Humanized L1CAM antibody drug conjugates

Abstract

The invention provides an anti-L1CAM antibody-drug conjugate as well as a preparation method and medical application thereof. In particular, the present invention provides an antibody-drug conjugate (ADC) comprising an anti-L1CAM antibody conjugated to MMAE or a derivative thereof, a pharmaceutical composition containing the ADC, and uses thereof in the preparation of drugs for treating L1CAM mediated diseases or symptoms, especially in the preparation of anti-cancer drugs.

Description

Humanized L1CAM antibody-drug conjugate

[0001] Cross-references

[0002] This application claims priority to Chinese patent application No. 202211317321.5 filed on October 26, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to a humanized L1CAM antibody-drug conjugate. Background Art

[0004] L1 cell adhesion molecule (L1CAM), also known as CD171, is a transmembrane glycoprotein with a molecular weight ranging from 200,000 to 220,000 [Miriam van der Maten et al., Int. J. Mol. Sci., 2019, 20:4180.]. In healthy individuals, L1CAM expression is primarily restricted to the nervous system, with small amounts expressed in kidney tissue and skin [Annette Kunkele et al., Clin. Cancer Res., 2016, 23:466-477.]. L1CAM is highly expressed in a variety of tumors and is closely associated with the proliferation and drug resistance of various tumor cells.

[0005] L1 cell adhesion molecule is a highly promising target for tumor therapy, but a therapeutic antibody targeting L1 cell adhesion molecule is still needed.

[0006] Antibody-drug conjugates (ADCs) are a new class of anticancer biopharmaceuticals composed of three components: an antibody, a cytotoxic agent, and a linker connecting the two. After chemically coupling a monoclonal antibody to a cytotoxic agent, the ADC leverages the monoclonal antibody's targeting properties to specifically recognize and bind to receptors on the surface of cancer cells. The ADC then enters the cell and utilizes intracellular proteases to release the cytotoxic agent, preventing and killing cancer cells. ADC technology integrates small molecule drugs with biological proteins, combining the strengths of both, significantly enhancing efficacy while minimizing toxic side effects, making it a next-generation therapeutic product.

[0007] The first clinical success of a targeted antibody-drug conjugate is gemtuzumab ozogamicin (trade name Mylotarg by Wyeth). Mylotarg is the first approved monoclonal antibody-drug conjugate. It consists of an anti-CD33 antibody, the DNA-degrading drug calicheamicin, and the chemical linker AcBut. Mylotarg is a humanized anti-CD33 IgG4 conjugated to the anti-tumor drug calicheamicin for the treatment of acute myeloid leukemia. Mylotarg, a first-generation monoclonal antibody-drug conjugate, suffered from three fatal technical flaws. First, the linker used to attach the toxin was extremely unstable, with a half-life of only two days. This resulted in severe toxin shedding and severe clinical side effects. Second, the antibody was conjugated to the linker via lysine amino groups, and an antibody has dozens of lysine residues on its surface, resulting in random conjugation sites, which partially affected drug efficacy. More importantly, the conjugation technology at the time was immature, with only 50% of antibodies being conjugated to the drug, resulting in suboptimal clinical efficacy. Third, the antibody used was IgG4, which lacked antibody-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). Consequently, ten years after its launch, Mylotarg was withdrawn from the market due to significant side effects and limited efficacy.

[0008] Another example of a successful clinical trial for a targeted antibody-drug conjugate is a new drug for the treatment of Hodgkin's lymphoma. Although the drug only underwent Phase II clinical trials, its exceptional efficacy earned it FDA approval in 2011. This drug, developed by Seattle Genetics, is a novel targeted antibody-drug conjugate (ADC) for the treatment of two types of lymphoma that express the CD30 antigen. Brentuximab, a drug conjugate composed of an anti-CD30 monoclonal antibody, a microtubule inhibitor (MMAE), and a dipeptide chemical linker, demonstrates low side effects and high efficacy in inhibiting lymphoma. In a Phase II, single-arm human clinical trial, 102 patients aged 15 to 77 years (median age, 31 years) with relapsed or refractory Hodgkin's lymphoma received brentuximab for a median of nine cycles. The overall response rate was 73%, and the median treatment duration was 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 adverse reaction was peripheral neuropathy. This success demonstrates the technical feasibility and promising future of targeted antibody-drug conjugates.

[0009] Another successful example of a targeted antibody-drug conjugate is T-DM1, developed by Genentech Inc. for the treatment of malignant breast cancer. The monoclonal antibody in this antibody-drug conjugate targets HER2 (ErbB2) on the surface of breast cancer cells, and the conjugated cytotoxic agent is the microtubule inhibitor DM1. Phase III clinical trial results for this drug demonstrated superior efficacy compared to chemotherapy with fewer side effects. Breast cancer patients who had previously received Herceptin and taxane chemotherapy, but whose disease still progressed, were able to significantly prolong their survival without disease progression. Based on the drug's promising efficacy, the U.S. Food and Drug Administration (FDA) approved it for marketing on February 22, 2013, for the treatment of patients with HER2-positive advanced metastatic breast cancer. Especially in 2019, polatuzumab vedotin (trade name Polivy), enfortumab vedotin (trade name Padcev) and fam-trastuzumab deruxtecan (trade name Enhertu) were successively approved for marketing, which further stimulated the enthusiasm of drug research and development companies.

[0010] Summary of the Invention

[0011] The present invention provides an antibody-drug conjugate targeting L1CAM, wherein the antibody-drug conjugate comprises 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 complementary determining regions LCDR1, LCDR2 and LCDR3 and / or a heavy chain variable region having heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, wherein the heavy chain variable region has HCDR1, HCDR2 and HCDR3 that are at least 80%, 90% or 100% identical to the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region sequence selected from SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 31, and the light chain variable region has HCDR1, HCDR2 and HCDR3 that are at least 80%, 90% or 100% identical to the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region sequence selected from SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: The LCDR1, LCDR2 and LCDR3 of the light chain variable region sequence of NO:32 have at least 80% identity with LCDR1, LCDR2 and LCDR3, wherein the therapeutic agent includes but is not limited to cytotoxic drugs, microtubule inhibitors, DNA damaging agents, immunopotentiators and radioactive isotopes, and wherein the therapeutic agent is coupled to the antibody or antigen-binding fragment via a linker.

[0012] In a specific embodiment, the heavy chain variable region and the light chain variable region have a CDR combination selected from the following:

[0013] (1) HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 21, and LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 22;

[0014] (2) HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 24;

[0015] (3) HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 25, and LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 26;

[0016] (4) HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 27, and LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 28;

[0017] (5) HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 29, and LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 30;

[0018] (6) HCDR1, HCDR2 and HCDR3 that are at least 80%, at least 90% or 100% identical to HCDR1, HCDR2 and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 31, and LCDR1, LCDR2 and LCDR3 that are at least 80%, at least 90% or 100% identical to LCDR1, LCDR2 and LCDR3 of the light chain variable region sequence of SEQ ID NO: 32.

[0019] In a specific technical solution, wherein:

[0020] (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;

[0021] (2) the HCDR2 has at least 80%, at least 90% or 100% identity to 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;

[0022] (3) the HCDR3 is at least 80%, at least 90% or 100% identical to 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;

[0023] (4) the LCDR1 has at least 80%, at least 90% or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 13;

[0024] (5) the LCDR2 has at least 80%, at least 90% or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 14, and SEQ ID NO: 19;

[0025] (6) The LCDR3 has at least 80%, at least 90% or 100% identity with a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO: 20.

[0026] In a specific technical solution, wherein:

[0027] (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1;

[0028] (2) the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2;

[0029] (3) the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0030] (4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4;

[0031] (5) the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5;

[0032] (6) The LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6.

[0033] In a specific technical solution, wherein:

[0034] (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0035] (2) the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 8;

[0036] (3) the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 9;

[0037] (4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4;

[0038] (5) the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5;

[0039] (6) The LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6.

[0040] In a specific technical solution, wherein:

[0041] (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10;

[0042] (2) the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0043] (3) the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12;

[0044] (4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13;

[0045] (5) the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14;

[0046] (6) The LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15.

[0047] In a specific technical solution, wherein:

[0048] (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16;

[0049] (2) the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17;

[0050] (3) the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18;

[0051] (4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13;

[0052] (5) the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19;

[0053] (6) The LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20.

[0054] In a specific technical solution, wherein: the heavy chain variable region comprises an amino acid sequence selected from any one of the following:

[0055] (1) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 21;

[0056] (2) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 23;

[0057] (3) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 25;

[0058] (4) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 27;

[0059] (5) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 29;

[0060] (6) An amino acid sequence having at least 90%, at least 95% or 100% sequence identity with SEQ ID NO: 31.

[0061] In a specific technical solution, the light chain variable region comprises an amino acid sequence selected from any one of the following:

[0062] (1) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 22;

[0063] (2) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 24;

[0064] (3) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 26;

[0065] (4) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 28;

[0066] (5) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 30;

[0067] (6) An amino acid sequence having at least 90%, at least 95% or 100% sequence identity with SEQ ID NO: 32.

[0068] In a specific technical solution, the antibody is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein.

[0069] In a specific technical solution, the linker is connected to the antibody via a sulfhydryl group.

[0070] In a specific technical solution, the therapeutic agent includes but is not limited to any one of auristatin-type cytotoxic molecules (such as MMAE, MMAF), maytansine-type cytotoxic molecules (such as DM1, DM4), anthramycin derivatives PBD, camptothecins and camptothecin derivatives (such as isotecan, Dxd) or any combination thereof.

[0071] The present invention provides an antibody drug conjugate having the following structure:

[0072] wherein Ab represents an antibody or antigen-binding fragment selected from any one of the above-mentioned embodiments of the present invention;

[0073] Wherein, L represents a degradable or non-degradable linker;

[0074] Wherein, D represents a therapeutic agent;

[0075] Wherein, n represents the drug loading value (Dar value), which is preferably 1-8.

[0076] In a preferred technical solution, L represents a degradable linker.

[0077] In a preferred technical solution, wherein L represents -L1-L2-L3-, wherein L1 represents: -(CH2) n -XY-(CH2) m -, -X(CH2) n -O-(CH2CH2O) p -(CH2) m -Y-, -(CH2) n -X-, -X-(CH2) m -Y-, wherein X and Y each independently represent -C(O)-, O, or -CR 1 R 2 -、-NR 1 -, S, or does not exist, where R1 、R 2 Each independently represents hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl; wherein m, n, and p each independently represent an integer of 0-10;

[0078] Wherein, L2 represents an amino acid residue;

[0079] Wherein, L3 represents absence, -C(O)- or -PAB-C(O);

[0080] Here, PAB represents p-aminobenzyloxy.

[0081] In a preferred technical solution, L2 represents any 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- or Ala-Leu-Ala-Leu, or any combination thereof.

[0082] In a preferred technical solution, L represents -(CH2)5-Val-Cit-PAB-C(O)-, -(CH2)5-Val-Ala-PAB-C(O)-.

[0083] In a preferred technical solution, the therapeutic agent is selected from any one of MMAE, MMAF, Dxd, DM1, DM4 or a combination thereof.

[0084] In addition, the present invention provides an antibody drug conjugate having the following structure:

[0085] Wherein, Ab represents an antibody or antigen-binding fragment selected from any one of the above items of the present invention, wherein n represents the drug loading value (Dar value), preferably 1-8.

[0086] In addition, the present invention also provides a pharmaceutical composition comprising the antibody-drug conjugate according to any one of the above items of the present invention, and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 Affinity of humanized L1CAM antibody-drug conjugate (anti-L1CAM ADC) for cell surface L1CAM

[0088] Figure 2 Binding specificity of humanized L1CAM antibody-drug conjugate (anti-L1CAM ADC) to cell surface L1CAM

[0089] Figure 3 Cytotoxicity of humanized L1CAM antibody-drug conjugate (anti-L1CAM ADC) on Hela cells

[0090] Figure 4 Cytotoxicity of humanized L1CAM antibody-drug conjugate (anti-L1CAM ADC) against Hela LICAM KO cells

[0091] Detailed Description of the Invention

[0092] In order to provide a more thorough understanding of the invention, some definitions are listed below. The above definitions are intended to include grammatical equivalents.

[0093] As used herein, "antibody" refers to a protein composed of one or more polypeptides encoded by all or part of substantially all recognized immunoglobulin genes. The recognized immunoglobulin genes, for example, in humans, include the kappa (κ), lambda (λ) and heavy chain loci, which contain numerous variable region genes, as well as the constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), and alpha (α), encoding the IgM, IgD, IgG, IgE, and IgA isotypes, respectively. Antibodies herein are meant to include full-length antibodies and antibody fragments, as well as natural antibodies from any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes as further specified below. The term "antibody" includes antibody fragments, as known in the art, such as Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies, or antibody fragments produced by modifying intact antibodies or those synthesized de novo using recombinant DNA technology. The term "antibody" includes monoclonal and polyclonal antibodies. The antibody can be an antagonist, an agonist, a neutralizing antibody, an inhibitory antibody, or a stimulatory antibody. The antibody of the present invention can be a non-human antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0094] The term "monoclonal antibody" as used herein refers to the antibody obtained from a substantially homogeneous antibody colony, that is, the colony comprising a single antibody is identical except the possible mutation (such as natural mutation) that may exist in a small amount. Therefore, the term "monoclonal" shows the character of the antibody, i.e., is not a mixture of irrelevant (discrete) antibodies. Contrary to the polyclonal antibody preparations that generally include different antibodies for different determinants (epi-topes), each monoclonal antibody of the monoclonal antibody preparation is directed to a single determinant on the antigen. Except for its specificity, the advantage of the monoclonal antibody preparation is that they are not contaminated by other immunoglobulins conventionally. The term "monoclonal" should not be construed as needing to produce the antibody by any specific method. The term monoclonal antibody specifically includes humanized antibodies.

[0095] As used herein, the term "complementarity determining region" (CDR, e.g., CDR1, CDR2, and CDR3) refers to the amino acid residues in the variable region of an antibody, the presence of which is essential for antigen binding. The "antigen binding region" of an antibody is typically present in one or more hypervariable regions of the antibody, such as the CDR regions of CDR1, CDR2, and CDR3. Each complementary determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat, e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region and 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). In some embodiments, the CDR regions herein are defined using the Kabat scheme. The CDR in the antibody sequence can also be defined according to other general rules (such as Chothia, IMGT, Contact numbering system, etc.) developed in the art or by comparing the sequence with the database of known variable regions. Kontermann and Dubel ed., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, described the method for identifying these regions in 2000. The same antibody may define different CDRs by different numbering schemes, and the disclosure of the CDR defined by one numbering scheme in this article means that the CDRs that may be defined by other numbering schemes are also disclosed. When the CDRs defined by the same scheme for two antibodies are identical, it is considered that the two antibodies have the same CDRs.

[0096] As used herein, "antigen" refers to a compound, composition or substance that can stimulate antibody production or T cell response in an animal, including compositions injected or absorbed into an animal, which can be proteins, carbohydrates, lipids or other pathogens.

[0097] A "functional fragment" or "antigen-binding fragment" of an antibody / immunoglobulin is defined herein as a fragment of an antibody / immunoglobulin that retains an antigen-binding region (e.g., the variable region of an IgG). The "antigen-binding region" of an antibody is typically found in one or more hypervariable regions of the antibody, such as CDR1, -2, and / or -3 regions; however, the variable "framework" region may also play an important role in antigen binding, for example by providing a scaffold for 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 VL and amino acid residues 4-111 of VH, and particularly preferably complete VL and VH chains (amino acids 1-109 of VL and amino acids 1-113 of VH; numbering is according to WO97 / 08320). The preferred immunoglobulin class used in the present invention is IgG.

[0098] As used herein, "amino acid" means one of the 20 naturally occurring amino acids or any non-natural analogs, which may be located at a specifically defined position. As used herein, "protein" means at least two covalently linked amino acids, which include proteins, polypeptides, oligopeptides, and peptides. Proteins can be composed of naturally occurring amino acids and peptide bonds, or of synthetic peptide mimetic structures, which are "analogs." Therefore, as used herein, "amino acid" or "peptide residue" means both naturally occurring and synthetic amino acids. For example, for the purposes of the present invention, homophenylalanine, citrulline, and norleucine are considered amino acids for the purposes of the present invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chains may be in the (R) or (S) configuration. In preferred embodiments, amino acids are present 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 vivo.

[0099] As used herein, "identity" refers to the similarity between nucleotide or amino acid sequences, alternatively referred to as sequence identity. Sequence identity is typically measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. When aligned using standard methods, homologs or variants will have a relatively high degree of sequence identity. Methods for sequence alignment for comparison are well known in the art. Various programs and alignment 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; and Altschul et al., Nature Genet., 1994, 6:119-129.

[0100] The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J. Mol. Biol., 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet for the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0101] " nucleic acid " used herein means the polymer consisting of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants and the analogs of the non-naturally occurring syntheses thereof) by phosphodiester bonds. Therefore, the term includes nucleotide polymers, wherein nucleotide and the bond therebetween include non-naturally occurring synthetic analogs, such as but not limited to phosphorothioate, phosphoramidate, methyl phosphonate, chiral methyl phosphonate, 2'-O-methyl ribonucleotides, peptide nucleic acid (PNA) etc. For example, automatic DNA synthesizer can be used to synthesize these polynucleotides. The term " oligonucleotide " generally refers to short polynucleotides, generally not more than about 50 nucleotides. It should be understood that when nucleotide sequence is represented by DNA sequence (i.e. A, T, G, C), this also includes the RNA sequence (i.e. A, U, G, C) in which " U " replaces " T ".

[0102] This article uses conventional notation to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5' end; the left-hand direction of a double-stranded nucleotide sequence is the 5' direction. The direction of addition of 5' to 3' nucleotides to the nascent RNA transcript is called the transcription direction. The DNA strand with the same sequence as the mRNA is called the coding strand.

[0103] As used herein, " encoding " means the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, used as a template for synthesizing other polymers and macromolecules in a biological process with a determined nucleotide sequence, or a determined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA produced by the gene produce a protein in a cell or other biological system, the gene encodes a protein. Coding strands (its nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and non-coding strands (used as transcription templates, genes or cDNAs) can be referred to as encoded proteins. Or other products of the gene or cDNA. Unless otherwise indicated, " nucleotide sequences encoding amino acid sequences " include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The nucleotide sequences of encoded proteins and RNA may include introns.

[0104] As used herein, "plasmid" refers to a plasmid that has been artificially constructed based on a natural plasmid to adapt to laboratory manipulation. A nucleic acid molecule can be introduced into a host cell to produce a transformed host cell. A vector can include nucleic acid sequences that permit replication in the host cell, such as an origin of replication, and can also include one or more selectable marker genes and other genetic elements known in the art.

[0105] As used herein, "host cell" is also called a recipient cell, and refers to a host cell that receives foreign genes during transformation and transduction (infection).

[0106] As used herein, "pharmaceutically acceptable carrier" refers to conventional pharmaceutically acceptable carriers. Remington's Pharmaceutical Sciences, E.W. Martin, Mack Publishing Co., Easton, Pa., 15th edition (1975), describes compositions and formulations suitable for the pharmaceutical delivery of one or more therapeutic compounds or molecules (e.g., one or more antibodies), as well as additional agents.

[0107] As used herein, "diagnosing" a disease means determining a patient's condition and its progression after examination. "Preventing" a disease means inhibiting the full development of a disease. "Treatment" means therapeutic intervention to ameliorate the signs or symptoms of a disease or pathological condition after it has begun to develop.

[0108] "Administering" herein means selecting an appropriate route to introduce the substance into a subject. For example, if the selected route is intravenous, the composition is administered by introducing the substance into the subject's vein.

[0109] As used herein, an "effective prophylactic / therapeutic dose" refers to an amount of a particular agent sufficient to achieve the desired effect in a subject being treated with the agent. The precise dosage will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques. Dosages may range from 0.01 to 100 mg / kg body weight or greater, such as 0.1, 1, 10, or 50 mg / kg body weight, preferably 1 to 10 mg / kg. As is known in the art, adjustments may be necessary for antibody or Fc fusion degradation, systemic or localized drug delivery, and new protease synthesis rates, as well as age, weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition, and can be determined by those skilled in the art through routine experimental methods. Such agents include the monomeric Fc domain molecules described herein. In a non-limiting example, this can be the amount of an HIV-specific monomeric Fc domain (or HIV-specific CH3 domain molecule) used to prevent, treat, or ameliorate HIV infection. Ideally, a therapeutically effective amount of an antibody is an amount sufficient to prevent, treat, or ameliorate an infection or disease, such as HIV infection, in a subject without causing significant cytotoxic effects in the subject. The therapeutically effective amount of an agent used to prevent, ameliorate, and / or treat a subject will depend on the subject being treated, the type and severity of the affliction, and the manner in which the therapeutic composition is administered.

[0110] "Cancer" herein is a solid tumor or a hematogenous cancer. Solid tumors of the present invention are sarcomas or cancers, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, or another sarcoma, synovioma, mesothelioma, Ewing's tumor, 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 carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, bladder cancer, or central nervous system tumors (such as gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal glands, hemangioblastomas, acoustic neuromas, oligodendrogliomas, hemangiomas, melanomas, neuroblastomas, or retinoblastomas). The blood-borne cancer described in the present invention is a leukemia, such as acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia or myelodysplasia.

[0111] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which this disclosure belongs. Unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" include plural indicators. It should also be understood that all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weights or molecular weight values ​​are approximate and are provided for description. Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprising" means "including". All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification (including term explanations) will prevail. In addition, materials, methods and examples are illustrative only and not restrictive.

[0112] Anti-L1CAM antibody

[0113] The antibodies of the present application 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.

[0114] Antibodies typically contain a heavy chain variable region (VH) and a light chain variable region (VL). The VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions (CDRs)) separated by relatively conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site.

[0115] In some embodiments, the antibody or its antigen-binding portion comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6 (as shown in antibody hz26A12C1H4L3).

[0116] In some embodiments, the antibody or its antigen-binding portion comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:7, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:8, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:9, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:6 (as shown in antibody hz46F1D5H4L3).

[0117] In some embodiments, the antibody or its antigen-binding portion comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15 (as shown in antibody hz49E10H1H5L6).

[0118] In some embodiments, the antibody or its antigen-binding portion comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15 (as shown in antibody hz49E10H1H5L7).

[0119] In some embodiments, the antibody or its antigen-binding portion comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 20 (as shown in antibody hz103E9B3H5L5 or hz103E9B3H6L5).

[0120] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL):

[0121] The VH comprises: (i) an amino acid sequence as shown in any one of SEQ ID NOs: 21, 23, 25, 27, 29 and 31; or (ii) an amino acid sequence that is at least 85%, 90% or 95% identical to the amino acid sequence as shown in any one of SEQ ID NOs: 21, 23, 25, 27, 29 and 31 and that, in combination with the VL region, retains specific binding affinity for L1CAM; and

[0122] The VL comprises: (i) an amino acid sequence as shown in any one of SEQ ID NOs: 22, 24, 26, 28, 30 and 32; or (ii) an amino acid sequence that is at least 85%, 90% or 95% identical to the amino acid sequence shown in any one of SEQ ID NOs: 22, 24, 26, 28, 30 and 32 and still retains specific binding affinity for L1CAM when combined with the VH region.

[0123] Preferably, the variants of the amino acid sequences described above that are at least 85%, 90% or 95% identical comprise mutations that occur in the framework regions rather than the CDR regions, for example, in the framework regions, there are one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid additions, deletions and / or substitutions. Preferably, the substitutions are conservative substitutions.

[0124] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted with another amino acid residue having a similar side chain, such as substitutions of physically or functionally similar residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.) to corresponding amino acid residues. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0125] The antibodies and antigen-binding fragments provided herein may further comprise a human IgG constant region comprising an Fc region and an optional 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 the Fc region may comprise one or more amino acid modifications (e.g., Leu234Ala / Leu235Ala or LALA substitutions) that alter antibody-dependent cellular cytotoxicity (ADCC) or other effector functions.

[0126] Linker-drug moiety

[0127] The use of antibody-drug conjugates for local delivery of cytotoxic or cytostatic agents, i.e., drugs that kill or inhibit tumor cells in cancer therapy, allows for targeted delivery of the drug moiety to the tumor, where it accumulates intracellularly, whereas systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells as well as the tumor cells being eliminated (Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), 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).

[0128] Drugs that can be used for ADCs include chemotherapeutic agents such as daunorubicin, doxorubicin, methotrexate, and vindesine; toxins, such as bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin, maytansinoids, and calicheamicin; auristatin peptides, auristatin E (AE) and monomethyl auristatin (MMAE), which are synthetic analogs of dolastatin. MMAE is a synthetic derivative of dolastatin 10, a natural cytostatic pseudopeptide. Toxins can achieve their cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when coupled to large antibodies or protein receptor ligands.

[0129] There are no specific limitations on the drugs and linkers that can be used in the conjugation methods of the present disclosure, as long as the drug molecule has anti-tumor, anti-viral, or anti-microbial effects and contains at least one substituent group or partial structure that allows for attachment 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 couple to the antibody. Preferably, the linker is susceptible to attack by the -SH group of the antibody and is capable of forming a bond with the antibody.

[0130] Depending on the desired drug and the selected linker, one skilled in the art can select an appropriate method to couple them together. For example, some conventional coupling methods, such as amine coupling methods, can be used to form the desired drug-linker complex, which still contains a reactive group for covalently coupling to the antibody. In the present disclosure, a drug-maleimide complex (i.e., a maleimide-linked drug) is an example of a loaded reactive group.

[0131] In one embodiment, the drug may include, but is not limited to, cytotoxic agents, such as chemotherapeutic agents, immunotherapeutic agents, etc., antiviral agents or antimicrobial agents. In one embodiment, the drug to be conjugated to the antibody may be selected from, but is not limited to, MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), MMAF (monomethyl auristatin F), etc.

[0132] In ADC preparation, the most common reactive group capable of binding to sulfhydryl groups is maleimide. In addition, organic bromides and iodides are also frequently used.

[0133] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes. See, for example, WO 93 / 21232 published on October 28, 1993. A variety of radionuclides can be used to produce radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. One or more small molecule toxins, such as calicheamicin, maytansines, dolastatins, auristatins, trichothecenes, and CC1065, as well as derivatives of these toxins having toxin activity, can be conjugated to antibodies using the methods disclosed herein.

[0134] The maytansine compound that is suitable for use as maytansine compound drug part is well-known in the art, and can be separated from natural source according to known method, use genetic engineering technology to produce (referring to Yu etc. (2002) PNAS 99:7968-7973), or according to known method synthetic preparation maytansinol and maytansinol analogue.Suitable maytansine compound is for example disclosed in United States Patent (USP) 5,208,020.Preferred maytansine compound is maytansinol and the maytansinol analogue modified at the aromatic ring or other positions of maytansinol molecule, for example various maytansinol esters.

[0135] Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, cell nucleus and cell division, and have anticancer and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). Dolastatins or auristatins drug moieties can be attached to antibodies via the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172). Exemplary embodiments including MMAE or MMAF and various linker components are shown below. For example, VcMMAE (Mc-vc-PAB-MMAE) is obtained by using MMAE that is connected to a lysosomal cleavable dipeptide valine-citrulline (vc) and a thiol-reactive maleimide-hexanoyl spacer (Mc) via p-aminobenzyloxycarbonyl ("PAB")

[0136] Conjugates of antibodies and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazido 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). For example, ricin 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 chelator for coupling radionucleotides to antibodies (WO 94 / 11026).

[0137] Also contemplated herein are exemplary embodiments comprising MMAE or MMAF and various linker components. For example, VcMMAE (Mc-vc-PAB-MMAE) is obtained by using MMAE linked via a p-aminobenzyloxycarbonyl group ("PAB") to a lysosomal cleavable dipeptide valine-citrulline (vc) and a thiol-reactive maleimidocaproyl spacer (Mc).

[0138] Purpose of ADC

[0139] In one aspect, the present disclosure relates to the use of an antibody-drug conjugate prepared by the aforementioned method in the preparation of a pharmaceutical composition or kit for treating a condition or disorder in a subject. In one aspect, the present disclosure also relates to a method for treating a subject suffering from cancer, comprising: administering a therapeutically effective amount of an antibody-drug conjugate to a subject in need thereof, thereby treating or preventing the condition or disorder. The subject can be a mammal, such as a human. The condition or disorder to be treated can be cancer, in particular a cancer associated with L1CAM. In certain embodiments, the cancer can be selected from breast cancer, bile duct cancer, melanoma, pancreatic cancer, glioma, lymphoma, lung cancer, kidney cancer, prostate cancer, fibrosarcoma, colon adenocarcinoma, liver cancer, or ovarian cancer. Example

[0140] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples.

[0141] Example 1: Production of monoclonal antibodies against L1CAM

[0142] To generate antibodies against L1CAM, Balb / c mice were immunized with recombinantly expressed human L1CAM antigen fragments. During the immunization procedure, mice were bled regularly to obtain serum samples and monitor the immune response. Mice with sufficient titers of anti-L1CAM antibodies were used in fusion experiments. Splenocytes and / or lymph node cells from the immunized mice were isolated and fused with a mouse myeloma cell line. Hybridomas producing L1CAM-specific antibodies were screened using HeLa cells overexpressing human L1CAM and HEK293 cells overexpressing monkey L1CAM by ELISA analysis using the human extracellular domain of L1CAM. Stable hybridoma clones were then subcloned. Following subcloning, monoclonal hybridoma cells were sequenced for antibody analysis for further analysis and testing.

[0143] Example 2: Production of chimeric antibodies against L1CAM

[0144] Based on the results of monoclonal hybridoma cell antibody sequencing, a human-mouse chimeric antibody was designed with the CH1, CH2, and CH3 of human IgG1 as constant regions and the VH and VL of mouse anti-L1CAM monoclonal antibody as variable regions. The antibody was recombinantly expressed in mammalian cells and purified for further analysis and testing.

[0145] Example 3: Production of humanized antibodies against L1CAM

[0146] A murine anti-L1CAM monoclonal antibody was selected for humanization. The antibody sequence was aligned with human germline sequences to determine the best-fit model. Based on homology to the original mouse antibody sequence, the closest matching human germline sequence was selected as the humanization template. The CDRs from the mouse antibody sequence were then grafted onto the template, along with residues that maintained the antibody's upper and central core structures. 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 mixed and matched to provide multiple humanized antibody clones. Recombinant expression was performed in mammalian cells and purified for further analysis and testing.

[0147] Example 4 Determination of humanized L1CAM antibody binding on Hela cell surface

[0148] The experimental principle is summarized as follows: Hela cells highly express L1CAM protein on their surface. After incubating the cells with humanized L1CAM antibodies, the antibody's binding ability to LICAM on the Hela cell surface can be determined by flow cytometry.

[0149] The experimental methods are outlined as follows:

[0150] The humanized antibody against L1CAM (Human IgG1-Kappa) was diluted 4-fold in series using complete culture medium, with a total of 7 concentration gradient points.

[0151] HeLa cells were cultured in complete DMEM (Corning, 10-013-CVR) supplemented with 10% FBS (Gibco, 10099-141) and a 100 U / mL penicillin-streptomycin mixture (Thermofisher, 15140122). When the cell confluence reached 80–90%, the HeLa cells were removed from the culture vessel using TripLE (Thermofisher, 12604-013) and seeded in 96-well plates (AXYGEN, P-96-450V-C) at a density of 2.2 × 10 6 cells / mL, with 100,000 cells per well (45 μL complete medium). Then, 5 μL of the antibody dilution was added to each well, mixed gently, and the 96-well plates were incubated at 4°C for 1 hour. 150 μL of PBS containing 2% FBS (Corning, 21-040-CVC) was then added to each well, followed by centrifugation at 1400 rpm for 4 minutes. The supernatant was removed and the plate was washed once more. 50 μL of a 1:800 dilution of Alexa Flour 647-conjugated goat anti-human fluorescent secondary antibody (Jackson Immuno, 109-605-088) was then added to each well, and the 96-well plate was incubated at 4°C for 0.5 hours. Cells were washed twice with PBS containing 2% FBS and resuspended in 120 μL of PBS. Cell surface fluorescence was measured using a flow cytometer (BD FACS Celesta) to obtain median fluorescence intensity (MFI). Dose-response curves were generated, and the median effective concentration (EC50) was calculated by nonlinear regression using GraphPad software. The results are shown in Table 1.

[0152] Table 1: Binding ability of humanized L1CAM antibodies to LICAM on the surface of Hela cells

[0153] Example 5 Conjugation of humanized anti-L1CAM monoclonal molecules with MMAE

[0154] Partially reduced antibody was obtained by mixing 10 mg / mL monoclonal antibody, 1 mmol / L diethylenetriaminepentaacetic acid (DTPA), and 200 μmol / L tris(2-carboxyethyl)-phosphine (TCEP) in PBS buffer and stirring at 37°C for 2 hours. The free thiol concentration and thus the degree of antibody reduction were determined using the DTNB assay.

[0155] After TCEP reduction, the antibody can be directly conjugated. A 10 mM maleimide-val-cit-PAB-MMAE (mc-vc-PAB-MMAE) drug solution was prepared in 20% DMSO. The drug was slowly added at a drug-to-antibody molar ratio of 8:1. The reaction was stirred at room temperature for 3 hours. Residual unreacted drug and free small molecules were purified using Sephadex G-25. The conjugation was analyzed by SDS-PAGE electrophoresis, hydrophobic high-performance liquid chromatography (HIC-HPLC), reversed-phase high-performance liquid chromatography (RP-HPLC), and size-exclusion high-performance liquid chromatography (SEC-HPLC).

[0156] Example 6 Determination of affinity of humanized L1CAM antibody-drug conjugate (anti-L1CAM ADC) for cell surface L1CAM

[0157] The experimental principle is summarized as follows: Hela cells (ATCC, CCL-2) express L1CAM protein on their surface. After incubating the cells with an anti-L1CAM ADC, the antibody's binding ability to cell surface LICAM can be measured by flow cytometry, thereby determining the antigen-antibody affinity.

[0158] The experimental methods are outlined as follows:

[0159] The anti-L1CAM ADC was diluted 4-fold in a series using complete culture medium, with a total of 10 concentration gradient points.

[0160] HeLa cells were cultured in complete DMEM (Corning, 10-013-CVR) containing 10% FBS (Gibco, 10099-141) and a mixture of 100 U / mL penicillin and streptomycin (Thermofisher, 15140122). When the cell confluence in the culture vessel reached 80-90%, the cells were digested from the culture vessel using TripLE (Thermofisher, 12604-013) and plated at 3.7 × 10 4Cells were seeded at a density of 50,000 cells / ml in a 96-well plate (1350 μL complete medium). 150 μL of the antibody dilution was added to each well, gently mixed, and the 96-well plate was incubated at 4°C for 1 hour. The plate was then centrifuged at 1400 rpm for 4 minutes, the supernatant removed, and 150 μL of PBS (Corning, 21-040-CVC) containing 2% FBS (Corning, 21-040-CVC) was added to each well and washed twice by centrifugation at 1400 rpm for 4 minutes. 50 μL of a 1:800 dilution of Alexa Flour 647-conjugated goat anti-human fluorescent secondary antibody (Jackson Immuno, 109-605-088) was then added to each well, and the 96-well plate was incubated at 4°C for 0.5 hour. The cells were washed twice with PBS containing 2% FBS and then resuspended in 120 μL of PBS. Flow cytometry (BD FACSCelesta) was used to measure cell surface fluorescence signals and calculate median fluorescence intensity (MFI). Dose-response curves were generated, and the equilibrium dissociation constant (KD) was calculated by nonlinear regression using GraphPad software. The results are shown in Table 2 and Figure 1.

[0161] Table 2 Affinity equilibrium dissociation constants of humanized L1CAM antibody-drug conjugates (anti-L1CAM ADC) for L1CAM on the surface of Hela cells

[0162] Example 7 Determination of Binding Specificity of Humanized L1CAM Antibody Drug Conjugate (anti-L1CAM ADC) to Cell Surface L1CAM

[0163] The experimental principle is summarized as follows: Hela cells (ATCC, CCL-2) highly express L1CAM protein on their surface. Since the L1CAM gene is knocked out in Hela L1CAM KO cells (Abcam, ab255401), L1CAM expression in Hela KO cells is significantly reduced compared to wild-type Hela cells. After incubating Hela and Hela KO cells with an anti-L1CAM ADC, the antibody's binding to cell surface LICAM is measured by flow cytometry, thereby confirming the binding specificity of the anti-L1CAM ADC.

[0164] The experimental methods are outlined as follows:

[0165] Hela and Hela L1CAM KO 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 confluence in the culture vessel reached 80-90%, the cells were digested from the culture vessel using TripLE (Thermofisher, 12604-013) and plated at 5.5 × 10 5 Cells were seeded at a density of 100,000 cells / ml in a 96-well plate (AXYGEN, P-96-450V-C) per well (90 μL complete medium). Then, 10 μL of the anti-L1CAM ADC dilution was added to each well, gently mixed, and the 96-well plate was incubated at 4°C for 1 hour. Then, 150 μL of PBS (Corning, 21-040-CVC) containing 2% FBS was added to each well, centrifuged at 1400 rpm for 4 minutes, the supernatant was removed, and the plate was washed once more. Then, 50 μL of a 1:800 dilution of Alexa Flour 647-conjugated goat 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 hour. The cells were washed twice with PBS containing 2% FBS and then resuspended in 120 μL of PBS. Flow cytometry (BD FACSCelesta) was used to measure the fluorescence signal on the cell surface and calculate the median fluorescence intensity (MFI). GraphPad software was then used to generate a dose-response curve. The experimental results are shown in Figure 2, demonstrating that the anti-L1CAM ADC has excellent binding specificity.

[0166] Example 8 Determination of the Cytotoxicity of Humanized L1CAM Antibody Drug Conjugates (anti-L1CAM ADCs) against Hela and Hela LICAM KO Cells

[0167] The experimental principle is summarized as follows: Hela cells (Nanjing Kebai, CBP62032) highly express the L1CAM protein on their surface. Hela L1CAM KO cells (Abcam, ab255401) have a knockout of the L1CAM gene, resulting in significantly reduced L1CAM expression in Hela KO cells compared to wild-type Hela cells. Incubation of the cells with an anti-L1CAM ADC leads to internalization of the antigen-antibody complex, followed by the release of toxins within the cells that kill tumor cells. The specific cytotoxicity of the ADC against Hela cells is measured using the CellTiter-Glo (CTG) chemiluminescent cell viability assay (Promega, G7573). Furthermore, the selectivity of the ADC is determined by measuring its cytotoxicity against Hela L1CAM KO cells.

[0168] The experimental methods are outlined as follows:

[0169] Hela and Hela L1CAM KO 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 confluence in the culture vessel reached 80-90%, Hela and Hela KO cells were digested from the culture vessel using TripLE (Thermofisher, 12604-013) and plated at 3.3 × 10 3 Cells were seeded at a density of 500 cells / ml in 96-well cell culture plates (Greiner, 655098), with 145 μL complete medium per well. The plates were incubated at 37°C overnight. The next day, a 4-fold serial dilution of the L1CAM humanized antibody-MMAE conjugate (ADC) was performed in complete medium, resulting in a total of nine concentrations. 5 μL of the ADC dilution was then added to each well of the 96-well cell culture plate, gently mixed, and the plates were incubated at 37°C for 6 days. Finally, 75 μL of CTG was added to each well, gently mixed, and the plates were incubated at room temperature for 10 minutes. Chemiluminescent signals were detected using a multi-function microplate reader (PerkinElmer Envision 2105). Dose-inhibition curves were generated, and the half-maximal inhibitory concentration (IC50) was calculated by nonlinear regression using GraphPad software. The results are shown in Table 3, Figures 3, and 4.

[0170] Table 3 Cytotoxicity test of humanized L1CAM antibody-drug conjugates (anti-L1CAM ADCs) against Hela and Hela LICAM KO cells

[0171] Table 4 Amino acid sequences of the complementary determining regions of the L1CAM antibodies of the present invention

[0172] Table 5 Amino acid sequences of the variable regions of the humanized L1CAM monoclonal antibody of the present invention

[0173] Table 6 Amino acid sequence of the humanized L1CAM monoclonal antibody of the present invention

[0174] Table 7 Nucleotide sequences encoding the L1CAM humanized monoclonal antibody of the present invention

Claims

1. An antibody-drug conjugate targeting L1CAM, wherein: The antibody-drug conjugate comprises 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 complementary determining regions LCDR1, LCDR2 and LCDR3 and / or a heavy chain variable region having heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, wherein the heavy chain variable region has HCDR1, HCDR2 and HCDR3 that are at least 80%, at least 90% or 100% identical to the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region sequence selected from SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 31, and the light chain variable region has HCDR1, HCDR2 and HCDR3 that are at least 80%, at least 90% or 100% identical to the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region sequence selected from SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: The LCDR1, LCDR2 and LCDR3 of the light chain variable region sequence of NO:32 have LCDR1, LCDR2 and LCDR3 that are at least 80%, at least 90% or 100% identical, wherein the therapeutic agent is selected from a cytotoxic drug, a microtubule inhibitor, a DNA damaging agent, an immunopotentiator and a radioactive isotope, and wherein the therapeutic agent is coupled to the antibody or antigen-binding fragment via a linker.

2. The antibody drug conjugate of claim 1, wherein the heavy chain variable region and the light chain variable region have a CDR combination selected from the following: (1) HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 21, and LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 22; (2) HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 24; (3) HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 25, and LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 26; (4) HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 27, and LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 28; (5) HCDR1, HCDR2, and HCDR3 that are at least 80%, at least 90%, or 100% identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 29, and LCDR1, LCDR2, and LCDR3 that are at least 80%, at least 90%, or 100% identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region sequence of SEQ ID NO: 30; (6) HCDR1, HCDR2 and HCDR3 that are at least 80%, at least 90% or 100% identical to HCDR1, HCDR2 and HCDR3 of the heavy chain variable region sequence of SEQ ID NO: 31, and LCDR1, LCDR2 and LCDR3 that are at least 80%, at least 90% or 100% identical to LCDR1, LCDR2 and LCDR3 of the light chain variable region sequence of SEQ ID NO:

32.

3. The 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 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 to 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 is at least 80%, at least 90% or 100% identical to 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) the LCDR1 has at least 80%, at least 90% or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 13; (5) the LCDR2 has at least 80%, at least 90% or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 14, and SEQ ID NO: 19; (6) The LCDR3 has at least 80%, at least 90% or 100% identity with a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 15, and SEQ ID NO:

20.

4. The antibody drug conjugate according to any one of claims 1 to 3, wherein: (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1; (2) the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2; (3) the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; (4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4; (5) the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5; (6) The LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

6.

5. The antibody drug conjugate according to any one of claims 1 to 3, wherein: (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 7; (2) the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 8; (3) the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 9; (4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4; (5) the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5; (6) The LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

6.

6. The antibody drug conjugate according to any one of claims 1 to 3, wherein: (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10; (2) the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11; (3) the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12; (4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13; (5) the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14; (6) The LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

15.

7. The antibody drug conjugate according to any one of claims 1 to 3, wherein: (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16; (2) the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17; (3) the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; (4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13; (5) the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19; (6) The LCDR3 comprises the amino acid sequence shown 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 an amino acid sequence selected from any one of the following: (1) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 21; (2) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to 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 acid sequence; (4) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 27; (5) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to 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 an amino acid sequence selected from any one of the following (1) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 22; (2) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 24; (3) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 26; (4) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 28; (5) an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to 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-idiotypic antibody or a fusion protein.

11. The antibody drug conjugate according to any one of claims 1 to 9, wherein: The linker is connected to the antibody via a sulfhydryl group.

12. The antibody-drug conjugate according to any one of claims 1 to 10, wherein the therapeutic agent is selected from any one of auristatin-type cytotoxic molecules (e.g., MMAE, MMAF), maytansine-type cytotoxic molecules (e.g., DM1, DM4), anthramycin derivatives PBD, camptothecins, and camptothecin derivatives (e.g., isotecan, Dxd), or any combination thereof.

13. An antibody drug conjugate having the following structure: in, Ab represents an antibody or antigen-binding fragment selected from any one of claims 1 to 9; Wherein, L represents a degradable or non-degradable linker; Wherein, D represents a therapeutic agent; Wherein, n represents the drug loading value (Dar value), which is preferably 1-8.

14. The antibody drug conjugate according to claim 13, wherein L represents a degradable linker.

15. The antibody drug conjugate according to claim 13 or 14, wherein L represents -L1-L2-L3-, wherein L1 represents: -(CH2) n -XY-(CH2) m -, -X(CH2) n -O-(CH2CH2O) p -(CH2) m -Y-, -(CH2) n -X-, -X-(CH2) m -Y-, wherein X and Y each independently represent -C(O)-, O, or -CR 1 R 2 -、-NR 1 -, S, or does not exist, where R 1 、R 2 Each independently represents hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl; wherein m, n, and p each independently represent an integer of 0-10; Wherein, L2 represents an amino acid residue; Wherein, L3 represents absence, -C(O)- or -PAB-C(O); Here, PAB represents p-aminobenzyloxy.

16. The antibody drug conjugate according to claim 15, wherein L2 represents any 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- or Ala-Leu-Ala-Leu, or any combination thereof.

17. The antibody drug conjugate according to claim 16, wherein L represents -(CH2)5-Val-Cit-PAB-C(O)- or -(CH2)5-Val-Ala-PAB-C(O)-.

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

19. Antibody-drug conjugates with the following structure: in, Ab represents an antibody or antigen-binding fragment selected from any one of claims 1 to 9, wherein n represents the drug loading value (Dar value), preferably 1-8.

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

21. Use of the antibody-drug conjugate according to any one of claims 1 to 19 in the preparation of a medicament for treating L1CAM-related cancers.

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

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