Humanized L1CAM antibody-drug conjugate

Abstract

The present invention provides anti-L1CAM antibody-drug conjugates, methods for producing the same, and their pharmaceutically acceptable uses. In particular, the present invention provides antibody-drug conjugates (ADCs) comprising an anti-L1CAM antibody conjugated to MMAE or a derivative thereof; pharmaceutical compositions comprising ADCs; and their use in the manufacture of agents for treating L1CAM-mediated diseases or symptoms, particularly their use in the manufacture of anticancer agents.

Description

[Technical Field]

[0001] cross reference This patent application relates to Chinese Patent Application No. 202211317321.5, filed on 26 October 2022, and claims the benefit of its priority, the contents of which are incorporated as a whole by reference.

[0002] Technical field This invention relates to the fields of biology and medicine, and more particularly to humanized anti-L1CAM antibody-drug conjugates. [Background technology]

[0003] background L1 cell adhesion molecule (L1CAM), also known as CD171, is a transmembrane glycoprotein with a molecular weight in the range of 200,000–220,000 daltons [Miriam van der Maten et al., Int. J. Mol. Sci., 2019, 20:4180]. In healthy individuals, L1 cell adhesion molecule expression is mainly limited to the nervous system, with small amounts also found in kidney tissue and skin [Annette Kunkele et al., Clin. Cancer Res., 2016, 23:466-477]. L1 cell adhesion molecule is highly expressed in various tumors and is closely associated with the proliferation and drug resistance of several types of tumor cells.

[0004] L1 cell adhesion molecules hold great potential as targets for tumor therapy, but the development of therapeutic antibodies that specifically target L1 cell adhesion molecules remains necessary. Antibody-drug conjugates belong to a new class of anti-cancer biomissile drugs, consisting of three components: an antibody, a cytotoxin, and a linker that connects the two. After chemically binding the cytotoxin to a monoclonal antibody, the antibody-drug conjugate utilizes the targeting ability of the monoclonal antibody to specifically recognize and bind to receptors on the surface of cancer cells. Once bound to the receptor, the antibody-drug conjugate is internalized within the cancer cell, where the cytotoxin is released by intracellular proteases, preventing cancer cell proliferation and killing the cancer cells. Antibody-drug conjugate technology combines small molecule drugs and biological proteins, combining the advantages of both (significantly increasing efficacy and reducing toxicity and side effects), making it a next-generation therapeutic product.

[0005] The first clinically successful example of a targeted antibody-drug conjugate is gemtuzumab ozogamicin (Wyeth, trade name: Mylotarg). Mylotarg is the first monoclonal antibody conjugate approved for market. This drug consists of an anti-CD33 antibody, the DNA degrading agent calicheamicin, and the chemical linker AcBut. Mylotarg is a humanized anti-CD33 IgG4 antibody conjugated with the antitumor drug calicheamicin for the treatment of acute myeloid leukemia. Although Mylotarg is a first-generation monoclonal antibody conjugate, it has three fatal technical flaws. Firstly, the linker used to conjugate the cytotoxin was highly unstable with a half-life of only two days, resulting in undesirable drug release and severe toxicity in clinical applications. Secondly, the antibody is conjugated to the linker via the amino group of lysine, but because there are dozens of lysine molecules on the surface of the antibody, the binding site is random and therefore partially affects efficacy. More importantly, the binding technology at the time was underdeveloped, and only 50% of the antibodies could successfully bind to the drug, resulting in less-than-ideal clinical efficacy. Thirdly, the antibody used was an IgG4 antibody, which lacked antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). As a result, Mylotarg was withdrawn from the market 10 years after its launch due to its severe toxicity, side effects, and limited therapeutic efficacy.

[0006] The second clinically successful example of a targeted antibody-drug conjugate is a new drug for the treatment of Hodgkin lymphoma. Although this drug has only recently completed Phase 2 clinical trials, its remarkable efficacy led to its approval by the US FDA in 2011. This novel targeted antibody-drug conjugate (ADC), developed by Seattle Genetics, can be used as a targeted therapy to treat patients with two types of lymphoma that express the CD30 antigen. The antibody-drug conjugate, brentuximab, consists of an anti-CD30 monoclonal antibody, a microtubule inhibitor (monomethyl auristatin E, MMAE), and a dipeptide chemical linker. This antibody-drug conjugate is characterized by fewer side effects and a high lymphoma-suppressing effect. In a Phase 2 single-arm clinical trial, 102 patients aged 15 to 77 years (median age 31 years) with relapsed or refractory Hodgkin lymphoma received treatment with brentuximab for a median of 9 cycles. The overall response rate was 73%, with a median treatment duration of 6.7 months. The complete response rate was 34%, with a median treatment duration of 20.5 months. 40% of treated patients achieved a partial response. The most common side effect was peripheral neuropathy. The success of this drug demonstrates the technical feasibility and promising future of targeted antibody-drug conjugates.

[0007] Another successful example of targeted antibody-drug conjugates is T-DM1, developed by Genentech Inc. for the treatment of malignant breast cancer. The monoclonal antibody in this conjugate targets HER2 (ErbB2) on the surface of breast cancer cells, while the conjugated cytotoxic substance is the microtubule inhibitor DM1. Phase 3 clinical trials of this drug demonstrated greater efficacy and fewer toxicity and side effects compared to chemotherapy. While breast cancer patients have previously been treated with chemotherapy drugs such as Herceptin and taxanes, their disease continues to progress. However, antibody-drug conjugate therapy has significantly extended the survival time of HER2-positive breast cancer patients without worsening their condition. Based on its promising efficacy, the U.S. Food and Drug Administration (FDA) approved T-DM1 on February 22, 2013, for the treatment of patients with HER2-positive advanced metastatic breast cancer. In particular, 2019 saw the approval of polatuzumab vedotin (brand name Polivy), enfortumab vedotin (brand name Padcev), and famtrastuzumab deruxtecan (brand name Enhertu), which further fueled the pharmaceutical industry's enthusiasm for developing new ADC therapies. [Overview of the project] [Problems that the invention aims to solve]

[0008] This disclosure provides an L1CAM-targeted antibody-drug conjugate comprising an antibody or antigen-binding fragment conjugated to one or more therapeutic agents, wherein the antibody or antigen-binding fragment comprises 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, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of heavy chain variable region sequences selected from the group consisting of SEQ ID NOs: 21, SEQ ID NOs: 23, SEQ ID NOs: 25, SEQ ID NOs: 27, SEQ ID NOs: 29, and SEQ ID NOs: 31 in a ratio of at least 80%, 90%, or 100% The light chain variable region has HCDR1, HCDR2, and HCDR3 having % identity, and the light chain variable region has LCDR1, LCDR2, and LCDR3 having at least 80% identity with the light chain variable region sequences selected from the group consisting of SEQ ID NOs: 22, SEQ ID NOs: 24, SEQ ID NOs: 26, SEQ ID NOs: 28, SEQ ID NOs: 30, and SEQ ID NOs: 32, wherein the therapeutic agent is conjugated to the antibody or antigen-binding fragment via a linker. [Means for solving the problem]

[0009] In one specific embodiment, the heavy chain variable region and the light chain variable region have a set of CDRs selected from the group consisting of: (1) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with the heavy chain variable sequence HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 21, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with the light chain variable region sequence LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 22; (2) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with the heavy chain variable sequence HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with the light chain variable region sequence LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 24; (3) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with the heavy chain variable sequence HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 25, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with the light chain variable region sequence LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 26; (4) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with HCDR1, HCDR2, and HCDR3 of the heavy chain variable sequence of SEQ ID NO: 27, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 28; (5) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with HCDR1, HCDR2, and HCDR3 of the heavy chain variable sequence of SEQ ID NO: 29, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 30; (6) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with HCDR1, HCDR2, and HCDR3 of the heavy chain variable sequence of SEQ ID NO: 31, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 32.

[0010] In one specific embodiment, (1) The HCDR1 has at least 80%, at least 90%, or 100% identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 16; (2) The HCDR2 has at least 80%, at least 90%, or 100% identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 17; (3) The HCDR3 has at least 80%, at least 90%, or 100% identity with a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 18; (4) LCDR1 has at least 80%, at least 90%, or 100% identity with a sequence selected from the group consisting of sequence number 4 and sequence number 13; (5) The LCDR2 has at least 80%, at least 90%, or 100% identity with a sequence selected from the group consisting of sequence number 5, sequence number 14, and sequence number 19; (6) LCDR3 has at least 80%, at least 90%, or 100% identity with a sequence selected from the group consisting of sequence number 6, sequence number 15, and sequence number 20.

[0011] In one specific embodiment, (1) The HCDR1 comprises the amino acid sequence described in Sequence ID No. 1; (2) The HCDR2 comprises the amino acid sequence described in Sequence ID No. 2; (3) The HCDR3 comprises the amino acid sequence described in Sequence ID No. 3; (4) The LCDR1 comprises the amino acid sequence described in Sequence ID No. 4; (5) The LCDR2 comprises the amino acid sequence described in Sequence ID No. 5; (6) The LCDR3 comprises the amino acid sequence described in Sequence ID No. 6.

[0012] In one specific embodiment, (1) The HCDR1 comprises the amino acid sequence described in Sequence ID No. 7; (2) The HCDR2 contains the amino acid sequence set forth in SEQ ID NO: 8; (3) The HCDR3 contains the amino acid sequence set forth in SEQ ID NO: 9; (4) The LCDR1 contains the amino acid sequence set forth in SEQ ID NO: 4; (5) The LCDR2 contains the amino acid sequence set forth in SEQ ID NO: 5; (6) The LCDR3 contains the amino acid sequence set forth in SEQ ID NO: 6.

[0013] In one specific embodiment, (1) The HCDR1 contains the amino acid sequence set forth in SEQ ID NO: 10; (2) The HCDR2 contains the amino acid sequence set forth in SEQ ID NO: 11; (3) The HCDR3 contains the amino acid sequence set forth in SEQ ID NO: 12; (4) The LCDR1 contains the amino acid sequence set forth in SEQ ID NO: 13; (5) The LCDR2 contains the amino acid sequence set forth in SEQ ID NO: 14; (6) The LCDR3 contains the amino acid sequence set forth in SEQ ID NO: 15.

[0014] In one specific embodiment, (1) The HCDR1 contains the amino acid sequence set forth in SEQ ID NO: 16; (2) The HCDR2 contains the amino acid sequence set forth in SEQ ID NO: 17; (3) The HCDR3 contains the amino acid sequence set forth in SEQ ID NO: 18; (4) The LCDR1 contains the amino acid sequence set forth in SEQ ID NO: 13; (5) The LCDR2 contains the amino acid sequence set forth in SEQ ID NO: 19; (6) The LCDR3 contains the amino acid sequence set forth in SEQ ID NO: 20.

[0015] In one specific embodiment, the heavy chain variable region contains any one amino acid sequence selected from the group consisting of: (1) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 21; (2) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 23; (3) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 25; (4) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 27; (5) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 29; (6) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 31.

[0016] In one specific embodiment, the light chain variable region includes one amino acid sequence selected from the group consisting of: (1) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 22; (2) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 24; (3) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 26; (4) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 28; (5) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 30; (6) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 32.

[0017] In one specific embodiment, the antibody is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a bivalent antibody, an anti-idiotype antibody, or a fusion protein.

[0018] In one specific embodiment, the linker is attached to the antibody via a thiol group.

[0019] In one specific embodiment, the therapeutic agent includes, but is not limited to, any one of auristatin cytotoxic molecules (e.g., MMAE and MMAF), maytansinoid cytotoxic molecules (e.g., DM1 and DM4), ansamycin derivative pyrrolobenzodiazepine (PBD), camptothecin and camptothecin derivatives (e.g., exatecan and Dxd), or any combination thereof.

[0020] The present invention provides an antibody-drug conjugate having the structure of formula (I) below: [Chemical formula] [Wherein, "Ab" refers to an antibody or antigen-binding fragment selected from any of the antibodies or antigen-binding fragments described herein; [[ID=1⑧]]Wherein, L refers to a cleavable or non-cleavable linker; Wherein, D refers to a therapeutic agent; Wherein, n preferably refers to a drug-to-antibody ratio (DAR) in the range of 1 to 8].

[0021] In a preferred embodiment, L refers to a cleavable linker.

[0022] In a preferred embodiment, L refers to -L1-L2-L3-, wherein L1 is -(CH2) n -X-Y-(CH2) m -, -X(CH2) n -O-(CH2CH2O) p -(CH2) m -Y-, -(CH2) n -X-, -X-(CH2) m -Y- or any combination thereof, wherein X and Y are each independently -C(O)-, O, -CR 1 R 2 -, -NR 1 -, S or does not exist, wherein R1 and R 2 Each of the following independently represents hydrogen, a C1-C6 alkyl group, or a C3-C6 cycloalkyl group, where m, n, and p each independently represent an integer between 0 and 10; In the formula, L2 refers to an amino acid residue; In the formula, L3 is either nonexistent or refers to -C(O)- or -PAB-C(O); In the formula, PAB refers to p-aminobenzyloxy.

[0023] In a preferred embodiment, L2 refers to one of -Val-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, Ala-Ala-Asn-, -Val-Lys, -Phe-Lys, -Phe-Cit, -Phe-Arg-, -Phe-Ala-, -Ala-Lys, -Leu-Cit-, -Ile-Cit-, -Trp-Cit-, -D-Phe-LPhe-Lys-, -Phe-Phe-Lys-, -D-Phe-Phe-Lys-, -Gly-Phe-Lys-, -Gly-Phe-Leu-Gly-, Ala-Leu-Ala-Leu, or any combination thereof.

[0024] In a preferred embodiment, L refers to -(CH2)5-Val-Cit-PAB-C(O)- and -(CH2)5-Val-Ala-PAB-C(O)-.

[0025] In a preferred embodiment, the therapeutic agent is selected from one of MMAE, MMAF, Dxd, DM1, DM4, or any combination thereof.

[0026] Furthermore, the present invention provides an antibody-drug conjugate having the following structure: [ka] or [ka] [In the formula, "Ab" refers to an antibody or antigen-binding fragment selected from any of the antibodies or antigen-binding fragments described above in the present invention, and in the formula, n refers to the drug-to-antibody ratio (DAR), which is preferably in the range of 1 to 8.]

[0027] Furthermore, the present invention also provides pharmaceutical compositions comprising an antibody-drug conjugate disclosed herein and a pharmaceutically acceptable carrier. [Brief explanation of the drawing]

[0028] [Figure 1] Figure 1. Affinity of humanized anti-L1CAM antibody-drug conjugates (anti-L1CAM ADCs) for L1CAM on the cell surface.

[0029] [Figure 2] Figure 2. Binding specificity of humanized anti-L1CAM antibody-drug conjugates (anti-L1CAM ADCs) to L1CAM on the cell surface.

[0030] [Figure 3] Figure 3. Cytotoxicity of humanized anti-L1CAM antibody-drug conjugates (anti-L1CAM ADCs) against HeLa cells.

[0031] [Figure 4] Figure 4. Cytotoxicity of humanized anti-L1CAM antibody-drug conjugates (anti-L1CAM ADCs) against HeLa L1CAM KO cells. [Modes for carrying out the invention]

[0032] Detailed explanation To better understand the disclosure, definitions and explanations of relevant terms are provided below. These definitions are intended to encompass grammatically equivalents and other variations.

[0033] As used herein, the term “antibody” refers to a protein consisting of one or more polypeptides encoded substantially all or part of a recognized immunoglobulin gene. Such immunoglobulin genes include, for example in humans, the kappa (κ), lambda (λ), and heavy chain loci, which include multiple genes encoding variable regions, as well as genes encoding the constant regions of IgM, IgD, IgG, IgE, and IgA isotypes mu (μ), delta (δ), gamma (γ), epsilon (ε), and alpha (α), respectively. Antibodies disclosed herein include full-length antibodies and antibody fragments, as well as naturally occurring antibodies of any biological origin, engineered antibodies, or recombinantly produced antibodies for experimental, therapeutic, or other purposes as further defined below. The term “antibody” includes Fab, Fab' 、 This includes antibody fragments known in the art, such as F(ab')2, Fv, scFv, or other subsequences of antibodies for antigen binding, or antibody fragments produced by modification of full-length antibodies, or antibody fragments newly synthesized using recombinant DNA technology. The term "antibody" includes monoclonal and polyclonal antibodies. The antibody may also be an antagonist, agonist, neutralizing antibody, inhibitory antibody, or stimulating antibody. The antibody of the present invention may be a non-human antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0034] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population; that is, a population containing unique antibodies is identical except for the presence of a few potential mutations (e.g., spontaneous mutations). Therefore, the term "monoclonal" indicates the nature of the antibody, i.e., it is not a mixture of unrelated (individual) antibodies. Typically, in contrast to polyclonal antibody preparations, which contain antibodies against different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single antigenic determinant on an antigen. In addition to specificity, an advantage of monoclonal antibody preparations is usually the avoidance of contamination by other immunoglobulins. The term "monoclonal" should not be interpreted as meaning that the antibody must be produced in a specific manner. The term monoclonal antibody specifically includes humanized antibodies.

[0035] As used in this disclosure, the term “complementarity-determining regions” (CDRs, e.g., CDR1, CDR2, and CDR3) refers to amino acid residues within the variable regions of an antibody, the presence of which these CDRs are essential for antigen binding. The “antigen-binding region” of an antibody is typically located within one or more ultra-high variable regions of the antibody, such as the CDR regions of CDR1, CDR2, and CDR3. Each complementarity-determining region may contain amino acid residues from “complementarity-determining regions” as defined by Kabat, including, for example, residues 24–34 (L1), 50–56 (L2), and 89–97 (L3) of the light chain variable region, and residues 26–33 (H1), 51–57 (H2), and 96–105 (H3) of the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, 1991). In some embodiments, the CDR regions as defined herein utilize the Kabat scheme. CDRs within an antibody sequence can also be identified according to other common definitions developed in the art (e.g., Chothia, IMGT, Contact numbering systems), or by comparing the sequence to a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel edited, Antibody Engineering, Springer, New York, NY, 2001; and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. The same antibody disclosed herein may have different sets of CDRs when determined by different numbering schemes. Disclosure of CDRs determined by one numbering scheme herein includes disclosure of CDRs that may be determined by other numbering schemes. Two antibodies are considered to have the same CDRs if the CDRs are identical when determined by the same scheme.

[0036] As used herein, the term “antigen” refers to a compound, composition, or substance containing a composition that is injected into or absorbed by an animal, which can stimulate the production of an antibody or T-cell response within the animal, and the antigen may be a protein, carbohydrate, lipid, or other pathogen.

[0037] A “functional fragment” or “antigen-binding fragment” of an antibody / immunoglobulin as defined herein is a fragment of an antibody / immunoglobulin that holds an antigen-binding region (e.g., the variable region of IgG). The “antigen-binding region” of an antibody is usually located within one or more hypervariable regions of the antibody, such as the CDR1, CDR2, and / or CDR3 regions. However, variable “framework” regions can also play an important role in antigen binding, for example, by providing a scaffold for the CDR. Preferably, the “antigen-binding region” includes 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 even more preferably the full-length VL and VH chains (amino acids 1-109 of the VL and amino acids 1-113 of the VH, numbered according to WO97 / 08320). The preferred immunoglobulin class used in the present invention is IgG.

[0038] As used herein, the term “amino acid” refers to either one of the 20 natural amino acids or one of its non-natural analogs, which can be positioned in specific locations. As used herein, the term “protein” refers to at least two covalently bonded amino acids, including proteins, polypeptides, oligopeptides, and peptides. Proteins may be composed of natural amino acids and peptide bonds, or they may be composed of synthetic peptide-mimicking structures, which are called “analogs.” Therefore, as used herein, “amino acid” or “peptide residue” refers to both natural and synthetic amino acids. For example, for the purposes of the present invention, homophenylalanine, citrulline, and norleucine are considered amino acids used for the purposes of the present invention. “Amino acids” also include imino acid residues such as proline and hydroxyproline. Side chains can take the (R) or (S) configuration. In preferred embodiments, amino acids exist in the (S) or (L) configuration. When non-natural side chains are used, non-amino acid substitutions may be used for purposes such as preventing or delaying degradation in vivo.

[0039] As used herein, the term “identity” is intended to refer to the similarity between two or more nucleotide or amino acid sequences, or to sequence identity. Sequence identity is usually measured as a percentage of identity (or similarity or homology). A higher percentage indicates greater similarity between the two sequences. When sequences are aligned using standard methods, homologous genes or variants exhibit a relatively high degree of sequence identity. Methods for aligning sequences for comparison are well established in the art. Various programs and sorting algorithms are described in Smith and Waterman, Adv Appl. Math., 2: 482, 1981; Needleman 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; and Altschul et al., Nature Genet., 1994, 6: 119-129.

[0040] The NCBI Basic Local Sorting Search Tool (BLAST®) (Altschul et al., J. Mol. Biol., 215: 403-410, 1990) is available from multiple sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet, as well as the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0041] As used herein, the term “nucleic acid” refers to polymers consisting of nucleotide units linked by phosphodiester bonds (ribonucleotides, deoxyribonucleotides, related natural structural variants, and unnatural synthetic analogs thereof). Therefore, this term includes nucleotide polymers, where the nucleotides and the bonds between them include, but are not limited to, unnatural synthetic analogs such as phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). These polynucleotides can be synthesized, for example, using automated DNA synthesizers. The term “oligonucleotide” usually refers to short polynucleotides with a length of approximately 50 nucleotides or less. It should be understood that when a nucleotide sequence is represented as a DNA sequence (i.e., A, T, G, C), this also includes the case where “T” is replaced by “U” in an RNA sequence (i.e., A, U, G, C).

[0042] In this specification, nucleotide sequences are described using conventional notation: the 5' end of a single-stranded nucleotide sequence is at the left end, and the 5' direction is called the leftward direction in a double-stranded nucleotide sequence. The direction in which nucleotides are added to a growing RNA transcript is called the transcription direction (5' to 3'). The DNA strand that has the same sequence as the mRNA is called the coding strand.

[0043] As used herein, the terms “coding,” “encoding,” or “encoded” refer to the inherent properties of a particular nucleotide sequence within a polynucleotide such as a gene, cDNA, or mRNA, which functions as a template for the synthesis of other polymers or macromolecules in biological processes having a distinct nucleotide sequence, or to a defined amino acid sequence and the resulting biological properties. Thus, if a protein is produced in a cell or other biological system by the transcription and translation of mRNA produced by a gene, that gene codes for that protein. The coding strand (having the same nucleotide sequence as the mRNA and usually provided in the sequence listing) and the non-coding strand (functioning as a transcription template, such as a gene or cDNA) refer to the gene or cDNA encoding a protein or other product. Unless otherwise specified, “nucleotide sequences encoding an amino acid sequence” include all nucleotide sequences that are degenerate in form and code for the same amino acid sequence. Protein and RNA-coding nucleotide sequences may contain introns.

[0044] As used herein, the term "plasmid" refers to a plasmid artificially constructed based on a naturally occurring plasmid for adaptation to an experimental procedure. A nucleic acid molecule can be introduced into a host cell, thereby generating a transformed host cell. A plasmid may contain a nucleic acid sequence that enables replication within the host cell, such as a replication origin, and may also contain one or more select marker genes as well as other genetic elements known in the art.

[0045] As used herein, the term "host cell," also known as a receptive cell, refers to a cell that receives foreign genes through transformation and transduction (infection).

[0046] As used herein, the term "pharmaceutically acceptable carrier" means a conventional pharmaceutically acceptable carrier known in the art. Compositions and formulations suitable for the delivery of one or more therapeutic compounds or molecules (e.g., one or more antibodies) and additional pharmaceuticals are described in Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975).

[0047] As used herein, “diagnosis” of a disease means determining the patient’s condition and progression after examination. With respect to a particular disease, the terms “prevent,” “preventing,” or “preventing” mean completely suppressing the progression of the disease. “Treatment,” “treating,” or “treated” means a therapeutic intervention aimed at improving the signs or symptoms of a disease or condition after they have begun to progress.

[0048] As used herein, the terms “administering,” “administering,” or “administered” refer to the process of introducing a substance into a subject through an appropriate route. For example, if the selected route of administration is intravenous, the composition is administered by introducing the substance into the subject’s vein.

[0049] As used herein, the terms “preventive / therapeutic effective dose / dosage” refer to the amount of a particular drug that is effective in achieving several desired effects in a subject being treated with the drug. The exact dose varies depending on the therapeutic purpose and can be determined by a person skilled in the art using well-established techniques. The dose range is 0.01 to 100 mg / kg body weight or more, for example, 0.1, 1, 10, or 50 mg / kg body weight, with a preferred range of 1 to 10 mg / kg body weight. As is known in the art, adjustments may be necessary with respect to the degradation of antibodies or Fc fusions, systemic or topical drug delivery, and the rate of nascent protein enzyme synthesis, as well as age, body weight, overall health, sex, diet, timing of administration, drug interactions, and severity of symptoms, which can be determined by a person skilled in the art using conventional experimental methods. Such drugs include monomer Fc domain molecules described herein. In one non-limiting example, this may be a certain amount of HIV-specific monomer Fc domain (or HIV-specific CH3 domain molecule) for use in the prevention, treatment, or improvement of HIV infection. Optimally, the therapeutically effective dose of an antibody is the amount sufficient to prevent, treat, or improve an infection or disease caused by HIV infection or other factors in the subject without causing significant cytotoxic effects in the subject. The therapeutically effective dose of a drug used for prevention, improvement, and / or treatment of a subject varies depending on the subject being treated, the type and severity of pain, and the method of administration of the therapeutic composition.

[0050] As used herein, the term "cancer" refers to a solid tumor or a hematogenous carcinoma. The solid tumors described herein are sarcomas or carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma or other sarcomas, synovial sarcoma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colorectal 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 carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, nephroblastoma, cervical cancer, testicular tumors, bladder cancer, or tumors of the central nervous system (for example, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, or retinoblastoma). The hematogenous carcinomas described herein include leukemia, for example, acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythrocytic leukemia); chronic leukemia (e.g., chronic granulocytic (granulocyte) leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia); polycythemia vera; lymphoma; Hodgkin's disease; non-Hodgkin lymphoma (low-grade and progressive); multiple myeloma; Waldenström macroglobulinemia; heavy chain disease; myelodysplastic syndrome; hairy cell leukemia; or myelodysplasia.

[0051] Unless otherwise defined, technical and scientific terms used in connection with this disclosure have meanings generally understood by those skilled in the art. Unless explicitly stated in the context, the singular terms “a,” “an,” and “the” used herein and in the appended claims refer to multiple subjects. It should also be understood that all base sizes or amino acid sizes, and all molecular weights or molecular weight values, of nucleic acids or polypeptides are approximations and are provided for illustrative purposes only. When carrying out or testing this disclosure, methods and materials similar to or equivalent to those described herein may be used, but suitable methods and materials are described below. The term “comprising” means “including.” All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety. In case of any conflict, this specification (including definitions of terms) shall prevail. Furthermore, the materials, methods, and examples provided herein are for illustrative purposes only and are not limiting.

[0052] Anti-L1CAM antibody The antibodies of the present invention include antibodies of all classes (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibodies may be chimeric or humanized monoclonal antibodies.

[0053] Antibodies typically consist of a heavy chain variable region (VH) and a light chain variable region (VL). The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) separated by relatively conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs arranged in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites.

[0054] In some embodiments, the antibody or its antigen-binding moiety includes: a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6 (as shown in antibody hz26A12C1H4L3).

[0055] In some embodiments, the antibody or its antigen-binding portion includes: a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 9, a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6 (as shown in antibody hz46F1D5H4L3).

[0056] In some embodiments, the antibody or its antigen-binding moiety includes: a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 12, a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 15 (as shown in antibody hz49E10H1H5L6).

[0057] In some embodiments, the antibody or its antigen-binding moiety includes: a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 16, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 18, a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 15 (as shown in antibody hz49E10H1H5L7).

[0058] In some embodiments, the antibody or its antigen-binding moiety includes: a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 16, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 18, a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 20 (as shown in the antibody hz103E9B3H5L5 or hz103E9B3H6L5).

[0059] In some embodiments, the antibody or its antigen-binding portion includes a heavy chain variable region (VH) and a light chain variable region (VL): VH includes: (i) the amino acid sequence described in any one of SEQ ID NOs: 21, 23, 25, 27, 29, or 31; or (ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to any one of the amino acid sequences described in SEQ ID NOs: 21, 23, 25, 27, 29, or 31, and that maintains specific binding affinity to L1CAM when bound to the VL region; and VL includes: (i) any one of the amino acid sequences described in SEQ ID NOs: 22, 24, 26, 28, 30, or 32; or (ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to any one of the amino acid sequences described in SEQ ID NOs: 22, 24, 26, 28, 30, or 32, and that retains specific binding affinity to L1CAM when bound to the VH region.

[0060] Preferably, the variants having at least 85%, 90%, or 95% identical amino acid sequences as described above include mutations located in the framework region outside the CDR region, for example, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions in the framework region. Preferably, the substitutions are conservative substitutions.

[0061] As used herein, the term “conservative substitution” refers to an amino acid substitution that does not adversely affect or alter the essential properties of a protein / polypeptide, including its amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, for example, a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., in terms of size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds). Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with alkaline side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0062] The antibodies and antigen-binding fragments provided herein may further comprise a human IgG constant region, the human IgG constant region comprising an Fc region and optionally a hinge region. The human IgG constant region may be a human IgG1, IgG2, IgG3, or IgG4 constant region. In some embodiments, the antibody comprises a human IgG1 or IgG4 Fc region. The Fc region may be a wild-type Fc region, or it may comprise one or more amino acid modifications that alter antibody-dependent cell-mediated cytotoxicity (ADCC) or other effector function (e.g., Leu234Ala / Leu235Ala or LALA substitution).

[0063] Linker - Drug part When antibody-drug conjugates are used for the local delivery of cytotoxic or cytostatic agents, i.e., drugs that kill or inhibit tumor cells in cancer treatment, targeted delivery of the drug portion to the tumor and intracellular accumulation there are possible. In contrast, systemic administration of the unconjugated drug can result in unacceptable levels of toxicity not only to the tumor cells being targeted but also to normal cells (Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”) in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506). Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87).

[0064] The drugs available for use in ADCs disclosed herein include chemotherapeutic agents, such as daunomycin, adriamycin, methotrexate, and vindesine; toxins, such as bacterial toxins like diphtheria toxin, plant toxins like lysine, and small molecule toxins like geldanamycin, meitansinoids, and calicheamicin; and auristatin peptides, auristatin E (AE), and monomethyl auristatin E (MMAE) (these are synthetic analogs of drastatin). MMAE is a synthetic derivative of drastatin 10, a naturally occurring cell growth inhibitory pseudopeptide. Toxins can exert cytotoxic and cytostatic effects through mechanisms such as tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to become inactive or lose activity when bound to large antibodies or protein receptor ligands.

[0065] The drugs and linkers that can be used in the binding process of this disclosure are not particularly limited, as long as the drug molecule has an antitumor, antiviral, or antibacterial effect and contains at least one substituent or substructure that enables connection to the linker structure, and the linker contains at least two reactive groups, one of which can covalently bind to the drug molecule and the other can covalently bind to the antibody. Preferably, the linker is susceptible to -SH attack from the antibody and can form a binding with the antibody.

[0066] Depending on the desired drug and the selected linker, those skilled in the art can select an appropriate method for linking them. For example, several conventional coupling methods, such as amine coupling, can be used to form a desired drug-linker complex that still contains a reactive group for binding to an antibody via covalent bond. A drug-maleimide complex (i.e., a maleimide-bound drug) is given as an example of a payload having a reactive group in this disclosure.

[0067] In one embodiment, the drug includes, but is not limited to, cytotoxic reagents such as chemotherapeutic agents and immunotherapy drugs, antiviral agents, and antibacterial agents. In one embodiment, the drug conjugated to the antibody can be selected from, but is not limited to, MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), MMAF (monomethyl auristatin F), and the like.

[0068] In ADC preparation, the most common reactive group that can be bonded to a thiol group is maleimide. Furthermore, organobromids and iodides are also frequently used.

[0069] Usable enzyme-active toxins and their fragments include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modexin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, geronin, mitogenin, restrictosin, phenomycin, enomycin, and trichothecenes. See, for example, WO93 / 21232, published on October 28, 1993. Various radioisotopes are available for the production of radiobound antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. One or more small molecule toxins, such as calicheamycin, meitansinoids, drastatin, auristatin, trichothecene, and CC1065, and derivatives of these toxins having toxic activity, can be conjugated to antibodies by the methods disclosed herein.

[0070] Suitable meitansin compounds for use as meitansinoid drug moieties are meitansinol and meitansinol analogs, which are known in the art and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or produced by synthesis according to known methods. Suitable meitansinoids are disclosed, for example, in U.S. Patent No. 5,208,020. Suitable meitansinoids include meitansinol and meitansinol analogs modified at the aromatic ring or other positions of the meitansinol molecule, such as various meitansinol esters.

[0071] Drastatin and auristatin have been shown to inhibit microtubule dynamics, GTP hydrolysis, and nuclear and cell division, as well as possessing anticancer and antifungal effects (Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965, 1998). The drug moiety of drastatin or auristatin can be conjugated to antibodies via the N (amino) or C (carboxyl) terminus of the peptide drug moiety (WO02 / 088172). Exemplary embodiments including MMAE or MMAF and various linker components are shown below. For example, VcMMAE (Mc-vc-PABC-MMAE) is obtained using MMAE conjugated via p-aminobenzyloxycarbonyl ("PABC") to valine-citrulline (vc), a lysosome-cleavable dipeptide, and a thiol-reactive maleimidocaproyl spacer (Mc).

[0072] Antibody-cytotoxic drug conjugates can be created using a variety of difunctional protein coupling agents, which include, for example, N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidylsberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bisactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene, etc.). For example, lysine immunotoxins can be prepared as described in Vitetta et al (1987) Science, 238:1098. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (WO94 / 11026).

[0073] This disclosure also encompasses exemplary embodiments including MMAE or MMAF and various linker components. For example, VcMMAE (Mc-vc-PABC-MMAE) is obtained by using MMAE conjugated via p-aminobenzyloxycarbonyl ("PABC") to valine-citrulline (vc), a lysosome-cleavable dipeptide, and a thiol-reactive maleimidocaproyl spacer (MC).

[0074] Applications of ADCs In one embodiment, the disclosure relates to the use of an antibody-drug conjugate prepared by the method described above in the manufacture of a pharmaceutical composition or kit for treating a symptom or disease in a subject. In one embodiment, the disclosure also relates to a method for treating a subject with cancer, the method comprising administering to a subject in need of treatment a therapeutically effective amount of an antibody-drug conjugate to treat or prevent a symptom or disease. The subject may be a mammal, such as a human. The symptom or disease to be treated may be cancer, particularly cancer associated with L1CAM. In certain embodiments, the cancer may be selected from breast cancer, cholangiocarcinoma, melanoma, pancreatic cancer, glioma, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer. [Examples]

[0075] The present invention will be further described by embodiments, but this does not limit the present invention to the scope of the embodiments described.

[0076] Example 1: Preparation of a monoclonal antibody against L1CAM Balb / c mice were immunized with recombinantly expressed human L1CAM antigen fragments to generate L1CAM antibodies. During the immunization process, blood samples were periodically collected from the mice to obtain serum samples and track the immune response. Mice with sufficient titers of anti-L1CAM antibodies were used in fusion experiments. Splenocytes and / or lymph node cells from immunized mice were isolated and fused with mouse myeloma cell lines. ELISA analysis was performed using recombinant proteins of the extracellular domain of human L1CAM. Hybridomas capable of producing L1CAM-specific antibodies were screened using HeLa cells highly expressing human L1CAM and HEK293 cells highly expressing monkey L1CAM, and subcloning was performed to obtain stable hybridoma clones. After subcloning, monoclonal hybridoma cells were sequenced for further analysis.

[0077] Example 2: Preparation of a chimeric antibody against L1CAM Based on the sequencing results of monoclonal hybridoma cell antibodies, human-mouse chimeric antibodies were designed with human IgG1 CH1, CH2, and CH3 as constant regions and mouse anti-L1CAM monoclonal antibody VH and VL as variable regions. These were recombinantly expressed in mammalian cells, purified, and further analyzed and tested.

[0078] Example 3: Preparation of a humanized antibody against L1CAM A mouse anti-L1CAM monoclonal antibody is selected for humanization. The antibody sequence is aligned with a human germline sequence to determine the best-fitting model. Based on homology with the original mouse antibody sequence, the most matching human germline sequence is selected as the humanization template. The CDR of the mouse antibody sequence is transplanted into the template along with residues that preserve the upper and central core structure of the antibody. Optimized mutations are introduced into the framework region to generate variants of the humanized heavy chain variable region and humanized light chain variable region, which are then mixed and matched to obtain multiple humanized antibody clones. These are recombinantly expressed in mammalian cells and purified for further analytical testing.

[0079] Example 4: Measurement of binding of humanized anti-L1CAM antibody to the surface of HeLa cells The experimental principle is as follows: HeLa cells express high levels of L1CAM protein on their surface. After incubating humanized L1CAM antibodies with the cells, the antibody's binding ability to L1CAM on the HeLa cell surface can be measured by flow cytometry.

[0080] The experimental method is as follows: Humanized anti-L1CAM antibody (human IgG1-kappa) is serially diluted fourfold in complete culture medium at seven concentration gradient points.

[0081] HeLa cells are cultured in DMEM complete medium (Corning, 10-013-CVR) containing 10% FBS (Gibco, 10099-141) and a 100 U / mL penicillin-streptomycin mixture (Thermofisher, 15140122). When the cell percentage in the culture vessel reaches 80-90%, the HeLa cells are digested from the culture vessel using TrypLE (Thermofisher, 12604-013) and transferred to a 96-well plate (AXYGEN, P-96-450V-C) in 2.2 × 10⁶ units. 6 Seed 100,000 cells per well (45 μL of complete medium) at a density of cells / ml. Next, add 5 μL of antibody diluent to each well, mix gently, and incubate the 96-well plate at 4°C for 1 hour. Next, add 150 μL of PBS (Corning, 21-040-CVC) containing 2% FBS to each well, centrifuge at 1400 rpm for 4 minutes, remove the supernatant, and wash again. Next, add 50 μL of Alexa Fluor 647-labeled goat anti-human fluorescent secondary antibody (Jacksonimmuno, 109-605-088) diluted 1:800 to each well, and incubate the 96-well plate at 4°C for 0.5 hours. Wash the cells twice with PBS solution containing 2% FBS, resuspend in 120 μL of PBS, and detect the fluorescence signal on the cell surface using a flow cytometer (BD FACS Celesta) to obtain the median fluorescence intensity (MFI). A dose-response curve was constructed, and the semi-maximal effective concentration (EC50) was calculated using nonlinear regression with GraphPad software. The results are shown in Table 1. [Table 1]

[0082] Example 5: Binding of humanized anti-L1CAM monoclonal molecule to MMAE A 10 mg / mL monoclonal antibody solution, 1 mmol / L diethylenetriaminepentaacetic acid (DTPA), and 200 μmol / L tris(2-carboxyethyl)phosphine (TCEP) are mixed in PBS buffer and stirred at 37°C for 2 hours to obtain a partially reduced antibody. The degree of antibody reduction is determined by measuring the concentration of free thiol groups using the DTNB assay.

[0083] Antibodies reduced with TCEP can be used directly for subsequent binding. A solution of 10 mM maleimide-val-cit-PABC-MMAE (mc-vc-PABC-MMAE) drug is prepared using 20% ​​DMSO, and the drug is slowly added in a drug-to-antibody molar ratio of 8:1. The mixture is stirred at room temperature for 3 hours, followed by purification with Sephadex G-25 to remove any remaining unreacted drug and free small molecules. Binding is then evaluated by SDS-PAGE electrophoresis, hydrophobic interaction high-performance liquid chromatography (HIC-HPLC), reverse-phase high-performance liquid chromatography (RP-HPLC), and size exclusion high-performance liquid chromatography (SEC-HPLC).

[0084] Example 6: Measurement of the binding affinity of a humanized anti-L1CAM antibody-drug conjugate (anti-L1CAM ADC) to L1CAM on the cell surface. The experimental principle is as follows: HeLa cells (ATCC, CCL-2) express the L1CAM protein on their surface. After incubating the cells with anti-L1CAM ADC, the ability of the antibody to bind to L1CAM on the cell surface is measured by flow cytometry, thereby allowing us to measure the affinity of the antibody to the antigen.

[0085] The experimental method is as follows: The anti-L1CAM ADC was sequentially diluted fourfold in complete culture medium at 10 concentration gradient points.

[0086] HeLa cells are cultured in DMEM complete medium (Corning, 10-013-CVR) containing 10% FBS (Gibco, 10099-141) and 100 U / mL penicillin-streptomycin mixture (Thermo Fisher, 15140122). When the cell percentage in the culture vessel reaches 80-90%, the HeLa cells are digested from the culture vessel using TrypLE (Thermo Fisher, 12604-013) and transferred to 3.7 × 10⁶ deep-well plates. 4Seed 50,000 cells per well (1350 μL of complete medium) at a density of cells / ml. Next, add 150 μL of diluted ADC to each well, mix gently, and incubate the 96-well plate at 4°C for 1 hour. Next, centrifuge the plate at 1,400 rpm for 4 minutes, remove the supernatant, add 150 μL of PBS solution containing 2% FBS (Corning, 21-040-CVC) to each well, centrifuge at 1,400 rpm for 4 minutes, and then wash twice. Next, add 50 μL of AlexaFluor 647-labeled goat anti-human fluorescent secondary antibody (JacksonImmuno, 109-605-088) diluted 1:800 to each well, and incubate the 96-well plate at 4°C for 0.5 hours. Cells were washed twice with a PBS solution containing 2% FBS, resuspended in 120 μL of PBS, and the fluorescence signal on the cell surface was detected using a flow cytometer (BD FACS Celesta) to obtain the median fluorescence intensity (MFI). Dose-response curves were constructed, and the equilibrium dissociation constant (KD) was calculated using nonlinear regression with GraphPad software. The results are shown in Table 2 and Figure 1. [Table 2]

[0087] Example 7: Measurement of the binding specificity of a humanized anti-L1CAM antibody-drug conjugate (anti-L1CAM ADC) to L1CAM on the cell surface. The experimental principle is as follows: HeLa cells (ATCC, CCL-2) express high levels of L1CAM protein on their surface, while HeLa L1CAM KO cells (Abcam, ab255401) have significantly reduced L1CAM expression levels due to L1CAM gene knockout. After incubating anti-L1CAM ADCs with HeLa cells and HeLa KO cells, the ability of the antibody to bind to L1CAM on the cell surface is detected using flow cytometry, thereby allowing for the measurement of the binding specificity of anti-L1CAM ADCs.

[0088] The experimental method is as follows: HeLa cells and HeLa L1CAM-KO cells are cultured in DMEM complete medium (Corning, 10-013-CVR) containing 10% FBS (Gibco, 10099-141) and a 100 U / mL penicillin-streptomycin mixture (Thermo Fisher, 15140122). When the cell percentage in the culture vessel reaches 80-90%, the HeLa cells and HeLa KO cells are digested from the culture vessel using TrypLE (Thermo Fisher, 12604-013) and transferred to 5.5 × 10⁶ cells on a 96-cell culture plate (AXYGEN, P-96-450V-C). 5 Seed 100,000 cells per well (in 90 μL of complete medium) at a cell / ml density. Next, add 10 μL of diluted anti-L1CAM ADC to each well, mix gently, and incubate the 96-well plate at 4°C for 1 hour. Next, add 150 μL of PBS (Corning, 21-040-CVC) containing 2% FBS to each well, centrifuge at 1,400 rpm for 4 minutes, remove the supernatant, and wash again. Next, add 50 μL of Alexa Fluor 647-labeled goat anti-human fluorescent secondary antibody (Jacksonimmuno, 109-605-088) diluted 1:800 to each well, and incubate the 96-well plate at 4°C for 0.5 hours. Cells were washed twice with a PBS solution containing 2% FBS, then resuspended in 120 μL of PBS. Fluorescence signals on the cell surface were detected using a flow cytometer (BD FACS Celesta), and the median fluorescence intensity (MFI) was obtained. Dose-response curves were constructed using GraphPad software, and the experimental results are shown in Figure 2. The results indicate that the anti-L1CAM ADC exhibits excellent binding specificity.

[0089] Example 8: Measurement of the cytotoxic activity of humanized anti-L1CAM antibody-drug conjugates (anti-L1CAM ADCs) against HeLa cells and HeLa L1CAM KO cells. The experimental principle is as follows: HeLa cells (Cobioer Biosciences Co., LTD, CBP62032) express high levels of L1CAM protein on their surface, while HeLa L1CAM KO cells (Abcam, ab255401) have significantly reduced L1CAM expression levels due to L1CAM gene knockout. When anti-L1CAM ADC is incubated with the cells, internalization of the antigen-antibody complex occurs, and the ADC molecule then releases toxins into the cells, killing tumor cells. The specific cytotoxicity of the ADC molecule against HeLa cells is measured by the CellTiter-Glo (CTG) luminescence cell viability assay (Promega, G7573), while the selectivity of the ADC molecule is measured by detecting its killing activity against HeLa L1CAM KO cells.

[0090] The experimental method is as follows: HeLa cells and HeLa L1CAM-KO cells are 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 percentage in the culture vessel reaches 80-90%, the HeLa cells and HeLa KO cells are digested from the culture vessel using TrypLE (Thermofisher, 12604-013) and transferred to 96-well cell culture plates (Greiner, 655098) in 3.3 × 10⁶ units. 3Cells were seeded at a density of cells / mL, 500 cells per well (145 μL of complete medium), and the 96-well plates were incubated overnight at 37°C. The following day, the humanized anti-L1CAM antibody-MMAE conjugate (ADC) was serially diluted fourfold in complete medium at nine concentration gradient points. Next, 5 μL of the diluted ADC molecule was added to each well of the 96-well cell culture plate, gently mixed, and the 96-well plates were incubated at 37°C for 6 days. Finally, 75 μL of CTG was added to each well, gently mixed, and the 96-well plates were incubated at room temperature for 10 minutes. Next, the luminescence signal was detected using a multimode microplate reader (PerkinElmer Envision 2105). Dose-inhibition curves were constructed, and the semi-maximal inhibitory concentration (IC50) was calculated using nonlinear regression with GraphPad software. The experimental results are shown in Table 3, Figure 3, and Figure 4. [Table 3] [Table 4] [Table 5] [Table 6-1] [Table 6-2] [Table 6-3] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] Table 7-6

Claims

1. An L1CAM-targeted antibody-drug conjugate comprising an antibody or antigen-binding fragment conjugated to one or more therapeutic agents, Here, the antibody or antigen-binding fragment includes 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, Here, the heavy chain variable region has HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of heavy chain variable region sequences selected from the group consisting of SEQ ID NOs: 21, 23, 25, 27, 29, and 31, and the light chain variable region has LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of light chain variable region sequences selected from the group consisting of SEQ ID NOs: 22, 24, 26, 28, 30, and 32, Here, the therapeutic agent is selected from cytotoxic agents, microtubule inhibitors, DNA damaging agents, immunostimulants, and radioisotopes. Here, the therapeutic agent is an antibody-drug conjugate, in which the therapeutic agent is bound to the antibody or antigen-binding fragment via a linker.

2. The antibody-drug conjugate according to claim 1, having a set of CDRs selected from the group consisting of the following: (1) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with the heavy chain variable sequence HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 21, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with the light chain variable region sequence LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 22; (2) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with HCDR1, HCDR2, and HCDR3 of the heavy chain variable sequence of Sequence ID No. 23, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of Sequence ID No. 24; (3) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with HCDR1, HCDR2, and HCDR3 of the heavy chain variable sequence of Sequence ID No. 25, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of Sequence ID No. 26; (4) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with HCDR1, HCDR2, and HCDR3 of the heavy chain variable sequence of Sequence ID No. 27, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of Sequence ID No. 28; (5) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with HCDR1, HCDR2, and HCDR3 of the heavy chain variable sequence of Sequence ID No. 29, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of Sequence ID No. 30; (6) HCDR1, HCDR2, and HCDR3 having at least 80%, at least 90%, or 100% identity with HCDR1, HCDR2, and HCDR3 of the heavy chain variable sequence of Sequence ID No. 31, and LCDR1, LCDR2, and LCDR3 having at least 80%, at least 90%, or 100% identity with LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of Sequence ID No.

32.

3. An antibody-drug conjugate according to claim 1 or 2, wherein, (1) The HCDR1 has at least 80%, at least 90%, or 100% identity with a sequence selected from sequence number 1, sequence number 7, sequence number 10, and sequence number 16; (2) The HCDR2 has at least 80%, at least 90%, or 100% identity with a sequence selected from sequence number 2, sequence number 8, sequence number 11, and sequence number 17; (3) The HCDR3 has at least 80%, at least 90%, or 100% identity with a sequence selected from sequence number 3, sequence number 9, sequence number 12, and sequence number 18; (4) The LCDR1 has at least 80%, at least 90%, or 100% identity with the sequence selected from sequence number 4 and sequence number 13; (5) The LCDR2 has at least 80%, at least 90%, or 100% identity with the sequence selected from sequence number 5, sequence number 14, and sequence number 19; (6) The LCDR3 is an antibody-drug conjugate having at least 80%, at least 90%, or 100% identity with a sequence selected from SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO:

20.

4. An antibody-drug conjugate according to any one of claims 1 to 3, wherein, (1) The HCDR1 comprises the amino acid sequence described in Sequence ID No. 1; (2) The HCDR2 comprises the amino acid sequence described in Sequence ID No. 2; (3) The HCDR3 comprises the amino acid sequence described in Sequence ID No. 3; (4) The LCDR1 comprises the amino acid sequence described in Sequence ID No. 4; (5) The LCDR2 comprises the amino acid sequence described in Sequence ID No. 5; (6) The LCDR3 is an antibody-drug conjugate comprising the amino acid sequence described in Sequence ID No.

6.

5. An antibody-drug conjugate according to any one of claims 1 to 3, wherein, (1) The HCDR1 comprises the amino acid sequence described in Sequence ID No. 7; (2) The HCDR2 comprises the amino acid sequence described in Sequence ID No. 8; (3) The HCDR3 comprises the amino acid sequence described in Sequence ID No. 9; (4) The LCDR1 comprises the amino acid sequence described in Sequence ID No. 4; (5) The LCDR2 comprises the amino acid sequence described in Sequence ID No. 5; (6) The LCDR3 is an antibody-drug conjugate comprising the amino acid sequence described in Sequence ID No.

6.

6. An antibody-drug conjugate according to any one of claims 1 to 3, wherein, (1) The HCDR1 comprises the amino acid sequence described in Sequence ID No. 10; (2) The HCDR2 comprises the amino acid sequence described in Sequence ID No. 11; (3) The HCDR3 comprises the amino acid sequence described in Sequence ID No. 12; (4) The LCDR1 comprises the amino acid sequence described in Sequence ID No. 13; (5) The LCDR2 comprises the amino acid sequence described in Sequence ID No. 14; (6) The LCDR3 is an antibody-drug conjugate comprising the amino acid sequence described in Sequence ID No.

15.

7. An antibody-drug conjugate according to any one of claims 1 to 3, wherein, (1) The HCDR1 comprises the amino acid sequence described in Sequence ID No. 16; (2) The HCDR2 comprises the amino acid sequence described in Sequence ID No. 17; (3) The HCDR3 comprises the amino acid sequence described in Sequence ID No. 18; (4) The LCDR1 comprises the amino acid sequence described in Sequence ID No. 13; (5) The LCDR2 comprises the amino acid sequence described in Sequence ID No. 19; (6) LCDR3 is an antibody-drug conjugate containing the amino acid sequence described in SEQ ID NO:

20.

8. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the heavy chain variable region comprises one amino acid sequence selected from the group consisting of the following: (1) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 21; (2) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 23; (3) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 25; (4) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 27; (5) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 29; (6) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO:

31.

9. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the light chain variable region comprises one amino acid sequence selected from the group consisting of the following: (1) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 22; (2) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 24; (3) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 26; (4) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 28; (5) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 30; (6) An amino acid sequence having at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO:

32.

10. The antibody-drug conjugate according to any one of claims 1 to 9, wherein the antibody is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a bivalent antibody, an anti-idiotype antibody, or a fusion protein.

11. The antibody-drug conjugate according to any one of claims 1 to 9, wherein the linker is bound to the antibody via a thiol group.

12. The antibody-drug conjugate according to any one of claims 1 to 10, wherein the therapeutic agent is selected from one of the following: auristatin cytotoxic molecules (e.g., MMAE, MMAF), maytansinoid cytotoxic molecules (e.g., DM1, DM4), ansamycin derivative PBD, camptothecin, and camptothecin derivatives (e.g., exatecan, Dxd), or any combination thereof.

13. An antibody-drug conjugate having the structure of formula (I) below: 【Chemistry 1】 [In the formula, "Ab" refers to an antibody or antigen-binding fragment selected from any of the antibodies or antigen-binding fragments of claims 1 to 9; In the formula, L refers to a linker that can be cut or cannot be cut; In the formula, D refers to the therapeutic drug; In the formula, n preferably refers to a drug-to-antibody ratio (DAR) in the range of 1 to 8.

14. The antibody-drug conjugate according to claim 13, wherein L refers to a cleavable linker.

15. L is -L １ -L ２ -L ３ - The antibody-drug conjugate according to claim 13 or 14, which refers to wherein, L １ is any one of -(CH ２ ), ｎ -X-Y-(CH ２ ), ｍ -X(CH ２ ), ｎ -O-(CH ２ CH ２ O) ｐ -(CH ２ ), ｍ -Y-, -(CH ２ ), ｎ -X-, -X-(CH ２ ), ｍ -Y- or any combination thereof, wherein X and Y are each independently -C(O)-, O, -CR １ R ２ , -NR １ -, S or do not exist, wherein R １ and R ２ are each independently hydrogen, C １ -C ６ alkyl or C ３ -C ６ cycloalkyl, wherein m, n, and p each independently refer to an integer from 0 to 10; In the ceremony, L ２ This refers to an amino acid residue; In the ceremony, L ３ Either it does not exist, or it refers to -C(O)- or -PAB-C(O); In the formula, PAB refers to p-aminobenzyloxy, an antibody-drug conjugate.

16. The antibody-drug conjugate according to claim 15, wherein L ２ An antibody-drug conjugate that refers to one of the following, or any combination thereof: -Val-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, Ala-Ala-Asn-, -Val-Lys, -Phe-Lys, -Phe-Cit, -Phe-Arg-, -Phe-Ala-, -Ala-Lys, -Leu-Cit-, -Ile-Cit-, -Trp-Cit-, -D-Phe-LPhe-Lys-, -Phe-Phe-Lys-, -D-Phe-Phe-Lys-, -Gly-Phe-Lys-, -Gly-Phe-Leu-Gly-, Ala-Leu-Ala-Leu.

17. L is - (CH ２ ) ５ -Val-Cit-PAB-C(O)- or -(CH ２ ) ５ The antibody-drug conjugate according to claim 16, which refers to -Val-Ala-PAB-C(O)-.

18. The antibody-drug conjugate according to claim 17, wherein the therapeutic agent is selected from one of MMAE, MMAF, Dxd, DM1, DM4, or any combination thereof.

19. Antibody-drug conjugate having the following structure: 【Chemistry 2】 or 【Transformation 3】 [In the formula, "Ab" refers to an antibody or antigen-binding fragment selected from the antibody or antigen-binding fragment described in any of claims 1 to 9, In the formula, n preferably refers to a drug-to-antibody ratio (DAR) in the range of 1 to 8.

20. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. Use of an antibody-drug conjugate according to any one of claims 1 to 19 in the manufacture of a therapeutic agent for L1CAM-related cancer.

22. A method for treating L1CAM-associated cancer, comprising administering an antibody-drug conjugate according to any one of claims 1 to 19 to a subject having cancer.

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