Anti-L1-CAM Antibodies and Their Use for Diagnostic and Therapeutic Applications

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to L1-CAM (CD171), polynucleotides encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence found in the antibodies or antigen-binding fragments thereof of the invention, host cells comprising the polynucleotides of the invention, immunoconjugates comprising the antibodies or antigen-binding fragments thereof of the invention and active agents, pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof or immunoconjugates of the invention, and their use in therapy and / or diagnosis. The antibodies, antigen-binding fragments thereof, immunoconjugates, and pharmaceutical compositions described herein are particularly useful for the treatment or diagnosis of L1-CAM (CD171)-associated cancers.

Description

[Technical Field]

[0001] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to L1-CAM (CD171), polynucleotides encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence found in the antibodies or antigen-binding fragments thereof of the invention, host cells comprising the polynucleotides of the invention, immunoconjugates comprising the antibodies or antigen-binding fragments thereof of the invention and active agents, pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof or immunoconjugates of the invention, and their use in therapy and / or diagnosis. The antibodies, antigen-binding fragments thereof, immunoconjugates, and pharmaceutical compositions described herein are particularly useful for the treatment or diagnosis of L1-CAM (CD171)-associated cancers. [Background technology]

[0002] With the advent of chimeric antigen receptor (CAR) technology (Sadelain M et al., Cancer Discov 3:388-98 (2013)), therapeutic research using anti-L1-CAM-redirected gene-modified T cells has rapidly expanded, and several clinical trials have used L1-CAM-CAR-modified T cells, such as NCT00889954 (all L1-CAM+ cancers) and NCT01935843 (L1-CAM+ solid tumors). See Hong H et al., J Immunother 37:93-104 (2014). While T cells can effectively target tumors with low levels of L1-CAM, concerns remain regarding potential bystander toxicity in normal tissues with low levels of L1-CAM expression.

[0003] Aside from toxicity, cell harvesting, processing, storage, transportation, and product release regulations for lymphocyte therapy can be challenging, especially if these cells must be genetically modified. T cell exhaustion, survival, and homing are suboptimal despite the infusion of billions of these T cells. Furthermore, CAR-modified T cells are no exception to the immunosuppressive tumor microenvironment, where Tregs, tumor-associated macrophages, and myeloid suppressor cells cooperate to circumvent the antitumor properties of CAR-modified T cells. Furthermore, CAR-modified T cells are subject to the same immunosuppressive constraints faced by classical T cells, including anergy following engagement of CTLA4 by B7 or PD-1 by PD-L1 (B7-H1) on tumor cells. Therefore, clinically effective alternatives to CAR-modified T cell therapy for the treatment of cancer are needed.

[0004] L1-CAM has been shown to be expressed in many human cancers and is often associated with poor prognosis, primarily due to the motility and invasion-promoting functions of L1-CAM (Altevogt et al., International Journal of Cancer, 138, 1565-1576 (2016)). Kiefel and colleagues (Cell Adhesion & Migration 6:4, 374-384) describe L1-CAM as a major driver of tumor cell invasion and motility.

[0005] Dobberstein et al. demonstrated that L1 CAM is expressed in triple-negative breast cancer and is inversely correlated with the androgen receptor (Dobberstein et al., BMC Cancer 2014, 14:958). Eliane Fischer et al. demonstrated the efficacy of L1-CAM-targeted antibody therapy for disseminated ovarian cancer and 177Lu-radioimmunotherapy has been described (E Fischer et al., Int. J. Cancer: 130, 2715-2721 (2012)). Wachowiak and colleagues have reported that increased L1-CAM levels are associated with glioblastoma and metastatic brain tumors (Wachowiak et al.; Medicine (2018) 97:38). Rached et al. showed that L1-CAM knockout radiosensitized neuroblastoma IMR-32 cells by simultaneously reducing MycN but increasing PTEN protein expression (Rached et al., International; Journal of Oncology, 49:1722-1730, 2016). L1-CAM has also been shown to increase adhesion-mediated growth and chemotherapy resistance in retinoblastoma (Jo et al., Oncotarget, 2017, Vol. 8, pp. 15441-15452). Terraneo et al. have shown that L1-CAM confers radioresistance to ovarian cancer and defines a new cancer stem cell population (Terraneo et al., Cancers 2020, 12, 217). Expression profile analysis in multiple human tumors identified L1-CAM as a molecular marker for differential diagnosis and targeted therapy (Huszar et al., Human Pathology (2006), 37, 1000-1008).

[0006] Therapeutic antibodies targeting L1-CAM have been previously described. Hoefnagel et al. (European Journal of Nuclear Medicine, 2001, 28:359-368) disclosed targeting of neuroblastoma with the anti-L1-CAM antibody mAb chCE7. Therapeutic efficacy in neuroblastoma xenograft models and imaging of neuroblastoma patients have been described. The same antibody, chCE7, has been shown to bind to an isoform of L1-CAM present in renal cancer cells (Meli et al., International Journal of Cancer, 83, 401-408, 1999). Novak-Hofer et al. (J. Nucl. Med., 1992, 33:231-236) described radioimmunolocalization of neuroblastoma xenografts using the chimeric antibody chCE7. L1-CAM has also been shown to define the regenerative origin of metastasis-initiating cells in colorectal cancer (Ganesh et al., 2020, 28-45).

[0007] Because L1-CAM is known to be expressed in peripheral nervous tissue, side effects of L1-CAM-targeted therapy are common. Specifically, these side effects include ADCC, CDC, and other immune reactions. Therefore, it is important to provide antibodies or fragments thereof that have reduced toxic effects. In the case of targeted radionuclide therapy, a low K associated with a low k-off upon binding to the tumor is essential. D It is particularly desirable to provide an antibody that has a .DELTA..times ...

[0008] WO 2018 / 232188 discloses certain anti-L1-CAM antibodies and uses thereof.

[0009] Amstutz et al. (Amstutz, Int. J. Cancer: 53, 147-152 (1993)) disclose the production and characterization of a mouse / human chimeric antibody against human neuroblastoma. Umana et al. (Nature Biotechnology, 1999, 176-180) disclose engineered glycoforms of anti-neuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxicity. The article "Improvement of Biophysical Properties and Affinity of a Human Anti-L1-CAM Therapeutic Antibody through Antibody Engineering Based on Computational Methods" in the International Journal of Molecular Sciences (Vol. 22, No. 13, pp. 6696-6796) describes certain variants of the antibody Ab417 (a humanized antibody that binds to L1-CAM) that have favorable properties in terms of production stability and antitumor activity. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2018 / 232188 [Non-patent literature]

[0011] [Non-Patent Document 1] Sadelain M et al., Cancer Discov 3:388-98(2013) [Non-patent document 2] Hong H et al., J Immunother 37:93-104(2014) [Non-patent document 3] Altevogt et al.,International Journal of Cancer,138,1565-1576(2016) [Non-patent document 4] Kiefel and colleagues (Cell Adhesion & Migration 6:4, 374-384 [Non-patent document 5] Dobberstein et al.,BMC Cancer 2014,14:958

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[0012] The present inventors have surprisingly found that the antibodies or antigen-binding fragments of the present invention exhibit improved affinity for binding to L1-CAM compared to the closely related antibodies from which they are derived, particularly the CE7 antibody (Amstutz et al.). Specifically, at least in part, the antibodies of the present invention are humanized antibodies, and one skilled in the art would expect that humanization of the antibody would negatively affect the antibody's binding affinity for its antigen. Thus, the present invention is based, at least in part, on the unexpected discovery that the antibodies of the present invention obtained when the CE7 antibody is humanized exhibit surprisingly improved binding affinity for L1-CAM compared to the CE7 antibody, as demonstrated, inter alia, in SPR studies performed by the present inventors (see Examples 6 and 7, below).

[0013] The inventors further observe that the antibodies or antigen-binding fragments thereof of the present invention exhibit at least equivalent (and no worse) affinity compared to the CE7 antibody from which they are derived via the humanization process.

[0014] The inventors have further demonstrated that the antibodies or antigen-binding fragments thereof of the present invention surprisingly exhibit improved stability compared to the related antibodies from which they are derived, in particular the CE7 antibody, which is manifested at least in a higher onset temperature of aggregation, as demonstrated in PANTA studies (see Example 23).

[0015] The present invention is summarized in the following embodiments.

[0016] In a first embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to L1-CAM (CD171), wherein the antibody or antigen-binding fragment thereof is: A variable heavy chain region comprising: CDR-H1, The sequence set forth in SEQ ID NO: 1 (GYWMH), The sequence set forth in SEQ ID NO: 2 (GYYMH), The sequence set forth in SEQ ID NO: 3 (GYFMH), and the sequence set forth in SEQ ID NO: 4 (GYLMH), and CDR-H2, The sequence set forth in SEQ ID NO: 5 (EINPSNGRTNYNERFQG), The sequence set forth in SEQ ID NO: 6 (EINPSNGRTNYNEKFQG), The sequence set forth in SEQ ID NO: 7 (EINPSNGRTNYNERFKS), The sequence set forth in SEQ ID NO: 8 (EINPSNGRTNYNERLKS), The sequence set forth in SEQ ID NO: 9 (EINPSNARTNYNERFQG), Sequence set forth in SEQ ID NO: 10 (EINPSNARTNYNEKFQG) The sequence set forth in SEQ ID NO: 11 (EINPSNARTNYNERFKS), and CDR-H2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 12 (EINPSNARTNYNERLKS), and a variable heavy chain region comprising a CDR-H3 characterized by the sequence set forth in SEQ ID NO: 13 (DYYGTSYNFDY); and / or A variable light chain region comprising: CDR-L1, The sequence set forth in SEQ ID NO: 14 (RANEDINNRLA), The sequence set forth in SEQ ID NO: 15 (KANEDINNRLA), The sequence set forth in SEQ ID NO: 16 (QANEDINNRLA), the sequence set forth in SEQ ID NO: 17 (RANEDINARLA), The sequence set forth in SEQ ID NO: 18 (KANEDINARLA), The sequence set forth in SEQ ID NO: 19 (QANEDINARLA), The sequence set forth in SEQ ID NO: 20 (RANEDINLRLA), The sequence set forth in SEQ ID NO: 21 (KANEDINLRLA), and CDR-L1 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 22 (QANEDINLRLA), and CDR-L2, The sequence set forth in SEQ ID NO: 23 (GATNLVT), and CDR-L2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 24 (GASNLVS), and CDR-L3, The sequence set forth in SEQ ID NO: 25 (QQYWSTPFT), The sequence set forth in SEQ ID NO: 26 (QQYYSTPFT), and and a variable light chain region comprising a CDR-L3 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 27 (QQYFSTPFT).

[0017] In a second embodiment, the present invention relates to a polynucleotide encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence according to the first embodiment.

[0018] In a third embodiment, the present invention relates to a host cell comprising the polynucleotide of the second embodiment.

[0019] In a fourth embodiment, the present invention relates to an immunoconjugate comprising the antibody or antigen fragment thereof of the first embodiment and an active agent, preferably wherein the active agent is a cytotoxic agent or a prodrug thereof.

[0020] In a fifth embodiment, the present invention relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the first embodiment, or the immunoconjugate of the fourth embodiment, and a pharmaceutically acceptable carrier.

[0021] In a sixth embodiment, the present invention relates to the antibody or antigen-binding fragment thereof of the first embodiment, or the immunoconjugate of the fourth embodiment, for use as a medicament.

[0022] In a seventh embodiment, the present invention relates to the antibody or antigen-binding fragment thereof of the first embodiment, or the immunoconjugate of the fourth embodiment, for use in the treatment of L1-CAM (CD171)-associated cancer.

[0023] In an eighth embodiment, the present invention relates to the antibody or antigen-binding fragment thereof of the first embodiment, or the immunoconjugate of the fourth embodiment, for use in diagnostics.

[0024] In a ninth embodiment, the present invention relates to the antibody or antigen-binding fragment thereof of the first embodiment, or the immunoconjugate of the fourth embodiment, for use in the diagnosis of L1-CAM (CD171)-associated cancer. [Brief explanation of the drawings]

[0025] The present invention is further described in the following figures / drawings, which should not be construed as limiting. [Figure 1-1] Figure 1 presents the purification profile of rAB, variants 1-18, as seen in Example 2. Non-reducing PAGE with Coomassie Blue staining. MW. Molecular weight marker. IN. Input. FT. Flow-through. W. Wash. E. Elution fractions. [Figure 1-2] Figure 1 presents the purification profile of rAB, variants 1-18, as seen in Example 2. Non-reducing PAGE with Coomassie Blue staining. MW. Molecular weight marker. IN. Input. FT. Flow-through. W. Wash. E. Elution fractions. [Figure 1-3] Figure 1 presents the purification profile of rAB, variants 1-18, as seen in Example 2. Non-reducing PAGE with Coomassie Blue staining. MW. Molecular weight marker. IN. Input. FT. Flow-through. W. Wash. E. Elution fractions. [Figure 2]Final sample QC in Example 2 is presented. Coomassie blue staining. Reducing and non-reducing PAGE analysis. MW. Molecular weight marker. 2 μg loaded per lane. [Figure 3] Figure 1 presents the purification profile of rAB, variants 19-28, as seen in Example 3. Non-reducing PAGE with Coomassie Blue staining. MW. Molecular weight marker. IN. Input. FT. Flow-through. W. Wash. E. Elution fractions. [Figure 4] Final sample QC in Example 3 is presented. Coomassie blue staining. Reducing and non-reducing PAGE analysis. MW. Molecular weight marker. 2 μg loaded per lane. [Figure 5] 1 presents the purification profile of rAB as seen in Example 4. Non-reducing PAGE with Coomassie Blue staining. MW. Molecular weight marker. IN. Input. FT. Flow-through. W. Wash. E. Elution fractions. [Figure 6] Final sample QC in Example 4 is presented. Coomassie blue staining. Reducing and non-reducing PAGE analysis. MW. Molecular weight marker. 2 μg loaded per lane. [Figure 7-1] Presented are the SEC-HPLC profiles obtained for CE7-variant 4_AG (part 1), CE7-variant 7_AG (part 2), CE7-variant 18_AG (part 3), standard application (part 4), and blank sample (part 5). [Figure 7-2] Presented are the SEC-HPLC profiles obtained for CE7-variant 4_AG (part 1), CE7-variant 7_AG (part 2), CE7-variant 18_AG (part 3), standard application (part 4), and blank sample (part 5). [Figure 7-3] Presented are the SEC-HPLC profiles obtained for CE7-variant 4_AG (part 1), CE7-variant 7_AG (part 2), CE7-variant 18_AG (part 3), standard application (part 4), and blank sample (part 5). [Figure 7-4]Presented are the SEC-HPLC profiles obtained for CE7-variant 4_AG (part 1), CE7-variant 7_AG (part 2), CE7-variant 18_AG (part 3), standard application (part 4), and blank sample (part 5). [Figure 7-5] Presented are the SEC-HPLC profiles obtained for CE7-variant 4_AG (part 1), CE7-variant 7_AG (part 2), CE7-variant 18_AG (part 3), standard application (part 4), and blank sample (part 5). [Figure 8-1] Blank-subtracted sensorgrams are presented for the binding of CE7-mutant 4-AG (part 1), CE7-mutant 7-AG (part 2), CE7-mutant 18-AG (part 3), chimeric HCE7 (part 4), and deglycosylated cHCE7 (part 5) to L1-CAM, respectively, as described in Example 6. [Figure 8-2] Blank-subtracted sensorgrams are presented for the binding of CE7-mutant 4-AG (part 1), CE7-mutant 7-AG (part 2), CE7-mutant 18-AG (part 3), chimeric HCE7 (part 4), and deglycosylated cHCE7 (part 5) to L1-CAM, respectively, as described in Example 6. [Figure 8-3] Blank-subtracted sensorgrams are presented for the binding of CE7-mutant 4-AG (part 1), CE7-mutant 7-AG (part 2), CE7-mutant 18-AG (part 3), chimeric HCE7 (part 4), and deglycosylated cHCE7 (part 5) to L1-CAM, respectively, as described in Example 6. [Figure 8-4] Blank-subtracted sensorgrams are presented for the binding of CE7-mutant 4-AG (part 1), CE7-mutant 7-AG (part 2), CE7-mutant 18-AG (part 3), chimeric HCE7 (part 4), and deglycosylated cHCE7 (part 5) to L1-CAM, respectively, as described in Example 6. [Figure 8-5]Blank-subtracted sensorgrams are presented for the binding of CE7-mutant 4-AG (part 1), CE7-mutant 7-AG (part 2), CE7-mutant 18-AG (part 3), chimeric HCE7 (part 4), and deglycosylated cHCE7 (part 5) to L1-CAM, respectively, as described in Example 6. [Figure 9-1] Blank-subtracted sensorgrams are presented for the binding of CE7-variant 7 (part 1), CE7-variant 26 (part 2), CE7-variant 27 (part 3), and CE7-variant 28 (part 4) to L1-CAM, respectively, as described in Example 7. [Figure 9-2] Blank-subtracted sensorgrams are presented for the binding of CE7-variant 7 (part 1), CE7-variant 26 (part 2), CE7-variant 27 (part 3), and CE7-variant 28 (part 4) to L1-CAM, respectively, as described in Example 7. [Figure 9-3] Blank-subtracted sensorgrams are presented for the binding of CE7-variant 7 (part 1), CE7-variant 26 (part 2), CE7-variant 27 (part 3), and CE7-variant 28 (part 4) to L1-CAM, respectively, as described in Example 7. [Figure 9-4] Blank-subtracted sensorgrams are presented for the binding of CE7-variant 7 (part 1), CE7-variant 26 (part 2), CE7-variant 27 (part 3), and CE7-variant 28 (part 4) to L1-CAM, respectively, as described in Example 7. [Figure 10] 1 presents the purification profile of rAB as seen in Example 8. Non-reducing PAGE with Coomassie Blue staining. MW. Molecular weight marker. IN. Input. FT. Flow-through. W. Wash. E. Elution fractions. [Figure 11] Final sample QC in Example 8 is presented. Coomassie blue staining. Reducing and non-reducing PAGE analysis. MW. Molecular weight marker. 2 μg loaded per lane. [Figure 12-1]SEC-HPLC profiles obtained for LV1-8 are presented. LV1-8 are based on the chimeric CE7 antibody containing mutations in the Fc portion (L234A, L235A, P331S, and N297A). The aim of this study was to identify the effect of sequence liability mutations on the parent mAb. [Figure 12-2] SEC-HPLC profiles obtained for LV1-8 are presented. LV1-8 are based on the chimeric CE7 antibody containing mutations in the Fc portion (L234A, L235A, P331S, and N297A). The aim of this study was to identify the effect of sequence liability mutations on the parent mAb. [Figure 13] Double-referenced solvent-corrected sensorgrams of the interaction between A) deglycosylated cHCE7 and L1-CAM, and B) deglycosylated cHCE7 and L1-CAM are shown, with the measured responses graphed. [Figure 14] 1 presents the purification profile of rAB as seen in Example 21. Non-reducing PAGE with Coomassie Blue staining. MW. Molecular weight marker. IN. Input. FT. Flow-through. W. Wash. E. Elution fractions. [Figure 15] Final sample QC in Example 21 is presented. Coomassie blue staining. Reducing and non-reducing PAGE analysis. MW. Molecular weight marker. 2 μg loaded per lane. [Figure 16] The SEC-HPLC profile obtained for LV29-32 is presented. [Figure 17] Embryo survival rates 3 days after intravenous injection of naked huCE7-V7AG are shown. The x-axis is shown as log [mg / mL]. [Figure 18] Biodistribution studies at 24, 48, 72, and 96 hours are presented for deglycosylated chCE7 and the variant 7AG according to the invention. [Figure 19]Biodistribution of 177Lu-labeled huCE7-LV40-(PEG-DOTA)2 and huCE7-LV40-(PEG-polymer-α-DOTA)2 in a mouse tumor xenograft model: CD1 nu / nu sc SKOVip3 (ovarian cancer, L1-CAM positive). [Figure 20-1] We present the efficacy of 4 Mbq of 177Lu-labeled huCE7-V7AG-(PEG4-DOTA)2 in a mouse tumor xenograft model. Mouse model: CD1 nu / nu sc SKOVip3 (ovarian cancer, L1-CAM positive). 12 days after tumor cell inoculation, mice were treated with a single injection of labeled mAb (treatment group, 10 mice) or PBS (negative control group, 10 mice). Tumor size, body weight, and survival rate were monitored throughout the study. Mice were euthanized when tumor size reached a threshold. [Figure 20-2] We present the efficacy of 4 Mbq of 177Lu-labeled huCE7-V7AG-(PEG4-DOTA)2 in a mouse tumor xenograft model. Mouse model: CD1 nu / nu sc SKOVip3 (ovarian cancer, L1-CAM positive). 12 days after tumor cell inoculation, mice were treated with a single injection of labeled mAb (treatment group, 10 mice) or PBS (negative control group, 10 mice). Tumor size, body weight, and survival rate were monitored throughout the study. Mice were euthanized when tumor size reached a threshold. [Figure 21] This paper presents the efficacy evaluation of huCE7-V7AG and HUCE7-V7AG-(MMAE)4 antibodies against MAXFTN-401 breast cancer cells in a zebrafish xenograft model. The antibodies were administered intravenously immediately after tumor implantation. Data are presented as mean ± SEM, followed by a Student's t-test (*p<0.05). [Figure 22] FACS analysis of H2171 SCLC cells (A) and the negative control cell line MDA-MB-468 (which does not express L1-CAM) (B) is presented. [Figure 23]Normalized primary tumor size and metastatic dissemination of H2171 cells in a zebrafish xenograft model 48 hours after intravenous treatment with HuCE7-V7AG and HuCE7-V7AG-(MMAE)4 antibodies are shown. Data are normalized to the negative control group. Data are presented as mean ± SEM. One-way ANOVA was performed (p<0.0001) followed by a two-tailed Student's t-test (*p<0.05 and ****p<0.0001). DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention is described in detail below, and it is to be understood that all disclosed features can be combined with each other, unless expressly stated to the contrary.

[0027] In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to L1-CAM (CD171).

[0028] L1-CAM refers to a transmembrane protein member of the L1 protein family, encoded by the L1-CAM gene, which is listed in the UniProt database under the reference number P32004. L1-CAM protein is a neuronal cell adhesion molecule that has a significant effect on cell migration, adhesion, neurite outgrowth, myelination, and neuronal differentiation. L1-CAM protein has also been found to play a role in therapy-resistant cancer. The term "L1-CAM" may be used interchangeably with the term "CD171," both of which are known to those skilled in the art.

[0029] Thus, an antibody or antigen-binding fragment thereof that specifically binds to L1-CAM can also be referred to as an antibody (or antigen-binding fragment thereof, respectively) that binds to an epitope within L1-CAM, preferably specifically binds to an epitope within L1-CAM. An "antibody that binds to an epitope" within a defined region of a protein is an antibody that requires the presence of one or more amino acids within that region for binding to that protein.

[0030] As understood herein, the antibodies and antigen-binding fragments thereof of the present invention preferably bind to the Ig6-like domain of L1-CAM and block binding of L1-CAM to integrins. Thus, the antibodies or antigen-binding fragments thereof preferably bind to the same epitope as the chCE7 antibody referred to herein.

[0031] As preferably referred to herein, specific binding to L1-CAM refers to a situation in which the antibody or fragment thereof can bind to L1-CAM with sufficient affinity that the antibody is useful as a diagnostic and / or therapeutic agent when targeting L1-CAM. Thus, preferably, the extent of binding of such an antibody to an unrelated, non-L1-CAM protein is less than about 10% of the antibody's binding to L1-CAM, as measured, for example, by immunoassay (e.g., radioimmunoassay) or SPR assay (surface plasmon resonance). More preferably, the extent of binding of such an antibody to an unrelated, non-L1-CAM protein is less than about 5% of the antibody's binding to L1-CAM, as measured, for example, by immunoassay (e.g., radioimmunoassay) or SPR assay (e.g., surface plasmon resonance performed using Biacore).

[0032] The term "binds to," when used in the context of the present invention, defines the binding (interaction) of at least two "antigen-interaction sites" with one another. The term "antigen-interaction site," according to the present invention, defines a polypeptide motif, i.e., a part of an antibody or antigen-binding fragment of the present invention, that exhibits the ability to specifically interact with a particular antigen or group of particular antigens of L1-CAM.

[0033] The cross-reactivity of an antibody or antigen-binding fragment thereof under investigation can be tested, for example, by assessing the binding of the antibody or a panel of antigen-binding fragments thereof to the (poly)peptide of interest and several (structurally and / or functionally) more or less closely related (poly)peptides under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)). Only constructs (i.e., antibodies, antigen-binding fragments thereof, etc.) that bind to a particular structure of L1-CAM as defined herein, e.g., a specific epitope or (poly)peptide / protein of L1-CAM as defined herein, but do not or essentially do not bind to any other epitope or (poly)peptide of the same L1-CAM, are considered specific for the epitope or (poly)peptide / protein of interest and are selected for further study according to the methods provided herein. These methods may include, inter alia, binding, blocking, and competition studies with structurally and / or functionally closely related molecules. These binding studies may also include those using FACS analysis, surface plasmon resonance (SPR, e.g., SPR using Biacore), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy, or radiolabeled ligand binding assays. Thus, specificity can be determined experimentally by methods known in the art and described herein. Such methods include, but are not limited to, Western blot, ELISA test, RIA test, ECL test, IRMA test, and peptide scan.

[0034] As preferably referred to herein, an antibody or antigen-binding fragment is defined below.

[0035] In general, the term "antibody" is used herein in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity. Unless expressly indicated to the contrary, whenever an antibody or its properties are discussed, reference is made to an antibody, its antigen-binding fragments.

[0036] As preferably referred to herein, an "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of the intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0037] Preferably, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a recombinant antibody, an antigen-binding fragment of a recombinant antibody, a single-chain antibody, a humanized antibody, a bispecific antibody, a multispecific antibody, or an antibody displayed on the surface of a phage or on the surface of a chimeric antigen receptor (CAR) T cell.

[0038] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they can be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The modified "monoclonal" refers to the character of the antibody as being within a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. As noted above, monoclonal antibodies used in accordance with the present invention can be produced by the hybridoma method described by Kohler (Nature 256 (1975), 495).

[0039] The term "chimeric antibody" refers to an antibody comprising a variable region of the present invention fused to or chimerized with an antibody region (e.g., constant region) from another human or non-human species (e.g., mouse, horse, rabbit, dog, cow, chicken).

[0040] The term "recombinant antibody" includes all antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes, antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial human antibody libraries, or antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Thus, the term antibody also relates to recombinant human antibodies, xenoantibodies, and heterohybrid antibodies. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. However, such antibodies can be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0041] A "heterologous antibody" is defined with respect to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or encoding nucleic acid sequence that corresponds to that found in an organism not consisting of the transgenic non-human animal and generally derived from a species other than the transgenic non-human animal species.

[0042] The term "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain combined with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric antibodies and humanized antibodies.

[0043] The term antibody also refers to humanized antibodies. "Humanized" forms of non-human (e.g., murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies), which contain minimal sequence derived from non-human immunoglobulin. Often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones Nature 321 (1986), 522-525; Reichmann Nature 332 (1998), 323-327; and Presta Curr Op Struct Biol 2 (1992), 593-596.

[0044] Thus, in the context of the present invention, the term "antibody" relates not only to complete immunoglobulin molecules but also to parts of such immunoglobulin molecules (i.e., "antigen-binding fragments thereof"). Furthermore, the term relates to modified and / or altered antibody molecules, as described above. The term also relates to recombinantly or synthetically produced / synthesized antibodies. The term relates not only to intact antibodies but also to antigen-binding fragments thereof, such as separated light and heavy chains, Fab, Fv, Fab', Fab'-SH, F(ab')2. The term antibody also includes, but is not limited to, fully human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, and antibody constructs such as single chain Fv (scFv) or antibody fusion proteins.

[0045] A single chain antibody, i.e., a "single chain Fv" or "scFv" antibody fragment, is defined in the context of the present invention as the V H Domain and V L domains, which are present in a single polypeptide chain. Generally, scFv polypeptides have V H Domains and V L It further comprises a polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding. Techniques described for the production of single-chain antibodies are described, for example, in Pluckthun in The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds. Springer-Verlag, NY (1994), 269-315.

[0046] As used herein, a "Fab fragment" refers to a fragment that contains one light chain and one heavy chain C. H Fab molecules are composed of a heavy chain and a variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0047] The "Fc" region is the C H 2 domain and C HThe two heavy chain fragments contain two heavy chain fragments each containing a C3 domain. These two heavy chain fragments are connected by two or more disulfide bonds and H The three domains are held together by hydrophobic interactions.

[0048] A "Fab' fragment" is a fragment of one light chain and one V H Domain and C H 1 domain and C H 1 Domain and C H and a portion of one heavy chain including the region between the two domains, which allows interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments to form the F(ab')2 molecule.

[0049] "F(ab')2 fragment" is a fragment of two light chains and a C H 1 Domain and C H The F(ab')2 fragment contains two heavy chains with a portion of the constant region between the two domains, which allows interchain disulfide bonds to form between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0050] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0051] As referred to herein, a bispecific antibody is an antibody that can simultaneously bind to two different types of antigens or to two different epitopes of the same antigen. During development, bispecific antibodies can be produced in several structural formats known to those skilled in the art. In the present invention, at least one of the antigens relates to L1-CAM, as defined herein.

[0052] A multispecific antibody, as referred to herein, is an antibody that can simultaneously bind to three or more different types of antigens or to three or more different epitopes of the same antigen.

[0053] The antibodies, antibody constructs, antibody fragments, antibody derivatives (all of which are Ig-derived) used according to the present invention, or their corresponding immunoglobulin chains, can be further modified using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, addition, and / or recombination, and / or any other modification known in the art, alone or in combination. Methods for introducing such modifications into the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to those skilled in the art; see, for example, Sambrook (1989) (ibid.). The term "Ig-derived domain" particularly relates to (poly)peptide constructs comprising at least one CDR. Fragments or derivatives of the listed Ig-derived domains define (poly)peptides that are part of the above-mentioned antibody molecules and / or that have been modified by chemical / biochemical or molecular biological methods. Corresponding methods are known in the art and are described, inter alia, in laboratory manuals (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and 3rd edition (2001); Gerhardt et al., Methods for General and Molecular Bacteriology ASM Press (1994); Lefkovits, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press (1997); Golemis, Protein-Protein Interactions: A Molecular Cloning Manual Cold Spring Harbor Laboratory Press (2002)).

[0054] More preferably, the antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0055] Antibodies referred to herein may be IgG1, IgG2a or IgG2b, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, or IgE. As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0056] Preferably, the monoclonal antibodies described herein are IgG1 antibodies.

[0057] Thus, the antibodies of the present invention can be full length, or can comprise only antigen-binding fragments such as antibody constant and / or variable domains of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, or IgE, or can consist of Fab, F(ab')2, and Fv fragments.

[0058] In the present invention, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region as described below and / or a variable light chain region as described below.

[0059] As encompassed by the present invention, the variable heavy chain region may be CDR-H1, The sequence set forth in SEQ ID NO: 1 (GYWMH), The sequence set forth in SEQ ID NO: 2 (GYYMH), The sequence set forth in SEQ ID NO: 3 (GYFMH), and the sequence set forth in SEQ ID NO: 4 (GYLMH), and CDR-H2, The sequence set forth in SEQ ID NO: 5 (EINPSNGRTNYNERFQG), The sequence set forth in SEQ ID NO: 6 (EINPSNGRTNYNEKFQG), The sequence set forth in SEQ ID NO: 7 (EINPSNGRTNYNERFKS), The sequence set forth in SEQ ID NO: 8 (EINPSNGRTNYNERLKS), The sequence set forth in SEQ ID NO: 9 (EINPSNARTNYNERFQG), Sequence set forth in SEQ ID NO: 10 (EINPSNARTNYNEKFQG) The sequence set forth in SEQ ID NO: 11 (EINPSNARTNYNERFKS), and CDR-H2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 12 (EINPSNARTNYNERLKS), and and CDR-H3 characterized by the sequence set forth in SEQ ID NO: 13 (DYYGTSYNFDY).

[0060] As encompassed by the present invention, the variable light chain region may comprise: CDR-L1, The sequence set forth in SEQ ID NO: 14 (RANEDINNRLA), The sequence set forth in SEQ ID NO: 15 (KANEDINNRLA), The sequence set forth in SEQ ID NO: 16 (QANEDINNRLA), the sequence set forth in SEQ ID NO: 17 (RANEDINARLA), The sequence set forth in SEQ ID NO: 18 (KANEDINARLA), The sequence set forth in SEQ ID NO: 19 (QANEDINARLA), The sequence set forth in SEQ ID NO: 20 (RANEDINLRLA), The sequence set forth in SEQ ID NO: 21 (KANEDINLRLA), and CDR-L1 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 22 (QANEDINLRLA), and CDR-L2, The sequence set forth in SEQ ID NO: 23 (GATNLVT), and CDR-L2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 24 (GASNLVS), and CDR-L3, The sequence set forth in SEQ ID NO: 25 (QQYWSTPFT), The sequence set forth in SEQ ID NO: 26 (QQYYSTPFT), and The sequence of SEQ ID NO: 27 (QQYFSTPFT) is selected from the group consisting of: CDR-L3;

[0061] As understood herein, the present invention is defined by several amino acid sequences, which are preferably provided using the single-letter code known to those skilled in the art. As defined herein, sequences are either provided directly in the body of the present application or by reference to the sequence listing attached hereto. In the event of a discrepancy between a sequence provided directly in the body of the present application and a sequence referred to in the sequence listing by a specific sequence identification number (SEQ ID NO), it is understood that the present invention relates to both sequences, i.e., the sequences provided herein or the sequences provided in the sequence listing, and preferably, the present invention relates to the sequences provided in the sequence listing.

[0062] The term "CDR" as used herein relates to the "complementarity determining region" as it is known in the art. CDR is the part of an immunoglobulin that determines the specificity of the molecule and contacts the specific ligand. CDR is the most variable part of the molecule and contributes to the diversity of these molecules. There are three CDR regions in each V domain: CDR1, CDR2, and CDR3. CDR-H denotes the CDR region of the variable heavy chain, and CDR-L relates to the CDR region of the variable light chain. VH means variable heavy chain, and VL means variable light chain. The CDR regions of Ig-derived regions can be determined as described in Kabat "Sequences of Proteins of Immunological Interest" (5th ed.), NIH Publication No. 91-3242 USDepartment of Health and Human Services (1991), Chothia J. Mol. Biol. 196 (1987), 901-917, or Chothia Nature 342 (1989), 877-883.

[0063] Thus, the present invention is based, at least in part, on the surprising discovery that the antibodies or fragments thereof of the present invention exhibit improved affinity for L1-CAM compared to prior art antibodies, and in particular compared to the CE7 antibody from which they are derived.

[0064] Preferably, in the antibody or antigen-binding fragment thereof of the present invention, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises a CDR-H1 characterized by a sequence selected from the sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. More preferably, in the antibody or antigen-binding fragment thereof of the present invention, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises a CDR-H1 characterized by a sequence selected from the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2. Even more preferably, in the antibody or antigen-binding fragment thereof of the present invention, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises a CDR-H1 characterized by the sequence set forth in SEQ ID NO: 1.

[0065] Preferably, in the antibody or antigen-binding or antigen-binding fragment thereof of the present invention, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises a CDR-H2 characterized by a sequence selected from the sequences set forth in SEQ ID NO: 5, 6, 9, or 10. More preferably, in the antibody or antigen-binding or antigen-binding fragment thereof of the present invention, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises a CDR-H2 characterized by the sequence set forth in SEQ ID NO: 5 or 6. In a specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises a CDR-H2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 8 or 12.

[0066] As understood herein, in the antibody or antigen-binding fragment thereof of the present invention, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises a CDR-H3 characterized by the sequence set forth in SEQ ID NO:13.

[0067] In a preferred embodiment, in the antibody or antigen-binding or antigen-binding fragment thereof of the present invention, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises the above-mentioned CDR-H1, the above-mentioned CDR-H2, and the above-mentioned CDR-H3.

[0068] Preferably, in the antibody or antigen-binding or antigen-binding fragment thereof of the present invention, the variable light chain region of the antibody or antigen-binding fragment thereof comprises a CDR-L1 characterized by the sequence set forth in SEQ ID NO: 14, 15, 17, 18, or 21. More preferably, in the antibody or antigen-binding or antigen-binding fragment thereof of the present invention, the variable light chain region of the antibody or antigen-binding fragment thereof comprises a CDR-L1 characterized by the sequence set forth in SEQ ID NO: 14 or 15.

[0069] Preferably, in the antibody or antigen-binding or antigen-binding fragment thereof of the present invention, the variable light chain region of the antibody or antigen-binding fragment thereof comprises a CDR-L2 characterized by the sequence set forth in SEQ ID NO:23.

[0070] Preferably, in the antibody or antigen-binding or antigen-binding fragment thereof of the present invention, the variable light chain region of the antibody or antigen-binding fragment thereof comprises a CDR-L3 characterized by the sequence set forth in SEQ ID NO: 25 or 26. More preferably, in the antibody or antigen-binding or antigen-binding fragment thereof of the present invention, the variable light chain region of the antibody or antigen-binding fragment thereof comprises a CDR-L3 characterized by the sequence set forth in SEQ ID NO: 25.

[0071] Preferably, in the antibody or antigen-binding or antigen-binding fragment thereof of the present invention, the variable light chain region of the antibody or antigen-binding fragment thereof comprises the above-mentioned CDR-L1, the above-mentioned CDR-L2, and the above-mentioned CDR-L3.

[0072] According to the present invention, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region of the antibody or antigen-binding fragment thereof comprising the above-mentioned CDR-H1, the above-mentioned CDR-H2, and the above-mentioned CDR-H3, and / or comprises a variable light chain region of the antibody or antigen-binding fragment thereof comprising the above-mentioned CDR-L1, the above-mentioned CDR-L2, and the above-mentioned CDR-L3.

[0073] Preferably, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain region of the antibody or antigen-binding fragment thereof comprising the above-mentioned CDR-H1, the above-mentioned CDR-H2, and the above-mentioned CDR-H3, and comprises a variable light chain region of the antibody or antigen-binding fragment thereof comprising the above-mentioned CDR-L1, the above-mentioned CDR-L2, and the above-mentioned CDR-L3.

[0074] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain region of the antibody or antigen-binding fragment thereof comprising the above-mentioned CDR-H1, the above-mentioned CDR-H2, and the above-mentioned CDR-H3, or comprises a variable light chain region of the antibody or antigen-binding fragment thereof comprising the above-mentioned CDR-L1, the above-mentioned CDR-L2, and the above-mentioned CDR-L3.

[0075] In one embodiment of the antibody or antigen-binding fragment thereof of the invention, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises a CDR-H2 characterized by the sequence set forth in SEQ ID NO: 9, 10, 11, or 12, and / or the variable light chain region of the antibody or antigen-binding fragment thereof comprises a CDR-L1 characterized by the sequence set forth in SEQ ID NO: 17, 18, or 19. Preferably, in this embodiment, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises a CDR-H2 characterized by the sequence set forth in SEQ ID NO: 9, 10, 11, or 12, and the variable light chain region of the antibody or antigen-binding fragment thereof comprises a CDR-L1 characterized by the sequence set forth in SEQ ID NO: 17, 18, or 19. Preferably, in this particular embodiment, the variable heavy chain region of the antibody or antigen-binding fragment thereof further comprises a CDR-H1 characterized by the sequence set forth in SEQ ID NO: 2 or 3, and / or the variable light chain region of the antibody or antigen-binding fragment thereof further comprises a CDR-L3 characterized by the sequence set forth in SEQ ID NO: 26 or 27. More preferably, in this particular embodiment, the variable heavy chain region of the antibody or antigen-binding fragment thereof further comprises a CDR-H1 characterized by the sequence set forth in SEQ ID NO: 2 or 3, and the variable light chain region of the antibody or antigen-binding fragment thereof further comprises a CDR-L3 characterized by the sequence set forth in SEQ ID NO: 26 or 27.

[0076] Preferably, the antibody or antigen-binding fragment thereof of the present invention is a humanized antibody.

[0077] Humanization approaches are well known in the art and have been particularly described for antibody molecules, e.g., Ig-derived molecules. The term "humanized" refers to humanized forms of non-human (e.g., murine) antibodies or fragments thereof (such as Fv, Fab, Fab', F(ab'), scFv, or other antigen-binding subsequences of antibodies) that contain some portion of sequence derived from the non-human antibody. Humanized antibodies include human immunoglobulins in which residues from a complementarity-determining region (CDR) of the human immunoglobulin are replaced by residues from a CDR of a non-human species, such as mouse, rat, or rabbit, having the desired binding specificity, affinity, and capacity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR (framework) regions are those of a human immunoglobulin consensus sequence. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin; see, inter alia, Jones et al., Nature 321 (1986), 522-525; Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acids introduced into it from a non-human source while still retaining the original binding activity of the antibody. Methods for humanizing antibodies / antibody molecules are further described in Jones et al., Nature 321 (1986), 522-525; Reichmann et al., Nature 332 (1988), 323-327; and Verhoeyen et al., Science 239 (1988), 1534-1536.Specific examples of humanized antibodies, e.g., antibodies against EpCAM, are known in the art, see, for example, (LoBuglio, Proceedings of the American Society of Clinical Oncology Abstracts (1997), 1562 and Khor, Proceedings of the American Society of Clinical Oncology Abstracts (1997), 847).

[0078] Preferably, as referred to herein, "framework region", also referred to as FR region, is a part of the variable domain of an antibody that is not a CDR. Thus, each variable domain sequence of an antibody has four framework regions, which are separated from each other by hypervariable region CDRs. Framework regions typically constitute up to 85% of the sequence of the variable domain and can act as a scaffold to expose CDRs so that they can interact with antigens. As known to those skilled in the art, mutations in framework regions can also affect the binding affinity of antibodies to CDR-specific antigens.

[0079] Thus, in the context of the present invention, there is provided an antibody molecule or antigen-binding fragment thereof that has been humanized and can be successfully used in a pharmaceutical composition.

[0080] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0081] Preferably, in the antibody or antigen-binding fragment thereof of the present invention, the variable heavy chain region comprises: The sequence set forth in SEQ ID NO: 28 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), The sequence set forth in SEQ ID NO: 29 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTVSS), The sequence set forth in SEQ ID NO: 30 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFTGYWMHWIRQPPGKGLEWIGEINPSNGRTNYNERLKSRVTLSVDKSKNQASLKLSSVTAADTAVYFCARDYYGTSYNFDYWGQGTLVTVSS), The sequence set forth in SEQ ID NO: 31 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), and The present invention is characterized by a sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, and even more preferably identical to a sequence selected from the sequence set forth in SEQ ID NO: 32 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWmGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS).

[0082] More preferably, in the antibody or antigen-binding fragment thereof of the invention, the variable heavy chain region is characterized by a sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, and even more preferably identical to a sequence selected from the sequence set forth in SEQ ID NO: 28 or the sequence set forth in SEQ ID NO: 32.

[0083] Even more preferably, in the antibody or antigen-binding fragment thereof of the present invention, the variable heavy chain region is characterized by a sequence identical to the sequence set forth in SEQ ID NO: 28 or a sequence selected from the sequence set forth in SEQ ID NO: 32.

[0084] Even more preferably, in the antibody or antigen-binding fragment thereof of the invention, the variable heavy chain region is characterized by a sequence identical to the sequence set forth in SEQ ID NO: 28. In one embodiment, in the antibody or antigen-binding fragment thereof of the invention, the variable heavy chain region is characterized by a sequence identical to the sequence set forth in SEQ ID NO: 32.

[0085] Preferably, in the antibody or antigen-binding fragment thereof of the present invention, the variable light chain region comprises: The sequence set forth in SEQ ID NO: 33 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), The sequence set forth in SEQ ID NO: 34 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDIATYYCQQYWSTPFTFGQGTKLEIK), The sequence set forth in SEQ ID NO: 35 (EIVMTQSPATLSVSPGERATLSCRANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEFTLTISSLQSEDFAVYYCQQYWSTPFTFGQGTKLEIK), The sequence set forth in SEQ ID NO: 36 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), and The present invention is characterized by a sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, and even more preferably identical to a sequence selected from the sequence set forth in SEQ ID NO: 37 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGAsNLVsGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK).

[0086] More preferably, in the antibody or antigen-binding fragment thereof of the present invention, the variable light chain region is characterized by a sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably identical to a sequence selected from the sequence set forth in SEQ ID NO: 33, the sequence set forth in SEQ ID NO: 36, and the sequence set forth in SEQ ID NO: 37, preferably to a sequence selected from the sequence set forth in SEQ ID NO: 33 and the sequence set forth in SEQ ID NO: 36.

[0087] Even more preferably, in the antibody or antigen-binding fragment thereof of the present invention, the variable light chain region is characterized by a sequence identical to a sequence selected from the sequence set forth in SEQ ID NO: 33, the sequence set forth in SEQ ID NO: 36 and the sequence set forth in SEQ ID NO: 37, preferably a sequence selected from the sequence set forth in SEQ ID NO: 33 and the sequence set forth in SEQ ID NO: 36.

[0088] Even more preferably, in the antibody or antigen-binding fragment thereof of the invention, the variable light chain region is characterized by a sequence identical to the sequence set forth in SEQ ID NO: 33. In one embodiment, in the antibody or antigen-binding fragment thereof of the invention, the variable light chain region is characterized by a sequence identical to the sequence set forth in SEQ ID NO: 36. In one embodiment, in the antibody or antigen-binding fragment thereof of the invention, the variable light chain region is characterized by a sequence identical to the sequence set forth in SEQ ID NO: 37.

[0089] As will be appreciated by those skilled in the art, a variable heavy chain sequence (or a variable light chain sequence) may be defined by its framework and its CDR sequences. Accordingly, further variable heavy or variable light chain sequences encompassed by the present invention are disclosed below.

[0090] In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the variable heavy chain region comprises the sequence: FH0-CDR-H1-FH1-CDR-H2-FH2-CDR-H3-FH3, wherein CDR-H1, CDR-H2 and CDR-H3 are as defined above, FH0 is characterized by the sequence set forth in SEQ ID NO: 38 (QVQLVQSGAEVKKPGASVKVSCKASGYTFT) or the sequence set forth in SEQ ID NO: 39 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFT), FH1 is characterized by the sequence set forth in SEQ ID NO: 40 (WVRQAPGQGLEWIG) or the sequence set forth in SEQ ID NO: 41 (WIRQPPGKGLEWIG), FH2 is characterized by the sequence set forth in SEQ ID NO: 42 (RVTLTVDKSISTAYMELSRLRSDDTAVYFCAR) or the sequence set forth in SEQ ID NO: 43 (RVTLSVDKSKNQASLKLSSVTAADTAVYFCAR), The present invention relates to an antibody or antigen-binding fragment thereof, wherein FH3 is characterized by a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 44 (WGQGTLVTVSS), preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, more preferably an identical sequence.

[0091] In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the variable light chain region comprises the sequence: FL0-CDR-L1-FL1-CDR-L2-FL2-CDR-L3-FL3, wherein CDR-L1, CDR-L2, and CDR-L3 are as defined above, FL0 is characterized by the sequence set forth in SEQ ID NO: 45 (DIQMTQSPSSLSASVGDRVTITC) or the sequence set forth in SEQ ID NO: 46 (EIVMTQSPATLSVSPGERATLSC), FL1 is characterized by the sequence set forth in SEQ ID NO: 47 (WYQQKPGKAPKLLIS) or the sequence set forth in SEQ ID NO: 48 (WYQQKPGQAPRLLIS), FL2 is characterized by the sequence set forth in SEQ ID NO: 49 (GVPSRFSGSGSGKDYTLTISSLQPEDIATYYC) or the sequence set forth in SEQ ID NO: 50 (GIPARFSGSGSGKEFTLTISSLQSEDFAVYYC), FL3 is characterized by a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 51 (FGQGTKLEIK), preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, more preferably an identical sequence.

[0092] In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, wherein the variable heavy chain region comprises the sequence: FH0-CDR-H1-FH1-CDR-H2-FH2-CDR-H3-FH3 wherein FH0, CDR-H1, FH1, CDR-H2, FH2, CDR-H3, and FH3 are as defined above, characterized by a sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, more preferably identical to and / or (preferably) The variable light chain region has the sequence: FL0-CDR-L1-FL1-CDR-L2-FL2-CDR-L3-FL3 (wherein FL0, CDR-L1, FL1, CDR-L2, FL2, CDR-L3, and FL3 are as defined above), the antibody or antigen-binding fragment thereof characterized by a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, more preferably identical to

[0093] The present invention encompasses antibodies or antigen-binding fragments thereof, which antibodies or antigen-binding fragments thereof include any combination of variable light chains as defined above and variable heavy chains as defined above.

[0094] For the design of a CDR-grafted version of the CE7 mouse VH, two human germline clones, IGHV1-2, were used. * 06 and IGHV4-34 * 01 was selected. These two human germlines share 66.3% and 53.1% sequence identity across the entire V gene, respectively. For the design of the CDR-grafted version of the CE7 murine VL, three human germlines, IGKV1-NL1 and IGKV1-NL1, were selected. * 01, IGKV1-33 * 01, and IGKV3-15 * 01 was selected. These three human germlines share 73.7%, 71.6%, and 60.0% sequence identity with the CE7 mouse VL across the entire V gene, respectively.

[0095] According to in silico immunogenicity modeling studies performed by the inventors, several mutations can be introduced into the above germlines to reduce their potential immunogenicity.

[0096] Therefore, CE7-CDR-grafted IGHV4-34 * In the VH of 01, the following mutations are predicted to be particularly beneficial in reducing immunogenicity: the M residue at position -34 may be replaced by a residue selected from C, D, and E; the M residue at position -35 may be replaced by C; the residue at position -37 may be replaced by a residue selected from C, D, and E; the R residue at position -63 may be replaced by a residue selected from D, E, and P; the L residue at position -64 may be replaced by a residue selected from A, C, D, E, G, H, K, N, P, Q, S, and T; the K residue at position -65 may be replaced by a residue selected from C, D, E, N, and P; the S residue at position -66 may be replaced by a residue selected from C and D; the R residue at position -67 may be replaced by a residue selected from D and P; the V residue at position -68 may be replaced by a residue selected from C, D, E, G, N, and Q; the T residue at position -69 may be replaced by a residue selected from D and E; the L residue at position -70 may be replaced by a residue selected from C, D, E, G, N, P, Q, S, and T; and / or The V residue at position -72 may be replaced by D.

[0097] Furthermore, IGKV1-NL1 with CE7-CDR grafted * In the VL of 01, the following mutations are predicted to be particularly beneficial in reducing immunogenicity: the L residue at position -46 may be replaced by a residue selected from A, D, E, G, H, N, P, Q, S, and T; the L residue at position -47 may be replaced by a residue selected from D, E, G, N, P, S, and T; the residue at position -51 may be replaced by E; the T residue at position -52 may be replaced by D; the N residue at position -53 may be replaced by D; the L residue at position -54 may be replaced by a residue selected from C, D, E, G, N, P, Q, S, and T; and / or The V residue at position -55 may be replaced by a residue selected from C, D, E, and G.

[0098] Furthermore, CE7-CDR-grafted IGHV1-2 * In the VH of 06, the following mutations are predicted to be particularly beneficial in reducing immunogenicity: The L residue at position -70 may be replaced by A, C, D, E, G, H, K, N, P, Q, S, and T; The T residue at position -71 may be replaced by C and D; the V residue at position -72 may be replaced by A, C, D, E, G, H, K, N, P, Q, S, T, and W; the K residue at position -74 may be replaced by C, D, E, G, P, Q, and T; The S residue at position -75 may be replaced by C, D, E, G, and Q; the I residue at position -76 may be replaced by C, D, E, G, K, N, P, Q, S, and T; The S residue at position -77 may be replaced by C or E; The T residue at position -78 may be replaced by D and E, and / or The residue at position -79 may be replaced with D.

[0099] Preferably, as encompassed by the present invention, an antibody (or antigen-binding fragment thereof, if applicable) according to any of the above embodiments comprises a heavy chain constant region sequence comprising an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 145, 146, 147, 148, 149, and 150, preferably the sequences set forth in SEQ ID NOs: 146, 147, 148, 149, and 150, more preferably the sequences set forth in SEQ ID NOs: 147, 148, 149, and 150. In one embodiment, an antibody of the invention comprises a heavy chain constant region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the sequences set forth in SEQ ID NOs: 145, 146, 147, 148, 149, and 150, preferably the sequences set forth in SEQ ID NOs: 146, 147, 148, 149, and 150, more preferably the sequences set forth in SEQ ID NOs: 147, 148, 149, and 150.

[0100] In one embodiment, the invention relates to an antibody or antigen-binding fragment thereof, wherein the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and still more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 151, 152, 153, and 154.

[0101] As shown in Example 26, antibody variants of the invention, particularly those of the embodiments described below, are characterized by reduced lymph node uptake. This represents an important aspect of the invention presented herein, as it can be hypothesized that reduced uptake of huCE7 variants in healthy lymph nodes would result in a better side effect profile. This effect may be the result of CE7 epitope and sequence changes during humanization that prevent the antibody from binding to the version of L1-CAM expressed in lymph nodes.

[0102] In one embodiment, it is particularly preferred that the heavy chain of the antibody or antigen-binding fragment thereof of the present invention comprises a sequence that is at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 155 to 163. In this embodiment, it is particularly preferred that the light chain of the antibody or antigen-binding fragment thereof of the present invention comprises a sequence that is at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 143.

[0103] In one specific embodiment, in the antibody or antigen-binding fragment thereof of the invention, the heavy chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 155, and the light chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 143.

[0104] In one specific embodiment, in the antibody or antigen-binding fragment thereof of the invention, the heavy chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 155, and the light chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 97.

[0105] In one specific embodiment, in the antibody or antigen-binding fragment thereof of the invention, the heavy chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 162, and the light chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 143.

[0106] In one specific embodiment, in the antibody or antigen-binding fragment thereof of the invention, the heavy chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 162, and the light chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 97.

[0107] Therefore, it is preferable that the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain region comprising CDR-H1 set forth in SEQ ID NO: 3, CDR-H2 set forth in SEQ ID NO: 9, and CDR-H3 set forth in SEQ ID NO: 13, and a variable light chain region comprising CDR-L1 set forth in SEQ ID NO: 18, CDR-L2 set forth in SEQ ID NO: 23, and CDR-L3 set forth in SEQ ID NO: 26.

[0108] In one specific embodiment, in the antibody or antigen-binding fragment thereof of the invention, the heavy chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 110 (CE7-1-2-VHB), and the light chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 97 (CE7-1-NL1-VLA).

[0109] In one specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain comprises a sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 142 (CE7-1-2-VHB+WH33F, GH55A), and the light chain comprises a sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 97 (CE7-1-NL1-VLA).

[0110] In one specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 110, and the light chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 143.

[0111] In a specific embodiment, the heavy chain of the antibody does not have a mutation that results in deglycosylation of the antibody. Thus, in a specific embodiment, in an antibody or antigen-binding fragment thereof of the present invention, the heavy chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 110, 142, 155-162, where the mutation N297A is reverted, and the light chain comprises a sequence at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 97 and 143.

[0112] Thus, in one specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain comprises a sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 95 and 164 to 172, and the light chain comprises a sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 97 and 143.

[0113] In one specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 110, 142, 155, 160, or 162, and the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 97 or 143.

[0114] In a specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 173 to 183, and the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 193.

[0115] In a specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 173 to 183, and the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 194.

[0116] In a specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 184 to 192, and the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 193.

[0117] In a specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 184 to 192, and the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to the sequence set forth in SEQ ID NO: 194.

[0118] It should be understood that whenever a heavy or light chain is described as comprising a particular sequence, a heavy or light chain, respectively, having or consisting of that sequence is also directly referenced.

[0119] As understood herein, preferably, the antibody or antigen-binding fragment thereof of the present invention is -11 Not exceeding M, more preferably 10 -12 The dissociation constant K for L1-CAM (CD171) does not exceed M D The K D It should be understood that is preferably measured in a Biacore-based assay.

[0120] Amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0121] In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Target sites for substitutional mutagenesis include the CDRs and FRs. Conservative substitutions are shown in Table D1 under the heading "Preferred Substitutions." More substantial changes are provided in Table D1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0122] [Table 1-1]

[0123] Amino acids can be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0124] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0125] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study have modified (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated, and the mutated antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0126] Modifications (e.g., substitutions) can be made to CDRs, for example, to improve antibody affinity. Such modifications can be made to CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDRs (α-CDRs), and the resulting mutant VH or VL are tested for binding affinity. Affinity maturation by constructing and then reselecting secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-specific approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR H3 and CDR L3 in particular are often targeted.

[0127] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made to a CDR. Such modifications may be outside of CDR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is either unaltered or contains no more than one, two, or three amino acid substitutions.

[0128] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and antigen is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Mutants can be screened to determine whether they contain desired properties.

[0129] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0130] In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0131] If the antibody contains an Fc region, the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally N-linked to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention can be made to generate antibody variants with certain improved properties.

[0132] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (Eu numbering of Fe region residues) in the Fc region; however, due to slight sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.) and 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication Nos. 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, U.S. Patent Application Publication Nos. 2003 / 0115614, 2002 / 0164328, 2004 / 0093621, and 2004 / 01 32140, 2004 / 0110704, 2004 / 0110282, 2004 / 0109865, WO 2003 / 085119, 2003 / 084570, 2005 / 035586, 2005 / 035778, 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986), U.S. Patent Application Publication No. 2003 / 0157108 (Al) (Presta, L), and WO 2004 / 056312 (Al) (Adams et al., especially Example 11)), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (e.g., Yamane-Ohnuki et al., Bioteeh. Bioeng. 87:614 (2004), Kanda, Y. et al., Bioteehnol. Bioeng., 94(4):680-688 (2006), and WO 2003 / 085 (See issue l07).

[0133] Further provided are antibody variants having bisected oligosaccharides, for example, where a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0134] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0135] In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for uses in which in vivo antibody half-life is important, but certain effector functions (such as complement and antibody-dependent cellular cytotoxicity) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC (complement-dependent cytotoxicity) and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express Fc(RIII) only, whereas monocytes express Fc(RI), Fc(RII, and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7063-7064 (1986)). 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).

[0136] Thus, preferably, in the antibody or antigen-binding fragment thereof according to the present invention (where applicable), the heavy chain comprises at least one point mutation in the Fc portion that affects the antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), serum half-life, and / or glycosylation status of the antibody. It should be understood by those skilled in the art that only antigen-binding fragments of such antibodies or fragments thereof comprising the heavy chain are encompassed herein. The at least one point mutation referred to herein is preferably selected from L234A, L234F, L235A, L235E, L235Q, G236A, M252Y, S254T, T256E, S267E, H268F, N297A, K322A, K322Q, S324T, P331S, and I332E. More preferably, the at least one point mutation is selected from L234A, L235A, P331S, and N297A. It should be understood herein that "at least one point mutation" may refer to two or more mutations. For example, in one preferred embodiment of the present invention, the heavy chain comprises L234A, L235A, P331S, and N297A point mutations. As preferably understood herein, the amino acid positions listed herein refer to the residue numbering as found in the IgG1 isotype. If any other antibody class or isotype is used, one skilled in the art is in a position to translate the amino acid positions to any other antibody class or isotype known in the art.

[0137] Those skilled in the art can also envisage the inclusion of at least one mutation that increases antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). One possible combination is at least one mutation selected from S267E / H268F / S324T / G236A / I332E (EFTAE modification), preferably the mutations S267E / H268F / S324T / G236A / I332E (EFTAE modification).

[0138] Those skilled in the art can further envision including at least one mutation that counteracts the negative effect of the N297A mutation (which allows for the production of a deglycosylated antibody). One possible combination is at least one mutation selected from F241K, L309D, T307R, and T307P, preferably the mutations F241K, L309D, T307R, and T307P. According to the present inventors, at least one mutation selected from F241K, L309D, T307R, and T307P, preferably the mutations F241K, L309D, T307R, and T307P, may result in increased thermal stability of the antibody. Without being bound by theory, this mutation increases the stability of the Fc portion of the heavy chain of the antibody.

[0139] The antibody or antigen-binding fragment thereof described herein further comprises one or more of the following mutations in its heavy chain: G31D, G31E, G31K, G31P, G31R, H35K, T28D, T28E, T28K, T28P, T28R, T30D, T30E, T30K, T30P, T30R, W33R, Y27D, Y27E, and Y27K. Further encompassed by the present invention are antibodies or antigen-binding fragments thereof described herein that further comprise a mutation selected from the group consisting of G31D, G31E, G31K, G31P, G31R, H35K, T28D, T28E, T28K, T28P, T28R, T30D, T30E, T30K, T30P, T30R, W33R, Y27D, Y27E, and Y27K. Preferably, the present invention encompasses antibodies or antigen-binding fragments thereof described herein that further comprise one or more of the following mutations in the heavy chain: T30P, T28K, and T30E. According to the inventors, at least one mutation selected from G31D, G31E, G31K, G31P, G31R, H35K, T28D, T28E, T28K, T28P, T28R, T30D, T30E, T30K, T30P, T30R, W33R, Y27D, Y27E, and Y27K, preferably selected from T30P, T28K, and T30E, may stabilize the adjacent region of CDR1 and / or result in improved affinity of the antibody or fragment thereof for L1-CAM.

[0140] The present invention also encompasses the antibodies or antigen-binding fragments thereof described herein, which further comprise one or more of the following mutations in the heavy chain: A40R, V68A, and L115T. Without being bound by theory, these mutations correspond to the incorporation of residues originally present in the mouse antibody, close to the aggregation-prone region. According to the inventors, at least one mutation selected from A40R, V68A, and L115T may result in increased thermal stability of the antibody. Without being bound by theory, the mutations increase the stability of the Fc portion of the heavy chain of the antibody.

[0141] The antibodies or antigen-binding antibodies of the present invention provided herein can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0142] Further suitable and preferred examples of water-soluble polymers, such as polymers containing an acrylic backbone, are described below.

[0143] The antibodies of the invention or antigen-binding fragments thereof provided herein can be further modified to contain additional moieties, thus providing immunoconjugates comprising the antibodies of the invention or antigen-binding fragments thereof, as described herein.

[0144] antibody production Antibodies can be produced using recombinant methods and compositions described, for example, in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acids encoding the antibodies described herein are provided. Such nucleic acids can encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody).

[0145] An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0146] An "isolated nucleic acid encoding an anti-L1-CAM antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including nucleic acid molecules in a single vector or separate vectors and present in one or more locations in a host cell.

[0147] In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. Accordingly, the present invention relates to polynucleotides encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence described herein. It should be understood that the terms polynucleotide and (isolated) nucleic acid may be used interchangeably, unless indicated to the contrary.

[0148] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0149] Accordingly, the present invention further relates to vectors comprising a polynucleotide encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence described herein.

[0150] In a further embodiment, a host cell containing such nucleic acid is provided.

[0151] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0152] In one such embodiment, the host cell contains (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, or a lymphoid cell (e.g., YO, NSO, Sp20). In one embodiment, a method of producing an antibody of the invention is provided, comprising culturing a host cell comprising a nucleic acid encoding an antibody provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0153] For recombinant production of an antibody of the invention, for example, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0154] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) Following expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction, which can be further purified.

[0155] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains in which the glycosylation pathway has been "humanized" thereby resulting in the production of antibodies with partial or fully human glycosylation patterns, are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0156] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0157] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0158] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed macaque kidney CV1 (COS-7), human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Viral. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), macaque kidney cells (CV1), African green monkey kidney cells (VER0-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT060562), TRI cells, e.g., Mather et al., Annals of NY Aead. Sei. 383:44-68 (1982), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl. Acad. cii. USA 77:4216 (1980)), and myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0159] In one embodiment, the antibody or antigen-binding fragment thereof may comprise an additional domain or additional amino acid sequence. For example, the antibody or antigen-binding fragment thereof may comprise a localization sequence. Further suitable examples include aldehyde tags and sortase recognition motifs. An aldehyde tag is an artificial peptide tag recognized by formylglycine-generating enzyme (FGE). A suitable example of an aldehyde tag is the tag set forth in the sequence LCTPSR (SEQ ID NO: 52), in which a cysteine ​​residue is converted to formylglycine when FGE acts on the sequence. A sortase recognition motif is set forth in the sequence LPXTG (SEQ ID NO: 53), in which X can be any naturally occurring amino acid residue. A sortase enzyme, for example, Staphylococcus aureus sortase, is a transpeptidase that attaches surface proteins to the cell wall. It cleaves between Gly and Thr in the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of the cell wall peptidoglycan. As known to those skilled in the art, the sortase recognition motif allows for the attachment of additional peptide moieties.

[0160] The antibody or antigen-binding fragment thereof may, in one embodiment, further comprise a domain or amino acid sequence used for simultaneous targeting of the tumor microenvironment (e.g., targeting fibroblast activation protein-α (FAP)), for overcoming the blood brain barrier (BBB) ​​(e.g., targeting transferrin receptor), or for overcoming the endothelial cell (EC) barrier (e.g., caveolae targeting of aminopeptidase P2 (APP2)). Such additional targeting domains may thereby be included directly at the C- or N-terminus of the antibody or antigen-binding fragment sequence using a spacer peptide (fusion construct), or may be subsequently attached, for example, by site-specific functionalization.

[0161] The antibody or antigen-binding fragment thereof may optionally contain one or more non-standard amino acids used to couple the antibody or antigen-binding fragment thereof to another chemical entity. The amino acids may contain residues reactive in an addition reaction known to those skilled in the art as click chemistry. Suitable examples of such residues include residues containing an azide moiety, a cyclooctyne moiety, or a moiety capable of undergoing an inverse demand Diels-Alder cycloaddition reaction, such as the trans-cyclooctene / tetrazine reactive pair. However, the present invention is not intended to be limited to any of these examples, and other such residues known to those skilled in the art may also be used. Methods for producing antibodies or fragments thereof containing non-standard amino acid residues using recombinant methods are known to those skilled in the art.

[0162] In one embodiment, the present invention further relates to an immunoconjugate comprising an antibody of the present invention or an antigen-binding fragment thereof and an active agent. Preferably, but not necessarily, the active agent is a cytotoxic agent or a prodrug thereof, preferably a cytotoxic agent. Thus, as understood herein, an "immunoconjugate" is an antibody (or antigen-binding fragment thereof) conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent. The cytotoxic agent or prodrug may also be referred to as a drug.

[0163] The immunoconjugates of the present invention may also be referred to as antibody-drug conjugates. Antibody-drug conjugates (ADCs) are targeted chemotherapy molecules that combine the properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor cells (Teicher, BA (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, and Senter PD (2008) The Cancer Jour. 14(3):154-169; Chari, RV (2008) Ace. Chem. Res. 41:98-107).

[0164] The ADC compounds of the present invention include those with anti-cancer activity. In some embodiments, the ADC compounds comprise an antibody conjugated, i.e., covalently linked, to a drug moiety. In some embodiments, the antibody is covalently linked to the drug moiety via a linker. The antibody-drug conjugates (ADCs) of the present invention selectively deliver an effective dose of the drug to tumor tissue, thereby increasing the therapeutic index ("therapeutic window") while at the same time achieving greater selectivity, i.e., a lower effective dose.

[0165] As referred to herein, the linker should not be particularly limited, and any linker that is considered by a person skilled in the art to be usable for immunoconjugates or antibody-drug conjugates can be used within the scope of the present invention.

[0166] In one embodiment of the present invention, the linker takes the form of a polymeric linker. Accordingly, the polymeric linker comprises at least two types of repeat units, preferably three or more types of repeat units. It should be understood that at least one type of repeat unit is covalently attached to an active agent, e.g., a cytotoxic agent. Therefore, at least one type of repeat unit does not comprise the active agent, and thus the polymeric carrier may incorporate the active agent into some of its repeat units. Furthermore, multiple molecules of the active agent may be consequently attached to a single attachment point on the antibody or antigen-binding fragment thereof by using a polymeric carrier.

[0167] Any polymeric carrier capable of binding to an antibody or a fragment thereof can be used in the immunoconjugates of the present invention. Examples of polymeric carriers particularly suitable for use in the immunoconjugates of the present invention are described in patent applications PCT / EP2020 / 080545 and PCT / EP2019 / 061769, each of which is incorporated herein by reference in its entirety. An exemplary polymeric carrier is defined below.

[0168] Preferably, the polymeric carrier has the formula (R1)

[0169] [ka] wherein R is -H, -CH3, -CH2-CH3, or -(CH2)2-CH3; X is -NH(CH2)4-, -NH(CH2)3-, -O-C6H4-CH2-, -O-CH2-, -O-CH(CH3)-, -S-CH2-, or -NH-C6H4-CH2-; and Z is H (when A is -O-) or -C n H 2n+1 (n is 1 to 8), A is -O- or -NH-, L is a spacer, and P comprises an activator).

[0170] As preferably understood herein, when A is -O-, Z is H or -C n H 2n+1 (n is 1 to 8), and when A is -NH-, Z is -C n H 2n+1 (n is 1 to 8). Alternatively, Z is preferably H or -C n H 2n+1 (n is 1 to 8). Preferably, when A is -O-, Z is preferably H. Preferably, the copolymer comprising a repeating unit of formula (R1a) or the copolymer comprising a repeating unit of formula (R1) is a copolymer of formula (R2):

[0171] [ka] a repeat unit of the formula: (wherein R is -H, -CH3, -CH2-CH3, or -(CH2)2-CH3; X is -NH(CH2)4-, -NH(CH2)3-, -O-C6H4-CH2-, -O-CH2-, -O-CH(CH3)-, -S-CH2-, or -NH-C6H4-CH2-; Y is H or -CO-CnH2n+1 (n is 1 to 8); or Y comprises a second payload molecule; and Z is H (when A is -O-) or -CnH2n+1 (n is 1 to 8); or Z comprises a second payload molecule; and A is -O- or -NH-; and / or formula (R3):

[0172] [ka] wherein R is -H, -CH3, -CH2-CH3, or -(CH2)2-CH3; Z is H (when A is O) or -CnH2n+1 (n is 1-8); or Z comprises a second payload molecule and A is -O- or -NH-.

[0173] As preferably understood herein, when A is -O-, Z is H or -CnH2n+1 (n is 1 to 8), and when A is -NH-, Z is -CnH2n+1 (n is 1 to 8). Alternatively, Z can be preferably defined as Z is H or -CnH2n+1 (n is 1 to 8). Preferably, when A is -O-, Z is preferably H.

[0174] Preferably, Z can be H and / or Y can be H. Alternatively, Z and / or Y can comprise a second payload molecule. Payload molecules are as defined herein. Alternatively, in certain embodiments, Z is H or -CnH2n+1 (n is 1 to 8), or Z comprises a second active agent molecule.

[0175] More preferably, the polymeric carriers described herein are prepared by polymerization of N,N-dimethyl-acrylamide, N-isobutyl-acrylamide, N-tert.butyl-acrylamide, N-hydroxyethyl-acrylamide, N-(2-hydroxypropyl)-acrylamide, N-(3-hydroxypropyl)-acrylamide, N-(3-hydroxypropyl)-methacrylamide, N-(2-hydroxypropyl)-methacrylamide, N-(3-aminopropyl)-acrylamide hydrochloride, or N-(3-aminopropyl)-methacrylamide hydrochloride. or repeating units obtained by polymerization of methacrylic acid, 2-hydroxyethyl-acrylate, 2-hydroxypropyl-acrylate, 3-hydroxypropyl-acrylate, 2-hydroxy-1-methylethyl-acrylate, 2-aminoethyl acrylate hydrochloride, 3-hydroxypropyl-methacrylate, 2-hydroxy-1-methylethyl-methacrylate, 2-hydroxyethyl-methacrylate, 2-hydroxypropyl-methacrylate, or 2-aminoethyl methacrylate hydrochloride.

[0176] More preferably, in the polymeric carriers described herein that are free of repeat units of formula (R2) and (R3), the average number of repeat units according to formula (R1) per copolymer molecule is 2 to 12, preferably 2 to 8, more preferably 2 to 6.

[0177] More preferably, in the polymeric carriers described herein in which the repeat units of formula (R2) or (R3) as defined herein are not functionalized, the average number of repeat units according to formula (R1), (R2), or (R3) per copolymer molecule is from 10 to 50, preferably from 10 to 40, more preferably from 10 to 30.

[0178] More preferably, in the polymeric carriers described herein, wherein the repeat units of formula (R2) or (R3) are functionalized with a second payload molecule, the average number of repeat units according to formula (R1), (R2), or (R3) per copolymer molecule is 4 to 20, preferably 4 to 15, and more preferably 4 to 10.

[0179] Particularly preferred examples of polymeric carriers for use in the immunoconjugates of the present invention include the following:

[0180] [ka] wherein each X is independently -NH(CH2)4- or -NH-C6H4-CH2-, n is 40 to 120, m is 4 to 16, o is 0 to 16, f is -H or -I (it is understood that I includes both stable and radioactive isotopes of iodine), and k and q are designated as active payloads or can be H.

[0181] As discussed and exemplified below, the end group e may be H or may be a modified end group.

[0182] Regarding end group modification, the end group e can be introduced via a thiol reactive group. Suitable reagents include, but are not limited to:

[0183] MC-DBCO N-[3-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]-2,5-dihydro-2,5-dioxo-1H-pyrrole-1-propanamide

[0184] Methyltetrazine-PEG4-maleimide 3-(2,5-Dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3yl)phenoxy]ethoxy]ethoxy]-ethoxy]ethyl]propanamide Cas1802908-02-6 It should be understood that the payload molecules k and q can be introduced via amino-reactive groups. Suitable reagents that can be used to introduce the payload molecules are selected from the following: p-SCN-Bn-CHX-A″-DTPA: [(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid; p-SCN-Bn-DTPA: S-2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid; DOTA-GA anhydride: 2,2',2"-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; p-SCN-Bn-DOTA: S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid; NHS DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester; p-SCN-Bn-NOTA: 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; NOTA-NHS ester: 2,2'-(7-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid; NODA-GA-NHS ester: 2,2'-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid; Paclitaxel NHS: (1S,2R)-1-benzamido-3-(((2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-6,12b-diacetoxy-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxet-9-yl)oxy)-3-oxo-1-phenylpropan-2-yl(2,5-dioxopyrrolidin-1-yl)succinate; MMAE-NHS: 4-(2-(2-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl(1-((1-((1-(2-(3-((1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate; MMAF-NHS: N-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropanoyl-L-phenylalanine; DM1-SMCC (maytansinoid-NHS-derivative): N2'-deacetyl-N2'-[3-[[1-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]cyclohexyl]methyl]-2,5-dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]-maytansine; SC-VC-PAB-DM1(CAS:2259318-47-1); Doxorubicin-SMCC: 7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-10-[[2,3,6-trideoxy-3-[[[4-[(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)methyl]cyclohexyl]carbonyl]amino]-α-L-lyxo-hexopyranosyl]oxy]-,(8S,10S)-5,12- Naphthacendione; Reagents suitable for introducing kinase inhibitors (preferably fasudil, sirolimus, imatinib, gefitinib, erlotinib, soragenib, sunitinib, dasatinib, lapatinib, nilotinib, temsirolimus, everolimus, pazopanib, ruxolitinib, vandetanib, vemurafenib, crizotinib, icotinib, axitinib, tofacitinib, bosutinib, cabozantinib, ponatinib, levothyroxine ... Gorafenib, Afatinib, Dabrafenib, Trametinib, Ibrutinib, Nintedanib, Idelasilib, Ceritinib, Apatinib, Ripasudil, Alectinib, Cobimetinib, Lenvatinib, Palbociclib, Radotinib, Osimertinib, Olmutinib, Neratinib, Ribociclib, Copanlisib, Abemaciclib, Acalabrunitib , Midostaurin, Brigatinib, Baricitinib, Netarsudil, Tivozanib, Simotinib, Fostamatinib, Encorafenib, Binimetinib, Catequentinib, Duvelisib, Dacomitinib, Lorlatinib, Larotrectinib, Gilteritinib, Pyrotinib, Fruquintinib, Erdafitinib, Alelisib, Umbralisib, Leniolisib, Pexidartinib, Entrek and selected from the group consisting of rituximab, ...

[0185] Suitable reagents for introducing doxorubicin or its derivatives. Preferably, derivatives of doxorubicin are understood as compounds comprising the following moieties:

[0186] [ka]

[0187] Preferably, the doxorubicin derivative comprises a DBCO moiety. PNU-159682; Alternatively, payload molecules k and q can be introduced in a two-step process via a Click reaction, where the amine-reactive reagent is an NHS ester of an azide-functionalized carboxylic acid and the payload molecule is, but is not limited to:

[0188] DBCO-DOTA 4,7,10-Tetraazacyclododecane-1,4,7-tris(acetic acid)-10-[3-oxo-3-(5-azadibenzocyclooxin)acetamide]

[0189] DBCO-PEG4-Val-Cit-PAB-MMAE [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-3-methylbutanoyl]amino]-5-(carbamoylamino)pentanoyl]amino]phenyl]methyl N-[(2S)- 1-[[(2S)-1-[[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-N-methylcarbamate DBCO-Val-Cit-PAB-MMAE DBCO-Val-Cit-PAB-MMAF DBCO-PEG4-MMAF(CAS:2360411-65-8) DM1-PEG4-DBCO DBCO-PEG4-Ahx-DM1 Reagents suitable for introducing kinase inhibitors (preferably fasudil, sirolimus, imatinib, gefitinib, erlotinib, soragenib, sunitinib, dasatinib, lapatinib, nilotinib, temsirolimus, everolimus, pazopanib, ruxolitinib, vandetanib, vemurafenib, crizotinib, icotinib, axitinib, tofacitinib, bosutinib, cabozantinib, ponatinib, levothyroxine ... Gorafenib, Afatinib, Dabrafenib, Trametinib, Ibrutinib, Nintedanib, Idelasilib, Ceritinib, Apatinib, Ripasudil, Alectinib, Cobimetinib, Lenvatinib, Palbociclib, Radotinib, Osimertinib, Olmutinib, Neratinib, Ribociclib, Copanlisib, Abemaciclib, Acalabrunitib , Midostaurin, Brigatinib, Baricitinib, Netarsudil, Tivozanib, Simotinib, Fostamatinib, Encorafenib, Binimetinib, Catequentinib, Duvelisib, Dacomitinib, Lorlatinib, Larotrectinib, Gilteritinib, Pyrotinib, Fruquintinib, Erdafitinib, Alelisib, Umbralisib, Leniolisib, Pexidartinib, Entrek and selected from the group consisting of rituximab, ...

[0190] Suitable reagents for introducing doxorubicin or its derivatives. Preferably, derivatives of doxorubicin are understood as compounds comprising the following moieties:

[0191] [ka]

[0192] Preferably, the doxorubicin derivative comprises a DBCO moiety. DBCO-PEG4-VC-PAB-DMEA-PNU-159682(CAS:2259318-56-2)

[0193] Suitable reagents for introducing AZD7648:

[0194] [ka] Those skilled in the art will understand that the phrase "derived from" recited herein preferably refers to the attachment of any of these compounds to the antibody or fragment thereof, for example, by a condensation reaction involving a free carboxylic acid group. It should be understood that the above formula preferably does not represent a block copolymer, but rather the statistical composition of the resulting polymer. However, it should be understood that the formation of block copolymers or gradient copolymers may also be feasible depending on the specific conditions in the reactor, and therefore block copolymers or gradient copolymers may also be encompassed by the definition provided herein.

[0195] The drug moiety (D) of an antibody-drug conjugate (ADC) can comprise any compound, moiety, or group that has a cytotoxic or cytostatic effect. Drug moieties may confer their cytotoxic and cytostatic effects by mechanisms including, but not limited to, tubulin binding, DNA binding, or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drug moieties include, but are not limited to, maytansinoids, calicheamicin, pyrrolobenzodiazepines (PBDs), nemorubicin and its derivatives, PNU-159682, anthracyclines, duocarmycins, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecin, deruxtecan, exatecan, elinafide, and their stereoisomers, isosteres, analogs, and derivatives that have cytotoxic activity. Non-limiting examples of such immunoconjugates are discussed in more detail below. In a preferred embodiment, drug moiety (D) is a topoisomerase inhibitor, i.e., a topoisomerase I and / or II inhibitor, such as a derivative of camptothecin, preferably selected from exatecan, deruxtecan, topotecan, irinotecan, SN38, and belotecan. In one embodiment, the active agent (i.e., drug moiety (D)) is selected from maytansinoids, calicheamicin, pyrrolobenzodiazepines (PBDs), nemorubicin and its derivatives, PNU-159682, anthracyclines, duocarmycins, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecin, efinafide, exatecan, deruxtecan, topotecan, irinotecan, SN38, and belotecan.

[0196] One skilled in the art can conjugate a drug moiety to an antibody. For example, exatecan can be conjugated as shown in the following scheme, where R generally represents the antibody or antigen-binding fragment thereof, including the remainder of the bond between the antibody or fragment thereof and the drug moiety (D).

[0197] [ka]

[0198] Another suitable example of a moiety that can be incorporated into the antibody-drug conjugates of the invention is a moiety derived from a compound according to the formula:

[0199] [ka]

[0200] It is understood that addition or click chemistry reactions are preferred for attaching the compounds provided herein to the present antibodies or antigen-binding fragments thereof.

[0201] Another suitable example of a drug moiety for incorporation into the antibody-drug conjugates of the invention is maytansine.

[0202] Exemplary embodiments of antibody-drug conjugate (ADC) compounds include an antibody (Ab) that targets a tumor cell, a drug moiety (D), and a linker moiety (L) that connects the Ab to D. In some embodiments, the antibody is attached to the linker moiety (L) through one or more amino acid residues, such as lysine and / or cysteine.

[0203] In one embodiment, the linker moiety is a polymeric carrier as described above.

[0204] In one embodiment, two or more linker moieties are attached to the antibody or antigen-binding fragment thereof. Thus, in such an embodiment, the immunoconjugate comprises A-(LD) i where i is an integer and i is greater than 1.

[0205] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Tb161 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, e.g., small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various anti-tumor or anti-cancer agents disclosed herein (e.g., topoisomerase inhibitors disclosed herein).

[0206] The term "radioisotope / radionuclide" within the scope of the present invention is preferably used synonymously and preferably refers to an atom that has excess nuclear energy, thereby making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferred to one of its electrons and emitted as a conversion electron; or used to generate and emit new particles (alpha or beta particles) from the nucleus. Radioisotope / radionuclide is herein preferably defined as an atom having a 10 19 Defined as an isotope with a half-life of less than a year.

[0207] Thus, in one embodiment, the present invention relates to an immunoconjugate, which comprises a radioisotope as an active agent, such a conjugate may also be referred to as an antibody-radionuclide conjugate.

[0208] The term "antibody radionuclide conjugate" (ARC) is preferably defined as a variant of ADC, where "drug molecule or active molecule" refers to a radionuclide / radioisotope, which is either covalently attached to the antibody-polymer conjugate, e.g., radioactive iodine, or covalently attached via a metal chelator complex with, e.g., radioactive lutetium, actinium, or terbium. ARCs thus formed can deliver large amounts of radiation to tumor tissue, thereby killing tumor cells due to damage to DNA, essential enzymes, etc. As will be apparent to those skilled in the art, the term ARC also encompasses antibody-radionuclide conjugates that do not include a polymer carrier but instead contain a PEG or peptide linker. One suitable example of such a peptide linker is (Ac)-Lys-Ala-Tyr-Ala-Lys(azido)-NH2 [SEQ ID NO: 54], which is useful for subsequent functionalization with DBCO-DOTA.

[0209] As will be appreciated by those skilled in the art, certain antibody-radionuclide conjugates may be used for diagnostic purposes. Certain radionuclides disclosed herein can be monitored; for example, terbium-161, due to its gamma emission, can be visualized with a gamma camera and therefore can be used to detect cancer tissues or cell types targeted by the antibody. Terbium-149, which can be used for targeted alpha therapy, is visible in PET scans and can therefore be monitored. According to the present disclosure, fluorine-18, scandium-43, scandium-44, copper-61, copper-64, gallium-68, zirconium-89, indium-111, iodine-123, terbium-152, and terbium-155 are particularly useful for the diagnostic applications described herein and may be referred to as diagnostically useful radionuclides. As known to those skilled in the art, diagnostically useful radionuclides can be monitored by using suitable methods, such as scintigraphy, Single Photon Emission Computed Tomography (SPE-CT), or Positron Emission Tomography Computed Tomography (PET-CT).

[0210] Those skilled in the art will appreciate that immunoconjugates in which the active agent comprises a radionuclide useful for therapeutic applications (these radionuclides may be referred to as therapeutically useful radionuclides), e.g., selected from copper-67, strontium-89, yttrium-90, iodine-131, samarium-153, lutetium-177, radium-223, and actinium-225, have substantially the same biodistribution as immunoconjugates in which the active agent is a radionuclide useful for diagnostic applications. Thus, the immunoconjugates of the present invention can preferably be used to monitor the biodistribution of therapeutic immunoconjugates during treatment. For example, immunoconjugates containing an active agent that is a therapeutically useful radionuclide can, for this purpose, preferably be supplemented with less than 10% by weight of immunoconjugates in which the active agent comprises a diagnostically useful radionuclide, as defined herein. More preferably, immunoconjugates of the present invention for use in combined therapeutic and diagnostic applications may contain two radionuclides, one therapeutically useful and one diagnostically useful, bound to different repeating units of a single polymeric carrier. Preferred combinations are those in which the therapeutically useful radionuclide and the diagnostically useful radionuclide are isotopes of the same element. Thus, preferred combinations include scandium-43 and scandium-47, copper-61 and copper-67, copper-64 and copper-67, iodine-123 and iodine-131, terbium-152 and terbium-161, and terbium-155 and terbium-161. Further preferred combinations include isotopes of two different elements, such as indium-111 and lutetium-177, and indium-111 and terbium-161.

[0211] Pharmaceutical formulations of the antibodies or immunoconjugates of the invention described herein are prepared by mixing such antibodies or immunoconjugates having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, proteins such as guanylate, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA or GLDA; sugars such as sucrose, mannitol, trehalose, or sorbitol; osmoprotectants such as ectoine; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0212] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0213] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0214] Exemplary lyophilized antibody or immunoconjugate formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody or immunoconjugate formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine-acetate buffer.

[0215] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.

[0216] The active ingredient may be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0217] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0218] The formulations to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0219] In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof of the present invention, an immunoconjugate of the present invention, or a pharmaceutical composition of the present invention for use as a medicament. In other words, the present invention relates to an antibody or antigen-binding fragment thereof of the present invention, an immunoconjugate of the present invention, or a pharmaceutical composition of the present invention for use in therapy. It will be understood that the antibody, immunoconjugate, or pharmaceutical composition of the present invention can be used to treat a disease or disorder.

[0220] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the disease in the treated individual, and can be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of the disease.

[0221] The antibodies or immunoconjugates of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as, if desired for localized treatment, intralesional, intrauterine, or intravesical administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time periods, a bolus dose, and pulse infusion.

[0222] The antibodies or immunoconjugates of the present invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to physicians. The antibodies or immunoconjugates are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody or immunoconjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and via the same routes of administration as those described herein, or at about 1-99% of the dosages described herein, or at any dosage and via any route empirically / clinically determined to be appropriate.

[0223] The appropriate dosage of the antibody or immunoconjugate of the invention for disease prevention or treatment (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of antibody or immunoconjugate, the severity and course of the disease, whether the antibody or immunoconjugate is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antibody or immunoconjugate, and the discretion of the attending physician. The antibody or immunoconjugate is preferably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage for administration to a patient may be about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg), whether by one or more separate administrations or by continuous infusion, for example. One typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment is generally sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody or immunoconjugate would be in the range of about 0.05 mg / kg to about 10 mg / kg. Accordingly, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives about two to about 20 doses, or for example, about six doses of the antibody). A higher initial loading dose, followed by one or more lower doses, may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0224] In one embodiment, the present invention relates to the antibody or antigen-binding fragment thereof of the present invention, the immunoconjugate of the present invention, or the pharmaceutical composition of the present invention for use in the treatment of an L1-CAM (CD171)-associated cancer.

[0225] Preferably, as understood herein, an L1-CAM (CD171) associated cancer is selected from the group consisting of leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, quadruple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, cholangiocarcinoma, carcinoid, neuroendocrine tumors, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, pancreatic neuroectodermal carcinoma, and the like. cancer), pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma, tumor vasculature, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine carcinoma, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granular cell tumor, Schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, prostate cancer, and head and neck squamous cell carcinoma.

[0226] The antibodies or immunoconjugates of the invention can be used in therapy alone or in combination with other agents, for example, the antibodies or immunoconjugates of the invention can be co-administered with at least one additional therapeutic agent.

[0227] Such combination therapy as described above encompasses combined administration (where two or more therapeutic agents are in the same formulation or in separate formulations) and separate administration, in which case the administration of the antibody or immunoconjugate of the invention can occur prior to, concurrently with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The antibody or immunoconjugate of the invention can also be used in combination with radiation therapy.

[0228] Preferably, the antibody or antigen-binding fragment thereof, or immunoconjugate will be administered to the subject along with an additional therapeutic agent selected from an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen, an aromatase inhibitor, an ovarian function suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, an immune checkpoint inhibitor, and a bisphosphonate therapy.

[0229] Preferably, the antibody or antigen-binding fragment thereof, or immunoconjugate of the present invention is to be administered to a subject together with an additional therapeutic agent, which may be, for example, an agent that sensitizes cells to radiation therapy, selected from a protein kinase inhibitor and a DNA intercalator. Preferably, the protein kinase inhibitor is selected from the group consisting of alisertib, MK1775, MK2206, saracatinib, temsirolimus, crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, vemurafenib, dabrafenib, ibrutinib, palbociclib, sorafenib, ribociclib, crizotinib, cabozantinib, gefitinib, erlotinib, lapatinib, vandetanib, afatinib, osimertinib, rucr ... It is selected from xolitinib, tofacitinib, trametinib, axitinib, gefitinib, imatinib, lenvatinib, nintedanib, pazopanib, regorafenib, sorafenib, sunitinib, vandetanib, bosutinib, dasatinib, ponatinib, vandetanib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib and sunitinib, more preferably selected from alisertib, MK1775, MK2206, saracatinib and temsirolimus.Preferably, the DNA intercalator is selected from doxorubicin and nemorubicin.

[0230] The drug that sensitizes cells to radiation therapy may be AZD7648:

[0231] [ka] As will be apparent to one of skill in the art in light of the above disclosure, the present invention further relates to an antibody or antigen-binding fragment thereof of the invention, an immunoconjugate of the invention, or a pharmaceutical composition of the invention for use in the manufacture of a medicament for treating an L1-CAM-associated cancer.

[0232] As will be further apparent to those skilled in the art in light of the above disclosure, the present invention further relates to a method for treating an L1-CAM-associated cancer, comprising administering to an individual in need thereof an antibody or antigen-binding fragment thereof of the present invention, an immunoconjugate of the present invention, or a pharmaceutical composition of the present invention. It should be understood that the antibody or antigen-binding fragment thereof, the immunoconjugate of the present invention, or the pharmaceutical composition of the present invention is to be administered in a therapeutically effective amount.

[0233] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as macaques), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0234] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0235] The present invention further relates to the antibody or antigen-binding fragment thereof, or the immunoconjugate of the present invention for use in diagnosis. The antibody, antigen-binding fragment thereof, and immunoconjugate of the present invention are particularly useful for diagnosing L1-CAM (CD171)-associated cancers, as described herein.

[0236] Preferably, the L1-CAM (CD171)-associated cancer is leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, quadruple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, cholangiocarcinoma, carcinoid, neuroendocrine tumor, gastrointestinal interstitial carcinoma, or the like. The tumor is selected from: gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, pancreatic neuroectodermal carcinoma, pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma, tumor vascular, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine carcinoma, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granular cell tumor, Schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, prostate cancer, and head and neck squamous cell carcinoma.

[0237] As will be appreciated by those skilled in the art, the present antibodies or antigen-binding fragments thereof may be used in diagnostic methods, particularly in vitro diagnostic methods, in which the antibodies or fragments thereof are used to detect L1-CAM-positive cells in tissue, which are then detected by a secondary antibody, preferably a monoclonal antibody.

[0238] As encompassed by the present invention, diagnostically useful immunoconjugates contain an active agent, which is a moiety that enables detection and localization of the immunoconjugate. Therefore, the active agent is not particularly limited as long as it is suitable for diagnostic purposes, but is preferably selected from radionuclides, MRI-active compounds, ultrasound contrast agents, fluorophores (preferably fluorophores in the far-infrared / near-infrared spectral region), markers for PET and SPECT, and Gd-based and Fe-particle-based MRI contrast agents. Suitable imaging methods that enable diagnostic applications are known to those skilled in the art. Preferably, the active agent is a radionuclide. Preferably, the radionuclide is a diagnostically useful radionuclide selected from fluorine-18, scandium-43, scandium-44, copper-61, copper-64, gallium-68, zirconium-89, indium-111, iodine-123, terbium-152, and terbium-155.

[0239] The antibody-radionuclide conjugates of the invention are particularly useful in diagnosis, and the radionuclide is, as described above, preferably a diagnostically useful radionuclide selected from fluorine-18, scandium-43, scandium-44, copper-61, copper-64, gallium-68, zirconium-89, indium-111, iodine-123, terbium-152, and terbium-155.

[0240] Further examples and / or embodiments of the present invention are disclosed in the following numbered items:

[0241] 1. An antibody or antigen-binding fragment thereof that specifically binds to L1-CAM (CD171), wherein the antibody or antigen-binding fragment thereof is: A variable heavy chain region comprising: CDR-H1, The sequence set forth in SEQ ID NO: 1 (GYWMH), The sequence set forth in SEQ ID NO: 2 (GYYMH), The sequence set forth in SEQ ID NO: 3 (GYFMH), and the sequence set forth in SEQ ID NO: 4 (GYLMH), and CDR-H2, The sequence set forth in SEQ ID NO: 5 (EINPSNGRTNYNERFQG), The sequence set forth in SEQ ID NO: 6 (EINPSNGRTNYNEKFQG), The sequence set forth in SEQ ID NO: 7 (EINPSNGRTNYNERFKS), The sequence set forth in SEQ ID NO: 8 (EINPSNGRTNYNERLKS), The sequence set forth in SEQ ID NO: 9 (EINPSNARTNYNERFQG), Sequence set forth in SEQ ID NO: 10 (EINPSNARTNYNEKFQG) The sequence set forth in SEQ ID NO: 11 (EINPSNARTNYNERFKS), and CDR-H2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 12 (EINPSNARTNYNERLKS), and a variable heavy chain region comprising a CDR-H3 characterized by the sequence set forth in SEQ ID NO: 13 (DYYGTSYNFDY); and / or A variable light chain region comprising: CDR-L1, The sequence set forth in SEQ ID NO: 14 (RANEDINNRLA), The sequence set forth in SEQ ID NO: 15 (KANEDINNRLA), The sequence set forth in SEQ ID NO: 16 (QANEDINNRLA), the sequence set forth in SEQ ID NO: 17 (RANEDINARLA), The sequence set forth in SEQ ID NO: 18 (KANEDINARLA), The sequence set forth in SEQ ID NO: 19 (QANEDINARLA), The sequence set forth in SEQ ID NO: 20 (RANEDINLRLA), The sequence set forth in SEQ ID NO: 21 (KANEDINLRLA), and CDR-L1 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 22 (QANEDINLRLA), and CDR-L2, The sequence set forth in SEQ ID NO: 23 (GATNLVT), and CDR-L2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 24 (GASNLVS), and CDR-L3, The sequence set forth in SEQ ID NO: 25 (QQYWSTPFT), The sequence set forth in SEQ ID NO: 26 (QQYYSTPFT), and An antibody or antigen-binding fragment thereof, comprising a CDR-L3 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 27 (QQYFSTPFT).

[0242] 2. The antibody or antigen-binding fragment thereof according to item 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a recombinant antibody, an antigen-binding fragment of a recombinant antibody, a single-chain antibody, a humanized antibody, a bispecific antibody, a multispecific antibody, or an antibody displayed on the surface of a phage or on the surface of a chimeric antigen receptor (CAR) T cell, and preferably the antibody or antigen-binding fragment thereof is a monoclonal antibody, preferably an IgG1 antibody.

[0243] 3. The antibody or antigen-binding fragment thereof according to item 1 or 2, wherein the variable heavy chain region comprises a CDR-H1 characterized by the sequence set forth in SEQ ID NO: 1.

[0244] 4. The antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the variable heavy chain region comprises a CDR-H2 characterized by the sequence set forth in SEQ ID NO: 5, 6, 9, or 10, preferably characterized by the sequence set forth in SEQ ID NO: 5 or 6.

[0245] 5. The antibody or antigen-binding fragment thereof according to any one of items 1 to 4, wherein the variable light chain region comprises a CDR-L1 characterized by the sequence set forth in SEQ ID NO: 14, 15, 17, 18, or 21, preferably characterized by the sequence set forth in SEQ ID NO: 14 or 15.

[0246] 6. The antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein the variable light chain comprises a CDR-L2 characterized by the sequence set forth in SEQ ID NO: 23.

[0247] 7. The antibody or antigen-binding fragment thereof according to any one of items 1 to 6, wherein the variable light chain comprises a CDR-L3 characterized by the sequence set forth in SEQ ID NO: 25.

[0248] 8. The variable heavy chain region is The sequence set forth in SEQ ID NO: 28 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), The sequence set forth in SEQ ID NO: 29 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTVSS), The sequence set forth in SEQ ID NO: 30 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFTGYWMHWIRQPPGKGLEWIGEINPSNGRTNYNERLKSRVTLSVDKSKNQASLKLSSVTAADTAVYFCARDYYGTSYNFDYWGQGTLVTVSS), The sequence set forth in SEQ ID NO: 31 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), and 3. The antibody or antigen-binding fragment thereof according to item 1 or 2, characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical to a sequence selected from the sequence set forth in SEQ ID NO: 32 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWmGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), and preferably characterized by the sequence set forth in SEQ ID NO: 28 or the sequence set forth in SEQ ID NO: 32.

[0249] 9. The variable light chain region is The sequence set forth in SEQ ID NO: 33 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), The sequence set forth in SEQ ID NO: 34 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDIATYYCQQYWSTPFTFGQGTKLEIK), The sequence set forth in SEQ ID NO: 35 (EIVMTQSPATLSVSPGERATLSCRANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEFTLTISSLQSEDFAVYYCQQYWSTPFTFGQGTKLEIK), The sequence set forth in SEQ ID NO: 36 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), and 3. The antibody or antigen-binding fragment thereof according to item 1 or 2, characterized in that the antibody or antigen-binding fragment thereof is at least 90% identical, preferably at least 95% identical, more preferably identical to a sequence selected from the sequence set forth in SEQ ID NO: 37 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGAsNLVsGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), and is preferably characterized in that the antibody or antigen-binding fragment thereof is selected from the sequence set forth in SEQ ID NO: 33, the sequence set forth in SEQ ID NO: 36, and the sequence set forth in SEQ ID NO: 37.

[0250] 10. the variable heavy chain region is characterized by the sequence set forth in SEQ ID NO: 28 and the variable light chain region is characterized by the sequence set forth in SEQ ID NO: 33; or the variable heavy chain region is characterized by the sequence set forth in SEQ ID NO: 32 and the variable light chain region is characterized by the sequence set forth in SEQ ID NO: 36, or 10. The antibody or antigen-binding fragment thereof according to any one of items 1 to 9, wherein the variable heavy chain region is characterized by the sequence set forth in SEQ ID NO: 14 and the variable light chain region is characterized by the sequence set forth in SEQ ID NO: 37.

[0251] 11. The variable heavy chain region has the sequence: FH0-CDR-H1-FH1-CDR-H2-FH2-CDR-H3-FH3 wherein CDR-H1, CDR-H2, and CDR-H3 are as defined in claim 1; FH0 is characterized by the sequence set forth in SEQ ID NO: 38 (QVQLVQSGAEVKKPGASVKVSCKASGYTFT) or the sequence set forth in SEQ ID NO: 39 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFT), FH1 is characterized by the sequence set forth in SEQ ID NO: 40 (WVRQAPGQGLEWIG) or the sequence set forth in SEQ ID NO: 41 (WIRQPPGKGLEWIG), FH2 is characterized by the sequence set forth in SEQ ID NO: 42 (RVTLTVDKSISTAYMELSRLRSDDTAVYFCAR) or the sequence set forth in SEQ ID NO: 43 (RVTLSVDKSKNQASLKLSSVTAADTAVYFCAR), FH3 is characterized by a sequence at least 90% identical, preferably at least 95% identical, more preferably identical to the sequence set forth in SEQ ID NO: 44 (WGQGTLVTVSS), and / or The variable light chain region has the sequence: FL0-CDR-L1-FL1-CDR-L2-FL2-CDR-L3-FL3 wherein CDR-L1, CDR-L2, and CDR-L3 are as defined in claim 1; FL0 is characterized by the sequence set forth in SEQ ID NO: 45 (DIQMTQSPSSLSASVGDRVTITC) or the sequence set forth in SEQ ID NO: 46 (EIVMTQSPATLSVSPGERATLSC), FL1 is characterized by the sequence set forth in SEQ ID NO: 47 (WYQQKPGKAPKLLIS) or the sequence set forth in SEQ ID NO: 48 (WYQQKPGQAPRLLIS), FL2 is characterized by the sequence set forth in SEQ ID NO: 49 (GVPSRFSGSGSGKDYTLTISSLQPEDIATYYC) or the sequence set forth in SEQ ID NO: 50 (GIPARFSGSGSGKEFTLTISSLQSEDFAVYYC), 3. The antibody or antigen-binding fragment thereof according to item 1 or 2, wherein FL3 is characterized by a sequence at least 90% identical, preferably at least 95% identical, more preferably identical to the sequence set forth in SEQ ID NO: 51 (FGQGTKLEIK).

[0252] 12. The antibody or antigen-binding fragment thereof according to any one of items 1 to 11, wherein the heavy chain further comprises at least one point mutation in the Fc portion that affects antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), serum half-life, and / or glycosylation status of the antibody.

[0253] 13. The antibody or antigen-binding fragment thereof according to item 11, wherein at least one point mutation is selected from L234A, L234F, L235A, L235E, L235Q, G236A, M252Y, S254T, T256E, S267E, H268F, N297A, K322A, K322Q, S324T, P331S, and I332E, preferably, at least one point mutation is selected from L234A, L235A, P331S, and N297A.

[0254] 14. Measured by Biacore-based assay, 10 -12 The dissociation constant K for L1-CAM (CD171) does not exceed M D 14. The antibody or antigen-binding fragment thereof according to any one of items 1 to 13,

[0255] 15. A polynucleotide encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence according to any one of items 1 to 11.

[0256] 16. A host cell containing the polynucleotide according to item 15.

[0257] 17. An immunoconjugate comprising the antibody or antigen fragment thereof conjugated thereto according to any one of items 1 to 14 and an active agent, preferably, the active agent is a cytotoxic agent or a prodrug thereof.

[0258] 18. The immunoconjugate of item 17, wherein the antibody or antigen-binding fragment thereof is linked to the active agent via a linker moiety, preferably the linker moiety comprises a polymeric carrier to which at least one active agent is attached.

[0259] 19. The immunoconjugate according to item 17 or 18, wherein the active agent is a radionuclide preferably selected from copper-67, strontium-89, yttrium-90, iodine-131, samarium-153, terbium-161, lutetium-177, astatine-211, radium-223, and actinium-225, preferably a radionuclide useful for therapeutic applications, and / or a radionuclide preferably useful for diagnostics selected from fluorine-18, scandium-43, scandium-44, copper-61, copper-64, gallium-68, zirconium-89, indium-111, iodine-123, terbium-152, and terbium-155.

[0260] 20. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 14, or the immune complex according to any one of items 17 to 19, and a pharmaceutically acceptable carrier.

[0261] 21. The antibody or antigen-binding fragment thereof according to any one of items 1 to 14, or the immune complex according to any one of items 17 to 19, for use as a pharmaceutical.

[0262] 22. The antibody or antigen-binding fragment thereof according to any one of items 1 to 14, or the immunoconjugate according to any one of items 17 to 19, for use in the treatment of L1-CAM (CD171)-associated cancer.

[0263] 23. L1-CAM (CD171)-associated cancers include leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, quadruple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, cholangiocarcinoma, carcinoid, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, and glioma. 23. The antibody or antigen-binding fragment thereof, or immunoconjugate for use according to item 22, wherein the antibody or antigen-binding fragment thereof is selected from pancreatic neuroectodermal carcinoma, pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma, tumor vasculature, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine carcinoma, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granular cell tumor, Schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, prostate cancer, and head and neck squamous cell carcinoma.

[0264] 24. The antibody or antigen-binding fragment thereof, or immunoconjugate for use according to item 22 or 23, wherein the antibody or antigen-binding fragment thereof, or immunoconjugate is to be administered to a subject in conjunction with an additional therapeutic agent selected from an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen, an aromatase inhibitor, an ovarian function suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, an immune checkpoint inhibitor, and a bisphosphonate therapy.

[0265] 25. The antibody or antigen-binding fragment thereof according to any one of items 1 to 14, or the immune complex according to any one of items 17 to 19, for use in diagnosis.

[0266] 26. The antibody or antigen-binding fragment thereof according to any one of items 1 to 14, or the immunoconjugate according to any one of items 17 to 19, for use in diagnosing L1-CAM (CD171)-associated cancer.

[0267] 27. L1-CAM (CD171)-associated cancers include leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, quadruple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, cholangiocarcinoma, carcinoid, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, and glioma. 27. The antibody or antigen-binding fragment thereof, or immunoconjugate for use according to Item 26, wherein the antibody or antigen-binding fragment thereof is selected from pancreatic neuroectodermal carcinoma, pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma, tumor vasculature, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine carcinoma, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granular cell tumor, Schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, prostate cancer, and head and neck squamous cell carcinoma.

[0268] Further examples and / or embodiments of the present invention are disclosed in the following numbered clauses.

[0269] 1. An antibody or antigen-binding fragment thereof that specifically binds to L1-CAM (CD171), wherein the antibody or antigen-binding fragment thereof is: A variable heavy chain region comprising: CDR-H1, The sequence set forth in SEQ ID NO: 1 (GYWMH), The sequence set forth in SEQ ID NO: 2 (GYYMH), The sequence set forth in SEQ ID NO: 3 (GYFMH), and the sequence set forth in SEQ ID NO: 4 (GYLMH), and CDR-H2, The sequence set forth in SEQ ID NO: 5 (EINPSNGRTNYNERFQG), The sequence set forth in SEQ ID NO: 6 (EINPSNGRTNYNEKFQG), The sequence set forth in SEQ ID NO: 7 (EINPSNGRTNYNERFKS), The sequence set forth in SEQ ID NO: 8 (EINPSNGRTNYNERLKS), The sequence set forth in SEQ ID NO: 9 (EINPSNARTNYNERFQG), Sequence set forth in SEQ ID NO: 10 (EINPSNARTNYNEKFQG) The sequence set forth in SEQ ID NO: 11 (EINPSNARTNYNERFKS), and CDR-H2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 12 (EINPSNARTNYNERLKS), and a variable heavy chain region comprising a CDR-H3 characterized by the sequence set forth in SEQ ID NO: 13 (DYYGTSYNFDY); and / or A variable light chain region comprising: CDR-L1, The sequence set forth in SEQ ID NO: 14 (RANEDINNRLA), The sequence set forth in SEQ ID NO: 15 (KANEDINNRLA), The sequence set forth in SEQ ID NO: 16 (QANEDINNRLA), the sequence set forth in SEQ ID NO: 17 (RANEDINARLA), The sequence set forth in SEQ ID NO: 18 (KANEDINARLA), The sequence set forth in SEQ ID NO: 19 (QANEDINARLA), The sequence set forth in SEQ ID NO: 20 (RANEDINLRLA), The sequence set forth in SEQ ID NO: 21 (KANEDINLRLA), and CDR-L1 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 22 (QANEDINLRLA), and CDR-L2, The sequence set forth in SEQ ID NO: 23 (GATNLVT), and CDR-L2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 24 (GASNLVS), and CDR-L3, The sequence set forth in SEQ ID NO: 25 (QQYWSTPFT), The sequence set forth in SEQ ID NO: 26 (QQYYSTPFT), and and a variable light chain region comprising a CDR-L3 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 27 (QQYFSTPFT), Preferably, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a recombinant antibody, an antigen-binding fragment of a recombinant antibody, a single-chain antibody, a humanized antibody, a bispecific antibody, a multispecific antibody, or an antibody displayed on the surface of a phage or on the surface of a chimeric antigen receptor (CAR) T cell, more preferably the antibody or antigen-binding fragment thereof is a monoclonal antibody, preferably an IgG1 antibody.

[0270] 2. The variable heavy chain region comprises a CDR-H1 characterized by the sequence set forth in SEQ ID NO: 1, and / or the variable heavy chain region comprises a CDR-H2 characterized by the sequence set forth in SEQ ID NO: 5, 6, 9 or 10, preferably characterized by the sequence set forth in SEQ ID NO: 5 or 6; and / or the variable light chain region comprises a CDR-L1 characterized by the sequence set forth in SEQ ID NO: 14, 15, 17, 18 or 21, preferably characterized by the sequence set forth in SEQ ID NO: 14 or 15; and / or the variable light chain comprises a CDR-L2 characterized by the sequence set forth in SEQ ID NO: 23, and / or 2. The antibody or antigen-binding fragment thereof of clause 1, wherein the variable light chain comprises a CDR-L3 characterized by the sequence set forth in SEQ ID NO:25.

[0271] 3. The variable heavy chain region is The sequence set forth in SEQ ID NO: 28 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), The sequence set forth in SEQ ID NO: 29 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTVSS), The sequence set forth in SEQ ID NO: 30 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFTGYWMHWIRQPPGKGLEWIGEINPSNGRTNYNERLKSRVTLSVDKSKNQASLKLSSVTAADTAVYFCARDYYGTSYNFDYWGQGTLVTVSS), The sequence set forth in SEQ ID NO: 31 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), and characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical to a sequence selected from the sequence set forth in SEQ ID NO: 32 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWmGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), and preferably characterized by the sequence set forth in SEQ ID NO: 28 or the sequence set forth in SEQ ID NO: 32, and / or The variable light chain region is The sequence set forth in SEQ ID NO: 33 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), The sequence set forth in SEQ ID NO: 34 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDIATYYCQQYWSTPFTFGQGTKLEIK), The sequence set forth in SEQ ID NO: 35 (EIVMTQSPATLSVSPGERATLSCRANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEFTLTISSLQSEDFAVYYCQQYWSTPFTFGQGTKLEIK), The sequence set forth in SEQ ID NO: 36 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), and 3. The antibody or antigen-binding fragment thereof according to clause 1 or 2, characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical to a sequence selected from the sequence set forth in SEQ ID NO: 37 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGAsNLVsGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), preferably characterized by a sequence selected from the sequence set forth in SEQ ID NO: 33, the sequence set forth in SEQ ID NO: 36, and the sequence set forth in SEQ ID NO: 37.

[0272] 4. the variable heavy chain region is characterized by the sequence set forth in SEQ ID NO: 28 and the variable light chain region is characterized by the sequence set forth in SEQ ID NO: 33; or the variable heavy chain region is characterized by the sequence set forth in SEQ ID NO: 32 and the variable light chain region is characterized by the sequence set forth in SEQ ID NO: 36, or 4. The antibody or antigen-binding fragment thereof of any one of clauses 1 to 3, wherein the variable heavy chain region is characterized by the sequence set forth in SEQ ID NO: 14 and the variable light chain region is characterized by the sequence set forth in SEQ ID NO: 37.

[0273] 5. The variable heavy chain region has the sequence: FH0-CDR-H1-FH1-CDR-H2-FH2-CDR-H3-FH3 wherein CDR-H1, CDR-H2, and CDR-H3 are as defined in clause 1; FH0 is characterized by the sequence set forth in SEQ ID NO: 38 (QVQLVQSGAEVKKPGASVKVSCKASGYTFT) or the sequence set forth in SEQ ID NO: 39 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFT), FH1 is characterized by the sequence set forth in SEQ ID NO: 40 (WVRQAPGQGLEWIG) or the sequence set forth in SEQ ID NO: 41 (WIRQPPGKGLEWIG), FH2 is characterized by the sequence set forth in SEQ ID NO: 42 (RVTLTVDKSISTAYMELSRLRSDDTAVYFCAR) or the sequence set forth in SEQ ID NO: 43 (RVTLSVDKSKNQASLKLSSVTAADTAVYFCAR), FH3 is characterized by a sequence at least 90% identical, preferably at least 95% identical, more preferably identical to the sequence set forth in SEQ ID NO: 44 (WGQGTLVTVSS), and / or The variable light chain region has the sequence: FL0-CDR-L1-FL1-CDR-L2-FL2-CDR-L3-FL3 wherein CDR-L1, CDR-L2, and CDR-L3 are as defined in claim 1; FL0 is characterized by the sequence set forth in SEQ ID NO: 45 (DIQMTQSPSSLSASVGDRVTITC) or the sequence set forth in SEQ ID NO: 46 (EIVMTQSPATLSVSPGERATLSC), FL1 is characterized by the sequence set forth in SEQ ID NO: 47 (WYQQKPGKAPKLLIS) or the sequence set forth in SEQ ID NO: 48 (WYQQKPGQAPRLLIS), FL2 is characterized by the sequence set forth in SEQ ID NO: 49 (GVPSRFSGSGSGKDYTLTISSLQPEDIATYYC) or the sequence set forth in SEQ ID NO: 50 (GIPARFSGSGSGKEFTLTISSLQSEDFAVYYC), The antibody or antigen-binding fragment thereof described in clause 1, wherein FL3 is characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical to the sequence set forth in SEQ ID NO: 51 (FGQGTKLEIK).

[0274] 6. The antibody or antigen-binding fragment thereof of any one of clauses 1 to 5, wherein the heavy chain further comprises at least one point mutation in the Fc portion that affects antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), serum half-life, and / or glycosylation status of the antibody, preferably wherein the at least one point mutation is selected from L234A, L234F, L235A, L235E, L235Q, G236A, M252Y, S254T, T256E, S267E, H268F, N297A, K322A, K322Q, S324T, P331S, and I332E, more preferably wherein the at least one point mutation is selected from L234A, L235A, P331S, and N297A.

[0275] 7. 10 as measured by Biacore-based assay -12 The dissociation constant K for L1-CAM (CD171) does not exceed M D 7. The antibody or antigen-binding fragment thereof according to any one of clauses 1 to 6, characterized in that

[0276] 8. A polynucleotide encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence according to any one of clauses 1 to 6.

[0277] 9. A host cell comprising a polynucleotide according to clause 8.

[0278] 10. An immunoconjugate comprising the antibody or antigen fragment thereof bound thereto according to any one of clauses 1 to 7 and an active agent, preferably the active agent being a cytotoxic agent or a prodrug thereof, Preferably, the antibody or antigen-binding fragment thereof is linked to the active agent via a linker moiety, preferably the linker moiety comprises a polymeric carrier to which at least one active agent is attached. and / or Preferably, the immunoconjugate wherein the active agent is a radionuclide preferably selected from copper-67, strontium-89, yttrium-90, iodine-131, samarium-153, terbium-161, lutetium-177, astatine-211, radium-223, and actinium-225, preferably a radionuclide useful for therapeutic applications, and / or a radionuclide preferably useful for diagnostics selected from fluorine-18, scandium-43, scandium-44, copper-61, copper-64, gallium-68, zirconium-89, indium-111, iodine-123, terbium-152, and terbium-155.

[0279] 11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of clauses 1 to 7, or the immune complex according to clause 10, and a pharmaceutically acceptable carrier.

[0280] 12. The antibody or antigen-binding fragment thereof according to any one of clauses 1 to 7, or the immune complex according to clause 10, for use as a pharmaceutical.

[0281] 13. Preferably, the L1-CAM (CD171)-associated cancer is leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, quadruple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, cholangiocarcinoma, carcinoid, neuroendocrine tumor, gastrointestinal interstitial carcinoma, or gastrointestinal interstitial carcinoma. selected from gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, pancreatic neuroectodermal carcinoma, pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma, tumor vascular, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine carcinoma, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granular cell tumor, Schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, prostate cancer, and head and neck squamous cell carcinoma; and / or The antibody or antigen-binding fragment thereof of any one of clauses 1 to 7, or the immunoconjugate of clause 10, for use in treating an L1-CAM (CD171)-associated cancer, wherein preferably the antibody or antigen-binding fragment thereof or immunoconjugate is to be administered to the subject in conjunction with an additional therapeutic agent selected from an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen, an aromatase inhibitor, an ovarian function suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, an immune checkpoint inhibitor, and a bisphosphonate therapy.

[0282] 14. The antibody or antigen-binding fragment thereof of any one of clauses 1 to 7, or the immune complex of clause 10, for use in diagnosis.

[0283] 15. Preferably, the L1-CAM (CD171)-associated cancer is leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, quadruple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, cholangiocarcinoma, carcinoid, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, pancreatic neuroectodermal carcinoma, pancreatic adenocarcinoma, or ovarian cancer. 11. The antibody or antigen-binding fragment thereof of any one of clauses 1 to 7, or the immunoconjugate of clause 10, for use in diagnosing an L1-CAM (CD171)-associated cancer selected from colorectal cancer, renal cell carcinoma, tumor vasculature, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine carcinoma, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granular cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, prostate cancer, and head and neck squamous cell carcinoma.

[0284] The present invention is illustrated in the following examples, which are not intended to be construed as limiting. Note that unless expressly indicated to the contrary, the present invention can relate to any and all antibodies or antigen-binding fragments thereof disclosed in the examples (particularly when defined by amino acid sequence), and / or any and all polynucleotides disclosed in the examples. [Example]

[0285] Example 1: Humanization of the CE7 mouse monoclonal antibody The CE7 mouse antibody was humanized by grafting three CDRs, defined by the Kabat nomenclature, from the light chain variable region (VL) onto a human germline VL that was as homologous as possible to the mouse antibody VL. Similarly, three CDRs from the heavy chain variable region (VH) were grafted onto a human germline VH that was as homologous as possible to the mouse antibody VH. In addition, several amino acid residues in the framework regions of selected human germline variable regions were changed to amino acid residues present in the mouse variable region (so-called back mutations). Based on information about the structure of immunoglobulin variable regions and by deriving a homology molecular model of the Fv of the CE7 mouse monoclonal antibody, these several residues in the framework regions were identified as having important roles in maintaining the CDRs in either the correct conformation or VH / VL packing. Therefore, they were retained in humanized version A or, if possible, replaced with their human germline counterparts in subsequent humanized versions. Under the guidance of homology molecular models, in the subsequent version B, where deemed possible, the CDR residues defined by Kabat were also replaced with their human germline counterparts (so-called germlining) in order to increase the degree of humanization (i.e., the percentage of sequence identity for both VH and VL between the humanized version and the closest human germline used as acceptor sequence for CDR grafting).

[0286] The added value of combining structural models with pure sequence analysis is the possibility of distinguishing between paratope-facing and non-paratope residues in the CDR regions. The purpose of structural models is to enable the expansion of the humanization process beyond simple CDR grafting. Structural models also allow for more intelligent selection of back mutations in light of the specific germline involved. Note that the Kabat CDR definition is not as strictly structural as that of other systems. Thus, for some germlines, the Kabat definition is too broad. For both heavy and light chains, we can usually be fairly confident that the assignment of residues from CDR1 and 2 as paratope and non-paratope is correct based on structural models. Similarly, light chain CDR3 is usually well-described with a high degree of certainty. The difficult case is always the heavy chain CDR3.

[0287] The humanized version A provided herein for both VH and VL are conservative versions that explicitly minimize / avoid alterations of CDR residues, and these versions are expected to confer similar binding / potency activity to the chimeric antibody used as the reference molecule (CE7 mouse VH and VL fused to human constant regions). Subsequent humanized version B for both VH and VL will be designed to reach a higher percentage of sequence identity with the closest human germline (as close to 85% as possible). This can be achieved by germlining framework and / or Kabat CDR amino acid residues (replacing mouse residues with the corresponding human germline residues).

[0288] For the design of a CDR-grafted version of the CE7 mouse VH, two human germline clones, IGHV1-2, were used. * 06 and IGHV4-34 * 01 was selected. These two human germlines share 66.3% and 53.1% sequence identity across the entire V gene, respectively.

[0289] For the design of a CDR-grafted version of the CE7 mouse VL, three human germline clones, IGKV1-NL1 * 01, IGKV1-33 * 01, and IGKV3-15 * 01 was selected. These three human germlines share 73.7%, 71.6%, and 60.0% sequence identity with the CE7 mouse VL across the entire V gene, respectively.

[0290] The first step in the humanization process should be to select the best combination of heavy and light chains between the humanized VH and VL versions. For VH, we selected two different human germlines, namely, IGHV1-2, which has four versions (A-D). * 06 and IGHV4-34 with three versions (A to C) * For the VL, we have three human germline-based humanized versions with four versions (A-D) for each of them. This creates a total of 12 different humanized VL. To have all possible combinations between humanized VH and VL, a total of 7 (VH) x 12 (VL) = 84 humanized variants need to be produced and purified.

[0291] This number is based on the human germline IGKV1-NL1 * 01, IGKV1-33 * 01, and IGKV3-15 * Human germline IGHV1-2 in combination with versions A and B of the VL humanized version based on 01 * For VH humanized versions based on 06, only versions A and B are available, and for human germline IGHV4-34 * For the humanized versions based on 01, this can be reduced to 18 by testing only version A. We will have 3 VH x 6 VL = 18 combinations.

[0292] From the results obtained with these 18 humanized mAbs, the best one or two VH / VL combinations can be selected and then subsequent versions B, C and D can be tested.

[0293] [Table 1-2]

[0294] The selection of the best heavy and light chain combination among the 18 variants should be done by evaluating the following criteria: ● Levels of transient expression of the humanized version produced in mammalian cells (HEK293 or preferably CHO) as human IgG1 / kappa (compared to the chimeric version). Use TC supernatants from transfected cells before harvesting for purification using ELISA or Octet Protein A measurements. - Binding ability (EC50 by ELISA or FACS, or preferably Kd by Biacore or Octet) compared to the CE7 chimeric human IgG1 / kappa version (chimeric means the combination of parental mouse VH and VL fused to a human constant region). • Biological activity of the humanized versions in relevant in vitro cellular assays compared to that of the reference CE7 chimeric antibody. • Cross-reactivity (in vitro binding activity) with related orthologous species, if relevant. Determining the biophysical properties of the humanized version compared to the CE7 chimera: SEC-HPLC profile to determine the level of high molecular weight soluble aggregates, SDS-PAGE under non-reducing and reducing conditions, Analysis by differential scanning calorimetry (DSC) using a Microcal VP-capillary DSC system to determine the Tm of Fab, CH2, and CH3.

[0295] Once the best two humanized VH and VL pairings have been identified, a second round of humanization can be initiated, where the consequences of sequence liability mutations (see the "Sequence Liability" section) are taken into account. Positive CDR mutations identified among the sequence liability mutations are introduced into the CDRs of the best two humanized VH / VL combinations to obtain the final humanized candidates. The VH and VL humanized versions (IGHV1-2) * For 01, C and D, IGHV4-34 * For 01, versions B and C, and for all light chains, versions C and D may also be tested. The best final candidates are selected after evaluation using the same criteria described above for the first round of candidates.

[0296] Humanization of the murine CE7 monoclonal antibody was performed using standard CDR grafting techniques. The principle of this method is to reconstruct a human antibody containing only the complementarity-determining regions (CDRs) from a murine monoclonal antibody, with the aim of reducing immunogenicity when used as a therapeutic agent in humans. Humanization by CDR grafting requires that the antigen-binding residues from the murine antibody be retained in the humanized antibody. Therefore, identifying these residues clearly plays a key role in the protocol. A homology molecular model of the Fv of the CE7 murine monoclonal antibody was constructed to guide the humanization process and help determine whether to preserve the parent murine residues or replace them with their human germline counterparts.

[0297] The CDR grafting protocol used is a modernized version of the approach developed by Greg Winter and colleagues at the Medical Research Council (Cambridge, UK). CDR definitions are based on the Kabat nomenclature. Selection of human framework acceptor regions onto which the CE7 mouse CDR regions were grafted was achieved by searching the IMGT mouse and human V gene databases using IgBLAST (http: / / www.ncbi.nlm.nih.gov / igblast / ), developed at NCBI to facilitate the analysis of immunoglobulin V region sequences, with the CE7 mouse variable region sequences as input. The strategy applied is to use human germline sequences, which are naturally occurring human sequences that do not contain the idiosyncratic somatic mutations found in individual human antibody sequences.

[0298] Sequence Liability Over the past decade, drug developers have become increasingly proactive in early identification and risk assessment to combat attrition. One commonly used strategy to achieve drug-like properties to avoid late-stage failure is the so-called developability or molecular evaluation approach, which aims to optimize the biophysical and chemical properties of molecules prior to product development. In addition to favorable biophysical properties (Jain et al., 2017 PNAS;114:944-949), mAbs must also possess sufficient chemical stability to meet stringent process development parameters. The main targeted chemical modifications for mAbs are oxidation (Met, M and Trp, W), deamidation (Asn, N), and isomerization (Asp, D). Therefore, identifying degradation sequence liability for redesign before committing resources to process and product development has the potential to reduce formulation development complexity and improve the likelihood of a successful liquid formulation. Based on a recent study of deamidation and isomerization liabilities of 131 clinical-stage antibodies published in MABS (Lu et al., 2019 MABS, 11:45-57), we analyzed the CDRs of CE7 VH and VL for deamidation and isomerization motifs that have been reported to be modified. The sequences of the CDRs as defined by Kabat nomenclature are underlined. Within the CDRs, potential sequence liabilities are in bold, and only potential sequence liabilities that are considered actual sequence liabilities are considered.

[0299] [ka]

[0300] In CDR1, there is a W at Kabat position H33. Examination of the molecular model shows that it is buried, with its side chain flush with the paratope surface and packing against VH-CDR3. The indole nitrogen may have access to the antigen, side chain contacts with VH-CDR2 residues E (H55), I (H56) (mc), N (H57), and R (H64), and VH-CDR3 residue Y (H109). The lowest-energy rotamer of substitution W(H33)Y induces excessive strain (30.5) and alters the contact geometry. W(H33)F is similarly negative, and W(H33)L induces moderate strain (17.6) but loses contact at R (H64), which may be a route to improved oxidation vulnerability. Therefore, F, Y, and L substitutions may be attempted, with the L having the best chance of functioning. See below for the sequences of the WH33F, WH33Y, and WH33L mutants that can be tested.

[0301] In CDR2, there is an NG motif at Kabat positions H54 and H55. Examination of molecular models shows that N H54 is surface-exposed, and its side chain can contact the antigen at the edge of the paratope surface. G H55 is surface-exposed, its side chain is located on the non-antigen-facing surface of VH-CDR2, and the alpha carbon does not make vernier or CDR contacts. G H55 to A substitution appears to be the best option for improving "NG" liability. Therefore, an A substitution at H55 should be attempted. See the sequences of the tested GH55A mutants below.

[0302] In CDR3, NF motifs at Kabat positions H100B and H100C. Inspection of molecular models shows that both residues are buried and therefore considered to be at low risk for deamidation.

[0303] CE7-VH-chimera-WH33Y QVQLQQPGAELVKPGASVKLSCKASGYTFTGYYMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSS [SEQ ID NO: 56]

[0304] CE7-VH-chimera-WH33F QVQLQQPGAELVKPGASVKLSCKASGYTFTGYFMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSS [SEQ ID NO: 57]

[0305] CE7-VH-chimera-WH33L QVQLQQPGAELVKPGASVKLSCKASGYTFTGYLMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSS [SEQ ID NO: 58]

[0306] CE7-VH-chimera-GH55A QVQLQQPGAELVKPGASVKLSCKASGYTFTGYWMHWVKQRPGHGLEWIGEINPSNARTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSS [SEQ ID NO: 59]

[0307] [ka]

[0308] CDR1 has the motif NN at Kabat positions L30 and L31. Examination of molecular models shows that NL30 is surface-exposed, with its side chain adjacent to the VL CDR3 at the edge of the paratope surface and potentially contacting the antigen. NL31 is surface-exposed, with its side chain at the edge of the paratope surface, facing away from the center, and its side chain contacts Vernier residues G (L80) and Y (L87). Substitution of NL31 with L or A appears to be the best option for NN liability. See the sequences of the NL31L and NL31A mutants to be tested below.

[0309] In CDR3, there is a W at Kabat position L92. Examination of molecular models indicates that W L92 is surface-exposed, with the side chain near the center of the paratope surface having a high probability of contacting the antigen. Oxidative liability can best be addressed by replacing W with Y (where the phenol ring overlaps with the indole ring, but binding is maintained) or F. See the sequences of the tested WL92Y and WL92F mutants below.

[0310] CE7-VL-chimera-NL31L DIQMTQSSSSFSVSLGDRVTITCKANEDINLRLAWYQQTPGNSPRLLISGATNLVT GVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYWSTPFTFGSGTELEIK [SEQ ID NO: 61]

[0311] CE7-VL-chimera-NL31A DIQMTQSSSSFSVSLGDRVTITCKANEDINARLAWYQQTPGNSPRLLISGATNLVT GVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYWSTPFTFGSGTELEIK [SEQ ID NO: 62]

[0312] CE7-VL-chimera-WL92Y DIQMTQSSSSFSVSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVT GVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYYSTPFTFGSGTELEIK [SEQ ID NO: 63]

[0313] CE7-VL-chimera-WL92F DIQMTQSSSSFSVSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVT GVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYFSTPFTFGSGTELEIK [SEQ ID NO: 64]

[0314] Heavy Chain Design Most homologous mouse germline, IGHV1S81 * Amino acid difference from 02 Amino acid sequence of VH of mouse CE7 hybridoma

[0315] [ka]

[0316] CE7 murine heavy chain variable (VH) region and murine germline immunoglobulin VH 1S81 * 02(IGHV1S81 * 02) is 91.8% identical (90 of a total of 98 residues in the V gene). The underlined parts in CE7 VH indicate that CE7 VH is identical to mouse germline IGHV1S81. * There are eight residues that differ between 02 and 03.

[0317] [ka]

[0318] The bolded residues are part of the classification of immunoglobulin VH domain frameworks defined by Honegger and Plückthun (J. Mol. Biol., 2001, 309:687-699). The authors report that immunoglobulin VH frameworks can be grouped into four clearly distinct types (I-IV) depending on the main-chain conformation of framework 1 (particularly that of Kabat residues 6, 7, 8, and 9). Mouse CE7 VH has a Gln at Kabat position H6 and a Pro at position H7, which is typical of Honegger type IV. Human germline IGHV1-2 used as acceptor sequences for CDR grafting * 06 and IGHV4-34 * 01 are both Honnerger type III (Gln at Kabat position H6, not Pro at position H7). See VH-IGHV1-2 columns N and O and VH-IGHV4-34 columns L and M of the form.

[0319] Selection of human framework acceptor VH regions Selection of the human framework acceptor VH region into which the CE7 mouse CDR regions would be grafted was achieved by searching the IMGT human VH gene database using IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / igblast.cgi) with the mouse CE7 VH amino acid sequence as input. The closest matching entry was identified based on sequence alignment of the parent antibody against the human germline. Identification of the optimal human germline as the acceptor was based on the following ordered criteria: sequence identity across the framework as defined by Kabat, and identity and / or compatibility of interchain interface residues and supporting loops with the canonical conformation of the parent CDR.

[0320] One of the important criteria in selecting a suitable human germline candidate is that the sequence identity between this particular germline and the designed humanized version can reach nearly 85%. We analyzed a large number of human germline sequences and selected two human germlines as the best candidates, one belonging to human family 1, IGHV1-2. * 06, and one belongs to human family 4, IGHV4-34 * 01. Human germline IGHV1-2 * 06 shares a high sequence identity of 66.3% with mouse CE7 VH (65 identical amino acid residues out of a total of 98), while human germline IGHV4-34 * 01 has 53.1% sequence identity (52 identical amino acid residues out of a total of 98).

[0321] In framework 4, the gene segment of mouse CE7 VH corresponding to the J gene was identified as most homologous to the mouse germline J2 gene (see VH in Table 1, columns E, F, and G of the VH-IGHV1-2 and VH-IGHV4-34 sheets). The mouse J2 segment gene was compared with the human J segment gene across CDR3 and FR4, and found to be highly homologous to the human J4 gene. * It was found to be most homologous to the 01 segment.

[0322] IGHV1-2 as framework acceptor region * 06 Designed to Use Human Germline Lines Mouse CE7 VH and human germline IGHV1-2 * Sequence alignment between 06 CE7 mouse heavy chain variable (VH) region and human germline immunoglobulin VH1-2 * 06(IGHV1-2 * 06) with 66.3% identity (65 identical residues out of a total of 98 residues in the V genes).

[0323] CDR residues as defined by Kabat nomenclature are highlighted in yellow. In the framework regions and CDRs, the mouse CE7 VH and human 13 germline IGHV1-2 * Residues that differ between 06 are in bold and underlined, respectively.

[0324] [ka]

[0325] Humanized Version A The mouse CDRs defined by the Kabat nomenclature were * 06, resulting in the following detailed sequence. The residues highlighted in green are framework murine residues (non-CDR residues) that are conserved from the parent murine CE7 VH sequence. They are conserved because they may be structurally important for maintaining full activity of the antibody.

[0326] [ka]

[0327] Humanized version A (CE7-1-2-VHA) and IGHV1-2 * 84.7% identity with human germline (83 identical residues out of a total of 98 residues in the V genes). Humanized version A and human germline IGHV1-2 in the framework regions and CDRs. * Residues that differ between 06 are in bold and underlined, respectively.

[0328] [ka]

[0329] For more information, see the full-length amino acid sequence of version A in Figure 1, the comments for each relevant amino acid position in the examination of the homology molecular model in the CE7 VH design in Table 3, and the VHs in Table 1 (mouse CE7 VH, IGHV1-2, respectively).* See sequence comparison in VH-IGHV1-2 columns E, G and I of the form, corresponding to 06 human germline and humanized version A.

[0330] Humanized version B In version B, three amino acid residues were germlined compared to version A (i.e., mouse residues were replaced with the corresponding IGHV1-2 * In CDR2, Kabat Lys(K)H64 and Kabat Ser(S)H65 were replaced by their corresponding human germline IGHV1-2 residues, respectively. * In FR3, Kabat Phe(F)H91 was replaced with the corresponding human germline IGHV1-2. * The 06 residue was replaced with Tyr (Y).

[0331] Amino acid residues that differ between humanized versions A and B are underlined.

[0332] [ka]

[0333] Humanized version B (CE7-1-2-VHB) and IGHV1-2 * 06 87.8% identity with human germline (86 identical residues out of a total of 98 residues in the V genes). Residues that differ between humanized versions A and B are underlined.

[0334] [ka]

[0335] Humanized Version C In version C, one amino acid residue was germlined compared to version B (i.e., the mouse residue was replaced with the corresponding IGHV1-2 *In CDR2, Kabat Arg(R)H62 was replaced with its corresponding human germline IGHV1-2 * The 06 residue was substituted with Lys (K).

[0336] Amino acid residues that differ between humanized versions B and C are underlined.

[0337] [ka]

[0338] Humanized version C (CE7-1-2-VHC) and IGHV1-2 * 06 88.8% identity with human germline (87 identical residues out of a total of 98 residues in the V genes). Residues that differ between humanized versions B and C are underlined.

[0339] [ka]

[0340] Humanized Version D In version D, one amino acid residue was germlined compared to version C (i.e., the mouse residue was replaced with the corresponding IGHV1-2 * In FR2, Kabat Ile(I)H48 was replaced with its corresponding human germline IGHV1-2 residue. * The 06 residue was replaced with Met (M).

[0341] Amino acid residues that differ between humanized versions D and E are underlined.

[0342] [ka]

[0343] Humanized version D (CE7-1-2-VHD) and IGHV1-2 *06 89.8% identity with human germline (88 identical residues out of a total of 98 residues in the V genes). Residues that differ between humanized versions C and D are underlined.

[0344] [ka]

[0345] IGHV4-34 as the framework acceptor region * 01 Designed to use human germline Mouse CE7 VH and human germline IGHV4-34 * Sequence alignment between 01 CE7 murine heavy chain variable (VH) region and human germline immunoglobulin VH4-34 * 01(IGHV4-34 * 01) 53.1% identity (52 identical residues out of a total of 98 residues in the V gene) between mouse CE7 VH and human germline IGHV4-34 in the framework and CDRs. * Residues that differ between 01 and 02 are in bold or underlined, respectively.

[0346] [ka]

[0347] Humanized Version A The mouse CDRs (highlighted in yellow) defined by the Kabat nomenclature are shown in Figure 1. * 01, resulting in the following detailed sequence: Framework murine residues (non-CDR residues) are conserved from the parent murine CE7 VH sequence. They are conserved because they may be structurally important for maintaining full activity of the antibody.

[0348] [ka]

[0349] Humanized version A (CE7-4-34-VHA) and IGHV4-34 * 01 83.7% identity with human germline (82 identical residues out of a total of 98 residues in the V gene). CE7-434-VHA and human germline IGHV4-34 in the framework regions and CDRs. * Residues that differ between 01 and 02 are in bold or underlined, respectively.

[0350] [ka]

[0351] Humanized version B In version B, three amino acid residues were germlined compared to version A (i.e., mouse residues were replaced with the corresponding IGHV4-34 * In framework 3 (FR3), Kabat Thr(T)H28 and Kabat Thr(T)H30 were both replaced with Ser(S), and in framework 3 (FR3), Kabat Phe(F)H91 was replaced with Tyr(Y).

[0352] [ka]

[0353] Humanized version B (CE7-434-VHB) and IGHV4-34 * 01 86.7% identity with human germline (85 identical residues out of a total of 98 residues in the V genes). Amino acid residues germlined in humanized version B compared to humanized version A are underlined.

[0354] [ka]

[0355] Humanized Version C In version C, two amino acid residues were germlined compared to version B (i.e., the murine residues were replaced with the corresponding IGHV4-34 residues). * In CDR2, Kabat Glu(E)H61 and Kabat Arg(R)H62 were replaced with Pro(P) and Ser(S), respectively.

[0356] [ka]

[0357] Humanized version C (CE7-434-VHC) and IGHV4-34 * 01 88.8% identity with human germline (87 identical residues out of a total of 98 residues in the V genes). Amino acid residues germlined in humanized version C compared to humanized version B are underlined.

[0358] [ka]

[0359] Light Chain Design Most homologous mouse germline IGKV13-84 * Amino acid difference from 01 Amino acid sequence of VL of mouse CE7 hybridoma (CDR regions defined by Kabat nomenclature are underlined)

[0360] [ka] CE7 murine light chain variable (VL) region and murine germline immunoglobulin IGKV13-84 * 01 (86 identical residues out of a total of 95 residues in the V gene). The underlined residues are the identity of the CE7 VL with the mouse germline IGKV13-84. * 01 are different residues.

[0361] [ka]

[0362] Selection of human framework acceptor VL regions Selection of the human framework acceptor VL region into which the CE7 mouse CDR region would be grafted was achieved by searching the IMGT human VL gene database using IgBLAST with the mouse VL region amino acid sequence as input. The closest matching entry was identified based on sequence alignment of the parent antibody against the human germline. The identification of the optimal human germline as the acceptor was based on the following ordered criteria: sequence identity across the framework as defined by Kabat, and identity and / or compatibility of interchain interface residues and support loops with the canonical conformation of the parent CDR. One of the important criteria in selecting a suitable human germline candidate is the ability to achieve nearly 85% sequence identity between this particular germline and the designed humanized version. We analyzed a large number of human germlines and selected three human germlines: IGKV1-NL1 * 01, IGKV1-33 * 01, and IGKV3-15 * 01 was selected for the design of a CDR-grafted humanized version.

[0363] The gene segment of mouse CE7 VL corresponding to the J gene is the most homologous mouse germline J4 gene (IGKJ4 * The mouse J4 gene segment was compared with the human J segment gene across CDR3 and FR4, and identified as the human J segment IGKJ2 (IGKJ2 * 01) was found to have the highest overall homology.

[0364] IGKV1-NL1 as the framework acceptor region * 01 Designed to use human germline Mouse CE7 VH and human germline IGKV1-NL1 * Sequence alignment between 01 CE7 mouse light chain variable (VL) region and human germline immunoglobulin VL1-NL1 * 01(IGKV1-NL1 * 01) (70 identical residues out of a total of 95 residues in the V genes). CDR residues as defined by Kabat nomenclature are highlighted in yellow. Mouse CE7 VL and human germline IGKV1-NL1 in the framework regions and CDRs. * Residues that differ between 01 and 02 are in bold or underlined, respectively.

[0365] [ka]

[0366] Humanized Version A The murine CDRs, defined by Kabat numbering, are * 01 to give the following detailed sequence:

[0367] [ka]

[0368] Humanized version A (CE7-1-NL1-VLA) and IGKV1-NL1 * 01 (80 identical residues out of a total of 95 residues in the V gene). Mouse framework residues (non-CDR residues - bold) were conserved from the parent mouse CE7 VL sequence. They are conserved because they may be structurally important to maintain full activity of the antibody. The underlined residues are those that are different within the CDRs between humanized version A and human germline IGKV1-NL1. * 01 and are in different positions.

[0369] [ka]

[0370] Humanized version B Compared to version A, version B has one amino acid residue germlined (i.e., replaced by the corresponding human germline residue). In CDR1, Lys(K) Kabat L24 was germlined and mutated to Arg(R). Examination of molecular models indicates that this residue is unlikely to make direct contact with the antigen and does not play an important structural role. Therefore, it is important to note that it is not a direct contact with its corresponding human germline IGKV1-NL1. * The primary reason for germlining this residue within Kabat CDR L1 is that this change is consistent with the human germline IGKV1-NL1 * This increases the identity percentage of humanized version B to 01 to 85.3%.

[0371] [ka]

[0372] Humanized version B (CE7-1-NL1-VLB) and IGKV1-NL1 * 01 85.3% identity with human kappa light chain germline (81 identical residues out of 95 total residues in the V gene). Amino acid residues germlined in humanized version B compared to humanized version A are underlined.

[0373] [ka]

[0374] Humanized Version C In version C compared to version B, two amino acid residues were germlined (i.e., replaced by the corresponding human germline residues). In CDR2, Kabat L52 and Thr (T) Kabat L56 were germlined and both were mutated to Ser (S). Examination of molecular models indicates that these residues are unlikely to make direct contact with the antigen and do not play an important structural role. Therefore, they are not identical to their corresponding human germline IGKV1-NL1 residues. *The primary reason for germlining residues within the Kabat CDRs is that these changes are substituted with the human germline IGKV1-NL1 * The goal is to increase the identity percentage of humanized version C to 01 to 87.4%. 24

[0375] [ka]

[0376] Humanized version C (CE7-1-NL1-VLC) and IGKV1-NL1 * 01 87.4% identity with human kappa light chain germline (83 identical residues out of 95 total residues in the V gene). Amino acid residues germlined in humanized version C compared to humanized version B are underlined.

[0377] [ka]

[0378] Humanized Version D Compared to version C, three amino acid residues were germlined (i.e., replaced by the corresponding human germline residues) in version D. In CDR1, TAsn(N)Kabat L26, Glu(E)L27, and Asp(D)Kabat L28 were germlined and mutated to Ser(S), Gln(Q), and Gly(G), respectively. Examination of molecular models indicates that these residues are unlikely to make direct contact with the antigen and do not play an important structural role. Therefore, they are not identical to their corresponding human germline IGKV1-NL1 residues. * The primary reason for germlining residues within the Kabat CDRs is that these changes are substituted with the human germline IGKV1-NL1 * The goal is to increase the identity percentage of humanized version D to O1 to 90.5%.

[0379] [ka]

[0380] Humanized version D (CE7-1-NL1-VLD) and IGKV1-NL1 * 01 90.5% identity with human kappa light chain germline (86 identical residues out of 95 total residues in the V gene). Amino acid residues germlined in humanized version D compared to humanized version C are underlined.

[0381] [ka]

[0382] IGKV1-33 as the framework acceptor region * 01 Designed to use human germline Mouse CE7 VL and human germline IGKV1-33 * Sequence alignment between 01 CE7 murine light chain variable (VL) region and human germline immunoglobulin VL1-33 * 01(IGKV1-33 * 01) (68 identical residues out of a total of 95 residues in the V genes). CDR residues as defined by Kabat nomenclature are highlighted in yellow. Mouse CE7 VL and human germline IGKV1-33 in the framework regions and CDRs. * Residues that differ between 01 and 02 are in bold or underlined, respectively.

[0383] [ka]

[0384] Humanized Version A The murine CDRs, as defined by Kabat numbering, were * 01 to give the following detailed sequence:

[0385] [ka]

[0386] Humanized version A (CE7-1-33-VLA) and IGKV1-33 * 01 (79 identical residues out of a total of 95 residues in the V gene). Bolded residues are murine framework residues (non-CDR residues) conserved from the parent murine CE7 VL sequence. They are conserved because they may be structurally important to maintaining full antibody activity. Underlined residues are those that are different within the CDRs between humanized version A and human germline IGKV1-33. * 01 and are in different positions.

[0387] [ka]

[0388] Humanized version B In version B compared to version A, two amino acid residues were germlined (i.e., replaced by the corresponding human germline residues). In CDR1, Lys (K) Kabat L24 was germlined and mutated to Gln (Q). In framework 3 (FR3), Leu (L) Kabat L73 was germlined and mutated to Phe (F). Examination of molecular models indicates that these residues are unlikely to make direct contact with the antigen and do not play an important structural role. Therefore, they are not identical to their corresponding human germline residues in IGKV1-33. * The primary reason for germlining residues within the Kabat CDRs is that these changes are consistent with the human germline IGKV1-33 * This increases the identity percentage of humanized version B to 01 to 85.3%.

[0389] [ka]

[0390] Humanized version B (CE7-1-33-VLB) and IGKV1-33 * 01 85.3% identity with human kappa light chain germline (81 identical residues out of 95 total residues in the V gene). Amino acid residues germlined in humanized version B compared to humanized version A are underlined.

[0391] [ka]

[0392] Humanized Version C In version C compared to version B, two amino acid residues were germlined (i.e., replaced by the corresponding human germline residues). In CDR2, Thr (T) Kabat L52 was germlined and mutated to Ser (S). In framework 3 (FR3), Tyr (Y) Kabat L71 was germlined and mutated to Phe (F). Examination of molecular models indicates that these residues are unlikely to make direct contact with the antigen and do not play an important structural role. Therefore, they are more closely related to their corresponding human germline IGKV1-33. * The primary reason for germlining residues within the Kabat CDRs is that these changes are consistent with the human germline IGKV1-33 * This increases the identity percentage of humanized version C to 01 to 87.4%.

[0393] [ka]

[0394] Humanized version C (CE7-1-33-VLC) and IGKV1-33 * 01 87.4% identity with human kappa light chain germline (83 identical residues out of 95 total residues in the V gene). Amino acid residues germlined in humanized version C compared to humanized version B are underlined.

[0395] [ka]

[0396] Humanized Version D In version D compared to version C, two amino acid residues were germlined (i.e., replaced by the corresponding human germline residues). In CDR1, Asn(N)Kabat L26 and Glu(E)Kabat L27 were germlined and mutated to Ser(S) and Gln(Q), respectively. Examination of molecular modeling indicates that these residues are unlikely to make direct contact with the antigen and do not play an important structural role. Therefore, they are not as similar to their corresponding human germline IGKV1-33 residues. * The primary reason for germlining residues within the Kabat CDRs is that these changes are consistent with the human germline IGKV1-33 * The goal is to increase the identity percentage of humanized version C to 01 to 89.5%.

[0397] [ka]

[0398] Humanized version D (CE7-1-33-VLD) and IGKV1-33 * 01 89.5% identity with human kappa light chain germline (85 identical residues out of 95 total residues in the V gene). Amino acid residues germlined in humanized version D compared to humanized version C are underlined.

[0399] [ka]

[0400] IGKV3-15 as the framework acceptor region * 01 Designed to use human germline Mouse CE7 VL and human germline IGKV3-15* Sequence alignment between 01 CE7 murine light chain variable (VL) region and human germline immunoglobulin VL3-15 * 01(IGKV3-15 * 01) (57 identical residues out of a total of 95 residues in the V gene). CDR residues as defined by Kabat nomenclature are shown. Mouse CE7 VL and human germline IGKV3-15 in the framework regions and CDRs. * Residues that differ between 01 and 02 are in bold or underlined, respectively.

[0401] [ka]

[0402] Humanized Version A The murine CDRs, as defined by Kabat numbering, were * 01 to give the following detailed sequence:

[0403] [ka]

[0404] Humanized version A (CE7-3-15-VLA) and IGKV3-15 * 01 (77 identical residues out of a total of 95 residues in the V gene). Bolded residues are murine framework residues (non-CDR residues) conserved from the parent murine CE7 VL sequence. They are conserved because they may be structurally important to maintaining full antibody activity. Underlined residues are those that are different within the CDRs between humanized version A and human germline IGKV3-15. * 01 and are in different positions.

[0405] [ka]

[0406] Humanized version B In version B compared to version A, two amino acid residues were germlined (i.e., replaced by the corresponding human germline residues). In CDR1, Lys (K) Kabat L24 was germlined and mutated to Arg (R). In framework 3 (FR3), Tyr (Y) Kabat L71 was germlined and mutated to Phe (F). Examination of molecular models indicates that these residues are unlikely to make direct contact with the antigen and do not play an important structural role. Therefore, they are more closely related to their corresponding human germline IGKV3-15. * The primary reason for germlining residues within the Kabat CDRs is that these changes are consistent with the human germline IGKV3-15 * This increases the identity percentage of humanized version B to 01 to 83.2%.

[0407] [ka]

[0408] Humanized version B (CE7-3-15-VLB) and IGKV3-15 * 01 (79 identical residues out of a total of 95 residues in the V gene). Bolded residues are mouse framework residues (non-CDR residues) conserved from the parent mouse CE7 VL sequence. They are conserved because they may be structurally important for maintaining full activity of the antibody. Amino acid residues germlined in humanized version B compared to humanized version A are underlined.

[0409] [ka]

[0410] Humanized Version C In version C compared to version B, two amino acid residues were germlined (i.e., replaced by the corresponding human germline residues). In CDR1, Asn(N)Kabat L24 was germlined and mutated to Ser(S). In CDR2, Thr(T)Kabat L52 was germlined and mutated to Ser(S). Examination of molecular models indicates that these residues are unlikely to make direct contact with the antigen and do not play an important structural role. Therefore, they are not comparable to their corresponding human germline IGKV3-15. * The primary reason for germlining residues within the Kabat CDRs is that these changes are consistent with the human germline IGKV3-15 * The aim is to increase the identity percentage of humanized version C to 01 to 85.3%.

[0411] [ka]

[0412] Humanized version B (CE7-3-15-VLC) and IGKV3-15 * 01 (81 identical residues out of a total of 95 residues in the V genes). Amino acid residues germlined in humanized version C compared to humanized version B are underlined.

[0413] [ka]

[0414] Humanized Version D In version D compared to version C, two amino acid residues were germlined (i.e., replaced by the corresponding human germline residues). In CDR1, Glu(E)Kabat L27 and Asp(D)Kabat L28 were germlined and mutated to Gln(Q) and Ser(S), respectively. Examination of molecular modeling indicates that these residues are unlikely to make direct contact with the antigen and do not play an important structural role. Therefore, they are not identical to their corresponding human germline IGKV3-15. * The primary reason for germlining residues within the Kabat CDRs is that these changes are consistent with the human germline IGKV3-15 * This increases the identity percentage of humanized version C to 01 to 87.4%.

[0415] [ka]

[0416] Humanized version D (CE7-3-15-VLD) and IGKV3-15 * 01 (83 identical residues out of a total of 95 residues in the V genes). Amino acid residues germlined in humanized version D compared to humanized version C are underlined.

[0417] [ka]

[0418] Example 2 - Recombinant antibody production - Variants 1-18. Genes and expression vectors Genes encoding rAbs Starting from the sequences of the heavy chain (HC) and light chain (LC) variable regions of 18 humanized mutant antibodies resulting from the in silico humanization step, a full-length human IgG1 kappa antibody with the mutations L234A, L235A, and P331S was designed.

[0419] cDNAs encoding the HC and LC variable regions were chemically synthesized with optimization for expression in CHO cells and subcloned into a proprietary mammalian cell expression vector from ProteoGenix, which contains the framework for the human IgG1 heavy chain constant region and the human kappa light chain constant region [https: / / www.proteogenix.science / product / xtencho-starter-kit / ]. A sequence encoding a signal peptide was added at the 5' / interposition. The sequences are shown below.

[0420] >CE7-1-2-VHA (L234A L235A + P331S) [SEQ ID NO: 85]

[0421] >CE7-1-2-VHB (L234A L235A + P331S) [SEQ ID NO: 86]

[0422] >CE7-4-34-VHA (L234A L235A + P331S) [SEQ ID NO: 87]

[0423] >CE7-1-NL1-VLA[SEQ ID NO: 88] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCTAGCTCCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACCTGCAAGGCCAATGAGGATATCAACAACCGGCTGGCTTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCTGCTGATCTCCGGCGCTACCAATCTGGTGACCGGCGTGCCCTCCAGGTTTAGCGGCAGCGGCTCCGGCAAGGATTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCTACCTACTATTGCCAGCAGTACTGGAGCACCCCTTTTACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0424] >CE7-1-NL1-VLB[SEQ ID NO: 89] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGATATCCAGATGACCCAGAGCCCTAGCAGCCTGTCCGCCAGCGTGGGCGATAGGGTGACCATCACCTGCAGGGCTAACGAGGACATCAATAACAGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCAGCGGCGCCACCAATCTGGTGACCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGAAGCGGCAAGGACTACACCCTGACCATCTCCTCCCTGCAGCCTGAGGACTTTGCCACCTATTATTGCCAGCAGTACTGGAGCACCCCTTTCACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0425] >CE7-1-33-VLA [Accession No. 90] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGTAAGGCTAATGAGGACATCAATAACCGGCTGGCTTGGTACCAGCAGAAGCCCGGCAAGGCTCCCAAGCTGCTGATCTCCGGCGCTACCAACCTGGTGACCGGCGTGCCTAGCAGGTTTTCCGGCTCCGGCAGCGGCAAGGATTATACCCTGACCATCTCCTCCCTGCAGCCCGAGGATATCGCTACCTACTACTGCCAGCAGTATTGGTCCACCCCTTTTACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0426] >CE7-1-33-VLB [Accession No. 91] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGATGACCCAGAGCCCCTCCAGCCTGTCCGCTTCCGTGGGCGATAGGGTGACCATCACCTGTCAGGCTAATGAGGACATCAACAACAGGCTGGCTTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCAGCGGCGCCACCAACCTGGTGACCGGCGTGCCAAGCAGGTTCAGCGGCAGCGGCAGCGGAAAGGACTATACCTTCACCATCTCCTCCCTGCAGCCTGAGGACATCGCTACCTATTACTGTCAGCAGTACTGGTCCACCCCCTTCACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0427] >CE7-3-15-VLA [Accession No. 92] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCAGCCTGCTGCTGTGGATCAGCGGCGCCTACGGCGAGATCGTGATGACCCAGTCCCCTGCCACCCTGTCCGTGTCCCCTGGCGAGAGGGCCACCCTGTCTTGCAAGGCTAATGAGGACATCAACAATAGGCTGGCCTGGTATCAGCAGAAGCCTGGCCAGGCTCCTCGGCTGCTGATCAGCGGCGCTACCAACCTGGTGACCGGCATCCCTGCCCGGTTCTCCGGCTCCGGAAGCGGAAAGGAGTATACCCTGACCATCAGCAGCCTGCAGAGCGAGGATTTTGCCGTGTACTATTGCCAGCAGTACTGGTCCACCCCCTTTACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0428] >CE7-3-15-VLB [SEQ ID NO: 93] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCAGCGGCGCCTACGGCGAGATCGTGATGACCCAGAGCCCTGCCACCCTGAGCGTGTCCCCTGGCGAGAGAGCCACCCTGTCCTGCCGGGCTAACGAGGACATCAACAACCGGCTGGCTTGGTATCAGCAGAAGCCTGGCCAGGCTCCTAGGCTGCTGATCTCCGGCGCTACCAACCTGGTGACCGGCATCCCTGCCAGGTTTAGCGGCAGCGGCTCCGGCAAGGAGTTCACCCTGACCATCAGCAGCCTGCAGTCCGAGGATTTCGCCGTGTATTATTGTCAGCAGTACTGGTCCACCCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0429] Sequence of the expressed protein >CE7-1–2-VHA (L234A L235A+P331S) [SEQ ID NO: 94] MKHLWFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0430] >CE7-1-2-VHB (L234A L235A+P331S) MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAR DYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0431] >CE7-4-34-VHA (L234A L235A + P331S) [SEQ ID NO: 96] MKHLWFFLLLVAAPRWVLSQVQLQQWGAGLLKPSETLSLTCAAYGYTFTGYWMHWIRQPPGKGLEWIGEINPSNGRTNYNERLKSRVTLSVDKSKNQASLKLSSVTAADTAVYFCAR DYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0432] >CE7-1-NL1-VLA [SEQ ID NO: 97] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0433] >CE7-1-NL1-VLB [SEQ ID NO: 98] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0434] >CE7-1-33-VLA [SEQ ID NO: 99] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDIATYYCQQYWSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0435] >CE7-1-33-VLB [SEQ ID NO: 100] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCQANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTFTISSLQPEDIATYYCQQYWSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0436] >CE7-3-15-VLA [SEQ ID NO: 101] MVLQTQVFISLLLWISGAYGEIVMTQSPATLSVSPGERATLSCKANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEYTLTISSLQSEDFAVYYCQQYWSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0437] >CE7-3-15-VLB [SEQ ID NO: 102] MVLQTQVFISLLLWISGAYGEIVMTQSPATLSVSPGERATLSCRANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEFTLTISSLQSEDFAVYYCQQYWSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0438] Small-scale production and purification tests Short Protocol Description Endotoxin-free DNA preparation was performed for the resulting constructs. Eighteen antibodies were expressed by combining HC and LC as shown in Table E1. Plasmids were transiently co-transfected into our proprietary Xten CHO cells using a proprietary Xten transfection protocol. Cell culture medium samples were collected when viability dropped below 50% (14 days after transfection), and the rAbs were then purified on Protein A resin using standard methods. -Clarification by 0.22μm filtration -Equilibration, binding, and washing with PBS (pH 7.5) -Elution by pH shift using citric acid -Neutralization with 1M Tris-HCl (pH 9.0) -Analysis by PAGE and pooling of the desired fractions - Final QC by PAGE: qualitative and quantitative by SDS-PAGE

[0439] The eluted fractions were pooled and buffer exchanged against PBS (pH 7.5) by dialysis. The final sample was filtered through a 0.22 μm Millipore filter. The purification profile and final QC are shown in Figures 1 and 2. The yields and purities obtained are summarized in Table E2.

[0440] [Table 2] * 30 ml culture was obtained after purification for testing. ** Based on the full-length antibody observed on non-reducing PAGE analysis in Figure 2.

[0441] Example 3 - Recombinant antibody production - Mutants 19-28. Genes and expression vectors Genes encoding rAbs Starting from the sequences of the heavy chain (HC) and light chain (LC) variable regions of 12 additional humanized mutant antibodies resulting from the in silico humanization step, a full-length human IgG1 kappa antibody with the mutations L234A, L235A, P331S, and N297A was designed.

[0442] The cDNAs encoding the HC and LC variable regions were chemically synthesized with optimization for expression in CHO cells and subcloned into a proprietary mammalian cell expression vector from ProteoGenix, which contains the framework for the human IgG1 heavy chain constant region and the human kappa light chain constant region (https: / / www.proteogenix.science / product / xtencho-starter-kit / ). A sequence encoding a signal peptide was added at the 5' / interposition. The sequences are shown below.

[0443] >CE7-1-2-VHB(L234A L235A+P331S)(14047) [SEQ ID NO: 86], as above

[0444] >CE7-1-2-VHB (L234A L235A + P331S N297A) [SEQ ID NO: 103]

[0445] >CE7-1-2-VHC(L234A L235A+P331S N297A) [SEQ ID NO: 104]

[0446] >CE7-1-2-VHD (L234A L235A + P331S N297A) [SEQ ID NO: 105]

[0447] >CE7-1-NL1-VLA[SEQ ID NO: 106] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGACATCCAGATGACCCAGAGCCCTTCCTCCCTGTCCGCCAGCGTGGGCGACAGGGTGACCATCACCTGTAAGGCCAATGAGGATATCAATAATCGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTCCGGCGCTACCAATCTGGTGACCGGCGTGCCTTCCCGGTTTTCCGGCTCCGGCAGCGGCAAGGATTATACCCTGACCATCAGCAGCCTGCAGCCCGAGGATTTTGCTACCTATTATTGCCAGCAGTATTGGTCCACCCCCTTCACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0448] >CE7-1-NL1-VLB[SEQ ID NO: 107] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCAGCTCCCTGTCCGCTAGCGTGGGCGACAGGGTGACCATCACCTGTAGGGCCAATGAGGACATCAACAACAGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCTCCTAAGCTGCTGATCTCCGGCGCTACCAATCTGGTGACCGGCGTGCCTTCCAGGTTCTCCGGCTCCGGCTCCGGAAAGGATTACACCCTGACCATCAGCTCCCTGCAGCCTGAGGACTTTGCTACCTATTACTGCCAGCAGTACTGGTCCACCCCCTTTACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0449] >CE7-1-NL1-VLC[SEQ ID NO: 108] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGATATCCAGATGACCCAGAGCCCCTCCAGCCTGAGCGCCTCCGTGGGAGACAGGGTGACCATCACCTGTAGGGCCAACGAGGATATCAACAACAGGCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCTCCTAAGCTGCTGATCAGCGGCGCTTCCAACCTGGTGAGCGGCGTGCCCTCCAGGTTTAGCGGCAGCGGCAGCGGAAAGGATTACACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACTATTGCCAGCAGTACTGGAGCACCCCTTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0450] >CE7-1-NL1-VLD[SEQ ID NO: 109] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGACATCCAGATGACCCAGTCCCCTAGCTCCCTGAGCGCCTCCGTGGGCGACAGGGTGACCATCACCTGCCGGGCCAGCCAGGGCATCAATAATCGGCTGGCTTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCAGCGGCGCCTCCAACCTGGTGAGCGGCGTGCCTTCCAGGTTCTCCGGCAGCGGCAGCGGCAAGGACTATACCCTGACCATCAGCAGCCTGCAGCCTGAGGACTTTGCCACCTACTATTGCCAGCAGTATTGGTCCACCCCCTTTACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0451] The sequence of the predicted protein produced is shown below. >CE7-1-2-VHB(L234A L235A+P331S)(14,047)[SEQ ID NO:94], as described above MKHLWFLLLVAAPRWVLSQVQLVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0452] >CE7-1-2-VHB (L234A L235A+P331S N297A) MKHLWFLLLVAAPRWVLSQVQLVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0453] >CE7-1-2-VHC (L234A L235A+P331S N297A) MKHLWFLLLVAAPRWVLSQVQLVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNEKFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0454] >CE7-1-2-VHD (L234A L235A+P331S N297A) MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWMGEINPSNGRTNYNEKFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAR DYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0455] >CE7-1-NL1-VLA [SEQ ID NO: 97] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0456] >CE7-1-NL1-VLB [SEQ ID NO: 98] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0457] >CE7-1-NL1-VLC [SEQ ID NO: 113] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGASNLVSGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0458] >CE7-1-NL1-VLD [SEQ ID NO: 114] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCRASQGINNRLAWYQQKPGKAPKLLISGASNLVSGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0459] Small-scale production and purification tests Short Protocol Description Endotoxin-free DNA preparations were performed on the resulting constructs as described herein. 12 antibodies were expressed by combining the HC and LC as follows:

[0460] [Table 3]

[0461] Small scale antibody production was performed as described in Example 2. The results are shown in Figures 3 and 4. The yields and purities obtained are summarized in Table E4.

[0462] [Table 4] * 30 ml culture was obtained after purification for testing. ** Based on the full-length antibody observed on non-reducing PAGE analysis in Figure 2. Final buffer: PBS (pH 7.5).

[0463] Example 4. Text results of expression and purification for variants 4, 7, 15, 17, and 18 in aglycosylated form. Starting from the sequences of the heavy chain (HC) and light chain (LC) variable regions of 18 humanized mutant antibodies resulting from the in silico humanization step, we designed a full-length human IgG1 kappa antibody with the mutations L234A, L235A, P331S, and N297A. The cDNAs encoding the HC and LC were chemically synthesized with optimization for expression in CHO cells and subcloned into a proprietary mammalian cell expression vector from ProteoGenix, which contains the framework for the human IgG1 heavy chain constant region and the human kappa light chain constant region (https: / / www.proteogenix.science / product / xtencho-starter-kit / ). A sequence encoding a signal peptide was added at the 5' / Nter position. The sequence is shown below.

[0464] >CE7-1-2-VHA (L234A L235A + P331S + N297A) [SEQ ID NO: 115]

[0465] The following sequences are as mentioned above: >CE7-1-2-VHB (L234A L235A + P331S + N297A) [SEQ ID NO: 103] >CE7-4-34-VHA (L234A L235A + P331S + N297A) [SEQ ID NO: 87] >CE7-1-NL1-VLA [SEQ ID NO: 88] >CE7-1-33-VLA [SEQ ID NO: 90] >CE7-1-33-VLB [SEQ ID NO: 91] >CE7-3-15-VLA [SEQ ID NO: 92] >CE7-3-15-VLB [SEQ ID NO: 93]

[0466] The expected protein products, depending on the sequence, are as follows or as defined herein above: >CE7-1-2-VHA (L234A L235A + P331S + N297A) [SEQ ID NO: 116] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCAR DYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0467] Small-scale production and purification tests Short Protocol Description Endotoxin-free DNA preparations were performed for the resulting constructs as described herein. Five antibodies were expressed by combining the HC and LC as follows:

[0468] [Table 5]

[0469] The results of small scale production are summarized in Figures 5 and 6. Yield and purity data are summarized in Table E6.

[0470] [Table 6] * 30 ml culture was obtained after purification for testing. ** Based on the full-length antibody observed on non-reducing PAGE analysis in FIG.

[0471] Example 5 - SEC HPLC Analysis SEC-HPLC analysis was performed to assess the aggregation levels of the top three candidates: CE7-variant4_AG, CE7-variant7_AG, and CE7-variant17_AG, starting with samples of each protein obtained after final conditioning and aliquoting as described in Example 4. The analysis was performed using an HPLC Waters 2695 device equipped with a photodiode array detector (2996).

[0472] Preparation of buffer solutions Mobile phase: 100 mM sodium sulfate, 100 mM phosphate, buffer (pH 6.7) (Na2SO4 14.2 g / L, Na2HPO4·12 H2O 15.6 g / L, NaH2P04 6.8 g / L). Air bubbles are removed from all buffers.

[0473] Sample preparation - Protein preparation: starting from one extra aliquot of each final sample stored overnight at -80°C, dilution of protein samples to 1 mg / ml with DD water - centrifugation at 12000 g for 10 minutes and collection of supernatant

[0474] Analysis conditions -Blank control: Dilution of sample with DD water (5 times) -Column: G3000SWXL, TOSOH, 7.8 x 300 mm -HPLC: Waters 2695 -Elution gradient: Mobile phase isocratic elution -Flow rate: 0.8ml / min -Temperature: 25℃ -Injection volume: 30μl -Detection wavelength: 280nm -Collection date and time: 25 minutes - Equilibrate with 100% mobile phase for 5 minutes - Running samples: one blank control injection, then sample injection -Data analysis

[0475] The results are shown in Table E7.

[0476] [Table 7] * The purity of the whole antibody single molecule detected in the sample.

[0477] If several peaks are detected, the data corresponding to the peak of a single intact antibody molecule are underlined. If a single peak is detected, an estimate of concentration (purity) is given.

[0478] The SEC-HPLC profiles obtained for the samples are also shown in Figure 7. SEC-HPLC analysis shows that all three antibody samples exhibit the best concentration of ideal heterotetrameric antibodies composed of two heavy chains and two light chains, and very little aggregates at <5%.

[0479] Example 6 - Determination of the KD of antibody / antigen interactions via surface plasmon resonance (SPR) technology (Biacore 8K) In this study, the Kd of three humanized antibodies against soluble antigens was determined via Biacore 8K. The L1-CAM recombinant protein (His tag) was obtained from Sinobiological under catalog number 10140-H08H (https: / / www.sinobiological.com / recombinant-proteins / human-l1cam-10140-h08h, evaluated on September 2, 2022). In the SPR experiment, the antigen was immobilized on a sensor chip, and solutions containing various concentrations of antibody were flowed over the antigen. Real-time measurements of kinetic parameters, including their on / off rates (ka and kd), were performed. Based on this, the binding affinity constant (KD) was determined. The materials used are summarized in Table E8.

[0480] [Table 8]

[0481] The following buffers were used in the experiments: Antigen dilution buffer: NaAc (pH4.5) (20mM NaAc (pH4.5)). Running buffer / antibody dilution buffer: HBS-EP+ (0.01M Hepes (pH7.4), 0.15M NaCl, 3mM EDTA, 0.01% Surfactant P20) Renaturation buffer: glycine (pH 1.5)

[0482] We used a CM5 sensor chip, a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached. While the gold is required for the generation of an SPR response, the dextran matrix allows for the covalent immobilization of biomolecules using maleimide chemistry.

[0483] method Channel 1: no coupling, used as negative control. Channel 2: Immobilization of L1-CAM (10 μg / ml) on a CM5 sensor chip using maleimide EDC / NHS coupling. The response measures the change in refractive index and is related to the change in mass near the sensor surface. The response is therefore proportional to the number of antibody molecules interacting with the antigen.

[0484] A defined concentration of antibody is flowed onto the CM5 chip, and the response is captured over time to indicate the progression of the interaction and association / dissociation cycle. Then, as described herein, regeneration is performed to remove any remaining bound antibody from the chip, and a new concentration is tested. After different concentrations are tested sequentially, kinetic parameters and affinity are calculated using BIA evaluation software.

[0485] The following concentrations were tested: 1.56 nM, 3.125 nM, 6.25 nM, 32 nM, 12.5 nM, and 25 nM. The sensorgram is shown in Figure 8 and the results are summarized in Table E9.

[0486] [Table 9]

[0487] The three humanized aglycosylated antibodies against L1-CAM and the two control antibodies show very high affinity for the antigen for the following reasons: -10 ~10 -13 Having a KD in the range of: - 10, indicating very strong interaction -13 Higher KD in the range, more favorable kinetic parameters indicating the absence of fast association and dissociation; -Sensorgram shape showing a clear concentration-response relationship.

[0488] Example 7 - Determination of the KD of antibody / antigen interactions via surface plasmon resonance (SPR) technology (Biacore8K) The KD of four monoclonal antibodies against soluble antigens was determined by using Biacore8K as in Example 6.

[0489] Table E10 summarizes the materials used in this study.

[0490] [Table 10]

[0491] Note that variants 26, 27, and 28 contain the N297A mutation and are therefore aglycosylated. Variant 7, as used herein, does not contain this mutation and is predicted to be glycosylated.

[0492] See Example 6 for a description of the experimental conditions.

[0493] The results are summarized in Figure 9 and Table E11.

[0494] [Table 11]

[0495] Example 8 - Production of liability mutants 1-8. Starting with the sequences of the heavy chain (HC) and light chain (LC) variable regions of eight additional liability mutant antibodies resulting from the in silico humanization step, we designed a full-length human IgG1 kappa antibody with the mutations L234A, L235A, P331S, and N297A. Note that these liability mutants contain mutations made on the chimeric CE7 antibody framework.

[0496] The cDNAs encoding the HC and LC variable regions were chemically synthesized with optimization for expression in CHO cells and subcloned into a proprietary mammalian cell expression vector from ProteoGenix, which contains the framework for the human IgG1 heavy chain constant region and the human kappa light chain constant region (https: / / www.proteogenix.science / product / xtencho-starter-kit). A sequence encoding a signal peptide was added at the 5' / Nter position. The sequences are shown below.

[0497] >CE7-VH-chimera-WH33Y(L234A L235A+P331S N297A) [SEQ ID NO: 117]

[0498] >CE7-VH-chimera-WH33F (L234A L235A + P331S N297A) [SEQ ID NO: 118]

[0499] >CE7-VH-chimera-WH33L (L234A L235A + P331S N297A) [SEQ ID NO: 119]

[0500] >CE7-VH-chimera-GH55A (L234A L235A + P331S N297A) [SEQ ID NO: 120]

[0501] >CE7-VH-chimera (L234A L235A + P331S N297A) [SEQ ID NO: 121]

[0502] >CE7-VL chimeric [SEQ ID NO: 122] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCAGCGGCGCCTACGGCGATATCCAGATGACCCAGAGCTCCAGCTCCTTTAGCGTGAGCCTGGGCGACCGGGTGACCATCACCTGTAAGGCTAATGAGGACATCAATAACAGGCTGGCTTGGTACCAGCAGACCCCCGGCAATAGCCCTAGGCTGCTGATCAGCGGCGCTACCAATCTGGTGACCGGCGTGCCTAGCAGGTTTAGCGGCTCCGGCTCCGGCAAGGATTACACCCTGACCATCACCTCCCTGCAGGCCGAGGATTTTGCTACCTACTATTGTCAGCAGTACTGGAGCACCCCTTTTACCTTTGGCTCCGGCACCGAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0503] >CE7-VL chimeric - NL31L [SEQ ID NO: 123] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGACATCCAGATGACCCAGAGCTCCTCCTCCTTTTCCGTGTCCCTGGGCGATCGGGTGACCATCACCTGTAAGGCCAATGAGGATATCAACCTGAGGCTGGCCTGGTACCAGCAGACCCCTGGCAACAGCCCTAGGCTGCTGATCTCCGGCGCTACCAATCTGGTGACCGGCGTGCCTAGCCGGTTTAGCGGCTCCGGCAGCGGCAAGGACTATACCCTGACCATCACCAGCCTGCAGGCCGAGGACTTTGCTACCTATTATTGTCAGCAGTACTGGAGCACCCCCTTTACCTTTGGCTCCGGCACCGAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0504] >CE7-VL-Chimeric-NL31A [SEQ ID NO: 124] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCTCCGGCGCTTATGGCGATATCCAGATGACCCAGAGCTCCAGCAGCTTCTCCGTGAGCCTGGGCGATAGGGTGACCATCACCTGTAAGGCCAACGAGGATATCAATGCTAGGCTGGCTTGGTATCAGCAGACCCCTGGCAATAGCCCCCGGCTGCTGATCTCCGGCGCCACAAACCTGGTGACCGGCGTGCCTTCCAGGTTCTCCGGCAGCGGCTCCGGCAAGGATTATACCCTGACCATCACCAGCCTGCAGGCTGAGGATTTTGCTACCTACTACTGTCAGCAGTATTGGAGCACCCCCTTTACCTTTGGCTCCGGCACCGAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0505] >CE7-VL-Chimeric-WL92Y[SEQ ID NO: 125] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCAGCCTGCTGCTGTGGATCTCCGGCGCTTATGGCGATATCCAGATGACCCAGAGCTCCTCCTCCTTTAGCGTGAGCCTGGGCGATCGGGTGACCATCACCTGTAAGGCCAACGAGGACATCAATAACAGGCTGGCTTGGTACCAGCAGACCCCTGGCAACAGCCCTCGGCTGCTGATCAGCGGCGCCACCAATCTGGTGACCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGATCCGGCAAGGACTACACCCTGACCATCACCAGCCTGCAGGCTGAGGACTTTGCCACCTATTACTGTCAGCAGTACTATTCCACCCCTTTCACCTTCGGCTCCGGCACCGAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0506] >CE7-VL-Chimeric-WL92F [SEQ ID NO: 126] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGACATCCAGATGACCCAGAGCAGCTCCAGCTTCTCCGTGTCCCTGGGCGACCGGGTGACCATCACCTGTAAGGCCAATGAGGATATCAACAACCGGCTGGCCTGGTATCAGCAGACCCCTGGCAACAGCCCCAGGCTGCTGATCTCCGGCGCCACCAATCTGGTGACCGGCGTGCCCTCCCGGTTTTCCGGCAGCGGAAGCGGCAAGGATTACACCCTGACCATCACCTCCCTGCAGGCTGAGGATTTCGCTACCTACTATTGCCAGCAGTACTTTTCCACCCCCTTTACCTTTGGCTCCGGCACCGAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0507] The sequence of the predicted protein produced is shown below. >CE7-VH-chimeric-WH33Y(L234A L235A+P331S N297A)[SEQ ID NO:127] MKHLWFFLLLVAAPRWVLSQVQLQQPGAELVKPGASVKLSCKASGYTFTGYYMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCAR DYYGTSYNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0508] >CE7-VH-chimera-WH33F (L234A L235A + P331S N297A) [SEQ ID NO: 128] MKHLWFFLLLVAAPRWVLSQVQLQQPGAELVKPGASVKLSCKASGYTFTGYFMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCAR DYYGTSYNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0509] >CE7-VH-chimera-WH33L (L234A L235A + P331S N297A) [SEQ ID NO: 129] MKHLWFFLLLVAAPRWVLSQVQLQQPGAELVKPGASVKLSCKASGYTFTGYLMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCAR DYYGTSYNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0510] >CE7-VH-chimera-GH55A (L234A L235A + P331S N297A) [SEQ ID NO: 130] MKHLWFFLLLVAAPRWVLSQVQLQQPGAELVKPGASVKLSCKASGYTFTGYWMHWVKQRPGHGLEWIGEINPSNARTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCAR DYYGTSYNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0511] >CE7-VH-chimera (L234A L235A + P331S N297A) [SEQ ID NO: 131] MKHLWFFLLLVAAPRWVLSQVQLQQPGAELVKPGASVKLSCKASGYTFTGYWMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCAR DYYGTSYNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0512] >CE7-VL-chimera [SEQ ID NO: 132] MVLQTQVFISLLLWISGAYGDIQMTQSSSSFSVSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYWSTPFT FGSGTELEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0513] >CE7-VL-chimera-NL31L [SEQ ID NO: 133] MVLQTQVFISLLLWISGAYGDIQMTQSSSSFSVSLGDRVTITCKANEDINLRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYWSTPFT FGSGTELEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0514] >CE7-VL-chimera-NL31A [SEQ ID NO: 134] MVLQTQVFISLLLWISGAYGDIQMTQSSSSFSVSLGDRVTITCKANEDINARLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYWSTPFT FGSGTELEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0515] >CE7-VL-chimera-WL92Y [SEQ ID NO: 135] MVLQTQVFISLLLWISGAYGDIQMTQSSSSFSVSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYYSTPFT FGSGTELEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0516] >CE7-VL-chimera-WL92F [SEQ ID NO: 136] MVLQTQVFISLLLWISGAYGDIQMTQSSSSFSVSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYFSTPFT FGSGTELEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0517] Endotoxin-free DNA preparation was performed on the resulting constructs as described above. Eight antibodies were expressed by combining HC and LC as follows:

[0518] [Table 12]

[0519] The methods described in Example 2 or Example 3 were used. The purification profile and final QC are shown in Figures 10 and 11. The antibody yields and purities obtained are summarized in Table E13.

[0520] [Table 13] * 30 ml culture was obtained after purification for testing. ** Based on the full-length antibody observed on non-reducing PAGE analysis in Figure 2. Final buffer: PBS (pH 7.5).

[0521] Example 9 - SEC-HPLC analysis of liability variants Short Protocol Description SEC-HPLC analysis was performed to assess purity levels and quantify the percentage of soluble aggregates for the liability antibody samples. Analysis was performed using an HPLC Waters 2695 device equipped with a photodiode array detector (2996).

[0522] Preparation of buffer solutions Mobile phase: 100 mM sodium sulfate, 100 mM phosphate, buffer (pH 6.7) (Na2SO4 14.2 g / L, Na2HPO4·12H2O 15.6 g / L, NaH2PO4 6.8 g / L) Remove air bubbles from all buffers.

[0523] Sample preparation - Protein preparation: Dilution of protein samples to 1 mg / ml with DD water, starting from one extra aliquot of each final sample stored overnight at -80°C - Centrifuge at 12000g for 10 minutes and collect the supernatant Analysis conditions Blank control: Dilution of sample with DD water (5 times) Column: G3000SWXL, TOSOH, 7.8 x 300 mm -HPLC: Waters 2695 -Elution gradient: Mobile phase isocratic elution -Flow rate: 0.8ml / min -Temperature: 25℃ -Injection volume: 30μl -Detection wavelength: 280nm -Collection date and time: 25 minutes - Equilibrate with 100% mobile phase for 5 minutes - Running samples: one blank control injection, then sample injection -Data analysis

[0524] The results are summarized in Table E14.

[0525] [Table 14] When several peaks were detected, the data corresponding to the peaks of the whole antibody single molecules are shown in bold. If a single peak is detected, an estimate of concentration (purity) is given.

[0526] Example 10. Differential Scanning Fluorimetry Short Protocol Description DSF analysis was performed to evaluate the thermal stability (unfolding) of 18 humanized variant antibodies. DSF runs were performed by using the Nanotemper-nanoDSF system with standard methods. mAbs were subjected to a linear thermal gradient from 40°C to 90°C at 1°C / min. Tryptophan fluorescence at 350 nm and 330 nm was collected at a rate of 10 data points per minute. The unfolding transition midpoint was automatically determined from the second derivative of the fluorescence ratio (F350 / F330). The samples are summarized in Table E15.

[0527] [Table 15]

[0528] The results of the analysis are shown in Table E16.

[0529] [Table 16]

[0530] All antibodies showed two unfolding transitions in the plot of fluorescence ratio (F330 / F350) versus temperature. The different unfolding events may be due to the different thermal stabilities of the Fab and Fc domains of the antibodies. DSF analysis shows that variants 5, 7, 8, 9, 10, and 11 have similar thermal stabilities, with Tm1 of approximately 68°C and Tm2 of approximately 80°C. Variants 13, 14, 15, 16, 17, and 18 show a slight decrease in Tm2 (approximately 74°C).

[0531] Example 11. Differential Scanning Fluorimetry Further analysis as in Example 10 was carried out on the samples summarized in Table E17.

[0532] [Table 17]

[0533] The results of the analysis are shown in Table E18.

[0534] [Table 18]

[0535] The DSF analysis is similar to that seen in Example 10, showing two unfolding events. The Tm1 and Tm2 of the chimeric antibody are comparable to those of the non-glycosylated variant. The aglycosylated chimeric antibody has a Tm1 value similar to the three humanized variants but lower than that of the chimeric antibody, which may correspond to an unfolding event of the Fc domain. The Tm2 values ​​for the three humanized aglycosylated variants are high and very similar to those obtained for the same non-glycosylated versions. This indicates that Tm2 may correspond to unfolding of the Fab domain.

[0536] Example 12. Differential Scanning Fluorimetry Further analysis as in Example 10 was performed on the samples summarized in Table E19. Note that all variants described herein, except for CE7-Variant 7, are aglycosylated (i.e., contain the mutation N297A in the heavy chain).

[0537] [Table 19]

[0538] The results of the analysis are shown in Table E20.

[0539] [Table 20]

[0540] All antibodies showed two unfolding transitions in the plot of fluorescence ratio (F330 / F350) versus temperature (Figure 1). The different unfolding events may be due to the different thermal stabilities of the Fab and Fc domains of the antibodies. DSF analysis shows that additional mutants 19-28 have similar thermal stabilities with Tm1 of approximately 57°C and Tm2 of approximately 80°C, very similar to mutant 7-AG. Liability mutants LV1-LV8 show a slight decrease in Tm2 (approximately 72°C).

[0541] Example 13 - Affinity of antibodies to L1-CAM - ELISA studies The affinity of the antibodies to L1-CAM was determined using an ELISA method according to the following protocol. 1. Antigen coating: human L1-CAM, 2 μg / ml, 100 μl / well, 37°C, 2 hours 2. Blocking: 3% BSA-PBS, 300 μl / well, 37°C, 1.5 hours 3. Wash: PBST, 300 μl / well, twice 4. Primary antibody (humanized variant), 100 μl / well, 37°C, 30 minutes 5. Wash: PBST, 300 μl / well, 3 times 6. Secondary antibody, 100 μl / well, 37°C, 30 minutes 7. Wash: PBST, 300 μl / well, 3 times 8. TMB substrate solution: 100 μl / well, 37°C, 5 to 10 minutes 9. Stop: 2M HCl, 50 μl / well 10. Reading: OD450~OD630

[0542] [Table 21]

[0543] Example 14 - GCI study of antibody binding In this study, the binding of certain antibodies of the invention to L1-CAM was investigated.

[0544] Rationale: This experiment should examine L1-CAM binding to Protein A / G capture antibodies in a multi-cycle kinetic experiment. See FAQ. • Ligands are captured anew for each analytical cycle. • Analyte is injected in increasing concentrations across the ligand and reference surfaces. After each analytical cycle, the ligand-analyte complex is removed from the capture surface by injection of a regeneration solution. ● A DMSO solvent calibration curve is established for solvent calibration. Please refer to the FAQ. • Raw sensorgrams are examined for non-specific and ligand-specific analyte binding. • Finally, the data are double-referenced and, where applicable, fitted to appropriate kinetic models.

[0545] [Table 22]

[0546] Figure 13A shows a double-referenced, solvent-corrected sensorgram of the interaction between mutant 7AG and L1-CAM, with the measured response shown graphically. Dose-responsive binding was detected. The data were fitted with a 1:1 interaction model, and the fitted curve is shown in black, resulting in a calculated affinity of approximately 4 nM. The ligand activity was calculated to be 28%. Lower concentrations were excluded to improve fitting quality.

[0547] Figure 13B shows a double-referenced, solvent-corrected sensorgram of the interaction between deglycosylated cHCE7 and L1-CAM, with the measured response shown graphically. Dose-responsive binding was detected. The data were fitted with a 1:1 interaction model, and the fitted curve is shown in black, resulting in a calculated affinity of approximately 4 nM. Ligand activity was calculated to be 28%. Lower concentrations were excluded to improve fitting quality.

[0548] Thus, this study confirms that the affinities of the humanized and chimeric antibodies are comparable.

[0549] Example 15 - Functionalization of antibodies of the invention with an azide linker using MTG The humanized antibody variant (5 mg / L in PBS, pH 7.5) was deglycosylated by mixing it with N-glycosidase (5.45 U per mg of antibody) (N-glycosidase F, 1 U / μL, REF: 11365177001, Sigma Aldrich, Germany) in a 1.5 mL reaction tube and incubated at 37°C for 24 hours with gentle shaking. The completion of the reaction was analyzed by LC-MS. The deglycosylated antibody was then buffer exchanged into Trizma buffer (50 mM, pH 7.5) using a PD-10 desalting column. The deglycosylated antibody (4 mg / ml) was then incubated in a 5 ml low protein binding reaction tube with a mixture of microbial transglutaminase (MTG) (2.5 U per mg of antibody [Andracon™, catalog number T-153, supplier Zedira, Germany]) and 80 equivalents per antibody of amino-PEG4-azide [supplier Broadpharm, catalog number BP-21615, USA] to modify residue Q295 in the antibody heavy chain (HC) with a click-reactive azide linker. The coupling reaction was carried out by incubation at 37°C for 24 hours. Completion of the reaction was monitored by LC-MS (HPLC 2795 Waters, ESI-TOF LCT). The coupling efficiency was calculated by comparing the AUC of HC (unmodified) with that of HC (linker-modified), and peaks were identified using the 245 Da linker mass difference. Residual enzyme and excess PEG linker were then removed by Protein A affinity chromatography (Protein Sepharose 4 Fast Flow, supplied by GE Healthcare (USA), catalog number 17.5280-20, equipped with the column Isolute® SPE, catalog number 120.1362, Biotage (Sweden)) using the manufacturer's protocol.In the final step, the purified antibody-linker-azide conjugate was buffer exchanged (5 times) into PBS (pH 7.5) using ultracentrifugation (Amicon® Ultra-15 centrifugal filter, 30 kDa MWCO, catalog number UFC903008, supplier Sigma Aldrich, Germany). The antibody-linker-azide conjugate was stored at -20°C until use.

[0550] Example 16 - Functionalization of an aglycosylated antibody of the invention with an azide linker using MTG An aglycosylated version of the humanized antibody of the invention was subjected to the same coupling protocol presented in Example 3, except that the enzymatic deglycosylation step was omitted. The coupling efficiency with MTG was comparable to that described in Example 15.

[0551] Example 17 - Functionalization of the mutant 7AG-Q295-NH-PEG4-azide with a metal chelator (DBCO DOTA) Azide-modified antibody-linker conjugates were functionalized with metal chelators to generate model antibody-radionuclide conjugates (ARCs) containing two DOTA chelators per mAb. Briefly, 23.2 μM of the mutant 7AG-[Q295-NH-PEG4-azide]2 (synthesized according to the protocol presented in Example 4) in PBS (pH 7.5) supplemented with 1 mM ammonium acetate (pH 5.5) was incubated with a 10-fold molar excess of click-reactive DBCO-DOTA (supplier: Macrocyclics, Texas, USA, catalog number B-283) at room temperature for 16 hours. Subsequently, excess DBCO-DOTA was removed by ultracentrifugation (Vivaspin 6, 30 kDa MWCO, catalog number VS0621, Bio-Rad, Germany).

[0552] Example 18 - Mouse biodistribution study using variant 7AG-[Q295-NH-PEG4-click-(DOTA)]2 The resulting huCE7-variant 7AG-[Q295-NH-PEG4-click-(DOTA)]2 from Example 5 was then labeled with radioactive lutetium-177 and used in a mouse biodistribution study to characterize tumor uptake.

[0553] [Table 23]

[0554] The reaction was carried out in a 1.5 mL reaction tube with gentle stirring at 37° C. for 1 hour. A 1 μL sample was taken and placed in an HPLC vial containing 100 μL of PBS (pH 7.5) and 2.5 μL of 1 mM DTPA, and analyzed by HPLC using a gamma counter as a detector to monitor the success of the labeling.

[0555] Cells for tumor models: Her2 / neu-positive SKOV3ip cells were maintained in DMEM medium at 37°C. The cell culture medium was supplemented with 10% FCS, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL Fungizone (BioConcept, Allschwil, Switzerland). The cell line was cultured in a humidified atmosphere containing 5% CO2. The cells were then used to inject mice (5 million cells per animal) to generate a Her2- and L1-CAM-positive ovarian cancer tumor model.

[0556] Mouse tumor model for biodistribution: A total of 32 mice bearing SKOV 3ip tumors were injected with 150 kBq of the huCE7 variant 7AG-[PEG-DOTA(Lu-177]2 (16 mice) in 100 µL of injection solution. In this study, cHCE7-[PEG-DOTA(Lu-177]2 (16 mice) served as a control for tumor uptake. Four mice from each group were sacrificed at 24, 48, 72, and 96 h postinfection, and organs / tissues were dissected, weighed, and measured for radioactivity. % iA / g was calculated and presented in Figure 18.

[0557] Tumor uptake between the humanized antibody huCE7 variant 7AG was approximately 68 ± 7.7% iA / g, comparable to chimeric cHCE7 over the 24-96 h monitoring period. The maximum activity per gram at 96 h was 77% ± 7.2% iA / g, indicating that the humanized framework did not interfere with tumor uptake.

[0558] The organ distribution of the humanized antibody huCE7 variant 7AG was within the literature range for typical ARC (Figure 18).

[0559] Example 19 - Functionalization of huCE7-mutant 7AG-[Q295-NH-PEG-Azide with Toxin (MMAE) Azide-modified antibody-linker conjugates were functionalized with the toxin monomethyl auristatin E (MMAE) to generate model antibody-drug conjugates containing four toxins per mAb. Briefly, mutant 7-[Q295-NH-PEG4-azide]2, synthesized according to the protocol presented in Example 4, was incubated with a 10-fold molar excess of click-reactive DBCO-(PEG2-vc-PAB-MMAE)2 (supplier: SyntaBio, USA) in PBS (pH 7.5) supplemented with 10% (m / v) dimethylacetamide for 16 hours at room temperature. The ADC was then purified on a Sephadex G25 column into PBS (pH 7.5), followed by charcoal depletion of residual toxin linker. The resulting ADC [huCE7-V7AG-(MMAE)4] was concentrated to approximately 3 mg / mL before a final 0.2 μm filtration.

[0560] Analysis of DAR in ADC: The monomer constituted 96.8% of the sample as determined by SEC. The total concentration as determined by SEC was 2.8 mg / mL. The drug-to-antibody ratio as determined by MS was 3.9, and the drug-to-antibody ratio as determined by HIC was 3.6. In total, 3.3 mg of huCE7-V7AG-(MMAE2)2 was produced.

[0561] Example 20 - Zebrafish Toxicity and Tumor Efficacy Studies with huCE7-Variant7-[Q295-NH-PEG4-Click-BranchedPEG-(MMAE)2]2 This study aimed to evaluate the toxicity and antitumor effects of the huCE7-V7AG (control = unmodified mAb) and huCE7-V7AG-(MMAE)4 antibodies on zebrafish embryos, primary tumor size, and metastasis formation. The MAXFTN-401 cell line (L1-CAM-positive TBNC) obtained from Charles River Laboratories (MA, USA) was used as a cancer model. The anticancer efficacy of the antibodies was determined by changes in primary tumor size (i.e., tumor growth or shrinkage) and the number of tumor cells disseminated into the distal caudal venous plexus (CVP) 3 days after implantation, as specified below.

[0562] The study design was divided into three parts and was carried out at BioReperia AB (Wahlbecksgatan 25,582 13 Linkoping). Part 1: Toxicity evaluation of the huCE7-V7AG antibody (reduced toxicity). Part 2: Toxicity evaluation of the HUCE7-V7AG-(MMAE)4 antibody (full toxicity evaluation). Part 3: Evaluation of efficacy using the ZTX-ONCOLEADS model (Bioreperia AB, Sweden).

[0563] Part 1: Toxicity evaluation of the huCE7-V7AG antibody A reduced toxicity study of the huCE7-V7AG antibody was conducted across five concentrations and a vehicle control (Table E24). The antibody was administered intravenously. Each group consisted of 20 zebrafish larvae that were 48 hours post-fertilization at the time of injection and incubated at 35.5°C for 3 days. Evaluation was performed 72 hours after treatment, and the outcome was assessed by the following parameters: live / dead and LD50 concentration.

[0564] [Table 24]

[0565] result: Toxicity evaluation of different concentrations of huCE7-V7AG "naked" was performed. The results showed that the antibody was very well tolerated by the embryos, with only one counted death (at 0.1 mg / mL concentration) among all experimental groups (Figure 17). Therefore, since none of the experimental groups had mortality rates exceeding 50%, the LD50 linear fit readout cannot be readily accepted.

[0566] Part 2: Toxicity evaluation of HUCE7-V7AG-(MMAE)4 antibody Following the toxicity results with the huCE7-V7AG antibody, a full toxicity evaluation of the HUCE7-V7AG-(MMAE)4 antibody was determined. A full toxicity study of the HUCE7-V7AG-(MMAE)4 antibody was conducted across five concentrations and a vehicle control (Table E25). The antibody was administered intravenously. Each group consisted of 20 zebrafish larvae, 48 hours post-fertilization at the time of injection, incubated at 35.5°C for 3 days. Evaluations were performed 24, 48, and 72 hours after treatment, and outcomes were assessed for the following parameters: survival / death, LT50, LD50, and non-lethal toxicity, such as pericardial edema, malformations, necrosis, and teratogenesis. Representative images of embryos were acquired at each time point.

[0567] [Table 25-1]

[0568] Results: Embryonic survival after exposure to intravenously injected HuCE7-V7Ag(MMAE)4

[0569] [Table 25-2]

[0570] [Table 25-3]

[0571] The MMAE-loaded ADC demonstrated good compatibility with non-tumor zebrafish models. Because 50% lethality was not reached (see Tables E25.2 and E25.3), the LT50 or LD50 concentrations could not be determined. In addition, all experimental groups were within background mortality for intravenously injected embryos. Regarding non-lethal toxicity, the 2.8 mg / mL concentration, as expected, showed the highest percentage of embryos with some toxicity, but still constituted an efficacy study. Nevertheless, the dose range for the efficacy study (Part 3) was set at 0.1–0.9 mg / mL.

[0572] Part 3: Evaluating efficacy using ZTX-ONCOLEADS The antitumor efficacy of the huCE7-V7AG and HUCE7-V7AG-(MMAE)4 antibodies was evaluated using MAXFTN-401 cancer cells. The study consisted of five experimental groups (Table E26). The antibody concentration to be tested was determined based on the results of toxicity assessment. Tumor cells were implanted subcutaneously, and antibody treatment was administered intravenously after tumor implantation. Twenty tumor-bearing embryos were included per experimental group and incubated at 35.5°C for three days. The antitumor efficacy of the antibodies was determined by the change in primary tumor size (i.e., tumor growth or shrinkage) and the number of tumor cells seeded into the CVP three days after implantation.

[0573] [Table 26]

[0574] Detailed results and description of the study are provided in Example 28.

[0575] Example 21 - Expression and purification of liability mutant LV29-32 cDNAs encoding the HC and LC sequences were chemically synthesized with optimization for expression in CHO cells and subcloned into a proprietary mammalian cell expression vector from ProteoGenix. The sequences are shown below.

[0576] >CE7-1-2-VHB-WH33Y-GH55A [SEQ ID NO: 137]

[0577] >CE7-1-2-VHB-WH33F-GH55A [SEQ ID NO: 138]

[0578] >CE7-1-NL1-VLA-NL31A-WL92Y [Accession No. 139] GAATTCGCCGCCACCATGGTGCTGCAGACCCAGGTGTTTATCAGCCTGCTGCTGTGGATCTCCGGCGCTTATGGCGATATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCTTCTGTGGGAGATAGGGTGACCATCACCTGTAAGGCTAATGAGGATATCAACGCTCGGCTGGCCTGGTACCAGCAGAAGCCTGGAAAGGCTCCTAAGCTGCTGATCTCCGGCGCCACAAACCTGGTGACCGGAGTTCCTTCCCGGTTCTCCGGAAGCGGATCCGGAAAGGACTATACCCTGACCATCTCCAGCCTGCAGCCCGAAGATTTCGCTACCTATTACTGCCAGCAGTATTATAGCACCCCCTTCACCTTCGGCCAGGGCACCAAACTGGAGATCAAGCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0579] >CE7-1-NL1-VLA-NL31A-WL92F [Accession No. 140] GAATTCGCCGCCACCATGGTGCTGCAGACCCAGGTGTTTATCAGCCTGCTGCTGTGGATCTCCGGCGCTTATGGCGATATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCTTCTGTGGGAGATAGGGTGACCATCACCTGTAAGGCTAATGAGGATATCAACGCTCGGCTGGCCTGGTACCAGCAGAAGCCTGGAAAGGCTCCTAAGCTGCTGATCTCCGGCGCCACAAACCTGGTGACCGGAGTTCCTTCCCGGTTCTCCGGAAGCGGATCCGGAAAGGACTATACCCTGACCATCTCCAGCCTGCAGCCCGAAGATTTCGCTACCTATTACTGCCAGCAGTATTTCAGCACCCCCTTCACCTTCGGCCAGGGCACCAAACTGGAGATCAAGCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0580] The sequence of the predicted protein produced is shown below. >CE7-1-2-VHB-WH33Y-GH55A(L234A L235A+P331S+N297A)[SEQ ID NO:141] MKHLWFLLLVAAPRWVLSQVQLVQLGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWIGEINPSNARTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0581] >CE7-1-2-VHB-WH33F-GH55A (L234A L235A+P331S+N297A) MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYFMHWVRQAPGQGLEWIGEINPSNARTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCAR DYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0582] >CE7-1-NL1-VLA-NL31A-WL92Y [SEQ ID NO: 143] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKANEDINARLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYYSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0583] >CE7-1-NL1-VLA-NL31A-WL92F [SEQ ID NO: 144] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKANEDINARLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYFSTPFT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0584] Production and purification Short Protocol Description Endotoxin-free DNA preparations were performed on the constructs obtained as described herein above.

[0585] Four antibodies were expressed by combining HC and LC as follows:

[0586] [Table 27]

[0587] The plasmids were transiently co-transfected into our proprietary Xten CHO cells using a proprietary Xten transfection protocol. Culture medium was collected when viability fell below 50% (14 days after transfection) and purified as follows. -Clarification by 0.22μm filtration -Equilibration, binding, and washing with PBS (pH 7.5) -Elution by pH shift using citric acid -Neutralization with 1M Tris-HCl (pH 9.0) -Analysis by PAGE and pooling of the desired fractions - Final QC by PAGE: qualitative and quantitative by SDS-PAGE

[0588] The eluted fractions were pooled and buffer exchanged against PBS (pH 7.5) by dialysis. The final sample was filtered through a 0.22 μm Millipore filter and assayed for endotoxin levels using a chromogenic LAL endotoxin assay kit according to the IFU (Genscript Toxinsensor kit #L00350). Purification test results and QC are shown in Figures 14 and 15.

[0589] The yields and purities obtained are summarized in Table E28.

[0590] [Table 28]

[0591] Example 22 - SEC-HPLC study of liability mutants Preparation of buffer solutions Mobile phase: 100 mM sodium sulfate, 100 mM phosphate, buffer (pH 6.7) (Na2SO4 14.2 g / L, Na2HPO4·12 H2O 15.6 g / L, NaH2PO4 6.8 g / L) Remove air bubbles from all buffers.

[0592] Sample preparation - Protein preparation: Dilution of protein samples to 1 mg / ml with DD water, starting from one extra aliquot of each final sample stored overnight at -80°C - Centrifuge at 12000g for 10 minutes and collect the supernatant

[0593] Analysis conditions -Blank control: Dilution of sample with DD water (5 times) -Column: G3000SWXL, TOSOH, 7.8 x 300 mm -HPLC:Waters 2695 -Elution gradient: Mobile phase isocratic elution -Flow rate: 1.0ml / min -Temperature: 25℃ -Injection volume: 30μl -Detection wavelength: 280nm -Collection date and time: 25 minutes - Equilibrate with 100% mobile phase for 5 minutes - Running samples: one blank control injection, then sample injection -Data analysis The results are shown in Table E29 and FIG.

[0594] [Table 29]

[0595] SEC-HPLC analysis shows that all Liability antibody variants exhibit very low aggregates of <5%.

[0596] Example 23 - PANTA studies of protein stability and aggregation Temperature-induced aggregation of target proteins was measured by monitoring sample turbidity changes using a Prometheus PANTA backreflected light. Because protein aggregation is usually a non-stimulative and not well-defined process, automated analysis of the turbidity curves determined only the so-called macroaggregation onset temperature (turbidity ON), defined by the region where a significant and continuous increase in turbidity signal was first observed.

[0597] For turbidity signals, fluorescence measurements are not possible; instead, reflection and scattering of excitation light are measured. Therefore, turbidity detection is not as sensitive as detecting thermal denaturation by fluorescence, especially at low target protein concentrations. This often results in broader, less pronounced peaks in the derivative curve. Furthermore, replicate measurements often differ by several degrees Celsius in the turbidity ON time because protein aggregation is often affected by the accumulation of local aggregation nuclei, which can vary between replicates.

[0598] In Prometheus Panta DLS, the sample is packed into a thin glass capillary. A low-wavelength laser light (λ = 405 nm) is focused onto the sample and backscattered by the particles in the sample. The scattered light intensity fluctuations over time are recorded. The intensity fluctuations are analyzed using an autocorrelation function, from which the diffusion coefficient is extracted by a cumulant or regularized fit. By applying the diffusion coefficient to the Stokes-Einstein equation, the hydrodynamic radius (r) of all particles in the solution is calculated. h ) is determined. The size distribution plot shows the radii of all particle populations and the relative proportions of these species. The larger species are more intense (10 6 ) scatters light, and therefore the intensity distribution is not a direct representation of the amount of different size species in the sample.

[0599] Details of the method are given in Table E30.

[0600] [Table 30]

[0601] The results of the measurements are shown in Table E31.

[0602] [Table 31-1]

[0603] [Table 31-2]

[0604] [Table 31-3]

[0605] T m: Melting temperature of a protein. This value is a thermodynamic parameter of a protein that can be rigorously determined only under thermodynamic equilibrium conditions and with well-known baseline parameters for the necessary thermodynamic fit. However, thermal unfolding experiments are often not at equilibrium because irreversible protein aggregation processes continue to remove unfolded protein from the reaction, making the unfolding process irreversible. In addition, heating rates faster than 1 °C / min reduce the equilibrium-like nature of the unfolding reaction.

[0606] Unfolding ON: Onset of thermal melting. This is the temperature at which thermal protein unfolding begins. It is determined from the first derivative curve of the thermal unfolding signal (350 / 330 nm ratio signal).

[0607] IP: Inflection point. Due to the above constraints, in nanoDSF, the thermal melting temperature of a protein is determined directly from the measured data by using a smoothing algorithm (polynomial fit) and detecting inflection points in the data. Assuming equilibrium, the determined IPs are the T of the protein. m Corresponds to the value.

[0608] Turbidity ON: Onset of protein macroaggregation. This is the temperature at which a protein sample begins to aggregate. It is determined from the first derivative curve of the turbidity signal. Typically, turbidity ON correlates with unfolding ON. For proteins that show more than one unfolding event, aggregation may begin due to the first unfolding event or a subsequent unfolding event.

[0609] summary: High sample uniformity was observed by DLS for all samples before and after incubation at 40°C. High thermal stability accompanied by a characteristic antibody unfolding profile was observed for samples measured using thermal gradient DLS + nanoDSF.

[0610] Example 24: In silico modeling of APR, design of APR liability mutants Here, the modelling of aggregation prone regions was carried out using the software Solubis and using the TANGO algorithm.

[0611] For modeling of the antibody wild type (cHCE7) structure, YASARA Structure: version 22.8.22 was used. Subsequently, the following software versions were used to identify suitable mutations:

[0612] To calculate the effect on thermodynamic stability, FoldX: version 3.0 beta 6 was used.

[0613] TANGO: version 2.2 was used to calculate the effect on aggregation tendency.

[0614] In the case of Solubis (which is essentially a combination of FoldX and TANGO to allow mutations in aggregation prone regions), the version was 1.0.

[0615] Further information about the program can be found here: https: / / switchlab.org / software (assessed November 7, 2022).

[0616] Table E32 presents the results of modeling using Solubis software based on the sequence of heavy chain LV31 (as in SEQ ID NO: 141).

[0617] [Table 32]

[0618] Table E33 shows the results of modelling using Solubis software based on the sequence of heavy chain LV32 (sequence as found in SEQ ID NO: 142).

[0619] [Table 33]

[0620] Table E34 shows the results of modeling using Solubis software based on the sequence of heavy chain chCE7 variant 7 (i.e., comprising the sequence obtained by linking the C-terminus of the sequence set forth in SEQ ID NO: 28 to the N-terminus of the sequence set forth in SEQ ID NO: 145).

[0621] [Table 34]

[0622] Table E35 presents the results of calculations using TANGO software for different mutations in the sequence of heavy chain LV31 (sequence as found in SEQ ID NO: 141), the sequence of heavy chain LV32 (sequence as found in SEQ ID NO: 142), and chCE7 variant 7 (i.e., comprising the sequence obtained by linking the C-terminus of the sequence set forth in SEQ ID NO: 28 to the N-terminus of the sequence set forth in SEQ ID NO: 145).

[0623] [Table 35-1]

[0624] Example 25: Production and in vitro characterization of huCE7 variants LV33-LV42 To implement the data for in silico modeling presented in Example 24, further mutants were generated and characterized. huCE7 mutants LV33-LV42 are based on the α-glycosylated huCE7 mutant 7AG with the following additional point mutations in the heavy chain: The generated mutants are summarized in Table E35.

[0625] [Table 35-2]

[0626] The sequences summarized in the table above are also provided below. Heavy chain of mutant _7_AG_L234A_L235A_P331S_N297A_+TH30P;WH33F;GH55A (also called CE7-1-2-VHB N297A(TH30P;WH33F;GH55A)): SEQ ID NO: 155 [TH30P, WH33F, GH55A] MKHLWFFLLL VAAPRWVLSQ VQLVQSGAEV KKPGASVKVS CKASGYTFPG YFMHWVRQAP GQGLEWIGEI NPSNARTNYN ERFQGRVTLT VDKSISTAYM ELSRLRSDDT AVYYCARDYY GTSYNFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYAS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PASIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0627] Heavy chain of mutant _7_AG_L234A_L235A_P331S_N297A_+WH33F, AH40R, GH55A (also called CE7-1-2-VHB N297A(WH33F, AH40R, GH55A)): SEQ ID NO: 156 [WH33F, AH40R, GH55A] MKHLWFFLLL VAAPRWVLSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTG YFMHWVRQRP GQGLEWIGEI NPSNARTNYN ERFQGRVTLT VDKSISTAYM ELSRLRSDDT AVYYCARDYY GTSYNFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYAS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PASIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0628] Heavy chain of mutant _7_AG_L234A_L235A_P331S_N297A_+WH33F, GH55A, VH68A (also called CE7-1-2-VHB N297A_(WH33F, GH55A, VH68A)): SEQ ID NO: 157 [WH33F, GH55A, VH68A] MKHLWFFLLL VAAPRWVLSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTG YFMHWVRQAP GQGLEWIGEI NPSNARTNYN ERFQGRATLT VDKSISTAYM ELSRLRSDDT AVYYCARDYY GTSYNFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYAS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PASIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0629] Heavy chain of mutant _7_AG_L234A_L235A_P331S_N297A_+WH33F GH55A, LH115T (also called CE7-1-2-VHB N297A(WH33F GH55A LH115T)): SEQ ID NO: 158 [WH33F, GH55A, LH115T] MKHLWFFLLL VAAPRWVLSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTG YFMHWVRQAP GQGLEWIGEI NPSNARTNYN ERFQGRVTLT VDKSISTAYM ELSRLRSDDT AVYYCARDYY GTSYNFDYWG QGTTVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYAS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PASIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0630] Heavy chain of mutant _7_AG_L234A_L235A_P331S_N297A_+WH33F, GH55A, FH241K (also called CE7-1-2-VHB N297A(WH33F, GH55A, FH241K)): SEQ ID NO: 159 [WH33F, GH55A, FH241K] MKHLWFFLLL VAAPRWVLSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTG YFMHWVRQAP GQGLEWIGEI NPSNARTNYN ERFQGRVTLT VDKSISTAYM ELSRLRSDDT AVYYCARDYY GTSYNFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPEAAGGP SVKLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYAS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PASIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0631] Heavy chain of mutant _7_AG_L234A_L235A_P331S_N297A_+WH33F, GH55A, LH309D (also called CE7-1-2-VHB N297A_(WH33F, GH55A, LH309D)): SEQ ID NO: 160 [WH33F, GH55A, LH309D] MKHLWFFLLL VAAPRWVLSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTG YFMHWVRQAP GQGLEWIGEI NPSNARTNYN ERFQGRVTLT VDKSISTAYM ELSRLRSDDT AVYYCARDYY GTSYNFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYAS TYRVVSVLTV DHQDWLNGKE YKCKVSNKAL PASIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0632] Heavy chain of mutant _7_AG_L234A_L235A_P331S_N297A_+WH33F,GH55A,TH307R (also called CE7-1-2-VHB N297A(WH33F,GH55A,TH307R)): SEQ ID NO: 161 [WH33F, GH55A, TH307R] MKHLWFFLLL VAAPRWVLSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTG YFMHWVRQAP GQGLEWIGEI NPSNARTNYN ERFQGRVTLT VDKSISTAYM ELSRLRSDDT AVYYCARDYY GTSYNFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYAS TYRVVSVLRV LHQDWLNGKE YKCKVSNKAL PASIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0633] Heavy chain of mutant _7_AG_L234A_L235A_P331S_N297A_+WH33F,GH55A,TH307P (also called CE7-1-2-VHB N297A(WH33F,GH55A,TH307P)): SEQ ID NO: 162 [WH33F, GH55A, TH307P] MKHLWFFLLL VAAPRWVLSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTG YFMHWVRQAP GQGLEWIGEI NPSNARTNYN ERFQGRVTLT VDKSISTAYM ELSRLRSDDT AVYYCARDYY GTSYNFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYAS TYRVVSVLPV LHQDWLNGKE YKCKVSNKAL PASIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0634] Heavy chain of mutant _7_AG_L234A_L235A_P331S_N297A_+TH307P (also called CE7-1-2-VHB N297A(TH307P)): SEQ ID NO: 163 [TH307P] MKHLWFFLLL VAAPRWVLSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTG YWMHWVRQAP GQGLEWIGEI NPSNGRTNYN ERFQGRVTLT VDKSISTAYM ELSRLRSDDT AVYYCARDYY GTSYNFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYAS TYRVVSVLPV LHQDWLNGKE YKCKVSNKAL PASIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

[0635] The above-mentioned mutants are also provided in their non-α-glycos...

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to L1-CAM (CD171), wherein the antibody or the antigen-binding fragment thereof is: A variable heavy chain region comprising: CDR-H1, The sequence set forth in SEQ ID NO: 1 (GYWMH), the sequence set forth in SEQ ID NO: 2 (GYYMH); The sequence set forth in SEQ ID NO: 3 (GYFMH), and CDR-H1 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 4 (GYLMH), and CDR-H2, The sequence set forth in SEQ ID NO: 5 (EINPSNGRTNYNERFQG), The sequence set forth in SEQ ID NO: 6 (EINPSNGRTNYNEKFQG), The sequence set forth in SEQ ID NO: 7 (EINPSNGRTNYNERFKS), The sequence set forth in SEQ ID NO: 8 (EINPSNGRTNYNERLKS), The sequence set forth in SEQ ID NO: 9 (EINPSNAARTNYNERFQG), Sequence set forth in SEQ ID NO: 10 (EINPSNAARTNYNEKFQG) The sequence set forth in SEQ ID NO: 11 (EINPSNAARTNYNERFKS), and CDR-H2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 12 (EINPSNAARTNYNERLKS), and a variable heavy chain region comprising a CDR-H3 characterized by the sequence set forth in SEQ ID NO: 13 (DYYGTSYNFDY); and A variable light chain region comprising: CDR-L1, The sequence set forth in SEQ ID NO: 14 (RANEDINNRLA), The sequence set forth in SEQ ID NO: 15 (KANEDINNRLA), The sequence set forth in SEQ ID NO: 16 (QANEDINNRLA), the sequence set forth in SEQ ID NO: 17 (RANEDINARLA); The sequence set forth in SEQ ID NO: 18 (KANEDINARLA), The sequence set forth in SEQ ID NO: 19 (QANEDINARLA), The sequence set forth in SEQ ID NO: 20 (RANEDINLRLA), The sequence set forth in SEQ ID NO: 21 (KANEDINLRLA), and CDR-L1 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 22 (QANEDINLRLA), and CDR-L2, The sequence set forth in SEQ ID NO: 23 (GATNLVT), and a CDR-L2 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 24 (GASNLVS), and CDR-L3, The sequence set forth in SEQ ID NO: 25 (QQYWSTPFT), The sequence set forth in SEQ ID NO: 26 (QQYYSTPFT), and and a variable light chain region comprising a CDR-L3 characterized by a sequence selected from the sequence set forth in SEQ ID NO: 27 (QQYFSTPFT).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a recombinant antibody, an antigen-binding fragment of a recombinant antibody, a single-chain antibody, a humanized antibody, a bispecific antibody, a multispecific antibody, or an antibody displayed on the surface of a phage or on the surface of a chimeric antigen receptor (CAR) T cell.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof is an IgG1 antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the variable heavy chain region comprises a CDR-H1 characterized by the sequence set forth in SEQ ID NO:

1.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the variable heavy chain region comprises a CDR-H2 characterized by the sequence set forth in SEQ ID NO: 5, 6, 9, or 10.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the variable heavy chain region comprises a CDR-H2 characterized by the sequence set forth in SEQ ID NO: 5 or 6.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the variable light chain region comprises a CDR-L1 characterized by the sequence set forth in SEQ ID NO: 14, 15, 17, 18, or 21.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the variable light chain region comprises a CDR-L1 characterized by the sequence set forth in SEQ ID NO: 14 or 15.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the variable light chain comprises a CDR-L2 characterized by the sequence set forth in SEQ ID NO:

23.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the variable light chain comprises a CDR-L3 characterized by the sequence set forth in SEQ ID NO:

25.

12. the variable heavy chain region Sequence set forth in SEQ ID NO: 28 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), Sequence set forth in SEQ ID NO: 29 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTVSS), Sequence set forth in SEQ ID NO: 30 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFTGYWMHWIRQPPGKGLEWIGEINPSNGRTNYNERLKSRVTLSVDKSKNQASLKLSSVTAADTAVYFCARDYYGTSYNFDYWGQGTLVTVSS), The sequence set forth in SEQ ID NO: 31 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), and The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical to a sequence selected from the sequence set forth in SEQ ID NO: 32 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWmGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS).

13. 13. The antibody or antigen-binding fragment thereof of claim 12, wherein the variable heavy chain region is characterized by the sequence set forth in SEQ ID NO: 28 or the sequence set forth in SEQ ID NO:

32.

14. the variable light chain region The sequence set forth in SEQ ID NO: 33 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), Sequence of SEQ ID NO: 34 DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDIATYYCQQYWSTPFTFGQGTKLEIK), Sequence of SEQ ID NO: 35 EIVMTQSPATTLSVSPGERATLSCRANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEFTLTISSLQSEDFAVYYCQQYWSTPFTFGQGTKLEIK), The sequence set forth in SEQ ID NO: 36 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), and The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical to a sequence selected from the sequence set forth in SEQ ID NO: 37 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGAsNLVsGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK).

15. 15. The antibody or antigen-binding fragment thereof of claim 14, wherein the variable light chain region is characterized by a sequence selected from the sequence set forth in SEQ ID NO: 33, the sequence set forth in SEQ ID NO: 36, and the sequence set forth in SEQ ID NO:

37.

16. the variable heavy chain region is characterized by the sequence set forth in SEQ ID NO: 28 and the variable light chain region is characterized by the sequence set forth in SEQ ID NO: 33; or the variable heavy chain region is characterized by the sequence set forth in SEQ ID NO: 32 and the variable light chain region is characterized by the sequence set forth in SEQ ID NO: 36; or 16. The antibody or antigen-binding fragment thereof of any one of claims 1 to 15, wherein the variable heavy chain region is characterized by the sequence set forth in SEQ ID NO: 14 and the variable light chain region is characterized by the sequence set forth in SEQ ID NO:

37.

17. The variable heavy chain region has the sequence: FH0-CDR-H1-FH1-CDR-H2-FH2-CDR-H3-FH3 wherein CDR-H1, CDR-H2, and CDR-H3 are as defined in claim 1; FH0 is characterized by the sequence set forth in SEQ ID NO: 38 (QVQLVQSGAEVKKPGASVKVSCKASGYTFT) or the sequence set forth in SEQ ID NO: 39 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFT), FH1 is characterized by the sequence set forth in SEQ ID NO: 40 (WVRQAPGQGLEWIG) or the sequence set forth in SEQ ID NO: 41 (WIRQPPGKGLEWIG), FH2 is characterized by the sequence set forth in SEQ ID NO: 42 (RVTLTVDKSISTAYMELSRLRSDDTAVYFCAR) or the sequence set forth in SEQ ID NO: 43 (RVTLSVDKSKNQASLKLSSVTAADTAVYFCAR), FH3 is characterized by a sequence at least 90% identical, preferably at least 95% identical, more preferably identical to the sequence set forth in SEQ ID NO: 44 (WGQGTLVTVSS), and The variable light chain region has the sequence: FL0-CDR-L1-FL1-CDR-L2-FL2-CDR-L3-FL3 wherein CDR-L1, CDR-L2, and CDR-L3 are as defined in claim 1; FL0 is characterized by the sequence set forth in SEQ ID NO: 45 (DIQMTQSPSSLSASVGDRVTITC) or the sequence set forth in SEQ ID NO: 46 (EIVMTQSPATTLSVSPGERATLSC), FL1 is characterized by the sequence set forth in SEQ ID NO: 47 (WYQQKPGKAPKLLIS) or the sequence set forth in SEQ ID NO: 48 (WYQQKPGQAPRLLIS), FL2 is characterized by the sequence set forth in SEQ ID NO: 49 (GVPSRFSGSGSGKDYTLTISSLQPEDIATYYC) or the sequence set forth in SEQ ID NO: 50 (GIPARFSGSGSGKEFTLTISSLQSEDFAVYYC), 5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein FL3 is characterized by a sequence at least 90% identical, preferably at least 95% identical, more preferably identical to the sequence set forth in SEQ ID NO: 51 (FGQGTKLEIK).

18. the heavy chain of the antibody or antigen-binding fragment thereof comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, still more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 110, 142, 155-163, preferably a sequence identical to the sequence set forth in SEQ ID NO: 110, 142, 155, 160, or 162; and The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the light chain of the antibody or antigen-binding fragment thereof comprises a sequence that is at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and still more preferably identical to the sequence set forth in SEQ ID NO: 97 or 143.

19. 5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably identical to the sequence set forth in SEQ ID NO: 155, and the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, still more preferably identical to the sequence set forth in SEQ ID NO:

143.

20. the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, still more preferably identical to a sequence set forth in SEQ ID NOs: 110, 142, 155-163; and 5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and still more preferably identical to the sequence set forth in SEQ ID NO: 97 or 143.

21. the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 95 or 164-172; and 5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and still more preferably identical to the sequence set forth in SEQ ID NO: 97 or 143.

22. the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 173-183; and 5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and still more preferably identical to the sequence set forth in SEQ ID NO:

193.

23. the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 173-183; and 5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and still more preferably identical to the sequence set forth in SEQ ID NO:

194.

24. the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 184-192; and 5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and still more preferably identical to the sequence set forth in SEQ ID NO:

193.

25. the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and even more preferably identical to a sequence selected from the sequences set forth in SEQ ID NOs: 184-192; and 5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, and still more preferably identical to the sequence set forth in SEQ ID NO:

194.

26. 5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the heavy chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably identical to the sequence set forth in SEQ ID NO: 175, and the light chain comprises a sequence at least 90% identical, preferably at least 95% identical, more preferably at least 98% identical, even more preferably at least 99% identical, still more preferably identical to the sequence set forth in SEQ ID NO:

194.

27. 27. The antibody or antigen-binding fragment thereof of any one of claims 1 to 26, wherein the heavy chain further comprises at least one point mutation in the Fc portion that affects antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), serum half-life, and / or glycosylation status of the antibody.

28. 28. The antibody or antigen-binding fragment thereof of claim 27, wherein the at least one point mutation is selected from L234A, L234F, L235A, L235E, L235Q, G236A, M252Y, S254T, T256E, S267E, H268F, N297A, K322A, K322Q, S324T, P331S, and I332E, more preferably, the at least one point mutation is selected from L234A, L235A, P331S, and N297A.

29. 10 as measured by a Biacore-based assay －１１ The dissociation constant K for L1-CAM (CD171) does not exceed M Ｄ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, characterized in that:

30. A polynucleotide encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence according to any one of claims 1 to 29.

31. A host cell comprising the polynucleotide of claim 30.

32. 30. An immunoconjugate comprising the antibody or antigen fragment thereof bound thereto according to any one of claims 1 to 29 and an active agent, preferably wherein said active agent is a cytotoxic agent or a prodrug thereof.

33. 33. The immunoconjugate of claim 32, wherein the antibody or antigen-binding fragment thereof is linked to the active agent via a linker moiety, preferably the linker moiety comprises a polymeric carrier to which at least one active agent is attached.

34. 34. The immunoconjugate of claim 32 or 33, wherein the active agent is a radionuclide preferably selected from copper-67, strontium-89, yttrium-90, iodine-131, samarium-153, terbium-161, lutetium-177, astatine-211, radium-223, and actinium-225, preferably a radionuclide useful for therapeutic applications, and / or a radionuclide preferably useful for diagnostics selected from fluorine-18, scandium-43, scandium-44, copper-61, copper-64, gallium-68, zirconium-89, indium-111, iodine-123, terbium-152, and terbium-155.

35. 34. The immunoconjugate of claim 32 or 33, wherein the active agent is selected from maytansinoids, calicheamicins, pyrrolobenzodiazepines (PBDs), nemorubicin and its derivatives, PNU-159682, anthracyclines, duocarmycins, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecins, efinafide, exatecan, deruxtecan, topotecan, irinotecan, SN38, and belotecan.

36. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, or the immunoconjugate according to any one of claims 32 to 35, and a pharmaceutically acceptable carrier.

37. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, or an immune complex according to any one of claims 32 to 35, for use as a medicament.

38. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, or an immunoconjugate according to any one of claims 32 to 35, for use in the treatment of L1-CAM (CD171)-associated cancer.

39. The L1-CAM (CD171)-associated cancer is leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, quadruple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, cholangiocarcinoma, carcinoid, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, pancreatic neuroectodermal carcinoma, 39. The antibody or antigen-binding fragment thereof for use according to claim 38, or the immunoconjugate for use according to claim 38, wherein the antibody or antigen-binding fragment thereof is selected from: pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma, tumor vasculature, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine carcinoma, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granular cell tumor, Schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, prostate cancer, and head and neck squamous cell carcinoma.

40. 40. The antibody or antigen-binding fragment thereof for use according to claim 38 or 39, or the immunoconjugate for use according to claim 38 or 39, wherein the antibody or antigen-binding fragment thereof, or the immunoconjugate is to be administered to a subject in conjunction with an additional therapeutic agent selected from an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen, an aromatase inhibitor, an ovarian function suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, an immune checkpoint inhibitor, and a bisphosphonate therapy.

41. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, or an immunoconjugate according to any one of claims 32 to 35, for use in diagnosis.

42. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 29, or an immunoconjugate according to any one of claims 32 to 35, for use in diagnosing L1-CAM (CD171)-associated cancer.

43. The L1-CAM (CD171)-associated cancer is leukemia, Ewing's sarcoma, neuroblastoma, osteosarcoma, glioblastoma multiforme, ovarian cancer, endometrial cancer, uterine cancer, triple-negative breast cancer, quadruple-negative breast cancer, melanoma, clear cell renal cell carcinoma, pheochromocytoma and paraganglioma, mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer, NSCLC, pancreatic ductal carcinoma, colon cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, cholangiocarcinoma, carcinoid, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, pancreatic neuroectodermal tumor, 43. The antibody or antigen-binding fragment thereof for use according to claim 42, or the immunoconjugate for use according to claim 42, wherein the cancer is selected from: pancreatic adenocarcinoma, colorectal cancer, renal cell carcinoma, tumor vasculature, chondrosarcoma, esophageal adenocarcinoma, oligodendroglioma, astrocytoma, ependymoma, pancreatic neuroendocrine carcinoma, adrenal adenoma, leiomyosarcoma, liposarcoma, ovarian granular cell tumor, Schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular salivary gland cancer, prostate cancer, and head and neck squamous cell carcinoma.

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