Novel binding molecule binding to l1cam

Abstract

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

Description

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

[0002] Monoclonal antibodies (mAbs) have become a new and important pillar of cancer therapy [1]. In the past two decades, molecular biology has provided methods for creating chimeric humanized or fully human antibodies for the treatment of major malignancies [2]. To date, many antibodies and antibody conjugates have been approved as cancer therapeutics in Europe and the United States [3, 4]. They include unmodified antibodies, antibody-drug conjugates, and conjugates with radionuclides and bispecific antibodies [5]. However, it is well known that mAbs directed against specific cancer antigens may have different abilities to target cancer cells.

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

[0004] WO 2008 / 151819 discloses an anti-L1CAM antibody, L1CAM mAb L1-9.3 (also referred to as mAb 9.3 or L9.3), which binds to an epitope within the first Ig domain of L1CAM. Results from xenograft [6] and human L1CAM transgenic mouse [7] models suggest that mAb 9.3 may be a promising tool for cancer therapy. Recent results indicate that this mAb, in the IgG2a version, is well suited to activate the immune system and recruit immune effector cells to eliminate cancer cells [6, 7].

[0005] WO 2016 / 050702 discloses binding molecules that bind to L1CAM, which are 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. Compared to mAb 9.3, the binding molecules of WO 2016 / 050702 have been shown to have improved internalization rates. It has been described that binding of L1CAM-specific antibodies will lead to L1CAM internalization, followed by recycling or degradation of the target molecule [8]. Internalization is a common feature of L1CAM with many other cell surface molecules. In fact, it has been described that L1CAM-mediated signal transduction and regulation of cell adhesion require L1CAM internalization [9-11].

[0006] Despite these findings, there is still a need for antibodies that exhibit specific binding with high affinity to L1CAM on the surface of human cancer cell lines, without exhibiting any reactivity to panel-coated human CHL1, NrCAM or neurofascin. In addition, there is a need for antibodies that inhibit the migration and proliferation of cancer cells, induce cytolysis of tumor cells, inhibit metastasis formation and reduce tumor burden and the development of ascites. Likewise, it would be advantageous to provide antibodies that improve the general clinical symptoms of the disease. Finally, it would generally be helpful if such antibodies had both high conformational and chemical stability and showed reduced formation of post-translational variants.

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

[0008] The antibodies of the invention which specifically bind to human L1CAM solve all of the above problems and have surprising advantages in the field of biotechnological research, diagnostics or therapy. Summary of the invention

[0009] The present invention provides an antibody that specifically binds to human L1CAM.

[0010] In addition, nucleic acids encoding all or part of an antibody that specifically binds to human L1CAM and host cells containing such nucleic acids are provided.

[0011] Also provided is a pharmaceutical composition comprising such antibody, nucleic acid or host cell.

[0012] Also provided are such antibodies, nucleic acids, host cells or pharmaceutical compositions for use as medicaments or diagnostic agents or for use in treating or preventing hyperproliferative disorders, neoplastic diseases, disorders associated with neovascularization and / or disorders associated with abnormal neurogenesis. DETAILED DESCRIPTION

[0013] The present invention provides a novel antibody which specifically binds to human L1CAM.

[0014] The novel antibodies that specifically bind to human L1CAM exhibit advantageous characteristics compared to prior art anti-L1CAM antibodies.

[0015] For example, the chimeric OV549.20 human IgG1 antibody of the present invention induced strong ADCC on Panc-1 target cells. In contrast, the chimeric human IgG1 version of the previously described antibody L9.3 that binds to the first Ig domain of L1CAM did not induce any ADCC on Panc-1 target cells (see Example 2, Figure 4Furthermore, the addition of the antibodies of the invention resulted in a decrease in proliferation of all three cancer cell lines tested (see Example 2, Figure 5 ). In contrast, the binding molecules of WO 2016 / 050702 that bind to L1CAM have no effect on the proliferation of cancer cell lines (see Example 2, Figure 5 In addition, the antibodies of the present 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 7 A).

[0016] The antibodies of the invention exhibit specific binding with high affinity to L1CAM on the surface of human cancer cell lines, but do not exhibit any reactivity with panel-coated human CHL1, NrCAM or neurofascin. In addition, the antibodies of the invention inhibit the migration and proliferation of a variety of different cancer cells, induce cytolysis of tumor cells, inhibit metastasis formation and reduce tumor burden and the development of ascites. This combination of properties makes the antibodies particularly suitable for improving clinical signs of neoplastic diseases in general. In addition, the antibodies of the invention have both high conformational and chemical stability and show reduced formation of post-translational variants, which makes them particularly suitable for large-scale manufacturing, clinical development, clinical safety and storage.

[0017] As used herein, the term "antibody that specifically binds to human L1CAM" means any polypeptide that has structural similarity to a naturally occurring antibody and is capable of binding to human L1CAM, wherein the binding specificity is determined by the CDR of the polypeptide. Therefore, "antibody that specifically binds to human L1CAM" is intended to be related to an immunoglobulin-derived structure that binds to human L1CAM.

[0018] L1CAM (also known as L1) is a transmembrane protein; it is a neuronal cell adhesion molecule belonging to the L1 protein family, has a molecular weight of 200-220 kDa, and is involved in axon guidance and cell migration, and is of great significance in treatment-resistant cancers.

[0019] According to the present invention, the term "human L1CAM" is preferably understood to mean the human L1CAM protein. The human L1CAM gene sequence has been assigned the gene ID: 3897. The Genbank entry for the isoform 1 precursor of the human L1CAM protein is NP_000416. L1CAM is also designated as CD171. The term "human L1CAM" describes any form of the protein known to be expressed by any cell type of the human being based on this L1CAM gene.

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

[0021] The term "antibody" generally describes any polypeptide having structural similarity to a naturally occurring antibody, such as a protein belonging to the immunoglobulin protein family. The term "antibody" includes full-length antibodies, antigen-binding fragments of antibodies, and molecules including antibody VH regions 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 including 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, intracellular antibodies, heterologous conjugated antibodies, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelid antibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of any of the above. The antibody can be part of a fusion protein or a conjugate. For example, antibodies can be included in chimeric antigen receptors (CARs).Antibodies 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 chain and each 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 constant region of the heavy chain can be one of the following five types of mammalian Ig heavy chains: α, δ, ε, γ and μ.The heavy chain types present generally define the categories (isotypes) of antibodies: IgA, IgD, IgE, IgG and IgM antibodies, respectively.Similarly, the constant region of the light chain can be one of the following two types of mammalian Ig light chains: κ and λ.The variable regions of heavy and light chains are generally composed of a unique combination of many protein sequences, thereby allowing binding to specific antigens. The term "antibody" further includes domain scaffolds such as affibodies, anticalins, affilins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centyrins), fynomers, Kunitz domains, pronectins and OBodys.

[0022] Preferably, the antibody described herein may be an IgG antibody or an isotype 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 antibody described herein is an IgG1 or IgG2 antibody.

[0024] Typically, in the case of a full-length intact antibody, each heavy chain is linked to one of the light chains, whereby the variable regions of the heavy and light chains combine to form one of two identical antigen binding sites, and their constant regions combine to form the constant region of the antibody. Additionally, two constructs of one heavy chain and one light chain can be linked via the constant regions of their heavy chains, thereby forming a "Y" shaped molecule, whereby the two arms depict the antigen binding variable regions and the stem depicts the constant region.

[0025] The antibody according to any of the aspects of the present invention may be a complete antibody, which means that it generally comprises a heavy chain of three or four constant domains and a light chain of one constant domain and corresponding variable domains, whereby each domain may comprise additional modifications, such as mutations, deletions or insertions, which do not alter the overall domain structure.

[0026] Typically, each heavy chain variable region and each light chain variable region of an antibody includes three non-contiguously arranged complementarity determining regions (CDRs).

[0027] As used herein, the term "CDR" or "complementarity determining region" refers to a non-continuous antigen binding site present in the variable region of both heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) (

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

[13] -

[15] ), wherein when compared to each other, the definition includes overlaps or subgroups of amino acid residues. The amino acid residues covering the CDR as defined by each of the references cited above are listed for comparison. Preferably, the term "CDR" is the CDR defined by Kabat based on sequence comparisons.

[0028] CDR is usually numbered as CDR1, CDR2 and CDR3 for the heavy chain variable region and the light chain variable region, respectively. Therefore, the arms of an antibody usually have 6 CDRs that together form an antigen binding site. Usually, the length of a CDR is usually 1 to 25 amino acids, preferably 3 to 20 amino acids in length, such as 3 to 16 amino acids in length. An antibody can include 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, the antibody comprising:

[0030] (a) a heavy chain variable region (VH) complementarity determining region (CDR) 1, wherein the VH CDR1 comprises the amino acid sequence of GYSITSDYX1WN (SEQ ID NO: 16), wherein:

[0031] X1 is A or T;

[0032] Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 16);

[0033] (b) VH CDR2, comprising the amino acid sequence of YISYSGSX1SYX2PSLKS (SEQ ID NO: 17), wherein

[0034] X1 is F or Y, and

[0035] X2 is H or N;

[0036] Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 17);

[0037] (c) a VH CDR3 comprising the amino acid sequence of SX1SYX2YGFAY ​​(SEQ ID NO: 18), wherein:

[0038] X1 is L or F, and

[0039] X2 is G, S or A;

[0040] Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 18);

[0041] (d) a light chain variable region (VL) CDR1 comprising the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4);

[0042] Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 4);

[0043] (e) a VL CDR2 comprising the amino acid sequence of SASYRYX1 (SEQ ID NO: 19), wherein:

[0044] X1 is T or I;

[0045] or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 19); and

[0046] (f) a VL CDR3 comprising the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6);

[0047] Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 6).

[0048] In one aspect, the present invention relates to an antibody that specifically binds to human L1CAM, the antibody comprising:

[0049] (a) a heavy chain variable region (VH) complementarity determining region (CDR) 1, wherein the VH CDR1 comprises the amino acid sequence of GYSITSDYX1WN (SEQ ID NO: 16), wherein:

[0050] X1 is A or T;

[0051] (b) VH CDR2, comprising the amino acid sequence of YISYSGSX1SYX2PSLKS (SEQ ID NO: 17), wherein

[0052] X1 is F or Y, and

[0053] X2 is H or N;

[0054] (c) a VH CDR3 comprising the amino acid sequence of SX1SYX2YGFAY ​​(SEQ ID NO: 18), wherein:

[0055] X1 is L or F, and

[0056] X2 is G, S or A;

[0057] (d) a light chain variable region (VL) CDR1 comprising the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4);

[0058] (e) a VL CDR2 comprising the amino acid sequence of SASYRYX1 (SEQ ID NO: 19), wherein:

[0059] X1 is T or I; and

[0060] (f) VL CDR3 comprising the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6).

[0061] In this regard, the variable "X1" or "X2" of a given SEQ ID NO: is independent of the "X1" and "X2" of different SEQ ID NOs:. "X1" or "X2" is defined independently for each SEQ ID NO:. If 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 are independent of the X1 of the mentioned sequences SEQ ID NO: 16 (VH CDR1), SEQ ID NO: 18 (VH CDR3) and SEQ ID NO: 19 (VLCDR2) and variants of X2 of SEQ ID NO: 18 (VH CDR3). For example, X1 and X2 of SEQ ID NO: 18 (VH CDR3) are typically selected independently of each other and independently of X1 of the mentioned sequences SEQ ID NO: 16 (VH CDR1), SEQ ID NO: 17 (VH CDR2) and SEQ ID NO: 19 (VL CDR2) and variants of X2 of SEQ ID NO: 17 (VH CDR2). Finally, for example, X1 of SEQ ID NO: 19 (VL CDR2) is typically selected independently of variants of X1 of the mentioned sequences SEQ ID NO: 16 (VHCDR1), SEQ ID NO: 17 (VH CDR2) and SEQ ID NO: 18 (VH CDR3) and variants of X2 of the mentioned sequences SEQ ID NO: 17 (VH CDR2) and SEQ ID NO: 18 (VH CDR3).

[0062] The remaining regions of the antibodies according to the invention, such as the framework regions of the variable heavy chain and the variable light chain and, where applicable, the constant domain of the heavy chain and the constant domain of the light chain, may be of any sequence, e.g. to the sequences known to the person skilled in the art.

[0063] The antibody of the present invention may further include two identical heavy chains and / or two identical light chains, or only include different heavy chains and light chains. It is also possible that the heavy chain variable region and the light chain variable region, respectively including the specific CDR1 to CDR3 sets mentioned above, form only one arm of the antibody, and the second (or additional) arm of the antibody includes different heavy chain variable regions and light chain variable regions.

[0064] Since the antibody according to the present invention may include two or more heavy chain variable regions and two or more light chain variable regions, there is further no need to connect the heavy chain variable region including the specific heavy chain CDR1 to CDR3 mentioned above with the light chain variable region including the specific light chain CDR1 to CDR3 mentioned above.

[0065] The antibodies of the present invention may be derived from mammals, such as rodents, for example mice, rabbits or rats. Each of the remaining regions of the antibodies according to the present invention, such as the framework regions, and where applicable, the constant domains of the heavy chain and the constant domains of the light chain, may include one or more modifications, such as mutations, including substitutions, deletions or insertions, which modifications do not alter the overall domain structure.

[0066] For example, the antibodies of the present invention may include 1 to 10 mutations (including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations, including any sub-range thereof) in the Fc domain of the antibody, such as substitutions, deletions and / or insertions, especially 1 to 10 substitutions (including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, including any sub-range thereof). Each mutation can be independently introduced into one chain of the Fc domain. Alternatively, each mutation can be independently and symmetrically introduced into two chains of the Fc domain.

[0067] For example, the antibodies of the invention may include 1 to 10 mutations (including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations, including any subranges thereof), such as substitutions, deletions and / or insertions, in particular 1 to 10 substitutions (including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, including any subranges thereof) in the CL and / or CH1 domains of the antibody.

[0068] In the context of various antibody examples, exemplary combinations of VH CDR1, VH CDR2 and VH CDR3 mentioned above are listed in Table 1 below, all of which correspond to antibodies according to the present invention. Among them, antibody OV549.20 represents a mouse example of an antibody according to the present invention. H1, H2, H3 and H4 are examples of VH regions of humanized variants of mouse antibody OV549.20. AFF1 to AFF10 are examples of antibodies according to the present invention, which have been further affinity matured. Among them, the combination of CDRH1, CDRH2 and CDRH3 of antibody "AFF4" is particularly preferred. The sequences highlighted in bold are particularly preferred sequences.

[0069] Table 1. Heavy chain CDR sequences of exemplary anti-L1CAM antibodies 1

[0070]

[0071]

[0072] 1 The CDRs in Table 1 are defined according to Kabat.

[0073] In the context of the various antibody examples already mentioned in Table 1, exemplary combinations of VL CDR1, VLCDR2 and VL CDR3 of the present invention mentioned above are listed in Table 2 below. L1 and L2 are examples of light chain variable regions of humanized antibodies related to the murine antibody OV549.20, which can be further combined with any one of the sets of VH CDR1, VH CDR2 and VHCDR3 of H1, H2, H3 and H4 of Table 1. The antibody may include the VH CDR of one antibody as depicted in Table 1 and the VL CDR of the same or different antibody as depicted in Table 2, preferably the VL CDR of the same antibody as depicted in Table 2. The sequences highlighted in bold are particularly preferred sequences. AFF1 to AFF10 are examples of antibodies according to the present invention, which have been further affinity matured. Among them, the combination of CDRL1, CDRL2 and CDRL3 of the antibody "AFF4" is particularly preferred.

[0074] Table 2. Light chain CDR sequences of exemplary anti-L1CAM antibodies 2

[0075]

[0076]

[0077] 2 The CDRs in Table 2 are defined according to Kabat.

[0078] 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.

[0079] In a preferred embodiment, the present invention relates to an antibody of the present invention that specifically binds to human L1CAM, wherein:

[0080] (a) the VH CDR1 comprises the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9) or a sequence comprising 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 9); and / or

[0081] (b) the VH CDR2 comprises the amino acid sequence of YISYSGSX1SYX2PSLKS (SEQ ID NO: 17), wherein

[0082] X1 is Y, and / or

[0083] X2 is N;

[0084] Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 17);

[0085] and / or

[0086] (c) the VH CDR3 comprises the amino acid sequence of SX1SYX2YGFAY ​​(SEQ ID NO: 18), wherein:

[0087] X1 is F, and / or

[0088] X2 is S;

[0089] Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 18);

[0090] and / or

[0091] (d) the VL CDR2 comprises the amino acid sequence of SASYRYT (SEQ ID NO: 5);

[0092] Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 5).

[0093] In a preferred embodiment, the present invention relates to an antibody of the present invention that specifically binds to human L1CAM, wherein:

[0094] (a) the VH CDR1 comprises the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9); and / or

[0095] (b) the VH CDR2 comprises the amino acid sequence of YISYSGSX1SYX2PSLKS (SEQ ID NO: 17), wherein

[0096] X1 is Y, and / or

[0097] X2 is N;

[0098] and / or

[0099] (c) the VH CDR3 comprises the amino acid sequence of SX1SYX2YGFAY ​​(SEQ ID NO: 18), wherein:

[0100] X1 is F, and / or

[0101] X2 is S;

[0102] and / or

[0103] (d) the VL CDR2 comprises the amino acid sequence of SASYRYT (SEQ ID NO: 5).

[0104] In a more preferred embodiment, the antibody specifically binding to human L1CAM of the present invention described herein comprises:

[0105] (a) a heavy chain variable region (VH), said VH comprising VH CDR1, VH CDR2 and VH CDR3, said VH CDR1 comprising the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO: 9), said VH CDR2 comprising the amino acid sequence of YISYSGSYSYNPSLKS (SEQ ID NO: 11) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO: 11), said VH CDR3 comprising the amino acid sequence of SFSYSYGFAY ​​(SEQ ID NO: 14) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO: 14); and

[0106] (b) a light chain variable region (VL), said VL comprising VL CDR1, VL CDR2 and VL CDR3, said VL CDR1 comprising the amino acid sequence of KASQDVSSAVA (SEQ ID NO:4) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO:4), said VL CDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO:5) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO:5), and said VL CDR3 comprising the amino acid sequence of QQHYSTPWT (SEQ ID NO:6) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO:6).

[0107] In a more preferred embodiment, the antibody specifically binding to human L1CAM of the present invention described herein comprises:

[0108] (a) a heavy chain variable region (VH), the VH comprising VH CDR1, VH CDR2 and VH CDR3, the VH CDR1 comprising the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9), the VH CDR2 comprising the amino acid sequence of YISYSGSYSYNPSLKS (SEQ ID NO: 11), and the VH CDR3 comprising the amino acid sequence of SFSYSYGFAY ​​(SEQ ID NO: 14); and

[0109] (b) a light chain variable region (VL), wherein the VL comprises VL CDR1, VL CDR2 and VL CDR3, wherein the VL CDR1 comprises the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4), the VL CDR2 comprises the amino acid sequence of SASYRYT (SEQ ID NO: 5), and the VL CDR3 comprises the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6).

[0110] In addition to the specific CDRs mentioned above, the heavy chain variable region and / or light chain variable region of the antibody of the present invention may also include one or more of the specific framework regions mentioned below.

[0111] Antibodies of the present invention may include framework sequences from any species. Preferably, they include mouse and / or human framework sequences or hybrids thereof. For example, the framework sequences may each be human, wherein there are optionally non-human positions, such as 1 to 10 (including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, including any subrange thereof) non-human positions.

[0112] The term "framework (FR) amino acid residues" as used herein refers to 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 not part of the CDR (e.g., using the Kabat definition of CDR). The framework region generally supports the binding of an antibody to an antigen 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 may be bound by an antibody that is specific for the antigen.

[0113] Methods for producing monoclonal antibodies using the above-mentioned CDR sequences are known in the art and include introducing the nucleic acid sequence encoding the CDR into a suitable expression vector encoding the desired framework sequence. Further methods are described below.

[0114] Suitable framework regions for the heavy chain variable region are, for example, SEQ ID NOs: 64-72. In detail, the heavy chain variable region of the antibody of the present invention may include one to four framework regions selected from the group of SEQ ID NOs: 64-72. Preferably, the heavy chain variable region of the antibody of the present invention includes one, two, three or four framework regions selected from the group of SEQ ID NOs: 64-72, more preferably four framework regions selected from the group of SEQ ID NOs: 64-72.

[0115] Following the Kabat numbering system for CDRs, the VH CDRs are defined by Kabat, and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format of FR1, CDRH1, FR2, CDRH2, FR3, CDRH3, and FR4.

[0116] Therefore, 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:66-69, 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.

[0117] Suitable framework regions for the light chain variable region are, for example, SEQ ID NOs: 73-82. The light chain variable region of the antibody of the present invention may include one to four framework regions selected from the group of SEQ ID NOs: 73-82. Preferably, the light chain variable region of the antibody of the present invention includes one, two, three or four framework regions selected from the group of SEQ ID NOs: 73-82, more preferably four framework regions selected from the group of SEQ ID NOs: 73-82.

[0118] Similarly, following the Kabat numbering system for CDRs, the VL CDRs are defined by Kabat, and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format of FR1, CDRL1, FR2, CDRL2, FR3, CDRL3, and FR4.

[0119] Therefore, 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.

[0120] Furthermore, FR1 to FR4 of the heavy chain variable region may be independently selected from FR1 to FR4 of the light chain variable region, and FR1 to FR4 of the light chain variable region may be independently selected from FR1 to FR4 of the heavy chain variable region.

[0121] Tables 3 and 4 below provide examples of antibodies according to the present invention, respectively, the antibodies further comprising SEQ ID NO: 64-72 as framework regions FR1 to FR4 of the heavy chain variable region, and comprising SEQ ID NO: 73-82 as framework regions FR1 to FR4 of the light chain variable region. Wherein, antibody OV549.20 represents a mouse example of an antibody according to the present invention. H1, H2, H3 and H4 are examples of VH regions of humanized variants of mouse antibody OV549.20. L1 and L2 are examples of VL regions of humanized variants of mouse antibody OV549.20. AFF1 to AFF10 are examples of antibodies according to the present invention, which have been further affinity matured.

[0122] Preferably, the set of VH framework regions FR1 to FR4 is selected from the set provided for specific antibodies in Table 3 below. Preferably, the set of VL framework regions FR1 to FR4 is selected from the set provided for specific antibodies in Table 4 below. Preferably, the set of VH framework regions FR1 to FR4 is selected from the set provided for specific antibodies in Table 3 below, and the set of VL framework regions FR1 to FR4 is selected from the set provided for the same specific antibodies in Table 4 below.

[0123] Table 3. VH framework (FR) sequences of exemplary anti-L1CAM antibodies 3

[0124]

[0125]

[0126] 3 The VH framework regions described in Table 3 are determined based on the boundaries of the Kabat numbering system of the CDRs. In other words, 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 of FR1, CDRH1, FR2, CDRH2, FR3, CDRH3, and FR4.

[0127] Table 4. VL framework (FR) sequences of exemplary anti-L1CAM antibodies 4

[0128]

[0129] 4The VL framework regions described in Table 4 are determined based on the boundaries of the Kabat numbering system of the CDRs. In other words, 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 of FR1, CDRL1, FR2, CDRL2, FR3, CDRL3, and FR4.

[0130] The antibody according to the present invention may further include a heavy chain variable region sequence and / or further include a light chain variable region sequence, wherein the heavy chain variable region sequence includes one or more framework regions in the framework region of the heavy chain variable region sequence of any one of SEQ ID NOs: 23-34, and the light chain variable region sequence includes one or more framework regions in the framework region of the light chain variable region sequence of any one of SEQ ID NOs: 20-22.

[0131] In another preferred embodiment, the antibody that specifically binds to human L1CAM of the present invention further comprises a heavy chain variable region sequence and / or further comprises a light chain variable region sequence, wherein the heavy chain variable region sequence comprises the framework region of the heavy chain variable region sequence of any one of SEQ ID NOs: 23-34, and the light chain variable region sequence comprises the framework region of the light chain variable region sequence of any one of SEQ ID NOs: 20-22.

[0132] The antibody according to the present invention may also include a heavy chain variable region sequence and / or a light chain variable region sequence, wherein the heavy chain variable region sequence includes an amino acid sequence selected from the group consisting of SEQ ID NO: 23-34, and the light chain variable region sequence includes an amino acid sequence selected from the group consisting of SEQ ID NO: 20-22. In this context, the heavy chain variable region can be independently selected from a selected light chain variable region, and the light chain variable region can be independently selected from a selected heavy chain variable region.

[0133] Thus, one of the heavy chain variable regions of the antibody may be selected from the group consisting of SEQ ID NOs: 23-34, while the sequence of the second heavy chain variable region may not be selected from this group.

[0134] Likewise, one of the light chain variable regions of the antibody according to one embodiment may be selected from the group consisting of SEQ ID NOs: 20-22, while the sequence of the second light chain variable region may not be selected from this group.

[0135] In addition, 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: 23-34 and a light chain variable region sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22, these may not necessarily be part of the same arm of an antibody comprising two or more arms, and may also be assigned to different arms.

[0136] 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 "comprise" or "consist of" the corresponding indicated sequences.

[0137] The antibody according to the present invention may further include one or two heavy chain variable region sequences and / or include one or two light chain variable region sequences, wherein the heavy chain variable region sequence includes an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 23-34, and the light chain variable region sequence includes an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 20-22.

[0138] In a preferred embodiment, the antibody that specifically binds to human L1CAM of the present invention comprises a heavy chain variable region sequence and / or a light chain variable region sequence, wherein the heavy chain variable region sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-34, and the light chain variable region sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22.

[0139] In a preferred embodiment, the antibody that specifically binds to human L1CAM of the present invention comprises a heavy chain variable region sequence and / or a light chain variable region sequence, wherein the heavy chain variable region sequence has an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-34, and the light chain variable region sequence has an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22.

[0140] The following Table 5 gives exemplary combinations of sequences of heavy chain variable regions and light chain variable regions. The names of the antibodies mentioned therein are related to the names of the antibodies mentioned and explained in Tables 1 to 4 above. The sequences highlighted in bold are particularly preferred sequences.

[0141] Preferably, in the context of the antibodies herein, the VH and / or VL sequences have the sequences shown in Table 5 below.

[0142] Table 5. VH and VL sequences of exemplary anti-L1CAM antibodies 5

[0143]

[0144]

[0145]

[0146] 5 The CDRs are shown underlined in Table 5. The CDRs in Table 5 were determined according to Kabat.

[0147] In a further preferred embodiment, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF1 to AFF10. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF1. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF2. ​​Thus, preferably, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF3. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF4. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF5. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF6. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF7. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF8. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF9. Thus, preferably, the antibody comprises at least one VH region and at least one VL region of an antibody designated herein as AFF10. The sequences of the VH and VL regions are shown in Table 5 above.

[0148] AFF4 is particularly preferably used. 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.

[0149] Therefore, the antibody may include a heavy chain variable region sequence and / or a light chain variable region sequence, wherein the heavy chain variable region sequence includes the amino acid sequence of SEQ ID NO: 30, and the light chain variable region sequence includes the amino acid sequence of SEQ ID NO: 20. Therefore, the antibody may include one or more heavy chain variable region sequences and / or one or more light chain variable region sequences, wherein the heavy chain variable region sequence includes the amino acid sequence of SEQ ID NO: 30, and the light chain variable region sequence includes the amino acid sequence of SEQ ID NO: 20.

[0150] In addition, when the antibody includes a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence includes the SEQ ID NO:30 amino acid sequence and the light chain variable region sequence includes the SEQ ID NO:20 amino acid sequence, these are not necessarily part of the same arm of the antibody including two or more arms, and may also be assigned to different arms.

[0151] In yet another preferred embodiment, the antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence, the heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 30, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 20.

[0152] 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 consisting of SEQ ID NOs: 35, 84 and 37.

[0153] The antibody of the present invention may further comprise a light chain sequence comprising or consisting of the amino acid sequences of SEQ ID NOs: 36 and 38.

[0154] In addition, the antibodies of the present invention may include one or two heavy chain sequences and / or include one or two light chain sequences, wherein the heavy chain sequence includes or consists of an amino acid sequence independently selected from the group of SEQ ID NOs: 35, 84 and 37, and the light chain sequence includes or consists of an amino acid sequence independently selected from the group of SEQ ID NOs: 36 and 38.

[0155] Preferably, the antibody comprises

[0156] (a) a heavy chain sequence and / or a light chain sequence, wherein the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 35, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 36; and / or

[0157] (b) a heavy chain sequence and / or a light chain sequence, wherein the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38; and / or

[0158] (c) a heavy chain sequence and / or a light chain sequence, wherein the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0159] In yet another preferred embodiment, the antibody comprises a heavy chain sequence and / or a light chain sequence, the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 37 or 84, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38. In yet another preferred embodiment, the antibody comprises a heavy chain sequence and a light chain sequence, the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 37 or 84, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0160] In yet another preferred embodiment, the antibody comprises a heavy chain sequence and / or a light chain sequence, the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38. In yet another preferred embodiment, the antibody comprises a heavy chain sequence and a light chain sequence, the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0161] In yet another preferred embodiment, the antibody comprises a heavy chain sequence and / or a light chain sequence, the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38. In yet another preferred embodiment, the antibody comprises a heavy chain sequence and a light chain sequence, the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0162] In another preferred embodiment, the antibody consists of a heavy chain sequence and / or a light chain sequence, the heavy chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 37 or 84, and the light chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 38. In another preferred embodiment, the antibody consists of a heavy chain sequence and a light chain sequence, the heavy chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 37 or 84, and the light chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 38.

[0163] In another preferred embodiment, the antibody consists of a heavy chain sequence and / or a light chain sequence, the heavy chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 38. In another preferred embodiment, the antibody consists of a heavy chain sequence and a light chain sequence, the heavy chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 38.

[0164] In another preferred embodiment, the antibody consists of a heavy chain sequence and / or a light chain sequence, the heavy chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 38. In another preferred embodiment, the antibody consists of a heavy chain sequence and a light chain sequence, the heavy chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 38.

[0165] In Example 5, humanized optimized antibodies were prepared and used. In Example 5, a humanized optimized antibody "AFF4" as an IgG type antibody is provided, wherein the heavy chain variable region sequence consists of the amino acid sequence of SEQ ID NO: 30, and the light chain variable region sequence consists of the amino acid sequence of SEQ ID NO: 20, connected to a human IgG1 heavy chain constant region, with 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.

[0166] In each embodiment, a humanized optimized antibody designated as "AFF4-WT" was also prepared and used. Antibody "AFF4-WT" is an IgG type antibody, wherein the heavy chain variable region sequence consists of the amino acid sequence of SEQ ID NO: 30, and the light chain variable region sequence consists of the amino acid sequence of SEQ ID NO: 20, connected to the 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.

[0167] Optionally, 1, 2 or 3 amino acids may be missing from the C-terminus of the full-length heavy chain. As is well known, such deletions do not affect the stability of the antibody. In addition, heavy chains comprising a C-terminal lysine of the Fc domain may also be used. Depending on the recombinant expression system used, the C-terminal lysine of the Fc domain may be present or absent.

[0168] Exemplary combinations of sequences of heavy chain variable regions and light chain variable regions are given in the following Table 6. The names of the antibodies mentioned therein are related to the names of the antibodies mentioned and explained in Tables 1 to 5 above.

[0169] Table 6. Heavy chain (HC) and light chain (LC) sequences of exemplary anti-L1CAM antibodies 6

[0170]

[0171]

[0172] 6 The CDRs are shown in underlined form in Table 6. The CDRs in Table 6 are determined according to Kabat. The FR1, FR2 and FR3 sequences of the corresponding variable domain heavy chain sequences and light chain sequences are shown in italics.

[0173] In yet another preferred embodiment, the antibody comprises:

[0174] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30; and

[0175] (b) a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 20.

[0176] Preferably, the antibody comprises a heavy chain variable region sequence and a light chain variable region sequence, the heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 30, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 20. Preferably, the antibody comprises a heavy chain variable region sequence and a light chain variable region sequence, the heavy chain variable region sequence has the amino acid sequence of SEQ ID NO: 30, and the light chain variable region sequence has the amino acid sequence of SEQ ID NO: 20.

[0177] The antibody can be further 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 including 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 intracellular antibody, a heteroconjugate antibody, a single domain antibody, a monovalent antibody, a single chain antibody or a single chain Fv (scFv), a camelid antibody, an affibody (affinity antibody) es), anticalins, affilins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centyrins), fynomers, Kunitz domains, pronectins and OBodys, 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 wherein the antibody is included in a chimeric antigen receptor (CAR).

[0178] The antibody may be selected from a monoclonal antibody. A monoclonal antibody is an identical monospecific antibody or an antibody having the same amino acid sequence because it is produced by one type of immune cell, and all of the monoclonal antibodies are clones of a single parent cell, for example, produced by a single clone of a B lymphocyte. "Monoclonal antibodies" and the production of monoclonal antibodies are currently state-of-the-art technologies. Typically, monoclonal antibodies can be prepared, for example, according to the known method of Winter and Milstein

[17] . As an alternative to preparing hybridomas that secrete monoclonal antibodies, monoclonal antibodies against the polypeptide of interest can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., Pharmacia recombinant phage antibody system, catalog number 27-9400-01; and Stratagene SurfZAP phage display kit, catalog number 240612). Additionally, examples of methods and reagents particularly useful for generating and screening antibody display libraries can be found in, e.g., U.S. Pat. No. 5,223,409; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; WO 90 / 02809;

[19] -

[22] .

[0179] The antibody may further be selected from synthetic antibodies. The term "synthetic antibody" describes any antibody produced completely in vitro that does not involve any animal. 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 (such as mammalian cell lines, insect cell lines or bacterial cell lines) to produce in animals or hybridoma cells. Suitable methods are well known to those skilled in the art.

[0180] The antibody can be further selected from recombinantly produced antibodies. The term "recombinantly produced antibody" thus includes any antibody produced in vitro using a DNA molecule produced by genetic recombination. Recombinant antibody production can be carried out in animals or hybridoma cells using cell lines (such as mammalian cell lines, insect cell lines or bacterial cell lines). The method for the recombinant production of antibodies is well known to those skilled in the art. For example, the antibody genes of immunospecific heavy and light antibody chains can be cloned into high-yield expression vectors, which are then introduced into expression hosts (such as bacteria, yeast, insects or mammalian cells) to produce recombinant antibodies.

[0181] The antibody may further be selected from a monovalent antibody, a monospecific antibody and / or a multispecific antibody, including a bispecific antibody.

[0182] The valency of an antibody describes the number of antigen binding sites present in each molecule of the antibody. 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, i.e., two or more, binding sites for an antigen, epitope, or cell type or tissue, such as two, three, four, or five.

[0183] The specificity of an antibody generally describes its ability to recognize a single antigenic epitope and distinguish it from other epitopes. The term "monospecific antibody" describes any antibody that is specific to an antigen, epitope, cell type or tissue. For example, a monoclonal antibody is monospecific because each of their two antigen-binding arms binds only to one epitope. As used herein, the term "bispecific antibody" can be understood in the broadest sense, describing an antibody that interacts with two different epitopes or alternatively two different epitopes on the same antigen, such as an antibody comprising two functional antigen-binding domains that are specific to two different antigens. The bispecific antibody can be derived from two monoclonal antibodies. Optionally, the two different epitopes can be located on the same antigen, but they can also be located on two different antigens. Bispecific antibodies can be produced using conventional techniques, and their specific methods include chemical production, or production from hybridomas and other techniques, including but not limited to providing techniques and knobs-into-holes engineering of molecules such as scFv, wherein the molecule has an antigen-binding region with different specificities from peptide linkers such as G4S linkers. As used herein, the term "multispecific" can be understood in the broadest sense, describing 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, there can be two or more, three or more functional antigen binding domains on a multispecific antibody, and can be specific for two or more, three or more different antigens or different epitopes. Therefore, bispecific and multispecific antibodies target two or more antigens or epitopes, respectively.

[0184] The antibody may further be a human antibody, a humanized antibody and / or a chimeric antibody. A chimeric antibody is an antibody in which at least one region of an immunoglobulin from one species is fused to another region of an immunoglobulin from another species through genetic engineering to reduce the immunogenicity of the antibody. For example, a mouse V L and V H The chimeric region can be fused to the rest of the human immunoglobulin. A special type of chimeric antibody is a humanized antibody. A humanized antibody is produced by merging the DNA encoding the CDR of a non-human antibody with the DNA producing a human antibody. The resulting DNA construct can then be used to express and produce antibodies, which are generally less immunogenic than the non-human parent antibody or chimeric antibody because only the CDR is non-human. In addition, the antibody can be a human antibody, i.e., the nucleic acid sequence of the antibody is completely of human origin.

[0185] The use of human, humanized or chimeric antibodies is preferably for in vivo applications, especially for human applications, such as for in vivo prophylaxis, therapy or diagnosis.

[0186] The antibody may further be selected from immunoglobulins. The term "immunoglobulin" describes any protein from the class of immunoglobulins produced by the immune system for neutralizing substances foreign to the body. An immunoglobulin comprises at least one immunoglobulin (Ig) domain.

[0187] The antibody may be further 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.

[0188] 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 include the complete heavy chain sequence and / or light chain sequence of a full-length antibody or only a portion thereof. A tetrameric antibody may particularly refer to a protein comprising two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, the protein comprising: (1) in the case of a heavy chain, a variable region and a domain comprising three C H 1. C H 2 and C H 3 heavy chain constant region; and (2) in the case of a light chain, a light chain variable region and a domain comprising a C L The light chain constant region of the antibody. With respect to the term "tetrameric antibody", any antibody is intended to have a typical overall domain structure of a naturally occurring antibody (i.e., a heavy chain including three or four constant domains and a light chain including a constant domain and corresponding variable domains). As described above, each domain may include additional modifications, such as mutations, including substitutions, deletions and / or insertions, which do not change the overall domain structure. For example, mAb OV549.20 is a tetrameric antibody.

[0189] The term "antibody light chain monomer" describes any antibody that includes only a light chain molecule but no heavy chain molecule. Thus, the term "antibody heavy chain monomer" describes any antibody that includes only one heavy chain molecule but no light chain molecule. Thus, the term "antibody light chain dimer" describes a complex of two light chain monomers, and "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 includes a light chain monomer and a heavy chain monomer pair.

[0190] The antibody may further be selected from "single domain antibodies". The term "single domain antibody" describes a single monomeric variable antibody domain, such as the variable domain of the light chain (V L ) or the variable domain of the heavy chain (V H ) composed of antibody fragments.

[0191] The antibody may further be selected from “single chain antibodies.” The term “single chain antibody” describes an antibody fragment of a single polypeptide chain.

[0192] The antibody may further be selected from intracellular antibodies and / or heteroconjugate antibodies. The term "intracellular antibody" describes an antibody that targets an intracellular protein in any target cell. Methods for transferring intracellular antibodies to target cells to allow the binding of intracellular target proteins are well known to those skilled in the art, such as the direct expression of intracellular antibodies by target cells used in gene therapy. "Heteroconjugate antibodies" are complexes of two or more covalently linked antibodies (e.g., monoclonal antibodies, Fabs, or scFvs) with different specificities.

[0193] The antibody may be further selected from camelid antibodies, affinity antibodies, anticalins, affilins, atrimers, DARPins, FN3 scaffolds (such as adnectin and centyrin), fynomers, Kunitz domains, pronectins and OBody and / or anti-idiotype (anti-Id) antibodies. The term "camelid antibody" describes any antibody having the structure of an antibody derived from a camelid mammal (such as llamas, camels and alpacas), such as an antibody lacking any light chain and consisting of two identical heavy chains. The term "affinity antibody" describes any antibody-mimicking protein that can bind to a large number of antigens with high affinity. For example, an affinity antibody can be based on the immunoglobulin binding domain of a protein, such as the Z domain of protein A from Staphylococcus aureus. Additional examples are well known to those skilled in the art. Anticalins, affilins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centyrins), fynomers, Kunitz domains, pronectins and OBodys are additional scaffolds known in the art and can be used according to the present invention. Such scaffolds are described, for example, in

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

[0194] The "antigen-binding fragment" of an antibody is a fragment of an antibody that preferably exhibits substantially the same function and specificity as the intact antibody from which the fragment is derived. Limited proteolytic digestion with papain usually cuts the Ig prototype into three fragments. Two identical amino-terminal fragments, each containing a complete L chain and about half of the H chain, are antigen-binding fragments (Fab). The third fragment is a crystallizable fragment (Fc), which is similar in size but contains the carboxyl-terminal half of the two heavy chains and their interchain disulfide bonds. Fc contains carbohydrates, complement binding, and FcR binding sites. Limited pepsin digestion produces a single F(ab')2 fragment containing both the Fab segment and the hinge region (including the HH interchain disulfide bond). F(ab')2 is divalent for antigen binding. The disulfide bonds of F(ab')2 can be cleaved to obtain Fab'. In addition, the variable regions of the heavy and light chains can be fused together to form a single-chain variable fragment (scFv).

[0195] Because first-generation full-size antibodies may have some problems, many second-generation antibodies may only include antibody fragments. L and a V H The smallest fragment of a complete antigen binding domain composed of. Such fragments with only binding domains can be produced by enzymatic methods or expression of related gene fragments, such as in bacteria and eukaryotic cells. Different methods can be used, such as a single Fv fragment or a Fab fragment comprising one of the upper arms of the "Y" comprising Fv plus the first constant domain. When only variable fragments are used, these fragments are usually stabilized by introducing a polypeptide connection between the two chains, thereby producing a single-chain Fv (scFv). Alternatively, a disulfide-linked Fv (dsFv) fragment can be used. The binding domain of the fragment can be combined with any constant domain to produce a full-length antibody, or can be fused with other proteins and polypeptides.

[0196] Preferred recombinant antibody fragments are single-chain Fv (scFv) fragments. Typically, scFv fragments have high affinity for their antigens and can be expressed in a variety of hosts. These and other properties make scFv fragments not only suitable for medical applications, but also have the potential for biotechnology applications. As described in detail above, in scFv fragments, V H and V L The domains are connected with a hydrophilic and flexible peptide linker, which improves expression and folding efficiency. A linker of about 15 amino acids is usually used, with the (Gly4Ser)3 linker being the most commonly used. ScFv molecules may be easily degraded by proteolysis, depending on the linker used. With the development of genetic engineering technology, these limitations can be practically overcome by focusing on research to improve function and stability. An example is the generation of disulfide-stabilized (or disulfide-linked) Fv fragments in which V H-V L The dimer is stabilized by interchain disulfide bonds. Cysteine ​​is introduced between V L Domain and V H The domains interact with each other at the interface, thereby forming a disulfide bond that links the two domains together.

[0197] scFv can be complexed into dimers (diabodies), trimers (tribodies) or larger aggregates such as TandAb and Flexibody.

[0198] Antibodies with two binding domains can be produced, for example, by the combination of two scFvs with a simple polypeptide chain (scFv)2 or by dimerization of two monomers (diabodies).The simplest design is a diabody with two functional antigen binding domains.

[0199] Likewise, antibody formats comprising four heavy chain variable domains and four light chain variable domains have been developed. Examples of these antibody formats include TandAb and flexibody (Affimed Therapeutics AG, Heidelberg. Germany). Due to its four binding domains, TandAb generally shows better binding properties than antibody formats (e.g., diabodies) comprising only two binding domains. Flexibody is a combination of scFv and diabody multimer motifs, producing highly flexible multivalent molecules for connecting two molecules that are far apart on the cell surface.

[0200] The antibody may also be selected from an antigen binding fragment of any of the molecules mentioned above. As specified above, an "antigen binding fragment" of an antibody is a fragment of an antibody that exhibits substantially the same antigen binding activity and specificity as the intact antibody from which the fragment is derived. An antigen binding fragment is generally understood to be a polypeptide that includes at least one antigen binding fragment of a full-length antibody. Typically, an antigen binding fragment consists of at least a heavy chain variable domain and a light chain variable domain, arranged in a manner that the two domains together are able to bind to a specific antigen.

[0201] 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 the incorporation of disulfide bonds to bring the VH and VL domains into line.

[0202] The antibody may be further included in a complex with other immunoglobulin molecules or fragments thereof (such as another antibody) or non-immunoglobulin molecules. For example, the antibody may form a homopolymer with other identical antibodies. Preferably, the antibody is included in a chimeric antigen receptor (CAR). The term "CAR" describes any receptor protein usually on T cells, which is specifically designed to allow T cells to target specific antigens. The method for preparing CAR is well known to those skilled in the art.

[0203] In yet another preferred embodiment, the antibody is selected from the group consisting of 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 including 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 intracellular antibody, a heteroconjugate antibody, a single domain antibody, a monovalent antibody, a single chain antibody or a single chain Fv (scFv), a camelid antibody, an affinity antibody, The invention relates to an antibody comprising a chimeric antigen receptor (CAR), a fynomer, a Kunitz domain, a probetin, and an OBody, and / or wherein the antibody is included in a chimeric antigen receptor (CAR).

[0204] In another yet 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 intracellular antibody, a heteroconjugate antibody, a single domain antibody, a monovalent antibody, a single chain antibody or a 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 wherein the antibody is included in a chimeric antigen receptor (CAR).

[0205] In addition, the antibody can include a heavy chain constant region and / or a light chain constant region. The heavy chain constant region can be selected from the human immunoglobulin group selected from IgA, IgD, IgE, IgG or IgM, including any subclass of these isotypes. Preferably, the heavy chain constant region is selected from the human immunoglobulin group consisting of IgG and IgA, and more preferably the heavy chain constant region is selected from the human immunoglobulin group consisting of IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Because the antibody can be recombinantly expressed and produced, the antibody can also include a heavy chain, such as an IgG1 and an IgG2 heavy chain, or two different constant regions of heavy chains from different species. However, the heavy chain is preferably from the same species. In addition, the antibody can include IgGκ or IgGλ light chain constant region. Preferably, the light chain constant region is selected from the human immunoglobulin group consisting of IgGκ and IgGλ.

[0206] In a yet 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 human immunoglobulin group consisting of IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, and / or wherein the light chain constant region is selected from the human immunoglobulin group consisting of IgGκ and IgGλ.

[0207] In a yet further preferred embodiment, 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.

[0208] In a preferred embodiment, the antibody comprises a heavy chain constant region, wherein the heavy chain constant region is a human IgG1 constant region.

[0209] The heavy chain constant region may further be a variant of a wild-type human IgG heavy chain constant region, preferably wherein the human IgG heavy chain constant region variant is bound to one or more human Fcγ receptors. Typically, 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 by the antibody types they interact with. Therefore, the term "Fcγ receptor" describes the Fc receptors that bind antibodies with IgG constant regions. The Fcγ receptor class further includes several subclasses, such as FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA or FcγRIIIB, and their structures and affinities to IgG and different IgG subclasses are generally different. Preferably, the human IgG heavy chain constant region variants of the antibodies of the present invention are bound to one or more human Fcγ receptors selected from the group consisting of FcγRI, FcγRIIA and FcγRIIIA. Also preferably, wherein the human IgG heavy chain constant region variant binds to one or more human Fcγ receptors with higher affinity than the wild-type human IgG heavy chain constant region binds to human Fcγ receptors, the one or more human Fcγ receptors being selected from the group consisting of FcγRI, FcγRIIA, FcγRIIIA. "Binding with higher affinity" means that the binding of the antibody of the present invention to one or more human Fcγ receptors is at least 2 times, preferably at least 3 times stronger than the binding of the wild-type human IgG heavy chain constant region to human Fcγ receptors, the one or more human Fcγ receptors being selected from the group consisting of FcγRI, FcγRIIA, FcγRIIIA, as determined, for example, by methods known to those skilled in the art (such as Western blot analysis, ELISA or surface plasmon resonance).

[0210] In a yet further preferred embodiment, the heavy chain constant region is a variant of a wild-type human IgG heavy chain constant region, preferably wherein the human IgG heavy chain constant region variant binds to one or more human Fcγ receptors with higher affinity than the wild-type human IgG heavy chain constant region binds to human Fcγ receptors, and the one or more human Fcγ receptors are selected from the group consisting of FcγRI, FcγRIIA, and FcγRIIIA.

[0211] On the other hand, 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 for binding to human L1CAM, preferably wherein the epitope is within the fibronectin type III domain 1-3 (FN III 1-3) of human L1CAM. All features of the antibodies of the present invention specifically described above are also considered to be related to the antibodies of the other aspects of the present invention.

[0212] The antibody may specifically bind to the same epitope of human L1CAM as the antibody of the present invention.

[0213] The term "epitope" describes the portion of an antigen that is recognized by an antibody or related binding molecule. For example, an epitope is a specific fragment of an antigen to which an antibody binds. An epitope can be a conformational epitope or a linear epitope. Conformational epitopes are usually composed of discontinuous portions of the amino acid sequence of an antigen. These epitopes interact with the antibody paratope (binding site on the antibody) based on the 3-D surface features and shape or tertiary structure of the antigen. The proportion of conformational epitopes is unknown. A linear epitope is an epitope that is usually recognized by an antibody through its amino acid sequence or primary structure.

[0214] Studies have shown that L1-OV549.20 binds to and recognizes an epitope within the fibronectin type III domain 1-3 (FN III 1-3) of L1CAM [6]. Methods for determining the epitope bound and recognized by a binding molecule are described in the prior art. The recognized epitope can be determined by constructing a series of L1CAM-Fc proteins carrying different Ig domains. For fine mapping, recombinant V5-tagged L1CAM fragments can be used, for example, as described in

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

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

[25] .

[0215] Said antibodies may alternatively or additionally compete with the antibodies of the invention for binding to human L1CAM, preferably wherein the binding epitope is within the fibronectin type III domain 1-3 (FN III 1-3) in human L1CAM.

[0216] Competition for antibody binding can generally be determined by assays known to those skilled in the art, such as competitive binding assays. Competitive binding assays are generally based on antibody-antigen interactions, where the number of antigen binding sites on the antibody is limited compared to the amount of different competing antibodies. For example, a competitive binding assay can take the form of an immunoassay.

[0217] Human L1CAM protein generally includes an N-terminal extracellular portion of six immunoglobulin domains (Ig I to Ig VI), followed by five fibronectin type III domains (FN III 1-5), a transmembrane helix, and a small C-terminal intracellular domain. Preferably, the epitope is within the fibronectin type III domains 1-3 (FN III 1-3) of human L1CAM.

[0218] Preferably, the antibody of any aspect of the present invention

[0219] (i) Binds to human L1CAM within the fibronectin type III domain 1-3 (FN III 1-3) of L1CAM.

[0220] Preferably, the antibody of any aspect of the present invention

[0221] (ii) binds to human L1CAM with an affinity (KD) of 20 nM or less, 10 nM or less, or 1 nM or less.

[0222] Preferably, the antibody of any aspect of the present invention

[0223] (iii) binds to cynomolgus monkey L1CAM with an affinity (KD) of 20 nM or less, 10 nM or less, or 1 nM or less.

[0224] Methods for determining the binding affinity of an antibody are well known to those skilled in the art and are also described exemplarily in the context of specific binding of an antibody. The surface plasmon resonance of the device is performed. For example, the affinity (KD) is determined at room temperature. For example, the binding affinity can be determined as described in Example 2 below.

[0225] Preferably, the antibody of any aspect of the present invention

[0226] (iv) Inhibition of tumor cell migration on fibronectin-coated surfaces in vitro.

[0227] The method for determining the migration of tumor cells on the surface of fibronectin coating in vitro is well known to those skilled in the art. For example, fluorescently labeled tumor cells can be seeded on the edge of the hole coated with fibronectin. After 48 hours, the migration of tumor cells towards the center of the hole can be determined. Preferably, when the antibody of any aspect of the various aspects of the present invention is applied to tumor cells, the migration of these tumor cells on the surface of fibronectin coating in vitro is suppressed.

[0228] In this context, inhibition of migration means that when an antibody of any of the aspects is applied to tumor cells, the migration of these 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.

[0229] In addition, the half maximum inhibitory concentration (IC50) of the antibody of the present invention for inhibiting cancer cell migration of HCT116 tumor cells is 50 ) can be 1x 10 -4 M to 1x 10 -8 M, and preferably 1x 10 -4 M to 1x 10 -7 M. Optionally, the half maximum inhibitory concentration (IC 50 ) can also be 2x 10 -5 M to 1x10 -6 M, preferably 2 x 10 -5 M to 4x 10 -6 M. Preferably, the tumor cell is an HCT116 tumor cell.

[0230] Preferably, the antibody of any aspect of the present invention

[0231] (v) inhibiting the proliferation of SKOV-3, Panc-1 and / or HCT-116 tumor cells in vitro.

[0232] Methods for determining the proliferation of SKOV-3, Panc-1 and / or HCT-116 tumor cells in vitro are well known to those skilled in the art. For example, such proliferation assays can be used 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 the confluency of cells over time by microscopic means, such as determining (e.g., by counting) the number of cells over time, for example after 24 hours, 48 ​​hours, 72 hours or 96 hours.

[0233] In this context, inhibition of proliferation in vitro means that when an antibody of any of the aspects of the invention is applied to tumor cells, the proliferation of these tumor cells after 24 hours, 48 ​​hours, 72 hours or 96 hours 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 proliferation of tumor cells of the same cell type treated with an isotype control antibody.

[0234] Preferably, the antibody of any aspect of the present invention

[0235] (vi) Inhibition of primary tumor growth in the mouse SKOV-3ip xenograft model.

[0236] Methods for determining primary tumor growth in the mouse SKOV-3ip xenograft model are well known to those skilled in the art. For example, in the SKOV-3ip xenograft model, 5 x 10 6 SKOV3 tumor cells are injected intraperitoneally (ip) into NMRI-nu / nu mice. After 5 days, mice can be randomly assigned to different groups and treatment can be started. Anti-L1CAM antibody or control antibody can be injected intraperitoneally three times a week at a dose of 10 mg / kg over a period of 6 weeks. After killing the mice, the treatment efficacy can be evaluated by comparing the tumor weight and ascites volume between the treated and control groups.

[0237] In this context, inhibition of primary tumor growth in a mouse model is understood to mean that when an antibody of any of the aspects of the invention is applied to a tumor mouse model, tumor growth at a time point that meets the endpoint requirement 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.

[0238] Preferably, the antibody of any aspect of the present invention

[0239] (vii) Reduced metastasis formation in the mouse MDA-MB-231 xenograft model.

[0240] MDA-MB-231 is a highly invasive model cell line derived from the metastatic site of breast cancer. The method for determining the metastasis formation in the MDA-MB-231 xenograft model is well known to those skilled in the art. For example, breast cancer cells expressing MDA-MB-231-luc2 luciferase can be injected into the tail vein of mice (e.g., about 5x 10 per mouse). 5 Cells). Mice can be further treated with antibodies as described herein (e.g., OV549.20) or vehicle (control), for example, starting 3 days before MDA-MB-231-luc2 injection, three times a week, at a dose of 10 mg / kg each time. In vivo imaging of luciferase activity can be performed every 7 days and compared between control and antibody-treated cells to monitor the formation of metastases in the lungs and other internal organs.

[0241] 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 aspects of the invention is applied to mice injected with MDA-MB-231-luc2, metastasis formation after 20 to 30 days of 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 control vehicle.

[0242] Preferably, the antibody of any aspect of the present invention

[0243] (viii) exhibit ADCC activity in vitro and / or bind to FcγRIIIa receptor in vitro.

[0244] The term "antibody-dependent cellular cytotoxicity (ADCC)" describes a mechanism by which target cells that have been bound by a specific antibody and thus labeled are lysed by effector cells of the immune system, such as natural killer (NK) cells. Preferably, the antibodies of the present invention exhibit ADCC activity in vitro, i.e., when bound to target cells in an in vitro assay, the antibodies are capable of mediating an ADCC immune response. Such in vitro assays are well known to those skilled in the art. For example, suitable assays employ effector cells, such as NK cells, which are capable of inducing lysis of target cells bound by antibodies applied at different effector to target (E:T) cell ratios. In such cases, for example, a real-time cell analysis device can be used for final analysis.

[0245] Alternatively or in combination, the antibodies of any of the aspects of the invention preferably bind to the FcγRIIIa receptor in vitro. Suitable in vitro antibody receptor binding assays that can be used to determine the binding between the antibodies of the invention and the FcγRIIIa receptor in vitro are well known to those skilled in the art, and are described, for example, in the specific binding section above. For example, Western blot analysis, ELISA or surface plasmon resonance can be used.

[0246] Preferably, the antibody of any aspect of the present invention

[0247] (ix) Exhibits binding to FcRn in vitro.

[0248] Neonatal Fc receptor (FcRn) is an Fc receptor protein that is capable of binding to, for example, IgG and is typically expressed by, for example, endothelial cells, where it supports the recycling of serum IgG and albumin. Suitable in vitro antibody receptor binding assays that can be used to determine in vitro binding between an antibody of the invention and FcRn are well known to those skilled in the art, or are described, for example, in the specific binding section above.

[0249] Preferably, the antibody of any aspect of the present invention

[0250] (x) Does not cross-react with human CHL1, human NrCAM and / or human neurofascin in vitro.

[0251] L1CAM belongs to the L1 protein family which includes a total of four different L1-like proteins, all of which are members of the cell adhesion molecule (CAM) and immunoglobulin superfamily. In addition to L1CAM, the other three members of the L1 family are L1CAM close homolog (CHL1), neuronal cell adhesion molecule (NrCAM) and neurofascin.

[0252] An antibody of any aspect of the present invention 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, but does not substantially bind to any of human CHL1, human NrCAM and / or human neurofascin in vitro. In one embodiment, an antibody of any aspect of the present invention that does not cross-react with human CHL1, human NrCAM and / or human neurofascin in vitro only shows very low or non-specific binding to human CHL1, human NrCAM and / or human neurofascin in vitro, with a binding affinity of K D The value is 1x 10 -7 M or higher, or 1x 10 -6 M or higher (until no more binding affinity is detected). In one embodiment, the binding affinity is determined using a standard binding assay, such as an ELISA assay in the examples, or surface plasmon resonance technology. For example, the binding affinity is determined at room temperature. Other suitable methods for determining that an antibody of any aspect of the present invention binds 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 changes in fluorescent signals in cytometer-based assays can be used.

[0253] In a yet further preferred embodiment, the antibody of any of the aspects of the invention described herein:

[0254] (i) binds to human L1CAM within the fibronectin type III domains 1-3 (FN III 1-3) of L1CAM, and / or

[0255] (ii) with an affinity (K of 20 nM or less, 10 nM or less, or 1 nM or less D ) binds to human L1CAM, and / or

[0256] (iii) with an affinity (K of 20 nM or less, 10 nM or less, or 1 nM or less D ) binds to cynomolgus monkey L1CAM, and / or

[0257] (iv) inhibiting the migration of tumor cells on fibronectin-coated surfaces in vitro, and / or

[0258] (v) inhibiting the proliferation of SKOV-3, Panc-1 and / or HCT-116 tumor cells in vitro; and / or

[0259] (vi) inhibiting primary tumor growth in a mouse SKOV-3ip xenograft model, and / or

[0260] (vii) reducing metastasis formation in the mouse MDA-MB-231 xenograft model, and / or

[0261] (viii) exhibit ADCC activity in vitro and / or bind to the FcγRIIIa receptor in vitro, and / or

[0262] (ix) exhibits binding to FcRn in vitro, and / or

[0263] (x) Does not cross-react with human CHL1, human NrCAM and / or human neurofascin in vitro.

[0264] In addition, the antibody according to any of the aspects of the present invention can be humanized. Preferably, the antibody according to the present invention is partially or completely humanized. The method for making the humanization of the antibody is well known to those skilled in the art, for example, by introducing the CDR sequence of the antibody of the present invention into the human antibody sequence. Optionally, in addition, one or more non-human positions, such as 1,2,3,4,5 or 6, such as up to 10 non-human positions can be reintroduced into the human framework sequence.

[0265] Preferably, the antibody according to any of the aspects of the present invention is humanized.

[0266] In another preferred embodiment of any of the various aspects of the present invention, the antibody is a multispecific antibody or a bispecific antibody and / or a humanized antibody.

[0267] Methods for producing antibodies (such as antibodies of the present invention) are well known to those skilled in the art. For example, antibodies can be produced by preparing hybridoma cells. Methods for producing hybridoma cells and methods for producing antibodies with the help of hybridoma cells are well known to those skilled in the art. Typically, mice are injected with the desired antigen and killed after a few days or weeks to separate splenocytes, which secrete antibodies against the desired antigen. Typically, these antibody-secreting splenocytes are fused with immortal non-secreting myeloma cells to produce hybridoma cells. Then, these hybridoma cells are usually screened and hybridomas that produce the desired antibodies are selected. Then, the selected hybridomas can be cultured in vivo or in vitro and the desired antibodies can be separated. Preferably, the antibodies herein are recombinantly produced in suitable host cells. The DNA encoding the antibody of interest can be placed in an expression vector, which is then transfected into cells such as Escherichia coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., from CHO GS System TM Antibody synthesis can be obtained by expressing the antibody in host cells such as CHO cells (Lonza) or myeloma cells that do not produce immunoglobulins.

[0268] Antibody Conjugates / Antibody-Drug Conjugates (ADC)

[0269] The antibody of any aspect of the present invention can be further connected to one or more chemical moieties to form an antibody conjugate. For example, the antibody can be connected to one or more chemical moieties by any chemical bond known to those skilled in the art, such as an ionic bond and / or a covalent bond and / or by any suitable intermolecular bond, such as a hydrogen bond and / or van der Waals force.

[0270] In addition, the chemical moiety to which the antibody of any aspect of the present invention can be connected can be any chemical moiety or substance suitable for application in antibody conjugates known to those skilled in the art. For example, the antibody can be connected to a therapeutically active substance, preferably a chemotherapeutic compound, a cytotoxic compound, a cytostatic compound, a cytokine, a nanoparticle, a radioactive isotope and / or an oncolytic virus.

[0271] The term "therapeutically active substance" describes any biologically active substance, ie a substance which produces an effect in living matter. When used in medicine, the therapeutically active substance is, for example, responsible for the activity of the medicine. Methods for determining the effect of substances on living matter are well known to the person skilled in the art.

[0272] The term "chemotherapeutic compound" describes any substance that can be used in the treatment of cancer as part of a standard chemotherapy regimen known to those skilled in the art.

[0273] The term "cytotoxic compound" describes any substance that is toxic to cells, for example by causing 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, small molecule toxins or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxins and exotoxins, Staphylococcus enterotoxin A), fungi (e.g., α-sarcin, restrictocin), or plants (e.g., abrin, ricin, modeccin, viscumin, pokeweed antiviral protein, saporin, gelonin, momordicin, trichosanthin, barley toxin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, saponaria officinalis officinalis inhibitors, gelonin, mitogellin, restrictocins, phenomycins, neomycins, and tricothecenes), mitotic inhibitors, or DNA damaging agents.

[0274] The term "cytostatic compound" describes any substance that is 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.

[0275] Suitable chemotherapeutic compounds include alkylating agents, alkyl sulfonates, aziridines, ethyleneimines and methylamelamines, acetogenins, camptothecins, bryostatins, ostatins, callystatins, CC-1065, cryptophycins, dolastatins, duocarmycins, eleutherobin, pancratistatins, sarcodictylns, spongistatins, nitrogen mustards, mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein diyne antibiotic chromophores, aciacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carahtein, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozoein, tubercidin, ubenimex, zinostatin, zorubicin;Antimetabolites, erlotinib, vemurafenib, erizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, antiadreners, folic acid supplements such as folinic acid, acegiatone, aldophosphamide glycoside, aminolevulinic acid, emluracil, anisacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate), epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine compounds, mitoguazone, mitoxantrone, mopidannioi, nitracrine, pentostatin, phenamet, pirarobicin, iosoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, Polysaccharide complexes, razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and anguidine; urethan; vindesine; dacarbazine; mannoniustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide ("etoposide"). side); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan, topoisomerase inhibitor RFS2000; difluoromethylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0276] 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.

[0277] The term "nanoparticle" describes any particle having a diameter between 1 nm and 100 nm. Methods for determining the diameter of a particle are also well known to those skilled in the art.

[0278] The term "radioisotope" describes any unstable or metastable isotope of a natural or artificial element. Suitable radioisotopes are preferably also suitable for use in the treatment of 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.

[0279] The term "oncolytic virus" describes any virus that preferably targets cancer cells, e.g., causing infection and killing of these cancer cells, typically by tumor cell lysis. Suitable oncolytic viruses are, e.g., genetically adapted herpes simplex viruses, adenoviruses, vaccinia viruses, vesicular stomatitis viruses, polioviruses, reoviruses, Senecaviruses, echoviruses, Semliki Forest virus, marabaviruses, and / or Coxsackieviruses.

[0280] Alternatively or in combination therewith, 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.

[0281] The term "diagnostic compound" describes any substance suitable for use in a diagnostic method for the human or animal body. For example, a diagnostic compound can be used to stain tissues, cells, or other materials in various analytical methods such as fluorescence imaging or spectroscopy. For example, a diagnostic compound can be used to specifically stain cancer cells. Additional diagnostic applications are well known to those skilled in the art.

[0282] The term "chemiluminescent compound" describes any substance that is able to emit light via a chemical reaction. Methods and devices for detecting chemiluminescence are well known to those skilled in the art.

[0283] The term "fluorescent compound" describes any substance that re-emits light in response to light excitation. Such substances are also commonly referred to as fluorophores. Suitable fluorescent compounds may, for example, be substrates for enzymes or probes. Methods and apparatus for detecting fluorescence are well known to those skilled in the art. Fluorescent compounds include, for example, reactive and coupled probes, for example, aminocoumarins, fluorescein and Texas Red, Alexa fluorescent dyes, Cy dyes and DyLight dyes.

[0284] The term "dye" describes any substance that is colored by not absorbing all wavelengths of visible light and that is able to chemically bind to a target molecule. Thus, dyes can be used to label and visualize normally colorless target molecules.

[0285] The term "enzyme" describes any protein capable of catalyzing a chemical reaction. Suitable enzymes are known in the art and include horseradish peroxidase (HRP).

[0286] In another preferred embodiment of any of the aspects of the present invention, the antibody:

[0287] (a) linked to a therapeutically active substance,

[0288] Preferably connected to

[0289] chemotherapeutic compounds,

[0290] Cytotoxic compounds,

[0291] Cytostatic compounds,

[0292] Cytokines,

[0293] Nanoparticles,

[0294] Radioisotopes, and / or

[0295] Oncolytic viruses,

[0296] and / or

[0297] (b) linked to a diagnostic compound, preferably selected from a radioisotope, a chemiluminescent compound, a fluorescent compound, a dye or an enzyme.

[0298] It is preferred that the therapeutically active substance of (a) and / or the diagnostic compound of (b) is selected from radioisotopes, chemotherapeutic compounds, cytotoxic compounds and / or cytostatic compounds.

[0299] Furthermore, the antibody of the present invention may be covalently linked to (a) a therapeutically active substance or a chelator thereof or (b) a diagnostic compound or a chelate thereof.

[0300] The term "covalent linkage" describes any chemical bond involving the sharing of electron pairs between atoms. Examples of such covalent bonds are cleavable bonds (such as disulfide bonds, hydrazone bonds or peptide bonds) or non-cleavable chains (such as thioether bonds). Preferably, the covalent bond is stable when applied to the body and circulates therein, and becomes cleavable only when it is inside the target cell or reaches the target tissue. The linker can be a cleavable linker or can be a non-cleavable linker.

[0301] The term "chelator" describes any substance that includes two or more free electron pairs and is therefore capable of forming two or more coordination bonds with metal ions. Chelators can be organic compounds (such as 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 aspect of the various aspects can be connected to a chelator, which is further connected to radioactive isotopes such as positron emission and gamma-emitting radioactive metals, so that sensitive and quantitative molecular positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging can be performed on the antibody distribution in vivo. In another example, an antibody according to any aspect of the various aspects can be connected to a chelator, which is further connected to a radioactive isotope, so as to allow targeted radioisotope delivery directly at a location of interest in the body (e.g., at a tumor location).

[0302] In addition, the antibody of the present invention can be covalently linked to the therapeutically active substance or its chelator of (a) or the diagnostic compound or its chelate of (b) through a linker. The antibody of the present invention connected to the therapeutically active substance or its chelator through a linker is also referred to as "antibody-drug conjugate", "antibody conjugate" or "ADC" herein.

[0303] In the context of "antibody-drug conjugate", "antibody conjugate" or "ADC" herein, the antibody of the present invention is also referred to as an antibody moiety, such as an "antibody portion".

[0304] The term "linker" describes any molecule suitable for linking an antibody of the invention to (a) a therapeutically active substance or a chelator thereof or (b) a diagnostic compound or a chelator thereof, wherein the linker is preferably linked 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.

[0305] Preferably, the linker provides a stable connection between the antibody of the invention and the therapeutically active substance or its chelator of (a) or the diagnostic compound or its chelator of (b) when applied to the body and circulates therein and only in the target cells or reaches the target tissue. For example, the linker can be a peptide with 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).

[0306] It is preferred that the antibody is covalently linked to (a) the therapeutically active substance or its chelator or (b) the diagnostic compound or its chelator, optionally via a linker.

[0307] A linker may include one coupling component or may include multiple components.

[0308] For example, the linker may include a spacer, which is a moiety that extends the drug linkage to avoid, for example, shielding the active site of an antibody or to improve the solubility of the ADC. Other examples of components of the linker include a stretcher unit and an amino acid unit.

[0309] Two approaches are commonly used to conjugate drugs to antibodies: alkylation of reduced interchain cysteine ​​disulfides via enzymatically non-cleavable maleimides or simple and cleavable disulfide linkers, and acylation of lysines via cleavable linear amino acids.

[0310] On the one hand, the linker covalently links the antibody to the therapeutically active substance. The same applies to diagnostic compounds. ADCs are prepared using linkers with reactive functions for binding to the antibody and the therapeutically active substance or diagnostic compound. For example, a cysteine ​​thiol or amine of an antibody, for example, the N-terminus or an amino acid side chain, such as lysine, can form a bond with a functional group of the linker.

[0311] In one embodiment, the linker has the function of being able to react with the free cysteine ​​present on the antibody to form a covalent bond. Non-limiting exemplary such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates.

[0312] In some embodiments, the connexon has a function that can react with the electrophilic group present on the antibody. Exemplary such electrophilic groups include but are not limited to aldehydes and ketone carbonyls. In some embodiments, the heteroatom of the reactive functional group of the connexon can react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Non-limiting exemplary such reactive functional groups include but are not limited to hydrazides, oximes, amino groups, hydrazine, thiosemicarbazide, carboxylic acid hydrazides and aromatic hydrazides.

[0313] Suitable linkers include, for example, cleavable and non-cleavable linkers. The linker can be a "cleavable linker" that promotes the release of the drug. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., including hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, glycosidase-sensitive (e.g., glucuronidase-sensitive) linkers, photolabile linkers, or linkers containing disulfides. Cleavable linkers are generally easy to cut under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cut by intracellular proteases (such as lysosomal proteases or endosomal proteases). In an exemplary embodiment, the linker can be a dipeptide linker, such as valine-citrulline (val-cit or "VC"), phenylalanine-lysine (phe-lys) or valine-alanine (val-ala or "VA") linkers.

[0314] For example, a PEG8-VA linker can be used, as shown in the examples for the conjugation of ADC with tesirine.

[0315] For example, the linker can contain a maleimide group for attachment to an antibody, a PEG8 linker, and a cleavable val-ala moiety to which a therapeutically active substance is attached, for example, a pyrrolobenzodiazepine (PBD) such as SG3199, a maytansinoid such as DM4, or an auristatin such as MMAE.

[0316] For example, the linker may contain a cleavable β-glucuronide moiety, such as MC-β-glucuronide in the examples, bound to a therapeutically active substance, such as a PDB such as SG3199, a maytansine such as DM4, or an auristatin such as MMAE.

[0317] For example, the linker can contain a cleavable val-cit ("VC") moiety bound to a therapeutically active substance, such as MC-VC-PABC in the examples. For example, the therapeutically active substance is a PDB such as SG3199, a maytansine such as DM4, or an auristatin such as MMAE.

[0318] For example, the linker may contain a cleavable sulfo-SPDB moiety, such as sulfo-SPDB in the examples, conjugated to a therapeutically active substance, such as a PDB such as SG3199, a maytansine such as DM4, or an auristatin such as MMAE.

[0319] The linker is preferably stable outside the cell in a sufficient manner to have a therapeutic effect. Prior to transport or delivery into the cell, the ADC is preferably stable and remains intact, i.e., the antibody remains coupled to the drug moiety. A linker that is stable outside the target cell may be cleaved at an effective rate once inside the cell. Thus, an effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow delivery of the therapeutically active substance, such as intracellular delivery; and (iii) maintain the therapeutic effect of the therapeutically active substance, such as a cytotoxic effect.

[0320] In one embodiment, the linker is cleavable under intracellular conditions, so that the cleavage of the linker is sufficient to release the drug from the antibody in the intracellular environment, thereby having a therapeutic effect. In some embodiments, the cleavable linker is pH sensitive, that is, sensitive to hydrolysis at certain pH values. Generally, pH-sensitive linkers can be hydrolyzed under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic acid amides, orthoesters, acetals, ketals, etc.) that can be hydrolyzed in lysosomes can be used. Such linkers are relatively stable under neutral pH conditions, such as those in blood, but are unstable at less than pH 5.5 or 5.0, the estimated pH of lysosomes. In some embodiments, the hydrolyzable linker is a linker containing a hydrazone.

[0321] In other embodiments, the linker can be cleaved under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the art, including, for example, disulfide linkers that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene).

[0322] In some embodiments, the linker can be cleaved by a cleavage agent (e.g., an enzyme) present in the intracellular environment (e.g., in a lysosome or endosome or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease (including but not limited to a lysosomal or endosomal protease). In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. The cleavage agent can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, thereby releasing active drugs in target cells. The most typical are peptidyl linkers that can be cleaved by enzymes present in cells expressing L1CAM. In a specific embodiment, the peptidyl linker that can be cleaved by intracellular proteases is a val-cit linker or a val-ala linker. One advantage of using intracellular proteolytic release of the therapeutic agent is that the therapeutic agent is usually attenuated when coupled and the serum stability of the conjugate is usually high.

[0323] In other embodiments, the linker is a malonate linker, a maleimidobenzoyl linker, or a 3'-N-amide analog.

[0324] In yet other embodiments, the linker unit is non-cleavable and the drug is released by, for example, antibody degradation.

[0325] In some embodiments, the linker is a substantially hydrophilic linker (e.g., PEG8, PEG 4- Mal and Sulfo-SPDB). Hydrophilic linkers can be used to reduce the extent of drug pumping out of drug-resistant cancer cells via MDR (multidrug resistance) or functionally similar transporters, or to improve the pharmacokinetic properties of ADCs.

[0326] In other embodiments, after cleavage, the role of the linker is to directly or indirectly inhibit cell growth and / or cell proliferation. For example, in some embodiments, the linker can act as an intercalator after cleavage, thereby inhibiting macromolecular biosynthesis (e.g., DNA replication, RNA transcription, and / or protein synthesis).

[0327] In other embodiments, the linker is designed to promote bystander killing (killing of neighboring cells) through diffusion of the linker-drug and / or drug alone to neighboring cells. In other embodiments, the linker promotes cellular internalization.

[0328] The presence of sterically hindered disulfides can increase the stability of specific disulfide bonds, thereby enhancing the effectiveness of ADCs. Therefore, in one embodiment, the linker includes a sterically hindered disulfide bond. A sterically hindered disulfide refers to a disulfide bond that is present in a specific molecular environment, wherein the environment is characterized by a specific spatial arrangement or orientation of atoms, typically within the same molecule or compound, which prevents or at least partially inhibits the reduction of the disulfide bond. Therefore, the presence of bulky (or sterically hindered) chemical moieties and / or bulky amino acid side chains near the disulfide bond prevents or at least partially inhibits the disulfide bond from having potential interactions that may lead to the reduction of the disulfide bond.

[0329] In a preferred embodiment of any of the aspects of the present invention, the antibody

[0330] (a) linked to a therapeutically active substance,

[0331] Preferably connected to

[0332] chemotherapeutic compounds,

[0333] Cytotoxic compounds,

[0334] Cytostatic compounds,

[0335] Cytokines,

[0336] Nanoparticles,

[0337] Radioisotopes, or

[0338] Oncolytic viruses,

[0339] and / or

[0340] (b) linked to a diagnostic compound, preferably selected from a radioisotope, a chemiluminescent compound, a fluorescent compound, a dye or an enzyme.

[0341] In a preferred embodiment, the therapeutically active substance in (a) and / or the diagnostic compound in (b) is selected from radioactive isotopes, chemotherapeutic compounds, cytotoxic compounds and / or cytostatic compounds, and / or wherein the antibody is covalently linked to the therapeutically active substance or its chelator in (a) or the diagnostic compound or its chelator in (b) optionally via a linker.

[0342] Preferably, the antibody according to any of the aspects of the present invention may be further linked to one or more molecules to form an antibody conjugate, in particular to a therapeutically active substance and / or a diagnostic compound.

[0343] The preferred embodiments of any of the various aspects of the present invention also apply to the antibody conjugates herein.

[0344] Therefore, preferably, the antibody portion of the antibody conjugate comprises:

[0345] (a) a heavy chain variable region (VH), the VH comprising VH CDR1, VH CDR2 and VH CDR3, the VH CDR1 comprising the GYSITSDYTWN (SEQ ID NO: 9) amino acid sequence, the VH CDR2 comprising the YISYSGSYSYNPSLKS (SEQ ID NO: 11) amino acid sequence, and the VH CDR3 comprising the SFSYSYGFAY ​​(SEQ ID NO: 14) amino acid sequence; and

[0346] (b) a light chain variable region (VL), wherein the VL comprises VL CDR1, VL CDR2 and VL CDR3, wherein the VL CDR1 comprises the KASQDVSSAVA (SEQ ID NO:4) amino acid sequence, the VL CDR2 comprises the SASYRYT (SEQ ID NO:5) amino acid sequence, and the VL CDR3 comprises the QQHYSTPWT (SEQ ID NO:6) amino acid sequence.

[0347] Therefore, preferably, the antibody portion of the antibody conjugate includes a heavy chain variable region sequence and / or includes a light chain variable region sequence, the heavy chain variable region sequence includes or consists of the amino acid sequence of SEQ ID NO: 30, and the light chain variable region sequence includes or consists of the amino acid sequence of SEQ ID NO: 20. In addition, preferably, the antibody portion of the antibody conjugate includes a heavy chain variable region sequence and includes a light chain variable region sequence, the heavy chain variable region sequence includes or consists of the amino acid sequence of SEQ ID NO: 30, and the light chain variable region sequence includes or consists of the amino acid sequence of SEQ ID NO: 20.

[0348] Preferably, the antibody portion of the antibody conjugate comprises a heavy chain sequence and / or a light chain sequence, the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38. In yet another preferred embodiment, the antibody portion of the antibody conjugate comprises a heavy chain sequence and a light chain sequence, the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0349] In another preferred embodiment, the antibody portion of the antibody conjugate consists of a heavy chain sequence and / or a light chain sequence, the heavy chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 38. In another preferred embodiment, the antibody portion of the antibody conjugate consists of a heavy chain sequence and a light chain sequence, the heavy chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 38.

[0350] Preferably, the antibody portion of the antibody conjugate comprises a heavy chain sequence and / or a light chain sequence, the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38. In yet another preferred embodiment, the antibody portion of the antibody conjugate comprises a heavy chain sequence and a light chain sequence, the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0351] In another preferred embodiment, the antibody portion of the antibody conjugate consists of a heavy chain sequence and / or a light chain sequence, the heavy chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 38. In another preferred embodiment, the antibody portion of the antibody conjugate consists of a heavy chain sequence and a light chain sequence, the heavy chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence includes or consists of the amino acid sequence of SEQ ID NO: 38.

[0352] In a preferred embodiment, the antibody of any aspect of the present invention is connected to at least one therapeutically active substance via a linker. Such compounds are also referred to as "antibody-drug conjugates", "antibody conjugates", "antibody drug conjugates" or "ADCs".

[0353] The antibodies of the invention described herein can be coupled to a drug moiety to form an anti-L1CAM antibody-drug conjugate (ADC). Due to the ability of ADC to selectively deliver one or more therapeutically active moieties to target tissues or cells (e.g., tumors expressing L1CAM or cells expressing L1CAM), antibody-drug conjugates (ADC) can increase the therapeutic efficacy of antibodies in treating diseases (e.g., cancer). Therefore, in certain embodiments, the present disclosure provides an anti-L1CAM ADC for therapeutic use (e.g., treatment of cancer).

[0354] The terms "therapeutically active substance," "therapeutically active substance moiety," "drug," "agent," and "drug moiety" are used interchangeably herein.

[0355] The terms "linked" and "coupled" are also used interchangeably herein and indicate that the antibody and the moiety are covalently linked.

[0356] In some embodiments, the ADC or the antibody of the present invention connected to at least one therapeutically active substance via a linker has the following formula (Formula I):

[0357] Ab-(LD)n(I)

[0358] Wherein Ab is an antibody of any aspect of the present invention described herein, and (LD) is a linker-drug moiety. The linker-drug moiety is composed of L- and -D, wherein L- is a linker, and the -D is a therapeutically active substance moiety (or drug moiety) that has cytostatic, cytotoxic or other therapeutic activity on target cells (e.g., cells 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.

[0359] Examples of therapeutically active substances that can be used in ADC, i.e., therapeutically active substances that can be coupled to the antibodies of the present invention, include mitotic inhibitors, antitumor antibiotics, immunomodulators, gene therapy vectors, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormonal agents, glucocorticoids, photosensitizing therapeutic agents, oligonucleotides, radioisotopes, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, and combinations thereof.

[0360] In one embodiment, the therapeutically active substance is selected from the group consisting of DNA damaging agents, anti-apoptotic agents, mitotic inhibitors, anti-tumor antibiotics, immunomodulators, nucleic acids for gene therapy, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormone agents, anti-hormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radioisotopes, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors.

[0361] Mitotic inhibitors

[0362] In one embodiment, the antibody of the present invention can be coupled with one or more mitotic inhibitors to form ADC for the treatment of hyperproliferative disorders, tumor diseases, disorders associated with neovascularization and / or disorders associated with abnormal neurogenesis. As used herein, the term "mitotic inhibitor" refers to a cytotoxic agent and / or therapeutic agent that blocks mitosis or cell division, which is a biological process that is particularly important for cancer cells. Mitotic inhibitors destroy microtubules, so that cell division is usually prevented by affecting microtubule polymerization (e.g., inhibiting microtubule polymerization) or microtubule depolymerization (e.g., stabilizing the microtubule cytoskeleton to prevent depolymerization). Therefore, in one embodiment, the antibody of the present invention is coupled with one or more mitotic inhibitors that destroy microtubule formation by inhibiting tubulin polymerization. In another embodiment, the antibody of the present invention is coupled with one or more mitotic inhibitors that stabilize the microtubule cytoskeleton to prevent depolymerization. In one embodiment, the mitotic inhibitor used in the ADC of the present invention is Ixempra (ixabepilone). The example of the mitotic inhibitor that can be used for the ADC of the present invention is provided below. The mitotic inhibitor genus includes the auristatins and maytansines, which are further described below.

[0363] Dolastatin

[0364] The antibodies of the present invention can be coupled to at least one dolastatin to form an ADC. Dolastatin is a short peptide compound isolated from the Indian Ocean sea hare, Dolabella auricularia. Examples of dolastatin include dolastatin 10 and dolastatin 15. Dolastatin 15 is a seven-subunit depsipeptide derived from Dolabella auricularia, a potent antimitotic agent that is structurally related to the five-subunit peptide anti-tubulin agent dolastatin 10 obtained from the same organism. Auristatin is a synthetic derivative of dolastatin 10.

[0365] Auristatin

[0366] The antibodies of the present invention may be coupled to at least one auristatin. Auristatin represents a group of dolastatin analogs that have generally been shown to have anticancer 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, which is a compound that inhibits tubulin polymerization by binding to the same site on tubulin as the anticancer drug vincristine. Dolastatin 10, auristatin PE and auristatin E are linear peptides with four amino acids, three of which are unique to dolastatin compounds. Exemplary embodiments of the auristatin subclass of mitotic inhibitors 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).

[0367] In one embodiment, the antibody of the present invention is coupled to at least one MMAE (monomethyl auristatin E). Monomethyl auristatin E (MMAE) inhibits cell division by blocking the polymerization of tubulin. Due to its toxicity, MMAE itself cannot be used as a drug. In the recent development of cancer therapy, MMAE is related to antibodies that recognize specific markers expressed in cancer cells and guide MMAE to cancer cells. In one embodiment, the linker connecting MMAE to the antibody of the present invention is stable in the extracellular fluid (i.e., the medium or environment outside the cell), but once the ADC binds to the specific cancer cell antigen and enters the cancer cell, the linker will be cleaved by cathepsin, thereby releasing toxic MMAE and activating an effective anti-mitotic mechanism. In one embodiment, the linker connecting MMAE to the antibody of the present invention is stable in the extracellular fluid (i.e., the medium or environment outside the cell), but once the ADC binds to the specific cancer cell antigen and enters the cancer cell, the linker will be cleaved by glucuronidase, thereby releasing toxic MMAE and activating an effective anti-mitotic mechanism.

[0368] Maytansinoids

[0369] The antibody of the present invention can be coupled to form ADC with at least one maytansine compound.Maytansine compounds are potent antitumor agents, which were originally isolated from higher plant families Celastraceae, Rhamnaceae and Euphorbiaceae and some moss species.There is evidence that maytansine compounds inhibit mitosis by inhibiting the polymerization of microtubule protein tubulin, thereby preventing the formation of microtubules.It has been shown that maytansine compounds use cell culture models in vitro and experimental animal systems in vivo to inhibit tumor cell growth.In addition, the cytotoxicity of maytansine compounds is 1,000 times the cytotoxicity of conventional chemotherapeutic agents (for example, methotrexate, daunorubicin and vincristine).Maytansine compounds include C-3 esters such as maytansine, maytansinol and maytansinol.

[0370] Suitable maytansine compounds for ADC of the present invention can be separated from natural sources, produced synthetically or semi-synthetically. In addition, maytansine compounds can be modified in any suitable manner, as long as enough cytotoxicity is retained in the final coupled molecule. In this respect, maytansine compounds lack the appropriate functional group that antibody can be connected. Ideally, linking moiety is utilized to connect maytansine compounds to antibody to form conjugate, and described in the embodiments.

[0371] Representative examples of maytansine compounds include, but are not limited to, DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine; also known as drug maytansine compound 1), DM2, DM3 (N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-maytansine), DM4 ((4-methyl-4-mercapto-1-oxopentyl)-maytansine) and maytansinol (a synthetic maytansine analog).

[0372] In one embodiment of the invention, the antibody of the invention is coupled to at least one DM1. In one embodiment, the antibody of the invention is coupled to at least one DM2. In one embodiment, the antibody of the invention is coupled to at least one DM3. In one embodiment, the antibody of the invention is coupled to at least one DM4.

[0373] Antitumor antibiotics

[0374] The antibody of the present invention can be coupled with one or more antitumor antibiotics. As used herein, the term "antitumor antibiotic" means an antitumor drug that blocks cell growth by interfering with DNA and is made of microorganisms. Typically, antitumor antibiotics destroy DNA chains or slow down or stop DNA synthesis. Examples of antitumor antibiotics that can be included in ADC include, but are not limited to, actinomycin (e.g., pyrrolo[2,1-c][1,4]benzodiazepine), anthracyclines, calicheamicin and duocarmycin. In addition to the above, other antitumor antibiotics that can be used include bleomycin, mitomycin and plicamycin (also known as mithramycin).

[0375] Immunomodulators

[0376] In one embodiment, the antibody of the present invention can be coupled with at least one immunomodulator. As used herein, the term "immunomodulator" refers to an agent that can stimulate or modify an immune response. In one embodiment, an immunomodulator is an immunostimulator that enhances the immune response of a subject. In another embodiment, an immunomodulator is an immunosuppressant that prevents or reduces the immune response of a subject. Immunomodulators can regulate bone marrow cells (monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) or lymphocytes (T cells, B cells and natural killer (NK) cells) and any further differentiated cells thereof. Representative examples include, but are not limited to, BCG (BCG) and levamisole (Ergamisol). Other examples of immunomodulators that can be used for ADC include, but are not limited to, cancer vaccines and cytokines.

[0377] Alkylating agents

[0378] The antibodies of the present invention can be coupled with one or more alkylating agents. Alkylating agents are a class of anti-tumor compounds that connect alkyl groups to DNA. Examples of alkylating agents that can be used in ADCs include, but are not limited to, alkyl sulfonates, ethyleneimines, methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas, triazines, and hydrazines.

[0379] DNA damaging agents

[0380] In one embodiment, the antibodies of the present invention can be coupled to one or more DNA damaging agents. As used herein, the term "DNA damaging agent" refers to an agent that can damage DNA and is well known to those of ordinary skill in the art. DNA damaging agents include DNA alkylating agents. DNA damaging agents also include indolin-benzodiazepine (IGN).

[0381] In one embodiment, the DNA damaging agent may also include pyrrolobenzodiazepine (PBD) or pyridobenzodiazepine (PDD) [26, 27].

[0382] For example, SG3199 or VA-SG3199 (ticillin) can be used. SG3199 is a pyrrolobenzodiazepine (PBD) dimer warhead component of the antibody-drug conjugate (ADC) payload ticillin.

[0383] Anti-angiogenic agents

[0384] In one embodiment, the antibodies of the invention described herein are coupled to at least one anti-angiogenic agent. Anti-angiogenic agents inhibit the growth of new blood vessels. Anti-angiogenic agents exert their effects in a variety of 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 main proteins involved in initiating angiogenesis by binding to specific receptors on the surface of cells. Therefore, certain anti-angiogenic agents that prevent VEGF from interacting with its cognate receptors prevent VEGF from initiating angiogenesis. In other embodiments, these agents interfere with intracellular signaling cascades. For example, once a specific receptor on the surface of a cell is triggered, a cascade of other chemical signals is initiated to promote the growth of blood vessels. Therefore, certain enzymes (e.g., some tyrosine kinases) that are known to promote intracellular signaling cascades that contribute to, for example, cell proliferation are targets for cancer treatment. In other embodiments, these agents interfere with intercellular signaling cascades. However, in other embodiments,These agents invalidate specific targets that activate and promote cell growth or invalidate by directly interfering with the growth of vascular cells. Angiogenesis inhibition properties have been found in more than 300 substances with many direct and indirect inhibitory effects. Representative examples of anti-angiogenic agents that can be used for ADC include but are not limited to 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 (Erlotinib) (tarceva), inhibitor protein, endostatin, BAY 12-9566 and fluorouracil or doxorubicin, canstatin, carboxyaminotriazole and paclitaxel, EMD121974, S-24, vitaxin, dimethylxanthenone acetic acid, IM862, interleukin-12, interleukin-2, NM-3, HuMV833, PTK787, RhuMab, angiozyme (ribozyme), IMC-1C11, Neovastat, marimstat, prinomastat, BMS-2752 91, COL-3, MM1270, SU101, SU6668, SU11248, SU5416 (containing paclitaxel, containing gemcitabine and cisplatin, containing irinotecan and cisplatin, and containing radiation), tecogalan, temozolomide, and PEG interferon α2b, tetrathiomolybdate, TNP-470, thalidomide, CC-5013, and containing taxotere, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors (rapamycin (deforolimus), everolimus (Afinitor, Novartis Pharmaceutical Corporation) and temsirolimus (Torisel, Pfizer, Inc.)), tyrosine kinase inhibitors (e.g., erlotinib (Tasiva, Genentech, Inc.), imatinib (Gleevec,Novartis Pharmaceuticals), gefitinib (Iressa, AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb), sunitinib (Sutent, Pfizer), nilotinib (Tasigna, Novartis Pharmaceuticals), lapatinib (Tykerb, GlaxoSmithKline Pharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), phosphoinositide 3-kinase (PI3K).

[0385] Antimetabolites

[0386] The antibody of the present invention can be coupled with at least one antimetabolite.Antimetabolites are various types of chemotherapy treatments that are very similar to normal substances in cells.When cells incorporate antimetabolites into cell metabolism, the result is negative for cells, such as cells cannot divide.Antimetabolites are classified according to the substances they interfere with.The examples of antimetabolites that can be used for ADC include but are not limited to folic acid antagonists (e.g., methotrexate), pyrimidine antagonists (e.g., 5-fluorouracil, floxuridine (Foxuridine), cytarabine (Cytarabine), capecitabine and gemcitabine), purine antagonists (e.g., 6-mercaptopurine and 6-thioguanine) and adenosine deaminase inhibitors (e.g., cladribine (Cladribine), fludarabine (Fludarabine), nelarabine (Nelarabine) and pentostatin).

[0387] Boron-containing agents

[0388] The antibodies of the present invention may be coupled to at least one boron-containing agent. Boron-containing agents include a class of cancer therapeutic compounds that interfere with cell proliferation. Representative examples of boron-containing agents include, but are not limited to, borophycin and bortezomib.

[0389] Chemical protective agent

[0390] The antibodies of the present invention may be coupled to at least one chemoprotectant.

[0391] Chemoprotective drugs are a class of compounds that help protect the body from the specific toxic effects of chemotherapy. Chemoprotectants can be used with various chemotherapy to protect healthy cells from the toxic effects of chemotherapy drugs, while allowing the use of the chemotherapeutic agents for treating cancer cells. Representative chemoprotectants include but are not limited to reducing the nephrotoxicity associated with the cumulative dose of cisplatin, dexrazoxane, for treating the extravasation caused by the administration of anthracyclines and for treating the heart-related complications caused by the administration of antitumor antibiotics doxorubicin, and for preventing hemorrhagic cystitis during chemotherapy with ifosfamide (mesna (Mesnex), Bristol-Myers Squibb Company (Bristol-Myers Squibb)).

[0392] Photoactive therapeutic agents

[0393] The antibodies of the present invention may be coupled to at least one photoactive therapeutic agent. Photoactive therapeutic agents include compounds that can be used to kill treated cells when exposed to electromagnetic radiation of a specific wavelength. The therapeutically relevant compound absorbs electromagnetic radiation of a wavelength that penetrates tissue. In a preferred embodiment, the compound is administered in a non-toxic form that is capable of producing a photochemical effect that is toxic to cells or tissues when fully activated. In other preferred embodiments, these compounds are retained by cancerous tissue and are easily cleared from normal tissue. Non-limiting examples include various chromophores and dyes.

[0394] In a preferred embodiment, the mitotic inhibitor is selected from maytansine compounds and auristatins.

[0395] In the examples, maytansinoid compound DM4 and auristatin MMAE were successfully used in the ADC of the present invention.

[0396] In another preferred embodiment, the DNA damaging agent is selected from pyrrolobenzodiazepine (PBD) and pyridobenzodiazepine (PDD).

[0397] In the Examples, the DNA damaging agent VA-SG3199 (ticillin) was successfully used in the ADCs of the present invention.

[0398] In another preferred embodiment, the linker is a non-cleavable linker.

[0399] In another preferred embodiment, the linker is a cleavable linker.

[0400] In yet another preferred embodiment, the therapeutically active substance is selected from monomethyl auristatin E (MMAE), 4-methyl-4-mercapto-1-oxopentyl-maytansine (DM4) and VA-SG3199 (ticillin).

[0401] As described above, any of the antibodies of the invention described herein can be used in ADCs.

[0402] In the Examples, ADCs comprising the AFF4-WT antibody were prepared and demonstrated to be effective in in vitro and / or in vivo tumor models.

[0403] Therefore, it is particularly preferred to provide an ADC comprising an antibody that includes 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 chain sequence and / or light chain sequence of the AFF4 or AFF4-VT antibody.

[0404] Preferably, an antibody or ADC of the present invention connected to at least one therapeutically active substance via a linker is provided, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and / or CDRL3 sequences of the AFF4 antibody or the VH and / or VL domain sequences of the AFF4 antibody, and wherein the antibody further comprises a human IgG1, IgG2, IgG3 or IgG4 constant region.

[0405] In one embodiment of the antibody of the present invention or the ADC of the present invention linked to at least one therapeutically active substance via a linker, the antibody further comprises a human IgG1 constant region.

[0406] 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 (including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations, including any sub-range thereof), such as substitutions, deletions and / or insertions, especially 1 to 10 substitutions (including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, including any sub-range thereof).

[0407] Each mutation can be introduced independently into one chain of the Fc domain. Alternatively, each mutation can be introduced independently and symmetrically into both chains of the constant region domain.

[0408] In a preferred embodiment, the antibody or ADC connected to at least one therapeutically active substance via a linker comprises a heavy chain variable region sequence and / or comprises a light chain variable region sequence, wherein the heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 30, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 20.

[0409] In a preferred embodiment, the antibody or ADC connected to at least one therapeutically active substance via a linker comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 30 and comprises a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 20.

[0410] In a preferred embodiment, the antibody or ADC connected to at least one therapeutically active substance via a linker comprises a heavy chain sequence and / or a light chain sequence, wherein the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 37, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0411] In a preferred embodiment, the antibody or ADC connected to at least one therapeutically active substance via a linker comprises a heavy chain sequence and / or a light chain sequence, wherein the heavy chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 84, and the light chain sequence comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0412] Nucleic acids, vectors and host cells

[0413] The term "nucleic acid" describes any form of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or artificial nucleic acid known to those skilled in the art.

[0414] The 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 specific amino acids are assembled in such a manner as to generate nucleic acids encoding antibodies. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides, which in brief involve the synthesis of overlapping oligonucleotides containing a portion of the sequence encoding the antibody, the ligation and connection of the oligonucleotides, and then the amplification of the ligated oligonucleotides by PCR.

[0415] When a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, nucleic acids encoding immunoglobulins can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from nucleic acids (preferably poly A+RNA) isolated from any tissue or cell expressing the antibody (e.g., a hybridoma cell selected for expression of an antibody described herein) by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence or by cloning using oligonucleotide probes specific for a particular gene sequence, thereby identifying, for example, a cDNA clone from a cDNA library encoding the antibody. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0416] DNA encoding the antibodies of the invention described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., from CHO GS System) that do not otherwise produce immunoglobulins. TM The antibody may be synthesized in recombinant host cells by expressing the antibody in CHO cells (Lonza) or in myeloma cells.

[0417] In order to generate complete antibodies, PCR primers including VH or VL nucleotide sequences, restriction sites and flanking sequences protecting the restriction sites can be used to amplify the VH or VL sequences in scFv clones or other clones. Using cloning techniques known to those skilled in the art, the VH domains amplified by PCR can be cloned into a vector expressing a heavy chain constant region (e.g., a human IgG1 constant region, a human IgG4 constant region, a human IgG2 constant region or a human IgG3 constant region), and the VL domains amplified by PCR can be cloned into a vector expressing a light chain constant region (e.g., a human κ or λ constant region). In certain embodiments, the vector for expressing the VH or VL domains includes a promoter, a secretion signal, a cloning site for the variable region, a constant domain and a selection marker (such as neomycin). The VH and VL domains can also be cloned into a vector expressing the necessary constant region. Then, the heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line using techniques known to those skilled in the art, thereby generating a stable or transient cell line expressing a full-length antibody (e.g., IgG). Alternatively, a vector expressing a heavy chain or light chain can be transfected into a separate cell, and the antibody can be assembled after recovery. In addition, alternatively, the nucleic acid encoding the antibody of the present invention can be expressed using a single vector for expression. Such vectors can encode the antibody of the present invention into a single molecule, such as scFv, or can encode two or more polypeptides that can be assembled into an antibody of the present invention.

[0418] The DNA may also be modified by, for example, replacing the murine sequences with coding sequences for human heavy and light chain constant domains, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0419] Site-directed mutagenesis or high-density mutagenesis or other mutagenesis methods of the variable region can be used to optimize the monoclonal antibody for specificity, affinity, etc. In particular, affinity maturation strategies and chain shuffling strategies

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

[0420] In another aspect, the present invention also relates to a nucleic acid

[0421] (i) which encodes an antibody that specifically binds to human L1CAM according to any one of the aspects of the present invention, and / or

[0422] (ii) it encodes at least one VH or HC and / or VL or LC of an antibody that specifically binds to human L1CAM according to any one of the aspects of the present invention, and / or

[0423] (iii) which encodes a sequence according to SEQ ID NO: 30 and / or according to SEQ ID NO: 20, and / or

[0424] (iv) It comprises a sequence encoding a complementarity determining region sequence of an antibody that specifically binds to human L1CAM according to any one of the aspects of the present invention.

[0425] In this context, 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, such as features relating to the antibodies encoded by said nucleic acids or the specific binding of such antibodies.

[0426] Preferably, the nucleic acid is part of a vector.

[0427] Typically, a vector (also referred to as an expression vector) is a plasmid used to introduce a desired nucleic acid sequence (such as a gene) into a target cell, thereby causing 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 typically includes regulatory sequences, such as promoter and enhancer regions, and polyadenylation sites to guide efficient transcription of the nucleic acid sequence on the expression vector. The expression vector may further include additional necessary or useful regions, such as a selective marker for selection in eukaryotic or prokaryotic cells, a purification tag for purification of the resulting protein, a multiple cloning site or an origin of replication.

[0428] Typically, the expression vector can be a viral vector or a non-viral vector. Typically, various viral vectors, such as retroviral vectors, such as lentiviral vectors or adenoviral vectors, or plasmids can be used.

[0429] Preferably, the nucleic acid is part of a vector.

[0430] Such vectors comprising the nucleic acid of the present invention can further be introduced into host cells.

[0431] Methods for introducing such vectors into host cells are well known to those skilled in the art, such as any known transfection method, such as any non-viral transfection method (e.g., chemical, non-chemical or particle-based) or any viral-based transfection method. Examples of suitable methods are transfection methods based on calcium phosphate precipitation, lipofection, cationic polymers, Fugene, dendrimers, nanoparticles, microinjection, cell squeezing, electroporation, particle guns (also known as gene guns), magnetic-assisted transfection, optical transfection, protoplast fusion, impalafection, hydrodynamic delivery, sonoporation, transferrin-based infection, antibody-mediated transfection, or viral-based transfection (e.g., adenoviral vectors or lentiviral vectors).

[0432] In another aspect, the invention relates to a host cell comprising the nucleic acid according to the invention.

[0433] Suitable host cells are well known to those skilled in the art, such as mammalian cells (such as human cells, mouse cells, rat cells or hamster cells), insect cells, bacterial cells or yeast cells. Such host cells can contain the nucleic acid of the present invention, for example, integrated into its genome or vector. Methods for introducing such nucleic acids into host cells are as described above, and are further known to those skilled in the art.

[0434] Compositions comprising the antibodies of the present invention

[0435] In another aspect, the present invention relates to a pharmaceutical composition comprising the antibody of the present invention or the nucleic acid of the present invention or the host cell of the present invention, and optionally one or more pharmaceutically acceptable carriers.

[0436] The content of the antibody, nucleic acid or host cell in the pharmaceutical composition is not limited as long as it can be used for treatment or prevention, but preferably contains 0.0000001 wt % to 10 wt % per the total composition.

[0437] Furthermore, the antibodies, nucleic acids or host cells described herein are preferably employed in one or more pharmaceutically acceptable carriers.

[0438] The term "carrier" describes any molecule that improves the selectivity, effectiveness and / or safety of administration of antibodies, nucleic acids or host cells to the human or animal body, eg by continuous or triggered release or by allowing membrane permeability of the antibodies, nucleic acids and host cells.

[0439] When the carrier has no or substantially no adverse unnecessary effects on the human or animal body, the carrier is further considered to be pharmaceutically acceptable, for example, it is considered to be generally safe, non-toxic and / or does not cause unnecessary biological side effects. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art. The choice of carrier can depend on the route of administration and the concentration of the active agent, and the carrier can be in the form of a lyophilized composition or an aqueous solution. Typically, an appropriate amount of pharmaceutically acceptable salt is used in the carrier so that the composition is isotonic. Examples of carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. Acceptable excipients, carriers or stabilizers are preferably nontoxic at the dosages and concentrations employed, and include buffers such as citric acid, phosphoric acid and other organic acids; salt-forming counterions such as sodium and potassium; low molecular weight (<10 amino acid residues) polypeptides; proteins such as serum albumin or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine or glycine; carbohydrates including glucose, mannose or dextrin; monosaccharides; disaccharides; other sugars such as sucrose; Sugar, mannitol, trehalose or sorbitol; chelating agents such as EDTA; nonionic surfactants such as Tween, Pluronics or polyethylene glycol; antioxidants including methionine, ascorbic acid and tocopherol; and / or preservatives such as octadecyldimethylbenzyl ammonium 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.

[0440] Therapeutic and diagnostic uses and applications

[0441] In another aspect, the 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 a diagnostic agent.

[0442] The term "drug" is a substance useful in the cure, treatment or prevention of disease. Similarly, the term "diagnostic agent" is a substance useful in diagnosing disease, ie determining whether the human or animal body suffers from a particular disease or condition.

[0443] In another aspect, the present invention relates to the use 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 for treating or preventing a hyperproliferative disorder, a tumor disease, a disorder associated with neovascularization and / or a disorder associated with abnormal neurogenesis.

[0444] The term "treatment" describes any form of improving the health status of the human or animal body with respect to a disease or condition. This may include alleviation of symptoms, slowing of progression, slight improvement, but may also include complete cure of the disease or condition of the human or animal body. Similarly, the term "prevention" describes maintaining the health status of the human or animal body from the effects of a disease or condition.

[0445] The term "hyperproliferative disorder" describes any disease or condition associated with hyperproliferative cells (such as tumors, cancers and neoplastic tissues in general, as well as precancerous and non-neoplastic or non-malignant hyperproliferative disorders), i.e., cells that display an abnormally high rate of cell proliferation. Examples are non-malignant (also non-neoplastic), precancerous or malignant tumors. Examples of precancerous and non-neoplastic or non-malignant hyperproliferative disorders are, for example, myelodysplastic disorders; carcinoma in situ of the cervix; familial intestinal polyposis such as Gardner syndrome; oral leukoplakia; histiocytosis; keloids; hemangiomas; hyperproliferative arterial stenosis, inflammatory arthritis; hyperkeratosis, papular rashes 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.

[0446] The term "neoplastic disease" describes any disease or condition associated with a tumor (also known as a neoplasm), which may be non-malignant (also known as non-neoplastic), pre-cancerous or malignant.

[0447] In an embodiment, the neoplastic disease is selected from ovarian cancer, breast cancer, endometrial cancer, melanoma, and neuroblastoma.

[0448] In one embodiment, 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 is used to treat or prevent a hyperproliferative disorder expressing L1CAM, a tumor disease expressing L1CAM, a disorder expressing L1CAM associated with neoangiogenesis and / or a disorder expressing L1CAM associated with abnormal neurogenesis.

[0449] A "hyperproliferative disorder expressing L1CAM" is understood to be a hyperproliferative disorder wherein at least a portion of the hyperproliferative cells express human L1CAM on the cell surface.

[0450] "L1CAM-expressing tumor diseases" are understood to be tumor diseases in which at least some of the tumor cells express human L1CAM on the cell surface.

[0451] In an embodiment, the L1CAM-expressing tumor disease is selected from the group consisting of L1CAM-expressing ovarian cancer, L1CAM-expressing breast cancer, L1CAM-expressing endometrial cancer, L1CAM-expressing melanoma, and L1CAM-expressing neuroblastoma.

[0452] "L1CAM-expressing disorder associated with neoangiogenesis" is understood as a neoangiogenesis-associated disorder in which at least a portion of the cells involved in neoangiogenesis express human L1CAM on the cell surface.

[0453] "L1CAM-expressing disorder associated with abnormal neurogenesis" is understood as a disorder associated with abnormal neurogenesis in which at least a portion of the cells involved in abnormal neurogenesis express human L1CAM on the cell surface.

[0454] In another preferred embodiment, an antibody of any aspect of the present invention linked to at least one therapeutically active substance 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 tumor disease, a disorder associated with neoangiogenesis, a disorder associated with abnormal neurogenesis, a hyperproliferative disorder expressing L1CAM, a tumor disease expressing L1CAM, a disorder associated with neoangiogenesis expressing L1CAM, or a disorder expressing L1CAM associated with abnormal neurogenesis by exhibiting a cytotoxic bystander effect. In an embodiment, the linker is a releasable pad.

[0455] The cytotoxic bystander effect is understood as an effect in which an ADC having such a bystander effect is taken up and processed by antigen-positive cancer cells in the form of a released cytotoxic payload that can diffuse freely to neighboring cells and thus has the ability to kill these cells independently of their antigen expression. Therefore, such ADCs are well suited for treating tumors with heterogeneous L1CAM target expression. In an embodiment, the linker is a releasable pad. Methods for determining the cytotoxic bystander effect are known in the art. Specifically, the assay described in Example 8 can be used.

[0456] "About" is understood to mean ±10% of the indicated value.

[0457] A disorder associated with neoangiogenesis describes any disease or condition associated with the formation of blood vessels in hyperproliferative tissues, such as non-malignant (also known as non-neoplastic), precancerous or malignant tumors and cancer.

[0458] A disorder associated with aberrant neurogenesis describes any disease or condition associated with the generation of abnormal cells of the nervous system (eg, abnormal neurons) from neural stem cells, such as abnormal (hippocampal) neurogenesis.

[0459] In another aspect, the present invention relates to the use 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 for diagnosing a hyperproliferative disorder, a tumor disease, a disorder associated with neoangiogenesis and / or a disorder associated with abnormal neurogenesis.

[0460] In another aspect, the present invention relates to the in vitro use 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 as a diagnostic agent, in particular for diagnosing hyperproliferative disorders, tumor diseases, disorders associated with neovascularization and / or disorders associated with abnormal neurogenesis.

[0461] On the other hand, the present invention relates to a method for treating or preventing a hyperproliferative disorder, a tumor disease, a disorder associated with neovascularization and / or a disorder associated with abnormal neurogenesis, the method comprising administering a pharmaceutically effective amount of an antibody of the present invention, a nucleic acid of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention to a patient in need thereof.

[0462] As used herein, in the context of administering a therapy to a subject, the term "pharmaceutically effective amount" refers to that amount of the therapy that achieves the desired prophylactic or therapeutic effect.

[0463] Those skilled in the art can determine the appropriate amount and dosage. For example, the antibody or pharmaceutical composition described herein can be administered 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 (e.g., by intravenous injection).

[0464] Thus, the administration of the antibodies, nucleic acids or host cells 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 dosing regimens are also well known to those skilled in the art. Preferably, the antibodies, nucleic acids or host cells of the present invention are administered in a pharmaceutically effective amount, i.e., in a dose or concentration that causes a biological response in the body to which the antibodies, nucleic acids or host cells of the present invention are administered.

[0465] In this context, all features described above for the antibodies of any aspect of the invention also apply (where applicable) to further aspects of the invention, such as features relating to antibody conjugates, nucleic acids, host cells or pharmaceutical compositions.

[0466] Generally, the disclosure is not limited to the particular methods, schemes and reagents described herein, as they may vary. In addition, the terms used herein are only used for the purpose of describing a particular embodiment, and are not intended to limit the scope of the disclosure. As used herein and in the appended claims, the singular forms "a / an" and "the" include plural referents, unless the context clearly indicates otherwise. Similarly, the words "comprise / include", "contain" and "cover" should be interpreted inclusively rather than exclusively.

[0467] 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 of the present disclosure. Although any methods and materials similar or equivalent to those described herein can be used in the practice described herein, specific methods and materials are described herein.

[0468] The following figures and examples further illustrate the present disclosure, but it should be understood that unless otherwise specifically stated, the figures and examples are for illustration purposes only and are not intended to limit the scope of the present disclosure.

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[0491] 23. Gouveia et al.: Production and purification of functional truncated soluble forms of human recombinant L1 cell adhesion glycoprotein from Spodoptera frugiperda Sf9 cells. Protein Expr. Purif. 52: 182-193 (2007)

[0492] 24. Epitope Mapping, A practical approach, 248 (Practical Approach Series); Oxford University Press, eds. Olwyn Westwood and Frank Hay (2001)

[0493] 25. Oleszewski, M. et al.: Characterization of the L1CAM-neurocan binding site. Implications for L1CAM-L1CAM homophilic binding. J. Biol. Chem. 275: 34478-34485 (2000).

[0494] 26. Veillard N. et al.: Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity. Cancer Res 78(13_Suppl):736.(2018)

[0495] 27. Stefano JE et al.: Micro- and Mid-Scale Maleimide-Based Conjugation of Cytotoxic Drugs to Antibody Hinge Region Thiols for Tumor Targeting. In: Ducry L. (ed.) Antibody-Drug Conjugates. Methods in Molecular Biology (Methods and Protocols), Vol. 1045. Humana Press, Totowa, NJ. (2013).

[0496] 28. Marks JD et al.: By-passing immunization: building high affinity human antibodies by chain shuffling. Biotechnology, 10: 779-783 (1992). BRIEF DESCRIPTION OF THE DRAWINGS

[0497] Figure 1 . Figure 1 A 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 light chain (glycosylated and non-glycosylated) are indicated by arrows. Lanes 1 to 3 include the following samples: Lane 1: molecular weight marker; Lane 2: OV549.20 mouse IgG2a; Lane 3: chimeric OV549.20 human IgG1. Figure 1 B shows mass spectrometry analysis of DTT-reduced chimeric OV549.20 human IgG1, indicating the presence of peaks corresponding to the heavy chain (50798.5 Da) as well as to glycosylated light chains (25885.5 Da) and non-glycosylated light chains (23533.2 Da).

[0498] Figure 2 Shown are the fluorescence signals detected on JIMT-1, SKOV-3 and Panc-1 cells using image-based cytometry after incubation with chimeric OV549.20 human IgG1 and secondary goat anti-human IgG antibody labeled with Alexa Fluor 488. A chimeric human IgG1 isotype control antibody was used for comparison.

[0499] Figure 3 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 are shown. Antibodies specific for commercially available human L1CAM, CHL1, NrCAM, and neurofascin were used as positive controls to demonstrate successful immobilization of all antigens on the ELISA plate and proper function of the detection system. Binding levels were quantified by evaluating the optical density (OD) at a wavelength of 450 nm in the ELISA assay.

[0500] Figure 4Shown is the dose-dependent induction of antibody-dependent cellular cytotoxicity (ADCC) by chimeric OV549.20 human IgG1 and Herceptin on Panc-1 pancreatic cancer cells. A chimeric version of human IgG1 that binds to the first Ig domain of L1CAM, previously described antibody L9.3, was tested in parallel, but did not induce ADCC on Panc-1 cells. Shown are mean values ​​± standard deviations from triplicates. GraphPad Prism software was used to fit the values ​​to a 4-parameter logistic curve.

[0501] Figure 5 Shown are the effects of OV549.20 mouse IgG2a, OV52.24 mouse IgG2a, and isotype control antibodies on the proliferation of HCT116, SKOV3, and Panc-1 cells as measured by confluency detection using an Incucyte device. Addition of OV549.20 mouse IgG2a reduced proliferation of all three cell lines, while OV52.24 mouse IgG2a and mouse IgG2a isotype control had no effect. Shown are mean ± standard deviation from duplicates.

[0502] Figure 6 It is shown that chimeric OV549.20 human IgG1 inhibits the migration of HCT116 cells on fibronectin-coated plates, while an irrelevant chimeric human IgG1 isotype control has no effect on cell migration. Shown are mean ± standard deviation from duplicates.

[0503] Figure 7 . Figure 7 A shows the inhibitory effect of OV549.20 mouse IgG2a on intraperitoneal tumor mass and ascites volume in the SKOV3 xenograft model of mouse ovarian cancer. SKOV3 cells were injected intraperitoneally and mice were treated five days later with 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). Treatments were repeated three times per week for a total of six weeks, followed by analysis of intraperitoneal tumor mass and ascites volume. Figure 7 B shows the results of the second SKOV3 xenograft experiment. SKOV3 cells were injected intraperitoneally and mice were treated five days later with 10 mg / kg OV549.20 mouse IgG2a (n=10) or vehicle (n=10). Treatments were repeated three times per week for a total of six weeks, followed by analysis of intraperitoneal tumor mass and ascites volume.

[0504] Figure 8The inhibitory effect of OV549.20 mouse IgG2a on metastasis formation in the intravenous MDA-MB-231-luc2 mouse xenograft model is shown. 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 injected intravenously with 5x 10 5 The body weight of the animals was recorded regularly, 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.

[0505] Fig. 9 Analysis of purified humanized antibody variants H1L1 to H4L2 by reducing SDS-PAGE is shown. 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, there are two forms of light chain, glycosylated and non-glycosylated.

[0506] Fig.10 . Fig.10 A shows the analysis of chimeric OV549.20 human IgG1 antibody and humanized variant H1L1 on a Tosoh TSKgelButyl-NPR hydrophobic interaction column. Fig.10 B shows analysis of the humanized antibody variant H1L1 on a Tosoh TSKgel Butyl-NPR column before ("unstressed H1L1") and after ("H1L1 stressed at pH 5.5") incubation for twelve days at 40°C in 20 mM sodium citrate pH 5.5. Fig.10 C shows analysis of antibody variant AFF4 on a Tosoh TSKgel Butyl-NPR column before ("unstressed AFF4") and after ("AFF4 stressed at pH 5.5") incubation for 14 days at 40°C in 20 mM sodium citrate pH 5.5.

[0507] Fig.11 Shown are the effects of AFF4 and a chimeric human IgG1 isotype control antibody on the proliferation of HCT116, SKOV3, and Panc-1 cells as measured by confluence detection using the Incucyte device. Addition of AFF4 reduced proliferation of all three cell lines compared to the isotype control antibody. Shown are mean ± standard deviation from duplicates.

[0508] Fig.12AFF4 is shown to inhibit the migration of HCT116 cells on fibronectin-coated plates, whereas a chimeric human IgG1 isotype control antibody had no effect on cell migration. Shown are mean ± standard deviation from duplicates.

[0509] Fig.13 The specific lysis % obtained for PC-03, Panc-1, HeLa and SKOV3 cancer cell lines by antibody-dependent cellular cytotoxicity mediated by AFF4 is shown. Cancer cells were seeded on xCelligence E-plates and allowed to settle and attach to the plates for 16-24 hours. Activated natural killer cells from three different donors (CD16 low affinity: FF; medium affinity: V / F; high affinity: VV) were each 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 with the xCelligence RTCA analyzer and the data were converted to specific cell lysis %.

[0510] Fig.14 The structures of four linker / payloads used for conjugation to AFF4-WT are shown. Fig.14 A: VA-SG3199 (MP-PEG8-VA-PABC-SG3199), CAS number: 1595275-62-9. IUPAC: (6S,6aS)-3-[5-[[(6aS)-2-methoxy-8-methyl-11-oxo-6a,7-dihydropyrrolo[2,1-c][1,4]benzodiazepine-3-yl]oxy]pentyloxy]-6-hydroxy-2-methoxy-8-methyl-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionylamino]-3-methylbutanoyl]amino]propionyl]amino]phenyl]methyl heptatriene-5-carboxylate; Fig.14B: VC-MMAE (MC-VC-PABC-MMAE), CAS number: 646502-53-6. IUPAC: 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-oxohept-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-N-methylcarbamic acid [4-[[(2S)-5-(carbamoylamino)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl ester; Fig.14 C: Gluc-MMAE (MC-β 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-phenyl [2-(4-(2-(4-(2-(4-(2-(4-(2-methyl-1-oxobutan-2-yl)-2-yl)amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxohept-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-methylcarbamoyl]oxymethyl]phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid; Fig.14 D: Sulfo-SPDB-DM4, CAS No. 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-dimethoxy Oxa-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-oxopropan-2-yl]dimercapto]-1-(2,5-dioxopyrrolidin-1-yl)oxy-1-oxobutane-2-sulfonic acid.

[0511] Fig.15The fluorescence signals detected on the cells by image-based cytometry after co-incubation of JIMT-1, OVCAR-3 and MeWo cells with AFF4-WT and human Fabfluor-pH red antibody labeling dye for 15 minutes or 20 hours, respectively, are shown. Chimeric human IgG1 isotype control antibody was used for comparison. Compared with isotype control / FabFluor treated cells, AFF4-WT / FabFluor treated cells had a strong increase in fluorescence after 20 hours, indicating efficient uptake and lysosomal localization of AFF4-WT.

[0512] Fig.16 Four AFF4-WT-conjugated drugs are shown for cytotoxic effects 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 for 6 days with JIMT-1 breast cancer cells, MeWo melanoma cells and OVCAR-3 ovarian cancer cells, respectively. Then, cell viability was quantitatively assessed by ATP, and untreated cells and cells incubated in the presence of 10 μM doxorubicin were used as 100% and 0% survival reference values, respectively, to calculate the survival rate of cells treated with AFF4-WT-conjugated drugs. Shown are individual values ​​from duplicates. The average value was fitted to a 4-parameter logistic curve using GraphPad Prism software.

[0513] Fig.17 . Fig.17 A 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 approximately 100 mm 3 The mice then received a single intravenous (iv) injection of the indicated AFF4-WT-conjugate drug or vehicle as control, and tumor growth was monitored over time. Fig.17 B 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 approximately 140 mm 3 The mice then received a first intravenous injection of the indicated AFF4-WT-conjugate drug or vehicle as control, followed by a second injection 14 days later. Tumor growth was monitored regularly. Fig.17 C shows the antitumor effects of four AFF4-WT-drug conjugates in a human melanoma xenograft model in mice. MeWo melanoma cells were implanted subcutaneously and tumors were allowed to grow to approximately 140 mm 3Mice then received a single intravenous injection of the indicated AFF4-WT-conjugate drug or vehicle as control, and tumor growth was monitored regularly.

[0514] Fig.18 The antitumor effect of different doses of AFF4-WT-VC-MMAE in a human breast cancer xenograft model in mice is shown. JIMT-1 breast cancer cells were implanted intramammarily and tumors were allowed to grow to approximately 100 mm 3 The mice then received a single intravenous injection of the indicated doses of AFF4-WT-VC-MMAE, and tumor growth was monitored over time.

[0515] Fig.19 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 is shown. PDX tumor fragments were implanted subcutaneously and allowed to grow to approximately 150-300 mm 3 Mice then received biweekly intravenous injections of AFF4-WT-VC-MMAE or vehicle, and tumor growth was monitored regularly for up to 60 days.

[0516] Fig. 20 AFF4-WT-VC-MMAE in vitro bystander cytotoxicity activity is shown. The serial dilutions of AFF4-WT-VC-MMAE are pre-incubated for 4 days on L1CAM high expression JIMT-1 cells, L1CAM low expression MDA-MB-468 cells or in the absence of cells. The supernatant from these pre-incubations was transferred to MDA-MB-468 cells by four times dilution and then incubated for 6 days. Then, the cell survival rate was quantitatively assessed by ATP, and the cells incubated in the presence of 10 μM doxorubicin were used as 100% and 0% survival rate reference values, respectively, to calculate the cell survival rate%. Shown are the mean and standard deviation from duplicates. The indicated AFF4-WT-VC-MMAE concentration refers to the final concentration of the MDA-MB-468 cells incubated with supernatant. The mean value was fitted to a 4-parameter logistic curve using GraphPad Prism software.

[0517] Example

[0518] Example 1

[0519] Generation of monoclonal antibody OV549.20 and its recombinant expression as a mouse IgG2a and human IgG1 chimeric antibody The mouse antibody OV549.20 was generated using hybridoma technology using SKOV3ip human ovarian cancer cells as immunogen.

[0520] The DNA sequences of the heavy chain variable domain and light chain variable domain of coding OV549.20 are cloned into the expression vectors of the heavy chain constant domain and light chain constant domain of coding mouse IgG2a or human IgG1 respectively in frame. The obtained protein sequences of the whole heavy chain and light chain of the antibody construct are shown in SEQ ID NO:35 and SEQ ID NO:36 of mouse antibody. The chimeric antibody has the same variable region, but contains human IgG1 constant domain. The plasmids of the heavy chain and light chain of the coding antibody are purified under low endotoxin conditions respectively, and transiently cotransfected into Chinese hamster ovary (CHO) K1 cells. The cells are grown in a chemically defined animal component-free culture medium and the supernatant containing the recombinant antibody is collected by centrifugation, and then filtered through a 0.2 μm filter. The antibody is purified from the supernatant by affinity chromatography using a protein A column (MabSelect SuRe, GE Healthcare), and the antibody is stored in phosphate buffered saline (PBS) containing 100 mM arginine.

[0521] Reducing SDS-PAGE analysis of purified OV549.20 mouse IgG2a and chimeric OV549.20 human IgG1 antibodies ( Figure 1 A) reveals bands at approximately 50 kDa for both antibodies, a faint band at approximately 25 kDa, and an additional strong band at approximately 30 kDa. Mass spectrometry analysis of chimeric OV549.20 human IgG1 ( Figure 1 B) confirmed the presence of predicted molecular weights of heavy chain (50798.5Da) and light chain (23533.2Da), and detected an extra mass of 25885.5Da, which greatly exceeded one of the predicted light chains. The molecular weight difference of 2352.3Da between the predicted light chain mass and the extra mass is consistent with the N-glycosylation reaction occurring in the light chain sequence, indicating that the extra mass corresponds to the glycosylation variant of the light chain. In fact, sequence analysis indicates the presence of a typical N-glycosylation motif (Asn-Ile-Thr) in the variable region of the OV549.20 light chain (SEQ ID NO: 36).

[0522] Example 2

[0523] In vitro pharmacology of OV549.20 mouse IgG2a and chimeric OV549.20 human IgG1

[0524] Expression of the extracellular domain of human and cynomolgus monkey L1CAM

[0525] In order to determine the target binding affinity of OV549.20 mouse IgG2a and chimeric OV549.20 human IgG1, the recombinant version of the extracellular part of human L1CAM and cynomolgus monkey L1CAM was first cloned, expressed and purified. To this end, the DNA sequence encoding amino acids 20 to 1120 of human L1CAM (Uniprot accession number P32004) and the corresponding sequence encoding amino acids 20 to 1120 of cynomolgus monkey L1CAM (NCBI accession number XP_005594994) were synthesized and cloned into a eukaryotic expression vector. The corresponding recombinant expression product was designed to include an N-terminal leader peptide for secretion into the cell supernatant and a C-terminal six-histidine 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 1L expression cultures, and supernatants were collected by centrifugation. Recombinant proteins were purified from the supernatant by protein A affinity chromatography and stored in PBS pH 7.4 until further analysis.

[0526] Determination of kinetic binding constants of OV549.20 mouse IgG2a and chimeric OV549.20 human IgG1 to human and cynomolgus monkey L1CAM

[0527] 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 by their fragment crystallizable (Fc) region on a CM5 protein A chip (GE Healthcare), and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), 0.05% Tween 20 were used as running buffer, and soluble His-tagged human or cynomolgus monkey L1CAM was injected as analyte at 5 different concentrations ranging from 1.25 nM to 20 nM. The detected resonance units (sensograms) were fitted to a 1:1 Langmuir binding model, and the association (k a ) and dissociation (k d ) rate constant and affinity constant (K D ). Table 7 shows that OV549.20 mouse IgG2a and chimeric OV549.20 human IgG1 bind to humans and cynomolgus monkeys with similar affinity:

[0528] Table 7: Binding affinity of OV549.20 mouse IgG2a and chimeric OV549.20 human IgG1 to human and cynomolgus monkey L1CAM

[0529]

[0530]

[0531] Binding of chimeric OV549.20 human IgG1 to human cancer cell lines expressing L1CAM

[0532] The breast cancer cell line JIMT-1 and the pancreatic cancer cell line Panc-1 were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal calf serum (FCS). The ovarian cancer cell line SKOV3 was cultured in McCoy's 5a medium containing 10% FCS. Cell separation solution (5 mL per T75 cell culture flask) was incubated for 10 minutes to separate the cells from the tissue culture plate, washed once with PBS, and incubated for 1 hour at 4°C with 2 μg / ml chimeric OV549.20 human IgG1 or 2 μg / ml of an unrelated chimeric human IgG1 isotype control antibody. Then, Alexa Fluor 488-labeled AffiniPure goat anti-human IgG antibody (Jackson Immuno Research) was added at a concentration of 5 μg / ml, and incubated for 1 hour at 4°C. Finally, the cells were incubated for 15 minutes at 37°C with 10 μg / ml Hoechst-33342 solution (ChemoMetec), and analyzed on an image-based cytometer (Nucleocounter NC-3000, ChemoMetec) using an excitation wavelength of 475 nm and an emission filter of 560 nm ± 35 nm. A total of 4'000 to 11'000 cells were analyzed for each condition and the detected fluorescence intensities were plotted as histograms on a semi-logarithmic scale.

[0533] Figure 2 It was shown that incubation of JIMT-1, Panc-1 and SKOV3 cells with chimeric OV549.20 human IgG1 resulted in a strong change in fluorescence signal compared to the isotype control antibody, indicating that chimeric OV549.20 human IgG1 specifically bound to L1CAM on the surface of these human cancer cell lines.

[0534] Determination of cross-reactivity of L1CAM homologs NrCAM, CHL1, and neurofascin

[0535] The binding ability of OV549.20 mouse IgG2a to other members of the L1 family adhesion molecules, i.e., L1 close homolog (CHL1), neurofascin, and neuronal 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 hexa-histidine-tagged versions of human CHL1 and neurofascin, were added to 50 mM carbonate buffer pH 9.6 in an ELISA plate at a concentration of 1 μg / mL (100 μL per well). The plate was incubated overnight at 4°C, washed 5 times with PBS / 0.05% Tween, and blocked by adding 200 μL PBS / 0.05% Tween / 1% bovine serum albumin (BSA) per well. OV549.20 mouse IgG2a was then serially diluted in PBS / 0.05% Tween / 1% BSA at a ratio of 1:3, starting from a maximum concentration of 10 μg / mL. As positive controls, identical dilution series were prepared for commercial mouse antibodies 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 plates were washed 5 times with PBS / 0.05% Tween, and 100 μL per well of the antibody dilution series was added to the plates. After incubation for 1 hour at room temperature, the plates were washed again, and 100 μL per well of horseradish peroxidase-conjugated polyclonal goat anti-mouse antibody (diluted 1:1000 in PBS / 0.05% Tween / 1% BSA) was added. After incubation for another 1 hour at room temperature, the plates were washed again and 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) substrate was added per well for detection. After incubation for 8 minutes at room temperature, the reaction was stopped by adding 50 μL of 0.16M H2SO4 per well and the absorbance at 450 nm was determined in a microplate reader.

[0536] Figure 3 The OD values ​​obtained in different ELISA assays at an antibody concentration of 1 μg / mL are shown. Control antibodies specific for human L1CAM, CHL1, NrCAM, and Neurofascin showed strong binding to their corresponding target antigens, demonstrating that all antigens were successfully immobilized on the ELISA plate and that the detection system was functioning properly. OV549.20 mouse IgG2a showed strong binding to plate-coated human L1CAM, but did not have any reactivity with plate-coated human CHL1, NrCAM, or Neurofascin.

[0537] Induction of antibody-dependent cellular cytotoxicity (ADCC) by chimeric OV549.20 human IgG1

[0538] Panc-1 human pancreatic cancer cells expressing L1CAM and Her2 on their surface were stained with Cr 51 Labeled and pre-incubated with 8 different concentrations of chimeric OV549.20 human IgG1 antibodies at room temperature for 20 minutes. Herceptin was used as a positive control, and the chimeric human IgG1 version of the anti-L1CAM antibody L9.3 bound to the first Ig domain of L1CAM was tested at the same time (WO 2008 / 151819). Then, human CD16 (158V isomeric form) transgenic T cells were added as effector cells at a ratio of 20:1 effector cells to target cells, and incubated for 4 hours at 37°C, 5% CO2 in a humidified atmosphere. After incubation, the cells were centrifuged and 25 μl of the supernatant was transferred to Lumplate (Perkin-Elmer). Target cell lysis was measured by supernatant γ counting and expressed as corrected counts per minute (CCPM) after detector normalization. Spontaneous lysis was determined by γ counting of the supernatant of Panc-1 cells incubated in a culture medium without effector cells and antibodies. Maximum lysis was determined by gamma counting of supernatants from Panc-1 cells lysed by addition of 0.75% Triton X-100. The percentage of specific lysis was calculated according to the following formula:

[0539]

[0540] CCPM Ab is the count of wells containing Panc-1 cells, effector cells, and antibodies, CCPM SL is the count of wells containing only Panc-1 cells in the absence of effector cells and antibody, and CCPM max is the count of wells of Panc-1 cells lysed with Triton X-100. After fitting the specific lysis values ​​to a 4-parameter logistic curve, the maximum lysis (E max ) and maximum half lysis (EC 50 )value.

[0541] Figure 4 The chimeric OV549.20 human IgG1 induced strong ADCC on Panc-1 target cells. The antibody induced a dose-dependent increase in specific lysis of Panc-1 cells, with E max is 36.8% and EC 50 The positive control antibody Herceptin mediated the lysis of Panc-1 cells, where Emax is 30.1% and EC 50 The chimeric human IgG1 version of the previously described antibody L9.3 that binds to the first Ig domain of L1CAM did not induce any ADCC on Panc-1 target cells.

[0542] Inhibition of cancer cell proliferation by OV549.20 mouse IgG2a

[0543] 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% confluence on 384-well plates, using 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 aforementioned antibody OV52.24 (WO 2016050702). The proliferation of cancer cells was monitored regularly over 96 hours by performing microscopic confluence assays using the Incucyte device.

[0544] Figure 5 It was shown that the mouse IgG2a isotype control antibody and the OV52.24 mouse IgG2a antibody had no effect on the proliferation of any cancer cell line, while the addition of the OV549.20 mouse IgG2a antibody resulted in a reduction in proliferation of all three cancer cell lines. Compared to cells treated with vehicle alone, by 96 hours, the addition of OV549.20 mouse IgG2a caused a decrease in the confluence of HCT116 cells from 45.7% to 37.3%, a decrease in the confluence of SKOV3 cells from 21.7% to 14.2%, and a decrease in the confluence of Panc-1 cells from 69.7% to 43.3%.

[0545] Inhibition of cancer cell migration by chimeric OV549.20 human IgG1

[0546] HCT116 colorectal cancer cells were cultured in DMEM supplemented with 10% FCS. Cell detachment solution (Capricorn) was collected and cultured at 4 × 10 4Cells were seeded at a density of 10 cells / 100 μL into plug-containing wells of fibronectin pre-coated ORIS-96 well plates (AMS-bio). Twenty-four hours later, the plug inserts were removed and the medium was replaced with 120 μL of fresh growth medium. Subsequently, 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 concentration of the antibody in the assay plate was 4 x 10 -5 M to 1.2x 10 -8 M. As a positive control for unlimited migration, cells were incubated in the absence of any antibody. As a negative control, migration was restricted by maintaining the plug in eight wells of each plate. After 48 hours, the culture medium was replaced with 75 μL of DMEM supplemented with 2 μg / ml calcein-AM (Life Technologies) without phenol red. The cells were incubated at 37 ° C for 15 minutes, and the fluorescent cells in the area defined by the insert were detected by a fluorescent microplate reader (Fluostar, BMG) using fluorescein isothiocyanate (FITC) settings (excitation: 485nm / emission: 520nm). The median of the positive control (removing the plug, not adding antibodies) was set to 100% (high control), and the median of the negative control (not removing the plug) was set to 0% (low control). The raw data was converted to cell migration % relative to the control, and a variable slope with a bottom constraint of 0 and a top constraint of 100 was used to fit to a 4-parameter logistic curve.

[0547] Figure 6 The results showed that the addition of chimeric human IgG1 isotype control antibody had a negligible effect on the migration of HCT116 cells, while the addition of chimeric OV549.20 human IgG1 antibody caused specific, dose-dependent inhibition of migration. The half-maximal inhibitory concentration (IC 50 ) is calculated as 4.2x 10 -6 M.

[0548] Example 3

[0549] In vivo pharmacology of OV549.20 mouse IgG2a

[0550] Inhibition of primary tumor growth in the SKOV3 ovarian cancer xenograft model

[0551] The ability of OV549.20 mouse IgG2a to inhibit primary tumor growth was investigated in the 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 with PBS containing 0.05% trypsin and 0.02% EDTA and injected intraperitoneally into 30 female NMRI:nu / nu mice (5 x 10 cells / mouse). 6 cells). Five days later, the mice were divided into three groups of 10 mice each, each of which received an intraperitoneal injection of OV549.20 mouse IgG2a antibody, L9.3 mouse IgG2a (WO 2008 / 151819), or a mouse IgG2a isotype control antibody at a dose of 10 mg / kg. The treatment was repeated three times a week for 6 weeks, then the animals were sacrificed and the SKOV3 tumor weight and the volume of ascites in the peritoneal cavity were determined.

[0552] Figure 7 A shows that treatment with OV549.20 mouse IgG2a antibody resulted in a 29% reduction in mean tumor mass compared to the isotype treated control group. In addition, the formation of ascites fluid was reduced by 88% in OV549.20 mouse IgG2a treated animals compared to the isotype treated control animals. In contrast, treatment with L9.3 mouse IgG2a did not induce a reduction in tumor mass and only a slight reduction in ascites fluid.

[0553] In the second experiment, 20 female NMRI:nu / nu mice were injected intraperitoneally with SKOV3 cells (5 10 cells / mouse). 6 cells). Five days after injection, the mice were divided into two groups of 10 mice each and injected intraperitoneally with OV549.20 mouse IgG2a (10 mg / kg) or vehicle, respectively. The treatment was repeated three times a week for a total of 6 weeks, and the tumor weight and ascites volume in the peritoneal cavity were subsequently analyzed.

[0554] Figure 7 B shows that treatment with OV549.20 mouse IgG2a resulted in a 35% reduction in median tumor burden and a 75% reduction in ascites volume compared to the vehicle-treated group.

[0555] Inhibition of metastasis formation in the MDA-MB-231 breast cancer xenograft model

[0556] The ability of OV549.20 mouse IgG2a to inhibit metastasis formation was tested in a mouse xenograft model of human MDA-MB-231-luc2 metastatic breast cancer cells. In this model, intravenously administered MDA-MB-231-luc2 cells metastasize to the lungs of mice and can be detected by whole-body luminescence after administration of a luminescent substrate.

[0557] Groups of female athymic nude-Foxn1nu mice (n=15 per group) were injected intravenously three times per week with OV549.20 mouse IgG2a antibody (10 mg / kg) or vehicle control for five weeks. Three days after the first administration, all animals were injected intravenously with 5x10 5 MDA-MB-231-luc2 cells.Clinical signs and body weight were recorded daily, and all animals were subjected to in vivo whole body luminescence imaging at 7, 14, 21 and 30 days after cell injection.For this reason, D-luciferin (150mg / kg) was administered to animals by intraperitoneal injection, and imaging was performed after 10 minutes under isoflurane anesthesia.Images were captured and the luminescent signal (total flux, in photons / second) of the chest region was measured.

[0558] Figure 8 It was shown that treatment with OV549.20 mouse IgG2a strongly inhibited metastasis formation, as determined by whole-body luminescence signals. On day 30, the total flux signal in mice treated with OV549.20 mouse IgG2a was reduced by 94.8% compared to vehicle controls. Although the vehicle control animals showed a significant and sustained decrease in body weight after day 20, mice treated with OV549.20 mouse IgG2a did not experience any detectable weight loss during the experiment. Therefore, treatment with OV549.20 mouse IgG2a effectively inhibited metastasis formation and improved clinical signs of the disease.

[0559] Example 4

[0560] Humanization of OV549.20

[0561] In silico transplantation of the CDRs of OV549.20 onto the human framework

[0562] Based on the computational modeling of OV549.20 Fv, the human VH sequence IGHV4 30 4 01 was identified as the most suitable acceptor framework for the CDR of the heavy chain of OV549.20. The VH CDRs of OV549.20 (SEQ ID NO: 1, 2 and 3; see Table 1) were transplanted to IGHV4 30 4 01 by computer simulation, and at the same time, certain positions in the IGHV4 30 4 01 human framework were reversely mutated to the corresponding mouse residues, as long as this was considered suitable for avoiding new contacts or maintaining existing contacts within the modeled OV549.20 Fv. In this way, four humanized variants of the heavy chain variable region of OV549.20 were generated, including different numbers of reverse mutant framework residues (H1-H4, SEQ ID NO 23, 24, 25, 26).

[0563] At the same time, the human VL sequence IGKV1 39 01 was identified as the most suitable human acceptor framework for the CDRs of the light chain of OV549.20. The VL CDRs of OV549.20 (SEQ ID NOs: 4, 5, and 6; see Table 2) were transplanted in silico onto IGKV139 01, and a series of reverse mutations of human residues to corresponding mouse residues were simultaneously introduced into the human framework as long as it was considered necessary to maintain structural and functional integrity. In addition, a putative N-glycosylation site (Asn-Ile-Thr) that may lead to light chain glycosylation when expressed in CHO cells (detectable as mass changes in SDS-PAGE and MS (see Figure 1 )) was mutated to a sequence that cannot support such glycosylation reactions (Thr-Ile-Thr). Four different humanized variants of the OV549.20 light chain variable domain were generated, each carrying a mutated N-glycosylation site and a different number of reverse-mutated framework residues. Among them, humanized light chain variable domains L1 and L2 (L1: SEQ ID NO: 20, L2: SEQ ID NO: 22) were further used

[0564] Expression and purification of humanized variants of OV549.20

[0565] The DNA sequences encoding four different humanized heavy chain variable domains (H1-H4) are each fused in the framework with the DNA sequence encoding human IgG1 constant domains, and used to produce the expression construct of full-length humanized IgG1 heavy chain. Similarly, the DNA sequences encoding both of the light chain variable domains (L1 and L2) are each fused in the framework with the sequence encoding the light chain constant domain, and used to produce the expression construct of two full-length humanized κ light chains. Each of the four heavy chain expression plasmids and one of the two light chain expression plasmids are transiently cotransfected into CHO cells, causing the expression of 8 different full-length humanized IgG1 antibody variants (H1L1 to H4L2). The cells are grown in a chemically defined animal component-free medium and the supernatant containing the recombinant antibody is collected by centrifugation, and then filtered 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.

[0566] Biophysical properties of humanized variants of OV549.20

[0567] All eight humanized antibodies were analyzed by reducing SDS-PAGE and compared to the parental chimeric OV549.20 human IgG1. Fig. 9 It was shown that all humanized antibodies showed 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. Compared to the parental chimeric OV549.20 human IgG1 antibody, no additional prominent band at 30 kDa that may represent a glycosylation variant of the light chain was detected in any of the humanized antibodies.

[0568] 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 showed a first melting point (T m 1), which is similar to the parent chimeric OV549.20 human IgG1 antibody. The second melting point (T m 2) depended on the corresponding heavy chain of the antibody variant and was above 83.8°C in all cases, indicating that the conformational stability of all humanized antibody variants was high.

[0569] Table 8: Comparison of the first and second melting points of humanized variants of OV549.20 with the parental chimeric OV549.20 human IgG1 and Rituximab

[0570]

[0571] 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 by their Fc region on a C1 protein A chip (GE Healthcare), and soluble His-tagged human L1CAM was injected as analyte at 3 different concentrations (10 nM, 20 nM, and 40 nM) using 10 mM HEPES / 150 mM NaCl / 3 mM EDTA / 0.05% Tween 20 as running buffer. The detected resonance units (sensograms) were fitted to a 1:1 Langmuir binding model, and the association (k a ) and dissociation (k d ) rate constant and affinity constant (K D ). Table 9 shows that all humanized antibody variants bind to human L1CAM with high affinity and display similar kinetic binding constants.

[0572] Table 9: Monovalent kinetic binding constants of humanized variants of OV549.20

[0573]

[0574]

[0575] All humanized antibody variants were analyzed by hydrophobic interaction chromatography on a Tosoh TSKgel Butyl-NPR column and compared to the parental chimeric OV549.20 human IgG1. Fig.10 The exemplary chromatogram in A shows that the parent chimeric antibody OV549.20 human IgG1 shows a main peak and an additional secondary peak / shoulder peak after the main peak, indicating that there are post-translational variants in the preparation. Humanized variant H1L1 shows the same peak pattern, but the additional peak is unexpectedly significantly reduced compared to the parent chimeric antibody, indicating that the presence of post-translational variants is reduced. The peak integration of the HIC chromatograms of all humanized antibody variants (Table 10) shows that the secondary peak areas of all variants are similarly reduced, indicating that compared to the parent chimeric antibody, transplantation to all human framework variants can reduce the formation of post-translational variants.

[0576] Table 10: Relative peak areas of humanized variants of OV549.20 in hydrophobic interaction chromatography

[0577]

[0578] 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. Fig.10As shown in B, incubation of H1L1 at 40°C in 20 mM sodium citrate pH 5.5 for twelve days significantly increased the amplitude of the second peak and resulted in the appearance of a prominent third peak. This indicates that post-translational modification reactions (such as isomerization reactions or similar reactions) that occur preferentially at low pH values ​​are the origin of the heterogeneity detected in the hydrophobic interaction chromatography. In fact, using AccuMAP TM Proteolytic digestion of unstressed and pH stressed H1L1 using the Low pH Protein Digestion Kit followed by reverse phase liquid chromatography mass spectrometry and MS-MS analysis of peptides corresponding to amino acids 1 to 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 modifications are consistent with an isomerization reaction of 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.

[0579] Example 5

[0580] Affinity improvement and optimization of biophysical properties and effector functions of humanized antibodies

[0581] The humanized antibody H1L1 was selected as the basis for further optimization of target affinity, biophysical properties and effector functions. Three different scFv phage display libraries were constructed, in which the selected amino acid positions in the CDRs of the heavy and light chains of H1L1 were randomly assigned using a degenerate oligonucleotide directed PCR mutagenesis method. After four rounds of translation of recombinant human L1CAM under various types of selection pressure, a total of 470 clones were randomly selected using the parental phage as a control and subjected to monoclonal phage ELISA on a plate coated with human L1CAM. Sixty-two clones with strongly enhanced binding signals in ELISA were selected and subjected to DNA sequencing. Twenty unique sequences were identified, 13 of which were expressed in soluble scFv form, and the sequences were subjected to ELISA again on a plate coated with human L1CAM. Among the 13 tested scFvs, 11 were detected to have increased binding compared to the parental scFv. Based on the sequences of the high binding clones, eight different modified heavy chain variable domains (SEQ ID NOs: 27-34) and one modified light chain variable domain (SEQ ID NO: 21) were designed in addition to the light chain variable domain SEQ ID NO: 20. The heavy and light chain variants were combined to produce 10 different heavy and light chain combinations, and the corresponding expression plasmids of the corresponding full-length human IgG1 antibodies were constructed. In order to improve the Fc-mediated effector function of the antibodies, 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 Fcγ receptors

[18] . As described in Example 4, the resulting full-length antibodies AFF1 to AFF10 were expressed and purified and subjected to detailed biophysical and in vitro pharmacological analyses.

[0582] The monovalent kinetic binding constants for 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 4 different concentrations (5 nM, 10 nM, 20 nM and 40 nM). Table 11 shows that all mutated variants showed improved binding to human L1CAM compared to the parent antibody H1L1, with affinity constants (K) of 0.1% and 0.2% in all cases. D ) are all in the sub-nanomolar range.

[0583] Table 11: Binding affinity of further mutated humanized antibodies (AFF1-AFF10)

[0584]

[0585]

[0586] The chemical stability of antibodies AFF1 to AFF10 was analyzed by hydrophobic interaction chromatography on a Tosoh TSKgel Butyl-NPR column and compared with the parent antibody H1L1. Surprisingly, none of the antibody variants showed a second peak on the hydrophobic interaction column compared to H1L1, and the area after the main peak of all variants (Table 12) was generally low, indicating that the post-translational variants derived from the isomerization of aspartic acid at position 32 were either absent or present in very low amounts in antibody variants AFF1 to AFF10.

[0587] Table 12 shows that exposure of the parent antibody H1L1 to high temperature and low pH stress (14 days at 40°C in 20 mM sodium citrate buffer pH 5.5) caused a significant increase in the area behind the main peak in hydrophobic interaction chromatography (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 variants AFF4 are shown in Table 12. Fig.10 Shown in C.

[0588] Table 12: Comparison of chemical stability of antibodies AFF1 to AFF10 and H1L1

[0589]

[0590]

[0591] Example 6

[0592] In vitro pharmacology of humanized optimized antibody AFF4

[0593] Inhibition of cancer cell proliferation

[0594] HCT116, Panc-1 and SKOV3 were cultured as described in Example 2 and seeded at 10%-15% confluence in 384-well plates in the presence of 50 μg / mL AFF4 or 50 μg / mL chimeric human IgG1 isotype control antibody. Cancer cell proliferation was monitored regularly over 120 hours by performing microscopic confluence assays using an Incucyte device.

[0595] Fig.11The addition of AFF4 antibody caused a decrease in proliferation of all three cell lines compared to the isotype control antibody. By 96 hours, the addition of AFF4 caused a decrease in confluence of HCT116 cells from 55.3% to 38.9%, a decrease in confluence of SKOV3 cells from 68.6% to 44.4%, and a decrease in confluence of Panc-1 cells from 67.4% to 42.7%, compared to cells treated with the isotype control antibody.

[0596] Inhibition of cancer cell migration

[0597] HCT116 colorectal cancer cells were cultured in DMEM supplemented with 10% FCS. Cell separation solution (Capricorn) was collected and cultured at 4 × 10 4 Cells were seeded at a density of 10 cells / 100 μL into plug-containing wells of fibronectin pre-coated ORIS-96 well plates (AMS-bio). Twenty-four hours later, the plug inserts were removed and the medium was replaced with 80 μL of fresh growth medium. Subsequently, 20 μL of a 5-fold concentrated serial dilution series of AFF4 or chimeric human IgG1 isotype control was added to the cells. The final concentration of the antibody in the assay plate was 4 x 10 -5 M to 1.2x10 -8 M. As a positive control for unlimited migration, cells were incubated in the absence of any antibody. As a negative control, migration was restricted by maintaining a stopper in eight wells of each plate. After 48 hours, the culture medium was replaced with 75 μL of DMEM supplemented with 2 μg / ml calcein-AM (Life Technologies) without phenol red. The cells were incubated at 37 ° C for 15 minutes, and the fluorescent cells in the area defined by the insert were detected by a fluorescent microplate reader (Fluostar, BMG) using FITC settings (excitation: 485nm / emission: 520nm). The median of the positive control (removing the stopper, not adding antibodies) was set to 100% (high control), and the median of the negative control (not removing the stopper) was set to 0% (low control). The raw data was converted to cell migration % relative to the control, and a variable slope with a bottom constraint of 0 and a top constraint of 100 was used to fit to a 4-parameter logistic curve.

[0598] Fig.12 The results showed that the addition of AFF4 antibody caused specific and dose-dependent inhibition of migration. The half maximum inhibitory concentration (IC 50 ) is calculated to be approximately 1.1x 10 -5 M.

[0599] Antibody-dependent cellular cytotoxicity (ADCC) induced by AFF4 against SKOV3, Panc-1, HeLa, and PC03 cancer cell lines

[0600] The ability of AFF4 to induce antibody-dependent cellular toxicity against L1CAM-expressing cancer cell lines SKOV3, HeLa, Panc-1 and PC03 was studied. In the first step, the expression level of L1CAM on the surface of different cell lines was evaluated by flow cytometry. Each cell line was grown in continuous culture and used 2 to 4 days after passage. Before the assay, the target cells were separated from the surface of the culture flask using a non-enzymatic cell dissociation buffer to limit the cleavage of surface-expressed L1CAM. Cells were stained using phycoerythrin (PE)-labeled anti-human L1CAM antibody (clone L1-OV198.5, BioLegend) or PE-labeled mouse IgG2a (κ) (clone MOPC-173, BioLegend) as the corresponding isotype control antibody. The stained cells were obtained on a Quanteon flow cytometer and analyzed using NovoExpress software. The median fluorescence intensity (MFI) value (triplicate) of each evaluated cell line was calculated and the average values ​​are listed in Table 13. To quantify expression levels, the fold increase of MFI values ​​obtained with anti-L1CAM antibody relative to isotype control antibody 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.

[0601] Table 13: L1CAM expression on the surface of different cell lines as determined by flow cytometry using PE-labeled anti-L1CAM antibody

[0602]

[0603] To evaluate ADCC, primary natural killer (NK) cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) from three different donors using an NK cell separation kit (Miltenyi Biotech). One of the donors was homozygous for the CD16 (FcγRIIIA) high affinity allele V158, one donor was homozygous for the CD16 low affinity allele F158, and the third donor was heterozygous, carrying both high affinity and low affinity alleles (V / F158). The purity of the enriched NK cells (>80% of CD3-CD56+ cells in the CD45+ cell population) was checked and incubated for 21±1 hours in NK92 culture medium+IL-2 (3ng / mL) at 36°C±1°C, 5%±1% CO2. At the same time, preheated cell culture medium was added to the wells of the xCelligence E-plate 96 (Agilent) (50 μL / well), and suspensions of SKOV3, HeLa, Panc-1, and PC-03 cells were added (100 μL / well) and allowed to stand at the bottom of the wells for 1 hour at room temperature. The E-plate was then transferred to the xCelligence Real-Time Cell Analyzer (RTCA) and incubated at 36°C ± 1°C, 5% ± 1% CO2 for 16-24 hours to allow cell attachment and proliferation. Impedance was continuously measured overnight to monitor cell proliferation. The next day, AFF4 was diluted to a concentration of 4 μg / mL in NK92 medium supplemented with 3 ng / mL IL-2. At the same time, NK cells were collected, counted, and resuspended in the same medium. The E-plate is taken out from the RTCA analyzer, the culture medium (50 μL / well) is drawn, and pre-diluted AFF4 (50 μL / well) and NK cells (50 μL / well) are added. NK cells are added at a density corresponding to the final effector cell to target cell (E:T) ratio of 5:1 or 10:1. The final concentration of AFF4 in the E-plate is 1 μg / mL. The plate is transferred to the RTCA analyzer, and impedance measurement is restarted to monitor effector cell-mediated target cell killing. Data is acquired for 72 hours and then analyzed with xCelligence immunotherapy software 1.0. The cell index data recorded by the RTCA analyzer are normalized relative to the last time point before the effector cells are added. The normalized cell index is converted to specific cell lysis% by comparing the normalized cell index obtained from the wells containing target cells, effector cells and treatment with the wells containing only target cells and effector cells. The xCELLigence RTCA software calculates specific cell lysis% using the following formula:

[0604]

[0605] NCIst is the normalized cell index of the sample, and NCI Rt is the mean of the normalized Cell Index of the matched reference wells (wells containing only target and effector cells).

[0606] Fig.13 It was shown that AFF4 activated effector cells from all three donors and induced specific cell lysis on all cell lines tested. The highest degree of cytotoxicity was observed on the HeLa cell line that highly expressed L1CAM, reaching 100% in most cases. When at a higher E:T ratio of 10:1, the degree of specific cell lysis was generally higher and was related to the corresponding CD16 (FcγRIIIA) affinity of the effector cells present in the donor cell preparation. The highest activity was observed in FcγRIIIA V158 homozygous (V / V) donors, moderate activity was observed in FcγRIIIA V / F158 heterozygous (V / F) donors, and the lowest activity was observed in FcγRIIIAF158 homozygous (F / F) donors. In the latter donor, the best correlation between L1CAM cell surface expression and the degree of specific cell lysis of different cell lines was observed, with the highest levels observed on HeLa and SKOV3 cells, moderate levels observed on Panc-1 cells, and the lowest levels observed on PC-03 cells.

[0607] Example 7

[0608] Generation and biophysical properties of AFF4-WT antibody-drug conjugates

[0609] For conjugation with cytotoxic payloads, a version of AFF4 was used that contained a wild-type human IgG1 constant domain and therefore did not carry the four mutations (G236A / S239D / A330L / I332E) present in the CH2 domain of AFF4. This human IgG1 wild-type version of AFF4 was designated AFF4-WT.

[0610] Conjugation of AFF4-WT with VA-SG3199 (MP-PEG8-VA-PABC-SG3199, Ticillin)

[0611] A solution containing AFF4-WT in PBS / 100 mM arginine was prepared by adding 5% v / v 0.5 M Tris-HCl / 25 mM EDTA pH 8.5 for reduction and then incubated at 30°C for 2 hours with 1.25 molar equivalents of TCEP (tris(2-carboxyethyl)phosphine, added from a 1 mM stock in water) to achieve an average of approximately 2 free thiols. The thiols were then removed by adding 5 molar equivalents of MP-PEG8-VA-PABC-SG3199 ( Fig.14 A) to couple the partially reduced AFF4-WT antibody. MP-PEG8-VA-PABC-SG3199 was added from DMA (N,N-dimethylacetamide) containing a 10 mM stock solution, and additional DMA was added during the coupling reaction to reach 5% v / v. After incubation for 2 hours at room temperature, the reaction was quenched with 5 molar equivalents of N-acetyl-cysteine ​​(added from a 10 mM stock solution in water) at room temperature for 30 minutes, and then desalted and buffer exchanged to 25 mM histidine / 0.2 M sucrose pH 6.0 by passing through a Sephadex G-25 column. In order to completely remove free toxins and other low molecular weight additives, the AFF4-WT-VA-SG3199 conjugate was subjected to 8 buffer exchanges of 25 mM histidine / 0.2 M sucrose pH 6.0 by discontinuous diafiltration using a PES (polyethersulfone) centrifugal concentrator (Vivaspin) with a molecular weight cutoff of 30 kDa. Polysorbate 20 was added from a 1% w / v stock solution 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.

[0612] Conjugation of AFF4-WT with VC-MMAE (MC-VC-PABC-MMAE, vedotin) and Gluc-MMAE (MC-β-glucuronide-MMAE)

[0613] A solution containing AFF4-WT in PBS / 100 mM arginine was prepared by adding 5% v / v 0.5 M Tris-HCl / 25 mM EDTA pH 8.5 for reduction and then incubated at 30°C for 2 hours with 2.5 molar equivalents of TCEP (added from a 1 mM stock in water) to achieve an average of approximately 4 free thiols. Fig.14 B) or MC-β-glucuronide-MMAE ( Fig.14C) to couple the partially reduced AFF4-WT antibody. MC-VC-PABC-MMAE and MC-β glucuronide-MMAE were added from DMA containing 10 mM stock solutions, with additional DMA added during the coupling reaction to reach 5% v / v. After incubation for 1 hour at room temperature, the reaction was quenched with 5 molar equivalents of N-acetyl-cysteine ​​(added from a 10 mM stock solution in water) at room temperature for 30 minutes, and then desalted and buffer exchanged to 25 mM histidine / 0.2 M sucrose pH 6.0 by passing through a Sephadex G-25 column. In order to completely remove free toxins and other low molecular weight additives, AFF4-WT-VC-MMAE and AFF4-WT-Gluc-MMAE conjugates were subjected to 8 buffer exchanges of 25 mM histidine / 0.2 M sucrose pH 6.0 by discontinuous diafiltration using a PES centrifugal concentrator (Vivaspin) with a molecular weight cutoff of 30 kDa. Polysorbate 20 was added from a 1% w / v stock solution to a final concentration of 0.02% w / v and the conjugate was 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.

[0614] Conjugation of AFF4-WT with Sulfo-SPDB-DM4

[0615] AFF4-WT (in PBS / 100 mM arginine) was applied to a protein A column disinfected 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 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 into 50 mM sodium phosphate / 150 mM NaCl / 2 mM EDTA pH 8.0 by passing through a Sephadex G25 column. AFF4-WT was then concentrated to 5 mg / ml using a PES centrifugal concentrator (Vivaspin) with a molecular weight cutoff of 30 kDa. The prepared antibody was then conjugated to ( Fig.14D). Sulfo-SPDB-DM4 was added from DMA containing a 10 mM stock solution, and additional DMA was added during the coupling reaction to reach 5% v / v. After incubation for 4 hours at room temperature, an additional 1 molar equivalent of Sulfo-SPDB-DM4 was added to the reaction solution and incubation was continued overnight. The coupling reaction was then quenched and desalted by adding 1% v / v 0.5M Tris pH 8.5 and buffer exchanged into 25mM histidine / 0.2M sucrose pH 6.0 by passing through a Sephadex G25 column. In order to completely remove free toxins and other low molecular weight additives, AFF4-WT-Sulfo-SPDB-DM4 was subjected to 10 buffer exchanges of 25mM histidine / 0.2M sucrose pH 6.0 by discontinuous diafiltration using a PES centrifugal concentrator with a molecular weight cutoff of 30 kDa (Vivaspin). Polysorbate 20 was added from a 1% w / v stock solution 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 frozen and stored at -80°C.

[0616] Biophysical properties of AFF4-WT-drug conjugates

[0617] All AFF4-WT-drug conjugates were analyzed for protein concentration and monomer content (by calculating the area under the curve in size exclusion chromatography at 214 nm), their endotoxin content (determined by horseshoe crab amebocyte lysate), and their free drug concentration (by reverse phase chromatography). The drug to antibody ratio of cysteine ​​conjugates (AFF4-WT-VC-MMAE, AFF4-WT-Gluc-MMAE, AFF4-WT-VA-SG3199) was determined by hydrophobic interaction chromatography, while the drug to antibody ratio of lysine conjugate (AFF4-WT-sulfo-SPDB-DM4) was determined by size exclusion chromatography by comparing the areas under the curve obtained at 252 nm (maximum absorbance of DM4) and 280 nm (maximum absorbance of 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:

[0618] Table 14: Biophysical properties of AFF4-WT-drug conjugates

[0619]

[0620] Example 8

[0621] Pharmacological properties of AFF4-WT antibody-drug conjugates

[0622] Internalization and lysosomal localization of AFF4-WT after binding to L1CAM on the surface of JIMT-1 breast cancer cells, OVCAR-3 ovarian cancer cells, and MeWo melanoma cells

[0623] In order to exert its cytotoxic effect, the antibody-drug conjugate needs to bind to and be effectively internalized in the lysosomal compartment of the cancer cell, where it is processed by specific proteases and releases its cytotoxic payload. To test the internalization ability of AFF4-WT and track its intracellular localization, human Fabfluor-pH red antibody labeling dye was used, which is an anti-human IgG (Fc-specific) Fab fragment that is coupled to a pH-sensitive dye that is almost non-fluorescent at neutral pH but becomes highly fluorescent at low pH. After binding to AFF4-WT, the Fabfluor labeling reagent is co-internalized with AFF4-VT and indicates whether the antibody has reached the low pH environment of the lysosomal pathway.

[0624] The lysosomal uptake of AFF4-WT was determined using JIMT-1 breast cancer cells, OVCAR-3 ovarian cancer cells, and MeWo melanoma cells expressing L1CAM. To this end, 20'000 cells / well (4x10 5 Cells were seeded at a density of 10 cells / ml, 50 μL / well). JIMT-1 cells were seeded in DMEM / 10% FCS, OVCAR-3 cells were seeded in RPMI1640 / 10% FCS, and MeWo cells were seeded in EMEM / 10% FCS. After incubation at 37°C / 5% CO2 for approximately 5 hours, AFF4-WT or chimeric human IgG1 isotype control antibody was added to Human Fabfluor-pH red antibody labeling dye (Sartorius, catalog number 4722) was mixed and diluted in RPMI164 / 10% FCS to a concentration of 2 μg / mL (the molar ratio of antibody to labeling dye was 1:3). After incubation at 37°C for 15 minutes, 50 μL / well of the mixture was added to each cell line so that the final well concentrations were 1 μg / mL of antibody and labeling dye, respectively.

[0625] The incubation lasted for 15 minutes or 20 hours, respectively. Thereafter, adherent cells were washed with PBS and then Cells were treated with cell detachment solution (Capricorn) for 5 minutes and then analyzed on an image-based cytometer (Nucleocounter NC-3000, ChemoMetec) using an excitation wavelength of 630 nm and an emission filter of 740 nm ± 60 nm.

[0626] Fig.15 It was shown that the fluorescence of AFF4-WT treated JIMT-1, OVCAR-3 and MeWo cells increased significantly after 20 hours compared with cells treated with chimeric human IgG1 isotype control antibody, indicating that AFF4-WT had been efficiently internalized by all cell lines and reached the lysosomal compartment.

[0627] In vitro cytotoxicity of cell lines expressing L1CAM

[0628] Then, the ability of different AFF4-WT conjugate drugs to induce cytotoxicity was tested in the cell-based in vitro killing assay. Using the same culture medium for internalization assay as described above, JIMT-1, OVCAR-3 and MeWo cells were inoculated into white 96-well culture plates with a total volume of 90 μL at 2'250, 2'100 and 2'000 cells / well, respectively. Cells were allowed to adhere for 6.5 to 7.5 hours, followed by the addition of 10 μL / wells of the AFF4-WT-conjugate drugs serially diluted in RPMI / 10% FCS. The final concentration of the conjugate in the assay is in the range of 100nM to 1.3pM (0.05pM 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. The assay plates were incubated at 37°C / 5% CO2 for 6 days and cell viability was assessed by ATP quantification and luminescence measurements using CellTiter Glo2.0 (Promega) according to the manufacturer's instructions. Luminescence readings were converted to viability values ​​in % using wells containing only cells and wells containing cells incubated in the presence of 10 μM doxorubicin, respectively, as 100% and 0% viability reference values. GraphPad Prism was used to fit the viability values ​​obtained at different AFF4-WT-conjugated drug concentrations to a 4-parameter logistic curve and used to calculate the corresponding IC values ​​for different conjugates. 50 concentration.

[0629] Fig.16 It was shown 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 cells, OVCAR-3 cells, and MeWo cells. The corresponding IC 50 The values ​​are summarized in Table 15 below.

[0630] Table 15: Cytotoxicity of AFF4-WT conjugates against different cancer cell lines 15

[0631]

[0632]

[0633] 15 Digital Indicator IC 50 The value is in nM.

[0634] Inhibition of tumor growth in vivo

[0635] 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.

[0636] JIMT-1 breast cancer cells (5 x 10 6 cells in 100 μL PBS) were injected into female athymic nude (Crl:NU(NCr)-Foxn1 nu ) mice. Tumors were measured with a caliper and tumor volume (TV) was calculated using the following formula: TV = (W 2 x L) / 2 (L=length of the tumor, W=vertical length of the tumor, L>W).

[0637] When the tumor reaches an average size of 100-150 mm 3 Mice were randomly assigned to 5 different groups at 4 pm. Four groups (n=6 per group) received a single intravenous (iv) injection of 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), while the fifth group (n=8) received vehicle as a control. Tumor volume was measured twice a week after injection.

[0638] like Fig.17 As shown in A, a single treatment with any 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.

[0639] OVCAR-3 ovarian cancer cells (1x 10 710 cells in Matrigel) were injected subcutaneously into female Jan:NMRI-nu / nu mice. Tumors were measured with a caliper and tumor volumes were calculated as described above. Once tumors reached an average volume of approximately 0.14 cm 3 , mice were randomly assigned to 5 groups (n=5) and treatment was started. Each group received two intravenous injections, two weeks apart, of 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 received vehicle as a control. Tumor volume was measured twice a week after randomization.

[0640] Fig.17 B shows that all AFF4-WT drug conjugates induced anti-tumor 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.

[0641] MeWo melanoma cells (1x 10 7 100 mg / ml of 100 μg / ml of 100 μg / ml of 100 μg / ml were injected subcutaneously into female Jan:NMRI-nu / nu mice. Tumor volume was determined with a caliper as described above. When tumors reached an average volume of approximately 0.14 cm 3 At 1 , mice were randomly assigned to 5 groups (n=5) and treated with a single intravenous injection of 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 control. Tumor volume was measured twice a week after treatment.

[0642] Fig.17 C shows that antitumor responses were observed in all AFF4-WT drug conjugates tested. Treatment with AFF4-WT-VC-MMAE or AFF4-WT-Gluc-MMAE induced tumor growth arrest, while treatment with AFF4-WT-VA-SG3199 or AFF4-WT-Sulfo-SPDB-DM4 caused a decrease in tumor growth.

[0643] Efficacy of different doses of AFF4-WT-VC-MMAE in the JIMT-1 breast cancer xenograft model

[0644] JIMT-1 breast cancer cells (5 x 10 6 cells in 100 μL PBS) were injected into female athymic nude (Crl:NU(NCr)-Foxn1 nu ) mice. When the tumors reached an average volume of approximately 100 mm 3 At 1 , mice were randomly assigned to 5 different groups. Then, four groups (n = 6 per group) received a single intravenous injection of AFF4-WT-VC-MMAE at 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg or 3.0 mg / kg, while the fifth group (n = 8) received the vehicle as a control. Tumor volume was measured twice a week after injection.

[0645] Fig.18 All doses of AFF4-WT-VC-MMAE inhibited JIMT-1 tumor growth, with a clear dose-response relationship between the dose levels tested and the extent of the antitumor effect.

[0646] Efficacy of AFF4-WT-VC-MMAE in a cancer patient-derived ovarian cancer xenograft model

[0647] The efficacy of AFF4-WT-VC-MMAE was investigated in four patient-derived human ovarian cancer xenograft (PDX) models with confirmed L1CAM expression. Female athymic nude-Foxn1nu mice were subcutaneously implanted with fragments of any of the four L1CAM-expressing PDX tumors. Tumor growth was monitored regularly and when the mean tumor volume reached 150-300 mm 3 At , animals were divided into treatment and control groups (n = 5 in each group) according to tumor volume. Then, AFF4-WT-VC-MMAE (3 mg / kg) was injected intravenously every two weeks to the treatment group, while the vehicle was injected every two weeks to the control group. Tumor volume was recorded twice a week, and the tumor growth inhibition degree (TGI) of the treatment group (T) relative to the control group (C) was calculated using the initial (i) and final (f) tumor measurements by the following formula:

[0648]

[0649] like Fig.19 As shown, AFF4-WT-VC-MMAE inhibited tumor growth in all four L1CAM-expressing ovarian cancer PDX models. At the last observed time point, the degree of tumor growth inhibition was 82% (CTG0868), 113% (CTG1086), 95% (CTG1649), and 74% (CTG3383), respectively.

[0650] In vitro cytotoxicity of AFF4-WT-VC-MMAE against L1CAM-expressing cell lines and its correlation with L1CAM expression levels

[0651] A series of 25 ovarian cancer, endometrial cancer, breast cancer, melanoma and neuroblastoma cell lines were inoculated into the wells of white 96-well cell culture plates at the desired density in a total volume of 90 μL of cell culture medium. In order to obtain the best assay window, the inoculation density and cell culture medium of each cell line were determined respectively. The cells were allowed to adhere for about 6 hours, followed by the addition of 10 μL / well of AFF4-WT-VC-MMAE serially diluted in RPMI / 10% FCS. The final concentration of AFF4-WT-VC-MMAE in the assay was in the range of 100nM to 1.3pM. As a positive control for 100% killing, doxorubicin was added to a separate well at a final concentration of 10 μM. The assay plate was incubated at 37°C / 5% CO2 for 5-12 days, and according to the manufacturer's instructions, cell viability was assessed by using CellTiter Glo2.0 (Promega) for ATP quantification and luminescence measurement. Luminescence readings obtained at different AFF4-WT-VC-MMAE concentrations were converted to % viability values ​​and fitted to a 4-parameter logistic curve using GraphPad Prism as described above. The IC values ​​for each individual cell line were derived. 50 concentration.

[0652] At the same time, determine the approximate number of L1CAM molecules expressed on the cell surface of each cancer cell line. To this end, first use Quantibrite beads labeled with four different densities of phycoerythrin (PE) molecules (BD Biosciences) to establish a calibration line. The beads are resuspended in 500 μL PBS, and 30 μL of this suspension is loaded on NC-slide A2 (ChemoMetec) and analyzed using an image-based cytometer (Nucleocounter, ChemoMetec). Use a green light source (LED530) and an exposure time of 500 milliseconds for analysis. After gating a single bead of four bead populations, determine the mean fluorescence intensity (MFI) and draw the corresponding number of PE molecules of each bead. Using GraphPad Prism, the data are fitted with a linear regression curve using the following equation:

[0653] Y=m×X+c,

[0654] Where y is the MFI and x is the number of PE molecules per bead provided by the manufacturer. The 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 plated at approximately 3 x 10 5 The density of each cell / well was transferred to two holes of a 96-well polypropylene plate. The plate was centrifuged at 300g 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 PE-labeled anti-L1CAM antibody or PE-labeled isotype control antibody. After incubation at 4°C for 1 hour in the dark, the cells were centrifuged again at 300g and 4°C for 5 minutes, and the supernatant was removed. Subsequently, the cells were resuspended in 300 μl PBS and washed twice, followed by centrifugation (5 minutes, 300g, 4°C) and the supernatant was removed. After the last centrifugation step, the cells were resuspended in 75 μl PBS, and 30 μl of cell suspension was analyzed on Nucleocounter, using the same settings as described above for determining the calibration line. A histogram of viable cells was generated, and the MFI value was inserted into the linear regression equation established with PE-labeled beads as a y value. Solve the equation for x (= number of PE molecules) and calculate the number of L1CAM molecules per cancer cell using the following formula:

[0655]

[0656] where DAR is the dye to antibody ratio of anti-L1CAM and isotype control antibodies, respectively, as reported by the manufacturers.

[0657] Table 16 below shows the number of L1CAM molecules on different cancer cell lines and the corresponding cytotoxic potency (IC 50 value):

[0658] Table 16: L1CAM expression levels on cancer cell lines from different sources and the corresponding cytotoxicity of AFF-WT-VC-MMAE 16

[0659]

[0660]

[0661] 16 SD: standard deviation, n: number of independent measurements

[0662] AFF4-WT-VC-MMAE induced strong cytotoxic effects on most of the cell lines tested. As shown in Table 16, the cytotoxic potency of AFF4-WT-VC-MMAE was closely related to the number of L1CAM molecules on the cell surface of the corresponding cell lines. Cell lines with high L1CAM expression were the most sensitive to the cytotoxic effects induced by AFF4-WT-VC-MMAE (= low IC 50 ), whereas cell lines with lower or absent L1CAM expression were less sensitive (high IC 50 ).

[0663] Bystander cytotoxicity of AFF4-WT-VC-MMAE

[0664] In human tumors, the expression of L1CAM may be heterogeneous, with some cancer cells expressing high L1CAM levels and other cells expressing low levels of L1CAM or not expressing it at all. Since the efficacy of AFF4-WT-VC-MMAE is related to the level of L1CAM expression (see Table 16), a question arises as to whether tumors with heterogeneous L1CAM expression can be effectively targeted by AFF4-WT-VC-MMAE. Therefore, it was investigated whether AFF4-WT-VC-MMAE exhibits a so-called cytotoxic bystander effect. ADCs with such a bystander effect are taken up and processed by antigen-positive cancer cells in the form of releasing a cytotoxic payload that can diffuse freely to neighboring cells and therefore have the ability to kill these cells independently of their antigen expression. Therefore, such ADCs are well suited for treating tumors with heterogeneous target expression.

[0665] To evaluate the bystander activity of AFF4-WT-VC-MMAE, L1CAM-high-expressing JIMT-1 breast cancer cells and L1CAM-low-expressing MDA-MB-468 optimal cancer cells (see Table 16) were inoculated at 5×10 4 A density of 10 cells / well was seeded into 6 wells of a 24-well cell culture plate in a total volume of 400 μL RPMI / 10% FCS. Six additional wells were filled with 400 μL RPMI / 10% FCS alone. After incubation for 5 hours 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 concentration of AFF4-WT-VC-MMAE in the assay ranged from 100 nM to 10 pM. One well each of the JIMT-1 seeded wells, MDA-MB-468 seeded wells, and wells containing cell culture medium alone was left untreated as a control. The plates were incubated at 37°C / 5% CO2 for 4 days,

[0666] L1CAM low-expressing MDA-MB468 cells were then seeded into wells of a white 96-well cell culture plate at a density of 1500 cells / well in a total volume of 75 μL RPMI / 10% FCS, followed by incubation at 37° C. / 5% CO 2 for 4 hours.

[0667] The supernatant in all holes of the 24-orifice plate of incubation for 4 days is transferred to a sterile tube, and centrifuged for 10 minutes at 2000rcf. Supernatant is transferred to a new sterile tube, and then every kind of supernatant of 25 μ L is added to the MDA-MB-468 cells of inoculation. As a negative control, fresh RPMI / 10% FCS culture medium is added to three holes, and as the positive control of maximum growth inhibition, doxorubicin is added to three holes with a final concentration of 10 μM. After adding supernatant, culture medium and doxorubicin, plate is incubated for 6 days at 37 ℃ / 5% CO2. Then, according to the explanation of the manufacturer, the ATP quantitative and luminescence measurement carried out by using CellTiter Glo2.0 (Puluomag company) is assessed cell survival rate. As described above, luminescence reading is converted to survival value %, and GraphPad Prism is used to be fitted to a 4-parameter logistic curve.

[0668] Fig. 20 The survival rate of MDA-MB-468 cells after incubation with supernatant from JIMT-1 or MDA-MB-468 cells is shown, and the JIMT-1 or MDA-MB-468 cells have been pre-incubated with serial dilutions of AFF4-WT-VC-MMAE. As a control, the survival rate obtained by the same serial dilutions of AFF4-WT-VC-MMAE pre-incubated in the absence of cells (only cell culture medium) is also shown. Consistent with the virtual insensitivity of MDA-MB-468 to AFF4-WT-VC-MMAE (Table 16), only marginal cytotoxic effects (IC) were observed when AFF4-WT-VC-MMAE was pre-incubated on MDA-MB-468 cells that lowly expressed L1CAM or in the absence of cells. 50 >5 nM). In contrast, AFF4-WT-VC-MMAE pre-incubated on JIMT-1 cells with high expression of L1CAM showed significantly stronger cytotoxicity against MDA-MB-468 cells, with IC 50The value is about 500pM. This result strongly indicates that JIMT-1 cells with high L1CAM expression take up and process AFF4-WT-VC-MMAE to produce free MMAE toxin, which is released into the supernatant where it can exert its cytotoxic effect. Therefore, these data support the view that AFF4-WT-VC-MMAE displays bystander cytotoxic activity. In tumors with heterogeneous L1CAM expression levels, this activity of AFF4-WT-VC-MMAE may ensure that not only high L1CAM expressing tumor cells are killed, but also neighboring low expressing tumor cells are effectively eliminated, and a good overall anti-tumor response is achieved.

Claims

1. An antibody that specifically binds to human L1CAM, comprising: (a) a heavy chain variable region (VH) complementarity determining region (CDR) 1, wherein the VH CDR1 comprises the amino acid sequence of GYSITSDYX1WN (SEQ ID NO: 16), wherein: X1 is A or T; Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 16); (b) VH CDR2, comprising the amino acid sequence of YISYSGSX1SYX2PSLKS (SEQ ID NO: 17), wherein X1 is F or Y, and X2 is H or N; Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 17); (c) a VH CDR3 comprising the amino acid sequence of SX1SYX2YGFAY ​​(SEQ ID NO: 18), wherein: X1 is L or F, and X2 is G, S or A; Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 18); (d) a light chain variable region (VL) CDR1 comprising the amino acid sequence of KASQDVSSAVA (SEQ ID NO: 4); Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (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; or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 19); and (f) a VL CDR3 comprising the amino acid sequence of QQHYSTPWT (SEQ ID NO: 6); Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 6).

2. An antibody that specifically binds to human L1CAM, comprising: (a) a heavy chain variable region (VH) complementarity determining region (CDR) 1, wherein the VH CDR1 comprises the amino acid sequence of GYSITSDYX1WN (SEQ ID NO: 16), wherein: X1 is A or T; (b) VH CDR2, comprising the amino acid sequence of YISYSGSX1SYX2PSLKS (SEQ ID NO: 17), wherein X1 is F or Y, and X2 is H or N; (c) a VH CDR3 comprising the amino acid sequence of SX1SYX2YGFAY ​​(SEQ ID NO: 18), wherein: X1 is L or F, and X2 is G, S or A; (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).

3. The antibody that specifically binds to human L1CAM according to claim 1 or 2, wherein: (a) the VH CDR1 comprises the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9) or a sequence comprising 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 9); and / or (b) the VH CDR2 comprises the amino acid sequence of YISYSGSX1SYX2PSLKS (SEQ ID NO: 17), wherein X1 is Y, and / or X2 is N; Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 17); and / or (c) the VH CDR3 comprises the amino acid sequence of SX1SYX2YGFAY ​​(SEQ ID NO: 18), wherein: X1 is F, and / or X2 is S; Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 18); and / or (d) the VL CDR2 comprises the amino acid sequence of SASYRYT (SEQ ID NO: 5); Or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence (SEQ ID NO: 5).

4. The antibody that specifically binds to human L1CAM according to any one of claims 1 to 3, wherein: (a) the VH CDR1 comprises the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9); and / or (b) the VH CDR2 comprises the amino acid sequence of YISYSGSX1SYX2PSLKS (SEQ ID NO: 17), wherein X1 is Y, and / or X2 is N; and / or (c) the VH CDR3 comprises the amino acid sequence of SX1SYX2YGFAY ​​(SEQ ID NO: 18), wherein: X1 is F, and / or X2 is S; and / or (d) the VL CDR2 comprises the amino acid sequence of SASYRYT (SEQ ID NO: 5).

5. The antibody that specifically binds to human L1CAM according to any one of claims 1 to 4, comprising: (a) a heavy chain variable region (VH), said VH comprising VH CDR1, VH CDR2 and VH CDR3, said VH CDR1 comprising the amino acid sequence of GYSITSDYTWN (SEQ ID NO: 9) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO: 9), said VH CDR2 comprising the amino acid sequence of YISYSGSYSYNPSLKS (SEQ ID NO: 11) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO: 11), said VH CDR3 comprising the amino acid sequence of SFSYSYGFAY ​​(SEQ ID NO: 14) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO: 14); and (b) a light chain variable region (VL), said VL comprising VL CDR1, VL CDR2 and VL CDR3, said VL CDR1 comprising the amino acid sequence of KASQDVSSAVA (SEQ ID NO:4) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO:4), said VL CDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO:5) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO:5), and said VL CDR3 comprising the amino acid sequence of QQHYSTPWT (SEQ ID NO:6) or a sequence containing 1, 2 or 3 amino acid substitutions in the sequence of (SEQ ID NO:6).

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

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

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

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

20.

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

38.

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

20.

12. The antibody according to any one of claims 1 to 11, wherein 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 including 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 intracellular antibody, a heterologous conjugate antibody, a single domain antibody, a monovalent antibody, a single chain antibody or a single chain Fv (scFv), a camelid antibody, an affinity antibody affibodies, anticalins, affilins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centyrins), fynomers, Kunitz domains, pronectins and OBodys, 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 wherein the antibody is included in a chimeric antigen receptor (CAR).

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 human immunoglobulin group consisting of IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, and / or wherein the light chain constant region is selected from the human immunoglobulin group consisting of IgGκ and IgGλ.

14. The antibody according to 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, preferably, wherein the human IgG heavy chain constant region variant binds to one or more human Fcγ receptors with higher affinity than the wild-type human IgG heavy chain constant region binds to human Fcγ receptors, and the one or more human Fcγ receptors are selected from the group consisting of FcγRI, FcγRIIA, and FcγRIIIA.

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

16. The antibody according to any one of claims 1 to 15, wherein the antibody (i) binds to human L1CAM within the fibronectin type III domains 1-3 (FNIII 1-3) of L1CAM, and / or (ii) binds to human L1CAM with an affinity (KD) of 20 nM or less, 10 nM or less, or 1 nM or less, and / or (iii) with an affinity (K of 20 nM or less, 10 nM or less, or 1 nM or less D ) binds to cynomolgus monkey L1CAM, and / or (iv) inhibiting the migration of tumor cells 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 the SKOV-3ip xenograft model, and / or (vii) reduce metastasis formation in the mouse xenograft model MDA-MB231, and / or (viii) exhibit ADCC activity in vitro and / or bind 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.

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

18. The antibody according to any one of claims 1 to 17, wherein the antibody (a) linked to a therapeutically active substance, Preferably connected to chemotherapeutic 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.

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

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

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, anti-tumor antibiotics, immunomodulators, nucleic acids for gene therapy, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormone agents, anti-hormones, corticosteroids, photoactive therapeutic agents, oligonucleotides, radioisotopes, radiosensitizers, topoisomerase inhibitors and tyrosine kinase inhibitors.

22. The antibody of claim 21, wherein the mitotic inhibitor is selected from the group consisting of maytansine compounds and auristatins.

23. The antibody of claim 21, wherein the DNA damaging agent is selected from pyrrolobenzodiazepine (PBD) and pyridobenzodiazepine (PDD).

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

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

38.

28. A nucleic acid: (i) which encodes the antibody according to any one of claims 1 to 27, and / or (ii) it encodes 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) it encodes a sequence according to SEQ ID NO: 30 and / or according to SEQ ID NO: 20, and / or (iv) it comprises a sequence encoding a complementarity determining region sequence of an antibody according to any one of claims 1 to 27, Preferably wherein the nucleic acid is part of a vector.

29. A host cell comprising the nucleic acid according to claim 28.

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

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

32. 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 in treating or preventing a hyperproliferative disorder, a tumor disease, a disorder associated with neovascularization, and / or a disorder associated with abnormal neurogenesis.

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