New anti-L1CAM antibody

Abstract

The present invention provides a fully human monoclonal antibody that binds highly specifically to human L1CAM, an antigen-binding fragment thereof, and a bispecific antibody thereof, etc. The antibody of the present invention can be used for the diagnosis and treatment of cancer.

Description

[Technical Field]

[0001] The present invention belongs to the field of biotechnology, and specifically relates to fully human-derived monoclonal antibodies, antigen-binding fragments, and chimeric antigen receptors that specifically bind to human L1CAM, as well as nucleic acids, plasmids, host cells, corresponding pharmaceutical compositions, and uses thereof that are necessary for constructing such antibodies, antigen-binding fragments, and chimeric antigen receptors. [Background technology]

[0002] The L1 cell adhesion molecule (L1CAM), also known as CD171, is a transmembrane glycoprotein with a molecular weight between 200,000 and 220,000 [Miriam van der Maten et al., Int. J. Mol. Sci., 2019, 20:4180.]. Expression of L1CAM in healthy individuals is primarily localized in the nervous system, with lower expression in kidney tissue and skin [Annette Kunkele et al., Clin. Cancer Res., 2016, 23:466-477.]. L1CAM is expressed at relatively high levels in various cancers and is closely associated with the proliferation and drug resistance of various cancer cells.

[0003] Although the L1 cell adhesion molecule is a cancer therapeutic target with enormous potential, there remains a need for therapeutic antibodies against the L1 cell adhesion molecule. Summary of the Invention

[0004] The inventors of the present invention have used hybridoma technology to screen and obtain anti-L1CAM monoclonal antibodies with high specificity, affinity and stability.

[0005] In a first aspect, the present invention provides an L1CAM-binding molecule comprising a fully human immunoglobulin that specifically binds to L1CAM.

[0006] In another aspect, nucleic acid molecules encoding the L1CAM-binding molecules of the invention, and expression vectors and host cells containing the nucleic acid molecules, are provided.

[0007] The present invention further provides nucleic acids encoding these fully cloned antibodies and antigen-binding fragments. In some embodiments, vectors containing these nucleic acids and host cells containing these vectors are disclosed. In further embodiments, pharmaceutical compositions containing these fully cloned antibodies, antigen-binding fragments, bispecific antibodies, immunoconjugates, fusion proteins, nucleic acids, and vectors are disclosed. In other embodiments, these fully cloned antibodies, antigen-binding fragments, bispecific antibodies, immunoconjugates, fusion proteins, nucleic acids, and vectors can be used to diagnose or treat diseases such as autoimmune diseases, chronic viral infections, and cancer.

[0008] The present invention discloses a novel, fully human monoclonal antibody that specifically binds to L1CAM. The CDR regions, portions of the antibody, or the entire gene can be used to engineer and produce various types of genetically engineered antibodies in any expression system, both prokaryotic and eukaryotic, for clinical use in the diagnosis or treatment of diseases such as autoimmune diseases, chronic viral infections, and cancer.

[0009] The present invention discloses fully human monoclonal antibodies that specifically bind to human L1CAM, and the CDR regions of their light chain variable regions and / or heavy chain variable regions. In some embodiments, the amino acid sequence of the light chain variable region and / or the heavy chain variable region of the monoclonal antibody or antigen-binding fragment can comprise at least one CDR region of the light chain and / or heavy chain variable region. The present invention also discloses antigen-binding fragments of these monoclonal antibodies, bispecific antibodies, chimeric antigen receptor (CAR), antibody complexes, corresponding nucleic acids, plasmids and host cells, corresponding pharmaceutical compositions, and uses in cancer diagnosis and treatment.

[0010] The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to L1CAM, comprising a light chain variable region having light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, and / or a heavy chain variable region having heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3. The antibody or antigen-binding fragment thereof can specifically bind to the Ig1 or FN-4 domain of L1-CAM and does not have the same or equivalent CDR combination as antibody L1-9.3 or UJ127.

[0011] In the technical solution of the present invention, the heavy chain variable region can comprise HCDR1, HCDR2 and HCDR3 having at least 80%, 90% or 100% identity to the HCDR1, HCDR2 and HCDR3 in the heavy chain variable region sequence selected from SEQ ID NOs: 43, 45, 47, 49 and 51, and the light chain variable region can comprise LCDR1, LCDR2 and LCDR3 having at least 80% identity to the LCDR1, LCDR2 and LCDR3 in the light chain variable region sequence selected from SEQ ID NOs: 44, 46, 48, 50 and 52.

[0012] The technical solution of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have a combination of CDRs selected from the group consisting of: (1) HCDR1, HCDR2, and HCDR3 having at least 80% identity to HCDR1, HCDR2, and HCDR3 in the heavy chain variable region sequence of SEQ ID NO: 43, and LCDR1, LCDR2, and LCDR3 having at least 80% identity to LCDR1, LCDR2, and LCDR3 in the light chain variable region sequence of SEQ ID NO: 44; (2) HCDR1, HCDR2, and HCDR3 having at least 80% identity to HCDR1, HCDR2, and HCDR3 in the heavy chain variable region sequence of SEQ ID NO: 45, and LCDR1, LCDR2, and LCDR3 having at least 80% identity to LCDR1, LCDR2, and LCDR3 in the light chain variable region sequence of SEQ ID NO: 46; (3) HCDR1, HCDR2, and HCDR3 having at least 80% identity to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 47, and LCDR1, LCDR2, and LCDR3 having at least 80% identity to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 48; (4) HCDR1, HCDR2, and HCDR3 having at least 80% identity to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 49, and LCDR1, LCDR2, and LCDR3 having at least 80% identity to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 50, and (5) HCDR1, HCDR2 and HCDR3 having at least 80% identity to HCDR1, HCDR2 and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 51, and LCDR1, LCDR2 and LCDR3 having at least 80% identity to LCDR1, LCDR2 and LCDR3 of the light chain variable region sequence of SEQ ID NO: 52.

[0013] In the technical solution of the present invention, HCDR1 has at least 80% identity to a sequence selected from the following: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, HCDR2 has at least 80% identity to a sequence selected from the following: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, HCDR3 has at least 80% identity to a sequence selected from the following: SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, LCDR1 has at least 80% identity to a sequence selected from the following: SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:40, LCDR2 has at least 80% identity to a sequence selected from the following: SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, LCDR3 has at least 80% identity to a sequence selected from the following: SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42.

[0014] In the technical solution of the present invention, (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1; (2) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2; (3) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3; (4) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4; (5) LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5; (6) LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0015] In the technical solution of the present invention, (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7; (2) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8; (3) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 9; (4) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10; (5) LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11; (6) LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12.

[0016] In the technical solution of the present invention, (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (2) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (3) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (4) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (5) LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (6) LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18.

[0017] In the technical solution of the present invention, (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19; (2) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20; (3) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21; (4) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22; (5) LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23; (6) LCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.

[0018] In the technical solution of the present invention, (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25; (2) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26; (3) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27; (4) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28; (5) LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29; (6) LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 30.

[0019] In the technical solution of the present invention, (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31; (2) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32; (3) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33; (4) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 34; (5) LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35; (6) LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 36.

[0020] In the technical solution of the present invention, (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 37; (2) HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 38; (3) HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 39; (4) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 40; (5) LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 41; (6) LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 42.

[0021] In the technical solution of the present invention, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:43, or an amino acid sequence having at least 90%, at least 95% or 100% identity to SEQ ID NO:43.

[0022] In the technical solution of the present invention, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:44, or an amino acid sequence having at least 90%, at least 95% or 100% identity to SEQ ID NO:44.

[0023] In the technical solution of the present invention, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:45, or an amino acid sequence having at least 90%, at least 95% or higher identity to SEQ ID NO:45.

[0024] In the technical solution of the present invention, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:46, or an amino acid sequence having at least 90%, at least 95% or higher identity to SEQ ID NO:46.

[0025] In the technical solution of the present invention, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:47, or an amino acid sequence having at least 90%, at least 95% or higher identity to SEQ ID NO:47.

[0026] In the technical solution of the present invention, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:48, or an amino acid sequence having at least 90%, at least 95% or higher identity to SEQ ID NO:48.

[0027] In the technical solution of the present invention, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:49, or an amino acid sequence having at least 90%, at least 95% or higher identity to SEQ ID NO:49.

[0028] In the technical solution of the present invention, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:50, or an amino acid sequence having at least 90%, at least 95% or higher identity to SEQ ID NO:50.

[0029] In the technical solution of the present invention, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:51, or an amino acid sequence having at least 90%, at least 95% or higher identity to SEQ ID NO:51.

[0030] In the technical solution of the present invention, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:52, or an amino acid sequence having at least 90%, at least 95% or higher identity to SEQ ID NO:52.

[0031] In the technical solution of the present invention, the antibody is a humanized antibody. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises one of the amino acid sequences set forth in SEQ ID NO:53 or SEQ ID NO:54 or SEQ ID NO:55 or SEQ ID NO:56, and the light chain variable region comprises one of the amino acid sequences set forth in SEQ ID NO:57 or SEQ ID NO:58 or SEQ ID NO:59.

[0033] The present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, wherein: 1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 53 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 57, or 2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 53 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 58, or 3) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 53 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, or 4) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 54 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 57, or 5) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 54 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 58, or 6) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 54 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, or 7) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 55 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 57, or 8) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 55 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 58, or 9) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 55 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, or 10) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 56 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 57, or 11) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 56 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 58, or 12) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59.

[0034] The present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises one of the amino acid sequences set forth in SEQ ID NO:60 or SEQ ID NO:61 or SEQ ID NO:62 or SEQ ID NO:63, and the light chain variable region comprises one of the amino acid sequences set forth in SEQ ID NO:64 or SEQ ID NO:65 or SEQ ID NO:66.

[0035] The present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, wherein: 1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64, or 2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 65, or 3) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 66, or 4) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 61 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64, or 5) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 61 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 65, or 6) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 61 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 66, or 7) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64, or 8) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 65, or 9) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 66, or 10) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 63 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64, or 11) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 63 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 65, or 12) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 63, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66.

[0036] The present invention provides an antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises one of the amino acid sequences set forth in SEQ ID NO:67 or SEQ ID NO:68 or SEQ ID NO:69 or SEQ ID NO:70 or SEQ ID NO:71 or SEQ ID NO:72 or SEQ ID NO:73 or SEQ ID NO:74, and the light chain variable region comprises one of the amino acid sequences set forth in SEQ ID NO:75 or SEQ ID NO:76 or SEQ ID NO:77 or SEQ ID NO:78 or SEQ ID NO:79 or SEQ ID NO:80 or SEQ ID NO:81.

[0037] The present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, wherein: 1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 67 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 75, or 2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 67 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76, or 3) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 67 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77, or 4) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 67 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78, or 5) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 67 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79, or 6) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 67 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80, or 7) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 67 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81, or 8) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 75, or 9) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76, or 10) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77, or 11) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78, or 12) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79, or 13) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80, or 14) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81, or 15) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 69 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 75, or 16) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 69 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76, or 17) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 69 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77, or 18) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 69 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78, or 19) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 69, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79, or 20) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 69 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80, or 21) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 69 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81, or 22) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 70 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 75, or 23) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 70 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76, or 24) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 70 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77, or 25) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 70, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78, or 26) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 70 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79, or 27) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 70, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80, or 28) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 70, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81, or 29) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 75, or 30) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76, or 31) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77, or 32) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78, or 33) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79, or 34) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80; or 35) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81, or 36) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 75, or 37) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76, or 38) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77, or 39) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78, or 40) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79, or 41) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80, or 42) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81; or 43) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 75, or 44) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76, or 45) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77, or 46) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78, or 47) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79, or 48) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80, or 49) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 73 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81, or 50) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 74 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 75, or 51) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 74 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76, or 52) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 74 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77, or 53) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 74 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78, or 54) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 74 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79, or 55) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 74 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80, or 56) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 74, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81.

[0038] The present invention provides an antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises one of the amino acid sequences set forth in SEQ ID NO:82 or SEQ ID NO:83 or SEQ ID NO:84 or SEQ ID NO:85 or SEQ ID NO:86 or SEQ ID NO:87 or SEQ ID NO:88 or SEQ ID NO:89 or SEQ ID NO:90, and the light chain variable region comprises one of the amino acid sequences set forth in SEQ ID NO:91 or SEQ ID NO:92 or SEQ ID NO:93 or SEQ ID NO:94 or SEQ ID NO:95 or SEQ ID NO:96.

[0039] The present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, wherein: 1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91, or 2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 92, or 3) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93, or 4) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 94, or 5) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 95, or 6) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 96, or 7) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91, or 8) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 92, or 9) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93, or 10) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 94, or 11) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 95, or 12) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 96, or 13) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 84 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91, or 14) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 84 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 92, or 15) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 84 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93, or 16) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 84 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 94, or 17) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 84 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 95, or 18) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 84 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 96, or 19) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 85 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91, or 20) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 85 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 92, or 21) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 85 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93, or 22) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 85 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 94, or 23) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 85 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 95, or 24) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 85 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 96, or 25) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91, or 26) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 92, or 27) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93, or 28) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 94, or 29) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 95, or 30) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 96, or 31) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91, or 32) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 92, or 33) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93, or 34) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 94, or 35) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 95, or 36) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 96, or 37) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91, or 38) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 92, or 39) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93, or 40) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 94, or 41) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 95, or 42) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 96, or 43) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 89 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91; or 44) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 89 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 92; or 45) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 89 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93, or 46) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 89 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 94, or 47) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 89 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 95, or 48) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 89 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 96, or 49) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91, or 50) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 92, or 51) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93, or 52) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 94, or 53) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 95, or 54) The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 96.

[0040] Additionally, the present invention provides fully human antibodies or antigen-binding fragments thereof, further comprising an Fc region, optionally comprising a human immune protein (Ig) Fc region, or optionally comprising the Fc region of a human IgG, wherein the Fc region is derived from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM.

[0041] The antigen-binding fragment of the present invention is an scFv, scFv-Fc, Fv, Fab, Fab', F(ab)2 or F(ab')2.

[0042] Preferably, the antigen-binding fragments according to the present invention are F(ab)2, Fab and scFv.

[0043] F(ab)2 fragments are "bivalent." "Bivalent" refers to the fact that antibodies and F(ab')2 fragments have two antigen-binding sites. Conversely, Fab, Fv, and scFv fragments are univalent and have only one antigen-binding site.

[0044] F(ab)2 is an antibody fragment with a molecular weight of approximately 50,000 that has antigen-binding activity and is obtained by treating an IgG antibody molecule with the protease papain (which cleaves the 224th amino acid residue of the H chain). Approximately half of the N-terminal end of the F(ab)2 antibody fragment is linked to the entire L chain via a disulfide bond (CAK Borrebaeck, editor (1995), Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001), Antibody Engineering (Springer Laboratory Manual), Springer Verlag).

[0045] Fab is an antibody fragment with a molecular weight of approximately 50,000 that has antigen-binding activity and is obtainable by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2. The Fab of the present invention can be produced by treating F(ab)2 with a reducing agent, such as dithiothretol.

[0046] "Single-chain Fv" or "scFv" refers to a molecule of an antibody heavy chain variable domain (or region, VH) and an antibody light chain variable domain (or region, VL) linked by a linker. Such scFv molecules can have the general structure H2-VL-linker-VH-COOH or H2-VH-linker-VL-COOH. Suitable prior art linkers consist of overlapping GGGGS amino acid sequences or variants thereof, e.g., 1-4 overlapping variants.

[0047] The antibody or antigen-binding fragment thereof according to the present invention may be a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a bivalent antibody, an anti-heterotypic antibody or a fusion protein.

[0048] Moreover, the present invention further provides an antibody-drug conjugate comprising the antibody or antigen-binding fragment according to any one of the above aspects of the present invention and another effector molecule conjugate.

[0049] Furthermore, the present invention further provides a nucleic acid molecule encoding the antibody or antigen-binding fragment according to any one of the above aspects of the present invention.

[0050] The nucleic acid molecule of the invention is operably linked to a promoter.

[0051] Additionally, the present invention further provides a recombinant expression vector containing a nucleic acid molecule according to the present invention.

[0052] Additionally, the present invention further provides a plasmid containing a nucleic acid molecule according to the present invention.

[0053] Moreover, the present invention further provides a host cell containing a nucleic acid molecule according to the invention, a recombinant expression vector according to the invention or a plasmid according to the invention.

[0054] Furthermore, the present invention further provides a method for detecting L1CAM expression in a biological sample, which method comprises the steps of contacting the biological sample with any one of the antibodies or antigen-binding fragments thereof described in the present invention and detecting the presence or absence of an immune complex, wherein the presence of the immune complex indicates the presence of L1CAM in the biological sample.

[0055] Additionally, the present invention further provides a pharmaceutical composition comprising any one of the antibodies or antigen-binding fragments thereof described in the present invention as an active substance, an antibody-drug conjugate, and a pharmaceutically acceptable carrier.

[0056] The present invention further provides pharmaceutical compositions prepared by mixing an effective prophylactic or therapeutic dose of the antibody, antigen-binding fragment, bispecific antibody, or chimeric antigen receptor of the present invention, or its nucleic acid molecule, plasmid, or cells expressing this chimeric antigen receptor, with a physiologically or pharmaceutically acceptable carrier, excipient, or stabilizer, and the compositions include, but are not limited to, lyophilized preparations, aqueous solution preparations, liposomes, or encapsulated preparations, etc. The concentration of the monoclonal antibody, antigen-binding fragment, bispecific antibody, or chimeric antigen receptor of the present invention, or its nucleic acid molecule, plasmid, or cells expressing this chimeric antigen receptor can vary from about 0.1% to 100% (by weight).

[0057] Additionally, the present invention further provides a test kit comprising any one of the antibodies or antigen-binding fragments thereof described in the present invention, the antibody-drug conjugates described in the present invention, and / or the pharmaceutical compositions described in the present invention.

[0058] Furthermore, the present invention further provides a method for treating cancer, comprising the step of administering to a subject in need of cancer treatment any one of the antibodies or antigen-binding fragments thereof described in the present invention, the antibody-drug conjugate described in the present invention, or the pharmaceutical composition of the present invention.

[0059] The above methods of the present invention further comprise the step of detecting the presence of L1-CAM in the cancer sample before administering the agent.

[0060] Preferably, the antibodies or antigen-binding fragments described in the present invention are fully human. Fully human means herein that the antibody or antigen-binding fragment is non-chimeric (e.g., non-"humanized") and not derived (in whole or in part) from a non-human species. A fully human antibody or antigen-binding fragment thereof may be of human origin or a synthetic human antibody. Another example of a fully human antibody or antigen-binding fragment thereof may be a human antibody encoded by nucleic acid isolated from a library of human-derived antibody sequences (e.g., a library based on naturally occurring human antibodies).

[0061] The antibody or antigen-binding fragment thereof of the present invention can be a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a bivalent antibody, an anti-heterotypic antibody or a fusion protein.

[0062] The present invention further provides antibodies or antigen-binding fragments that bind to L1CAM, which are bispecific antibodies or bispecific antigen-binding fragments. In some embodiments, the bispecific antibodies or bispecific antigen-binding fragments can be separated. The term "bispecific" refers to an antigen-binding molecule that specifically binds to at least two different antigenic determinants. In some embodiments, the bispecific antibodies and bispecific antigen-binding fragments comprise antigen-binding fragments according to the present invention. In some embodiments, the bispecific antibodies and bispecific antigen-binding fragments comprise antigen-binding fragments capable of binding to L1CAM, and the antigen-binding fragments capable of binding to L1CAM comprise antigen-binding fragments according to the present invention. In some embodiments, the bispecific antibodies and bispecific antigen-binding fragments comprise a fragment capable of binding to an L1CAM-binding antigen and a fragment capable of binding to an antigen capable of binding to the other target protein. In some embodiments, the bispecific antibodies or antigen-binding fragments comprise an antigen-binding molecule capable of binding to L1CAM and an antigen-binding molecule capable of binding to an antigen other than L1CAM. In some embodiments, the antigen other than L1CAM is an immune cell surface molecule. In some embodiments, the antigen other than L1CAM is a cancer cell antigen. In some embodiments, the antigen other than L1CAM is a receptor molecule, eg, a cell surface receptor.

[0063] The present invention provides nucleic acids encoding monoclonal antibodies, antigen-binding fragments, bispecific antibodies, or chimeric antigen receptors. The present invention provides plasmids containing the nucleic acids, which can optionally be operably linked to regulatory sequences, such as promoters, enhancers, etc. The present invention provides host cells containing the plasmids and methods for producing and optionally recovering the monoclonal antibodies, antigen-binding fragments, bispecific antibodies, or chimeric antigen receptors. Host cells according to the present invention can be any prokaryotic or eukaryotic cell, including, but not limited to, bacterial cells (e.g., Escherichia coli, Bacillus subtilis), insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells (e.g., CHO or BHK cell lines). Other suitable host cells will be known to those of skill in the art.

[0064] Preferably, the disease according to the present invention is cancer, including but not limited to lymphoma, germinoma, sarcoma (including liposarcoma), neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenoma, melanoma and non-leukemic leukemia or lymphoid malignancies. More specific examples of the above cancers include squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, pancreatic cancer, malignant brain tumor, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct cancer, head cancer, neck cancer, myeloma, osteoclastoma, multiple myeloma, osteolytic bone cancer, and the like. cancers), central nervous system tumors, brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ventricular leukemia, and retinoblastoma), nasopharyngeal carcinoma, basal cell carcinoma, cholangiocarcinoma, Kaposi's sarcoma, primary liver or endometrial cancer, and vascular tumors (angiosarcoma and hemagiopericytoma), hematoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (Burkitt's lymphoma, small lymphocytic lymphoma / chronic lymphocytic leukemia, mycosis fungoides, ectodermal cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal lymphoma, hairy cell leukemia, and lymphoplasmacytic leukemia), lymphoid precursor tumors, B-cell acute lymphoblastoma These include hematologic cancers such as myeloid non-leukemia / lymphoma, T-cell acute lymphoblastic non-leukemia / lymphoma, thymoma, mature T-cell and NK-cell lymphoma, peripheral T-cell non-leukemia, mature T-cell non-leukemia / T-cell lymphoma, large granular lymphocytic leukemia, Langerhans cell histiocytosis, acute myeloid granulocytic leukemia myeloma, mature acute myeloid leukemia (AML), differentiated acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute mononuclear cell leukemia, myelodysplastic syndrome, chronic myeloproliferative disorders, chronic myelogenous leukemia, multiple myeloma (MM), myeloproliferative dysplasia syndrome (MDS) and acute myeloid leukemia (AML).

[0065] The antibodies, antigen-binding fragments, bispecific antibodies, or chimeric antigen receptors, or nucleic acid molecules thereof, plasmids, or cells expressing the chimeric antigen receptors, and pharmaceutical compositions of the present invention can be administered to human or animal subjects via a variety of different administration routes, which are determined by the characteristics of the disease being treated. Generally, the methods of the present invention can be carried out using any medically acceptable form of administration, including oral, rectal, topical, intraocular, intraocular, intracisternal, intraventricular, intratracheal, intranasal infusion, transdermal, subcutaneous, intravitreal, intramuscular, intraperitoneal, intraperitoneal, intracranial, or intravenous injection.

[0066] In order to provide a more complete understanding of the present invention, several definitions are provided below. The above definitions include all grammatical equivalents.

[0067] As used herein, the term "antibody" refers essentially to a protein composed of one or more polypeptides encoded by all or part of known immunoglobulin genes. These known immunoglobulin genes include, for example, the kappa (κ), lambda (λ), and heavy chain loci in humans, which contain numerous variable region genes and the constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), and alpha (α) that encode IgM, IgD, IgG, IgE, and IgA isoforms. As used herein, "antibody" includes full-length antibodies and antibody fragments, as well as natural antibodies from any organism, engineered antibodies, or antibodies produced by genetic engineering for testing, therapy, or other purposes described below. The term "antibody" includes antibody fragments, including, for example, Fab, Fab', F(ab'), Fv, scFv, or other subsequences of an antibody that bind to an antigen, or antibody fragments produced by modifying intact antibodies or resynthesizing these antibodies using recombinant DNA technology. The term "antibody" includes monoclonal and polyclonal antibodies. The antibody may be an antagonist, an agonist, a neutralizing or inhibitory antibody, or a stimulatory antibody. The antibody of the invention may be a non-human antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population; i.e., a population containing the monoclonal antibody is identical except for minor variations (e.g., natural mutations). The term "monoclonal" thus refers to the nature of the antibody, i.e., it is not a mixture of unrelated (distinct) antibodies. In contrast to polyclonal antibody preparations, which typically contain different antibodies targeting different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation targets only one single determinant on the antigen. Besides this specificity, an advantage of monoclonal antibody preparations is that they are typically uncontaminated by other immunoglobulins. The term "monoclonal" should not be construed as requiring the antibody to be produced by any particular method. The term monoclonal antibody specifically includes fully human antibodies (see CN201280067865 - Anti-FGFR2 antibodies and their uses - Patent granted).

[0069] As used herein, the term "complementarity-determining region" (CDR, e.g., CDR1, CDR2, and CDR3) refers to amino acid residues in an antibody variable region, and the presence of a complementarity-determining region is essential for antigen binding. The "antigen-binding region" of an antibody is typically located in one or more hypermutable regions of the antibody, such as the CDR regions CDR1, CDR2, and CDR3. Each complementarity-determining region can include amino acid residues derived from the "complementarity-determining region" defined by Kabat, e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and residues 26-33 (H1), 51-57 (H2), and 96-105 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, 1991).

[0070] As used herein, "antigen" refers to a compound, composition, or substance that stimulates antibody production or a T-cell response in an animal, including compositions injected or absorbed into an animal, and may be a protein, sugar, lipid, or other pathogen.

[0071] A "functional fragment" or "antigen-binding fragment" of an antibody / immunoglobulin is defined herein as a fragment of an antibody / immunoglobulin (e.g., the variable region of an IgG) that retains the antigen-binding region. The "antigen-binding region" of an antibody is typically located in one or more hypermutable regions of the antibody, such as CDR-1, -2, and / or -3, although variable "frame" regions also play an important role in antigen binding and can be achieved, for example, by providing the structure of the CDRs. Preferably, the "antigen-binding region" comprises at least amino acid residues 4-103 of the variable light chain (VL) and amino acid residues 5-109 of the variable heavy chain (VH), more preferably amino acid residues 3-107 of the VL and amino acid residues 4-111 of the VH, and particularly preferably the complete VL and VH chains (amino acid residues 1-109 of the VL and amino acid residues 1-113 of the VH, numbering according to WO 97 / 08320). The preferred immunoglobulin for use in the present invention is IgG.

[0072] As used herein, "amino acid" refers to one of the 20 naturally occurring amino acids or any non-natural analog, which may be located at a specifically defined position. As used herein, "protein" refers to at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides, and peptides. Proteins can be composed of naturally occurring amino acids and peptide bonds or synthetic peptide analog structures, which are therefore "analogs." Thus, as used herein, "amino acid" or "peptide residue" refers to naturally occurring or synthetic amino acids. For example, for purposes of the present invention, phenylalanine, citrulline, and norleucine may be amino acids of interest. "Amino acid" may also include imino acid residues such as proline and hydroxyproline. Side chains may be in the (R) or (S) configuration. In preferred embodiments, amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substitutions may be used, for example, to prevent or delay degradation in the body.

[0073] As used herein, "identity" refers to the similarity between nucleotide or amino acid sequences, or is referred to as sequence identity. Sequence identity is usually measured based on percentage identity (or similarity or homology), and the higher the percentage, the more similar the two sequences are. When compared using standard methods, homologous organisms or variants have a relatively high degree of sequence identity. Control methods for comparing sequences are well known in the art. Various programs and sequence algorithms are described in Smith and Waterman, Adv Appl. Math., 2: 482, 1981; Needlema and Wunsch, J. Mol. Biol. 48: 443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins and Sharp, Gene 73: 237-244, 1988; Higgins and Sharp, CABIOS 5: 151-153, 1989; Corpet et al., Nucleic Acids Research 16: 10881-10890, 1988; Altschul et al., Nature Genet., 1994, 6: 119-129.

[0074] These can be obtained from a variety of sources, including the NCBI Basic Local Comparison Search Tool (BLAST™) (Altschul et al., J. Mol. Biol., 215:403-410, 1990), the National Center for Biotechnology Information (NCBI, Bethesda, Md.), and the programs blastp, blastn, blastx, tblastn, and tblastx used for sequence analysis on the Internet.

[0075] As used herein, a "nucleic acid" refers to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic, non-naturally occurring analogs thereof) and phosphodiester bonds between them. The term thus encompasses nucleotide polymers in which the nucleotides and the linkages between them are non-naturally occurring synthetic analogs, including, but not limited to, thiophosphates, phosphoramidates, methylphosphates, chiral methylphosphates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). These polynucleotides are synthesized using an automated DNA synthesizer. The term "oligonucleotide" generally refers to short polynucleotides, generally less than about 50 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also encompasses RNA sequences in which "U" replaces "T" (i.e., A, U, G, C).

[0076] This specification utilizes common symbols to describe nucleotide sequences, with the left-hand end of a single-stranded nucleotide sequence referred to as the 5' end and the left-hand direction of a double-stranded nucleotide sequence referred to as the 5' direction. The direction of adding 5' to 3' nucleotides to a nascent RNA transcript is referred to as the transcription direction. The DNA strand with the same sequence as the mRNA is referred to as the coding strand.

[0077] As used herein, "encoding" refers to the unique property of a nucleotide sequence in a polynucleotide, e.g., a gene, cDNA, or mRNA, that is used as a template for the synthesis of other polymers or macromolecules in biological processes and has a defined nucleotide sequence or a defined amino acid sequence and the biological characteristics produced thereby. Thus, a gene encodes a protein if transcription and translation of the mRNA produced by that gene produces the protein in a cell or other biological system. The coding strand (whose nucleotide sequence is the same as the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand (used as the gene or cDNA transcription template) are referred to as the encoded protein or other product of that gene or cDNA. Unless otherwise specified, "a nucleotide sequence encoding an amino acid sequence" refers to all nucleotide sequences that encode the same amino acid sequence, in overlapping form. Protein- and RNA-encoding nucleotide sequences can include intervening sequences.

[0078] As used herein, "plasmid" refers to a plasmid that is artificially constructed based on a natural plasmid and adapted for laboratory manipulation. The nucleic acid molecule is introduced into a host cell to produce a transformed host cell. A vector contains a nucleic acid sequence that allows replication in the host cell, such as an origin of replication, and also contains one or more selectable marker genes and other genetic elements of the field.

[0079] As used herein, "host cells," also called recipient cells, refer to host cells that receive foreign genes during transformation and transduction (infection).

[0080] As used herein, "pharmaceutically acceptable carrier" refers to a typical pharmaceutically acceptable carrier. Remington's Pharmaceutical Sciences, E.W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975) discloses compositions and formulations of one or more therapeutic compounds or molecules (e.g., one or more antibodies) and other agents for drug delivery applications.

[0081] As used herein, "diagnosis" of a disease refers to determining a patient's condition and progression of symptoms after testing the patient. "Prevention" of a disease refers to inhibiting the full development of a disease. "Treatment" refers to therapeutic intervention that ameliorates the signs or symptoms of a disease or medical condition after symptoms have begun to develop.

[0082] As used herein, "administration" refers to introducing the substance into a subject by an appropriate route. For example, if the selected route is intravenous, the composition is administered by introducing the substance into the subject's vein.

[0083] As used herein, an "effective prophylactic / therapeutic dose" refers to a specific amount of a particular drug that achieves the desired effect in a subject treated with that drug. The exact dosage depends on the purpose of treatment and can be determined by those skilled in the art using known techniques. The dosage range can be 0.01 to 100 mg / kg body weight or more, for example, 0.1, 1, 10, or 50 mg / kg body weight, preferably 1 to 10 mg / kg body weight. As known in the art, adjustments are necessary based on antibody or Fc fusion degradation, systemic or local drug delivery, and new protein enzyme synthesis rates, as well as age, body weight, general health status, sex, diet, administration time, drug interactions, and severity of the disease, and can be determined by those skilled in the art using routine experimental methods. Such reagents include the monomeric Fc domain molecules described herein. In a non-limiting example, this may be the amount of an HIV-specific monomeric Fc domain (or an HIV-specific CH3 domain molecule) that can prevent, treat, or ameliorate HIV infection. Ideally, a therapeutically effective amount of an antibody is an amount sufficient to prevent, treat, or ameliorate infection or disease resulting from, for example, HIV infection in a subject, without causing significant cytotoxicity to the subject. The amount of an agent effective to prevent, ameliorate, and / or treat in a subject is determined by the subject being treated, the type and severity of the affliction, and the form of administration of the therapeutic composition.

[0084] As used herein, "cancer" refers to a solid tumor or a blood cancer. The solid tumor of the present invention is a sarcoma or carcinoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, or other types of sarcoma, such as synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary epithelial carcinoma, papillary adenocarcinoma, cavernous carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, nephroblastoma, cervical cancer, testicular tumor, bladder cancer, or a tumor of the central nervous system (such as colloid carcinoma, astrocytoma, medulloblastoma, craniopharyngioma, ventriculoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, or retinoblastoma). Hematological cancers according to the present invention are leukemias, such as acute leukemias (e.g. acute lymphocytic leukemia, acute myeloid cell leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic, mononuclear and erythrocytic), chronic leukemias (e.g. chronic granulocytic (granulocytic) leukemia, chronic granulocytic leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (myeloid and high-grade), multiple myeloma, Bartenslom's macroglobulinemia, heavy chain coccosis, myeloproliferative disorder syndrome, multiple leukemia or myeloproliferative disorder.

[0085] Unless otherwise specified, the technical and scientific terms used above have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise clearly stated in the context, the singular terms "a," "one," and "the" include plural referents. It should also be understood that all base or amino acid sizes and all molecular weights or molecular weight values ​​described or provided in the description of nucleic acids or polypeptides are similar and are provided for descriptive purposes. Although methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, suitable methods and materials are described below. The term "comprises" means "including." All publications, patent applications, patents, and other references discussed above are incorporated by reference in their entirety. In case of conflict, the present specification (including interpretations of terms) will control. Furthermore, the materials, methods, and examples are illustrative only and not limiting. [Brief explanation of the drawings]

[0086] [Figure 1] Binding curve of mouse anti-L1CAM monoclonal antibody to HeLa cells. [Figure 2] Binding curves of anti-L1CAM human-mouse chimeric antibodies to HeLa cells and HeLa KO cells. [Example]

[0087] 1. Definition Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those of ordinary skill in the art.In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all terms and common procedures widely used in the field.At the same time, in order to better understand the present invention, the definitions and interpretations of related terms are provided below.

[0088] As used herein, the term "L1CAM" refers to any variant, homolog, or species homolog of L1CAM. L1CAM is a transmembrane glycoprotein with a molecular weight between 200,000 and 220,000 and is also known as CD171. Structurally, the extracellular portion of L1CAM contains, from the amino terminus of the protein, six immunoglobulin domains and five fibronectin type III overlapping sequences. Furthermore, L1CAM contains one transmembrane domain and one intracellular domain. In some embodiments, L1CAM is human L1CAM. Exemplary sequences of human L1CAM proteins are disclosed in NCBI Ref Seq No. NP_001265045.1, NCBI Ref Seq No. NP_000416.1, NCBI Ref Seq No. NP_076493.1, and NCBI Ref Seq No. NP_001137435.1. Exemplary predicted sequences for monkey L1CAM proteins are disclosed in NCBI Ref Seq No. XP_005594990.1, Ref Seq No. XP_005594993.1, Ref Seq No. XP_005594994.1, Ref Seq No. XP_005594995.1, and Ref Seq No. XP_005594996.1.

[0089] The term "antibody" as used herein refers to an immunoglobulin that binds to any specific antigen. A natural intact antibody contains two heavy (H) chains and two light (L) chains. Each mammalian heavy chain is composed of a variable region (VH) and first, second, third, and fourth (optionally) constant regions (CH1, CH2, CH3, and CH4, respectively). Mammalian light chains are classified as lambda or kappa, each composed of a variable region (VL) and a constant region. The variable regions of the light and heavy chains determine antigen binding. The variable region of each chain contains three highly variable regions called complementarity-determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR variants of the antibodies and antigen-binding fragments disclosed herein can be named and identified using the Kabat nomenclature system.

[0090] As used herein, the term "monoclonal antibody" refers to a population of identical antibodies, where each individual antibody molecule in the monoclonal antibody population is identical to the other antibody molecules. This property is in contrast to that of a polyclonal antibody population, which contains antibodies with many different sequences.

[0091] The term "bispecific antibody" as used herein refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and can bind to two different epitopes, which may be present on the same antigen or on two different antigens.

[0092] As used herein, the term "multispecific antibody" refers to an artificial antibody that has fragments derived from multiple different monoclonal antibodies and can bind to multiple different epitopes. The multiple epitopes may be present on the same antigen or on multiple different antigens.

[0093] The term "recombinant antibody" as used herein refers to an antibody that has been produced through recombinant genetic engineering techniques and expressed in an edited cell line or strain.

[0094] The term "chimeric antibody" as used herein refers to an antibody molecule in which the antibody constant region or a part thereof is replaced so that the antigen-binding site (variable region, CDR or part thereof) is connected to the constant region of a different species.

[0095] As used herein, the term "amino" refers to an organic compound containing an amino group (-NH) and a carboxyl group (-COOH) functional group and the characteristic side chain of each amino acid. The names of amino acids may be represented herein by standard single-letter or three-letter codes recognized in the art.

[0096] As used herein, "treatment" or "therapy" of a disease, condition or condition includes preventing or reducing the occurrence of the disease, condition or condition, reducing the rate of onset or development of the disease, condition or condition, reducing the risk of developing a disease, condition or condition, preventing or delaying the development of symptoms associated with the disease, condition or condition, reducing or halting symptoms associated with the disease, condition or condition, fully or partially reversing the development of the disease, condition or condition, curing the disease, condition or condition, or any combination thereof.

[0097] As used herein, the term "diagnosis" refers to the testing of a pathological state, disease or condition, for example, the testing of a cancer-related disease, or the testing of a subject that would benefit from a particular method of treatment.

[0098] II. Example The present invention will be further described below through examples, but the present invention is not limited to the scope of the examples described.

[0099] Example 1: Production of monoclonal antibodies against L1CAM To generate antibodies against L1CAM, Balb / c mice are immunized with recombinantly expressed human L1CAM antigen fragments. During the immunization procedure, blood is collected periodically to obtain serum samples and monitor the immune response. Mice with sufficient titers of anti-L1CAM antibodies are used in fusion experiments. Spleen cells and / or lymph node cells from the immunized mice are isolated and fused with mouse myeloma cell lines. ELISA analysis is performed using the human L1CAM extracellular domain recombinant protein to screen for hybridomas capable of producing L1CAM-specific antibodies from HeLa cells highly expressing human L1CAM and HEK293 cells highly expressing monkey L1CAM. These hybridomas are then subcloned to obtain stable hybridoma clones. After subcloning, the monoclonal hybridoma cells are subjected to antibody sequencing and submitted for further analytical testing.

[0100] In the present invention, mouse anti-L1CAM monoclonal antibodies are designated by "hybridoma clone number." For example, a mouse anti-L1CAM monoclonal antibody derived from hybridoma clone 25F8H8 is designated as "25F8H8."

[0101] Example 2: Production of chimeric antibodies against L1CAM Based on the results of the monoclonal hybridoma cell antibody sequencing, a human-mouse chimeric antibody was designed, with the CH1, CH2, and CH3 constant regions of human IgG1 and the VH and VL variable regions of a mouse anti-L1CAM monoclonal antibody. The chimeric antibody was recombinantly expressed in mammalian cells and purified for further analysis. In this specification, the chimeric antibody number is represented as "ch + hybridoma clone number." For example, a chimeric antibody derived from hybridoma clone 25F8H8 is represented as "ch25F8H8."

[0102] Example 3: Binding of mouse anti-L1CAM monoclonal antibodies to HeLa cells In this example, the binding of mouse anti-L1CAM monoclonal antibody to endogenously expressed human L1CAM on the cell surface of HeLa cells was measured. HeLa cells were cultured in DMEM complete medium (purchased from Corning) containing 10% FBS (purchased from Gibco) and 100 U / mL penicillin-streptomycin mixture (Thermofisher, 15140122). When the cell coverage in the culture vessel reached 80-90%, the HeLa cells were dissociated from the culture vessel using TrypLE (purchased from Thermofisher) and 4 x 10 6Cells were seeded into a 96-well plate (purchased from AXYGEN) at a density of 100,000 cells / ml per well (25 μL DMEM complete medium). 25 μL of diluted antibody-containing hybridoma supernatant was added to each well, mixed gently, and the 96-well plate was incubated at 4°C for 1 hour. Then, 150 μL of 2% FBS-containing PBS buffer (purchased from Corning) was added to each well, and the plate was centrifuged at 1400 rpm for 4 minutes. The supernatant was removed and the plate was washed once more. Next, 50 μL of a 1:800 diluted Alexa Flour 647-conjugated sheep anti-mouse fluorescent secondary antibody (purchased from Jackson Immuno) was added to the medium-containing wells, and the 96-well plate was incubated at 4°C for 0.5 hours. After washing the cells twice with 2% FBS-containing PBS, the cells were resuspended in 120 μL of PBS and the cell surface fluorescent signal was detected using a flow cytometer (BD FACS Celesta) to obtain the median fluorescence intensity (MFI). The mouse IgG concentration in the supernatant of the antibody-containing hybridoma was measured using an anti-mouse ELISA assay. Finally, a dose-response curve was generated and the half maximal effective concentration (EC50) was calculated using nonlinear regression using GraphPad software.

[0103] [Table 1]

[0104] Example 4: Binding of anti-L1CAM human-mouse chimeric antibody to human L1CAM endogenously expressed on HeLa cells In this example, we measured the binding of mouse anti-L1CAM monoclonal antibodies to endogenously expressed human L1CAM on the cell surface using HeLa cells and human L1CAM knockout HeLa cells (purchased from Abcam). Human L1CAM knockout HeLa cells are abbreviated as HeLa KO cells in this invention.

[0105] HeLa cells were cultured in DMEM complete medium (purchased from Corning) containing 10% FBS (purchased from Gibco) and 100 U / mL penicillin-streptomycin mixture (Thermofisher, 15140122). When the cell coverage in the culture vessel reached 80-90%, HeLa cells and Hela KO cells were dissociated from the culture vessel using TrypLE (purchased from Thermofisher). 4 x 10 6 Cells were seeded into a 96-well plate (purchased from AXYGEN) at a density of 100,000 cells / ml in each well (25 μL DMEM complete medium). A human-mouse chimeric antibody was then serially diluted in DMEM complete medium, and 25 μL of the diluted chimeric antibody solution was added to each well, mixed gently, and the 96-well plate was incubated at 4°C for 1 hour. Next, 150 μL of 2% FBS-containing PBS buffer (purchased from Corning) was added to each well, and the plate was centrifuged at 1400 rpm for 4 minutes. The supernatant was removed and the plate was washed once more. Next, 50 μL of a 1:800 diluted Alexa Flour 647-conjugated sheep anti-mouse fluorescent secondary antibody (purchased from Jackson Immuno) was added to the wells containing the medium, and the 96-well plate was incubated at 4°C for 0.5 hours. After washing the cells twice with 2% FBS-containing PBS, the cells were resuspended in 120 μL of PBS and the cell surface fluorescence signal was detected using a flow cytometer (BD FACS Celesta) to obtain the median fluorescence intensity (MFI). A dose-response curve was then generated and the half-effective concentration (EC50) was calculated using nonlinear regression in GraphPad software. HeLa cells endogenously express high levels of L1CAM, and because the L1CAM gene was knocked out in HeLa KO cells, L1CAM expression was significantly reduced compared to wild-type HeLa. Therefore, antibody binding to L1CAM on the HeLa cell surface was measured by flow cytometry, and antibody specificity was assessed by detecting the fluorescent signal on the HeLa KO cell surface.

[0106] [Table 2]

[0107] Example 5: Production of humanized antibodies against L1CAM The mouse anti-L1CAM monoclonal antibodies 46F1D5, 49E10H1, and 103E9B3 were selected for humanization. The antibody sequences were compared with the human sequences to determine the optimal model. Based on the homology of the original mouse antibody sequence, the most closely matched human sequence was used as the humanization template. Next, the CDRs from the mouse antibody sequence and residues that maintain the supracluster and central core structures of the antibody were grafted onto the template. Optimized mutations were introduced into the framework regions to generate humanized heavy chain variable region variants and humanized light chain variable region variants, which were then mixed and matched to provide a large number of humanized antibody clones. Recombinant expression was carried out in mammalian cells, followed by purification and further analytical testing.

[0108] Example 6 Measurement of humanized L1CAM antibody binding to HeLa cells The experimental principle is as follows: HeLa cells highly express L1CAM protein on their surface, so after culturing the cells with a humanized L1CAM antibody, the antibody's binding ability to L1CAM on the HeLa cell surface is measured by flow cytometry.

[0109] The experimental method was outlined as follows: A humanized antibody to L1CAM (human IgG1-Kappa) was serially diluted 4-fold using complete medium to obtain a total of seven concentration gradient points.

[0110] HeLa cells were cultured in DMEM complete medium (Corning, 10-013-CVR) containing 10% FBS (Gibco, 10099-141) and 100 U / mL penicillin-streptomycin mixture (Thermofisher, 15140122). When the cell coverage in the culture vessel reached 80-90%, the HeLa cells were dissociated from the culture vessel using TrypLE (Thermofisher, 12604-013). 2.2 × 10 6Cells were seeded into a 96-well plate (AXYGEN, P-96-450V-C) at a density of 100,000 cells / ml in 45 μL of complete medium per well. 5 μL of antibody dilution was then added to each well, mixed gently, and the 96-well plate was incubated at 4°C for 1 hour. Next, 150 μL of 2% FBS-containing PBS (Corning, 21-040-CVC) was added to each well, and the plate was centrifuged at 1400 rpm for 4 minutes. The supernatant was removed and the plate was washed once more. Next, 50 μL of a 1:800 diluted Alexa Flour 647-conjugated sheep anti-human fluorescent secondary antibody (Jackson Immuno, 109-605-088) was added to each well, and the 96-well plate was incubated at 4°C for 0.5 hours. After washing the cells twice with 2% FBS-containing PBS, resuspend the cells in 120 µL of PBS and detect the cell surface fluorescent signal using flow cytometry (BD FACS Celesta) to obtain the median fluorescence intensity (MFI). Then, generate a dose-response curve and calculate the half maximal effective concentration (EC50) using nonlinear regression using GraphPad software.

[0111] [Table 3]

[0112] [Table 4]

[0113] [Table 5]

[0114] In the present invention, the humanized antibody number is represented by "hz + hybridoma clone number + heavy chain number + light chain number", for example, the humanized antibody "hz46F1D5H1L2" refers to a humanized antibody derived from the mouse anti-L1CAM monoclonal antibody 46F1D5, and the variable region heavy chain is VH1 (i.e., SEQ ID NO: 60) and the variable region light chain is VL2 (i.e., SEQ ID NO: 65).

[0115] In the present invention, the human-mouse chimeric antibody number is indicated by "ch + hybridoma clone number." For example, the human-mouse chimeric antibody "ch46F1D5" refers to a human-mouse chimeric antibody derived from the mouse anti-L1CAM monoclonal antibody 46F1D5, and the variable heavy chain is the heavy chain variable region of the mouse monoclonal antibody 46F1D5, the variable light chain is the light chain variable region of the mouse monoclonal antibody 46F1D5, and the constant region is the constant region of human IgG1.

[0116] Antibody sequence information: [Table 6]

[0117] [Table 7]

[0118] [Table 8] TIFF0007755062000009.tif238167TIFF0007755062000010.tif242168TIFF0007755062000011.tif238167TIFF000 7755062000012.tif239167TIFF0007755062000013.tif241169TIFF0007755062000014.tif242170TIFF00077550620 00015.tif238169TIFF0007755062000016.tif238167TIFF0007755062000017.tif237170TIFF0007755062000018.t if238167TIFF0007755062000019.tif243167TIFF0007755062000020.tif239167TIFF0007755062000021.tif125169

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to L1CAM, comprising heavy chain CDRs and light chain CDRs selected from the group consisting of: (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; (b) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; and (c) HCDR1 comprising the amino acid sequence of SEQ ID NO: 31, HCDR2 comprising the amino acid sequence of SEQ ID NO: 32, HCDR3 comprising the amino acid sequence of SEQ ID NO: 33, LCDR1 comprising the amino acid sequence of SEQ ID NO: 34, LCDR2 comprising the amino acid sequence of SEQ ID NO: 35, and LCDR3 comprising the amino acid sequence of SEQ ID NO:

36.

2. 2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:49, or an amino acid sequence having at least 90%, at least 95% or more identity to SEQ ID NO:49, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:50, or an amino acid sequence having at least 90%, at least 95% or more identity to SEQ ID NO:

50. An antibody or antigen-binding fragment thereof, 3. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:49 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:50; or the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 72 to 74, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 70, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80 or 81; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 71, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 79 or 80. An antibody or antigen-binding fragment thereof,

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising an Fc region, optionally comprising a human immunoglobulin (Ig) Fc region, or optionally comprising a human IgG Fc region, wherein the Fc region is derived from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that the antigen-binding fragment is scFv, scFv-Fc, Fv, Fab, Fab', F(ab)2 or F(ab')2.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that it is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a bivalent antibody or a fusion protein.

7. An antibody-drug conjugate, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and another effector molecule conjugate.

8. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

9. A recombinant expression vector comprising the nucleic acid molecule of claim 8.

10. A plasmid comprising the nucleic acid molecule of claim 8.

11. A host cell characterized in that it contains the nucleic acid molecule of claim 8.

12. An ex vivo method for detecting L1CAM expression in a biological sample, comprising the steps of contacting the biological sample with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 3 and detecting the presence or absence of an immune complex, wherein the presence of the immune complex indicates the presence of L1CAM in the biological sample.

13. A pharmaceutical composition comprising, as an active substance, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

14. A test kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 for treating cancer in a subject.

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