substrate. The kit may also contain a control sample or a series of control samples, which can be assayed and compared with the test sample. Each component of the kit can be encapsulated in a separate container, and all the different containers can be placed in a single package together with instructions for interpreting the results of the assay using the kit. The kit of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, for example, for detecting L1-CAM protein in vitro or in vivo, or for treating L1-CAM-related cancers in subjects in need. In certain embodiments, the use of the reagents can be according to the methods of the present technology.

[0344] Example

[0345] The present technology is further illustrated by the following examples, which should not be construed as limiting in any manner. The following examples demonstrate the preparation, characterization, and use of illustrative L1-CAM antibodies of the present technology. Examples 1-11 demonstrate the generation of chimeric, humanized, and bispecific antibodies of the present technology, as well as the characterization of their binding specificity and in vivo biological activity.

[0346] Example 1: Materials and Methods for Producing and Characterizing Anti-L1-CAM Antibodies of the Present Technology

[0347] Immunohistochemistry (IHC). The use of human tumors and normal tissues for IHC was approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center. Five to seven micron sections of snap-frozen tissue were fixed in acetone at -20°C for 30 minutes. Endogenous biotin binding activity was blocked by treating with avidin and biotin (Vector Avidin-Biotin Blocking Kit; Invitrogen, Carlsbad, CA) for 20 minutes, followed by blocking with 10% horse serum for 1 hour at room temperature. The sections were then reacted with primary antibodies, biotinylated horse anti-mouse IgG (H+L) (Vector Laboratories, Inc., Burlingame, CA) and avidin-biotin complex (Vectastain ABC Kit, Vector Vector Laboratories, Inc., Burlingame, CA) for 60 minutes at room temperature, and washed between each reaction. Subsequently, sections were stained with dye (DAB Peroxidase Substrate Kit, Vector Laboratories, Burlingame, CA) for 2 min, washed, counterstained with Myer's hematoxylin, washed, and dehydrated in 95% ethanol.

[0348] Construction of chimeric and humanized versions of E71 and E72. Based on the human homologs, the CDR sequences of both the heavy and light chains of E71 and E72 were transplanted into the human IgG1 framework and optimized. The huE71 and huE72 genes were synthesized (Blue Heron Biotechnology, Bothell, WA; or Genscript, Piscataway, NY) and incorporated into mammalian expression vectors (Eureka, CA) and transfected into DG44 cells or CHO-S cells for stable production. Similarly, human V H and V L The sequences were transplanted onto the human IgG4 framework to produce IgG4 recombinant antibodies. Finally, these genes were transfected into DG44 cells lacking the GnT1 enzyme to produce specific IgG1 glycoforms.

[0349] Purification of chimeric or humanized E71 and E72. HuE71 and huE72 producer lines were cultured in Opticho serum-free medium (Invitrogen, Carlsbad, California) or PowerCHO-2 (Lonza, Basel, Switzerland), and mature supernatants were harvested. Protein A affinity columns were pre-balanced with 25mM sodium citrate buffer and 0.15M NaCl (pH 8.2). The bound huE71 or huE72 was eluted with 0.1M citric acid / sodium citrate buffer (pH 3.9) and alkalized in 25mM sodium citrate (pH 8.5) (1: 10 v / v ratio). It was passed through a Sartobind-Q membrane and concentrated to 5-10 mg / mL in 25mM sodium citrate, 0.15M NaCl (pH 8.2). 2 μg of each protein was analyzed by SDS-PAGE using a 4%-15% Tris-Glycine Ready-to-Use Gel System (Bio-Rad, Hercules, CA) under non-reducing or reducing conditions. Invitrogen SeeBlue Plus2 prestained standards (Invitrogen, Carlsbad, CA) were used as protein molecular weight markers. After electrophoresis, the gel was stained using GelCode blue staining reagent (Pierce Biotechnology, Waltham, MA). The gel was scanned using a Bio-Rad Fluor-S MultiImager (Bio-Rad, Hercules, CA) and band intensities were quantified using Quantity One software (Bio-Rad, Hercules, CA).

[0350] According to the in vitro binding kinetics of Biacore T-100 biosensor (GE Healthcare, Uppsala, Sweden). CM5 sensor chips (research grade) and related reagents were purchased from Biacore USA (Piscataway, New Jersey). According to the manufacturer's instructions, the antigen L1-CAM-Fc or L1-CAM-His was directly immobilized on the CM5 sensor chip. Before analysis, the purified monoclonal antibodies (MoAb) (mouse chimeric IgG1 and IgG4 of E71 and E72 and humanized IgG1 and IgG4 forms, and their IgG1n glycoforms) were diluted in HBS-EP buffer at different concentrations (41.7-666.7nM). The sample was injected onto the sensor surface at a flow rate of 30μl / min within 1min. The association time was set to 1min and the dissociation time was set to 5min to 15min. At the end of each cycle, the surface was regenerated using 50mM NaOH at a flow rate of 50μl / min within 1min. The data were analyzed using the Biacore T-100 evaluation software with a bivalent analyte model and default parameter settings for rate constants, and the apparent association rate constants (k on ), dissociation rate constant (k off ) and equilibrium dissociation constant (K d =k off / k on ).

[0351] 51 Chromium release assay. For ADCC analysis, effector cells were peripheral blood mononuclear cells (PBMCs) from healthy donors. The effector cell:target cell (E:T) ratio was 50:1. For T cell cytotoxicity assay, effector T cells were cultured in vitro for approximately 14 days in the presence of anti-CD3 and anti-CD28 and used at an E:T ratio of 10:1. All target tumor cells were harvested with 2 mM EDTA in PBS and 51 Cr (Amersham, Arlington Heights, IL) was added at 100 μCi / 10 6 5000 target cells were added to each well of a 96-well polystyrene round-bottom plate (BD Biosciences, Bedford, MA) along with the antibodies to a final volume of 250 μl / well. 10 units / ml of IL2 were added for ADCC assay. The plates were incubated at 37°C for 4 hours. The released IgG in the supernatant was counted in a gamma counter (Packed Instrument, Denison, IL). 51The percentage of specific release was calculated using the following formula: (experimental cpm-background cpm) / (total cpm-background cpm) × 100%, where cpm represents cpm released per minute. 51 Cr was counted. Total release was assessed by lysis with 10% SDS (Sigma, St. Louis, MO), and background release was measured in the absence of effector cells. EC50 was calculated using Sigmaplot or Graphpad Prism software.

[0352] Biodistribution of MoAb in xenograft mice. Female athymic nude mice were purchased from Harlan SpragueDawley, Inc (Indianapolis, IN). All procedures were performed in accordance with protocols approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee and institutional guidelines for the appropriate and humane use of animals in research. Tumor cells were harvested and resuspended in Matrigel (BD Biosciences, Bedford, MA). Cells (2-10 × 10 6 ) were implanted subcutaneously (sc) into the flank of mice in a volume of 0.1 ml. Tumors were allowed to grow to approximately 200 mm before treatment was initiated. 3 The size of the tumor. Mice with established tumors were randomly divided into treatment groups. Each mouse was injected intravenously with 100 μCi of radioiodinated antibody, and the animals were usually killed at 48 hours, their organs were removed, and counted in a gamma counter (Packard Instruments, Perkin Elmer, Waltham, Massachusetts). These organs include skin, liver, spleen, kidney, adrenal gland, stomach, small intestine, large intestine, bladder, femur, muscle, tumor, heart, lung, spine and brain. Based on the μCi accumulated in the organ and the organ weight, the injected dose % (%ID) / gram mouse was calculated. The %ID / g ratio of tumor to non-tumor was also calculated.

[0353] huE71-BsAb design, production and purification analysis. The huE71-BsAb format was designed using a 15 amino acid linker (G 4 S) 3(SEQ ID NO:77) Fusion of huOKT3 scFv with the C-terminus of the light chain of huE71-IgG1 (huE71-huOKT3). N297A and K322A mutations were introduced in the IgG1 Fc region. The nucleotide sequence was synthesized by GenScript (Piscataway, NY) with appropriate flanking restriction sites and subcloned into a standard mammalian expression vector. The linearized plasmid DNA was used to transfect CHO-S cells (Invitrogen, Carlsbad, CA) for stable production of BsAb. 2×10 6 Cells were transfected with 5 μg of plasmid DNA by nucleofection (Lonza, Basel, Switzerland) and then recovered for 2 days in CD OptiCHO medium supplemented with 8 mM L-glutamine (Invitrogen, Carlsbad, CA) in 6-well culture plates at 37°C. Stable pools were selected for approximately two weeks with 500 μg / mL hygromycin, and individual clones were selected by limiting dilution. huE71-BsAb titers were determined by L1-CAM (+) tumor cell and CD3 (+) Jurkat cell ELISA, respectively, and the highest expressing stable clones were selected. BsAb producer lines were cultured in PowerCHO-2 medium and mature supernatants were harvested. Protein A affinity columns (GE Healthcare, Chicago, IL) were pre-equilibrated with 25 mM sodium citrate buffer and 0.15 M NaCl (pH 8.2). The bound BsAb was eluted with 0.1M citric acid / sodium citrate buffer (pH 3.9) and neutralized with 25mM sodium citrate (pH 8.5, 1: 10v / v ratio). For storage, the BsAb was dialyzed into 25mM sodium citrate, 0.15M NaCl (pH 8.2) and frozen at -80°C in aliquots. Two micrograms of protein were analyzed by SDS-PAGE under reducing conditions using a 4%-15% Tris-glycine ready-to-use gel system (Bio-Rad, Hercules, California). The purity of huE71-BsAb was also evaluated by size exclusion high performance liquid chromatography (SE-HPLC). About 20 μg of protein was injected into a TSK-GEL G3000SWXL 7.8mm×30cm, 5μm column (TOSOH Bioscience, Tokyo, Japan), in which 0.4M NaCl was used. 4 , 0.05M NaH 2 PO 4 (pH 6.0) buffer with a flow rate of 0.5 mL / min and UV detection at 280 nm. Ten microliters of MW markers of gel filtration standards (Bio-Rad, Hercules, CA) were analyzed in parallel.

[0354] FACS analysis. Cells were incubated with different concentrations of primary antibodies (chimeric or humanized huE71 and huE72 and their IgG4 subclass and IgG1n variants, as well as BsAb formats) in PBS at 4°C for 30 minutes, and a phycoerythrin-labeled secondary antibody specific for human Fc was used after washing away excess primary antibodies. Cells were fixed with 1% paraformaldehyde (PFA) before analysis on a FACS Calibur cytometer (BD Biosciences, Bedford, MA). Controls were cells incubated with rituximab or palivizumab, with a mean fluorescence intensity (MFI) set to 5.

[0355] For quantification, the Quantum Simply Cellular anti-mouse IgG kit (Bons Laboratory, Fisher, Indiana) was used. In brief, the kit contains five microsphere populations; one blank, four labeled with increasing amounts of anti-mouse IgG. The beads and tumor cells (37°C for 5 hours) were then labeled on ice with the same fluorescently conjugated mouse E71 for 30 minutes. The cells were then washed with PBS and analyzed on a BD FACS Calibur (BD Biosciences, Bedford, Massachusetts) along with the labeled beads. The Excel-based QuickCal analysis template provided with each kit helps to correlate fluorescence intensity with antigen density on tumor cells. Each data point represents the average of duplicates.

[0356] In vivo anti-tumor assay. For in vivo studies, BALB-Rag2-KO-IL-2R-γc-KO (DKO) mice (derived from Dr. Mamoru Ito, CIEA, Kawasaki, Japan) were used. See Koo GC et al., Expert Rev Vaccines 8:113-20 (2009). Tumor cells were subcutaneously implanted with a 1:1 mixture of fresh culture medium / BD Matrigel (BD Biosciences, Bedford, Massachusetts). For tumor volume measurement, all tumors were measured using a handheld TM900 scanner (Peira, Brussels, Belgium).

[0357] General Laboratory Procedures for Radiolabeled Antibody Studies Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO) and used as received, and all instruments were calibrated and maintained according to standard procedures. 89 Zr was generated at the Memorial Sloan Kettering Cancer Center on a TR19 / 9 cyclotron (Ebco Industries Inc., Richmond, British Columbia, Canada). 89 Y(p,n)89 Zr was produced by reaction and purified to produce a specific activity of 196-496 MBq / mg 89 Zr. Use CRC-15R dose calibrator (Capintec, Inc., Florham Park, New Jersey) to carry out activity measurement. In order to quantify activity, samples are counted on an automatic Wizard gamma counter (PerkinElmer, Waltham, Massachusetts). The radioactive labeling of the ligand is monitored using instant thin layer chromatography paper (Agilent Technologies, Santa Clara, California) and analyzed on a Bioscan AR-2000 radioactive ITLC plate reader using Winscan Radio-TLC software (Bioscan Inc., Washington, D.C.). All in vivo experiments are carried out according to the protocol (Protocol 08-07-013) approved by the Memorial Sloan Kettering Animal Protection and Use Committee.

[0358] Cell culture. T-75 flasks (Corning, Corning, NY) containing cells were maintained at 37°C and 5% CO 2 The SKOV3 human epithelial ovarian cancer cell line (ATCC, Manassas, VA) was cultured in RPMI McCoy's 5A medium (Thermo Fisher Scientific, Waltham, MA) modified to contain 1.5 mM L-glutamine, 100 units / mL penicillin G and 100 μg / mL streptomycin, and 10% fetal bovine serum. The SKOV3 cell line was subcultured once a week using 0.25% trypsin / 0.53 mM EDTA in Hank's buffered salt solution without calcium and magnesium and passaged 1:5 for standard cell line passaging.

[0359] Xenograft Mouse Model. Eight to ten-week-old athymic nu / nu female mice were purchased from Charles River Laboratories (Kingston, NY). Animals were housed in ventilated cages with food and water ad libitum and allowed to acclimate for approximately 1 week prior to inoculation. 5.0 × 10 cells / mL of 150 μL cell suspension in a 1:1 mixture of fresh culture medium / BD Matrigel (BD Biosciences, Bedford, MA) were injected subcutaneously. 6SKOV-3 tumors were induced on the right shoulder with 100 cells. Experiments were performed approximately 2-3 weeks after cancer cell injection. For tumor volume measurement, all tumors were measured using a handheld TM900 scanner (Peira, Brussels, Belgium). After the initial tumor measurement, mice were randomized into groups (n=8-9 per group) to ensure that all groups had approximately equal mean tumor volumes at the start.

[0360] Antibody bioconjugation. Antibodies were obtained in citrate buffer (25 mM sodium citrate, 150 mM sodium chloride) at an average concentration of 4-5 mg / mL. Antibodies were concentrated to a final concentration of 12-15 mg / mL using a centrifugal filter unit with a molecular weight cutoff of 50,000 (Amicon Ultra 4 Centrifugal Filter Unit, Millipore Corp., Billerica, MA). After concentration, the antibodies were washed with 0.1 M Na 2 CO 3 The pH of the antibody was adjusted to 8.5-9.0, and then 10 equivalents of p-SCN-Bn-DFO or p-SCN-Bn-DOTA (Macrocyclics, Inc., Dallas, Texas) dissolved in DMSO were added. The reaction was incubated at 37°C for 1 hour, shaking continuously at 500 rpm. The antibody was purified using a centrifugal filter unit with a molecular weight cutoff of 50,000 (Amicon Ultra 4 centrifugal filter unit, Millipore Corp., Billerica, Massachusetts) to purify the ligand antibody conjugate. The final bioconjugate was aliquoted and stored in PBS (pH 7.4) at -80°C.

[0361] Radiolabeled. After target treatment, the 89 Zr-oxalate 89 The solution was neutralized with 1.0 M sodium carbonate to a pH of about 7. The DFO antibody was mixed with the labeled neutralized 89 Zr was incubated at 37°C for 60 min. 177LuCl (specific activity: 170MBq / mg, Perkin Elmer, Waltham, Massachusetts), and diluted in acetic acid ammonium buffer (200mmol / L, pH 5.4), and incubated with DOTA antibody at 42°C for 1 hour. Via radioactive ITLC, silica gel-impregnated glass microfiber paper strips (ITLC-SG, Varian, Forest Lake, California) (analyzed by the AR-2000 of Bioscan Inc. in Washington, D.C.), 50mM EDTA of pH 5 was used as mobile phase to monitor the progress of the reaction. The antibody complex was retained at the starting point, while the free radionuclides were absorbed by the EDTA in the mobile phase and migrated along the solvent front. The crude radiochemical yield was calculated using radioactive ITLC data. The radiolabeled antibody was subsequently purified using size exclusion chromatography (PD10), and the final volume was then concentrated using centrifugal filtration for preparation. Before use, the radiochemical purity of the final purified radiolabeled antibody was confirmed to be >99% by radioactive ITLC.

[0362] Serum stability. 89 Aliquots of the Zr-antibody complex (100 μL) were incubated with 900 μL of human serum and stirred constantly on a thermomixer at 37°C. Samples were taken from each microcentrifuge tube in triplicate on days 0, 1, 3, 5, and 7 and analyzed using radio-ITLC. The stability of the complex was calculated as the number of cells remaining at the starting point of the radio-ITLC strip. 89 The percentage of Zr is reported as % complete.

[0363] Immunoreactivity was determined using the Lindmo cell binding assay 89 The immunoreactivity score of Zr-DFO-antibody was determined (Ben QW et al., Ann Surg Oncol 17:2213-21 (2010)). For this purpose, SKOV3 cells were cultured at 5.0×10 5 -5.0×10 6 Cells were suspended in a concentration range of 10 cells / mL in 500 μL PBS, 1% BSA (pH 7.4) in a microcentrifuge tube. 89Aliquots of Zr-DFO-antibody (50 μL of 1 μCi / mL stock solution) were added to each test tube to a final volume of 500 μL. The samples were incubated on a thermal mixer at 37°C for 60 min. The treated cells were then precipitated via centrifugation (1400 RPM for 4 min), the supernatant was aspirated, and washed three times with cold PBS, then the supernatant was removed and the radioactivity associated with the cells was counted. The activity data were background corrected and compared with the total number of counts in the appropriate control samples. Immunoreactivity scores were determined by linear regression analysis of the graph of (total / bound) activity versus (1 / [normalized cell concentration]).

[0364] PET imaging. PET imaging experiments were performed on an Inveon PET / CT scanner (Siemens Healthcare Global, Malvern, PA). Female athymic nude mice bearing SKOV3 xenografts on the right shoulder were given 89 Zr-antibody (192-214 μCi in 150 μL PBS). Animals were anesthetized by inhalation of a 2% isoflurane (Baxter Healthcare, Deerfield, IL) and medical air gas mixture and placed in the scanner and maintained anesthesia using a 2% isoflurane and medical air gas mixture. PET data for each mouse were recorded via static scans at 24, 48, 72, and 96 hours after injection. The energy window used was 350-700 keV, and the coincidence timing window was 6 ns. The data were classified into 2D histograms by Fourier binning, and the transverse images were reconstructed into a 128×128×63 (0.72×0.72×1.3 mm3) matrix by filtered back projection (FBP). The count rates in the reconstructed images were converted to activity concentrations (percentage of injected dose per gram of tissue, %ID / g) by using a system calibration factor derived from the analysis of the PET images containing 89 Imaging of a mouse-sized water-equivalent phantom of Zr. Images were analyzed using ASIPro VM software (Concorde Microsystems, Knoxville, TN).

[0365] Biodistribution. Use 89 Biodistribution studies of Zr antibody were performed in female athymic nude mice bearing SKOV3 tumors. Animals were given 150 μL of PBS via intravenous tail vein injection. 89 Each of the Zr-antibodies (17-26 μCi). 96 hours after injection, CO 2Animals were euthanized by asphyxiation (n=4 per group). After euthanasia, 17 organs (blood, tumor, heart, lung, liver, spleen, stomach, large intestine, small intestine, pancreas, ovary, kidney, bone, muscle, lymph, skin, tail) were collected, weighed, and analyzed in 89 Radioactivity is measured on a Zr-calibrated γ counter. Counts are converted to activity using a calibration curve generated from known standards. Count data is background, and decay correction is performed for the time of injection, and the injected dose percentage (% ID / g) per gram of each tissue sample is calculated by normalizing the total activity injected. The data described in this study are expressed as mean ± standard deviation. GraphPad Prism 7 software is used to analyze statistical differences by unpaired two-tailed Student's t-test. P value <0.05 is considered statistically significant and indicated with an asterisk.

[0366] Example 2: Structure of humanized anti-L1-CAM antibodies of the present technology

[0367] Humanized E71 and E72 anti-L1-CAM antibodies were generated. Sequence design was based on human IgG homology calculations while retaining key mouse amino acid residues. Figure 6 and Figure 7 The V H and V L (expressed as SEQ ID NOs: 1 and 3) and V of mouse E72 H and V L (Shown as SEQ ID NO: 5 and 7). Figure 6 ) and E72 (see Figure 7 The CDRs of the heavy and light chains of E71 were grafted onto the human IgG1 framework based on their homology to the human framework. For E71, the homologous human sequence used for the heavy chain was IGHV7-4-1 (SEQ ID NO: 2), and the homologous human sequence used for the light chain was IGKV-58 (SEQ ID NO: 4) ( Figure 6 ). For E72, the homologous human sequences used for the heavy and light chains were IGHV1-2*02|66.3|IGHJ4*01|85.7 (SEQ ID NO: 6) and IGKV1-NL1*01|73.7|IGKJ2*02|81.8 (SEQ ID NO: 8), respectively ( Figure 7 ).

[0368] Two different heavy chain sequences (H1 and H2 for huE71; see for the corresponding amino acid sequences Figure 8 , and for cDNA sequences see Fig. 9 ) and two different light chain sequences (L1 and L2; for the corresponding amino acids see Fig.10, and for cDNA sequences see Fig.11 ) was expressed as intact IgG of huE71 and tested for binding and stability. Two different heavy chain sequences (H1 and H2 for huE72; see for the corresponding amino acid sequences Fig.12 , and for cDNA sequences see Fig.13 ) and two different light chain sequences (L1 and L2; for the corresponding amino acid sequences see Fig.14 , and for cDNA sequences see Fig.15 ) were expressed as intact IgG to huE72 and tested for binding and stability.

[0369] E71 was also prepared (for the corresponding amino acid sequence see Fig.16 , and for cDNA sequences see Fig.17 ) and E72 (for the corresponding amino acid sequences see Fig.18 , and for cDNA sequences see Fig.19 ) chimeric forms for comparison. For the final forms of huE71 and huE72, the most stable combination was selected: H1 and L1 of E71 (respectively Fig. 20 and Fig.21 ) and H1 and L2 of E72 (respectively Fig. 22 and Fig.23 ) was used for the remaining experiments. Additional constructs were prepared replacing the heavy chain sequence of chE71 with a human IgG4 framework ( Fig.24 The amino acid sequence and Fig.25 ), and the heavy chain sequence of huE71 was replaced with the human IgG4 framework (respectively Fig.26 The amino acids and Fig. 27 cDNA of ). HuE71 was packaged into a single vector (for balanced heavy and light chain secretion) that was transduced into DG44 cells. HuE71-IgG1n is another huE71-IgG1 glycoform expressed in CHO cells with variant glycosylation caused by GnT1 deficiency (Jefferis R, Nat Rev Drug Discov 8:226-34 (2009)). HuE71-IgG1, huE71-IgG4, and huE71-IgG1n were purified using standard protein A affinity chromatography. Sugar analysis confirmed that huE71-IgG1n had 71.1% (Mol%) mannose, 26.3% (Mol%) N-acetylglucosamine, and 2.6% (Mol%) glucose. On SDS gels, huE71 and huE72 migrated as IgGs with heavy and light chains of appropriate size; and they were all eluted as intact IgGs by HPLC, with <5% aggregates formed.

[0370] Example 3: Antigen binding of HuE71 and HuE72 and their derivatives

[0371] Antigens (L1-CAM-Fc and L1-CAM-His) were immobilized on a CM5 chip and the kinetics of antibody binding (k) were compared by surface plasmon resonance (SPR) using Biacore T-100. on , k off and K d ) (For L1-CAM-Fc see Fig.28 The binding kinetics of chimeric and humanized antibodies on L1-CAM-Fc compared with other L1-CAM mouse antibodies are summarized in Fig.29 and Fig.30 The binding kinetics of chimeric and humanized antibodies on L1-CAM-His are summarized in Fig.31 middle.

[0372] Antigen binding was also analyzed by FACS analysis using L1-CAM(+) tumor cells. Data are expressed as mean fluorescence intensity (MFI) determined by flow cytometry and normalized to the highest antibody concentration used (1 μg / 10 6 cells):Mesothelioma ( Fig.32 ), leukemia, breast cancer, and the Ewing family of tumors ( Fig.33 ) and melanoma, neuroblastoma, osteosarcoma, rhabdomyosarcoma, small cell lung cancer ( Fig.34 )'s binding percentage.

[0373] HuE71-IgG1 is stable, with an EC50 of 0.03 μg / million cells for antigen binding after five freeze and thaw cycles. An ELISA method using tumor cells coated on plastic plates as antigens was also used to determine huE71-IgG1 and huE71-IgG1n binding; both huE71-IgG1 and huE72-IgG1 showed comparable binding to tumor cells. Binding of huE71-IgG1n to tumor cells was unchanged after five repeated freeze and thaw cycles.

[0374] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically bind to L1-CAM with high binding affinity. Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect L1-CAM protein in a sample.

[0375] Example 4: ADCC mediated by HuE71-IgG1n and HuE72-IgG1n

[0376] ADCC (antibody-dependent cell-mediated cytotoxicity) was evaluated using PBMC as effectors in normal volunteers. In contrast to the specific glycoforms, chE71, huE71, chE72 and all wild-type human IgG1 forms of huE72 were unable to mediate ADCC. Two target cell lines, LAN-1 and NB1691, were tested. Only huE71-IgG1n was able to mediate ADCC (EC50 of 0.06 μg / ml for LAN-1 cells and 0.1 μg / ml for NB1691 cells). See Fig.61 and Fig.62 .

[0377] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology can induce ADCC. Therefore, the immunoglobulin-related compositions disclosed herein can be used to treat L1-CAM positive cancers.

[0378] Example 5: Design of HuE71-BsAb and HuE72-BsAb and expression in CHO-S cells

[0379] Using the IgG-scFv format ( Fig.35 )and Fig.36 BsAbs were designed based on the amino acid sequences shown in . For the huE71-huOKT3 format, the heavy chain was identical to that of huE71 IgG1, and the light chain was synthesized by using a C-terminal (G 4 S) 3 The huE71 light chain was extended with a linker (SEQ ID NO: 77) followed by a huOKT3 scFv. The design of HuE72-huOKT3 is similar. For both antibodies, N297A and K322A mutations were introduced into the hIgG1 Fc region to remove glycosylation and complement activation. The DNA encoding the heavy and light chains of the BsAb was inserted into a mammalian expression vector, transfected into CHO-S cells, and stable clones showing the highest expression were selected. The supernatant was collected from the shake flask and purified on protein A affinity chromatography. The protein was further purified to >90% monomer by size exclusion chromatography.

[0380] Under reducing SDS-PAGE conditions, both BsAbs produced two bands at about 50 KDa due to the increase in MW to about 50 KDa from the fusion of huOKT3 scFv with the light chain. SEC-HPLC showed a major peak with a MW of about 210 KDa for both antibodies (97% by UV analysis) and a minor peak of multimers that could be removed by gel filtration. When measured by SPR on L1-CAM-Fc, the affinity of the BsAbs for L1-CAM was comparable to that of the parental IgG for L1-CAM ( Fig.37 ).

[0381] These results demonstrate that the antibodies or antigen-binding fragments thereof of the present technology specifically bind to L1-CAM with high binding affinity. Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect L1-CAM protein in a sample.

[0382] Example 6: Binding of anti-L1-CAM BsAb of the present technology to both tumor cells and T cells

[0383] Fig.28 and Fig.37 The binding kinetics of the two BsAbs on L1-CAM-Fc measured by SPR are shown. The binding of huE71-BsAb and huE72-BsAb to both target and effector cells was tested by FACS immunostaining. huE71-BsAb was as efficient as the parent huE71 in binding to L1-CAM(+) neuroblastoma BE(2)C, while the binding of huE72-BsAb to BE(2)C was half of the binding observed in the case of huE71-BsAb (Figure 38(A)). For binding to CD3(+) T cells, both huE71-BsAb and huE72-BsAb were less efficient than huOKT3 IgG1; about 20 times weaker than huOKT3 (Figure 38(B)). The reduced affinity of the light chain anchored scFv of the BsAb for T cells is partly due to the spatial restriction of this format, but is beneficial for minimizing cytokine release, especially when target cells are not present.

[0384] These data demonstrate that binding to CD3 is functionally monovalent, thereby precluding spontaneous activation of T cells in the absence of tumor targets.

[0385] Example 7: Th1 cytokine release induced by HuE71-BsAb or HuE72-BsAb

[0386] After 24 hours of activation with the cell line IMR32-luciferase, both huE71-BsAb and huE72-BsAb induced the release of Th1 cytokines (such as TNFα) from PBMCs. IFNγ and IL2 levels were relatively low. Both huE71-BsAb and huE72-BsAb induced the release of Th2 cytokines, IL6 and IL10. However, the levels of these cytokines were lower than those induced by the control hu3F8-BsAb ( Figure 39(A)-Figure 39(E) ).

[0387] Example 8: HuE71-BsAb and HuE72-BsAb redirected T cell killing of human tumor cell lines

[0388] In the standard 4 h with activated T cells (E:T ratio = 10:1) 51HuE71-BsAb or huE72-BsAb-mediated T cell cytotoxicity against L1-CAM positive cancer cell lines was tested in a Cr release assay. Both BsAbs induced T cell cytotoxicity against tumor cells, and huE72-BsAb was more effective than huE71-BsAb (EC50 of 50.0 pM and 62.9 pM, respectively, in the case of IMR32-luciferase cell line) ( Fig.40 When an expanded panel of human tumor cell lines (including neuroblastoma, melanoma, ovarian cancer, breast cancer, cervical adenocarcinoma) was tested, huE71-BsAb or huE72-BsAb-mediated T cell cytotoxicity correlated with tumor L1-CAM expression assessed by FACS ( Fig.41 and Figure 42(A)-Figure 42(B) ).

[0389] Example 9: Efficacy of HuE71-BsAb and HuE72-BsAb on xenografts in humanized mice

[0390] For in vivo therapy studies, BALB-Rag2-KO-IL-2R-γc-KO (DKO) mice were used (Koo GC et al., Expert Rev Vaccines 8: 113-20 (2009); Andrade D et al., Arthritis Rheum 63: 2764-73 (2011)). In separate humanized mouse xenograft models (subcutaneous (sc) tumor plus subcutaneous effector cells, subcutaneous tumor plus intravenous (iv) effector cells), intravenous L1-CAM-BsAb showed high activity against established tumors in the presence of PBMCs from healthy donors.

[0391] For in vivo studies, L1-CAM(+) neuroblastoma patient-derived xenografts (PDX) ( Fig.43 ) were transplanted subcutaneously into DKO mice. HuE71-BsAb or huE72-BsAb treatment was initiated on day 15 when tumors were measurable, using a dosing schedule of twice weekly injections for five weeks. PBMCs (7.5×10 6 ), and then injected once a week for 3 weeks. The size of the tumor was measured once a week. The tumor growth of mice treated with huE72-BsAb and PBMC was equal to that of the control. Therefore, although huE72-BsAb has high binding affinity and excellent in vitro tumor killing efficacy, its anti-tumor response in vivo is still not obvious. In contrast, HuE71-BsAb together with PBMC significantly inhibited tumor growth.

[0392] exist Fig.44In this study, IMR32 neuroblastoma cell line was used instead of PDX and mixed with an equal number of PBMCs and implanted subcutaneously. HuE71-BsAb or huE71-IgGn significantly inhibited tumor growth, while huE72-BsAb had no effect on tumor volume.

[0393] Example 10: Use 89 Zr-huE71-IgG1n and 89 Tumor imaging of Zr-huE72-IgG1n

[0394] use 89 Zr-huE71-IgG1n and 89 Zr-huE72-IgG1n was used to image nude mice implanted subcutaneously with SKOV3 human ovarian xenografts (see Fig.45 and Fig.46 Although huE71 has a lower affinity than huE72, 89 Zr-huE71-IgG1n had higher in vivo targeting efficiency.

[0395] Example 11: L1-CAM Antibody Fc Variants

[0396] The following study evaluated the effects of modifications in the Fc region of humanized antibodies on their in vivo biodistribution. Human IgG antibodies have four subclasses (IgG1, IgG2, IgG3, and IgG4), which can be broken down into further subtypes. In addition, there are several Fc receptors that bind to IgG antibodies, namely the Fcγ receptors.

[0397] Fcγ receptors are further subdivided into categories that are differentially expressed on immune cells, with Fcγ receptor I having the highest affinity for IgG antibodies (Nimmerjahn F, Ravetch JV. Nat Rev Immunol 8:34-47 (2008)). IgG1 antibodies are most abundant in humans and have the highest affinity to activate Fcγ receptors (Bruhns P et al., Blood 113:3716-25 (2009)). In addition, the sugar moiety or glycosylation state of IgG plays an important role in binding to Fcγ receptors. Specifically, for IgG1 antibodies, the sugar moiety attached to the N297 residue on the constant region interacts with the antibody and changes the Fc binding characteristics of the antibody (Sazinsky SL et al., Proc Natl Acad Sci USA 105:20167-72 (2008)). The complex sugar moieties attached to this residue include mannose and N-acetylglucosamine (GlcNAc) as well as other branched sugars including fucose, galactose and sialic acid, and changes in this glycosylation site can greatly affect binding (Maverakis E et al., J Autoimmun 57: 1-13 (2015); Arnold JN et al., Annu Rev Immunol 25: 21-50 (2007)). IgG antibodies with all these sugars missing show a significant reduction in Fcγ receptor binding without affecting their FcR (n) affinity. On the other hand, removing fucose residues from glycosylation sites greatly improves the affinity of IgG for Fcγ receptors (Maverakis E et al., J Autoimmun 57: 1-13 (2015)). Although IgG1 antibodies have the highest affinity for Fcγ receptors, IgG4 antibodies, which represent the lowest abundance of IgG in humans, have a significantly lower affinity for Fcγ receptors and are generally considered to be an anti-inflammatory IgG that inhibits immune responses as a regulatory mechanism. Without wishing to be bound by theory, it is believed that this phenomenon is due in part to an intrinsic property of IgG4 antibodies known as dynamic Fab arm exchange (Van der Neut Kolfschoten M et al., Science 317: 1554-7 (2007)).

[0398] All subclasses of IgG antibodies are composed of two heavy-light chain pairs, which are bound together by disulfide bonds formed by cysteine ​​residues in the antibody hinge region on the heavy chain. Depending on the subclass of IgG, the length of this hinge region and the number of disulfide bonds are variable. These features affect the flexibility of the hinge region and play a role in binding affinity to Fcγ receptors (Rispens T et al., J Biol Chem 289: 6098-109 (2014)). In the case of IgG4 antibodies, two half molecules (heavy-light chain pairs) can dissociate from each other in the hinge region and spontaneously reorganize with other half molecules, thereby forming bispecific monovalent antibodies in vitro and in vivo (Rispens T et al., J Biol Chem 289: 6098-109 (2014); Vidarsson G et al., Front Immunol 5: 520 (2014)). This dynamic Fab arm exchange property can be disabled in IgG4 antibodies by introducing a serine to proline mutation (S228P) in the antibody hinge region (Silva JP et al., J Biol Chem 290:5462-9 (2015)). The experiments conducted in this study utilized humanized antibodies in mice expressing mouse Fcγ receptors. Although the homology of Fcγ receptors in humans and mice is only 60%-70%, recent studies have shown that human IgG binds to orthologous mouse Fcγ receptors with very similar affinity, indicating similar biological activity (Overdijk MB et al., J Immunol 189:3430-8 (2012)).

[0399] L1-CAM Antibody Modifications. To investigate the effect of the Fc region on the in vivo biodistribution of radioimmunoconjugates, a panel of humanized IgG1 and IgG4 antibodies were developed that all have exactly the same antigen binding site but have modifications in the Fc region that alter their affinity for Fcγ receptors ( Fig.47). HuE71-1 MAGE is an IgG1 antibody against L1-CAM with glycans lacking α-1,6-fucose, which is produced in CHO cells that lack the enzyme UDP-N-acetylglucosamine:α-3-d-mannoside-β-1,2-N-acetylglucosamine transferase I (GnT1), thereby enhancing the Fc region (Xu H et al., Cancer Immunol Res 4:631-8 (2016)). HuE71-1 WT is a wild-type IgG1 antibody against L1-CAM produced in wild-type CHO cells, while deglycosylated HuE71-1 is a deglycosylated variant that contains an N297A mutation that reduces its Fcγ receptor binding. HuE71-4 WT is an IgG4 variant of this antibody, while the HuE71-4 mutant contains an S228P mutation that prevents dynamic Fab arm exchange from occurring. Finally, HuCtrl-4 is an IgG4 wild-type antibody targeting GD2, a nonspecific antigen in the animal model used in this study.

[0400] Conjugation and radiolabeling. To conjugate the L1-CAM antibodies disclosed herein with a functionalized deferoxamine isothiocyanate chelator (p-SCN-Bn-DFO), a concentrated solution of each antibody dissolved in PBS was adjusted to pH 8.5-9 and incubated with the chelator dissolved in DMSO at a 10:1 molar excess at 37°C for 1 hour. Following spin filter purification of the antibodies (Amicon Ultra 50 kDa), aliquots were frozen and samples of the starting material (unconjugated antibody) and conjugated antibody were evaluated using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-ToF MS) to determine the number of chelates for each antibody. Because a nonspecific conjugation method was used, it is possible that the DFO moiety formed multiple thiourea bonds on lysine residues randomly distributed on the antibody. MALDI-ToF MS analysis of the antibodies before and after conjugation revealed that each 89 Zr-HuE71-1 MAGE has 2.38 chelates, each 89 Zr-HuE71-1 WT has 1.41 chelates, each 89 Zr-deglycosylated HuE71-1 has 1.07 chelates, each 89 Zr-HuE71-4 WT has 1.30 chelates, each 89 The Zr-HuE71-4 mutant had 0.63 chelates and each 89 Zr-HuCtrl-4 WT has 1.12 chelates. These values ​​represent the average number of chelates per antibody molecule. 89The value of 1.41 DFO chelate molecules for Zr-HuE71-1 WT indicated that 1 or 2 chelators were bound per antibody molecule. Each antibody was then incubated in PBS at 37°C with 89 Zr radiolabeling was performed for 60 min.

[0401] Radiochemical yield and specific activity were calculated using instant thin layer chromatography (ITLC) and EDTA mobile phase (pH 5.0, 50 mM), where free 89 Zr migrates with the solvent front, while the chelated 89 Zr remained at baseline. After size exclusion chromatography purification (PD-10 column), the radiochemical yield of all antibodies was greater than 95%, and the radiochemical purity of all antibodies was greater than 99%. After radiolabeling, all antibodies (excluding 89 Zr-HuE71-4 mutant) had a specific activity between 6.18 and 6.89 μCi / μg. 89 Many optimization attempts were made for the Zr-HuE71-4 mutant, but specific activities above 2.14 μCi / μg were never achieved, which is consistent with the MALDI-ToF MS data, which suggests that 89 The Zr-HuE71-4 mutant had the lowest average number of chelates, 0.63.

[0402] In vitro characterization. To assess the radiochemical stability of the tracers, a serum stability challenge was performed. An aliquot (100uL) of each radioimmunoconjugate was incubated in human serum (900μL) at 37°C for seven days. Samples were collected at 0, 1, 3, 5, and 7 days and evaluated via radioactive instant thin layer chromatography to determine the percentage of activity still bound to the antibody. The study showed that all tracers remained stable for the full seven days, 89 The value of Zr-HuE71-1 MAGE was 97.3%±0.8%, 89 The value of Zr-HuE71-1 WT was 97.7%±0.7%, 89 The value of Zr-deglycosylated HuE71-1 was 96.6%±0.8%, 89 The value of Zr-HuE71-4 WT was 96.9%±0.3%, 89 The value for the Zr-HuE71-4 mutant was 97.5% ± 0.3% and 89 The value for Zr-HuCtrl-4 WT was 96.7% ± 1.1%. These values ​​were above 95% integrity within seven days, consistent with 89 The expected values ​​were consistent with those of the Zr-DFO radiolabeled antibody.

[0403] To determine the extent to which these antibodies were able to bind to their targets after radiolabeling, immunoreactivity assays were performed. 89 Zr-labeled antibodies were incubated with increasing concentrations of target-positive ovarian cancer cells (SKOV3), and after washing, cell-bound activity was counted and used to calculate immunoreactivity scores. These experiments yielded immunoreactivity greater than 85% for all antibodies, specifically, for 89 Zr-HuE71-1 MAGE was 89.5%±1.5%, 89 Zr-HuE71-1 WT was 93.1%±2.2%, 89 Zr-deglycosylated HuE71-1 was 85.8%±2.9%, 89 Zr-HuE71-4 WT was 86.7% ± 1.9% and for 89 The Zr-HuE71-4 mutant was 88.6% ± 2.8% ( Figure 48(A)-Figure 48(E) ). Since the immunoreactivities of all tracers were within the range of each other, it can be assumed that any observable differences in the imaging and biodistribution patterns of the radioimmunoconjugates were not due to differences in their binding abilities to the target antigen (L1-CAM).

[0404] Serial PET Imaging. Serial PET imaging studies were performed on female athymic nude mice bearing subcutaneous SKOV3 tumors on the right shoulder to determine how modifications to the Fc region of antibodies of the present technology would alter the biodistribution pattern of these radioimmunoconjugates. Radiolabeled immunoconjugates were injected via the tail vein and PET images were acquired at 4 time points (24, 48, 72, and 96 hours) post-injection. Presented in all imaging figures are coronal sections of the mouse through the tumor tissue (all ranging from 0 to 30% injected dose per gram) and maximum intensity projections (MIPs) representing whole-body images.

[0405] for 89 Zr-HuE71-1 MAGE( Fig.45 ), high radioactivity concentrations of the tracer were observed in tumor tissue 24 hours after injection (white arrows), with minimal radioactivity in non-targeted organs. Since no heart or major vessels were visible, the tracer was rapidly cleared from the blood pool. Interestingly, four bilateral structures in the upper part of the animal (grey arrows) also showed high radioactivity concentrations. These PET-positive tissues were harvested and identified as the submandibular and axillary lymph nodes. In addition, lymph nodes showed a significant decrease in radioactivity after injection of the radiotracer. 89 The absorption of Zr-HuE71-1 MAGE lasted for 96 hours. 89PET images of SKOV-3 xenografts of Zr-HuE71-1 WT revealed high tracer uptake within tumor tissue at 24 h, which continued to accumulate up to 96 h ( Fig.49 ). The liver had a high concentration of radioactivity, quite intense at 24 hours, and then gradually washed out at 96 hours. Again, the four upper lymph nodes of the mouse as well as the lower lymph nodes were illuminated by PET at 24 hours, but they showed slight disappearance at 96 hours.

[0406] exist 89 Zr-deglycosylated HuE71-1 images ( Fig.50 ), high radioactive concentrations of the tracer in tumor tissue were observed at 24 hours, which continued to accumulate for up to 96 hours. The blood pool activity of this tracer was quite high at 24 hours and was clearly visible in the heart, descending aorta, and left and right common carotid arteries, but this activity was rapidly cleared 72 hours after injection. According to PET imaging data of IgG1 radiolabeled antibodies, 89 Zr-deglycosylated HuE71-1 had the cleanest biodistribution profile, as the radioactivity concentration in tumor tissue was the highest from 24 to 96 h after injection, and the amount of non-target tissue activity was the lowest.

[0407] for 89 Zr-HuE71-4 WT( Fig.51 ), at 24 hours most of the activity was in the tumor tissue, and in the maximum intensity projection, blood pool activity could be seen very clearly. Two bilateral structures (grey arrows) could also be seen in the middle of the animal, and they have been confirmed to be kidneys. High activity in the tumor tissue as well as kidney activity persisted until 96 hours post-injection.

[0408] exist 89 Images of the Zr-HuE71-4 mutant ( Fig.52 ), slight tumor uptake was observed at 24 hours, with gradual accumulation continuing for up to 96 hours. Here, there was blood pool activity, which in this case showed a partial reduction over the full 96 hours. However, the most noteworthy 89 The Zr-HuE71-4 mutant has no renal uptake, which is 89 This was clearly observed in Zr-HuE71-4 WT.

[0409] The final tracer imaged in this study was a nonspecific control antibody 89 Zr-HuCtrl-4 WT( Fig.53). Activity within tumor tissue was very low at all time points, consistent with the fact that the antibody's target, GD2, is not highly expressed by the SKOV-3 cell line. Liver activity was seen at all time points, as well as robust kidney activity.

[0410] Except for control antibody ( 89 Zr-HuCtrl-4 WT) all other tracers have exactly the same antigen binding site targeting L1-CAM. The only difference between the five L1-CAM tracers is within the Fc region, which has a major impact on in vivo biodistribution. Based on PET images, the antibody variant with lower Fc receptor binding ( 89 Zr-deglycosylated HuE71-1, 89 Zr-HuE71-4 WT, 89 Zr-HuE71-4 mutant) showed no lymph node uptake. In contrast, antibodies with normal or enhanced Fcγ receptor binding ( 89 Zr-HuE71-1 WT, 89 Zr-HuE71-1 MAGE) had strong lymph node uptake. Taken together, the data suggest that local expression of antigen does not play a role in lymph node uptake because the antigen binding region was identical for all L1-CAM tracers. Furthermore, in the immunoreactivity assay, the L1-CAM radioimmunoconjugates produced immunoreactivity scores greater than 85%, which ruled out differences in antigen binding as a cause of differential lymph node uptake. Because normal and enhanced Fcγ receptor binding antibodies ( 89 Zr-HuE71-1MAGE and 89 Zr-HuE71-1 WT) exhibited low Fcγ receptor binding antibody ( 89 Zr-deglycosylated HuE71-1, 89 Zr-HuE71-4 WT, 89 Zr-HuE71-4 mutant), the observed high lymph node uptake was likely due to differences in Fc binding.

[0411] With respect to the IgG4 tracer, high renal uptake was observed in the wild-type variant. The S228P mutation in this tracer was able to reduce renal uptake to a level that it was not visible on PET at any time. As previously mentioned, IgG4 antibodies undergo dynamic Fab arm exchange and are able to spontaneously cleave at the hinge region (forming an approximately 75 kDa construct) and then reassemble with the other half molecules. Without wishing to be bound by theory, it is believed that the renal uptake observed in the wild-type IgG4 L1-CAM antibody may be a secondary effect of the dynamic Fab arm exchange property, as the S228P mutation prevents this renal accumulation from occurring. In addition, the 75 kDa half antibody may be further reduced into 25 and 50 kDa fragments, allowing them to pass through the glomeruli within the kidney. Nonspecific 89 Zr-HuCtrl-4 WT IgG4 antibody showed high accumulation in kidney tissue, which is consistent with the theory of IgG4 renal uptake.

[0412] In vitro biodistribution. To confirm the PET imaging results and verify the specificity of the tracer, an in vitro biodistribution study was performed ( Fig.54 and Fig.55 ). Radiolabeled immunoconjugates were injected into female athymic nude mice bearing SKOV3 tumors via the tail vein, and various tissues were collected 96 hours after injection for gamma count analysis and normalized to body weight. The results of these studies matched the trends revealed by the PET imaging experiments. Antibodies known to have normal or enhanced Fcγ receptor binding ( 89 Zr-HuE71-1 WT, 89 Zr-HuE71-1 MAGE) showed high lymph node uptake (16.85% ± 1.39% ID / g, 17.16% ± 0.41% ID / g). In contrast, the deglycosylated variants of the antibody 89 Zr-deglycosylated HuE71-1 showed a basal level of lymph node uptake of 5.59% ± 0.49% ID / g. The values ​​obtained by ex vivo biodistribution correlated with the PET imaging data and further confirmed the hypothesis that the increased lymph node uptake was due to binding of the Fc region of the antibody rather than the variable region.

[0413] For IgG4 antibodies, 89 Zr-HuE71-4 WT showed lymph node uptake of 3.98% ± 0.969% ID / g, while 89 Zr-HuE71-4 mutant was 3.70% ± 0.975% ID / g ( Fig.56 and Fig.57 ). No significant difference was found between the two antibodies in lymph nodes. 89Zr-HuE71-4 WT accumulated in kidney with a value of 12.06% ± 1.23% ID / g, while 89 The Zr-HuE71-4 mutant was significantly lower at 5.83% ± 0.537% ID / g. These data recapitulate what was observed in the PET imaging data and further suggest that the IgG4 nature of dynamic Fab arm exchange plays a role in renal uptake of the tracer.

[0414] To evaluate the specificity of these tracers for the target, a blocking group was included in the ex vivo biodistribution study. A 50-fold excess of cold tracer was co-injected with the radiolabeled tracer via the tail vein. Theoretically, the addition of cold tracer would significantly reduce the specific activity of the hot tracer and a significant reduction in the uptake of the hot tracer would be observed. This was observed for all five L1-CAM targeting antibodies. Tumor tissue uptake values ​​were 89 Zr-HuE71-1 MAGE decreased from 16.15%±0.85% ID / g to 5.87%±0.76% ID / g. 89 Zr-HuE71-1 WT decreased from 19.67%±1.23% ID / g to 6.82%±0.809% ID / g. 89 Zr-deglycosylated HuE71-1 decreased from 32.02%±2.99% ID / g to 10.58%±1.52% ID / g. 89 Zr-HuE71-4 WT decreased from 27.29% ± 1.55% ID / g to 10.65% ± 0.472% ID / g, and for 89 The Zr-HuE71-4 mutant decreased from 16.90% ± 1.21% ID / g to 9.04 ± 1.42% ID / g. 89 Zr-HuCtrl-4 WT, tumor uptake was very low, 6.02% ± 0.90% ID / g. This uptake in tumor tissue can be attributed to the enhanced permeability and retention (EPR) effect. See Heneweer C et al., J Nucl Med 52:625-33 (2011).

[0415] Ex vivo histology. Lymph nodes and tumors were harvested and analyzed histologically to determine whether the lymph node uptake observed in PET imaging studies with the IgG1 tracer was actually indicative of metastatic disease ( Fig.58). Hematoxylin and eosin staining of the tumor tissue confirmed that the mass was an undifferentiated carcinoma. The tumor tissue was described as a well-circumscribed, nonencapsulated, densely cellular nodular neoplastic mass composed of nests of neoplastic epithelial cells supported by tightly packed lobules and fine vascular septa. The neoplastic cells were polygonal to spindle-shaped with distinct cell borders, abundant eosinophilic cytoplasm that was occasionally vacuolated, and round nuclei with finely granular to cystic chromatin and 1–2 prominent magenta nucleoli. Severe anisocytosis and anisocytosis of the nuclei were present. An average of 4–6 mitoses were present per high-power field (40×). Multifocal areas with coagulative necrosis were present. Hematoxylin and eosin staining of the left and right axillary and mesenteric lymph nodes revealed histiocytes and plasmacytosis within the medullary sinuses, a common finding in drainage reactions, and no signs of neoplastic cells.

[0416] 177 Lu radioimmunotherapy study. As shown above, 89 Zr-deglycosylated HuE71-1 exhibited high tumor active concentration of 32.02% ± 2.99% ID / g at 96 h after injection and low background tissue uptake (especially kidney and blood). 89 Zr-deglycosylation of HuE71-1 using metal radionuclides 177 Lu labeling because of its radiochemical properties (half-life 6.7 days; mean range 0.2 mm; %β = 100), and 177 Lu is widely used in imaging and radioimmunotherapy applications. Deglycosylated HuE71-1 antibody was conjugated with DOTA and 3.94 mCi / mg 177 Lu specific activity was labeled (radiochemical purity >99%) and imaging studies and ex vivo biodistribution studies were performed in athymic nude mice bearing SKOV-3 subcutaneous tumors ( Figure 59(A)-Figure 59(C) These studies confirm that 89 A similar biodistribution profile was observed for Zr-deglycosylated HuE71-1, with the majority of activity localized to tumor tissue as seen in SPECT / CT images and Cerenkov images, and confirmed by ex vivo biodistribution 168 h after injection.

[0417] Will 177 The efficacy of Lu-deglycosylated HuE71-1 as a radioimmunotherapy agent was evaluated at 3 doses (800 μCi, 800 μCi divided doses (2 doses, staggered for 14 days), and 400 μCi) and compared with cold antibody, 800 μCi nonspecific antibody control, and saline (n=8-9 per group) in the same animal model ( Fig.60). After the mean tumor volume at day 80 (60 days after initial treatment), a significant differential response was observed between the control group and the radiolabeled treatment group. Notably, none of the control groups showed signs of tumor regression beyond the starting volume. 177 The Lu-deglycosylated HuE71-1 group showed a significant growth delay effect. However, at later time points studied, 177 There were no significant differences in tumor volume between the Lu-deglycosylated HuE71-1 groups, and the mean tumor volumes of these groups were equal within the error range at day 80. In addition, within the first 2 weeks after treatment, the highest treatment group (800 μCi, 177 Signs of toxicity (>80% weight loss and petechiae) were observed in 4 / 8 mice injected with 800 μCi of nonspecific control antibody, most likely due to differences in tracer clearance / elimination. These data suggest that a single dose of 400 μCi 177 Lu-deglycosylated HuE71-1 was sufficient to delay tumor growth in this model while avoiding toxicity issues.

[0418] In summary, these results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit the progression of tumor growth and / or metastasis. Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect and treat L1-CAM positive cancers in subjects in need thereof.

[0419] Equivalent

[0420] The technology of the present invention is not limited to the specific embodiments described in this application, and it is intended that the embodiments be used as a single description of the individual aspects of the technology of the present invention. As will be apparent to those skilled in the art, various modifications and changes can be made to the technology of the present invention without departing from the spirit and scope of the technology of the present invention. It will be clear to those skilled in the art from the foregoing description that, in addition to the methods and devices listed herein, functionally equivalent methods and devices within the scope of the technology of the present invention. The modifications and changes are intended to fall within the scope of the technology of the present invention. It should be understood that the technology of the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, but can certainly be changed. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments, and are not intended to be restrictive.

[0421] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0422] Those skilled in the art will appreciate that, for any and all purposes, particularly in providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily identified as fully describing the same range and enabling the same range to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will be appreciated by those skilled in the art, all such as "up to", "at least", "greater than", "less than", etc., include the numbers and relate to the ranges that can subsequently be decomposed into sub-ranges as described above. Finally, those skilled in the art will appreciate that the range includes each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0423] All patents, patent applications, pending applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are consistent with the explicit teachings of this specification.

[0424] In summary, the present invention includes but is not limited to the following:

[0425] 1. A method comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ) or an antigen-binding fragment thereof, wherein:

[0426] (a) V H Contains V of GYTFTSYWMQ H -CDR1 sequence (SEQ ID NO: 53), V H -CDR2 sequence (SEQ ID NO: 54) and V of YDGYYAMDY H - CDR3 sequence (SEQ ID NO: 55); and / or

[0427] (b) V L Contains V of KSSQSLLYSSNQKNYLA L -CDR1 sequence (SEQ ID NO: 56), V L -CDR2 sequence (SEQ ID NO: 57) and V of QQYHSYPFT L - CDR3 sequence (SEQ ID NO: 58).

[0428] 2. The antibody or antigen-binding fragment according to claim 1, further comprising an Fc domain of an isotype selected from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE.

[0429] 3. The antibody according to item 2, comprising an IgG1 constant region comprising one or more amino acid substitutions selected from N297A and K322A.

[0430] 4. The antibody according to item 2, comprising an IgG4 constant region comprising an S228P mutation.

[0431] 5. The antigen-binding fragment according to item 1, wherein the antigen-binding fragment is selected from Fab, F(ab') 2 , Fab', scF v and F v .

[0432] 6. The antibody or antigen-binding fragment according to any one of items 1 to 5, wherein the antibody or antigen-binding fragment binds to an epitope of L1-CAM protein, the epitope comprising at least five to eight consecutive amino acid residues of SEQ ID NO: 74.

[0433] 7. The antibody according to any one of items 1 to 4 or 6, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody or a bispecific antibody.

[0434] 8. An antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:51, or a variant thereof having one or more conservative amino acid substitutions, and / or a light chain (LC) amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:52, or a variant thereof having one or more conservative amino acid substitutions.

[0435] 9. The antibody according to item 8, comprising a HC amino acid sequence and a LC amino acid sequence selected from the following:

[0436] SEQ ID NO:9 and SEQ ID NO:13 (huE71 H1 / L1);

[0437] SEQ ID NO:9 and SEQ ID NO:14 (huE71 H1 / L2);

[0438] SEQ ID NO: 10 and SEQ ID NO: 13 (huE71 H2 / L1);

[0439] SEQ ID NO: 10 and SEQ ID NO: 14 (huE71 H2 / L2);

[0440] SEQ ID NO: 17 and SEQ ID NO: 21 (huE72 H1 / L1);

[0441] SEQ ID NO:17 and SEQ ID NO:22 (huE72 H1 / L2);

[0442] SEQ ID NO: 18 and SEQ ID NO: 21 (huE72 H2 / L1);

[0443] SEQ ID NO: 18 and SEQ ID NO: 22 (huE72 H2 / L2);

[0444] SEQ ID NO:26 and SEQ ID NO:25 (chE71 IgG1);

[0445] SEQ ID NO:30 and SEQ ID NO:29 (chE72 IgG1);

[0446] SEQ ID NO:42 and SEQ ID NO:41 (chE71 IgG4);

[0447] SEQ ID NO:46 and SEQ ID NO:45 (huE71 IgG4);

[0448] SEQ ID NO:49 and SEQ ID NO:50 (huE71 BsAb); and

[0449] SEQ ID NO:51 and SEQ ID NO:52 (huE72 BsAb).

[0450] 10. An antibody comprising (a) a light chain immunoglobulin variable domain sequence that is at least 95% identical to the light chain immunoglobulin variable domain sequence present in any one of SEQ ID NO: 13, 14, 21, 22, 25, 29, 41, 45, 50 or 52; and / or

[0451] (b) a heavy chain immunoglobulin variable domain sequence that is at least 95% identical to the heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 9, 10, 17, 18, 26, 30, 42, 46, 49 and 51.

[0452] 11. An antibody comprising (a) an LC sequence that is at least 95% identical to the LC sequence present in any one of SEQ ID NO: 13, 14, 21, 22, 25, 29, 41, 45, 50 or 52; and / or

[0453] (b) a HC sequence that is at least 95% identical to the HC sequence present in any one of SEQ ID NOs: 9, 10, 17, 18, 26, 30, 42, 46, 49 and 51.

[0454] 12. The antibody according to any one of items 8 to 11, wherein the antibody is a chimeric antibody, a humanized antibody or a bispecific antibody.

[0455] 13. The antibody according to any one of items 8 to 12, wherein the antibody binds to an epitope of L1-CAM protein comprising at least five to eight consecutive amino acid residues of SEQ ID NO: 74 or SEQ ID NO: 75.

[0456] 14. The antibody according to any one of items 8 to 13, wherein the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from N297A and K322A.

[0457] 15. An antibody according to any one of items 8 to 13, wherein the antibody comprises an IgG4 constant region comprising an S228P mutation.

[0458] 16. A recombinant nucleic acid sequence encoding the antibody according to any one of items 8 to 15.

[0459] 17. A recombinant nucleic acid sequence selected from the group consisting of SEQ ID NO: 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 43, 44, 47 and 48.

[0460] 18. A host cell or vector comprising the recombinant nucleic acid sequence according to item 16 or item 17.

[0461] 19. A composition comprising an antibody or antigen-binding fragment according to any one of items 1 to 7 and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the following: an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA or any combination thereof.

[0462] 20. A composition comprising an antibody according to any one of items 8 to 15 and a pharmaceutically acceptable carrier, wherein the antibody is optionally conjugated to an agent selected from the following: an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA or any combination thereof.

[0463] 21. The antibody according to any one of items 1 to 4, 6 or 7, wherein the antibody lacks α-1,6-fucose modification.

[0464] 22. The antibody according to any one of items 8 to 15, wherein the antibody lacks α-1,6-fucose modification.

[0465] 23. The bispecific antibody of item 7 or 12, wherein the bispecific antibody binds to T cells, B cells, bone marrow cells, plasma cells or mast cells.

[0466] 24. The bispecific antibody of claim 7 or 12, wherein the bispecific antibody binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, KIR or a small molecule DOTA hapten.

[0467] 25. A method of treating L1-CAM-associated cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody comprising a HC amino acid sequence and a LC

[0468] Amino Acid Sequence:

[0469] SEQ ID NO:9 and SEQ ID NO:13 (huE71 H1 / L1);

[0470] SEQ ID NO:9 and SEQ ID NO:14 (huE71 H1 / L2);

[0471] SEQ ID NO: 10 and SEQ ID NO: 13 (huE71 H2 / L1);

[0472] SEQ ID NO: 10 and SEQ ID NO: 14 (huE71 H2 / L2);

[0473] SEQ ID NO: 17 and SEQ ID NO: 21 (huE72 H1 / L1);

[0474] SEQ ID NO:17 and SEQ ID NO:22 (huE72 H1 / L2);

[0475] SEQ ID NO: 18 and SEQ ID NO: 21 (huE72 H2 / L1);

[0476] SEQ ID NO: 18 and SEQ ID NO: 22 (huE72 H2 / L2);

[0477] SEQ ID NO:26 and SEQ ID NO:25 (chE71 IgG1);

[0478] SEQ ID NO:30 and SEQ ID NO:29 (chE72 IgG1);

[0479] SEQ ID NO:42 and SEQ ID NO:41 (chE71 IgG4);

[0480] SEQ ID NO:46 and SEQ ID NO:45 (huE71 IgG4);

[0481] SEQ ID NO:49 and SEQ ID NO:50 (huE71 BsAb); and

[0482] SEQ ID NO:51 and SEQ ID NO:52 (huE72 BsAb),

[0483] The antibody specifically binds to L1-CAM and neutralizes the activity of L1-CAM.

[0484] 26. The method of claim 25, wherein the L1-CAM-associated cancer is leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, bile duct cancer, carcinoid, neuroendocrine tumor, gastrointestinal stromal tumor (GIST). T), pheochromocytoma, glioma, pancreatic neuroectodermal cancer, pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma, tumor vascular, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine cancer, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, nasal glioma, medulloblastoma, capillary hemangioma, Kaposi sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, or head and neck squamous cell carcinoma.

[0485] 27. A method according to item 25 or 26, wherein the antibody and the additional therapeutic agent are administered to the subject separately, sequentially or simultaneously.

[0486] 28. The method of claim 27, wherein the additional therapeutic agent is one or more of the following: an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen drug, an aromatase inhibitor, an ovarian inhibitor, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, a bisphosphonate therapeutic agent.

[0487] 29. A method for detecting a tumor in a subject in vivo, comprising

[0488] (a) administering to the subject an effective amount of an antibody according to any one of items 1 to 15, wherein the antibody is configured to localize to a tumor expressing L1-CAM and is labeled with a radioactive isotope;

[0489] as well as

[0490] (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the antibody above a reference value.

[0491] 30. A method according to claim 29, wherein the subject is diagnosed with or suspected of having cancer.

[0492] 31. A method according to item 29 or 30, wherein the level of radioactivity emitted by the antibody is detected using positron emission tomography or single photon emission computed tomography.

[0493] 32. The method according to any one of items 29 to 31, further comprising administering to the subject an effective amount of an immunoconjugate comprising the antibody according to any one of items 1 to 15 conjugated to a radionuclide.

[0494] 33. A method according to claim 32, wherein the radionuclide is an alpha particle emitting isotope, a beta particle emitting isotope, an Auger emitter or any combination thereof.

[0495] 34. A method according to claim 33, wherein the isotope emitting beta particles is selected from 86 Y. 90 Y. 89 Sr. 165 Dy, 186 Re, 188 Re, 177 Lu and 67 Cu.

Claims

1. An antibody or antigen-binding fragment thereof, comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ),in: The V L comprising a sequence identical to the light chain immunoglobulin variable domain sequence shown in any one of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:29 and SEQ ID NO:52, and The V H comprising a sequence identical to the heavy chain immunoglobulin variable domain sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 30 and SEQ ID NO: 51, The antibody or antigen-binding fragment thereof binds to the 6th Ig-like domain of the L1-CAM protein, wherein the 6th Ig-like domain has an amino acid sequence of SEQ ID NO:

75.

2. The antibody according to claim 1, comprising a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 21 (huE72 H1 / L1); SEQ ID NO:17 and SEQ ID NO:22 (huE72 H1 / L2); SEQ ID NO: 18 and SEQ ID NO: 21 (huE72 H2 / L1); SEQ ID NO: 18 and SEQ ID NO: 22 (huE72 H2 / L2); SEQ ID NO:30 and SEQ ID NO:29 (chE72 IgG1); and SEQ ID NO:51 and SEQ ID NO:52 (huE72 BsAb).

3. The antibody or antigen-binding fragment thereof according to claim 1, further comprising an Fc domain of a subtype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD and IgE. 4 . The antibody or antigen-binding fragment thereof according to claim 3 , comprising an IgG1 constant region into which amino acid substitutions of N297A and K322A are introduced, or an IgG4 constant region into which amino acid mutation S228P is introduced.

5. The antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', scF v and F v .

6. The antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody is a chimeric antibody, a humanized antibody or a bispecific antibody.

7. The bispecific antibody of claim 6, wherein the bispecific antibody binds to T cells, B cells, bone marrow cells, plasma cells, or mast cells.

8. The bispecific antibody of claim 6, wherein the bispecific antibody binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCRγ / δ, NKp46, KIR or a small molecule DOTA hapten.

9. The antibody of any one of claims 1-8, wherein the antibody lacks α-1,6-fucose modification.

10. The antibody or antigen-binding fragment of any one of claims 1-9, wherein the antibody or antigen-binding fragment is optionally conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

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