A novel binding molecule that binds to L1CAM

Abstract

The present invention relates to antibodies that specifically bind to human L1CAM, related nucleic acids, host cells, and pharmaceutical compositions, and related methods and uses, as defined in the claims.

Description

[Technical Field]

[0001] The present invention relates to antibodies that specifically bind to human L1CAM, related nucleic acids, host cells, pharmaceutical compositions, as well as related methods and uses, as defined in the claims. [Background technology]

[0002] Monoclonal antibodies (mAbs) have emerged as a new and important pillar of cancer therapy [1]. Over the past two decades, molecular biology has provided the means to generate chimeric, humanized, or fully human antibodies for the treatment of major malignancies [2]. To date, many antibodies and antibody conjugates have been approved for sale as cancer therapeutics in Europe and the United States [3, 4]. These include unmodified antibodies, antibody-drug conjugates, as well as conjugates with radionuclides and bispecific antibodies [5]. However, it is well known that mAbs against a given cancer antigen can differ in their ability to target cancer cells.

[0003] Recent studies have shown that L1CAM (neural cell adhesion molecule L1, also called L1) may be an excellent target molecule for human cancers. L1CAM is a transmembrane glycoprotein that is normally involved in the development of the nervous system by affecting cell adhesion and cell motility. L1CAM is overexpressed in many human cancers, confers poor prognosis, and enhances cell motility, invasion, and metastasis.

[0004] WO2008 / 151819 discloses an anti-L1CAM antibody, L1CAM mAb L1-9.3 (also called mAb 9.3 or L9.3), which binds to an epitope within the first Ig domain of L1CAM. Results from xenograft models [6] and human L1CAM transgenic mouse models [7] suggest that mAb 9.3 may be a promising tool for cancer therapy. Recent results indicate that this mAb in its IgG2a form is sufficient to activate the immune system and recruit immune effector cells, leading to the elimination of cancer cells [6, 7].

[0005] WO2016 / 050702 discloses a binding molecule that binds to L1CAM, capable of binding to the same L1CAM epitope recognized by monoclonal antibody L1-OV52.24 and / or competing with monoclonal antibody L1-OV52.24 for binding to L1CAM. The binding molecule of WO2016 / 050702 was shown to have an improved internalization rate compared to mAb 9.3. It has been reported that binding of an L1CAM-specific antibody leads to L1CAM internalization, followed by recycling or degradation of the target molecule [8]. Internalization is a feature that L1CAM shares with many other cell surface molecules. Indeed, it has been reported that L1CAM internalization is required for signal transduction and regulation of L1CAM-mediated cell adhesion [9-11].

[0006] Despite these discoveries, there remains a need for antibodies that exhibit high-affinity, specific binding to L1CAM on the surface of human cancer cell lines but that do not exhibit any reactivity with plate-coated human CHL1, NrCAM, or neurofascin. Furthermore, there is a need for antibodies that inhibit cancer cell migration and proliferation, induce tumor cell cytolysis, inhibit metastasis formation, and reduce tumor burden and ascites development. It would also be advantageous to provide antibodies that ameliorate common clinical signs of disease. Finally, it would be generally useful if such antibodies simultaneously possessed high conformational and chemical stability and exhibited reduced formation of post-translational variants.

[0007] Although some features of the antibodies of the present invention have been described (see [6]), the antibodies themselves or the sequences of their complementarity-determining regions (CDRs) have never been published or made publicly available.

[0008] The antibodies of the present invention that specifically bind to human L1CAM solve all the problems mentioned above and are surprisingly advantageous in the fields of biotechnology research, diagnostics, or therapy. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2008 / 151819 [Patent Document 2] WO2016 / 050702 [Non-patent literature]

[0010] [Non-Patent Document 1] Galluzzi L et al.: Trial Watch: Monoclonal antibodies in cancer therapy. Oncoimmunology 1(1):28-37 (2012) [Non-patent document 2] Presta LG: Molecular engineering and design of therapeutic antibodies. Curr. Opin. Immunol. 20(4):460-470 (2008) [Non-patent document 3] Reichert JM: Marketed therapeutic antibodies compendium. MAbs 2012, 4(3):413-415 (2012) [Non-patent document 4] Reichert JM: Antibodies to watch in 2014: mid-year update. mAbs 6(4):799-802 (2014) [Non-patent document 5] Reichert JM: Antibody-based therapeutics to watch in 2011. MAbs 3(1):76-99 (2011) [Non-patent document 6] Wolterink S et al.: Therapeutic antibodies to human L1CAM: functional characterization and application in a mouse model for ovarian carcinoma. Cancer Res. 70(6):2504-2515 (2010) [Non-Patent Document 7] Doberstein K et al.: Antibody therapy to human L1CAM in a transgenic mouse model blocks local tumor growth but induces EMT. Int. J. Cancer 136(5):E326-339 (2015) [Non-patent document 8] Novak-Hofer I et al. : Internalization and degradation of monoclonal antibody chCE7 by human neuroblastoma cells. Int. J. Cancer 57(3):427-432 (1994) [Non-Patent Document 9] Schaefer AW et al. : Activation of the MAPK signal cascade by the neural cell adhesion molecule L1 requires L1 internalization. J. Biol. Chem. 274(53):37965-37973 (1999) [Non-Patent Document 10] Long KE et al.: The role of endocytosis in regulating L1-mediated adhesion. J. Biol. Chem. 276(2):1285-1290 (2001) [Non-Patent Document 11] Schaefer AW et al.: L1 endocytosis is controlled by a phosphorylation-dephosphorylation cycle stimulated by outside-in signaling by L1. J. Cell Biol. 157(7):1223-1232 (2002) Summary of the Invention [Means for solving the problem]

[0011] Antibodies that specifically bind to human L1CAM are provided.

[0012] Additionally provided are nucleic acids encoding all or part of an antibody that specifically binds human L1CAM, as well as host cells containing such nucleic acids.

[0013] Pharmaceutical compositions comprising such antibodies, nucleic acids, or host cells are also provided.

[0014] Also provided are such antibodies, nucleic acids, host cells, or pharmaceutical compositions for use as pharmaceuticals or diagnostics, or in treating or preventing hyperproliferative disorders, neoplastic diseases, disorders involving angiogenesis, and / or disorders involving abnormal neurogenesis.

[0015] The present invention The present invention provides novel antibodies that specifically bind to human L1CAM.

[0016] The novel antibodies that specifically bind to human L1CAM exhibit advantageous characteristics compared to prior art anti-L1CAM antibodies. For example, the chimeric OV549.20 human IgG1 antibody of the present invention induced robust ADCC to Panc-1 target cells. In contrast, a chimeric human IgG1 version of the previously described antibody L9.3, which binds to the first Ig domain of L1CAM, did not induce any ADCC to Panc-1 target cells (see Example 2, Figure 4). Furthermore, addition of the antibodies of the present invention resulted in a decrease in the proliferation of all three cancer cell lines tested (see Example 2, Figure 5). In contrast, the binding molecule of WO2016 / 050702 that binds to L1CAM did not affect the proliferation of the cancer cell lines (see Example 2, Figure 5). Furthermore, the antibodies of the invention reduced tumor mass and ascites volume in a xenograft model of human SKOV3 ovarian cancer cells in mice, whereas the previously described antibody L9.3 had no effect (see Example 3, Figure 7A).

[0017] The antibodies of the present invention specifically bind to L1CAM on the surface of human cancer cell lines with high binding affinity, but do not exhibit any reactivity with plate-coated human CHL1, NrCAM, or neurofascin. Furthermore, the antibodies of the present invention inhibit the migration and proliferation of a variety of different cancer cells, induce tumor cell cytolysis, inhibit metastasis formation, and reduce tumor burden and ascites development. This combination of properties makes the antibodies particularly suitable for ameliorating clinical symptoms of tumor diseases in general. Additionally, the antibodies of the present invention simultaneously possess high conformational and chemical stability and exhibit reduced formation of post-translational variants, making them particularly suitable for large-scale manufacturing, clinical development, clinical safety, and storage.

[0018] As used herein, the term "antibody that specifically binds to human L1CAM" refers to any polypeptide that has structural similarity to naturally occurring antibodies and is capable of binding to human L1CAM, where the binding specificity is determined by the CDRs of the polypeptide. Thus, "antibody that specifically binds to human L1CAM" is intended to refer to an immunoglobulin-derived structure that has binding to human L1CAM.

[0019] L1CAM (also called L1) is a transmembrane protein; it is a 200-220 kDa member of the L1 protein family, a neural cell adhesion molecule, involved in axon guidance and cell migration, and has been implicated in treatment-resistant cancers. The term "human L1CAM" according to the present invention is preferably understood as the human L1CAM protein. The human L1CAM gene sequence has been assigned Gene ID: 3897. The Genbank entry for the isoform 1 precursor of the human L1CAM protein is NP_000416. L1CAM has also been named CD171. The term "human L1CAM" describes any type of protein known to be expressed by any human cell type based on this L1CAM gene.

[0020] "Specific binding" is understood to mean that the binding of a binding molecule to L1CAM is at least 50-fold, preferably at least 100-fold, stronger than the binding to a control protein such as albumin, as determined by methods known to those skilled in the art, such as, for example, surface plasmon resonance-based kinetic binding analysis. Alternatively, methods such as determining a shift in fluorescent signal in Western blot analysis, enzyme-linked immunosorbent assay (ELISA), or a cytometer-based assay can also be used. Such specific binding can be based on any interaction between an antibody and its antigen known to those skilled in the art, such as non-covalent binding (e.g., van der Waals contacts, hydrogen bond formation, or hydrophobic interactions).

[0021] The term "antibody" generally describes any polypeptide having structural similarity to naturally occurring antibodies, such as proteins belonging to the immunoglobulin protein family. The term "antibody" includes full-length antibodies, antigen-binding fragments of antibodies, and molecules comprising antibody VH and / or VL regions. Antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies, including bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFvs), camelid antibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of any of the above. The antibody can be part of a fusion protein or conjugate. For example, the antibody can be included in a chimeric antigen receptor (CAR). The antibody can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subisotype (e.g., IgG2a or IgG2b). Each heavy and light chain can have a variable region and a constant region or a portion thereof. If the antibody contains a heavy chain constant region or a portion thereof, the heavy chain constant region can be one of the five types of mammalian Ig heavy chains: α, δ, ε, γ, and μ. The type of heavy chain present usually defines the antibody class (isotype): IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Similarly, the light chain constant region can be one of the two types of mammalian Ig light chains: κ and λ. The variable regions of the heavy and light chains are usually composed of a unique combination of multiple protein sequences, enabling them to bind to a specific antigen.The term "antibody" further includes domain scaffolds such as affibodies, anticalins, affilins, atrimers, DARPins, FN3 scaffolds such as adnectins and sentinels, fynomers, Kunitz domains, pronectins, and OBodies.

[0022] Preferably, the antibodies described herein may be IgG antibodies or isotypes thereof, such as human IgG1, human IgG2, or human IgG4.

[0023] Preferably, the antibody is a humanized monoclonal antibody. Alternatively, the antibody can be a chimeric antibody. Alternatively, the antibody can be a human antibody. For example, the antibodies described herein are IgG1 or IgG2 antibodies.

[0024] Generally, in a full-length, intact antibody, each heavy chain is connected to one light chain, such that the variable regions of the heavy and light chains combine to form one of the two identical antigen-binding sites of the antibody, and their constant regions combine to form the antibody's constant region. Furthermore, both heavy and light chain constructs can be connected via the constant regions of their heavy chains to form a "Y" shaped molecule, such that the two arms delineate the antigen-binding variable regions and the stem delineates the constant region.

[0025] The antibodies according to any of the aspects of the invention herein are intact antibodies, which means that they usually comprise a heavy chain of three or four constant domains, and a light chain of one constant domain, as well as each variable domain, whereby each domain may contain further modifications such as mutations, deletions, or insertions that do not change the overall domain structure.

[0026] Generally, each heavy chain variable region and each light chain variable region of an antibody contains three non-contiguous complementary determining regions (CDRs).

[0027] As used herein, the term "CDR" or "complementarity-determining region" refers to the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions are described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) (

[12] ) and Kabat et al., Sequences of proteins of immunological interest. (1991), as well as by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996) (

[13] -

[15] ), in which the definitions include overlapping or subsets of amino acid residues when compared with each other. The amino acid residues encompassing the CDRs as defined by each of the above-cited references are shown for comparison. Preferably, the term "CDR" refers to a CDR as defined by Kabat, based on sequence comparison.

[0028] CDRs are typically numbered CDR1, CDR2, and CDR3 for the heavy chain variable region and the light chain variable region, respectively. As a result, an antibody arm typically has six CDRs, which together form the antibody-binding site. In general, each CDR is typically 1 to 25 amino acids long, preferably 3 to 20 amino acids long, for example, 3 to 16 amino acids long. An antibody may contain one, two, or more arms, i.e., one, two, or three antigen-binding sites.

[0029] In one aspect, the present invention relates to an antibody that specifically binds to human L1CAM, comprising: (a) the amino acid sequence of GYSITSDYX1WN (SEQ ID NO: 16), wherein X1 is A or T; or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 16) heavy chain variable region (VH) complementarity-determining region (CDR) 1 comprising: (b) YISYSGSX1SYX2PSLKS (SEQ ID NO: 17) (In the formula, X1 is F or Y, and X2 is H or N), or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 17) VH CDR2 comprising: (c) SX1SYX2YGFAY ​​(SEQ ID NO: 18) (In the formula, X1 is L or F, and X2 is G, S, or A; or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 18) a VH CDR3 comprising: (d) the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4); or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 4) Light chain variable region (VL) CDR1 containing (e) the amino acid sequence of SASYRYX1 (SEQ ID NO: 19), wherein X1 is T or I; or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 19) a VL CDR2 comprising: (f) the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6); or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 6) VL CDR3 comprising:

[0030] In one aspect, the present invention relates to an antibody that specifically binds to human L1CAM, comprising: (a) a heavy chain variable region (VH) complementarity-determining region (CDR) 1 comprising the amino acid sequence of GYSITSDYX1WN (SEQ ID NO: 16) (wherein X1 is A or T); (b) YISYSGSX1SYX2PSLKS (SEQ ID NO: 17) (In the formula, X1 is F or Y, and X2 is H or N) a VH CDR2 comprising the amino acid sequence of (c) SX1SYX2YGFAY ​​(SEQ ID NO: 18) (In the formula, X1 is L or F, and X2 is G, S, or A) a VH CDR3 comprising the amino acid sequence of: (d) a light chain variable region (VL) CDR1 comprising the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4); (e) a VL CDR2 comprising the amino acid sequence of SASYRYX1 (SEQ ID NO: 19), wherein X1 is T or I; and (f) VL CDR3 comprising the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6).

[0031] In this regard, the variable "X1" or "X2" for a given SEQ ID NO is independent of the "X1" or "X2" of a different SEQ ID NO. "X1" or "X2" is defined independently for each SEQ ID NO. When a sequence contains "X1" and "X2", "X1" and "X2" are selected independently of each other. For example, X1 and X2 of SEQ ID NO: 17 (VH CDR2) are usually selected independently of each other and independently of the X1 of the sequences of SEQ ID NO: 16 (VH CDR1), SEQ ID NO: 18 (VH CDR3), and SEQ ID NO: 19 (VL CDR2) and the X2 of the sequence of SEQ ID NO: 18 (VH CDR3), respectively. For example, X1 and X2 of SEQ ID NO: 18 (VH CDR3) are typically selected independently of each other and independently of the mentioned variants of X1 of the sequences of SEQ ID NO: 16 (VH CDR1), SEQ ID NO: 17 (VH CDR2), and SEQ ID NO: 19 (VL CDR2) and X2 of the sequences of SEQ ID NO: 17 (VH CDR2), respectively. Finally, for example, X1 of SEQ ID NO: 19 (VL CDR2) is typically selected independently of the mentioned variants of X1 of the sequences of SEQ ID NO: 16 (VH CDR1), SEQ ID NO: 17 (VH CDR2), and SEQ ID NO: 18 (VH CDR3) and X2 of the sequences of SEQ ID NO: 17 (VH CDR2) and SEQ ID NO: 18 (VH CDR3).

[0032] The remaining regions of the antibody according to the invention, e.g., the framework regions of the heavy and light chain variable regions, as well as, if applicable, the heavy and light chain constant domains, may be of any sequence, e.g., sequences known to those skilled in the art.

[0033] The antibodies of the present invention may further comprise two identical heavy chains and / or two identical light chains, or may simply comprise non-identical heavy and light chains. It is further possible that a heavy chain variable region and a light chain variable region, each comprising the above-mentioned specific set of CDR1-CDR3, form only one arm of the antibody, and that a second (or even further) arm of the antibody comprises different heavy and light chain variable regions.

[0034] Because antibodies according to the present invention may contain two or more heavy chain variable regions and two or more light chain variable regions, it is also not necessary that a heavy chain variable region containing the above-mentioned specific heavy chain CDR1 to CDR3 be linked to a light chain variable region containing the above-mentioned specific light chain CDR1 to CDR3.

[0035] The antibodies of the present invention may originate from a mammal, such as a rodent, e.g., a mouse, a rabbit, or a rat. Each of the remaining regions of the antibodies of the present invention, e.g., the framework regions, as well as the heavy chain constant domain and the light chain constant domain, if applicable, may contain one or more modifications, such as mutations including substitutions, deletions, or insertions, that do not change the overall domain structure.

[0036] For example, an antibody of the present invention may include 1 to 10 mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations, including any subrange thereof), e.g., substitutions, deletions, and / or insertions, particularly 1 to 10 substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, including any subrange thereof), in the Fc domain of the antibody. Each mutation may be introduced independently in one chain of the Fc domain. Alternatively, each mutation may be introduced independently symmetrically in both chains of the Fc domain.

[0037] For example, an antibody of the invention may comprise 1 to 10 mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations, including any subrange thereof), e.g., substitutions, deletions, and / or insertions, particularly 1 to 10 substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, including any subrange thereof), in the CL and / or CH1 domain of the antibody.

[0038] Exemplary combinations of the VH CDR1, VH CDR2, and VH CDR3 mentioned above are listed in Table 1 below in the context of various antibody examples, all of which correspond to antibodies according to the invention. In that table, antibody OV549.20 represents a murine example of an antibody according to the invention. H1, H2, H3, and H4 are examples of VH regions of humanized variants of murine antibody OV549.20. AFF1 to AFF10 are examples of antibodies according to the invention that have further undergone affinity maturation. Of these, the combination of CDRH1, CDRH2, and CDRH3 of antibody "AFF4" is particularly preferred. Sequences highlighted in bold are particularly preferred sequences.

[0039] [Table 1] JPEG2025532716000002.jpg113161

[0040] Exemplary combinations of the above-mentioned VL CDR1, VL CDR2, and VL CDR3 of the present invention are listed in Table 2 below in the context of various exemplary antibodies already mentioned in Table 1. L1 and L2 are examples of light chain variable regions of a humanized antibody related to the murine antibody OV549.20, which may be further combined with any of the VH CDR1, VH CDR2, and VH CDR3 sets of H1, H2, H3, and H4 in Table 1. An antibody may comprise the VH CDRs of one antibody as depicted in Table 1 and the VL CDRs of the same or different antibodies as depicted in Table 2, preferably the VL CDRs of the same antibody as depicted in Table 2. Sequences highlighted in bold are particularly preferred sequences. AFF1 to AFF10 are examples of antibodies according to the present invention that have further undergone affinity maturation. Of these, the combination of CDRL1, CDRL2, and CDRL3 of antibody "AFF4" is particularly preferred.

[0041] [Table 2] JPEG2025532716000004.jpg52156

[0042] Preferably, X1 of SEQ ID NO: 16 (VH CDR1) is T; and / or X1 of SEQ ID NO: 17 (VH CDR2) is Y, and / or X2 of SEQ ID NO: 17 (VH CDR2) is N; and / or X1 of SEQ ID NO: 18 (VH CDR3) is F, and / or X2 of SEQ ID NO: 18 (VH CDR3) is S; and / or X1 of SEQ ID NO: 19 (VL CDR1) is T.

[0043] In a preferred embodiment, the present invention relates to an antibody that specifically binds to human L1CAM of the present invention, which is: (a) VH CDR1 comprises the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9), or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 9); and / or (b) VH CDR2 is YISYSGSX1SYX2PSLKS (SEQ ID NO: 17) wherein X1 is Y, and / or X2 is N), or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 17) and / or (c) VH CDR3 is SX1SYX2YGFAY ​​(SEQ ID NO: 18) wherein X1 is F, and / or X2 is S) The amino acid sequence of or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 18) and / or (d) VL CDR2 has the amino acid sequence of SASYRYT (SEQ ID NO: 5); or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 5) Includes.

[0044] In a preferred embodiment, the present invention relates to an antibody that specifically binds to human L1CAM of the present invention, which is: (a) VH CDR1 comprises the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9); and / or (b) VH CDR2 is YISYSGSX1SYX2PSLKS (SEQ ID NO: 17) wherein X1 is Y, and / or X2 is N) and / or (c) VH CDR3 is SX1SYX2YGFAY ​​(SEQ ID NO: 18) wherein X1 is F, and / or X2 is S) and / or (d) VL CDR2 comprises the amino acid sequence of SASYRYT (SEQ ID NO: 5).

[0045] In a more preferred embodiment, the antibodies that specifically bind to human L1CAM of the present invention described herein include: (a) a heavy chain variable region (VH) comprising a VH CDR1 comprising the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 9), a VH CDR2 comprising the amino acid sequence of YISYSGSYSYNPSLKS (SEQ ID NO: 11) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 11), and a VH CDR3 comprising the amino acid sequence of SFSYSYGFAY ​​(SEQ ID NO: 14) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 14); and (b) a light chain variable region (VL) comprising a VL CDR1 comprising the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 4), a VL CDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 5) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 5), and a VL CDR3 comprising the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 6).

[0046] In a more preferred embodiment, the antibodies that specifically bind to human L1CAM of the present invention described herein include: (a) a heavy chain variable region (VH) comprising a VH CDR1 comprising the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9), a VH CDR2 comprising the amino acid sequence of YISYSGSYSYNPSLKS (SEQ ID NO: 11), and a VH CDR3 comprising the amino acid sequence of SFSYSYGFAY ​​(SEQ ID NO: 14); and (b) a light chain variable region (VL) comprising a VL CDR1 having the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4), a VL CDR2 having the amino acid sequence of SASYRYT (SEQ ID NO: 5), and a VL CDR3 having the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6).

[0047] In addition to the specific CDRs mentioned above, the heavy and / or light chain variable regions of an antibody of the invention may also comprise one or more of the specific framework regions mentioned below.

[0048] The antibody of the present invention can comprise framework sequences from any species. Preferably, it comprises murine and / or human framework sequences or hybrids thereof. For example, the framework sequences can each be human, and optionally, there can be between 1 and 10 non-human positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, including any subrange thereof).

[0049] As used herein, the term "framework (FR) amino acid residues" refers to those amino acids in the framework region of an immunoglobulin chain. As used herein, the term "framework region" or "FR region" includes amino acid residues that are part of the variable region but are not part of the CDRs (e.g., using the Kabat definition of CDR). Framework regions typically support the binding of an antibody to an antigen, either by supporting the structure of the antibody (and not contacting the antigen) or by directly contacting the antigen. The term "antigen" describes any molecule or molecular structure that can be bound by an antibody specific for that antigen.

[0050] Methods for producing monoclonal antibodies with CDR sequences as referred to above are known in the art and involve introducing nucleic acid sequences encoding the CDRs into an appropriate expression vector encoding the desired framework sequences. Further methods are described below.

[0051] Suitable framework regions for the heavy chain variable region are, for example, SEQ ID NOs: 64 to 72. Specifically, the heavy chain variable region of the antibody of the present invention may comprise one to four framework regions selected from the group of SEQ ID NOs: 64 to 72. Preferably, the heavy chain variable region of the antibody of the present invention comprises one, two, three, or four framework regions selected from the group of SEQ ID NOs: 64 to 72, more preferably four framework regions selected from the group of SEQ ID NOs: 64 to 72.

[0052] According to the Kabat numbering system for CDRs, the VH CDRs are determined by Kabat, and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format FR1, CDRH1, FR2, CDRH2, FR3, CDRH3, and FR4.

[0053] Thus, FR1 of the heavy chain variable region can be independently selected from SEQ ID NO: 64 or 65, FR2 of the heavy chain variable region can be independently selected from SEQ ID NO: 66 to 69, FR3 of the heavy chain variable region can be independently selected from SEQ ID NO: 70 or 72, and / or FR4 of the heavy chain variable region can be SEQ ID NO: 71.

[0054] Suitable framework regions for the light chain variable region are, for example, SEQ ID NOs: 73 to 82. The light chain variable region of the antibody of the present invention may comprise one to four framework regions selected from the group of SEQ ID NOs: 73 to 82. Preferably, the light chain variable region of the antibody of the present invention comprises one, two, three, or four framework regions selected from the group of SEQ ID NOs: 73 to 82, more preferably four framework regions selected from the group of SEQ ID NOs: 73 to 82.

[0055] Similarly, according to the Kabat numbering system for CDRs, the VL CDRs are determined by Kabat, and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format FR1, CDRL1, FR2, CDRL2, FR3, CDRL3, and FR4.

[0056] Thus, FR1 of the light chain variable region can be independently selected from SEQ ID NO: 76, 80, or 82; FR2 of the light chain variable region can be independently selected from SEQ ID NO: 73 or 77; FR3 of the light chain variable region can be independently selected from SEQ ID NO: 74, 78, or 81; and / or FR4 of the light chain variable region can be independently selected from SEQ ID NO: 75 or 79.

[0057] Furthermore, FR1 to FR4 of the heavy chain variable region can be selected independently of FR1 to FR4 of the light chain variable region, and FR1 to FR4 of the light chain variable region can be selected independently of FR1 to FR4 of the heavy chain variable region.

[0058] Tables 3 and 4 below provide examples of antibodies according to the present invention, further comprising SEQ ID NOS: 64-72 as framework regions FR1-FR4 of the heavy chain variable region and SEQ ID NOS: 73-82 as framework regions FR1-FR4 of the light chain variable region, respectively. In the tables, antibody OV549.20 represents a murine example of an antibody according to the present invention. H1, H2, H3, and H4 are examples of VH regions of humanized variants of murine antibody OV549.20. L1 and L2 are examples of VL regions of humanized variants of murine antibody OV549.20. AFF1-AFF10 are examples of antibodies according to the present invention that have further undergone affinity maturation.

[0059] Preferably, the set of VH framework regions FR1-FR4 is selected from the set provided for a particular antibody in Table 3 below. Preferably, the set of VL framework regions FR1-FR4 is selected from the set provided for a particular antibody in Table 4 below. Preferably, the set of VH framework regions FR1-FR4 is selected from the set provided for a particular antibody in Table 3 below, and the set of VL framework regions FR1-FR4 is selected from the set provided for the same particular antibody in Table 4 below.

[0060] [Table 3]

[0061] [Table 4]

[0062] The antibody according to the present invention may further comprise a heavy chain variable region sequence comprising one or more of the framework regions of the heavy chain variable region sequence of any one of SEQ ID NOs: 23 to 34, and / or may further comprise a light chain variable region sequence comprising one or more of the framework regions of the light chain variable region sequence of any one of SEQ ID NOs: 20 to 22.

[0063] In a further preferred embodiment, the antibody of the present invention that specifically binds to human L1CAM further comprises a heavy chain variable region sequence comprising the framework region of the heavy chain variable region sequence of any one of SEQ ID NOs: 23 to 34, and / or a light chain variable region sequence comprising the framework region of the light chain variable region sequence of any one of SEQ ID NOs: 20 to 22.

[0064] The antibody according to the present invention may also comprise a heavy chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 34, and / or a light chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 22. In this regard, the heavy chain variable region may be selected independently of the selected light chain variable region, and the light chain variable region may be selected independently of the selected heavy chain variable region.

[0065] Therefore, one of the heavy chain variable regions of the antibody can be selected from the group consisting of SEQ ID NOs: 23 to 34, but the sequence of the second heavy chain variable region does not have to be selected from this group.

[0066] Furthermore, one of the light chain variable regions of an antibody according to an embodiment may be selected from the group consisting of SEQ ID NOs: 20 to 22, but the sequence of the second light chain variable region does not have to be selected from this group.

[0067] Furthermore, when an antibody according to the present invention comprises a heavy chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 34, and a light chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 22, these do not necessarily have to be part of the same arm of an antibody comprising two or more arms, and may be assigned to different arms.

[0068] It is further disclosed herein that for all embodiments, the heavy chain variable region sequence, light chain variable region sequence, complementarity determining region, light chain sequence, and heavy chain sequence "comprises" or "consists of" the respective indicated sequence.

[0069] The antibody according to the present invention may further comprise one or two heavy chain variable region sequences comprising an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 23 to 34, and / or one or two light chain variable region sequences comprising an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 20 to 22.

[0070] In a preferred embodiment, the antibody of the present invention that specifically binds to human L1CAM comprises a heavy chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 34, and / or a light chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 22.

[0071] In a preferred embodiment, the antibody of the present invention that specifically binds to human L1CAM comprises a heavy chain variable region sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 34, and / or a light chain variable region sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 22.

[0072] Exemplary combinations of heavy and light chain variable region sequences are shown below in Table 5. The antibody names mentioned herein relate to the names mentioned and explained in connection with Tables 1-4 above. Sequences highlighted in bold are particularly preferred sequences.

[0073] Preferably, in the context of antibodies herein, the VH and / or VL sequences have the sequences as shown in Table 5 below.

[0074] [Table 5] JPEG2025532716000008.jpg236166JPEG2025532716000009.jpg235166JPEG2025532716000010.jpg233165JPEG2025532716000011.jpg169165

[0075] In further preferred embodiments, the antibody comprises at least one VH region and at least one VL region of the antibodies designated herein as AFF1-AFF10. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of the antibody designated herein as AFF1. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of the antibody designated herein as AFF2. ​​Thus, preferably, the antibody comprises at least one VH region and at least one VL region of the antibody designated herein as AFF3. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of the antibody designated herein as AFF4. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of the antibody designated herein as AFF5. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of the antibody designated herein as AFF6. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of the antibody designated herein as AFF7. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of the antibody designated herein as AFF8. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of the antibody designated herein as AFF9. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of the antibody designated herein as AFF10. The sequences of the VH and VL regions are shown in Table 5 above.

[0076] The use of AFF4 is particularly preferred. The VH region of AFF4 has the amino acid sequence of SEQ ID NO: 30, and the VL region of AFF4 has the amino acid sequence of SEQ ID NO: 20.

[0077] Thus, an antibody may comprise a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 30, and / or may comprise a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 20. Thus, an antibody may also comprise one or more heavy chain variable region sequences comprising the amino acid sequence of SEQ ID NO: 30, and / or may comprise one or more light chain variable region sequences comprising the amino acid sequence of SEQ ID NO: 20.

[0078] Furthermore, if an antibody comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 30 and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 20, these are not necessarily part of the same arm of an antibody comprising two or more arms, but may be assigned to different arms.

[0079] In still further preferred embodiments, the antibody comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO:30, and / or a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO:20.

[0080] Furthermore, the antibody of the present invention may comprise a heavy chain sequence comprising or consisting of an amino acid sequence selected from the group of SEQ ID NOs: 35, 84, and 37.

[0081] The antibody of the invention may further comprise a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NOs:36 and 38.

[0082] In addition, the antibodies of the present invention may comprise one or two heavy chain sequences comprising or consisting of an amino acid sequence independently selected from the group of SEQ ID NOs: 35, 84, and 37, and / or may comprise one or two light chain sequences comprising or consisting of an amino acid sequence independently selected from the group of SEQ ID NOs: 36 and 38.

[0083] Preferably, the antibody comprises: (a) a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 35, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 36; and / or (b) a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38; and / or (c) a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0084] In still further preferred embodiments, the antibody comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37 or 84, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38. In still further preferred embodiments, the antibody comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37 or 84, and a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0085] In yet a further preferred embodiment, the antibody comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38. In yet a further preferred embodiment, the antibody comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0086] In yet a further preferred embodiment, the antibody comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38. In yet a further preferred embodiment, the antibody comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0087] In another further preferred embodiment, the antibody consists of a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37 or 84, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38. In another further preferred embodiment, the antibody consists of a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37 or 84, and a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0088] In another further preferred embodiment, the antibody consists of a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38. In another further preferred embodiment, the antibody consists of a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0089] In another further preferred embodiment, the antibody consists of a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38. In another further preferred embodiment, the antibody consists of a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0090] A humanized, optimized antibody was prepared and used in Example 5. In Example 5, a humanized, optimized antibody "AFF4" is provided, which is an IgG-type antibody whose heavy chain variable region sequence consists of the amino acid sequence of SEQ ID NO: 30 and whose light chain variable region sequence consists of the amino acid sequence of SEQ ID NO: 20 linked to a human IgG1 heavy chain constant region having G236A / S239D / A330L / I332E mutations (EU numbering) in the human IgG1 constant region. The heavy chain of the antibody consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence consists of the amino acid sequence of SEQ ID NO: 38.

[0091] In the examples, a humanized, optimized antibody designated "AFF4-WT" was also prepared and used. The antibody "AFF4-WT" is an IgG-type antibody whose heavy chain variable region sequence consists of the amino acid sequence of SEQ ID NO: 30 and whose light chain variable region sequence consists of the amino acid sequence of SEQ ID NO: 20 linked to a wild-type human IgG1 constant region. The heavy chain of AFF4-WT consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence consists of the amino acid sequence of SEQ ID NO: 38.

[0092] Optionally, one, two, or three amino acids may be deleted from the C-terminus of the full-length heavy chain. Such deletions are known not to affect the stability of the antibody. Furthermore, it is also possible to use a heavy chain containing the C-terminal lysine of the Fc domain. Depending on the recombinant expression system used, the C-terminal lysine of the Fc domain may or may not be present.

[0093] Exemplary combinations of heavy and light chain variable region sequences are shown below in Table 6. The antibody names mentioned therein relate to the names mentioned and described in connection with Tables 1-5 above.

[0094] [Table 6] JPEG2025532716000013.jpg237161JPEG2025532716000014.jpg132161

[0095] In yet a further preferred embodiment, the antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20.

[0096] Preferably, the antibody comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 30, and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 20. Preferably, the antibody comprises a heavy chain variable region sequence having the amino acid sequence of SEQ ID NO: 30, and a light chain variable region sequence having the amino acid sequence of SEQ ID NO: 20.

[0097] Antibodies may further include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies including bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, univalent antibodies, single chain antibodies or single chain Fvs (scFvs). Fv), camelid antibodies, affibodies, anticalins, affilins, atrimers, DARPins, FN3 scaffolds such as adnectins and centilins, fynomers, Kunitz domains, pronectins, and OBodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFv), anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of any of the above, and / or the antibody is comprised in a chimeric antigen receptor (CAR).

[0098] The antibody can be selected from monoclonal antibodies. Monoclonal antibodies are identical, monospecific antibodies, or antibodies with the same amino acid sequence because they are all produced by one type of immune cell that is a clone of a single parent cell, e.g., a single clone of B lymphocytes. "Monoclonal antibodies" and the production of monoclonal antibodies are at the forefront of technology. Generally, monoclonal antibodies can be prepared according to known methods, for example, those of Winter and Milstein

[17] . As an alternative to preparing hybridomas that secrete monoclonal antibodies, monoclonal antibodies directed against a polypeptide of interest can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) for the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly suitable for use in generating and screening antibody display libraries can be found, for example, in U.S. Pat. No. 5,223,409; WO92 / 18619; WO91 / 17271; WO92 / 20791; WO92 / 15679; WO93 / 01288; WO92 / 01047; WO92 / 09690; WO90 / 02809;

[19] -

[22] .

[0099] The antibody may also be selected from synthetic antibodies. The term "synthetic antibody" describes any antibody that is produced entirely in vitro without any involvement of animals. Methods for producing synthetic antibodies are well known to those skilled in the art, such as recombinant protein production. However, although synthetic antibodies are produced in vitro, they can still be produced in vivo, such as using cell lines (e.g., mammalian, insect, or bacterial cell lines) in animals or hybridoma cells. Suitable methods are well known to those skilled in the art.

[0100] The antibody may further be selected from recombinantly produced antibodies. Accordingly, the term "recombinantly produced antibody" includes any antibody produced in vitro using recombinantly engineered DNA molecules. Recombinant antibody production can be carried out using cell lines (e.g., mammalian, insect, or bacterial cell lines) in animals or hybridoma cells. Methods for the recombinant production of antibodies are well known to those skilled in the art. For example, antibody genes for immunospecific antibody heavy and light chains can be cloned into high-yield expression vectors, which are subsequently introduced into expression hosts (e.g., bacterial, yeast, insect, or mammalian cells) to produce the recombinant antibody.

[0101] The antibody may further be selected from a monovalent antibody, a monospecific antibody, and / or a multispecific antibody, including a bispecific antibody.

[0102] The valency of an antibody describes the number of antigen binding sites present per antibody molecule. Thus, the term "monovalent antibody" describes any antibody that has one binding site for an antigen, epitope, or cell type or tissue. A bivalent antibody has two binding sites for an antigen, epitope, or cell type or tissue. A multivalent antibody has multiple binding sites for an antigen, epitope, or cell type or tissue, i.e., two or more, for example, two, three, four, or five, for an antigen, epitope, or cell type or tissue.

[0103] The specificity of an antibody generally describes its ability to recognize and distinguish a single antigen epitope from other epitopes. The term "monospecific antibody" describes any antibody that has specificity for one antigen, epitope, cell type, or tissue. For example, monoclonal antibodies are monospecific because they bind to only one epitope with each of their two antigen-binding arms. As used herein, the term "bispecific antibody" describes, in the broadest sense, an antibody that interacts with two different epitopes, such as an antibody containing two functional antigen-binding domains with specificity for two different antigens, or alternatively, two different epitopes on the same antigen. A bispecific antibody can be derived from two monoclonal antibodies. Optionally, these two different epitopes may be located on the same antigen, but they may also be located on two different antigens. Bispecific antibodies can be produced using conventional technologies, including chemical or hybrid hybridoma production, as well as other technologies, including, but not limited to, technologies that provide molecules such as scFvs with antigen-binding regions of different specificities using peptide linkers such as G4S linkers, and knobs-into-holes engineering. As used herein, the term "multispecific" can be understood in its broadest sense to describe antibodies that interact with two or more different types of epitopes. Optionally, these epitopes can be located on the same antigen or on two or more antigens. For example, in multispecific antibodies, two or more or three or more functional antigen-binding domains can be present, with specificity for two or more or three or more distinct antigens or distinct epitopes. Thus, bispecific and multispecific antibodies target two and more antigens or epitopes, respectively.

[0104] The antibody may also be a human antibody, a humanized antibody, and / or a chimeric antibody. Chimeric antibodies are antibodies in which at least one region of an immunoglobulin from one species is fused to another region of an immunoglobulin from another species by genetic engineering to reduce the immunogenicity of the antibody. For example, mouse V L and V H The region can be fused with the remaining portion of a human immunoglobulin. A particular type of chimeric antibody is a humanized antibody. Humanized antibodies are produced by merging DNA encoding the CDRs of a non-human antibody with human antibody-producing DNA. The resulting DNA construct can then be used to express and produce antibodies that are typically not as immunogenic as the non-human parent antibody or as chimeric antibodies, since only the CDRs are non-human. Furthermore, the antibody can be a human antibody, i.e., the nucleic acid sequence of the antibody is entirely of human origin.

[0105] The use of human, humanized, or chimeric antibodies is preferred for in vivo, particularly human, applications, eg, for prevention, treatment, or in vivo diagnosis.

[0106] The antibody may further be selected from an immunoglobulin. The term "immunoglobulin" describes any protein from the immunoglobulin class that is produced by the immune system to neutralize substances foreign to the body. An immunoglobulin contains at least one immunoglobulin (Ig) domain.

[0107] The antibody may further be selected from a tetrameric antibody comprising two heavy chain molecules and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, and / or an antibody light chain-antibody heavy chain pair.

[0108] The term "tetrameric antibody comprising two heavy chain molecules and two light chain molecules" describes any antibody complex comprising two heavy chain molecules and two light chain molecules. These may comprise the complete heavy and / or light chain sequences of a full-length antibody, or only portions thereof. Tetrameric antibodies are particularly characterized by: (1) a heavy chain comprising a variable region and three domains, C;H 1. C H 2, and C H (2) for the light chain, a heavy chain constant region comprising a light chain variable region and one domain C L The term "tetrameric antibody" may refer to a protein comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, including a light chain constant region comprising: a heavy chain constant region comprising: ...

[0109] The term "antibody light chain monomer" describes any antibody that contains only light chain molecules but no heavy chain molecules. Accordingly, the term "antibody heavy chain monomer" describes any antibody that contains only one heavy chain molecule but no light chain molecules. Accordingly, the term "antibody light chain dimer" describes a complex of two light chain monomers, and the term "antibody heavy chain dimer" describes a complex of two heavy chain monomers. The term "antibody light chain-antibody heavy chain pair" describes any complex that contains a pair of light chain monomers and heavy chain monomers.

[0110] The antibody may further be selected from "single domain antibodies". The term "single domain antibody" refers to an antibody comprising a variable domain (V) of the light chain. L ) or the variable domain of the heavy chain (V H ) describes antibody fragments consisting of a single monomeric variable antibody domain.

[0111] The antibody may further be selected from "single chain antibodies." The term "single chain antibody" describes antibody fragments consisting of a single polypeptide chain.

[0112] The antibody may further be selected from intrabody and / or heteroconjugate antibody. The term "intrabody" describes any antibody that targets an intracellular protein within a cell. Methods for transferring intrabody into target cells to enable binding to intracellular target proteins are well known to those skilled in the art, such as direct expression of intrabody by target cells, as applied in gene therapy. A "heteroconjugate antibody" is a complex of two or more antibodies (e.g., monoclonal antibodies, Fab, or scFv) of different specificities that are covalently linked.

[0113] The antibody may further be selected from camelid antibodies, affibodies, anticalins, affilins, atrimers, DARPins, FN3 scaffolds such as adnectins and centilins, finomers, Kunitz domains, pronectins, and OBodies, and / or anti-idiotypic (anti-Id) antibodies. The term "camelid antibody" describes any antibody having the structure of an antibody derived from the mammalian Camelidae family (e.g., llamas, camels, and alpacas), e.g., an antibody lacking any light chains and consisting of two identical heavy chains. The term "affibody" describes any antibody-mimetic protein capable of binding multiple antigens with high affinity. For example, an affibody may be based on the immunoglobulin-binding domain of a protein, such as the Z domain of protein A from Staphylococcus aureus. Further examples will be familiar to those skilled in the art. FN3 scaffolds, such as anticalins, affilins, atrimers, DARPins, adnectins and centrins, finomers, Kunitz domains, pronectins, and OBodies are further scaffolds known in the art and can be used in accordance with the present invention. Scaffolds are described, for example, in

[16] . The term "anti-idiotypic (anti-Id) antibody" describes any antibody capable of binding to the idiotype of another antibody.

[0114] An "antigen-binding fragment" of an antibody is preferably a fragment of an antibody that exhibits essentially the same function and specificity as the intact antibody from which it is derived. Limited proteolysis with papain typically cleaves Ig phenotypes into three fragments. Two identical amino-terminal fragments, each containing one entire light chain and approximately half of a heavy chain, are the antigen-binding fragment (Fab). The third fragment, similar in size but containing the carboxy-terminal halves of both heavy chains along with their interchain disulfide bonds, is the crystallizable fragment (Fc). The Fc contains carbohydrate chains, a complement-binding site, and an FcR-binding site. Limited pepsin digestion produces a single F(ab')2 fragment containing both Fab pieces and the hinge region, including the interchain disulfide bond. F(ab')2 is bivalent for antigen binding. The disulfide bond of F(ab')2 can be cleaved to obtain Fab'. Additionally, the variable regions of the heavy and light chains can be fused together to form a single-chain variable fragment (scFv).

[0115] Because the first generation of full-sized antibodies can present some problems, many second generation antibodies may contain only fragments of antibodies. The variable domain (Fv) is a single V L and one V H The smallest fragments contain an intact antigen-binding domain consisting of the Fv fragment (Fv) and the Fab fragment (Fv). Such fragments containing only the binding domain can be produced by enzymatic methods or by expression of relevant gene fragments, for example, in bacterial and eukaryotic cells. Various approaches can be used, for example, either Fv fragments alone or Fab fragments containing one of the upper arms of the "Y" comprising the Fv and the first constant domain. When only variable fragments are used, they can usually be stabilized by introducing a polypeptide linkage between the two chains, resulting in the generation of a single-chain Fv (scFv). Alternatively, disulfide-linked Fv (dsFv) fragments can be used. The binding domain of the fragment can be combined with any constant domain to generate a full-length antibody or fused to other proteins and polypeptides.

[0116] A preferred recombinant antibody fragment is a single-chain Fv (scFv) fragment. Generally, it has high affinity to its antigen and can be expressed in various hosts. These and other properties make scFv fragments not only applicable in medicine but also potentially applicable in biotechnology. As detailed above, in scFv fragments, V H Domains and V L The domains are linked by hydrophilic and flexible peptide linkers, which improve expression and folding efficiency. Linkers of about 15 amino acids are usually used, of which the (Gly4Ser)3 linker is the most frequently used. Depending on the linker used, scFv molecules may be easily proteolytically degraded. With the development of genetic engineering techniques, these limitations could be virtually overcome by research focused on improving function and stability. Examples include V H -V L The creation of disulfide-stabilized (or disulfide-linked) Fv fragments in which the dimer is stabilized by an interchain disulfide bond. L Domains and V H Cysteines are introduced at the interface between the domains to form disulfide bridges that hold the two domains together.

[0117] scFvs can be complexed into dimers (diabodies), trimers (triabodies), or larger aggregates such as TandAbs and Flexibodies.

[0118] Antibodies with two binding domains can be generated, for example, either through the association of two scFvs with a simple polypeptide linkage (scFv)2 or through the dimerization of two monomers (diabodies). The simplest design is a diabody, which has two functional antigen-binding domains.

[0119] Antibody formats containing four variable domains in the heavy chain and four variable domains in the light chain have also been developed. Examples of these include TandAb and flexibody (Affimed Therapeutics AG, Heidelberg, Germany). Due to its four binding domains, TandAb usually exhibits superior binding properties compared to antibody formats containing only two binding domains, such as diabodies. Flexibodies combine the diabody multimer motif of scFv, resulting in a highly flexible multivalent molecule that can link two molecules that are quite separate from each other on the cell surface.

[0120] Antibodies may also be selected from any of the antigen-binding fragments of the above-mentioned molecules.As specified above, the "antigen-binding fragment" of an antibody is a fragment of an antibody, which essentially exhibits the same antigen-binding activity and specificity as the complete antibody from which the fragment is derived.An antigen-binding fragment is generally understood as a polypeptide that contains at least one antigen-binding fragment of a full-length antibody.Generally, antigen-binding fragments consist of at least the heavy chain variable domain and the light chain variable domain, arranged in such a way that both domains can cooperate to bind to specific antigens.

[0121] Furthermore, certain binding molecules or antigen-binding fragments of monoclonal antibodies, including but not limited to Fv, scFv, diabody molecules, or domain antibodies (Domantis), can be stabilized by incorporating disulfide bridges to reinforce the VH and VL domains.

[0122] The antibody may further be included in a complex with additional immunoglobulin molecules or fragments thereof (such as additional antibodies) or non-immunoglobulin molecules. For example, the antibody may form a homomultimer with additional identical antibodies. Preferably, the antibody is included in a chimeric antigen receptor (CAR). The term "CAR" describes any receptor protein, usually on T cells, that is specifically designed to enable T cells to target specific antigens. Methods for preparing CARs are well known to those skilled in the art.

[0123] In still further preferred embodiments, the antibody is a monoclonal antibody, a recombinantly produced antibody, a monospecific antibody, a multispecific antibody including a bispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, an immunoglobulin, a synthetic antibody, a tetrameric antibody comprising two heavy chain molecules and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, an intrabody, a heteroconjugate antibody, a single domain antibody, a univalent antibody, a single chain antibody or is selected from single chain Fv (scFv), camelid antibodies, affibodies, anticalins, affilins, atrimers, DARPins, FN3 scaffolds such as adnectins and centilins, fynomers, Kunitz domains, pronectins, and OBodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of any of the above, and / or the antibody is comprised in a chimeric antigen receptor (CAR).

[0124] In another still further preferred embodiment, the antibody is selected from a monoclonal antibody, a recombinantly produced antibody, a monospecific antibody, a multispecific antibody, including a bispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, an immunoglobulin, a synthetic antibody, a tetrameric antibody comprising two heavy chain molecules and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, an intrabody, a heteroconjugate antibody, a single domain antibody, a monovalent antibody, a single chain antibody or single chain Fv (scFv), a camelid antibody, a Fab fragment, a F(ab')2 fragment, a disulfide-linked Fv (dsFv), an anti-idiotypic (anti-Id) antibody, and an antigen-binding fragment of any of the above, and / or the antibody is comprised in a chimeric antigen receptor (CAR).

[0125] Furthermore, the antibody may comprise a heavy chain constant region and / or a light chain constant region. The heavy chain constant region may be selected from the group of human immunoglobulins selected from IgA, IgD, IgE, IgG, or IgM (including any subclass of these isotypes). Preferably, the heavy chain constant region is selected from the group of human immunoglobulins consisting of IgG and IgA, and more preferably, the heavy chain constant region is selected from the group of human immunoglobulins consisting of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Because antibodies can be recombinantly expressed and produced, the antibody may also comprise two different heavy chain constant regions, for example, one IgG1 heavy chain and one IgG2 heavy chain, or heavy chains derived from different species. However, the heavy chains are preferably derived from the same species. Furthermore, the antibody may comprise either an IgGκ light chain constant region or an IgGλ light chain constant region. Preferably, the light chain constant region is selected from the group of human immunoglobulins consisting of IgGκ and IgGλ.

[0126] In yet a further preferred embodiment, the antibody comprises a heavy chain constant region and / or a light chain constant region, preferably wherein the heavy chain constant region is selected from the group of human immunoglobulins consisting of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, and / or the light chain constant region is selected from the group of human immunoglobulins consisting of IgGκ and IgGλ.

[0127] In still further preferred embodiments, the antibody comprises a heavy chain constant region, wherein the heavy chain constant region is selected from the group of human immunoglobulins consisting of IgG1, IgG2, IgG3, and IgG4.

[0128] In a preferred embodiment, the antibody comprises a heavy chain constant region, wherein the heavy chain constant region is a human IgG1 constant region.

[0129] The heavy chain constant region may also be a variant of the wild-type human IgG heavy chain constant region, and preferably, the variant human IgG heavy chain constant region binds to one or more human Fc gamma receptors. Generally, Fc receptors are surface proteins of certain cells that contribute to the immune system. There are several classes of Fc receptors, which can be distinguished, inter alia, by the antibody type with which they interact. Thus, the term "Fc gamma receptor" describes an Fc receptor that binds an antibody having an IgG constant region. This class of Fc gamma receptor further includes several subclasses, such as FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB, which generally differ in structure and affinity for IgG and different IgG subclasses. Preferably, the variant human IgG heavy chain constant region of the antibody of the present invention binds to one or more human Fc gamma receptors selected from the group consisting of FcγRI, FcγRIIA, and FcγRIIIA. Also preferably, the variant human IgG heavy chain constant region binds to one or more human Fc gamma receptors selected from the group consisting of FcγRI, FcγRIIA, and FcγRIIIA with higher affinity than the wild-type human IgG heavy chain constant region binds to the human Fc gamma receptor. "Higher affinity binding" means that the binding of the antibody according to the present invention to one or more human Fc gamma receptors selected from the group consisting of FcγRI, FcγRIIA, and FcγRIIIA is at least two-fold, and preferably at least three-fold, stronger than the binding of the wild-type human IgG heavy chain constant region to the human Fc gamma receptor, as determined, for example, by methods known to those skilled in the art, such as Western blot analysis, ELISA, or surface plasmon resonance.

[0130] In yet a further preferred embodiment, the heavy chain constant region is a variant of the wild-type human IgG heavy chain constant region, and preferably the variant human IgG heavy chain constant region binds to one or more human Fc gamma receptors selected from the group consisting of FcγRI, FcγRIIA, and FcγRIIIA with higher affinity than the wild-type human IgG heavy chain constant region binds to the human Fc gamma receptor.

[0131] In another aspect, the present invention relates to an antibody that specifically binds to the same epitope of human L1CAM as the antibody of the present invention and / or competes with the antibody of the present invention in binding to human L1CAM, preferably an antibody whose epitope is within fibronectin type III domains 1 to 3 (FN III 1 to 3) of human L1CAM.

[0132] All of the above-identified features of the antibodies of the invention are considered to relate to the antibodies of the further aspects of the invention as well.

[0133] The antibody can specifically bind to the same epitope of human L1CAM as the antibody of the present invention.

[0134] The term "epitope" describes the portion of an antigen recognized by an antibody or related binding molecule. For example, an epitope is a specific piece of an antigen to which an antibody binds. Epitopes can be conformational or linear epitopes. Conformational epitopes are usually composed of non-contiguous stretches of the antigen's amino acid sequence. These epitopes interact with the paratope (the binding site on the antibody) based on the antigen's 3D surface features and shape or tertiary structure. The percentage of epitopes that are conformational is unknown. Linear epitopes are usually epitopes recognized by antibodies through their amino acid sequence or primary structure.

[0135] L1-OV549.20 has been shown to bind to and recognize an epitope within the fibronectin type III domains 1-3 (FN III 1-3) of L1CAM [6]. Methods for determining the epitope bound and recognized by a binding molecule have been described in the prior art. The recognized epitope can be determined by constructing a series of L1CAM-Fc proteins with distinct Ig domains. For fine mapping, recombinant V5-tagged L1CAM fragments can be used, as described, for example, in

[23] . The recombinant proteins can be used in ELISA or Western blot analysis for epitope mapping. In general, methods for determining the epitope of a given antibody are known in the art and involve preparing synthetic linear peptides of a given region of interest and then testing whether the antibody binds to the peptide (see

[24] ). Alternatively, different recombinant proteins covering the region of interest can be generated and tested for antibody binding

[25] .

[0136] Alternatively or additionally, the antibody may compete with the antibody of the invention for binding to human L1CAM, preferably the epitope is within fibronectin type III domains 1-3 (FN III 1-3) of human L1CAM.

[0137] The competition in antibody binding can generally be determined by assays known to those skilled in the art, such as competitive binding assays.Competitive binding assays are usually based on antibody-antigen interaction, where the number of antigen binding sites on antibody is limited compared to the amount of different competing antibodies.For example, competitive binding assays can take the form of immunoassays.

[0138] The human L1CAM protein typically contains six immunoglobulin domains (Ig I-Ig VI), followed by an N-terminal extracellular portion consisting of five fibronectin type III domains (FN III 1-5), a transmembrane helix, and a small C-terminal intracellular domain. Preferably, the epitope is located within fibronectin type III domains 1-3 (FN III 1-3) of human L1CAM.

[0139] Preferably, the antibody of any of the aspects of the invention comprises: (i) It binds to human L1CAM within the fibronectin type III domains 1 to 3 (FN III 1 to 3) of L1CAM.

[0140] Preferably, the antibody of any of the aspects of the invention comprises: (ii) an affinity (K) of 20 nM or less, 10 nM or less, or 1 nM or less with human L1CAM; D ) to join them.

[0141] Preferably, the antibody of any of the aspects of the invention comprises: (iii) an affinity (K) of 20 nM or less, 10 nM or less, or 1 nM or less with cynomolgus monkey L1CAM; D ) to join them.

[0142] Methods for determining the binding affinity of an antibody are well known to those skilled in the art and are exemplarily described above in the context of the specific binding of an antibody. For example, surface plasmon resonance using a Biacore® device can be used. For example, the affinity (K D ) is determined at room temperature. For example, binding affinity can be determined as described in Example 2 below.

[0143] Preferably, the antibody of any of the aspects of the invention comprises: (iv) inhibiting tumor cell migration on fibronectin-coated surfaces in vitro;

[0144] Methods for determining tumor cell migration on fibronectin-coated surfaces in vitro are well known to those skilled in the art. For example, fluorescently labeled tumor cells can be seeded on the edge of a fibronectin-coated well. After 48 hours, migration of tumor cells to the center of the well can be determined. Preferably, when an antibody according to any of the embodiments of the present invention is applied to tumor cells, migration of these tumor cells on fibronectin-coated surfaces in vitro is inhibited.

[0145] In this context, inhibition of migration means that when any of the antibodies of the present embodiments are applied to tumor cells, the migration of those tumor cells on a fibronectin-coated surface in vitro after 48 hours is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to the migration of tumor cells of the same cell type treated with an isotype control antibody on a fibronectin-coated surface in vitro.

[0146] Additionally, the half-maximal inhibitory concentrations (IC) for the antibodies of the present invention for inhibiting cancer cell migration of HCT116 tumor cells were calculated. 50 ) is 1×10 -4 M to 1 x 10 -8 up to 1 x 10 M, preferably 1 x 10 -4 M to 1 x 10 -7 Optionally, the half-maximal inhibitory concentration (IC) for the antibody of the invention for inhibiting cancer cell migration of HCT116 tumor cells may be up to 100 M. 50 ) is also 2×10 -5 M to 1 x 10 -6 Up to 2 x 10 M, preferably 2 x 10 -5 M to 4 x 10 -6 The tumor cells may be up to M. Preferably, the tumor cells are HCT116 tumor cells.

[0147] Preferably, the antibody of any of the aspects of the invention comprises: (v) inhibiting the proliferation of SKOV-3, Panc-1, and / or HCT-116 tumor cells in vitro;

[0148] The method for determining the proliferation of SKOV-3, Panc-1, and / or HCT-116 tumor cells in vitro is well known to those skilled in the art.For example, such proliferation assays can be applied to determine the number of cells, the number of cell divisions, metabolic activity, or DNA synthesis over time.Preferably, tumor cell proliferation is monitored by detecting cell confluence over time using a microscopic device, for example, by determining (for example, counting) the number of cells over time, for example, after 24 hours, 48 ​​hours, 72 hours, or 96 hours.

[0149] In this context, inhibiting proliferation in vitro means that when an antibody of any of the embodiments of the invention is applied to tumor cells, the proliferation of those tumor cells 24 hours, 48 ​​hours, 72 hours, or 96 hours later is reduced by at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to the in vitro growth of tumor cells of the same cell type treated with an isotype control antibody.

[0150] Preferably, the antibody of any of the aspects of the invention comprises: (vi) inhibiting primary tumor growth in a mouse SKOV-3ip xenograft model;

[0151] Methods for determining primary tumor growth in a murine SKOV-3ip xenograft model are well known to those skilled in the art. For example, in a SKOV-3ip xenograft model, 5×10 6 SKOV3 tumor cells (intraperitoneally) can be injected into NMRI-nu / nu mice. Five days later, the mice can be randomized into different groups and treatment can begin. Anti-L1CAM antibody or control antibody can be injected intraperitoneally at a dose of 10 mg / kg three times a week for six weeks. Upon sacrificing the mice, treatment efficacy can be evaluated by comparing tumor weight and ascites volume between the treatment and control groups.

[0152] In this context, inhibition of primary tumor growth in a mouse model is understood to mean that when an antibody of any of the embodiments of the invention is applied to a tumor mouse model, tumor growth at the time endpoint requirements are met is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to tumor growth in the same model treated with an isotype control antibody or vehicle.

[0153] Preferably, the antibody of any of the aspects of the invention comprises: (vii) Reduces metastasis formation in a murine MDA-MB-231 xenograft model.

[0154] MDA-MB-231 is a highly invasive model cell line derived from metastatic sites of breast cancer. Methods for determining metastasis formation in MDA-MB-231 xenograft models are well known to those skilled in the art. For example, MDA-MB-231-luc2 luciferase-expressing breast cancer cells can be injected into the tail vein of mice (e.g., approximately 5 x 10 per mouse). 5 Mice can be further treated with an antibody according to the invention (e.g., OV549.20) or vehicle (control), e.g., at a dose of 10 mg / kg, three times a week, starting three days before MDA-MB-231-luc2 injection. To monitor the formation of metastases in the lungs and other visceral organs, in vivo imaging of luciferase activity can be performed every seven days and compared in control-treated and antibody-treated cells.

[0155] In this context, a reduction in metastasis formation in a mouse MDA-MB-231 xenograft model is understood to mean that when an antibody of any of the embodiments of the invention is applied to MDA-MB-231-luc2 injected mice, metastasis formation 20-30 days after exposure to MDA-MB231-luc2 cells is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 94%, at least 95%, or at least 99% compared to metastasis formation in the same model treated with a vehicle control.

[0156] Preferably, the antibody of any of the aspects of the invention comprises: (viii) exhibits ADCC activity in vitro and / or binds to the FcγRIIIa receptor in vitro;

[0157] The term "antibody-dependent cellular cytotoxicity (ADCC)" describes the mechanism by which target cells bound and marked by a specific antibody are consequently lysed by effector cells of the immune system (e.g., natural killer (NK) cells). Preferably, the antibody of the present invention exhibits ADCC activity in vitro, i.e., it is capable of mediating an ADCC immune response when bound to target cells in an in vitro assay. Such in vitro assays are well known to those skilled in the art. For example, a suitable assay utilizes effector cells, such as NK cells, capable of inducing lysis of target cells bound by the antibody applied at various effector-to-target (E:T) cell ratios. In such cases, final analysis can be performed, for example, using a real-time cell analysis device.

[0158] Alternatively, or in combination with exhibiting ADCC activity, the antibody of any of the embodiments of the present invention preferably binds to the FcγRIIIa receptor in vitro. Suitable in vitro binding assays for antibodies and receptors that can be used to determine the binding between the antibody of the present invention and the FcγRIIIa receptor in vitro are well known to those skilled in the art, and are described, for example, in the context of specific binding above. For example, Western blot analysis, ELISA, or surface plasmon resonance may be used.

[0159] Preferably, the antibody of any of the aspects of the invention comprises: (ix) Demonstrates binding to FcRn in vitro.

[0160] Fetal Fc receptor (FcRn) is an Fc receptor protein that is capable of binding, for example, IgG and is normally expressed, for example, by endothelial cells, where it assists in the recycling of serum IgG and albumin. Suitable in vitro binding assays for antibodies and receptors that can be used to determine binding between an antibody of the invention and FcRn in vitro are well known to those skilled in the art or are described, for example, in the context of specific binding, above.

[0161] Preferably, the antibody of any of the aspects of the invention comprises: (x) does not cross-react with human CHL1, human NrCAM, and / or human neurofascin in vitro;

[0162] L1CAM belongs to the L1 family of proteins, which includes a total of four different L1-like proteins, all of which are cell adhesion molecules (CAMs) and members of the immunoglobulin superfamily. Besides L1CAM, three additional members of the L1 family are a close homolog of L1CAM (CHL1), neural cell adhesion molecule (NrCAM), and neurofascin.

[0163] An antibody of any of the present aspects that "does not cross-react" with human CHL1, human NrCAM, and / or human neurofascin in vitro is understood to be an antibody that binds to human L1CAM in vitro but does not substantially bind to any one of human CHL1, human NrCAM, and / or human neurofascin in vitro. In one embodiment, an antibody of any of the present aspects that does not cross-react with human CHL1, human NrCAM, and / or human neurofascin in vitro binds to human CHL1, human NrCAM, and / or human neurofascin in vitro at a concentration of 1×10 -7 M or more or 1×10 -6 K above M (to the point where binding affinity is no longer detectable) D The binding affinity of the antibody of the present invention shows very low or non-specific binding with a binding affinity of 0.05%. In one embodiment, the binding affinity is determined by a standard binding assay such as an ELISA assay as in the Examples or surface plasmon resonance. For example, the binding affinity is determined at room temperature. Further suitable methods for determining the binding of any of the antibodies of the present invention to CHL1, NrCAM, and / or neurofascin are well known to those skilled in the art. For example, methods such as Western blot analysis or determining a shift in the fluorescent signal in a cytometer-based assay can be used.

[0164] In still further preferred embodiments, the antibody of any of the aspects of the invention described herein comprises: (i) binds to human L1CAM within fibronectin type III domains 1-3 (FN III 1-3) of L1CAM; and / or (ii) an affinity (K) of 20 nM or less, 10 nM or less, or 1 nM or less with human L1CAM; D ) and / or (iii) an affinity (K) of 20 nM or less, 10 nM or less, or 1 nM or less with cynomolgus monkey L1CAM; D ) and / or (iv) inhibiting tumor cell migration on fibronectin-coated surfaces in vitro; and / or (v) inhibiting the growth of SKOV-3, Panc-1, and / or HCT-116 tumor cells in vitro; and / or (vi) inhibiting primary tumor growth in a mouse SKOV-3ip xenograft model; and / or (vii) reducing metastasis formation in a murine MDA-MB-231 xenograft model; and / or (viii) exhibits ADCC activity in vitro and / or binds to the FcγRIIIa receptor in vitro; and / or (ix) exhibits binding to FcRn in vitro; and / or (x) does not cross-react with human CHL1, human NrCAM, and / or human neurofascin in vitro;

[0165] Furthermore, an antibody according to any of the aspects of the present invention may be humanized. Preferably, the antibody according to the present invention is partially or fully humanized. Methods for humanizing antibodies are well known to those skilled in the art, for example, by introducing the sequences of the CDRs of the antibody of the present invention into the sequence of a human antibody. Optionally, in addition, one or more non-human positions, for example, 1, 2, 3, 4, 5, or 6, for example, up to 10, non-human positions, may be reintroduced into the human framework sequence.

[0166] Preferably, the antibody according to any of the aspects of the invention is humanized.

[0167] In another preferred embodiment of any of the aspects of the invention, the antibody is a multispecific or bispecific antibody and / or is a humanized antibody.

[0168] Methods for producing antibodies, such as the antibody of the present invention, are well known to those skilled in the art. For example, antibodies can be produced by generating hybridoma cells. Methods for generating hybridoma cells and for producing antibodies with the help of hybridoma cells are well known to those skilled in the art. Generally, mice are injected with a desired antigen and killed several days or weeks later to isolate spleen cells secreting antibodies against the desired antigen. Generally, these antibody-secreting spleen cells are fused with immortalized non-secretory myeloma cells to produce hybridoma cells. These hybridoma cells are then usually screened to select hybridomas that produce the desired antibody. The selected hybridomas can then be cultured in vivo or in vitro, and the desired antibody can be isolated. Preferably, the antibodies herein are produced recombinantly in suitable host cells. DNA encoding the antibody of interest can be placed into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells derived from the CHO GS System™ (Lonza)), or myeloma cells, to obtain antibody synthesis.

[0169] Antibody conjugates / antibody-drug conjugates (ADCs) The antibodies of any of the embodiments of the present invention may further be linked to one or more chemical moieties to form antibody conjugates. For example, the antibody may be linked to one or more such chemical moieties by any chemical bond known to those skilled in the art, such as ionic and / or covalent bonds, and / or by any suitable intermolecular bond, such as hydrogen bonds and / or van der Waals forces.

[0170] Furthermore, the chemical moiety to which the antibody of any of the embodiments of the invention may be linked may be any chemical moiety or substance known to those skilled in the art suitable for application in antibody conjugates. For example, the antibody may be linked to a therapeutically active substance, preferably a chemotherapeutic compound, a cytotoxic compound, a cytostatic compound, a cytokine, a nanoparticle, a radioisotope, and / or an oncolytic virus.

[0171] The term "therapeutically active substance" describes any biologically active substance, i.e., a substance that causes an effect in a living organism. When used in reference to pharmaceutical drugs, a therapeutically active substance is responsible for, for example, the activity of a drug. Methods for determining the effect of a substance on a living organism are well known to those skilled in the art.

[0172] The term "chemotherapeutic compound" describes any substance that can be used in the treatment of cancer as part of a standard chemotherapy regimen as known to those skilled in the art.

[0173] The term "cytotoxic compound" describes any substance that is toxic to cells, for example, by inducing apoptosis or necrosis. Suitable examples of cells are known to those skilled in the art, such as immune cells. The cytotoxicity of a compound can be measured by common cytotoxicity assays known to those skilled in the art. Examples of cytotoxic agents include, for example, bacterial (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A), fungal (e.g., alpha-sarcin, restrictocin), or plant (e.g., abrin, ricin, modeccin, viscumin, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, neomycin, and trichothecene small molecule or enzymatically active toxins, mitotic inhibitors, or DNA damaging agents.

[0174] The term "cytostatic compound" describes any substance capable of inhibiting cell growth. The ability of a compound to inhibit cell growth can be measured by common cell growth assays known to those skilled in the art.

[0175] Suitable chemotherapeutic compounds include alkylating agents, alkyl sulfonates, aziridines, ethyleneimines and methylamelamine, acerogenin, camptothecin, bryostatin, ostatin, kallistatin, CC-1065, cryptophycin, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyln, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemycin, bisphosphonates, esperamicin, chromoprotein enediyne Antibiotic chromophores, aciacinomycins, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carahtein, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid acid), nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamyein, rodorubicin, streptonigrin, streptozoein, tubercidin, ubenimex, zinostatin, zorubicin;Antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogues, purine analogues, androgens, anti-adrenals, folic acid replenishers such as folinic acid, acegiatone, aldophosphamide glycosides, aminolevulinic acid, emururacil, anisacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, eflornithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mito Xantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, iosoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK® polysaccharide complex, razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracrine A) A), roridin A and anguidine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan, topoisomerase inhibitor RFS 2000;Difluoromethylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0176] The term "cytokine" describes a substance that has the ability to affect cell growth and / or cell differentiation. Suitable cytokines are well known to those skilled in the art, such as interferons, interleukins, colony-stimulating factors, tumor necrosis factors, or chemokines. Methods for measuring cell growth or cell differentiation are well known to those skilled in the art. Suitable cytokines include, for example, IL-2, G-CSF, GM-CSF, and TNF-α.

[0177] The term "nanoparticle" describes any particle having a diameter between 1 and 100 nm. Methods for determining the diameter of a particle are also well known to those skilled in the art.

[0178] The term "radioisotope" describes any substance that is an unstable or metastable isotope of a natural or artificial element. Suitable radioisotopes are preferably applicable for treatment in the human or animal body. Suitable radioisotopes include: 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186 Re is an example.

[0179] The term "oncolytic virus" preferably describes any virus that targets cancer cells, e.g., causes infection and killing of these cancer cells, usually by oncolysis. Suitable oncolytic viruses are, for example, genetically adapted herpes simplex viruses, adenoviruses, vaccinia viruses, vesicular stomatitis viruses, polioviruses, reoviruses, Seneca viruses, echoviruses, Semliki Forest viruses, Maraba viruses, and / or coxsackie viruses.

[0180] Alternatively, or in combination, the antibody may be linked to a diagnostic compound, preferably selected from a radioisotope, a chemiluminescent compound, a fluorescent compound, a dye, or an enzyme.

[0181] The term "diagnostic compound" describes any substance suitable for application in diagnostic methods for the human or animal body. For example, diagnostic compounds can be used to stain tissues, cells, or other materials in various analytical methods, such as fluorescence imaging or spectroscopy. For example, diagnostic compounds can be used to specifically stain cancer cells. Further diagnostic applications are well known to those skilled in the art.

[0182] The term "chemiluminescent compound" describes any substance capable of emitting light as a result of a chemical reaction. Methods and devices for detecting chemiluminescence are well known to those skilled in the art.

[0183] The term "fluorescent compound" describes any substance that re-emits light in response to light excitation. Usually, such substances are also called fluorophores. Suitable fluorescent compounds can be, for example, substrates for enzymes or probes. Methods and devices for detecting fluorescence are well known to those skilled in the art. Fluorescent compounds include, for example, reactive and conjugated probes, such as aminocoumarins, fluorescein, and Texas Red, Alexa Fluor dyes, Cy dyes, and DyLight dyes.

[0184] The term "dye" describes any substance that is colored by absorbing less than all wavelengths of visible light and that is capable of chemically binding to a target molecule, thereby allowing it to be used to label and visualize normally colorless target molecules.

[0185] The term "enzyme" describes any protein capable of catalyzing a chemical reaction. Suitable enzymes are known in the art and include horseradish peroxidase (HRP).

[0186] In another preferred embodiment of any of the aspects of the invention, the antibody (a) a therapeutically active substance; Preferably, chemotherapy compounds, cytotoxic compounds, cytostatic compounds, cytokines, nanoparticles, Radioisotopes, and / or Oncolytic viruses and / or (b) linked to a diagnostic compound, preferably selected from a radioisotope, a chemiluminescent compound, a fluorescent compound, a dye, or an enzyme.

[0187] Preferably, the (a) therapeutically active substance and / or (b) diagnostic compound is selected from a radioisotope, a chemotherapeutic compound, a cytotoxic compound, and / or a cytostatic compound.

[0188] Additionally, the antibodies of the present invention may be covalently linked to (a) a therapeutically active agent or a chelator thereof, or (b) a diagnostic compound or a chelator thereof.

[0189] The term "covalently linked" describes any chemical bond involving the sharing of electron pairs between atoms. Examples of such covalent bonds are cleavable bonds (e.g., disulfide bonds, hydrazone-based bonds, or peptide bonds) or non-cleavable bonds (e.g., thioether bonds). Preferably, the covalent bond is stable when applied to the body and circulation therein, and only becomes cleavable within the target cells or upon reaching the target tissue. The linker may be a cleavable linker or a non-cleavable linker.

[0190] The term "chelator" describes any substance that contains two or more lone pairs of electrons and is therefore capable of forming two or more coordinate bonds with a metal ion. The chelator can be an organic compound (e.g., ethylenediaminetetraacetic acid (EDTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)). For example, an antibody according to any of the present embodiments can be linked to a chelator that is further linked to a radioisotope, e.g., a positron-emitting and gamma-emitting radiometal, enabling sensitive and quantitative molecular positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging of antibody distribution in vivo. In another example, an antibody according to any of the present embodiments can be linked to a chelator, which is further linked to a radioisotope, allowing for targeted delivery of the radioisotope directly to a desired location in the body (e.g., a tumor site).

[0191] Furthermore, the antibodies of the present invention may be covalently linked to (a) a therapeutically active agent or a chelator thereof, or (b) a diagnostic compound or a chelator thereof via a linker. Such antibodies of the present invention linked to a therapeutically active agent or a chelator thereof via a linker are also referred to herein as "antibody-drug conjugates," "antibody conjugates," "antibody drug conjugates," or "ADCs."

[0192] In the context of "antibody-drug conjugate," "antibody conjugate," "antibody drug conjugate," or "ADC" herein, the antibody portion is also referred to as the "antibody moiety."

[0193] The term "linker" describes any molecule suitable for connecting an antibody of the invention to (a) a therapeutically active substance or a chelator thereof or (b) a diagnostic compound or a chelator thereof, and the linker is preferably connected to the antibody of the invention and / or (a) a therapeutically active substance or a chelator thereof, or (b) a diagnostic compound or a chelator thereof by a covalent bond.

[0194] Preferably, the linker provides a stable connection between the antibody of the invention and (a) the therapeutically active agent or its chelator or (b) the diagnostic compound or its chelator when applied to the body and circulation therein, and is only cleavable within the target cell or upon reaching the target tissue. For example, the linker can be a peptide having a length of 2 to 50 amino acids, such as a dipeptide, or an organic compound such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0195] Preferably, the antibody is covalently linked, optionally via a linker, to (a) a therapeutically active agent or chelator thereof or (b) a diagnostic compound or chelator thereof.

[0196] A linker may contain one conjugating component or may contain multiple components.

[0197] For example, a linker may comprise a spacer, which is a moiety that extends the drug linkage, e.g., to avoid blocking the active site of the antibody or improving the solubility of the ADC. Other examples of linker components include stretcher units and amino acid units.

[0198] Two methods are commonly used to conjugate drugs to antibodies: alkylation of reduced interchain cysteine ​​disulfides through enzymatically non-cleavable maleimide or simple cleavable disulfide linkers, and acylation of lysines with cleavable linear amino acids.

[0199] In one embodiment, the linker covalently attaches the antibody to the therapeutically active substance. The same applies to diagnostic compounds. ADCs are prepared using a linker with a reactive functional group for binding the antibody to the therapeutically active substance or diagnostic compound. For example, the cysteine ​​thiol or amine, for example, the N-terminus or amino acid side chain such as lysine of the antibody can form a bond with the functional group of the linker.

[0200] In one embodiment, the linker has a functional group capable of reacting with a free cysteine ​​present on an antibody to form a covalent bond. Non-limiting exemplary such reactive functional groups include activated esters such as maleimide, haloacetamide, α-haloacetyl, succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydride, acid chloride, sulfonyl chloride, isocyanate, and isothiocyanate.

[0201] In some embodiments, the linker has a functional group capable of reacting with an electrophilic group present on an antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linker can react with an electrophilic group on an antibody to form a covalent bond with an antibody unit. Non-limiting exemplary such reactive functional groups include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0202] Suitable linkers include, for example, cleavable and non-cleavable linkers. The linker may be a "cleavable linker" that facilitates release of the drug. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, glycosylase-sensitive (e.g., glucuronidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers. Cleavable linkers are typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that are cleavable by intracellular proteases, such as lysosomal or endosomal proteases. In exemplary embodiments, the linker may be a dipeptide linker, such as a valine-citrulline (val-cit or "VC"), phenylalanine-lysine (phe-lys), or valine-alanine (val-ala or "VA") linker.

[0203] For example, a PEG8-VA linker can be used, as shown in the examples for the ADC conjugate with Tecilin.

[0204] For example, the linker may contain a maleimide group for attachment to the antibody, a PEG linker, and a cleavable val-ala moiety that is coupled to a therapeutically active agent, e.g., a pyrrolobenzodiazepine (PBD) such as SG3199, a maytansinoid such as DM4, or an auristatin such as MMAE.

[0205] For example, the linker may contain a cleavable beta-glucuronide moiety, such as, in the examples, MC-beta-glucuronide, which is linked to a therapeutically active agent, for example, a PDB such as SG3199, a maytansinoid such as DM4, or an auristatin such as MMAE.

[0206] For example, the linker may contain a cleavable val-cit ("VC") moiety, such as in the examples MC-VC-PABC, which is linked to a therapeutically active agent, e.g., the therapeutically active agent is a PDB such as SG3199, a maytansinoid such as DM4, or an auristatin such as MMAE.

[0207] For example, the linker may contain a cleavable sulfo-SPDB moiety, such as sulfo-SPDB in the examples, which is coupled to a therapeutically active agent, for example, the therapeutically active agent being a PDB such as SG3199, a maytansinoid such as DM4, or an auristatin such as MMAE.

[0208] The linker is preferably stable outside the cell in a manner sufficient to be therapeutically effective.Before transport or delivery into cells, the ADC is preferably stable and remains intact, i.e., the antibody remains conjugated with the drug moiety.A linker that is stable outside the target cell can be cleaved at a fairly effective rate once inside the cell.Therefore, an effective linker (i) will maintain the specific binding property of the antibody; (ii) will enable the delivery, for example, intracellular delivery, of the therapeutically active substance; and (iii) will maintain the therapeutic effect, for example, cytotoxic effect, of the therapeutically active substance.

[0209] In one embodiment, the linker is cleavable under intracellular conditions such that cleavage of the linker is sufficient to release the drug from the antibody in the intracellular environment and be therapeutically effective. In some embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) that is hydrolyzable in lysosomes can be used. Such linkers are relatively stable under neutral pH conditions, such as the neutral pH conditions in blood, but are unstable at pHs below 5.5 or 5.0, which are the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a hydrazone-containing linker.

[0210] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including those that can be formed using, for example, SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).

[0211] In some embodiments, the linker is cleavable by a cleaving agent, e.g., an enzyme, present in the intracellular environment (e.g., in a lysosome, endosome, or caveolae). The linker can be, for example, a peptidyl linker cleaved by an intracellular peptidase or protease enzyme, such as, but not limited to, a lysosomal protease or an endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives in target cells, resulting in the release of the active drug. Most typical are peptidyl linkers cleavable by enzymes present in L1CAM-expressing cells. In certain embodiments, the peptidyl linker cleavable by an intracellular protease is a val-cit linker or a val-ala linker. One advantage of using intracellular proteolytic release of a therapeutic agent is that the therapeutic agent is typically attenuated when conjugated, and the serum stability of the conjugate is typically high.

[0212] In other embodiments, the linker is a malonate linker, a maleimidobenzoyl linker, or a 3'-N-amide analog.

[0213] In still other embodiments, the linker unit is non-cleavable and the drug is released, for example, by antibody degradation.

[0214] In some embodiments, the linker is a substantially hydrophilic linker (e.g., PEG, PEG 4- Hydrophilic linkers can be used to reduce the extent to which drugs can be pumped out of resistant cancer cells through MDR (multidrug resistance) or functionally similar transporters, or to improve the pharmacokinetic properties of the ADC.

[0215] In other embodiments, upon cleavage, the linker functions to directly or indirectly inhibit cell growth and / or cell proliferation. For example, in some embodiments, the linker, upon cleavage, can function as an intercalator, thereby inhibiting macromolecular biosynthesis (e.g., DNA replication, RNA transcription, and / or protein synthesis).

[0216] In other embodiments, the linker is designed to promote bystander killing through diffusion of the linker-drug and / or drug alone to neighboring cells. In other embodiments, the linker promotes cellular internalization.

[0217] The presence of a sterically hindered disulfide can increase the stability of a particular disulfide bond and enhance the potency of an ADC. Thus, in one embodiment, the linker comprises a sterically hindered disulfide linkage. A sterically hindered disulfide refers to a disulfide bond that exists within a particular molecular environment, which is characterized by a particular spatial arrangement or orientation of atoms, typically within the same molecule or compound, that prevents or at least partially inhibits reduction of the disulfide bond. Thus, the presence of bulky (or sterically hindering) chemical moieties and / or bulky amino acid side chains in proximity to the disulfide bond prevents or at least partially inhibits the disulfide bond from entering into potential interactions that would result in reduction of the disulfide bond.

[0218] In a preferred embodiment of any of the aspects of the invention, the antibody (a) a therapeutically active substance; Preferably, chemotherapy compounds, cytotoxic compounds, cytostatic compounds, cytokines, nanoparticles, radioisotopes, or Oncolytic viruses in conjunction with, and / or (b) linked to a diagnostic compound, preferably selected from a radioisotope, a chemiluminescent compound, a fluorescent compound, a dye, or an enzyme.

[0219] In a preferred embodiment, the (a) therapeutically active substance and / or the (b) diagnostic compound are selected from a radioisotope, a chemotherapeutic compound, a cytotoxic compound, and / or a cytostatic compound, and / or the antibody is covalently linked to the (a) therapeutically active substance or its chelator or the (b) diagnostic compound or its chelator, optionally via a linker.

[0220] Preferably, the antibody according to any of the aspects of the present invention may be further linked to one or more molecules, particularly therapeutically active substances and / or diagnostic compounds, to form an antibody conjugate.

[0221] Preferred embodiments of any of the aspects of the invention also apply to the antibody conjugates herein.

[0222] Thus, preferably, the antibody portion of the antibody conjugate comprises: (a) a heavy chain variable region (VH) comprising a VH CDR1 comprising the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9), a VH CDR2 comprising the amino acid sequence of YISYSGSYSYNPSLKS (SEQ ID NO: 11), and a VH CDR3 comprising the amino acid sequence of SFSYSYGFAY ​​(SEQ ID NO: 14); and (b) a light chain variable region (VL) comprising a VL CDR1 having the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4), a VL CDR2 having the amino acid sequence of SASYRYT (SEQ ID NO: 5), and a VL CDR3 having the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6).

[0223] Thus, preferably, the antibody portion of the antibody conjugate comprises a heavy chain variable region sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 30, and / or a light chain variable region sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 20. Furthermore, preferably, the antibody portion of the antibody conjugate comprises a heavy chain variable region sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 30, and a light chain variable region sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 20.

[0224] Preferably, the antibody portion of the antibody conjugate comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38. In yet a further preferred embodiment, the antibody portion of the antibody conjugate comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0225] In another further preferred embodiment, the antibody portion of the antibody conjugate consists of a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38. In another further preferred embodiment, the antibody portion of the antibody conjugate consists of a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0226] Preferably, the antibody portion of the antibody conjugate comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38. In yet a further preferred embodiment, the antibody portion of the antibody conjugate comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0227] In another further preferred embodiment, the antibody portion of the antibody conjugate consists of a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38. In another further preferred embodiment, the antibody portion of the antibody conjugate consists of a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0228] In one preferred embodiment, an antibody of any of the present aspects is linked to at least one therapeutically active agent via a linker. Such compounds are also referred to as "antibody-drug conjugates," "antibody conjugates," "antibody drug conjugates," or "ADCs."

[0229] The antibodies of the present invention described herein can be conjugated with a drug moiety to form an anti-L1CAM antibody-drug conjugate (ADC). Antibody-drug conjugates (ADCs) can increase the therapeutic efficacy of antibodies in treating diseases, such as cancer, due to the ability of the ADC to selectively deliver one or more therapeutically active agent moieties to target tissues or cells, such as L1CAM-expressing tumors or L1CAM-expressing cells. Thus, in certain embodiments, the present disclosure provides anti-L1CAM ADCs for therapeutic use, such as for the treatment of cancer.

[0230] The terms "therapeutically active agent," "therapeutically active agent moiety," "drug," "agent," and "drug moiety" are used interchangeably herein.

[0231] The terms "linked" and "conjugated" are also used interchangeably herein to indicate that an antibody and a moiety are covalently linked together.

[0232] In some embodiments, an ADC, or an antibody of the invention linked to at least one therapeutically active agent via a linker, has the following formula (Formula I): Ab-(LD)n (I) wherein Ab is an antibody according to any of the aspects of the invention described herein, and (LD) is a linker-drug moiety. The linker-drug moiety is composed of a linker, L-, and a therapeutically active agent moiety (or drug moiety), -D, which has, for example, cytostatic, cytotoxic, or otherwise therapeutic activity against a target cell, e.g., a cell expressing L1CAM; and n is an integer from 1 to 20. In some embodiments, n is in the range of 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or is 1.

[0233] Examples of therapeutically active agents that can be used in ADCs, i.e., that can be conjugated to the antibodies of the invention, include antimitotic agents, antitumor antibiotics, immunomodulatory agents, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormonal agents, glucocorticoids, photoactive therapeutic agents, oligonucleotides, radioisotopes, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, and combinations thereof.

[0234] In one embodiment, the therapeutically active agent is selected from the group consisting of DNA damaging agents, anti-apoptotic agents, antimitotic agents, antitumor antibiotics, immunomodulatory agents, nucleic acids for gene therapy, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormonal agents, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radioisotopes, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors.

[0235] Mitotic inhibitors In one embodiment, the antibodies of the present invention may be conjugated to one or more antimitotic agents to form ADCs for the treatment of hyperproliferative disorders, neoplastic diseases, disorders involving angiogenesis, and / or disorders involving abnormal neurogenesis. As used herein, the term "antimitotic agent" refers to a cytotoxic and / or therapeutic agent that blocks mitosis, or cell division, a biological process particularly important to cancer cells. Antimitotic agents disrupt microtubules, often by affecting microtubule polymerization (e.g., inhibiting microtubule polymerization) or microtubule depolymerization (e.g., stabilizing the microtubule cytoskeleton against depolymerization), resulting in the prevention of cell division. Thus, in one embodiment, the antibodies of the present invention are conjugated to one or more antimitotic agents that disrupt microtubule formation by inhibiting tubulin polymerization. In another embodiment, the antibodies of the present invention are conjugated to one or more antimitotic agents that stabilize the microtubule cytoskeleton, preventing it from depolymerization. In one embodiment, the antimitotic agent used in the ADCs of the present invention is Ixempra (ixabepilone). Examples of antimitotic agents that can be used in the ADCs of the invention are provided below. The class of antimitotic agents includes the auristatins and maytansinoids, which are described further below.

[0236] Dolastatin The antibodies of the present invention can be conjugated with at least one dolastatin to form an ADC. Dolastatins are short peptide compounds isolated from the Indian Ocean sea hare Dolabella auricularia. Examples of dolastatins include dolastatin 10 and dolastatin 15. Dolastatin 15, a seven-subunit depsipeptide derived from Dolabella auricularia, is a potent antimitotic agent structurally related to dolastatin 10, an antitubulin agent, a five-subunit peptide obtained from the same organism. Auristatins are synthetic derivatives of dolastatin 10.

[0237] Auristatin The antibody of the present invention can be conjugated with at least one auristatin. Auristatins represent a group of dolastatin analogs that have generally been shown to have anti-cancer activity by interfering with microtubule dynamics and GTP hydrolysis, thereby inhibiting cell division. For example, auristatin E is a synthetic analog of the marine natural product dolastatin 10, a compound that inhibits tubulin polymerization by binding to the same site on tubulin as the anti-cancer drug vincristine. Dolastatin 10, auristatin PE, and auristatin E are linear peptides with four amino acids, three of which are unique to the dolastatin class of compounds. Exemplary embodiments of the auristatin subclass of antimitotic agents include, but are not limited to, monomethyl auristatin D (MMAD or auristatin D derivatives), monomethyl auristatin E (MMAE or auristatin E derivatives), monomethyl auristatin F (MMAF or auristatin F derivatives), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid-AE ester (AEVB).

[0238] In one embodiment, the antibody of the present invention is conjugated with at least one MMAE (monomethyl auristatin E). Monomethyl auristatin E (MMAE) inhibits cell division by blocking tubulin polymerization. Due to its toxicity, it cannot be used as a drug by itself. In recent cancer treatment developments, it is linked to an antibody that recognizes a specific marker expressed on cancer cells and directs MMAE to cancer cells. In one embodiment, the linker linking MMAE to the antibody of the present invention is stable in extracellular fluid (i.e., the medium or environment outside the cell), but is cleaved by cathepsin once the ADC binds to a specific cancer cell antigen and enters the cancer cell, thereby releasing the toxic MMAE and activating a potent antimitotic mechanism. In one embodiment, the linker connecting MMAE to the antibody of the invention is stable in extracellular fluids (i.e., the medium or environment outside the cell), but is cleaved by glucuronidase once the ADC binds to a specific cancer cell antigen and enters the cancer cell, thus liberating the toxic MMAE and activating a potent antimitotic mechanism.

[0239] Maytansinoids The antibodies of the present invention can be conjugated with at least one maytansinoid to form an ADC. Maytansinoids are potent antitumor agents originally isolated from members of the higher plant families Celastraceae, Rhamnaceae, and Euphorbiaceae, as well as several species of moss. Evidence suggests that maytansinoids inhibit mitosis by inhibiting the polymerization of the microtubule protein tubulin, thereby preventing the formation of microtubules. Maytansinoids have been shown to inhibit tumor cell growth in vitro using cell culture models and in vivo using experimental animal systems. Furthermore, the cytotoxicity of maytansinoids is 1,000-fold higher than that of conventional chemotherapeutic agents such as methotrexate, daunorubicin, and vincristine. Maytansinoids include, for example, maytansine, maytansinol, and C-3 esters of maytansinol.

[0240] Suitable maytansinoids for use in the ADCs of the present invention can be isolated from natural sources, synthetically produced, or semisynthetically produced. Furthermore, maytansinoids can be modified in any suitable manner, so long as sufficient cytotoxicity is preserved in the final conjugate molecule. In this regard, maytansinoids lack a suitable functional group to which an antibody can be linked. To link a maytansinoid to an antibody to form a conjugate, a linking moiety is desirably utilized, as described in the Examples.

[0241] Representative examples of maytansinoids include, but are not limited to, DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), also known as drug maytansinoid 1, DM2, DM3 (N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-maytansine), DM4 (4-methyl-4-mercapto-1-oxopentyl)-maytansine), and maytansinol (a synthetic maytansinoid analogue).

[0242] In one embodiment of the present invention, the antibody of the present invention is conjugated to at least one DM1. In one embodiment, the antibody of the present invention is conjugated to at least one DM2. In one embodiment, the antibody of the present invention is conjugated to at least one DM3. In one embodiment, the antibody of the present invention is conjugated to at least one DM4.

[0243] Antitumor antibiotics The antibody of the present invention can be conjugated with one or more antitumor antibiotics. As used herein, the term "antitumor antibiotic" refers to an anti-cancer drug that blocks cell growth by interfering with DNA and is produced by microorganisms. Often, antitumor antibiotics either break DNA strands or slow or stop DNA synthesis. Examples of antitumor antibiotics that can be included in ADCs include, but are not limited to, actinomycin (e.g., pyrrolo[2,1-c][1,4]benzodiazepine), anthracycline, calicheamicin, and duocarmycin. In addition to the above, additional antitumor antibiotics that can be used include bleomycin, mitomycin, and plicamycin (also known as mithramycin).

[0244] immunomodulators In one embodiment, the antibody of the present invention may be conjugated to at least one immunomodulator. As used herein, the term "immunomodulator" refers to an agent capable of stimulating or modifying an immune response. In one embodiment, an immunomodulator is an immunostimulatory agent that enhances a subject's immune response. In another embodiment, an immunomodulator is an immunosuppressant that blocks or reduces a subject's immune response. An immunomodulator may regulate myeloid cells (monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) or lymphoid cells (T cells, B cells, and natural killer (NK) cells), and any further differentiated cells thereof. Representative examples include, but are not limited to, bacillus calmette-guerin (BCG) and levamisole (ergamisole). Other examples of immunomodulators that may be used in ADCs include, but are not limited to, cancer vaccines and cytokines.

[0245] Alkylating agents The antibody of the present invention can be conjugated with one or more alkylating agents. Alkylating agents are a class of anticancer compounds that attach alkyl groups to DNA. Examples of alkylating agents that can be used in ADCs include, but are not limited to, alkyl sulfonates, ethylenimines, methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas, triazines, and hydrazines.

[0246] DNA damaging agents In one embodiment, the antibody of the present invention can be conjugated with one or more DNA damaging agents.As used herein, the term "DNA damaging agent" refers to an agent capable of damaging DNA, and is well known to those skilled in the art.DNA damaging agents include DNA alkylating agents.DNA damaging agents also include indolino-benzodiazepines (IGNs).

[0247] In one embodiment, DNA damaging agents may also include pyrrolobenzodiazepines (PBDs) or pyridinobenzodiazepines (PDDs) [26,27].

[0248] For example, SG3199 or VA-SG3199 (Tesirin) can be used. SG3199 is a pyrrolobenzodiazepine (PBD) dimer, the warhead component of the antibody-drug conjugate (ADC) payload Tesirin.

[0249] Antiangiogenic agents In one embodiment, the antibodies of the present invention described herein are conjugated to at least one anti-angiogenic agent. Anti-angiogenic agents inhibit the growth of new blood vessels. Anti-angiogenic agents exert their effects in various ways. In some embodiments, these agents interfere with the ability of growth factors to reach their targets. For example, vascular endothelial growth factor (VEGF) is one of the major proteins involved in initiating angiogenesis by binding to specific receptors on the cell surface. Therefore, certain anti-angiogenic agents that block the interaction of VEGF with its cognate receptor prevent VEGF from initiating angiogenesis. In other embodiments, these agents interfere with intracellular signaling cascades. For example, once a specific receptor on the cell surface is triggered, a cascade of other chemical signals is initiated, promoting blood vessel growth. Therefore, certain enzymes, such as several tyrosine kinases, known to promote intracellular signaling cascades that contribute to cell proliferation are targets for cancer treatment. In other embodiments, these agents interfere with intracellular signaling cascades. In yet other embodiments, these agents disable specific targets that activate and promote cell growth or by directly interfering with the growth of vascular cells. Angiogenesis inhibition has been found in over 300 substances, with numerous direct and indirect inhibitory effects. Representative examples of antiangiogenic agents that can be used in ADCs include angiostatin, ABX EGF, C1-1033, PKI-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD 55900, CP358, PD153035, AG1478, IMC-C225 (Erbitux), ZD1839 (Iressa), OSI-774, erlotinib (Tarceva), arrestin, endostatin, BAY12-9566 and fluorouracil or doxorubicin, Canstatin, carboxyamidotriozole and paclitaxel combination, EMD121974, S-24, vitaxin, dimethylxanthenone acetic acid, IM862, interleukin-12, interleukin-2, NM-3, HuMV833, PTK787, RhuMab, angiozyme (ribozyme), IMC-1C11, Neovastat, marimastat, prinomastat, BMS-275291, C OL-3, MM1270, SU101, SU6668, SU11248, SU5416, combinations with paclitaxel, combinations with gemcitabine and cisplatin, and combinations with irinotecan and cisplatin, and combinations with radiation, tecogalan, temozolomide and PEG-interferon alpha 2b, tetrathiomolybdate, TNP-470, thalidomide, combinations of CC-5013 and taxotere, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors (deforolimus, everolimus (Afinitor), Novartis) Pharmaceutical Corporation), and temsirolimus (Torisel, Pfizer, Inc.), tyrosine kinase inhibitors (e.g., erlotinib (Tarceva, Genentech, Inc.), imatinib (Gleevec, Novartis Pharmaceutical Corporation), gefitinib (Iressa, AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb), sunitinib (Sutent, Pfizer, Inc.), nilotinib (Tasigna, Novartis Pharmaceutical Corporation), lapatinib (Tykerb, GlaxoSmithKlinePharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), phosphoinositide 3-kinase (PI3K).

[0250] Antimetabolites The antibodies of the present invention may be conjugated to at least one antimetabolite. Antimetabolites, which closely resemble normal substances within cells, are a type of chemotherapy treatment. When cells incorporate an antimetabolite into their metabolism, the results are negative for the cell, for example, the cell cannot divide. Antimetabolites are classified by the substances they interfere with. Examples of antimetabolites that can be used in ADCs include, but are not limited to, folate antagonists (e.g., methotrexate), pyrimidine antagonists (e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine), purine antagonists (e.g., 6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nelarabine, and pentostatin).

[0251] Boron-containing agents The antibody of the present invention can be conjugated with at least one boron-containing agent.Boron-containing agents include a class of cancer treatment compounds that interfere with cell proliferation.Representative examples of boron-containing agents include, but are not limited to, borophycin and bortezomib.

[0252] Chemical protectants The antibodies of the present invention may be conjugated to at least one chemoprotectant.

[0253] Chemoprotective agents are a class of compounds that help protect the body from certain toxic effects of chemotherapy. Chemoprotective agents can be administered with various chemotherapy drugs to protect healthy cells from the toxic effects of chemotherapy drugs, while allowing cancer cells to be treated with the administered chemotherapy. Representative chemoprotective agents include, but are not limited to, amifostine (used to reduce the nephrotoxicity associated with the cumulative dose of cisplatin, dexrazoxane, to treat the extravasation caused by the administration of anthracyclines, and to treat cardiac complications caused by the administration of the antitumor antibiotic doxorubicin), and mesna (Mesnex, Bristol-Myers Squibb) (used to prevent hemorrhagic cystitis during chemotherapy treatment with ifosfamide).

[0254] Photoactive Therapeutic Agents The antibodies of the present invention may be conjugated with at least one photoactive therapeutic agent. Photoactive therapeutic agents include compounds that can be configured to kill treated cells upon exposure to electromagnetic radiation of a specific wavelength. The therapeutically relevant compound absorbs electromagnetic radiation at a wavelength that is transparent to tissue. In preferred embodiments, the compound is administered in a non-toxic form that is capable of producing a photochemical effect that is toxic to cells or tissues upon sufficient activation. In other preferred embodiments, these compounds are retained by cancerous tissue and easily cleared from normal tissue. Non-limiting examples include various chromagens and dyes.

[0255] In preferred embodiments, the antimitotic agent is selected from maytansinoids and auristatins.

[0256] The maytansinoid DM4 and the auristatin MMAE were successfully used in the ADCs of the invention in the Examples.

[0257] In another preferred embodiment, the DNA damaging agent is selected from pyrrolobenzodiazepines (PBDs) and pyridinobenzodiazepines (PDDs).

[0258] In the examples, the DNA damaging agent VA-SG3199 (Tesirin) was successfully used with the ADCs of the invention.

[0259] In another preferred embodiment, the linker is a non-cleavable linker.

[0260] In another preferred embodiment, the linker is a cleavable linker.

[0261] In yet another preferred embodiment, the therapeutically active agent is selected from monomethylauristatin E (MMAE), 4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4), and VA-SG3199 (Tesirin).

[0262] As noted above, any of the antibodies of the invention described herein can be used in ADCs.

[0263] In the Examples, ADCs comprising the AFF4-WT antibody were prepared and shown to be effective in in vitro and / or in vivo tumor models.

[0264] Therefore, it is particularly preferred to provide ADCs comprising antibodies comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 sequences of the AFF4 antibody, the VH and / or VL domain sequences of the AFF4 or AFF4-WT antibody, and / or the heavy and / or light chain sequences of the AFF4 or AFF4-WT antibody.

[0265] Preferably, an antibody of the present invention is provided, i.e., an ADC, linked via a linker to at least one therapeutically active substance, the antibody comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 sequence of the AFF4 antibody, or the VH and / or VL domain sequence of the AFF4 antibody, wherein the antibody further comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0266] In one embodiment of the antibody of the present invention linked to at least one therapeutically active agent via a linker, or the ADC of the present invention, the antibody further comprises a human IgG1 constant region.

[0267] For example, the human IgG constant region can be a wild-type human IgG constant region. Alternatively, the human IgG constant region can be a wild-type human IgG constant region containing 1 to 10 mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations, including any subrange thereof) in the constant region, such as substitutions, deletions, and / or insertions, particularly 1 to 10 substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, including any subrange thereof).

[0268] Each mutation can be introduced independently into one chain of the Fc domain, or each mutation can be introduced independently and symmetrically into both chains of the constant region domain.

[0269] In one preferred embodiment, an antibody linked to at least one therapeutically active agent via a linker, i.e., an ADC, comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 30 and / or a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 20.

[0270] In one preferred embodiment, an antibody linked to at least one therapeutically active agent via a linker, i.e., an ADC, comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO:30, and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO:20.

[0271] In one preferred embodiment, the antibody linked to at least one therapeutically active agent via a linker, i.e., the ADC, comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37 and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0272] In one preferred embodiment, an antibody linked to at least one therapeutically active agent via a linker, i.e., an ADC, comprises a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84 and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38.

[0273] Nucleic acids, vectors, and host cells The term "nucleic acid" describes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or any form of artificial nucleic acid known to those skilled in the art.

[0274] Nucleotide sequences encoding the antibodies described herein and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled to generate a nucleic acid encoding the antibody. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides, which briefly involves synthesizing overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0275] If a clone containing nucleic acid encoding a particular antibody is not available but the sequence of the antibody molecule is known, nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library made from any tissue or cell that expresses the antibody, such as hybridoma cells selected to express an antibody described herein, i.e., made from nucleic acid, preferably polyA+ RNA, isolated from that tissue or cell), for example, by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence, to identify a cDNA clone encoding the antibody from a cDNA library. Amplified nucleic acids produced by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0276] DNA encoding the antibodies of the present invention described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which can then be transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells derived from the CHO GS System™ (Lonza)), or myeloma cells, to obtain antibody synthesis in the recombinant host cells.

[0277] To generate whole antibodies, the VH or VL sequence can be amplified in an scFv clone or other clone using PCR primers containing the VH or VL nucleotide sequence, restriction sites, and flanking sequences to protect the restriction sites. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as a human IgG1 constant region, a human IgG4 constant region, a human IgG2 constant region, or a human IgG3 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as a human kappa or lambda constant region. In certain embodiments, vectors for expressing VH or VL domains contain a promoter, a secretion signal, cloning sites for the variable regions, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a single vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line to generate stable or transient cell lines expressing full-length antibodies, such as IgG, using techniques known to those skilled in the art. Alternatively, vectors expressing the heavy and light chains can be transfected into separate cells, and the antibodies can be recovered and then assembled. Furthermore, alternatively, nucleic acids encoding antibodies of the invention can be expressed using a single vector for expression. Such a vector may encode an antibody of the invention as a single molecule, e.g., an scFv, or may encode two or more polypeptides that can assemble into an antibody of the invention.

[0278] The DNA can also be modified, for example, by substituting coding sequences for human heavy and light chain constant regions for the murine sequences, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0279] Site-directed or high-density mutagenesis of the variable regions or other mutagenesis methods can be used to optimize the specificity, affinity, etc. of monoclonal antibodies. In particular, affinity maturation and chain shuffling strategies

[28] are known in the art and can be used to generate high-affinity human antibodies.

[0280] In another aspect, the present invention also provides a method for producing a pharmaceutical composition comprising: (i) a nucleic acid encoding an antibody that specifically binds human L1CAM according to any of the embodiments of the present invention, and / or (ii) a nucleic acid encoding at least one VH or HC and / or VL or LC of an antibody that specifically binds human L1CAM according to any of the embodiments of the present invention, and / or (iii) a nucleic acid encoding a sequence according to SEQ ID NO: 30 and / or a sequence according to SEQ ID NO: 20, and / or (iv) a nucleic acid comprising a sequence encoding a complementarity-determining region sequence of an antibody that specifically binds to human L1CAM according to any of the aspects of the present invention. Regarding.

[0281] In this regard, all features described above for the antibodies of any of the aspects of the invention also apply, where applicable, to the nucleic acids of the invention, e.g., to features related to the antibodies they encode, or to the specific binding of such antibodies.

[0282] Preferably, the nucleic acid is part of a vector.

[0283] Generally, a vector (also an expression vector) is a plasmid used to introduce a desired nucleic acid sequence, such as a gene, into a target cell, resulting in the transcription and translation of a protein, such as a chimeric antigen receptor, an antibody, or a binding molecule, encoded by the nucleic acid sequence. Therefore, an expression vector generally contains regulatory sequences, such as a promoter and an enhancer region, as well as a polyadenylation site, to direct the efficient transcription of the nucleic acid sequence on the expression vector. An expression vector may further contain additional necessary or useful regions, such as a selection marker for selection in eukaryotic or prokaryotic cells, a purification tag for purifying the resulting protein, a multiple cloning site, or an origin of replication.

[0284] Generally, the expression vector can be a viral vector or a non-viral vector. Generally, various types of viral vectors, such as retroviral vectors, for example, lentiviral or adenoviral vectors, or plasmids can be used.

[0285] It is preferred that the nucleic acid is part of a vector.

[0286] Such vectors containing the nucleic acids of the invention can then be introduced into host cells.

[0287] Methods for introducing such vectors into host cells are well known to those skilled in the art and include, for example, any known transfection method, such as any non-viral transfection method (e.g., chemical-based, non-chemical- or particle-based) or any viral-based transfection method. Examples of suitable methods are calcium phosphate precipitation, lipofection, cationic polymers, Fugene, dendrimers, nanoparticles, microinjection, cell squeezing, electroporation, particle guns (also known as gene guns), magnet-assisted transfection, optical transfection, protoplast fusion, impalafection, hydrodynamic delivery, sonoporation, transferrin-based infection, antibody-based transfection, or viral-based transfection (e.g., based on adenoviral or lentiviral vectors).

[0288] In another aspect, the present invention relates to a host cell comprising a nucleic acid according to the invention.

[0289] Suitable host cells are well known to those skilled in the art, and include, for example, mammalian cells (e.g., human, mouse, rat, or hamster cells), insect cells, bacterial cells, or yeast cells. Such host cells can contain, for example, a nucleic acid of the invention integrated into their genome or in a vector. Methods for introducing such nucleic acids into host cells are described above and are further well known to those skilled in the art.

[0290] Compositions Comprising Antibodies of the Invention In another aspect, the invention relates to a pharmaceutical composition comprising an antibody of the invention, or a nucleic acid of the invention, or a host cell of the invention, and optionally one or more pharmaceutically acceptable carriers.

[0291] The content of the antibody, nucleic acid, or host cell in the pharmaceutical composition is not limited as long as it is useful for treatment or prevention, but preferably contains 0.0000001 to 10% by weight of the total composition.

[0292] Furthermore, the antibodies, nucleic acids, or host cells described herein are preferably employed in one or more pharmaceutically acceptable carriers.

[0293] The term "carrier" describes any molecule that improves the selectivity, efficacy, and / or safety of administration of an antibody, nucleic acid, or host cell to the human or animal body, for example, by sustained or triggered release or by allowing membrane permeation of the antibody, nucleic acid, or host cell.

[0294] A carrier is also considered pharmaceutically acceptable when it does not cause any or substantial harmful undesirable effects on the human or animal body, for example, when it is generally considered safe and non-toxic, and / or does not cause undesirable biological side reactions.Suitable pharmaceutically acceptable carriers are well known to those skilled in the art.The selection of a carrier can depend on the administration route and concentration of the active substance, and the carrier can be in the form of a lyophilized composition or an aqueous solution.Generally, an appropriate amount of pharmaceutically acceptable salt is used in the carrier to make the composition isotonic.Examples of carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. Preferably, acceptable excipients, carriers, or stabilizers are non-toxic at the dosages and concentrations employed and include buffers such as citrate, phosphate, and other organic acids; salt-forming counterions, e.g., sodium and potassium; low molecular weight (<10 amino acid residues) polypeptides; proteins, e.g., serum albumin or gelatin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, such as histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates, including glucose, mannose, or dextrins; monosaccharides; disaccharides; other sugars, e.g., sucrose, mannitol, trehalose, or Include sorbitol; chelating agents such as EDTA; nonionic surfactants such as Tween, Pluronic® or polyethylene glycol; antioxidants such as methionine, ascorbic acid and tocopherol; and / or preservatives such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol.Suitable carriers and their formulations are described in more detail in Remington's Pharmaceutical Sciences, 17th Edition, 1985, Mack Publishing Co.

[0295] Therapeutic and Diagnostic Uses and Applications In another aspect, the present invention relates to the antibody of the invention, or the nucleic acid of the invention, or the host cell of the invention, or the pharmaceutical composition of the invention for use as a medicament or as a diagnostic agent.

[0296] The term "drug" is a substance that can be applied to cure, treat, or prevent a disease. Similarly, the term "diagnostic agent" is a substance that can be applied to diagnose a disease, i.e., to determine whether the human or animal body has a particular disease or condition.

[0297] In another aspect, the invention relates to an antibody of the invention, or a nucleic acid of the invention, or a host cell of the invention, or a pharmaceutical composition of the invention for use in treating or preventing a hyperproliferative disorder, a tumor disease, a disorder involving angiogenesis, and / or a disorder involving abnormal neurogenesis.

[0298] The term "treating" describes any manner of improving the health of the human or animal body against a disease or condition. This can include alleviating symptoms, slowing progression, slight improvement, but can also include complete cure of the human or animal body against a disease or condition. Similarly, the term "preventing" describes the health of the human or animal body so that it does not suffer from a disease or condition.

[0299] The term "hyperproliferative disorder" describes any disease or condition associated with hyperproliferative cells, i.e., cells that exhibit an abnormally high rate of cell proliferation (e.g., tumors, cancers, and neoplastic tissues in general, as well as premalignant and non-neoplastic or non-malignant hyperproliferative disorders). Examples are non-malignant (also non-neoplastic), pre-malignant, or malignant tumors. Examples of premalignant and non-neoplastic or non-malignant hyperproliferative disorders include, for example, myelodysplastic disorders; cervical intraepithelial neoplasia; familial intestinal polyposis such as Gardner's syndrome; oral leukoplakia; histiocytosis; keloids; hemangiomas; hyperproliferative arterial stenosis; inflammatory arthritis; hyperkeratosis, papular-scaling eruptions including arthritis, and hyperproliferative skin disorders such as chronic inflammatory skin diseases (e.g., psoriasis), as well as virally induced hyperproliferative diseases such as warts and EBV-induced diseases (i.e., infectious mononucleosis), scarring, and the like.

[0300] The term "tumor disease" describes any disease or condition associated with a tumor (also called a neoplasm), which may be non-malignant (also non-neoplastic), pre-malignant, or malignant.

[0301] In embodiments, the neoplastic disease is selected from ovarian cancer, breast cancer, endometrial cancer, melanoma, and neuroblastoma.

[0302] In embodiments, the antibody of the present invention, or the nucleic acid of the present invention, or the host cell of the present invention, or the pharmaceutical composition of the present invention is used to treat or prevent L1CAM-expressing hyperproliferative disorders, L1CAM-expressing tumor diseases, L1CAM-expressing disorders associated with angiogenesis, and / or L1CAM-expressing disorders associated with abnormal neurogenesis.

[0303] An "L1CAM-expressing hyperproliferative disorder" is understood as a hyperproliferative disorder in which at least some of the hyperproliferative cells express human L1CAM on the cell surface.

[0304] An "L1CAM-expressing tumor disease" is understood as a tumor disease in which at least a portion of the tumor cells express human L1CAM on the cell surface.

[0305] In embodiments, the L1CAM-expressing tumor disease is selected from L1CAM-expressing ovarian cancer, L1CAM-expressing breast cancer, L1CAM-expressing endometrial cancer, L1CAM-expressing melanoma, and L1CAM-expressing neuroblastoma.

[0306] An "L1CAM-expressing disorder associated with angiogenesis" is understood as a disorder associated with angiogenesis in which at least some of the cells involved in angiogenesis express human L1CAM on the cell surface.

[0307] An "L1CAM-expressing disorder associated with abnormal neurogenesis" is understood as a disorder associated with abnormal neurogenesis, in which at least some of the cells involved in the abnormal neurogenesis express human L1CAM on the cell surface.

[0308] In another preferred embodiment, an antibody of any of the present embodiments linked to at least one therapeutically active agent via a linker (also referred to as an "antibody-drug conjugate," "antibody conjugate," "antibody drug conjugate," or "ADC") is used to treat or prevent a hyperproliferative disorder, a neoplastic disease, a disorder associated with angiogenesis, a disorder associated with abnormal neurogenesis, an L1CAM-expressing hyperproliferative disorder, an L1CAM-expressing neoplastic disease, an L1CAM-expressing disorder associated with angiogenesis, or an L1CAM-expressing disorder associated with abnormal neurogenesis, by exhibiting a cytotoxic bystander effect. In an embodiment, the linker is a releasable linker.

[0309] The cytotoxic bystander effect is understood as the effect that an ADC endowed with such a bystander effect is taken up by antigen-positive cancer cells and processed to be released in the form of a cytotoxic payload, which can freely diffuse to neighboring cells and therefore has the ability to kill those cells regardless of their antigen expression. Therefore, such an ADC is well suited for treating tumors with heterogeneous L1CAM target expression. In an embodiment, the linker is a releasable linker. Methods for determining the cytotoxic bystander effect are known in the art. In particular, an assay such as that described in Example 8 can be used.

[0310] "About" is understood to mean ±10% of the indicated value.

[0311] Disorders involving angiogenesis describe any disease or condition associated with blood vessel formation in hyperproliferative tissue, such as non-malignant (also non-neoplastic), pre-malignant, or malignant tumors and cancers.

[0312] A disorder involving abnormal neurogenesis describes any disease or condition associated with the production of abnormal cells of the nervous system (eg, abnormal neurons) by neural stem cells, eg, abnormal (hippocampal) neurogenesis.

[0313] In another aspect, the present invention relates to an antibody of the present invention, or a nucleic acid of the present invention, or a host cell of the present invention, or a pharmaceutical composition of the present invention for use in diagnosing a hyperproliferative disorder, a tumor disease, a disorder involving angiogenesis, and / or a disorder involving abnormal neurogenesis.

[0314] In another aspect, the present invention relates to the in vitro use of an antibody of the present invention, or a nucleic acid of the present invention, or a host cell of the present invention, or a pharmaceutical composition of the present invention as a diagnostic agent, in particular for diagnosing hyperproliferative disorders, tumor diseases, disorders involving angiogenesis, and / or disorders involving abnormal neurogenesis.

[0315] In another aspect, the present invention relates to a method for treating or preventing a hyperproliferative disorder, a neoplastic disease, a disorder involving angiogenesis, and / or a disorder involving abnormal neurogenesis, comprising the step of administering to a patient in need thereof a pharmaceutically effective amount of the antibody of the present invention, or the nucleic acid of the present invention, or the host cell of the present invention, or the pharmaceutical composition of the present invention.

[0316] As used herein, the term "pharmaceutically effective amount" in the context of administration to a subject of treatment refers to that amount of treatment that achieves the desired prophylactic or therapeutic effect.

[0317] Appropriate amounts and dosages can be determined by one of skill in the art. For example, an antibody or pharmaceutical composition described herein can be administered (e.g., by intravenous injection) to a subject at about 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg, or about 10 mg / kg.

[0318] Administration of the antibody, nucleic acid, or host cell of the present invention refers to any drug administration route known to those skilled in the art, such as intravenous, intraperitoneal, subcutaneous, oral, intranasal, or sublingual administration. Suitable administration regimens are also well known to those skilled in the art. Preferably, the antibody, nucleic acid, or host cell of the present invention is administered in a pharmaceutically effective amount, i.e., at a dose or concentration that elicits a biological response in the body to which the antibody, nucleic acid, or host cell of the present invention is administered.

[0319] In this regard, all features described above for the antibodies of any aspect of the invention also apply, where applicable, to features related to further aspects of the invention, such as antibody conjugates, nucleic acids, host cells, or pharmaceutical compositions.

[0320] In general, the present disclosure is not limited to the particular methodologies, protocols, and reagents described herein, as these may vary. Furthermore, the terminology used herein is intended to describe particular embodiments only and is not intended to limit the scope of the present disclosure. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the words "comprise," "contain," and "include" should be interpreted inclusively rather than exclusively.

[0321] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice as presented herein, particular methods and materials are described herein.

[0322] The present disclosure is further illustrated by the following figures and examples, which, unless expressly indicated otherwise, are understood to be included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0323] JPEG2025532716000015.jpg227156JPEG2025532716000016.jpg234155 [Brief explanation of the drawings]

[0324] [Figure 1]Figure 1A shows the analysis of purified OV549.20 mouse IgG2a and chimeric OV549.20 human IgG1 by reducing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The heavy chain and two forms of the light chain (glycosylated and nonglycosylated) are indicated by arrows. Lanes 1-3 contain the following samples: Lane 1: molecular weight marker; Lane 2: OV549.20 mouse IgG2a; Lane 3: chimeric OV549.20 human IgG1. Figure 1B shows the mass spectrometry analysis of DTT-reduced chimeric OV549.20 human IgG1, demonstrating the presence of peaks corresponding to the heavy chain (50,798.5 Da) and the glycosylated (25,885.5 Da) and nonglycosylated (23,533.2 Da) light chains. [Figure 2] Figure 1 shows the fluorescence signal detected by an image-based cytometer in JIMT-1, SKOV-3, and Panc-1 cells after incubation with chimeric OV549.20 human IgG1 and a secondary Alexa Fluor 488-labeled goat anti-human IgG antibody. A chimeric human IgG1 isotype control antibody was used for comparison. [Figure 3] Figure 1 shows the binding of OV549.20 mouse IgG2a to human L1CAM in an ELISA assay and its lack of cross-reactivity with other members of the L1 family. To demonstrate successful immobilization of all antigens on the ELISA plate and proper functionality of the detection system, commercially available antibodies specific for human L1CAM, CHL1, NrCAM, and neurofascin were used as positive controls. The level of binding was quantified by assessing the optical density (OD) at 450 nm wavelength in the ELISA assay. [Figure 4]Figure 1 shows the dose-dependent induction of antibody-dependent cellular cytotoxicity (ADCC) on Panc-1 pancreatic cancer cells by chimeric OV549.20 human IgG1 and Herceptin. Binding of a human IgG1 chimeric version of the previously described antibody L9.3 to the first Ig domain of L1CAM was tested in parallel but did not induce ADCC on Panc-1 cells. Mean values ​​± standard deviation from triplicates are shown. Values ​​were fitted to a four-parameter logistic curve using GraphPad Prism software. [Figure 5-1] Figure 1 shows the effect of OV549.20 mouse IgG2a, OV52.24 mouse IgG2a, and an isotype control antibody on the proliferation of HCT116, SKOV3, and Panc-1 cells, as measured by confluency detection on an Incucyte device. Addition of OV549.20 mouse IgG2a reduced proliferation of all three cell lines, while OV52.24 mouse IgG2a and the mouse IgG2a isotype control had no effect. Mean values ​​± standard deviation from duplicates are shown. [Figure 5-2] Figure 1 shows the effect of OV549.20 mouse IgG2a, OV52.24 mouse IgG2a, and an isotype control antibody on the proliferation of HCT116, SKOV3, and Panc-1 cells, as measured by confluency detection on an Incucyte device. Addition of OV549.20 mouse IgG2a reduced proliferation of all three cell lines, while OV52.24 mouse IgG2a and the mouse IgG2a isotype control had no effect. Mean values ​​± standard deviation from duplicates are shown. [Figure 6] 1 shows that chimeric OV549.20 human IgG1 inhibited migration of HCT116 cells on fibronectin-coated plates, whereas an irrelevant chimeric human IgG1 isotype control had no effect on cell migration. Mean values ​​± standard deviations from duplicates are shown. [Figure 7A]Figure 7A shows the inhibitory effect of OV549.20 mouse IgG2a on intraperitoneal tumor mass and ascites volume in a SKOV3 xenograft model of ovarian cancer in mice. SKOV3 cells were injected intraperitoneally, and five days later, mice were treated with either 10 mg / kg OV549.20 mouse IgG2a (n=10), 10 mg / kg L9.3 mouse IgG2a, or 10 mg / kg mouse IgG2a isotype control (n=10). Treatment was repeated three times a week for a total of 6 weeks, and then tumor mass in the peritoneal cavity and ascites volume were analyzed. [Figure 7B] Figure 7B shows the results of a second SKOV3 xenograft experiment. SKOV3 cells were injected intraperitoneally, and five days later, mice were treated with either 10 mg / kg OV549.20 mouse IgG2a (n=10) or vehicle (n=10). Treatment was repeated three times a week for a total of 6 weeks, followed by analysis of tumor mass and ascites volume in the peritoneal cavity. [Figure 8] Figure 1 shows the inhibitory effect of OV549.20 mouse IgG2a on metastasis formation in an intravenous MDA-MB-231-luc2 mouse xenograft model. Mice (n=15 per group) received intravenous injections of OV549.20 mouse IgG2a antibody (10 mg / kg) or vehicle (three times a week for five weeks). Three days after the first administration, all animals were intravenously injected with 5 x 10 MDA-MB-231-luc2 cells. Animal weights were recorded at regular intervals, and whole-body luminescence imaging was performed on days 7, 14, 21, and 30 after cell injection to monitor metastatic colonization of organs in the thoracic region. [Figure 9] Figure 1 shows the analysis of purified humanized antibody variants H1L1-H4L2 by reducing SDS-PAGE. For comparison, the parent chimeric OV549.20 human IgG1 antibody was included in the analysis. The heavy chain (HC) and light chain (LC) are indicated by arrows. In the case of chimeric OV549.20 human IgG1, two forms of the light chain exist: a glycosylated form and a non-glycosylated form. [Figure 10]Figure 10A shows the analysis of the chimeric OV549.20 human IgG1 antibody and humanized variant H1L1 on a Tosoh TSKgel Butyl-NPR hydrophobic interaction column. Figure 10B shows the analysis of the humanized antibody variant H1L1 on a Tosoh TSKgel Butyl-NPR column before ("unstressed H1L1") and after ("pH 5.5-stressed H1L1") incubation in 20 mM sodium citrate, pH 5.5, at 40°C for 12 days. Figure 10C shows the analysis of the antibody variant AFF4 on a Tosoh TSKgel Butyl-NPR column before ("unstressed AFF4") and after ("pH 5.5-stressed AFF4") incubation in 20 mM sodium citrate, pH 5.5, at 40°C for 14 days. [Figure 11-1] Figure 1 shows the effect of AFF4 and a chimeric human IgG1 isotype control antibody on the proliferation of HCT116, SKOV3, and Panc-1 cells, as measured by confluency detection on an Incucyte device. Addition of AFF4 reduced proliferation of all three cell lines compared to the isotype control antibody. Mean values ​​± standard deviations from duplicates are shown. [Figure 11-2] Figure 1 shows the effect of AFF4 and a chimeric human IgG1 isotype control antibody on the proliferation of HCT116, SKOV3, and Panc-1 cells, as measured by confluency detection on an Incucyte device. Addition of AFF4 reduced proliferation of all three cell lines compared to the isotype control antibody. Mean values ​​± standard deviations from duplicates are shown. [Figure 12] 1 shows that AFF4 inhibits migration of HCT116 cells on fibronectin-coated plates, while a chimeric human IgG1 isotype control antibody had no effect on cell migration. Mean values ​​± standard deviations from duplicates are shown. [Figure 13]This figure shows the % specific lysis achieved through AFF4-mediated antibody-dependent cellular cytotoxicity in PC-03, Panc-1, HeLa, and SKOV3 cancer cell lines. Cancer cells were seeded onto xCelligence E-Plates and allowed to settle and adhere to the plate for 16–24 hours. Activated natural killer cells (CD16 low affinity: FF; medium affinity: V / F; high affinity: VV) from three different donors were added to the cancer cells at two different effector-to-target (E:T) cell ratios (5:1 or 10:1), along with AFF4 at a final concentration of 1 μg / mL. Impedance measurements were recorded over 72 hours using an xCelligence RTCA analyzer, and the data were converted to % specific cell lysis. [Figure 14]Figure 14A shows the structures of the four linkers / payloads used for conjugation with AFF4-WT. Figure 14B shows the structures of the four linkers / payloads used for conjugation with AFF4-WT. ... 2-Methoxy-8-methyl-11-oxo-6a,7-dihydropyrrolo[2,1-c][1,4]benzodiazepine-3-yl]oxy]pentoxy]-6-hydroxy-2-methoxy-8-methyl-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate; Figure 14B: VC-MMAE (MC-VC-PABC-MMAE), CAS number: 646502-53-6. IUPAC: [4-[[(2S)-5-(carbamoylamino)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl N-[(2S)-1-[[(2S)-1-[[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-N-methylcarbamate; Figure 14C: Gluc-MMAE (MC-beta glucuronide-MMAE), CAS number: 1703778-92-0.IUPAC: (2S,3S,4S,5R,6S)-6-[2-[3-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]propanoylamino]-4-[[[(2S)-1-[[(2S)-1-[[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino ]-1-Methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-methylcarbamoyl]oxymethyl]phenoxy]-3,4,5-trihydroxyoxane-2-carbamoyl carboxylic acid; Figure 14D: Sulfo-SPDB-DM4, CAS number: 1626359-59-8; IUPAC: 4-[[5-[[(2S)-1-[[(1S,2R,3S,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22- Diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl]oxy]-1-oxopropan-2-yl]-methylamino]-2-methyl-5-oxopentan-2-yl]disulfanyl]-1-(2,5-dioxopyrrolidin-1-yl)oxy-1-oxobutane-2-sulfonic acid. [Figure 15] Figure 1 shows the fluorescence signal detected by an image-based cytometer after 15 minutes or 20 hours of co-incubation of JIMT-1, OVCAR-3, and MeWo cells with AFF4-WT and human Fabfluor-pH Red antibody-labeled dye. A chimeric human IgG1 isotype control antibody was used for comparison. The strongly enhanced fluorescence of AFF4-WT / FabFluor-treated cells after 20 hours compared to isotype control / Fabfluor-treated cells indicates efficient uptake and lysosomal localization of AFF4-WT. [Figure 16]Figure 1 shows the cytotoxic effects of four AFF4-WT-drug conjugates on three different cancer cell lines. Serial dilutions of AFF4-WT-VC-MMAE, AFF4-WT-Gluc-MMAE, AFF4-WT-VA-SG3199, and AFF4-WT-sulfo-SPDB-DM4 were incubated in duplicate with JIMT-1 breast cancer cells, MeWo melanoma cells, and OVCAR-3 ovarian cancer cells for 6 days. Cell viability was then assessed by ATP quantification, and the percent viability of cells treated with AFF4-WT-drug conjugates was calculated using untreated cells and cells incubated in the presence of 10 μM doxorubicin as reference values ​​(100% and 0%, respectively). Individual values ​​from duplicates are shown. Average values ​​were fitted to a four-parameter logistic curve using GraphPad Prism software. [Figure 17-1] Figure 17A shows the antitumor effects of four AFF4-WT-drug conjugates in a human breast cancer xenograft model in mice. JIMT-1 breast cancer cells were implanted intramammarily, and tumors were allowed to grow to an average size of approximately 100 mm. Mice then received a single intravenous injection of the indicated AFF4-WT-drug conjugate or vehicle as a control, and tumor growth was monitored over time. Figure 17B shows the antitumor effects of four AFF4-WT-drug conjugates in a human ovarian cancer xenograft model in mice. OVCAR-3 ovarian cancer cells were implanted subcutaneously, and tumors were allowed to grow to an average size of approximately 140 mm. Mice then received a first intravenous injection of the indicated AFF4-WT-drug conjugate or vehicle as a control, followed by a second injection 14 days later. Tumor growth was monitored at regular intervals. [Figure 17-2]Figure 17C shows the antitumor effects of four AFF4-WT-drug conjugates in a human melanoma xenograft model in mice. MeWo melanoma cells were subcutaneously implanted, and tumors were allowed to grow to approximately 140 mm. Mice then received a single intravenous injection of the indicated AFF4-WT-drug conjugate or vehicle as a control, and tumor growth was monitored at regular intervals. [Figure 18]

[0023] Figure 1 shows the antitumor effect of different doses of AFF4-WT-VC-MMAE in a human breast cancer xenograft model in mice. JIMT-1 breast cancer cells were implanted intramammarily, and tumors were allowed to grow to an average size of approximately 100 mm. Mice then received a single intravenous injection of the indicated dose of AFF4-WT-VC-MMAE, and tumor growth was monitored over time. [Figure 19-1] Figure 1 shows the antitumor effect of treatment with AFF4-WT-VC-MMAE in four different L1CAM-expressing patient-derived xenograft (PDX) models of human ovarian cancer in mice. PDX tumor fragments were implanted subcutaneously and allowed to grow to an average size of approximately 150-300 mm. Mice then received intravenous injections of AFF4-WT-VC-MMAE or vehicle every two weeks, and tumor growth was monitored at regular intervals for up to 60 days. [Figure 19-2] Figure 1 shows the antitumor effect of treatment with AFF4-WT-VC-MMAE in four different L1CAM-expressing patient-derived xenograft (PDX) models of human ovarian cancer in mice. PDX tumor fragments were implanted subcutaneously and allowed to grow to an average size of approximately 150-300 mm. Mice then received intravenous injections of AFF4-WT-VC-MMAE or vehicle every two weeks, and tumor growth was monitored at regular intervals for up to 60 days. [Figure 20]Figure 1 shows the bystander cytotoxic activity of AFF4-WT-VC-MMAE in vitro. Serial dilutions of AFF4-WT-VC-MMAE were preincubated for 4 days on either L1CAM-high-expressing JIMT-1 cells, L1CAM-low-expressing MDA-MB-468 cells, or in the absence of cells. Supernatants from these preincubations were diluted 4-fold and transferred to MDA-MB-468 cells, followed by incubation for 6 days. Cell viability was then assessed by ATP quantification, and % cell viability was calculated using untreated cells and cells incubated in the presence of 10 μM doxorubicin as reference values ​​of 100% and 0%, respectively. Mean values ​​and standard deviations from duplicates are shown. The indicated AFF4-WT-VC-MMAE concentrations refer to the final concentration on MDA-MB-468 cells incubated with the supernatant. Mean values ​​were fitted to a four-parameter logistic curve using GraphPad Prism software. Example 1

[0325] Generation of monoclonal antibody OV549.20 and its recombinant expression as a murine IgG2a and human IgG1 chimeric antibody The murine antibody OV549.20 was generated using hybridoma technology with SKOV3ip human ovarian cancer cells as the immunogen. DNA sequences encoding the heavy and light chain variable domains of OV549.20 were cloned in frame into expression vectors encoding the heavy and light chain constant domains of either mouse IgG2a or human IgG1, respectively. The resulting protein sequences of the entire heavy and light chains of the antibody constructs are shown in SEQ ID NOs: 35 and 36 for the mouse antibody. The chimeric antibody has the same variable regions but contains a human IgG1 constant domain. Plasmids encoding the antibody heavy and light chains, respectively, were purified under low-endotoxin conditions and transiently co-transfected into Chinese hamster ovary (CHO) K1 cells. Cells were grown in chemically defined, animal-component-free medium, and the supernatant containing the recombinant antibody was collected by centrifugation followed by filtration through a 0.2 μm filter. The antibody was purified from the supernatant by affinity chromatography using a Protein A column (MabSelect SuRe, GE Healthcare) and stored in phosphate-buffered saline (PBS) containing 100 mM arginine. Reducing SDS-PAGE analysis of purified OV549.20 murine IgG2a and chimeric OV549.20 human IgG1 antibodies (Figure 1A) revealed a band at approximately 50 kDa, a faint band at approximately 25 kDa, and an additional strong band at approximately 30 kDa for both antibodies. Mass spectrometry analysis of chimeric OV549.20 human IgG1 (Figure 1B) confirmed the presence of the predicted molecular masses of the heavy chain (50,798.5 Da) and light chain (23,533.2 Da), and detected an additional mass of 25,885.5 Da, which greatly exceeded the amount of the predicted light chain. The molecular weight difference of 2,352.3 Da between the predicted light chain mass and the additional mass is consistent with N-glycosylation occurring within the light chain sequence, indicating that the additional mass corresponds to a glycosylation variant of the light chain. Indeed, sequence analysis revealed the presence of a canonical N-glycosylation motif (Asn-Ile-Thr) within the variable region of the OV549.20 light chain (SEQ ID NO: 36). Example 2

[0326] In vitro pharmacology of OV549.20 murine IgG2a and chimeric OV549.20 human IgG1 Expression of the extracellular domain of human and cynomolgus monkey L1CAM To determine the target binding affinity of OV549.20 mouse IgG2a and chimeric OV549.20 human IgG1, recombinant versions of the extracellular portions of human L1CAM and cynomolgus monkey L1CAM were first cloned, expressed, and purified. To achieve this goal, DNA sequences encoding amino acids 20–1120 of human L1CAM (Uniprot accession number P32004) and cynomolgus monkey L1CAM (NCBI accession number XP_005594994) were synthesized and cloned into eukaryotic expression vectors. Each recombinant expression product was designed to contain an N-terminal leader peptide for secretion into the cell supernatant and a C-terminal hexahistidine tag for subsequent affinity purification. Plasmid DNA was purified under low-endotoxin conditions and used for transient transfection of human embryonic kidney (HEK) cells. Transfected HEK cells were grown in 1 L expression cultures, and the supernatant was collected by centrifugation. The recombinant protein was purified from the supernatant by Protein A affinity chromatography and stored in PBS, pH 7.4, until further analysis.

[0327] Determination of the kinetic binding constants of OV549.20 murine IgG2a and chimeric OV549.20 human IgG1 to human and cynomolgus monkey L1CAM The monovalent kinetic binding constants of OV549.20 mouse IgG2a and chimeric OV549.20 human IgG1 to human and cynomolgus monkey L1CAM were determined by surface plasmon resonance using a Biacore T200 instrument. The antibodies were captured via their fragment crystallizable (Fc) regions on a CM5 Protein A chip (GE Healthcare). Soluble His-tagged human or cynomolgus monkey L1CAM, respectively, was injected at five different concentrations ranging from 1.25 nM to 20 nM as analyte, using 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% Tween 20 as the running buffer. The detected resonance units (sensorgrams) were fitted to a 1:1 Langmuir binding model to determine the association rate constant (k a ) and dissociation rate constant (k d ), plus the affinity constant (K D ) was calculated. Table 7 shows that OV549.20 murine IgG2a and chimeric OV549.20 human IgG1 bound to both humans and cynomolgus monkeys with similar affinities.

[0328] [Table 7]

[0329] Binding of chimeric OV549.20 human IgG1 to human cancer cell lines expressing L1CAM The breast cancer cell line JIMT-1 and pancreatic cancer cell line Panc-1 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). The ovarian cancer cell line SKOV3 was cultured in McCoy's 5a medium containing 10% FCS. Cells were detached from tissue culture plates by incubation with Accutase® cell detachment solution (5 mL per T75 cell culture flask) for 10 minutes, washed once with PBS, and incubated with 2 μg / ml chimeric OV549.20 human IgG1 or 2 μg / ml of an irrelevant chimeric human IgG1 isotype control antibody at 4°C for 1 hour. Alexa Fluor 488-labeled AffiniPure goat anti-human IgG antibody (Jackson Immuno Research) was then added at a concentration of 5 μg / ml, and incubation continued for 1 hour at 4°C. Finally, the cells were incubated with 10 μg / ml Hoechst-33342 solution (ChemoMetec) for 15 min at 37°C and analyzed in an image-based cytometer (Nucleocounter NC-3000, ChemoMetec) using an excitation wavelength of 475 nm and an emission filter at 560 nm ± 35 nm. A total of 4,000–11,000 cells were analyzed per condition, and the detected fluorescence intensities were plotted as histograms on a semi-logarithmic scale. Figure 2 shows that incubation of JIMT-1, Panc-1, and SKOV3 cells with chimeric OV549.20 human IgG1 resulted in a strong shift in the fluorescent signal compared to the isotype control antibody, demonstrating the specific binding of chimeric OV549.20 human IgG1 to L1CAM on the surface of these human cancer cell lines.

[0330] Determination of cross-reactivity with L1CAM homologues NrCAM, CHL1 and neurofascin The ability of OV549.20 mouse IgG2a to bind to other members of the L1 family of adhesion molecules, namely, the close L1 homolog (CHL1), neurofascin, and neural cell adhesion molecule (NrCAM), was tested in a direct ELISA. Recombinant human L1CAM-human Fc and human NrCAM-human Fc fusion proteins, as well as recombinant hexahistidine-tagged versions of human CHL1 and neurofascin, were added to ELISA plates at a concentration of 1 μg / mL in 50 mM carbonate buffer, pH 9.6 (100 μL per well). Plates were incubated overnight at 4°C, washed five times with PBS / 0.05% Tween, and blocked by adding 200 μL per well of PBS / 0.05% Tween / 1% bovine serum albumin (BSA). OV549.20 mouse IgG2a was then serially diluted 1:3 in PBS / 0.05% Tween / 1% BSA, starting at a maximum concentration of 10 μg / mL. As positive controls, the same dilution series was prepared for commercially available mouse antibodies directed against L1CAM (clone UJ127.11, NovusBio), human NrCAM (clone 308000, Creative Diagnostics), human CHL1 (clone 6E6E4, Sino Biological), and human neurofascin (clone 727030, R&D systems). The plate was washed five times with PBS / 0.05% Tween, and 100 μL of the antibody dilution series was added per well to the plate. After 1 hour of incubation at room temperature, the plate was washed again, and 100 μL of horseradish peroxidase-conjugated polyclonal goat anti-mouse antibody (1:1000 dilution in PBS / 0.05% Tween / 1% BSA) was added per well. After an additional 1 hour of incubation at room temperature, the plate was washed again, and 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) substrate was added per well for detection. After 8 minutes of incubation at room temperature, the reaction was stopped by adding 50 μL of 0.16 M H2SO4 per well, and the absorbance at 450 nm was determined in a microplate reader. Figure 3 shows the OD values ​​obtained in different ELISA assays at an antibody concentration of 1 μg / mL. Control antibodies specific for human L1CAM, CHL1, NrCAM, and neurofascin showed strong binding to their respective target antigens, demonstrating successful immobilization of all antigens on the ELISA plate and proper functionality of the detection system. OV549.20 mouse IgG2a showed strong binding to plate-coated human L1CAM but did not show any reactivity with plate-coated human CHL1, NrCAM, or neurofascin.

[0331] Induction of antibody-dependent cellular cytotoxicity (ADCC) by chimeric OV549.20 human IgG1 Panc-1 human pancreatic cancer cells, which express L1CAM and Her2 on their surface, were cultured in Cr 51 The cells were labeled with β-glucan and preincubated with eight different concentrations of the chimeric OV549.20 human IgG1 antibody for 20 minutes at room temperature. Herceptin was used as a positive control, and a chimeric human IgG1 version of the anti-L1CAM antibody L9.3 (WO2008 / 151819), which binds to the first Ig domain of L1CAM, was tested in parallel. Human CD16 (158V alloform) transgenic T cells were then added as effector cells at an effector-to-target ratio of 20:1, and incubation continued for 4 hours at 37°C in a humidified atmosphere with 5% CO2. After incubation, the cells were centrifuged, and 25 μl of the supernatant was transferred to a Lumaplate (Perkin Elmer). Target cell lysis was measured by gamma counting of the supernatant and expressed as corrected counts per minute (CCPM) after detector normalization. Spontaneous lysis was determined by gamma counting of supernatants from Panc-1 cells incubated in culture medium in the absence of effector cells and antibodies. Maximum lysis was determined by gamma counting of supernatants from Panc-1 cells lysed by the addition of 0.75% Triton X-100. The percentage of specific lysis was calculated according to the following formula:

[0332]

number

[0333] Inhibition of cancer cell growth by OV549.20 murine IgG2a HCT116 colorectal cancer cells (in DMEM:Ham's F12 supplemented with 0.5% FBS), Panc-1 pancreatic cancer cells (in DMEM supplemented with 4.5 g / L glucose, glutamine, and 0.5% FBS), and SKOV3 ovarian cancer cells (in McCoys 5a supplemented with 1.5 mM glutamine, 2.2 g / L sodium bicarbonate, and 0.5% FBS) were seeded at 10-15% confluency in 384-well plates with either vehicle alone or in the presence of 50 μg / mL OV549.20 mouse IgG2a antibody, 50 μg / mL mouse IgG2a isotype control antibody, or the mouse IgG2a version of the previously described antibody OV52.24 (WO2016050702). Cancer cell growth was monitored at regular intervals over 96 hours by microscopic confluency detection using an Incucyte device. Figure 5 shows that the mouse IgG2a isotype control antibody and the OV52.24 mouse IgG2a antibody had no effect on the growth of any of the cancer cell lines, whereas the addition of the OV549.20 mouse IgG2a antibody resulted in a decrease in the growth of all three cancer cell lines. By 96 hours, the addition of OV549.20 mouse IgG2a caused a decrease in confluency from 45.7% to 37.3% for HCT116 cells, from 21.7% to 14.2% for SKOV3 cells, and from 69.7% to 43.3% for Panc-1 cells, compared to cells treated with vehicle alone.

[0334] Inhibition of cancer cell migration by chimeric OV549.20 human IgG1 HCT116 colorectal cancer cells were cultured in DMEM supplemented with 10% FCS, collected with Accutase® cell detachment solution (Capricorn), and plated at 4 × 10 cells onto stopper-containing wells of fibronectin-precoated ORIS-96-well plates (AMS-bio). 4Cells were seeded at a density of 100 μL of cells / 100 μL. After 24 hours, the stopper insert was removed and the medium was replaced with 120 μL of fresh growth medium. Then, 30 μL of a 5-fold concentrated serial dilution series of chimeric OV549.20 human IgG1 or chimeric human IgG1 isotype control was added to the cells. The final antibody concentration in the assay plate was 4 × 10 -5 M to 1.2 x 10 -8 The median values ​​ranged from 100% to 100%. As a positive control for unrestricted migration, cells were incubated without any antibody. As a negative control, migration was restricted by maintaining stoppers in eight wells per plate. After 48 h, the medium was replaced with 75 μL of phenol red-free DMEM supplemented with 2 μg / ml calcein-AM (Life Technologies). Cells were incubated at 37°C for 15 min, and fluorescent cells in the area defined by the insert were detected using a fluorescence microplate reader (Fluostar, BMG) with fluorescein isothiocyanate (FITC) settings (excitation: 485 nm / emission: 520 nm). The median value of the positive control (stopper removal, no antibody addition) was set to 100% (high control), and the median value of the negative control (no stopper removal) was set to 0% (low control). Raw data were converted to % cell migration relative to the control and fitted to a four-parameter logistic curve with a variable slope and a bottom constraint of 0 and a top constraint of 100. Figure 6 shows that addition of the chimeric human IgG1 isotype control antibody had negligible effect on migration of HCT116 cells, while addition of the chimeric OV549.20 human IgG1 antibody resulted in specific dose-dependent inhibition of migration. The half-maximal inhibitory concentrations (IC) for chimeric OV549.20 human IgG1 were 50 ) is 4.2 × 10 -6 It was calculated as M. Example 3

[0335] In vivo pharmacology of OV549.20 murine IgG2a Inhibition of primary tumor growth in the SKOV3 ovarian cancer xenograft model The ability of OV549.20 murine IgG2a to inhibit primary tumor growth was examined in a SKOV3 ovarian cancer xenograft model. SKOV3 ovarian cancer cells were cultured in DMEM containing 10% FCS and 1 mM glutamine. At 60-70% confluence, cells were harvested using 0.05% trypsin and 0.02% EDTA in PBS and injected intraperitoneally into 30 female NMRI:nu / nu mice (5 x 10 per mouse). 6 Cells). Five days later, the mice were divided into three groups of 10 animals each, and the animals in the three groups received an intraperitoneal injection of 10 mg / kg of OV549.20 mouse IgG2a antibody, L9.3 mouse IgG2a (WO2008 / 151819), or a mouse IgG2a isotype control antibody, respectively. Treatment was repeated three times a week for a period of six weeks, and after the animals were sacrificed, the weight of the SKOV3 tumor in the peritoneal cavity, as well as the volume of ascites, was determined. Figure 7A shows that treatment with the OV549.20 murine IgG2a antibody caused a 29% reduction in mean tumor mass compared to the isotype-treated control group. Furthermore, the occurrence of ascites was reduced by 88% in OV549.20 murine IgG2a-treated animals compared to isotype-treated control animals. In contrast, treatment with L9.3 murine IgG2a induced no reduction in tumor mass and only a slight reduction in ascites. In a second experiment, 20 female NMRI:nu / nu mice were inoculated with SKOV3 cells (5 x 10 per mouse). 6 OV549.20 mouse IgG2a (10 mg / kg) or vehicle was injected intraperitoneally into the mice. Five days after injection, the mice were divided into two groups of 10 mice each, and each group was injected intraperitoneally with OV549.20 mouse IgG2a (10 mg / kg) or vehicle. Treatment was repeated three times a week for a total of six weeks, followed by analysis of tumor weight and ascites volume in the peritoneal cavity. FIG. 7B shows that treatment with OV549.20 murine IgG2a caused a 35% reduction in median tumor burden and a 75% reduction in ascites volume when compared to the vehicle-treated group.

[0336] Inhibition of metastasis formation in an MDA-MB-231 breast cancer xenograft model The ability of OV549.20 murine IgG2a to inhibit metastasis formation was tested in a mouse xenograft model of human MDA-MB-231-luc2 metastatic breast cancer cells, in which intravenously administered MDA-MB-231-luc2 cells metastasize to the lungs of mice, where they can be detected by whole-body luminescence after administration of a luminescent substrate. Groups of female athymic nude-Foxn1 mice (n=15 per group) were intravenously injected with either OV549.20 murine IgG2a antibody (10 mg / kg) or vehicle control, three times a week for five weeks. Three days after the first injection, all animals received 5x10 5 MDA-MB-231-luc2 cells were intravenously injected into the thoracic region. Clinical signs and body weight were recorded daily, and in vivo whole-body luminescence imaging was performed on all animals on days 7, 14, 21, and 30 after cell injection. To this end, animals were administered D-luciferin (150 mg / kg) by intraperitoneal injection and imaged 10 minutes later under isoflurane anesthesia. Images were acquired, and the luminescence signal (total flux in p / s) was measured in the thoracic region.

[0337] Figure 8 shows that treatment with OV549.20 mouse IgG2a strongly inhibited metastasis formation, as determined by whole-body luminescence signal. At day 30, the total flux signal in OV549.20 mouse IgG2a-treated mice was reduced by 94.8% compared to vehicle controls. Vehicle control animals showed a strong and sustained loss of body weight after day 20, whereas OV549.20 mouse IgG2a-treated mice did not show any detectable weight loss over the course of the experiment. Thus, treatment with OV549.20 mouse IgG2a strongly inhibited metastasis formation and ameliorated clinical signs of disease. Example 4

[0338] Humanization of OV549.20 In silico grafting of the CDRs of OV549.20 into human frameworks Based on computational modeling of the OV549.20 Fv, the human VH sequence IGHV4 30 4 01 was identified as the most suitable acceptor framework for the CDRs of the OV549.20 heavy chain. The OV549.20 VH CDRs (SEQ ID NOS: 1, 2, and 3; see Table 1) were grafted onto IGHV4 30 4 01 in silico, while specific positions in the human framework of IGHV4 30 4 01 were backmutated to the corresponding murine residues whenever deemed appropriate to avoid new contacts or maintain existing contacts within the modeled OV549.20 Fv. In this manner, four humanized variants of the OV549.20 heavy chain variable region were generated (H1-H4, SEQ ID NOS: 23, 24, 25, and 26), each containing a different number of backmutated framework residues. In parallel, the human VL sequence IGKV1 39 01 was identified as the most suitable human acceptor framework for the light chain CDRs of OV549.20. The VL CDRs of OV549.20 (SEQ ID NOS: 4, 5, and 6; see Table 2) were grafted in silico onto IGKV1 39 01, and simultaneously, a series of backmutations of human residues to corresponding murine residues were introduced into the human framework whenever deemed necessary to maintain structural and functional integrity. In addition, a putative N-glycosylation site (Asn-Ile-Thr), detectable as a mass shift in SDS-PAGE and MS (see Figure 1), which likely leads to glycosylation of the light chain upon expression in CHO cells, was mutated to a sequence (Thr-Ile-Thr) that cannot support such glycosylation. Four different humanized variants of the OV549.20 light chain variable domain were generated, each with a mutated N-glycosylation site and a varying number of back-mutated framework residues, of which the humanized light chain variable domains L1 and L2 (L1: SEQ ID NO: 20, L2: SEQ ID NO: 22) were further used.

[0339] Expression and purification of humanized variants of OV549.20 DNA sequences encoding four different humanized heavy chain variable domains (H1-H4) were each fused in-frame with a DNA sequence encoding a human IgG1 constant domain and used to generate expression constructs for full-length humanized IgG1 heavy chains. Similarly, DNA sequences encoding two of the light chain variable domains (L1 and L2) were each fused in-frame with a sequence encoding a light chain constant domain and used to generate expression constructs for two full-length humanized kappa light chains. Each of the four heavy chain expression plasmids was transiently cotransfected into CHO cells with one of the two light chain expression plasmids, resulting in the expression of eight different full-length humanized IgG1 antibody variants (H1, L1-H4, L2). Cells were grown in a chemically defined, animal-component-free medium, and the supernatant containing the recombinant antibodies was collected by centrifugation followed by filtration through a 0.2 μm filter. Antibodies were purified from the supernatant by affinity chromatography using a Protein A column (MabSelect SuRe, GE Healthcare) and stored in PBS containing 100 mM arginine.

[0340] Biophysical characterization of humanized variants of OV549.20 All eight humanized antibodies were analyzed by reducing SDS-PAGE and compared to the parent chimeric OV549.20 human IgG1. Figure 9 shows that all humanized antibodies exhibited a single band at approximately 50 kDa corresponding to the heavy chain and a single band at approximately 25 kDa corresponding to the light chain. In contrast to the parent chimeric OV549.20 human IgG1 antibody, an additional prominent band at 30 kDa, likely representing a glycosylation variant of the light chain, was not detected in any of the humanized antibodies. The conformational stability of the humanized antibodies was analyzed by differential scanning fluorimetry using Sypro Orange. As shown in Table 8, all humanized antibody variants exhibited a first melting temperature (T) of approximately 70°C, similar to that of the parent chimeric OV549.20 human IgG1 antibody. m 1) was shown. The second melting point (T m2) depends on the respective heavy chain of the antibody variant and is above 83.8°C in all cases, indicating high conformational stability of all humanized antibody variants.

[0341] [Table 8] The monovalent kinetic binding constants of eight humanized antibody variants to human L1CAM were determined by surface plasmon resonance using a Biacore T200 instrument. Antibodies were captured via their Fc regions on a C1 Protein A chip (GE Healthcare), and soluble His-tagged human L1CAM was injected as the analyte at three different concentrations (10 nM, 20 nM, and 40 nM) using 10 mM HEPES / 150 mM NaCl / 3 mM EDTA / 0.05% Tween 20 as the running buffer. The detected resonance units (sensorgrams) were fitted to a 1:1 Langmuir binding model to determine the association rate constant (k a ) and dissociation rate constant (k d ), plus the affinity constant (K D ) was calculated. Table 9 shows that all humanized antibody variants bound to human L1CAM with high affinity and exhibited similar kinetic binding constants.

[0342] [Table 9] All humanized antibody variants were analyzed by hydrophobic interaction chromatography on a Tosoh TSKgel Butyl-NPR column and compared with the parent chimeric OV549.20 human IgG1. The exemplary chromatogram in Figure 10A shows that the parent chimeric antibody OV549.20 human IgG1 exhibited a major peak and an additional minor peak / shoulder after the major peak, indicating the presence of post-translational variants in the preparation. The humanized variant H1L1 exhibited the same peak pattern, but the additional peak was surprisingly significantly reduced compared to the parent chimeric antibody, indicating a reduced presence of post-translational variants. Peak integration of the HIC chromatograms of all humanized antibody variants (Table 10) showed that all variants produced similar reductions in the area of ​​the minor peaks, indicating that grafting on all human framework variants reduced the formation of post-translational variants compared to the parent chimeric antibody.

[0343] [Table 10]

[0344] The nature of the post-translational variants detected by hydrophobic interaction chromatography was investigated in detail by exposing the humanized antibody variant H1L1 to high temperature and low pH stress. As shown in Figure 10B, incubation of H1L1 in 20 mM sodium citrate, pH 5.5, at 40°C for 12 days significantly increased the magnitude of the second minor peak and resulted in the appearance of a prominent third peak. This indicated that post-translational modification reactions, such as isomerization or similar reactions, that occur preferentially at low pH values ​​were responsible for the heterogeneity detected by hydrophobic interaction chromatography. Indeed, protein digestion of unstressed and pH-stressed H1L1 with the AccuMAP™ Low pH Protein Digestion Kit, followed by reversed-phase liquid chromatography-mass spectrometry and MS-MS analysis of the peptide corresponding to amino acids 1–39 of the H1L1 heavy chain, revealed that low pH stress resulted in a mass loss of 18 Da at a position between amino acids 31 and 34. Such a modification is consistent with an isomerization reaction at the aspartic acid residue at position 32, resulting in the loss of a water molecule (-18 Da) and the formation of a stable succinimide intermediate. Example 5

[0345] Affinity improvement and optimization of biophysical properties and effector functions of humanized antibodies The humanized antibody H1L1 was selected as the basis for further optimization of target affinity, biophysical properties, and effector function. Three different scFv phage display libraries were constructed, and selected amino acid positions in the CDRs of the H1L1 heavy and light chains were randomized using a degenerate oligonucleotide-directed PCR mutagenesis approach. After four rounds of panning on recombinant human L1CAM under various selection pressures, a total of 470 clones were randomly selected and subjected to monoclonal phage ELISA on human L1CAM-coated plates, using the parental phage as a control. 62 clones with strongly enhanced binding signals in ELISA were selected and subjected to DNA sequencing. 20 unique sequences were identified, 13 of which were expressed in soluble scFv format and again subjected to ELISA on human L1CAM-coated plates. Increased binding compared to the parental scFv was detected for 11 of the 13 tested scFvs. Subsequently, based on the sequence of the high-binding clone, we designed eight different modified heavy chain variable domains (SEQ ID NOs: 27-34) and one modified light chain variable domain (SEQ ID NO: 21), in addition to the light chain variable domain SEQ ID NO: 20. The heavy and light chain variants were combined to generate 10 different heavy and light chain combinations, and expression plasmids for the corresponding full-length human IgG1 antibodies were constructed. To improve the antibodies' Fc-mediated effector function, i.e., their ability to induce antibody-dependent cellular cytotoxicity (ADCC), four mutations (G236A / S239D / A330L / I332E, EU numbering) were introduced into the CH2 domain of the heavy chain. These mutations have been described to selectively enhance the affinity of the Fc domain for activating Fc gamma receptors

[18] . The resulting full-length antibodies, AFF1-AFF10, were expressed, purified, and subjected to detailed biophysical and in vitro pharmacological analysis, as described in Example 4. The monovalent kinetic binding constants to human L1CAM were determined by surface plasmon resonance using a Biacore T200 instrument as described in Example 4, except that soluble His-tagged human L1CAM was injected at four different concentrations (5 nM, 10 nM, 20 nM, and 40 nM). Table 11 shows that all mutational variants exhibited affinity constants (K) in the subnanomolar range in all cases. D ) showing improved binding to human L1CAM compared to the parent antibody H1L1.

[0346] [Table 11]

[0347] The chemical stability of antibodies AFF1 to AFF10 was analyzed by hydrophobic interaction chromatography on a Tosoh TSKgel Butyl-NPR column and compared with that of the parent antibody H1L1. Surprisingly, in contrast to H1L1, none of the antibody variants showed a second peak on the hydrophobic interaction column, and the area after the main peak (Table 12) was overall very low for all variants, indicating that post-translational variants resulting from aspartic acid isomerization at position 32 were either absent or present in very low amounts in antibody variants AFF1 to AFF10. Table 12 shows that exposure of the parent antibody H1L1 to high temperature and low pH stress (40°C in 20 mM sodium citrate buffer, pH 5.5, for 14 days) caused a significant increase in the area behind the main peak in hydrophobic interaction chromatography (from 19.5% to 76.2%). This increase was significantly reduced for antibody variants AFF1 to AFF10, indicating that the introduced mutations strongly inhibited the isomerization reaction at position 32. Exemplary chromatograms of stressed and unstressed antibody variant AFF4 are shown in Figure 10C.

[0348] [Table 12] Example 6

[0349] In vitro pharmacology of the humanized optimized antibody AFF4 Inhibition of cancer cell growth HCT116, Panc-1, and SKOV3 cells were cultured as described in Example 2 and seeded onto 384-well plates at 10-15% confluence in the presence of 50 μg / mL AFF4 or 50 μg / mL chimeric human IgG1 isotype control antibody. Cancer cell growth was monitored at regular intervals over 120 hours by microscopic confluency detection using an Incucyte device. Figure 11 shows that addition of AFF4 antibody resulted in a decrease in proliferation of all three cell lines compared to the isotype control antibody. By 96 hours, addition of AFF4 caused a decrease in confluency from 55.3% to 38.9% for HCT116 cells, from 68.6% to 44.4% for SKOV3 cells, and from 67.4% to 42.7% for Panc-1 cells when compared to cells treated with the isotype control antibody.

[0350] Inhibition of cancer cell migration HCT116 colorectal cancer cells were cultured in DMEM supplemented with 10% FCS, collected with Accutase® cell detachment solution (Capricorn), and plated at 4 × 10 cells onto stopper-containing wells of fibronectin-precoated ORIS-96-well plates (AMS-bio). 4 Cells were seeded at a density of 100 μL of cells / 100 μL. After 24 hours, the stopper insert was removed and the medium was replaced with 80 μL of fresh growth medium. Then, 20 μL of a 5-fold concentrated serial dilution series of AFF4 or a chimeric human IgG1 isotype control was added to the cells. The final antibody concentration in the assay plate was 4 × 10 -5 M to 1.2 x 10 -8The median values ​​ranged from 100 to 1000 M. As a positive control for unrestricted migration, cells were incubated without any antibody. As a negative control, migration was restricted by maintaining stoppers in eight wells per plate. After 48 h, the medium was replaced with 75 μL of phenol red-free DMEM supplemented with 2 μg / ml calcein-AM (Life Technologies). Cells were incubated at 37°C for 15 min, and fluorescent cells in the area defined by the insert were detected using a fluorescence microplate reader (Fluostar, BMG) with FITC settings (excitation: 485 nm / emission: 520 nm). The median value of the positive control (stopper removal, no antibody addition) was set to 100% (high control), and the median value of the negative control (no stopper removal) was set to 0% (low control). Raw data were converted to % cell migration relative to the control and fitted to a four-parameter logistic curve with a variable slope and a bottom constraint of 0 and a top constraint of 100. Figure 12 shows that addition of AFF4 antibody resulted in specific, dose-dependent inhibition of migration. The half-maximal inhibitory concentration (IC) for AFF4 50 ) is approximately 1.1 × 10 -5 It was calculated as M.

[0351] AFF4 induces antibody-dependent cellular cytotoxicity (ADCC) in SKOV3, Panc-1, HeLa, and PC03 cancer cell lines The ability of AFF4 to induce antibody-dependent cellular cytotoxicity was examined in the L1CAM-expressing cancer cell lines SKOV3, HeLa, Panc-1, and PC03. In the first step, the level of L1CAM expression on the surface of the different cell lines was assessed by flow cytometry. Each cell line was grown in continuous culture and used 2–4 days after passage. To limit cleavage of surface-expressed L1CAM, target cells were detached from the surface of the culture flask using a non-enzymatic cell dissociation buffer prior to the assay. Cells were stained with phycoerythrin (PE)-labeled anti-human L1CAM antibody (clone L1-OV198.5, BioLegend) or PE-labeled mouse IgG2a(κ) (clone MOPC-173, BioLegend) as the respective isotype control antibody. Stained cells were acquired on a Quanteon flow cytometer and analyzed using NovoExpress software. Median fluorescence intensity (MFI) was calculated for each cell line evaluated (in triplicate), and the average values ​​are tabulated in Table 13. To quantify expression levels, the fold increase in MFI values ​​obtained with anti-L1CAM antibodies relative to isotype control antibodies was calculated. All cell lines expressed L1CAM on their surface, with SKOV3 and HeLa showing the highest expression levels, and Panc-1 and PC03 showing intermediate and low expression levels, respectively.

[0352] [Table 13]

[0353] For ADCC evaluation, primary natural killer (NK) cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) of three different donors using an NK cell isolation kit (Miltenyi Biotech). One donor was homozygous for the CD16 (FcγRIIIA) high-affinity allele V158, one was homozygous for the CD16 low-affinity allele F158, while the third was heterozygous for both the high-affinity and low-affinity alleles (V / F158). Enriched NK cells were checked for purity (CD45+ >80% CD3 in the cell population - CD56 + The cells were incubated in NK92 medium plus IL-2 (3 ng / mL) at 36±1°C and 5±1% CO2 for 21±1 hours. In parallel, prewarmed cell culture medium was added (50 μL / well) to the wells of an xCelligence E-Plate 96 (Agilent), and SKOV3, HeLa, Panc-1, and PC-03 cell supernatants (100 μL / well) were added and allowed to settle to the bottom of the wells at room temperature for 1 hour. The E-Plate was then transferred to an xCelligence Real-Time Cell Analyzer (RTCA) and incubated at 36±1°C and 5±1% CO2 for 16–24 hours to allow cells to attach and proliferate. Impedance was measured continuously overnight to monitor cell proliferation. The following day, AFF4 was diluted to a concentration of 4 μg / mL in NK92 medium supplemented with 3 ng / mL IL-2. In parallel, NK cells were collected, counted, and resuspended in the same medium. The E-plate was removed from the RTCA analyzer, the medium was aspirated (50 μL / well), and prediluted AFF4 (50 μL / well) and NK cells (50 μL / well) were added. NK cells were added at a density corresponding to a final effector-to-target (E:T) cell ratio of either 5:1 or 10:1. The final concentration of AFF4 in the E-plate was 1 μg / mL. The plate was transferred to the RTCA analyzer, and impedance measurements were again initiated to monitor effector cell-mediated killing of target cells. Data were acquired for 72 hours and then analyzed with xCelligence Immunotherapy Software 1.0. Cell index data recorded by the RTCA analyzer were normalized to the last time point before the addition of effector cells. The normalized cell index obtained from wells containing target cells, effector cells, and treatment was compared to wells containing only target and effector cells to convert the normalized cell index to % specific cell lysis. The following formula was used by the xCELLigence RTCA software to calculate % specific cell lysis:

[0354]

number

[0355] Figure 13 shows that AFF4 activated effector cells from all three donors and induced specific cytolysis in all tested cell lines. The highest degree of cytotoxicity was observed in the L1CAM-highly expressing HeLa cell line, reaching 100% in the majority of cases. Specific cytolysis was generally higher at higher E:T ratios of 10:1 and correlated with the CD16 (FcγRIIIA) affinity of the effector cells present in the donor cell preparation. The highest activity was observed in the FcγRIIIA V158 homozygous (V / V) donor, intermediate activity in the FcγRIIIA V / F158 heterozygous (V / F) donor, and lowest activity in the FcγRIIIA F158 homozygous (F / F) donor. In this latter donor, the best correlation between cell surface expression of L1CAM and specific cell lysis in different cell lines was observed, with the highest levels observed in HeLa and SKOV3 cells, intermediate levels in Panc-1 cells, and the lowest levels in PC-03 cells. Example 7

[0356] Preparation and biophysical characterization of AFF4-WT antibody-drug conjugates For conjugation with cytotoxic payloads, a version of AFF4 was used that contains a wild-type human IgG1 constant domain and therefore lacks the four mutations (G236A / S239D / A330L / I332E) present in the CH2 domain of AFF4. This human IgG1 wild-type version of AFF4 is designated AFF4-WT.

[0357] Conjugation of AFF4-WT with VA-SG3199 (MP-PEG8-VA-PABC-SG3199, Tecilin) A solution containing AFF4-WT in PBS / 100 mM arginine was prepared for reduction by adding 5% v / v of 0.5 M Tris-HCl / 25 mM EDTA, pH 8.5, and then incubated with 1.25 molar equivalents of TCEP (tris(2-carboxyethyl)phosphine, added from a 1 mM stock in water) at 30°C for 2 hours, yielding an average of approximately two free thiols. The partially reduced AFF4-WT antibody was then conjugated by adding 5 molar equivalents of MP-PEG8-VA-PABC-SG3199 (Figure 14A). MP-PEG8-VA-PABC-SG3199 was added from a 10 mM stock in DMA (N,N-dimethylacetamide), with additional DMA added to reach 5% v / v during the conjugation reaction. After 2 hours of incubation at room temperature, the reaction was quenched with 5 molar equivalents of N-acetylcysteine ​​(added from a 10 mM stock in water) for 30 minutes at room temperature, followed by desalting and buffer exchange into 25 mM histidine / 0.2 M sucrose, pH 6.0, by passage through a Sephadex G-25 column. For complete removal of free toxins and other low-molecular-weight additives, the AFF4-WT-VA-SG3199 conjugate underwent eight buffer exchanges into 25 mM histidine / 0.2 M sucrose, pH 6.0, by intermittent diafiltration using PES (polyethersulfone) centrifugal concentrators (Vivaspin) with a 30 kDa molecular weight cutoff. Polysorbate 20 was added from a 1% w / v stock to a final concentration of 0.02% w / v, and the conjugate was sterile filtered using a 0.22 μm PVDF (polyvinylidene fluoride) membrane (Millipore Durapore). Aliquots of AFF4-WT-VA-SG3199 were frozen at −80°C.

[0358] Conjugation of AFF4-WT with VC-MMAE (MC-VC-PABC-MMAE, Vedotin) and Gluc-MMAE (MC-beta-glucuronide-MMAE) A solution containing AFF4-WT in PBS / 100 mM arginine was prepared for reduction by adding 5% v / v of 0.5 M Tris-HCl / 25 mM EDTA, pH 8.5, and then incubated with 2.5 molar equivalents of TCEP (added from a 1 mM stock in water) at 30°C for 2 hours, yielding an average of approximately four free thiols. The partially reduced AFF4-WT antibody was then conjugated by adding 10 molar equivalents of MC-VC-PABC-MMAE (Figure 14B) or MC-beta-glucuronide-MMAE (Figure 14C), respectively. MC-VC-PABC-MMAE and MC-beta-glucuronide-MMAE were added from 10 mM stocks in DMA, with additional DMA added to reach 5% v / v in the conjugation reaction. After 1 hour of incubation at room temperature, the reaction was quenched with 5 molar equivalents of N-acetylcysteine ​​(added from a 10 mM stock in water) for 30 minutes at room temperature, followed by desalting and buffer exchange into 25 mM histidine / 0.2 M sucrose, pH 6.0, by passage through a Sephadex G-25 column. For complete removal of free toxins and other low-molecular-weight additives, the AFF4-WT-VC-MMAE and AFF4-WT-Gluc-MMAE conjugates underwent eight buffer exchanges into 25 mM histidine / 0.2 M sucrose, pH 6.0, by intermittent diafiltration using a PES centrifugal concentrator (Vivaspin) with a 30 kDa molecular weight cutoff. Polysorbate 20 was added from a 1% w / v stock to a final concentration of 0.02% w / v, and the conjugates were sterile filtered using a 0.22 μm PVDF membrane (Millipore Durapore). Aliquots of AFF4-WT-VC-MMAE and AFF4-WT-Gluc-MMAE were frozen at −80°C.

[0359] Conjugation of AFF4-WT with sulfo-SPDB-DM4 AFF4-WT (in PBS / 100 mM arginine) was applied to a Protein A column that had been sterilized with 0.1 M NaOH and equilibrated with PBS, pH 7.4. After an extensive wash step with 20 column volumes of PBS, pH 7.4 (to remove the primary amine arginine), bound AFF4-WT was eluted by adding 0.1 M sodium citrate, pH 3.0. The eluted antibody was immediately desalted and buffer-exchanged to 50 mM sodium phosphate / 150 mM NaCl / 2 mM EDTA, pH 8.0, by passage through a Sephadex G25 column. AFF4-WT was then concentrated to 5 mg / ml using a PES centrifugal concentrator (Vivaspin) with a 30 kDa molecular weight cutoff. The prepared antibody was then conjugated by adding 7 molar equivalents of sulfo-SPDB-DM4 (Figure 14D). Sulfo-SPDB-DM4 was added from a 10 mM stock in DMA, and additional DMA was added to reach 5% v / v in the conjugation reaction. After 4 hours of incubation at room temperature, an additional 1 molar equivalent of sulfo-SPDB-DM4 was added to the reaction, and incubation was continued overnight. The conjugation reaction was then quenched by the addition of 1% v / v 0.5 M Tris, pH 8.5, and desalted and buffer-exchanged to 25 mM histidine / 0.2 M sucrose, pH 6.0, by passage through a Sephadex G25 column. To completely remove free toxins and other low-molecular-weight additives, AFF4-WT-sulfo-SPDB-DM4 underwent 10 buffer exchanges to 25 mM histidine / 0.2 M sucrose, pH 6.0, with intermittent diafiltration using a PES centrifugal concentrator (Vivaspin) with a 30 kDa molecular weight cutoff. Polysorbate 20 was added from a 1% w / v stock to a final concentration of 0.02% w / v, and the conjugate was sterile filtered using a 0.22 μm PES membrane (Millipore Express). Aliquots of AFF4-WT-Sulfo-SPDB-DM4 were stored frozen at −80°C.

[0360] Biophysical characterization of AAF4-WT-drug conjugates All AFF4-WT-drug conjugates were analyzed for their protein concentration and monomer content (by calculating the area under the curve in size-exclusion chromatography at 214 nm), their endotoxin content (by limulus amebocyte lysate assay), and their free drug concentration (by reverse-phase chromatography). The drug-to-antibody ratios of the cysteine ​​conjugates (AFF4-WT-VC-MMAE, AFF4-WT-Gluc-MMAE, and AFF4-WT-VA-SG3199) were determined by hydrophobic interaction chromatography, while the drug-to-antibody ratio of the lysine conjugate (AFF4-WT-sulfo-SPDB-DM4) was determined by size-exclusion chromatography through comparison of the areas under the curve obtained at 252 nm (absorbance maximum of DM4) and 280 nm (absorbance maximum of the antibody), respectively. The monovalent kinetic binding constants of different AFF4-WT-drug conjugates to human L1CAM were determined by surface plasmon resonance, essentially as described in Example 4. The results are summarized in Table 14:

[0361] [Table 14] Example 8

[0362] Pharmacological characterization of AFF4-WT antibody-drug conjugates Internalization and lysosomal localization of AFF4-WT upon binding to L1CAM on the surface of JIMT-1 breast cancer cells, OVCAR-3 ovarian cancer cells, and MeWo melanoma cells To exert their cytotoxic effects, antibody-drug conjugates must bind to and be efficiently internalized in the lysosomal compartment of cancer cells, where they are processed by specific proteases to liberate their cytotoxic payload. To test the internalization ability of AFF4-WT and track its intracellular localization, we used a human Fabfluor-pH Red antibody-labeling dye (an anti-human IgG (Fc-specific) Fab fragment conjugated to a pH-sensitive dye that is barely fluorescent at neutral pH but becomes highly fluorescent at low pH). Upon binding to AFF4-WT, the Fabfluor-labeled reagent is internalized along with AFF4-WT, indicating whether the antibody has reached the low-pH environment of the lysosomal pathway. The lysosomal uptake of AFF4-WT was determined using L1CAM-expressing JIMT-1 breast cancer cells, OVCAR-3 ovarian cancer cells, and MeWo melanoma cells. To this end, cells were cultured in 96-well cell culture plates at 20,000 cells / well (4 × 10 5 JIMT-1 cells were seeded in DMEM / 10% FCS, OVCAR-3 cells in RPMI1640 / 10% FCS, and MeWo cells in EMEM / 10% FCS. After approximately 5 hours of incubation at 37°C / 5% CO2, AFF4-WT or chimeric human IgG1 isotype control antibody was mixed with Incucyte® human Fabfluor-pH Red antibody labeling dye (Sartorius catalog no. 4722) and diluted to a concentration of 2 μg / mL (1:3 molar ratio of antibody to labeling dye) in RPMI164 / 10% FCS. After 15 minutes of incubation at 37°C, 50 μL / well of the mixture was added to each cell line, resulting in a final well concentration of 1 μg / mL of antibody and labeling dye, respectively. Incubation was continued for 15 minutes or 20 hours, respectively, after which the adherent cells were washed with PBS and then treated with Accutase® cell detachment solution (Capricorn) for 5 minutes, followed by analysis in an image-based cytometer (Nucleocounter NC-3000, ChemoMetec) using an excitation wavelength of 630 nm and an emission filter at 740 nm ± 60 nm. FIG. 15 shows that the fluorescence of AFF4-WT-treated JIMT-1, OVCAR-3, and MeWo cells was strongly increased after 20 hours compared to cells treated with the chimeric human IgG1 isotype control antibody, indicating that AFF4-WT was efficiently internalized by all cell lines and reached the lysosomal compartment.

[0363] In vitro cytotoxicity to L1CAM-expressing cell lines The ability of different AFF4-WT-drug conjugates to induce cytotoxicity was then tested in an in vitro cell-based killing assay. JIMT-1, OVCAR-3, and MeWo cells were seeded at 2,250 cells / well, 2,100 cells / well, and 2,000 cells / well, respectively, in a total volume of 90 μL in white 96-well culture plates using the same medium as described above for the internalization assay. Cells were allowed to adhere for 6.5–7.5 hours, after which 10 μL / well of AFF4-WT-drug conjugates serially diluted in RPMI / 10% FCS were added. The final concentrations of the conjugates in the assay ranged from 100 nM to 1.3 pM (0.05 pM for AFF4-WT-VA-SG3199). As a positive control for 100% killing, doxorubicin was added to a separate well at a final concentration of 10 μM. Assay plates were incubated at 37°C / 5% CO2 for 6 days, and cell viability was assessed by ATP quantification using CellTiter Glo 2.0 (Promega) and luminescence measurement according to the manufacturer's instructions. Luminescence readings were converted to % viability values ​​using wells with cells alone and wells with cells incubated in the presence of 10 μM doxorubicin as 100% and 0% viability reference values, respectively. The % viability values ​​obtained at different AFF4-WT-drug conjugate concentrations were fitted to a four-parameter logistic curve using GraphPad Prism, which was used to calculate the respective IC of the different conjugates. 50 The concentration was calculated. Figure 16 shows that AFF4-WT-VC-MMAE, AFF4-WT-Gluc-MMAE, AFF4-WT-VA-SG3199, and AFF4-WT-Sulfo-SPDB-DM4 all induced dose-dependent killing of JIMT-1, OVCAR-3, and MeWo cells. 50 The values ​​are summarized in Table 15 below.

[0364] [Table 15]

[0365] Inhibition of tumor growth in vivo The in vivo efficacy of the AFF4-WT drug conjugate was determined in mouse xenograft models of human cancer cell lines JIMT-1, OVCAR-3, and MeWo. JIMT-1 breast cancer cells (5 × 10 in 100 μL PBS) were cultured in 100 μL of PBS. 6 cells) were cultured in female athymic nude (Crl:NU(NCr)-Foxn1 nu ) mice were injected into the left mammary fat pad. Tumors were measured with a vernier caliper and tumor volume (TV) was calculated using the following formula: TV = (W 2 × L) / 2 (L = tumor length, W = tumor perpendicular length, L > W). The tumor is 100-150 mm 3 When tumors reached an average volume of 1000 mg / kg, mice were randomized into five different groups. Four groups (n = 6 each) received a single intravenous injection of either AFF4-WT-VC-MMAE (5 mg / kg), AFF4-WT-Gluc-MMAE (5 mg / kg), AFF4-WT-VA-SG3199 (1 mg / kg), or AFF4-WT-Sulfo-SPDB-DM4 (5 mg / kg), while the fifth group (n = 8) received vehicle as a control. Tumor volumes were measured twice weekly after injection. As shown in Figure 17A, a single treatment with either of the AFF4-WT drug conjugates induced a strong antitumor response. Tumor regression was observed in mice treated with AFF4-WT-VC-MMAE and AFF4-WT-Gluc-MMAE, respectively, while tumor growth arrest was observed in mice treated with AFF4-WT-VA-SG3199 or AFF4-WT-sulfo-SPDB-DM4. OVCAR-3 ovarian cancer cells (1 × 10 in Matrigel 7 Cells) were injected subcutaneously into female Jan:NMRI-nu / nu mice. Tumors were measured with calipers and tumor volumes were calculated as described above. Once tumors reached approximately 0.14 cm 3Once tumor volumes reached a mean volume of 1000 mg / kg, mice were randomized into five groups (n=5) and treatment was initiated. Each group received two intravenous injections, 2 weeks apart, of either AFF4-WT-VC-MMAE (5 mg / kg), AFF4-WT-Gluc-MMAE (5 mg / kg), AFF4-WT-VA-SG3199 (1 mg / kg), AFF4-WT-Sulfo-SPDB-DM4 (5 mg / kg), or vehicle as a control. Tumor volumes were measured twice weekly after randomization. Figure 17B shows that all AFF4-WT-drug conjugates induced antitumor responses in the OVCAR-3 xenograft model. Treatment with AFF4-WT-VC-MMAE, AFF4-WT-Gluc-MMAE, or AFF4-WT-VA-SG3199 induced tumor growth arrest, while treatment with AFF4-WT-sulfo-SPDB-DM4 caused a decrease in tumor growth compared to the vehicle control group. MeWo melanoma cells (1 × 10 in Matrigel 7 ) were injected subcutaneously into female Jan:NMRI-nu / nu mice. Tumor volumes were determined with calipers as described above. Tumors were approximately 0.14 cm 3 When tumors reached a mean tumor volume of 1000 mg / kg, mice were randomized into five groups (n=5) and treated with a single intravenous injection of either AFF4-WT-VC-MMAE (5 mg / kg), AFF4-WT-Gluc-MMAE (5 mg / kg), AFF4-WT-VA-SG3199 (1 mg / kg), AFF4-WT-Sulfo-SPDB-DM4 (5 mg / kg), or vehicle as a control. Tumor volumes were measured twice weekly after treatment. Figure 17C shows that antitumor responses were observed for all tested AFF4-WT-drug conjugates. Treatment with AFF4-WT-VC-MMAE or AFF4-WT-Gluc-MMAE induced tumor growth arrest, whereas treatment with AFF4-WT-VA-SG3199 or AFF4-WT-sulfo-SPDB-DM4 caused a decrease in tumor growth.

[0366] Efficacy of different doses of AFF4-WT-VC-MMAE in the JIMT-1 breast cancer xenograft model JIMT-1 breast cancer cells (5 x 10 in 100 μL PBS 6 cells) were cultured in female athymic nude (Crl:NU(NCr)-Foxn1 nu ) mice were injected into the left mammary fat pad. 3 When tumors reached an average volume of 1000 mg / kg, mice were randomized into five different groups. Four groups (n=6 each) then received a single intravenous injection of AFF4-WT-VC-MMAE at either 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, or 3.0 mg / kg, while the fifth group (n=8) received vehicle as a control. Tumor volumes were measured twice weekly after injection. FIG. 18 shows that all doses of AFF4-WT-VC-MMAE inhibited JIMT-1 tumor growth, with a clear dose-response relationship between the tested dose levels and the degree of antitumor efficacy.

[0367] Efficacy of AFF4-WT-VC-MMAE in a patient-derived xenograft model of ovarian cancer The efficacy of AFF4-WT-VC-MMAE was investigated in four patient-derived xenograft (PDX) models of human ovarian cancer with confirmed L1CAM expression. Female athymic nude-Foxn1 mice were subcutaneously implanted with fragments of one of the four L1CAM-expressing PDX tumors. Tumor growth was monitored at regular intervals until the mean tumor volume reached 150–300 mm. 3 When tumor volume reached 100 μg / day, animals were matched into treatment and control groups (n=5 each) by tumor volume. Thereafter, the treatment group received intravenous injections of AFF4-WT-VC-MMAE (3 mg / kg) every two weeks, while the control group received vehicle injections every two weeks. Tumor volumes were recorded twice weekly, and tumor growth inhibition (TGI) was calculated for the treatment group (T) relative to the control group (C) using the initial (i) and final (f) tumor measurements according to the following formula:

[0368]

number

[0369] As shown in Figure 19, AFF4-WT-VC-MMAE inhibited tumor growth in all four L1CAM-expressing ovarian cancer PDX models. The tumor growth inhibition rates at the last observation time point were 82% (CTG0868), 113% (CTG1086), 95% (CTG1649), and 74% (CTG3383), respectively.

[0370] In vitro cytotoxicity of AFF4-WT-VC-MMAE to L1CAM-expressing cell lines and correlation with L1CAM expression levels A series of 25 ovarian, endometrial, breast, melanoma, and neuroblastoma cell lines were seeded into wells of a white 96-well cell culture plate at the required density in a total volume of 90 μL of cell culture medium. The seeding density and cell culture medium were determined individually for each cell line to obtain the optimal assay window. Cells were allowed to adhere for approximately 6 hours, followed by the addition of 10 μL / well of AFF4-WT-VC-MMAE serially diluted in RPMI / 10% FCS. The final concentrations of AFF4-WT-VC-MMAE in the assay ranged from 100 nM to 1.3 pM. As a positive control for 100% killing, doxorubicin was added to a separate well at a final concentration of 10 μM. Assay plates were incubated at 37°C / 5% CO2 for 5-12 days, and cell viability was assessed by ATP quantification using CellTiter Glo 2.0 (Promega) and luminescence measurement according to the manufacturer's instructions. Luminescence readings obtained at different AFF4-WT-VC-MMAE concentrations were converted to % viability values ​​and fitted to a four-parameter logistic curve using GraphPad Prism, as detailed above. IC 50 Concentrations were derived for each individual cell line.

[0371] In parallel, the approximate number of L1CAM molecules expressed on the cell surface of each cell line was determined. To achieve this, a calibration line was first established using Quantibrite beads labeled with four different concentrations of phycoerythrin (PE) molecules (BD Biosciences). The beads were resuspended in 500 μL PBS, and 30 μL of this suspension was loaded onto an NC-Slide A2 (ChemoMetec) and analyzed using an image-based cytometer (Nucleocounter, ChemoMetec). A green light source (LED530) and a 500 ms exposure time were used for analysis. After gating on single beads among the four bead populations, the mean fluorescence intensity (MFI) was determined and plotted against the respective number of PE molecules per bead. Using GraphPad Prism, a linear regression curve was fitted to the data using the following equation: y=m×x+c where y is the MFI and x is the number of PE molecules per bead, as provided by the manufacturer. Different cancer cell lines were then grown in their respective cell culture media until they reached approximately 80% confluence. The cells were then detached with Accutase, resuspended in PBS, and each weighed approximately 3 x 10 5The cells were transferred to two wells of a 96-well polypropylene plate at a density of 100 cells / well. The plate was centrifuged at 300 g for 5 minutes at 4°C, the supernatant was removed, and the cell pellet was resuspended in 300 μL PBS / 1% FBS containing 1 μg / mL of either PE-labeled anti-L1CAM antibody or PE-labeled isotype control antibody. After 1 hour of incubation at 4°C in the dark, the cells were centrifuged again at 300 g for 5 minutes at 4°C, and the supernatant was removed. Subsequently, the cells were washed twice by resuspending in 300 μL PBS, followed by centrifugation (5 minutes at 300 g at 4°C) and removing the supernatant. After the final centrifugation step, the cells were resuspended in 75 μL PBS, and 30 μL of the cell suspension was analyzed in a Nucleocounter using the same settings as described above for the determination of the calibration line. Histogram plots of live cells were made, and the MFI values ​​were inserted as y values ​​into the linear regression equation established with the PE-labeled beads. The equation was solved for x (=number of PE molecules), and the number of L1CAM molecules per cancer cell was calculated using the following formula:

[0372]

number

[0373] [Table 16]

[0374] AFF4-WT-VC-MMAE induced potent cytotoxic effects on most of the cell lines tested. As shown in Table 16, the cytotoxic potency of AFF4-WT-VC-MMAE closely correlated with the number of L1CAM molecules on the cell surface of each cell line. Cell lines with high L1CAM expression were most sensitive to AFF4-WT-VC-MMAE-induced cytotoxic effects (=low IC 50 ), whereas cell lines with lower or absent L1CAM expression were less sensitive (high IC 50 ).

[0375] Bystander cytotoxicity of AFF4-WT-VC-MMAE Within human tumors, L1CAM expression can be heterogeneous, with some cancer cells expressing high levels of L1CAM, while others express lower levels or none at all. Because the potency of AFF4-WT-VC-MMAE correlates with the level of L1CAM expression (see Table 16), the question arises as to whether tumors with heterogeneous L1CAM expression can be efficiently targeted by AFF4-WT-VC-MMAE. Therefore, we investigated whether AFF4-WT-VC-MMAE exhibits a so-called cytotoxic bystander effect. ADCs endowed with such a bystander effect are taken up by antigen-positive cancer cells and processed to release a cytotoxic payload, which can freely diffuse to neighboring cells and thus kill those cells regardless of their antigen expression. Therefore, such ADCs are well suited for treating tumors with heterogeneous target expression.

[0376] To evaluate the bystander activity of AFF4-WT-VC-MMAE, L1CAM-highly expressing JIMT-1 breast cancer cells and L1CAM-lowly expressing MDA-MB-468 breast cancer cells (see Table 16) were cultured at 5 × 10 in six wells of a 24-well cell culture plate. 4Cells were seeded at a density of 100 cells / well in a total volume of 400 μL in RPMI / 10% FCS. Six additional wells were filled with 400 μL RPMI / 10% FCS alone. After 5 hours of incubation at 37°C / 5% CO2, AFF4-WT-VC-MMAE was serially diluted in RPMI / 10% FCS, and 9.8 μL / well of the serial dilutions were added to the seeded wells and wells containing cell culture medium alone. The concentrations of AFF4-WT-VC-MMAE in the assay ranged from 100 nM to 10 pM. One well each from the JIMT-1-seeded wells, MDA-MB-468-seeded wells, and wells containing cell culture medium alone were left untreated as controls. The plate was incubated at 37°C / 5% CO2 for 4 days.

[0377] L1CAM-low expressing MDA-MB468 cells were then seeded into wells of white 96-well cell culture plates at a density of 1500 cells / well in a total volume of 75 μL in RPMI / 10% FCS, followed by incubation for 4 hours at 37°C / 5% CO2.

[0378] Supernatants from all wells of the 24-well plate incubated for 4 days were transferred to sterile tubes and centrifuged at 2000 rcf for 10 minutes. The supernatants were transferred to new sterile tubes, and then 25 μL of each supernatant was added to the plated MDA-MB-468 cells. As a negative control, fresh RPMI / 10% FCS medium was added to three wells. As a positive control for maximal growth inhibition, doxorubicin was added to three wells at a final concentration of 10 μM. After addition of supernatant, medium, and doxorubicin, the plates were incubated at 37°C / 5% CO2 for 6 days. Cell viability was then assessed by ATP quantification using CellTiter Glo 2.0 (Promega) and luminescence measurement according to the manufacturer's instructions. Luminescence readings were converted to % viability values ​​and fitted to a four-parameter logistic curve using GraphPad Prism, as detailed above.

[0379] Figure 20 shows the % viability of MDA-MB-468 cells after incubation with supernatants from JIMT-1 or MDA-MB-468 cells preincubated with serial dilutions of AFF4-WT-VC-MMAE. As a control, the % viability obtained with the same serial dilutions of AFF4-WT-VC-MMAE preincubated in the absence of cells (cell culture medium only) is also shown. Consistent with the virtual insensitivity of MDA-MB-468 to AFF4-WT-VC-MMAE (Table 16), AFF4-WT-VC-MMAE had little cytotoxic effect (IC) when preincubated on L1CAM-low-expressing MDA-MB-468 cells or in the absence of cells. 50 In contrast, AFF4-WT-VC-MMAE pre-incubated on JIMT-1 cells with high L1CAM expression exhibited only an IC of approximately 500 pM on MDA-MB-468 cells. 50 The results showed significantly stronger cytotoxic effects than the control group. These results strongly suggest that the uptake and processing of AFF4-WT-VC-MMAE by JIMT-1 cells with high L1CAM expression generates free MMAE toxin, which is released into the supernatant where it can exert its cytotoxic effects. Thus, the data support the idea that AFF4-WT-VC-MMAE exhibits bystander cytotoxic activity. In the context of tumors with heterogeneous levels of L1CAM expression, this activity of AFF4-WT-VC-MMAE appears to ensure that not only high-L1CAM-expressing tumor cells are killed, but also that adjacent tumor cells with lower L1CAM expression are efficiently eliminated, achieving a good overall antitumor response.

Claims

1. Antibodies that specifically bind to human L1CAM, including: (a) GYSITSDYX １ WN (SEQ ID NO: 16) １ is A or T), or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 16) heavy chain variable region (VH) complementarity determining region (CDR) 1 comprising: (b) YISYSGSX １ SYX ２ PSLKS (SEQ ID NO: 17) (In the formula, X １ is F or Y, and X ２ is H or N), or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 17) a VH CDR2 comprising: (c) SX １ SYX ２ YGFAY ​​(SEQ ID NO: 18) (In the formula, X １ is L or F, and X ２ is G, S, or A, or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 18) a VH CDR3 comprising: (d) the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4); or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 4) Light chain variable region (VL) CDR1 comprising (e) SASYRYX １ (SEQ ID NO: 19) (wherein X １ is T or I, or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 19) and a VL CDR2 comprising: (f) the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6); or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 6) and a VL CDR3 comprising:

2. Antibodies that specifically bind to human L1CAM, including: (a) GYSITSDYX １ WN (SEQ ID NO: 16) １ a heavy chain variable region (VH) complementarity-determining region (CDR) 1 comprising the amino acid sequence of (b) YISYSGSX １ SYX ２ PSLKS (SEQ ID NO: 17) (In the formula, X １ is F or Y, and X ２ is H or N) a VH CDR2 comprising the amino acid sequence of: (c) SX １ SYX ２ YGFAY ​​(SEQ ID NO: 18) (In the formula, X １ is L or F, and X ２ is G, S, or A) a VH CDR3 comprising the amino acid sequence of: (d) a light chain variable region (VL) CDR1 comprising the amino acid sequence KASQDVSSAVA (SEQ ID NO: 4); (e) SASYRYX １ (SEQ ID NO: 19) (wherein X １ is T or I; and (f) a VL CDR3 comprising the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6).

3. 3. The antibody that specifically binds to human L1CAM according to claim 1 or 2, which is: (a) VH CDR1 comprises the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9), or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 9); and / or (b) VH CDR2 is YISYSGSX １ SYX ２ PSLKS (SEQ ID NO: 17) (In the formula, X １ is Y, and / or X ２ is N), or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 17) and / or (c) VH CDR3 is SX １ SYX ２ YGFAY ​​(SEQ ID NO: 18) (In the formula, X １ is F, and / or X ２ is S) The amino acid sequence of or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 18) and / or (d) VL CDR2 has the amino acid sequence of SASYRYT (SEQ ID NO: 5); or a sequence containing one, two or three amino acid exchanges in the sequence of (SEQ ID NO: 5) Includes.

4. The antibody that specifically binds to human L1CAM according to any one of claims 1 to 3, which is: (a) VH CDR1 comprises the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9); and / or (b) VH CDR2 is YISYSGSX １ SYX ２ PSLKS (SEQ ID NO: 17) (In the formula, X １ is Y, and / or X ２ is N) and / or comprising the amino acid sequence (c) VH CDR3 is SX １ SYX ２ YGFAY ​​(SEQ ID NO: 18) (In the formula, X １ is F, and / or X ２ is S) and / or comprising the amino acid sequence (d) VL CDR2 comprises the amino acid sequence of SASYRYT (SEQ ID NO: 5).

5. An antibody that specifically binds to human L1CAM according to any one of claims 1 to 4, comprising: (a) a heavy chain variable region (VH) comprising a VH CDR1 comprising the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 9), a VH CDR2 comprising the amino acid sequence of YISSYSGSYSYNPSLKS (SEQ ID NO: 11) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 11), and a VH CDR3 comprising the amino acid sequence of SFSYSYGFAY ​​(SEQ ID NO: 14) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 14); and (b) a light chain variable region (VL) comprising a VL CDR1 comprising the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 4), a VL CDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 5) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 5), and a VL CDR3 comprising the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6) or a sequence containing one, two, or three amino acid exchanges in the sequence of (SEQ ID NO: 6).

6. An antibody that specifically binds to human L1CAM according to any one of claims 1 to 5, comprising: (a) a heavy chain variable region (VH) comprising a VH CDR1 comprising the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9), a VH CDR2 comprising the amino acid sequence of YISSYSGSYSYNPSLKS (SEQ ID NO: 11), and a VH CDR3 comprising the amino acid sequence of SFSYSYGFAY ​​(SEQ ID NO: 14); and (b) a light chain variable region (VL) comprising a VL CDR1 comprising the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4), a VL CDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 5), and a VL CDR3 comprising the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6).

7. The antibody of any one of claims 1 to 6, further comprising a heavy chain variable region sequence comprising the framework region of a heavy chain variable region sequence of any one of SEQ ID NOs: 23 to 34, and / or a light chain variable region sequence comprising the framework region of a light chain variable region sequence of any one of SEQ ID NOs: 20 to 22.

8. The antibody of any one of claims 1 to 7, comprising a heavy chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 34, and / or a light chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 22.

9. The antibody of any one of claims 1 to 8, comprising a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 30 and / or a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO:

20.

10. The antibody of any one of claims 1 to 9, comprising a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37 or 84, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO:

38.

11. The antibody of any one of claims 1 to 10, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

20.

12. Monoclonal antibodies, recombinantly produced antibodies, single-specific antibodies, multispecific antibodies including bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, univalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelid antibodies, a The antibody of any one of claims 1 to 11, which is selected from FN3 scaffolds such as fibrobodies, anticalins, affilins, atrimers, DARPins, adnectins and centilins, phynomers, Kunitz domains, pronectins, and OBodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of any of the above, and / or is comprised in a chimeric antigen receptor (CAR).

13. 13. The antibody according to any one of claims 1 to 12, further comprising a heavy chain constant region and / or a light chain constant region, preferably wherein the heavy chain constant region is selected from the group of human immunoglobulins consisting of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, and / or the light chain constant region is selected from the group of human immunoglobulins consisting of IgGκ and IgGλ.

14. The antibody of any one of claims 1 to 13, wherein the heavy chain constant region is a variant of a wild-type human IgG heavy chain constant region, and preferably the variant human IgG heavy chain constant region binds to one or more human Fc gamma receptors selected from the group consisting of FcγRI, FcγRIIA, and FcγRIIIA with higher affinity than the wild-type human IgG heavy chain constant region binds to said human Fc gamma receptors.

15. An antibody that binds to the same epitope of human L1CAM as the antibody of any one of claims 1 to 14 and / or competes with the antibody of any one of claims 1 to 14 in binding to human L1CAM, preferably wherein the epitope is within fibronectin type III domains 1 to 3 (FN III 1 to 3) of human L1CAM.

16. (i) binds to human L1CAM within fibronectin type III domains 1-3 (FN III 1-3) of L1CAM; and / or (ii) an affinity (K) of 20 nM or less, 10 nM or less, or 1 nM or less with human L1CAM; Ｄ ) and / or (iii) an affinity (K) of 20 nM or less, 10 nM or less, or 1 nM or less with cynomolgus monkey L1CAM; Ｄ ) and / or (iv) inhibiting tumor cell migration on fibronectin-coated surfaces in vitro; and / or (v) inhibiting the proliferation of SKOV-3, Panc-1, and / or HCT-116 tumor cells in vitro; and / or (vi) inhibiting primary tumor growth in a SKOV-3ip xenograft model; and / or (vii) reducing metastasis formation in the mouse xenograft model MDA-MB-231; and / or (viii) exhibits ADCC activity in vitro and / or binds to the FcγRIIIa receptor in vitro; and / or (ix) exhibits binding to FcRn in vitro; and / or (x) does not cross-react with human CHL1, human NrCAM, and / or human neurofascin in vitro; An antibody described in any one of claims 1 to 15.

17. The antibody of any one of claims 1 to 16, which is a multispecific or bispecific antibody and / or a humanized antibody.

18. (a) a therapeutically active substance; Preferably, chemotherapy compounds, cytotoxic compounds, cytostatic compounds, cytokines, nanoparticles, Radioisotopes, and / or Oncolytic viruses and / or (b) linked to a diagnostic compound, preferably selected from a radioisotope, a chemiluminescent compound, a fluorescent compound, a dye, or an enzyme; An antibody described in any one of claims 1 to 17.

19. 19. The antibody of claim 18, wherein the therapeutically active substance (a) and / or the diagnostic compound (b) are selected from a radioisotope, a chemotherapeutic compound, a cytotoxic compound, and / or a cytostatic compound, and / or the antibody is covalently linked to the therapeutically active substance (a) or a chelator thereof or the diagnostic compound (b) or a chelator thereof, optionally via a linker.

20. The antibody of any one of claims 1 to 17, which is linked to at least one therapeutically active substance via a linker.

21. 21. The antibody of any one of claims 18 to 20, wherein the therapeutically active substance is selected from the group consisting of DNA damaging agents, anti-apoptotic agents, mitotic inhibitors, antitumor antibiotics, immunomodulatory agents, nucleic acids for gene therapy, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormonal agents, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radioisotopes, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors.

22. 22. The antibody of claim 21, wherein the antimitotic agent is selected from maytansinoids and auristatins.

23. 22. The antibody of claim 21, wherein the DNA damaging agent is selected from pyrrolobenzodiazepines (PBDs) and pyridinobenzodiazepines (PDDs).

24. The antibody of any one of claims 18 to 23, wherein the linker is a non-cleavable linker.

25. The antibody of any one of claims 18 to 23, wherein the linker is a cleavable linker.

26. 26. The antibody of any one of claims 18 to 25, wherein the therapeutically active substance is selected from monomethylauristatin E (MMAE), 4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4), and VA-SG3199 (Tesirin).

27. (a) comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 30, and / or comprises a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 20; or (b) comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 30, and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 20; or (c) a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38; or (d) comprising a heavy chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and / or a light chain sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 38; An antibody described in any one of claims 18 to 26.

28. (i) encoding an antibody according to any one of claims 1 to 27, and / or (ii) encoding at least one VH or HC and / or VL or LC of an antibody according to any one of claims 1 to 27; and / or (iii) encoding a sequence according to SEQ ID NO: 30 and / or a sequence according to SEQ ID NO: 20, and / or (iv) a sequence encoding the complementarity determining region sequence of the antibody according to any one of claims 1 to 27; A nucleic acid, preferably part of a vector.

29. 29. A host cell comprising the nucleic acid of claim 28.

30. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 27, or a nucleic acid according to claim 28, or a host cell according to claim 29, and optionally one or more pharmaceutically acceptable carriers.

31. An antibody according to any one of claims 1 to 27, or a nucleic acid according to claim 28, or a host cell according to claim 29, or a pharmaceutical composition according to claim 30, for use as a medicament or as a diagnostic agent.

32. 31. The antibody of any one of claims 1 to 27, or the nucleic acid of claim 28, or the host cell of claim 29, or the pharmaceutical composition of claim 30, for use in treating or preventing a hyperproliferative disorder, a tumor disease, a disorder involving angiogenesis, and / or a disorder involving abnormal neurogenesis.

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