Anti-L1-CAM antibodies and their use in diagnostic and therapeutic applications

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds L1-CAM (CD171), a polynucleotide encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence as in the antibody of the invention or antigen-binding fragment thereof, a host cell comprising the polynucleotide of the invention, and a method for preparing the same. The present invention relates to antibodies or antigen-binding fragments thereof of the invention, immunoconjugates comprising the antibodies or antigen-binding fragments thereof of the invention and an active agent, pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof of the invention 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 in the treatment or diagnosis of L1-CAM (CD171) associated cancers.

Description

Technical Field

[0001] The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to L1-CAM (CD171), a polynucleotide encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence as in the antibody or antigen-binding fragment of the present invention, a host cell comprising the polynucleotide of the present invention, an immunoconjugate comprising the antibody or antigen-binding fragment of the present invention and an active agent, a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present invention or the immunoconjugate of the present invention, and their use in treatment and / or diagnosis. The antibodies, antigen-binding fragments, immunoconjugates and pharmaceutical compositions described herein are particularly useful in the treatment or diagnosis of L1-CAM (CD171)-related cancers. Background Art

[0002] The emergence of chimeric antigen receptor (CAR) technology (Sadelain M et al., Cancer Discov 3:388-98 (2013)) has rapidly expanded the therapeutic research of L1-CAM redirected gene-modified T cells, and there are currently several clinical trials using L1-CAM-CAR modified T cells: NCT00889954 (all L1-CAM(+) cancers), NCT01935843 (L1-CAM(+) solid tumors). See Hong H et al., J Immunother 37:93-104 (2014). Although T cells can effectively target tumors with low levels of L1-CAM, there are concerns about potential indirect toxicity in normal tissues with lower L1-CAM expression levels.

[0003] In addition to toxicity issues, the regulations regarding cell harvesting, processing, storage, transportation and product release involved in lymphocyte therapy can be challenging, especially when the cells have to undergo genetic modification. Despite the infusion of billions of T cells, the depletion, survival and homing of these cells are not ideal. In addition, CAR-modified T cells are no exception in the immunosuppressive tumor microenvironment, where Treg cells, tumor-associated macrophages and myeloid-derived suppressor cells work together to block the anti-tumor properties of CAR-modified T cells. In addition, CAR-modified T cells are subject to the same immunosuppressive limitations as classical T cells, including unresponsiveness to tumor cells after engagement of CTLA4 by B7 or PD-1 by PD-L1 (B7-H1). Therefore, clinically effective alternatives to CAR-modified T cell therapy for treating cancer are needed.

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

[0005] Doberstein et al. have shown that L1 CAM is expressed in triple-negative breast cancer and is negatively correlated with androgen receptor (Doberstein et al., BMC Cancer 2014, 14:958). Eliane Fischer et al. described antibody therapy targeting L1-CAM and 177 Lu radioimmunotherapy for diffuse ovarian cancer (E Fischer et al., Int.J.Cancer: 130, 2715-2721 (2012)). According to Wachowiak and colleagues, elevated L1-CAM levels are associated with glioblastoma and metastatic brain tumors (Wachowiak et al.; Medicine (2018) 97:38). Rached et al. have shown that L1-CAM gene knockout increases PTEN protein expression while decreasing MycN protein expression, making neuroblastoma IMR-32 cells more sensitive to radiotherapy (Rached et al., International; Journal of Oncology, 49:1722-1730, 2016). L1-CAM has also been shown to increase the proliferation and chemoresistance of adhesion-mediated retinoblastoma (Jo et al., Oncotarget, 2017, Vol. 8, pp. 15441-15452). Terraneo et al. have shown that L1-CAM renders ovarian cancer radioresistant and defines a new cancer stem cell population (Terraneo et al., Cancers 2020, 12, 217). Expression profiling in multiple human tumors has 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 the use of the anti-L1-CAM antibody mAb chCE7 to target neuroblastoma. Therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients were disclosed. The same antibody chCE7 has been shown to bind to an L1-CAM isoform 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 the radioimmunolocalization of neuroblastoma xenografts using the chimeric antibody chCE7. L1-CAM has also been shown to identify the regenerative origin of metastatic initiating cells in colorectal cancer (Ganesh et al., 2020, 28-45).

[0007] Since L1-CAM is known to be expressed in peripheral nerve tissue, treatment side effects against L1-CAM are common. Specifically, the side effects include ADCC, CDC and other immune responses. Therefore, it is crucial to provide an antibody or its fragment in which the toxic effects are reduced. In the case of radionuclide therapy, it is particularly desirable to provide an antibody with a low K D which is related to the low k-off of the antibody after binding to the tumor, thus avoiding its diffusion into healthy tissues.

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

[0009] Amstutz et al. (Amstutz, Int. J. Cancer: 53, 147-152 (1993)) disclosed the production and characterization of a mouse / human chimeric antibody against human neuroblastoma.

[0010] Umana et al. (Nature Biotechnology, 1999, 176-180) disclosed an engineered glycosylated form of anti-neuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxicity activity.

[0011] The document "Improvement of Biophysical Properties and Affinity of a Human Anti-L1-CAM Therapeutic Antibody through Antibody Engineering Based on Computational Methods", International Journal of Molecular Sciences, Vol. 22, No. 13, p. 6696 describes certain variants of antibody Ab417 (a humanized antibody that binds L1-CAM) with good properties in terms of production stability and anti-tumor activity. Summary of the Invention

[0012] The inventors have surprisingly found that, compared to closely related antibodies, particularly antibodies derived from the CE7 antibody (Amstutz et al.), the antibodies or antigen-binding fragments of the present invention exhibit improved affinity when binding to L1-CAM. Specifically, at least a portion of the antibodies of the present invention are humanized antibodies, and those skilled in the art would expect that humanization of an antibody would have a negative impact on the binding affinity of the antibody to its antigen. Thus, the present invention is at least partially based on an unexpected finding: namely, that compared to the CE7 antibody, the antibodies of the present invention obtained after humanization of the CE7 antibody exhibit surprisingly improved binding affinity for L1-CAM, as confirmed in the SPR studies conducted by the inventors (see Examples 6 and 7 below).

[0013] It is further noted that, according to the inventors, the antibodies or antigen-binding fragments of the present invention exhibit at least comparable (rather than worse) affinity compared to antibodies derived from the CE7 antibody through the humanization process.

[0014] The inventors have further demonstrated that, compared to closely related antibodies, particularly antibodies derived from the CE7 antibody, the antibodies or antigen-binding fragments of the present invention surprisingly exhibit improved stability. The improved stability is at least reflected in a higher aggregation onset temperature, as confirmed by the PANTA study (see Example 23).

[0015] The present invention will be outlined in the following embodiments.

[0016] In a first embodiment, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to L1-CAM (CD171), the antibody or antigen-binding fragment thereof comprising:

[0017] A variable heavy chain region, which comprises:

[0018] CDR-H1 characterized by a sequence selected from the following:

[0019] The sequence according to SEQ ID NO.: 1 (GYWMH),

[0020] The sequence according to SEQ ID NO.: 2 (GYYMH),

[0021] The sequence according to SEQ ID NO.: 3 (GYFMH), and

[0022] The sequence according to SEQ ID NO.: 4 (GYLMH); and

[0023] CDR-H2 characterized by a sequence selected from the following:

[0024] The sequence according to SEQ ID NO.: 5 (EINPSNGRTNYNERFQG),

[0025] The sequence according to SEQ ID NO.: 6 (EINPSNGRTNYNEKFQG),

[0026] The sequence according to SEQ ID NO.: 7 (EINPSNGRTNYNERFKS),

[0027] The sequence according to SEQ ID NO.: 8 (EINPSNGRTNYNERLKS),

[0028] The sequence according to SEQ ID NO.: 9 (EINPSNARTNYNERFQG),

[0029] The sequence according to SEQ ID NO.: 10 (EINPSNARTNYNEKFQG),

[0030] The sequence according to SEQ ID NO.: 11 (EINPSNARTNYNERFKS) and

[0031] The sequence according to SEQ ID NO.: 12 (EINPSNARTNYNERLKS); and

[0032] CDR-H3 characterized by the sequence according to SEQ ID NO.: 13 (DYYGTSYNFDY); and / or

[0033] A variable light chain region, which comprises:

[0034] CDR-L1 characterized by a sequence selected from the following:

[0035] The sequence according to SEQ ID NO.: 14 (RANEDINNRLA),

[0036] The sequence according to SEQ ID NO.: 15 (KANEDINNRLA),

[0037] The sequence according to SEQ ID NO.: 16 (QANEDINNRLA),

[0038] The sequence according to SEQ ID NO.: 17 (RANEDINARLA),

[0039] The sequence according to SEQ ID NO.: 18 (KANEDINARLA),

[0040] The sequence according to SEQ ID NO.: 19 (QANEDINARLA),

[0041] The sequence according to SEQ ID NO.: 20 (RANEDINLRLA),

[0042] The sequence according to SEQ ID NO.: 21 (KANEDINLRLA), and

[0043] The sequence according to SEQ ID NO.: 22 (QANEDINLRLA); and

[0044] CDR-L2 characterized by a sequence selected from the following:

[0045] The sequence according to SEQ ID NO.: 23 (GATNLVT) and

[0046] The sequence according to SEQ ID NO.: 24 (GASNLVS); and

[0047] CDR-L3 characterized by a sequence selected from the following:

[0048] The sequence according to SEQ ID NO.: 25 (QQYWSTPFT),

[0049] The sequence according to SEQ ID NO.: 26 (QQYYSTPFT) and

[0050] The sequence according to SEQ ID NO.: 27 (QQYFSTPFT).

[0051] In a second embodiment, the present invention relates to a polynucleotide that encodes at least one variable heavy chain sequence and / or at least one variable light chain sequence as described in the first embodiment.

[0052] In a third embodiment, the present invention relates to a host cell comprising the polynucleotide of the second embodiment.

[0053] In a fourth embodiment, the present invention relates to an immunoconjugate comprising the antibody of the first embodiment or an antigen-binding fragment thereof and an active agent, preferably, wherein the active agent is a cytotoxic agent or a prodrug thereof.

[0054] In a fifth embodiment, the present invention relates to a pharmaceutical composition comprising the antibody of the first embodiment or an antigen-binding fragment thereof, or the immunoconjugate of the fourth embodiment, and a pharmaceutically acceptable carrier.

[0055] In a sixth embodiment, the present invention relates to the antibody of the first embodiment or an antigen-binding fragment thereof or the immunoconjugate of the fourth embodiment for use as a medicament.

[0056] In a seventh embodiment, the present invention relates to the antibody of the first embodiment or an antigen-binding fragment thereof or the immunoconjugate of the fourth embodiment for the treatment of L1-CAM (CD171)-related cancer.

[0057] In an eighth embodiment, the present invention relates to the antibody of the first embodiment or an antigen-binding fragment thereof or the immunoconjugate of the fourth embodiment for use in diagnosis.

[0058] In a ninth embodiment, the present invention relates to the antibody of the first embodiment or an antigen-binding fragment thereof or the immunoconjugate of the fourth embodiment for the diagnosis of L1-CAM (CD171)-related cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The present invention is further described in the following drawings, which should not be construed as limiting.

[0060] Figure 1 presents the purification profiles of the rAbs (i.e., variants 1 to 18) as in Example 2. Non-reducing PAGE stained with Coomassie blue. MW: molecular weight marker. IN: input. FT: flow-through. W: wash. E: elution fraction.

[0061] Figure 2 shows Figure 2 presents the final sample QC of Example 2. Coomassie blue staining. Reduced and non-reduced PAGE analysis. MW: molecular weight marker. 2 μg was loaded per lane.

[0062] Figure 3 shows Figure 3 presents the purification profiles of the rAbs (i.e., variants 19 to 28) as in Example 3. Non-reducing PAGE stained with Coomassie blue. MW: molecular weight marker. IN: input. FT: flow-through. W: wash. E: elution fraction.

[0063] Figure 4 shows The final sample QC in Example 3 was presented. Coomassie blue staining. Simplified and non-simplified PAGE analysis. MW: molecular weight marker. 2 μg was loaded per lane.

[0064] Figure 5 shows The purification profile of rAb as in Example 4 was presented. Non-reducing PAGE stained with Coomassie blue. MW: molecular weight marker. IN: input. FT: flow-through. W: wash. E: elution fraction.

[0065] Figure 6 shows The final sample QC in Example 4 was presented. Coomassie blue staining. Simplified and non-simplified PAGE analysis. MW: molecular weight marker. 2 μg was loaded per lane.

[0066] Figure 7 presents the SEC-HPLC profiles obtained for CE7 variant 4_AG (part 1), CE7 variant 7_AG (part 2), CE7 variant 18_AG (part 3), the applied standard (part 4), and the blank sample (part 5).

[0067] Figure 8 presents the blank-subtracted sensorgrams of the binding of CE7 variant 4-AG (part 1), CE7 variant 7-AG (part 2), CE7 variant 18-AG (part 3), chimeric HCE7 (part 4), and deglycosylated cHCE7 (part 5) to L1-CAM as described in Example 6.

[0068] Figure 9 presents the blank-subtracted sensorgrams of 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 as described in Example 7.

[0069] Figure 10 shows The purification profile of rAb as in Example 8 was presented. Non-reducing PAGE stained with Coomassie blue. MW: molecular weight marker. IN: input. FT: flow-through. W: wash. E: elution fraction.

[0070] Figure 11 shows The final sample QC in Example 8 was presented. Coomassie blue staining. Simplified and non-simplified PAGE analysis. MW: molecular weight marker. 2 μg was loaded per lane.

[0071] Figure 12 presents the SEC-HPLC profile obtained for LV1-8. LV 1-8 is a chimeric CE7 antibody containing mutations (L234A, L235A, P331S, and N297A) in the FC portion. The aim of this study was to identify the effect of sequence susceptibility mutations on the parental mAb.

[0072] Figure 13 showsPresented are the double-reference processed, solvent-corrected sensorgrams of A) deglycosylated cHCE7 and L1-CAM and B) the interaction between deglycosylated cHCE7 and L1-CAM, and the measured response values are shown on the figure.

[0073] Figure 14 shows Showed the purification profile of the rAb as in Example 21. Non-reducing PAGE stained with Coomassie blue. MW: molecular weight marker. IN: input. FT: flow-through. W: wash. E: elution fraction.

[0074] Figure 15 shows Showed the final sample QC in Example 21. Coomassie blue staining. Reduced and non-reduced PAGE analysis. MW: molecular weight marker. 2 μg was loaded per lane.

[0075] Figure 16 shows Showed the SEC-HPLC profile obtained for LV29-32.

[0076] Figure 17 shows Showed the survival of embryos 3 days after intravenous injection of naked huCE7-V7AG. The X-axis is shown in log[mg / mL].

[0077] Figure 18 shows Showed the biodistribution study of deglycosylated chCE7 and variant 7AG according to the present invention at 24 hours, 48 hours, 72 hours, and 96 hours.

[0078] Figure 19 shows Presented 177 The biodistribution of Lu-labeled huCE7-LV40-(PEG4-DOTA)2 and huCE7-LV40-(PEG4-polymer-α-DOTA4)2 in a mouse tumor xenograft model. Mouse model: CD1 nu / nu sc SKOVip3 (ovarian cancer, L1-CAM positive).

[0079] Figure 20 Presented the efficacy of Lu-labeled huCE7-V7AG-(PEG4-DOTA)2 in a mouse tumor xenograft model using 4 Mbq 177 Mouse model: CD1 nu / nu sc SKOVip3 (ovarian cancer, L1-CAM positive). On day 12 after tumor cell inoculation, mice were treated with a single injection of the labeled mAb (treatment group, 10 mice) or PBS (negative control group, 10 mice). Tumor size, body weight, and survival were monitored throughout the study. Once the tumor size reached the threshold, the mice were euthanized.

[0080] Figure 21 showsEfficacy evaluation of the huCE7-V7AG and HUCE7-V7AG-(MMAE)4 antibodies against MAXFTN-401 breast cancer cells in a zebrafish xenograft model was demonstrated. The antibodies were administered by intravenous injection immediately after tumor implantation. Data are presented as mean ± SEM, followed by Student's t-test, *p < 0.05.

[0081] Figure 22 shows A. FACS analysis of H2171 SCLC cells and B. FACS analysis of the negative control cell line MDA-MB-468 (which does not express L1-CAM) are shown.

[0082] Figure 23 shows Normalized primary tumor size and metastatic dissemination of H2171 cells in a zebrafish xenograft model were demonstrated 48 hours after treatment with intravenous injection of the HuCE7-V7AG and HuCE7-V7AG-(MMAE)4 antibodies. Normalized data relative to the negative control group are shown. Data are presented as mean ± SEM; one-way ANOVA (p < 0.0001) was performed, followed by a two-tailed Student's t-test,

[0083] *p < 0.05 and ****p < 0.0001. Detailed Description

[0084] The present invention will be described in detail hereinafter. It should be understood that all disclosed features can be combined with each other unless the contrary is explicitly stated.

[0085] In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to L1-CAM (CD171).

[0086] L1-CAM refers to a transmembrane protein member of the L1 protein family encoded by the L1-CAM gene, as described in the UniProt database under the reference number P32004. The L1-CAM protein is a neuronal cell adhesion molecule that plays important roles in cell migration, adhesion, neurite outgrowth, myelination, and neuronal differentiation. The L1-CAM protein has also been found to play a role in treatment-resistant cancers. The term "L1-CAM" can be used interchangeably with the term "CD171", both of which are known to those skilled in the art.

[0087] 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) 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 in order to bind to the protein.

[0088] As understood herein, the antibodies and antigen-binding fragments of the invention preferably bind to the Ig6-like domain of L1-CAM and prevent L1-CAM from binding integrin. Accordingly, these antibodies or antigen-binding fragments thereof preferably bind to the same epitope as the chCE7 antibody mentioned herein.

[0089] As preferably mentioned herein, specific binding to L1-CAM refers to a situation in which an antibody or a fragment thereof is capable of binding L1-CAM with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting L1-CAM. Accordingly, and preferably, the degree of binding of such an antibody to an irrelevant non-L1-CAM protein is less than about 10% of the degree of binding of the antibody to L1-CAM, as measured, for example, in an immunoassay (e.g., radioimmunoassay) or in an SPR assay (surface plasmon resonance). More preferably, the degree of binding of such an antibody to an irrelevant non-L1-CAM protein is less than about 5% of the degree of binding of the antibody to L1-CAM, as measured, for example, in an immunoassay (e.g., radioimmunoassay) or in an SPR assay (e.g., surface plasmon resonance using Biacore).

[0090] The term "binding" as used in the context of the present invention defines the binding (interaction) of at least two "antigen interaction sites" with each other. According to the present invention, the term "antigen interaction site" defines a motif of a polypeptide, i.e., a part of an antibody or antigen-binding fragment of the invention, which demonstrates the ability to specifically interact with a specific antigen or a specific group of antigens of L1-CAM.

[0091] The cross-reactivity of the antibody or antigen-binding fragment thereof under investigation can be tested, for example, by evaluating the binding of the antibody population or antigen-binding fragments thereof to the (poly)peptide of interest and to many more or less (structurally and / or functionally) closely related (poly)peptides under conventional conditions (see, for example, 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, their antigen-binding fragments, etc.) that bind to a specific structure of L1-CAM as defined herein (e.g., a specific epitope of L1-CAM or a (poly)peptide / protein as defined herein) but do not bind or bind substantially not 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 investigation according to the methods provided herein. These methods can in particular include binding studies, blocking and competition studies with molecules that are closely related structurally and / or functionally. These binding studies also include FACS analysis, surface plasmon resonance (SPR, e.g., performed using Biacore), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy measurements, fluorescence spectroscopy or ligand-binding assays by radiolabeling. Specificity can thus be determined experimentally by methods known in the art and methods described herein. Such methods include, but are not limited to, Western blotting, ELISA tests, RIA tests, ECL tests, IRMA tests and peptide scanning.

[0092] As preferably mentioned herein, an antibody or antigen-binding fragment is defined as follows.

[0093] In general, the term "antibody" is used herein in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies and antibody fragments, provided that they exhibit the desired antigen-binding activity. Unless otherwise explicitly stated to the contrary, whenever an antibody or its properties are discussed and the antibody is mentioned, it is meant the antibody and its antigen-binding fragments.

[0094] As preferably mentioned herein, an "antigen-binding fragment" of an antibody refers to a molecule other than the intact antibody that comprises a part 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.

[0095] Preferably, the antibody or antigen-binding fragment thereof is a monoclonal antibody, chimeric antibody, recombinant antibody, antigen-binding fragment of a recombinant antibody, single-chain antibody, humanized antibody, bispecific antibody, multispecific antibody, or an antibody displayed on the surface of a phage or an antibody displayed on the surface of a chimeric antigen receptor (CAR) T cell.

[0096] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor variations that may occur naturally. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they can be synthesized by hybridoma cultures and are substantially free of contamination by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being a member of a substantially homogeneous population of antibodies and is not to 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 prepared by the hybridoma method described by Kohler, Nature 256(1975), 495.

[0097] The term "chimeric antibody" refers to an antibody that comprises a variable region of the invention fused or chimerized with an antibody region (e.g., constant region) from another human or non-human species (e.g., mouse, horse, rabbit, dog, bovine, chicken).

[0098] The term "recombinant antibody" includes all antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from transgenic animals (e.g., mice) expressing 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 that involve splicing human immunoglobulin gene sequences to other DNA sequences. Thus, the term antibody also encompasses recombinant human antibodies, xenogenic antibodies, and xenogenic hybrid antibodies. Such recombinant human antibodies have variable and constant regions (if any) derived from human germline immunoglobulin sequences. However, such antibodies can be mutagenized in vitro (or, when transgenic animals expressing human Ig sequences are used, by in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, while derived from and related to human germline VH and VL sequences, may not be sequences that occur naturally in the human antibody germline repertoire in vivo.

[0099] “Xenogeneic antibody” is defined in relation to the transgenic non-human organism from which such antibodies are produced. The term refers to an antibody having an amino acid sequence or a coding nucleic acid sequence corresponding to an amino acid sequence or a coding nucleic acid sequence found in an organism not consisting of a transgenic non-human animal (and typically an organism of a different species from the transgenic non-human animal).

[0100] The term “xenoheterologous antibody” refers to an antibody having light and heavy chains of different biological origin. For example, an antibody having a human heavy chain that binds to a murine light chain is a xenoheterologous antibody. Examples of xenoheterologous antibodies include chimeric antibodies and humanized antibodies.

[0101] The term antibody also encompasses humanized antibodies. A “humanized” form of a non-human (e.g., murine or rabbit) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab′, F(ab′)2, or other antigen-binding subsequences of an antibody) that contains minimal sequences derived from a non-human immunoglobulin. Ordinarily, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the complementarity-determining regions (CDRs) of the recipient have been replaced by residues from the CDRs 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, a humanized antibody may contain residues that are neither present in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further improve and optimize antibody performance. Generally, a humanized antibody will contain substantially all of the sequence of at least one (and typically two) variable domains, wherein 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 FR regions of human immunoglobulin consensus sequences. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), usually 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.

[0102] Thus, in the context of the present invention, the term "antibody" refers to intact immunoglobulin molecules as well as portions of such immunoglobulin molecules (i.e., "antigen-binding fragments thereof"). Additionally, as described above, the term refers to modified and / or altered antibody molecules. The term also refers to recombinantly or synthetically produced / synthesized antibodies. The term also refers to intact antibodies and antibody fragments such as isolated 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.

[0103] In the context of the present invention, single-chain antibodies, i.e., "single-chain Fv" or "scFv" antibody fragments, have the V H domain and the V L domain, where these domains are present in a single polypeptide chain. Typically, the scFv polypeptide also contains a polypeptide linker between the V H domain and the V L domain, which enables the scFv to form the required structure for antigen binding. Techniques for producing single-chain antibodies are described, for example, in "The Pharmacology of Monoclonal Antibodies" by Plückthun, edited by Rosenberg and Moore, Springer-Verlag, N.Y. (1994), 269-315.

[0104] As used herein, a "Fab fragment" consists of a light chain and the C H 1 and the variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0105] The "Fc" region contains two heavy chain fragments comprising the C H 2 domain and the C H 3 domain of the antibody. These two heavy chain fragments are bound together by two or more disulfide bonds and hydrophobic interactions of the C H 3 domain.

[0106] A "Fab' fragment" contains a light chain and a portion of one heavy chain that contains the V H domain and the C H 1 domain as well as the region between the C H 1 domain and the C H 2 domain, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0107] The "F(ab')2 fragment" contains two light chains and two heavy chains, and these two heavy chains contain a part of the constant region between the C H 1 domain and the C H 2 domain, such that an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments bound together by a disulfide bond between the two heavy chains.

[0108] The "Fv region" contains the variable regions from both the heavy chain and the light chain, but lacks the constant region.

[0109] The bispecific antibodies referred to herein are antibodies that are capable of binding two different types of antigens or two different epitopes of the same antigen simultaneously. After development, bispecific antibodies can be manufactured in several structural forms known to those skilled in the art. In the present invention, at least one antigen relates to L1-CAM as defined herein.

[0110] The multispecific antibodies referred to herein are antibodies that are capable of binding more than two different types of antigens or more than two different epitopes of the same antigen simultaneously.

[0111] The antibodies, antibody constructs, antibody fragments, antibody derivatives (all Ig-derived), or their corresponding immunoglobulin chains employed according to the invention can be further modified using conventional techniques known in the art, e.g., amino acid deletions, insertions, substitutions, additions, and / or recombinations and / or any other modifications known in the art, either alone or in combination. Methods for introducing such modifications into the underlying DNA sequence of the amino acid sequence of the immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook (1989), loc. cit. The term "Ig-derived domain" specifically refers to a (poly)peptide construct comprising at least one CDR. Fragments or derivatives of said Ig-derived domains define (poly)peptides that are part of the above-described antibody molecules and / or are 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 ed. (1989) and 3rd ed. (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)).

[0112] More preferably, the antibody or its antigen-binding fragment is a monoclonal antibody.

[0113] The antibodies referred to herein can refer to IgG1, IgG2a or IgG2b, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE. As used herein, "isotype" is the class of antibody encoded by the heavy chain constant region gene (e.g., IgM or IgG1). The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these antibodies can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively referred to as α, δ, ε, γ, and μ.

[0114] Preferably, the monoclonal antibody as described herein is an IgG1 antibody.

[0115] Thus, the antibodies of the present invention can be full-length or can contain only antigen-binding fragments, such as the antibody constant domains and / or variable domains of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, or IgE, or can be composed of Fab fragments, F(ab')2 fragments, and Fv fragments.

[0116] In the present invention, the antibody or its antigen-binding fragment contains a variable heavy chain region as described below and / or a variable light chain region as described below.

[0117] As covered by the present invention, the variable heavy chain region contains:

[0118] CDR-H1 characterized by a sequence selected from:

[0119] The sequence according to SEQ ID NO.:1 (GYWMH),

[0120] The sequence according to SEQ ID NO.:2 (GYYMH),

[0121] The sequence according to SEQ ID NO.:3 (GYFMH), and

[0122] The sequence according to SEQ ID NO.:4 (GYLMH); and

[0123] CDR-H2 characterized by a sequence selected from:

[0124] The sequence according to SEQ ID NO.:5 (EINPSNGRTNYNERFQG),

[0125] The sequence according to SEQ ID NO.:6 (EINPSNGRTNYNEKFQG),

[0126] The sequence according to SEQ ID NO.: 7 (EINPSNGRTNYNERFKS),

[0127] The sequence according to SEQ ID NO.: 8 (EINPSNGRTNYNERLKS),

[0128] The sequence according to SEQ ID NO.: 9 (EINPSNARTNYNERFQG),

[0129] The sequence according to SEQ ID NO.: 10 (EINPSNARTNYNEKFQG),

[0130] The sequences according to SEQ ID NO.: 11 (EINPSNARTNYNERFKS) and

[0131] The sequence according to SEQ ID NO.: 12 (EINPSNARTNYNERLKS); and

[0132] A CDR-H3 characterized by the sequence according to SEQ ID NO.: 13 (DYYGTSYNFDY).

[0133] As covered by the present invention, the variable light chain region comprises:

[0134] A CDR-L1 characterized by a sequence selected from:

[0135] The sequence according to SEQ ID NO.: 14 (RANEDINNRLA),

[0136] The sequence according to SEQ ID NO.: 15 (KANEDINNRLA),

[0137] The sequence according to SEQ ID NO.: 16 (QANEDINNRLA),

[0138] The sequence according to SEQ ID NO.: 17 (RANEDINARLA),

[0139] The sequence according to SEQ ID NO.: 18 (KANEDINARLA),

[0140] The sequence according to SEQ ID NO.: 19 (QANEDINARLA),

[0141] The sequence according to SEQ ID NO.: 20 (RANEDINLRLA),

[0142] The sequence according to SEQ ID NO.: 21 (KANEDINLRLA), and

[0143] The sequence according to SEQ ID NO.: 22 (QANEDINLRLA); and

[0144] CDR-L2 characterized by a sequence selected from the following:

[0145] The sequence according to SEQ ID NO.: 23 (GATNLVT) and

[0146] The sequence according to SEQ ID NO.: 24 (GASNLVS); and

[0147] CDR-L3 characterized by a sequence selected from the following:

[0148] The sequence according to SEQ ID NO.: 25 (QQYWSTPFT),

[0149] The sequence according to SEQ ID NO.: 26 (QQYYSTPFT) and

[0150] The sequence according to SEQ ID NO.: 27 (QQYFSTPFT).

[0151] As understood herein, the present invention is defined by a number of amino acid sequences, which are preferably provided using the single-letter code known to those skilled in the art. As defined herein, these sequences should be given directly in the text of the present application or by reference to the sequence listing attached to the present application. In the case where there is a difference between the sequence given directly in the text of the present application and the sequence mentioned in the sequence listing by a specific sequence identifier number (SEQ ID NO.), the present invention should be understood as relating to both sequences, i.e., the sequence given in the specification or the sequence given in the sequence listing. Preferably, the present invention should be understood as relating to the sequence given in the sequence listing.

[0152] As used herein, the term "CDR" refers to the "complementary determining region" well known in the art. A CDR is a part of an immunoglobulin that determines the specificity of the molecule and contacts a specific ligand. The CDR is the most variable part of the molecule and contributes to the diversity of these molecules. There are three CDR regions, CDR1, CDR2, and CDR3, in each V domain. CDR-H denotes the CDR regions of the variable heavy chain, and CDR-L denotes the CDR regions 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 the following references: Kabat, "Sequences of Proteins of Immunological Interest", 5th Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services (1991); Chothia J. Mol. Biol. 196 (1987), 901-917 or Chothia Nature 342 (1989), 877-883.

[0153] Accordingly, the present invention is at least in part based on the surprising finding that, compared to antibodies of the prior art, and in particular compared to antibodies derived from the CE7 antibody, the antibodies or fragments thereof of the present invention exhibit improved affinity for L1-CAM.

[0154] 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 following: the sequences according to 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 following: the sequences according to SEQ ID NO.:1 and SEQ ID NO.:2. 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 according to SEQ ID NO.:1.

[0155] 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 or antigen-binding fragment thereof comprises a CDR-H2 characterized by a sequence selected from the following: the sequences according to 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 or antigen-binding fragment thereof comprises a CDR-H2 characterized by the sequence according to SEQ ID NO.: 5 or 6. In a specific 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 or antigen-binding fragment thereof comprises a CDR-H2 characterized by a sequence selected from the following: the sequences according to SEQ ID NO.: 8 or 12.

[0156] As understood herein, 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 or antigen-binding fragment thereof comprises a CDR-H3 characterized by the sequence according to SEQ ID NO.: 13.

[0157] 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 or antigen-binding fragment thereof comprises the CDR-H1 as described above, the CDR-H2 as described above, and the CDR-H3 as described above.

[0158] 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 or antigen-binding fragment thereof comprises a CDR-L1 characterized by a sequence according to 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 or antigen-binding fragment thereof comprises a CDR-L1 characterized by the sequence according to SEQ ID NO.: 14 or 15.

[0159] 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 or antigen-binding fragment thereof comprises a CDR-L2 characterized by the sequence according to SEQ ID NO.: 23.

[0160] 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 or antigen-binding fragment thereof comprises a CDR-L3 characterized by a sequence according to 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 or antigen-binding fragment thereof comprises a CDR-L3 characterized by the sequence according to SEQ ID NO.: 25.

[0161] 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 CDR-L1 as described above, CDR-L2 as described above, and CDR-L3 as described above.

[0162] According to the present invention, the antibody or antigen-binding fragment thereof of the present invention comprises the variable heavy chain region of the antibody or antigen-binding fragment thereof and / or the variable light chain region of the antibody or antigen-binding fragment thereof. The variable heavy chain region comprises CDR-H1 as described above, CDR-H2 as described above, and CDR-H3 as described above. The variable light chain region comprises CDR-L1 as described above, CDR-L2 as described above, and CDR-L3 as described above.

[0163] Preferably, the antibody or antigen-binding fragment thereof of the present invention comprises the variable heavy chain region of the antibody or antigen-binding fragment thereof and the variable light chain region of the antibody or antigen-binding fragment thereof. The variable heavy chain region comprises CDR-H1 as described above, CDR-H2 as described above, and CDR-H3 as described above. The variable light chain region comprises CDR-L1 as described above, CDR-L2 as described above, and CDR-L3 as described above.

[0164] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises the variable heavy chain region of the antibody or antigen-binding fragment thereof and the variable light chain region of the antibody or antigen-binding fragment thereof. The variable heavy chain region comprises CDR-H1 as described above, CDR-H2 as described above, and CDR-H3 as described above. The variable light chain region comprises CDR-L1 as described above, CDR-L2 as described above, and CDR-L3 as described above.

[0165] In one embodiment of 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 the sequence according to 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 according to SEQ ID NO.: 17, 18 or 19. Preferably, in the said embodiment, the variable heavy chain region of the antibody or antigen-binding fragment thereof comprises a CDR-H2 characterized by the sequence according to 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 according to SEQ ID NO.: 17, 18 or 19. Preferably, in the said specific embodiment, the variable heavy chain region of the antibody or antigen-binding fragment thereof further comprises a CDR-H1 characterized by the sequence according to 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 according to SEQ ID NO.: 26 or 27. More preferably, in the said specific embodiment, the variable heavy chain region of the antibody or antigen-binding fragment thereof further comprises a CDR-H1 characterized by the sequence according to 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 according to SEQ ID NO.: 26 or 27.

[0166] Preferably, the antibody or antigen-binding fragment thereof of the present invention is a humanized antibody.

[0167] Humanization methods are well known in the art and have been particularly described for antibody molecules, such as Ig-derived molecules. The term "humanized" refers to a humanized form of a non-human (e.g., murine) antibody or a fragment thereof (such as an Fv, Fab, Fab', F(ab'), scFv or other antigen-binding portion sequence of an antibody) that contains certain portions of the sequence derived from the non-human antibody. A humanized antibody includes a human immunoglobulin in which the residues of the complementarity-determining regions (CDRs) from the human immunoglobulin are replaced with residues of CDRs from a non-human species such as a mouse, rat or rabbit that have the desired binding specificity, affinity and capacity. Generally, a humanized antibody will contain substantially all of the sequence of at least one (and usually two) variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin, and all or substantially all of the FR (framework) regions are FR regions of human immunoglobulin consensus sequences. Desirably, a humanized antibody will also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin; see in particular 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. Typically, a humanized antibody has one or more amino acids introduced therein from a non-human source that still retain the original binding activity of the antibody. Methods for humanizing antibodies / antibody molecules are further described in detail in the following references: 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 (such as antibodies against EpCAM) are known in the art, see for example LoBuglio, "Proceedings of the American Society of Clinical Oncology Abstract (1997)", page 1562, and Khor, "Proceedings of the American Society of Clinical Oncology Abstract (1997)", page 847.

[0168] Preferably, the "framework region" referred to herein, also known as the FR region, is the non-CDR portion of the antibody variable domain. Thus, there are four framework regions in each variable domain sequence of an antibody, and these four framework regions are separated from each other by the hypervariable regions - CDRs. The framework regions typically account for about 85% of the variable domain sequence and serve as a scaffold to expose the CDRs such that these CDRs can interact with an antigen. As is known to those skilled in the art, variations in the framework region can also affect the binding affinity of the antibody for the CDR-specific antigen.

[0169] Accordingly, in the context of the present invention, there are provided humanized antibody molecules or antigen-binding fragments thereof that can be successfully used in pharmaceutical compositions.

[0170] "Percent amino acid sequence identity (%)" is defined with respect to a reference polypeptide sequence as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and any conservative substitutions are not counted towards the sequence identity. The alignment for determining percent amino acid sequence identity can be achieved in a variety of ways known to those skilled in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0171] Preferably, in the antibody or antigen-binding fragment thereof of the present 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:

[0172] The sequence according to SEQ ID NO.:28 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS),

[0173] The sequence according to SEQ ID NO.:29 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTVSS),

[0174] The sequence according to SEQ ID NO.:30 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFTGYWMHWIRQPPGKGLEWIGEINPSNGRTNYNERLKSRVTLSVDKSKNQASLKLSSVTAADTAVYFCARDYYGTSYNFDYWGQGTLVTVSS),

[0175] The sequence according to SEQ ID NO.:31 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), and

[0176] The sequence according to SEQ ID NO.:32 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWmGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS).

[0177] More preferably, in the antibody or antigen-binding fragment thereof of the present 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 according to SEQ ID NO.:28 or the sequence according to SEQ ID NO.:32.

[0178] 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 that is identical to a sequence selected from the sequence according to SEQ ID NO.:28 or the sequence according to SEQ ID NO.:32.

[0179] 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 that is identical to the sequence according to SEQ ID NO.:28. In one embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the variable heavy chain region is characterized by a sequence that is identical to the sequence according to SEQ ID NO.:32.

[0180] 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, and even more preferably identical to a sequence selected from the following:

[0181] The sequence according to SEQ ID NO.:33 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK),

[0182] The sequence according to SEQ ID NO.:34 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDIATYYCQQYWSTPFTFGQGTKLEIK),

[0183] The sequence according to SEQ ID NO.:35 (EIVMTQSPATLSVSPGERATLSCRANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEFTLTISSLQSEDFAVYYCQQYWSTPFTFGQGTKLEIK),

[0184] The sequence according to SEQ ID NO.:36 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), and

[0185] The sequence according to SEQ ID NO.:37 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGAsNLVsGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK).

[0186] 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, and even more preferably identical to a sequence selected from the following: the sequence according to SEQ ID NO.: 33, the sequence according to SEQ ID NO.: 36, and the sequence according to SEQ ID NO.: 37, preferably the sequence according to SEQ ID NO.: 33 and the sequence according to SEQ ID NO.: 36.

[0187] 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 that is identical to a sequence selected from the following: the sequence according to SEQ ID NO.: 33, the sequence according to SEQ ID NO.: 36, and the sequence according to SEQ ID NO.: 37, preferably the sequence according to SEQ ID NO.: 33 and the sequence according to SEQ ID NO.: 36.

[0188] 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 that is identical to the sequence according to SEQ ID NO.: 33. In one embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the variable light chain region is characterized by a sequence that is identical to the sequence according to SEQ ID NO.: 36. In one embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the variable light chain region is characterized by a sequence that is identical to the sequence according to SEQ ID NO.: 37.

[0189] As will be understood by those skilled in the art, the sequence of the variable heavy chain (or the sequence of the variable light chain) can also be defined by its framework and its CDR sequences. Accordingly, other sequences of the variable heavy chain or variable light chain covered by the present invention are disclosed hereinafter.

[0190] In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, wherein the variable heavy chain region is characterized by a sequence that is at least 80% identical to the following sequence, preferably at least 85% identical to the following sequence, more preferably at least 90% identical to the following sequence, even more preferably at least 95% identical to the following sequence, and more preferably identical to the following sequence:

[0191] FH0-CDR-H1-FH1-CDR-H2-FH2-CDR-H3-FH3.

[0192] CDR-H1, CDR-H2, and CDR-H3 are as defined above.

[0193] FH0 is characterized by the sequence according to SEQ ID NO.: 38 (QVQLVQSGAEVKKPGASVKVSCKASGYTFT) or the sequence according to SEQ ID NO.: 39 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFT).

[0194] FH1 is characterized by the sequence according to SEQ ID NO.: 40 (WVRQAPGQGLEWIG) or the sequence according to SEQ ID NO.: 41 (WIRQPPGKGLEWIG).

[0195] FH2 is characterized by the sequence according to SEQ ID NO.: 42 (RVTLTVDKSISTAYMELSRLRSDDTAVYFCAR) or the sequence according to SEQ ID NO.: 43 (RVTLSVDKSKNQASLKLSSVTAADTAVYFCAR).

[0196] FH3 is characterized by the sequence according to SEQ ID NO.: 44 (WGQGTLVTVSS).

[0197] In one embodiment, the present invention relates to an antibody or an antigen-binding fragment thereof, wherein 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, and most preferably identical to the following sequence:

[0198] FL0-CDR-L1-FL1-CDR-L2-FL2-CDR-L3-FL3.

[0199] CDR-L1, CDR-L2 and CDR-L3 are as defined above.

[0200] FL0 is characterized by the sequence according to SEQ ID NO.: 45 (DIQMTQSPSSLSASVGDRVTITC) or the sequence according to SEQ ID NO.: 46 (EIVMTQSPATLSVSPGERATLSC).

[0201] FL1 is characterized by the sequence according to SEQ ID NO.: 47 (WYQQKPGKAPKLLIS) or the sequence according to SEQ ID NO.: 48 (WYQQKPGQAPRLLIS).

[0202] FL2 is characterized by the sequence according to SEQ ID NO.: 49 (GVPSRFSGSGSGKDYTLTISSLQPEDIATYYC) or the sequence according to SEQ ID NO.: 50 (GIPARFSGSGSGKEFTLTISSLQSEDFAVYYC).

[0203] FL3 is characterized by the sequence according to SEQ ID NO.: 51 (FGQGTKLEIK).

[0204] In one embodiment, the present invention relates to an antibody or an antigen-binding fragment thereof, wherein the variable heavy chain region is characterized by a sequence that is at least 80% identical to the following sequence, preferably at least 85% identical to the following sequence, more preferably at least 90% identical to the following sequence, even more preferably at least 95% identical to the following sequence, and even more preferably identical to the following sequence:

[0205] FH0-CDR-H1-FH1-CDR-H2-FH2-CDR-H3-FH3

[0206] wherein FH0, CDR-H1, FH1, CDR-H2, FH2, CDR-H3, and FH3 are as defined above,

[0207] and / or (preferably and)

[0208] wherein the variable light chain region is characterized by a sequence that is at least 80% identical to the following sequence, preferably at least 85% identical to the following sequence, more preferably at least 90% identical to the following sequence, even more preferably at least 95% identical to the following sequence, and even more preferably identical to the following sequence:

[0209] FL0-CDR-L1-FL1-CDR-L2-FL2-CDR-L3-FL3

[0210] wherein FL0, CDR-L1, FL1, CDR-L2, FL2, CDR-L3, and FL3 are as defined above.

[0211] The present invention encompasses an antibody or an antigen-binding fragment thereof, wherein any combination of the variable light chain as defined above and the variable heavy chain as defined above is encompassed.

[0212] For the design of the CDR-grafted version of CE7 murine VH, two human germlines were selected, namely IGHV1-2*06 and IGHV4-34*01. The sequence identities of these two human germlines across the entire V gene were 66.3% and 53.1% respectively. For the design of the CDR-grafted version of CE7 murine VL, three human germlines were selected; IGKV1-NL1*01, IGKV1-33*01, and IGKV3-15*01. The sequence identities of these three human germlines with CE7 murine VL across the entire V gene were 73.7%, 71.6%, and 60.0% respectively.

[0213] According to computer immunogenicity modeling studies conducted by the present inventors, a number of mutations can be introduced into these germlines as described above to reduce their potential immunogenicity.

[0214] Thus, in the VH of IGHV4-34*01 onto which CE7-CDR is grafted, the following mutations are expected to be particularly beneficial for reducing immunogenicity:

[0215] - The M residue at position 34 can be replaced with a residue selected from C, D, and E;

[0216] - The M residue at position 35 can be replaced with C;

[0217] - The I residue at position 37 can be replaced with a residue selected from C, D, and E;

[0218] - The R residue at position 63 can be replaced with a residue selected from D, E, and P;

[0219] - The L residue at position 64 can be replaced with a residue selected from A, C, D, E, G, H, K, N, P, Q, S, and T;

[0220] - The K residue at position 65 can be replaced with a residue selected from C, D, E, N, and P;

[0221] - The S residue at position 66 can be replaced with a residue selected from C and D;

[0222] - The R residue at position 67 can be replaced with a residue selected from D and P;

[0223] - The V residue at position 68 can be replaced with a residue selected from C, D, E, G, N, and Q;

[0224] - The T residue at position 69 can be replaced with a residue selected from D and E;

[0225] - The L residue at position 70 can be replaced with a residue selected from C, D, E, G, N, P, Q, S, and T; and / or

[0226] - The V residue at position 72 can be replaced with D.

[0227] Furthermore, in the VL of IGKV1-NL1*01 with CE7-CDR grafted thereon, the following mutations are predicted to be particularly beneficial for reducing immunogenicity:

[0228] - The L residue at position 46 can be replaced by a residue selected from A, D, E, G, H, N, P, Q, S, and T;

[0229] - The L residue at position 47 can be replaced by a residue selected from D, E, G, N, P, S, and T;

[0230] - The A residue at position 51 can be replaced by E;

[0231] - The T residue at position 52 can be replaced by D;

[0232] - The N residue at position 53 can be replaced by D;

[0233] - The L residue at position 54 can be replaced by a residue selected from C, D, E, G, N, P, Q, S, and T; and / or

[0234] - The V residue at position 55 can be replaced by a residue selected from C, D, E, and G.

[0235] Furthermore, in the VH of IGHV1-2*06 with CE7-CDR grafted thereon, the following mutations are expected to be particularly beneficial for reducing immunogenicity:

[0236] - The L residue at position 70 can be replaced by A, C, D, E, G, H, K, N, P, Q, S, and T;

[0237] - The T residue at position 71 can be replaced by C and D;

[0238] - The V residue at position 72 can be replaced by A, C, D, E, G, H, K, N, P, Q, S, T, and W;

[0239] - The K residue at position 74 can be replaced by C, D, E, G, P, Q, and T,

[0240] - The S residue at position 75 can be replaced by C, D, E, G, and Q;

[0241] - The I residue at position 76 can be replaced by C, D, E, G, K, N, P, Q, S, and T;

[0242] - The S residue at position 77 can be replaced by C and E;

[0243] - The T residue at position 78 can be replaced by D and E; and / or

[0244] - The A residue at position 79 can be replaced by D.

[0245] Preferably, as encompassed by the present invention, an antibody (or, if applicable, an antigen-binding fragment thereof) according to any of the above-described embodiments comprises a heavy-chain constant region sequence that comprises an amino acid sequence selected from the group consisting of: the sequences according to SEQ ID NOs.: 145, 146, 147, 148, 149, and 150, preferably the sequences according to SEQ ID NOs.: 146, 147, 148, 149, and 150, more preferably the sequences according to SEQ ID NOs.: 147, 148, 149, and 150. In one embodiment, an antibody of the present invention comprises a heavy-chain constant region sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of: the sequences according to SEQ ID NOs.: 145, 146, 147, 148, 149, and 150, preferably the sequences according to SEQ ID NOs.: 146, 147, 148, 149, and 150, more preferably the sequences according to SEQ ID NOs.: 147, 148, 149, and 150.

[0246] In one embodiment, the present invention relates to an antibody or an antigen-binding fragment thereof, wherein the heavy chain 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, still more preferably identical to a sequence selected from the group consisting of: the sequences according to SEQ ID NOs.: 151, 152, 153, and 154.

[0247] As shown in Example 26, the antibody variants of the present invention, particularly the antibody variants of the embodiments described below, are characterized by a reduced uptake in lymph nodes. This represents an important aspect of the present invention because it is assumed that a reduced uptake of the huCE7 variant in healthy lymph nodes will result in a better side-effect profile. This effect may be due to changes in the CE7 epitope and sequence during humanization, such that the antibody no longer binds to the version of L1-CAM expressed in lymph nodes.

[0248] In one embodiment, it is particularly preferred that in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain 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, still more preferably identical, to a sequence selected from the sequences according to SEQ ID NOs.: 155 to 163. In this embodiment, it is particularly preferred that in the antibody or antigen-binding fragment thereof of the present invention, the light chain 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, still more preferably identical, to the sequence according to SEQ ID NO.: 143.

[0249] In a 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, still even more preferably identical, to the sequence according to SEQ ID NO.: 155, 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, more preferably at least 98% identical, even more preferably at least 99% identical, still even more preferably identical, to the sequence according to SEQ ID NO.: 143.

[0250] In a 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, still even more preferably identical, to the sequence according to SEQ ID NO.: 155, 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, more preferably at least 98% identical, even more preferably at least 99% identical, still even more preferably identical, to the sequence according to SEQ ID NO.: 97.

[0251] In a 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 still even more preferably identical to the sequence according to SEQ ID NO.: 162, 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, more preferably at least 98% identical, even more preferably at least 99% identical, and still even more preferably identical to the sequence according to SEQ ID NO.: 143.

[0252] In a 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 still even more preferably identical to the sequence according to SEQ ID NO.: 162, 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, more preferably at least 98% identical, even more preferably at least 99% identical, and still even more preferably identical to the sequence according to SEQ ID NO.: 97.

[0253] Therefore, preferably, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain region and a variable light chain region, the variable heavy chain region comprising CDR-H1 according to SEQ ID NO.: 3, CDR-H2 according to SEQ ID NO.: 9, and CDR-H3 according to SEQ ID NO.: 13, and the variable light chain region comprising CDR-L1 according to SEQ ID NO.: 18, CDR-L2 according to SEQ ID NO.: 23, and CDR-L3 according to SEQ ID NO.: 26.

[0254] In a 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 still even more preferably identical to the sequence according to SEQ ID NO.: 110 (CE7-1-2-VHB), 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, more preferably at least 98% identical, even more preferably at least 99% identical, and still even more preferably identical to the sequence according to SEQ ID NO.: 97 (CE7-1-NL1-VLA).

[0255] In a 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, still even more preferably identical to the sequence according to 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, more preferably at least 98% identical, even more preferably at least 99% identical, still even more preferably identical to the sequence according to SEQ ID NO.: 97 (CE7-1-NL1-VLA).

[0256] In a 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, still even more preferably identical to the sequence according to SEQ ID NO.: 110, 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, more preferably at least 98% identical, even more preferably at least 99% identical, still even more preferably identical to the sequence according to SEQ ID NO.: 143.

[0257] In a specific embodiment, the heavy chain of the antibody does not carry a mutation that causes the antibody to be deglycosylated. Thus, in a 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, still even more preferably identical to a sequence selected from the following: the sequences according to SEQ ID NO.: 110, 142, 155 to 162, wherein the mutation N297A has been reversed, 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, more preferably at least 98% identical, even more preferably at least 99% identical, still even more preferably identical to a sequence selected from the following: the sequences according to SEQ ID NO.: 97 and 143.

[0258] Thus, in a 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 still even more preferably identical to a sequence selected from: the sequences according to SEQ ID NO.: 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, more preferably at least 98% identical, even more preferably at least 99% identical, and still even more preferably identical to a sequence selected from: the sequences according to SEQ ID NO.: 97 and 143.

[0259] In a specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain 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 a sequence according to SEQ ID NO.: 110, 142, 155, 160 or 162, and the light chain 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 a sequence according to SEQ ID NO.: 97 or 143.

[0260] In a specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain 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 a sequence selected from: the sequences according to SEQ ID NO.: 173 to 183, and the light chain 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 a sequence according to SEQ ID NO.: 193.

[0261] In a specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain 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 a sequence selected from: the sequences according to SEQ ID NO.: 173 to 183, and the light chain 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 a sequence according to SEQ ID NO.: 194.

[0262] In a specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain 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, still more preferably identical to a sequence selected from the sequences according to SEQ ID NO.: 184 to 192, and the light chain 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, still more preferably identical to the sequence according to SEQ ID NO.: 193.

[0263] In a specific embodiment, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain 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, still more preferably identical to a sequence selected from the sequences according to SEQ ID NO.: 184 to 192, and the light chain 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, still more preferably identical to the sequence according to SEQ ID NO.: 194.

[0264] It should be understood that when the heavy chain or light chain is described as comprising a specific sequence, it also directly refers to the heavy chain or light chain having or consisting of the said sequence.

[0265] As understood herein, preferably, the antibody or antigen-binding fragment thereof of the present invention is characterized by a dissociation constant K D not exceeding 10 -11 M, more preferably not exceeding 10 -12 M. It should be understood that the K D is preferably measured in a Biacore-based assay.

[0266] 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 the 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 and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0267] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the CDRs and FRs. Conservative substitutions are shown below the heading "Preferred Substitutions" in Table D1. More distinct changes are provided below the heading "Exemplary Substitutions" in Table D1 and are further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activities such as retained / improved antigen binding, reduced immunogenicity or improved ADCC or CDC.

[0268] Table D1 .

[0269]

[0270]

[0271] Amino acids can be grouped according to common side chain properties:

[0272] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0273] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0274] (3) Acidic: Asp, Glu;

[0275] (4) Basic: His, Lys, Arg;

[0276] (5) Residues affecting chain orientation: Gly, Pro;

[0277] (6) Aromatic: Trp, Tyr, Phe.

[0278] Non-conservative substitutions will entail replacing a member of one class with a member of another class.

[0279] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., improvement) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parental antibody and / or will substantially retain certain biological properties of the parental antibody. Exemplary substitution variants are affinity matured antibodies which can be readily generated using, for example, affinity maturation techniques based on phage display such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0280] Alterations (e.g., substitutions) can occur in the CDRs, e.g., to improve antibody affinity. Such alterations can occur in CDR “hot spots” (i.e., residues encoded by codons that mutate at high frequency during somatic maturation) (see, e.g., Chowdhury, Methods Mol.Biol. 207:179-196 (2008)) and / or SDRs (α-CDRs), and the resulting variant VH or VL is tested for binding affinity. For example, affinity maturation has been described in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, (2001)) by constructing a secondary library and rescreening therefrom. 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 created. The library is then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity includes methods directed to the CDRs, where several CDR residues (e.g., 4 to 6 residues at a time) are randomized. CDR residues involved in antigen binding can be identified by specific methods, e.g., using alanine-scanning mutagenesis or by modeling. In particular, CDR H3 and CDR-L3 are often targeted.

[0281] In certain embodiments, substitutions, insertions, or deletions can occur within one or more CDRs, provided that such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can occur in the CDRs. Such alterations can occur outside of CDR “hot spots” or SDRs. In certain embodiments providing variant VH and VL sequences as described above, each CDR is unaltered, or contains no more than one, two, or three amino acid substitutions.

[0282] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine-scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or a set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial replacement. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they possess the desired properties.

[0283] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions (ranging in length from one residue to polypeptides containing one hundred or more residues), as well as insertions of single or multiple amino acid residues within the sequence. Examples of terminal insertions include antibodies having an N-terminal methionine. Other insertion variants of antibody molecules include fusions of the N-terminal or C-terminal of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide, which prolongs the serum half-life of the antibody.

[0284] In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. The glycosylation sites of the antibody can be conveniently increased or deleted by altering the amino acid sequence, thereby creating or removing one or more glycosylation sites.

[0285] When the antibody contains an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide can include various carbohydrates (e.g., 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, the oligosaccharides in the antibodies of the invention can be modified to produce antibody variants having certain improved properties.

[0286] In one embodiment, there is provided an antibody variant having a carbohydrate structure that lacks fucose linked (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all glycan structures linked to Asn 297 (such as complex-type structures, hybrid-type structures, and high-mannose-type structures), as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of the Fc region residues); however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297 (i.e., between positions 294 and 300), due to minor sequence variations in the antibody. Such fucosylation variants can have improved ADCC activity. See, for example, U.S. Patent Publication No. US2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US 2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; W02005 / 053742; W02002 / 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 Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotechnol. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0287] Antibody variants having a bisecting oligosaccharide are also provided, e.g., antibody variants in which the biantennary oligosaccharide linked 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 in, e.g., WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described in, e.g., WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0288] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein to generate Fc region variants. The Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0289] In certain embodiments, the present invention contemplates an antibody variant that has partial but not all effector functions, making it a desirable candidate for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and antibody-dependent cell cytotoxicity) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC (complement-dependent cytotoxicity) and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcyR binding ability (and thus may lack ADCC activity), but retains FcRn binding ability. The primary cells that mediate ADCC (i.e., NK cells) express only Fc(RIII, while monocytes express Fc(RI, Fc(RII, and Fc(RIII. The 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 for assessing the ADCC activity of a molecule of interest are described in the following references: U.S. Patent 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 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).

[0290] Thus, and 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, which at least one point mutation affects antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), serum half-life, and / or the glycosylation status of the antibody. It should be understood by those skilled in the art that antigen-binding fragments of the antibody comprising only the heavy chain or fragments thereof may be encompassed herein. The at least one point mutation mentioned 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 the at least one point mutation may refer to more than one mutation. For example, in a preferred embodiment of the present invention, the heavy chain comprises the L234A, L235A, P331S, and N297A point mutations. As preferably understood herein, the amino acid positions described herein refer to residue numbering as in the IgG1 isotype. In the case of using any other antibody class or isotype, those skilled in the art will be able to translate the amino acid positions to any other antibody class or isotype known in the art.

[0291] Those skilled in the art can also envision including 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 mutation S267E / H268F / S324T / G236A / I332E (EFTAE modification).

[0292] Those skilled in the art can also envision including at least one mutation that counteracts the negative effects of the N297A mutation (which allows the production of deglycosylated antibodies). 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, the at least one mutation is selected from F241K, L309D, T307R, and T307P, preferably the mutations F241K, L309D, T307R, and T307P that result in an increase in the thermal stability of the antibody. Without being bound by theory, the mutations increase the stability of the Fc portion of the antibody heavy chain.

[0293] The present invention also encompasses an antibody or antigen-binding fragment thereof as described herein, which further comprises in its heavy chain one or more of the following mutations: G31D, G31E, G31K, G31P, G31R, H35K, T28D, T28E, T28K, T28P, T28R, T30D, T30E, T30K, T30P, T30R, W33R, Y27D, Y27E, and Y27K, preferably comprising a 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, the present invention encompasses an antibody or antigen-binding fragment thereof as described herein, which further comprises in its heavy chain one or more of the following mutations: 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, stabilizes the flanking region of CDR1 and / or may result in an improved affinity of the antibody or its fragment for L1-CAM.

[0294] The present invention also encompasses an antibody or antigen-binding fragment thereof as described herein, which further comprises in its heavy chain one or more of the following mutations: A40R, V68A, and L115T. Without being bound by theory, these mutations correspond to the incorporation of residues initially present in a murine antibody that are near regions prone to aggregation. According to the inventors, at least one mutation selected from A40R, V68A, and L115T may result in an increase in the thermal stability of the antibody. Without being bound by theory, the mutations increase the stability of the Fc portion of the antibody heavy chain.

[0295] The antibodies of the invention provided herein, or antigen-binding portions thereof, can be further modified to contain additional non-protein moieties that are known and readily available in the art. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propanediol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacture due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers linked to the antibody can vary, and in the case of linking more than one polymer, these antibodies can be the same or different molecules. In general, the number and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, and the like.

[0296] Other suitable and preferred examples of water-soluble polymers, such as polymers containing an acrylic backbone, are described hereinbelow.

[0297] The antibodies of the invention provided herein, or antigen-binding portions thereof, can also be further modified to contain additional moieties, thereby producing an immunoconjugate comprising the antibody of the invention or an antigen-binding fragment thereof. Such immunoconjugates are described herein.

[0298] Production of antibodies

[0299] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such nucleic acids can encode an amino acid sequence containing the VL of the antibody and / or an amino acid sequence containing the VH of the antibody (e.g., the light and / or heavy chains of the antibody).

[0300] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0301] "Isolated nucleic acids encoding anti-L1-CAM antibodies" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules present in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0302] In another embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. Accordingly, the invention relates to a polynucleotide encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence as described herein. It should be understood that the terms polynucleotide and (isolated) nucleic acid are used interchangeably unless otherwise indicated.

[0303] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures, as well as vectors that are integrated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked to them. Such vectors are referred to herein as "expression vectors".

[0304] Accordingly, the invention also relates to a vector containing a polynucleotide encoding at least one variable heavy chain sequence and / or at least one variable light chain sequence as described herein.

[0305] In another embodiment, a host cell containing such nucleic acids is provided.

[0306] 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 progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical in nucleic acid composition to the parental cell, but may contain mutations. Mutant progeny having the same function or biological activity as the originally transformed cell are included herein.

[0307] In one such embodiment, the host cell comprises (e.g., is obtained by transformation): (1) a vector that comprises nucleic acids encoding an amino acid sequence of a VL that contains an antibody and an amino acid sequence of a VH that contains an antibody, or (2) a first vector and a second vector, the first vector comprising nucleic acids encoding an amino acid sequence of a VL that contains an antibody, and the second vector comprising nucleic acids encoding an amino acid sequence of a VH that contains an antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., YO, NSO, Sp20). In one embodiment, a method of preparing an antibody of the invention is provided, wherein the method comprises culturing a host cell that contains nucleic acids encoding an antibody as provided above under conditions suitable for expressing the antibody, and optionally harvesting the antibody from the host cell (or the host cell culture medium).

[0308] For recombinant production of an antibody of the invention, nucleic acids encoding the antibody (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

[0309] 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 when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (edited by B.K.C. Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be separated from the bacterial cell paste in soluble fractions and further purified.

[0310] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains in which the glycosylation pathways have been "humanized" to produce antibodies with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0311] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains that can be used with insect cells have been identified, and are particularly suitable for transfection of Spodoptera frugiperda cells.

[0312] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES TM technology) for producing antibodies in transgenic plants).

[0313] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension growth may be useful. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney cell CV1 line (COS-7); the human embryonic kidney cell line (293 or 293 cells, as described, for example, by Graham et al., J. Gen. Viral. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, by Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VER0-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, by Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 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. Sci. 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, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by B.K.C. Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0314] In one embodiment, the antibody or antigen-binding fragment thereof may comprise additional domains or additional amino acid sequences. For example, the antibody or antigen-binding fragment thereof may comprise a localization sequence. Suitable other 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 a tag conforming to the sequence LCTPSR (SEQ ID NO.:52), wherein when FGE acts on the sequence, the cysteine residue is converted to formylglycine. A sortase recognition motif conforms to the sequence LPXTG (SEQ ID NO.:53), wherein X can be any natural amino acid residue. A sortase, such as Staphylococcus aureus sortase, is a transpeptidase that attaches surface proteins to the cell wall; it cleaves between Gly and Thr of 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 the attachment of additional peptide moieties.

[0315] In one embodiment, the antibody or antigen-binding fragment thereof may also comprise a domain or amino acid sequence for the following functions: co-targeting the tumor microenvironment (e.g., targeting fibroblast activation protein-α (FAP)); breaching the blood-brain barrier (BBB) (e.g., targeting the transferrin receptor); or breaching the endothelial cell (EC) barrier (e.g., targeting aminopeptidase P2 (APP2) via caveolae). Thus, the additional targeting domain may be incorporated into the sequence of the antibody or antigen-binding fragment by fusing the C-terminus or N-terminus using a spacer peptide (fusion construct), or by subsequent ligation, for example, by site-specific functionalization.

[0316] The antibody or antigen-binding fragment thereof may also optionally comprise one or more unnatural amino acids for conjugating the antibody or fragment thereof to another chemical entity. The amino acids may include residues that are reactive in addition reactions known to those skilled in the art, such as click chemistry. Suitable examples of such residues include residues containing an azide moiety, or a cyclooctyne moiety, or a moiety capable of undergoing an inverse electron demand Diels-Alder cycloaddition reaction, such as a trans-cyclooctene / tetrazine reaction pair. However, the present invention is not meant 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 unnatural amino acid residues using recombinant methods are known to those skilled in the art.

[0317] In one embodiment, the present invention also relates to an immunoconjugate comprising an antibody or an antigen-binding fragment thereof of the present invention 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 an antigen-binding fragment thereof) conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents. The cytotoxic agent or prodrug may also be referred to as a drug.

[0318] The immunoconjugates of the present invention may also be referred to as antibody-drug conjugates. Antibody-drug conjugates (ADCs) are targeted chemotherapeutic molecules that combine the properties of both antibodies and cytotoxic drugs by targeting effective cytotoxic drugs to antigen-expressing tumor cells (Teicher, B.A. (2009) Current Cancer Drug Targets 9:982-1004), thereby improving the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter and Senter P.D. (2008) The Cancer Jour. 14(3):154-169; Chari, R.V. (2008) Ace. Chem. Res. 41:98-107).

[0319] The ADC compounds of the present invention include those having anti-cancer activity. In some embodiments, the ADC compounds include an antibody conjugated (i.e., covalently linked) to a drug moiety. In some embodiments, the antibody is covalently linked to the drug moiety through a linker. The antibody-drug conjugates (ADCs) of the present invention selectively deliver an effective dose of the drug to tumor tissue, thereby enabling greater selectivity (i.e., a lower effective dose), while increasing the therapeutic index ("therapeutic window").

[0320] The linker mentioned herein is not particularly limited, and any linker that can be conceived by those skilled in the art and can be used for immunoconjugates or antibody-drug conjugates can be used within the scope of the present invention.

[0321] In one embodiment of the present invention, the linker takes the form of a polymeric linker. Thus, the polymeric linker includes at least two types of repeating units, preferably more than two types of repeating units. It should be understood that at least one type of repeating unit is covalently linked to an active agent (such as a cytotoxic agent). Thus, at least one type of repeating unit does not contain the active agent, and correspondingly, the polymeric carrier can incorporate the active agent into some of its repeating units. Further, by using the polymeric carrier, multiple molecules of the active agent can be linked to a single attachment point on the antibody or an antigen-binding fragment thereof.

[0322] Any polymer carrier capable of being linked to an antibody or a fragment thereof can be used in the immunoconjugates of the present invention. Examples of polymer carriers particularly suitable for 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 polymer carrier will be defined below.

[0323] Preferably, the polymer carrier comprises a repeating unit of formula (R1)

[0324]

[0325] 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-; Z is H (if A is -O-) or -C n H 2n+1 (where n = 1-8); and A is -O- or -NH-; L is an α / spacer, and P comprises an active agent.

[0326] As preferably understood herein, if A is -O-, then Z is H or -C n H 2n+1 (where n = 1-8), while if A is -NH-, then Z is -C n H 2n+1 (where n = 1-8). Alternatively, Z can also preferably be defined as Z is H or -C n H 2n+1 (where n = 1-8). Preferably, if A is -O-, then Z is preferably H. Preferably, the copolymer comprising a repeating unit of formula (R1a) or the copolymer comprising a repeating unit of formula (R1) further comprises a repeating unit of formula (R2):

[0327]

[0328] 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 (where n = 1-8), or Y comprises a second payload molecule; Z is H (if A is -O-) or -CnH2n+1 (where n = 1-8), or Z comprises a second payload molecule; and A is -O- or -NH-,

[0329] and / or repeating unit of formula (R3):

[0330]

[0331] wherein: R is -H, -CH3, -CH2-CH3 or -(CH2)2-CH3; Z is H (if A is O) or -CnH2n+1 (where n = 1 - 8), or Z comprises a second payload molecule; and A is -O- or -NH-.

[0332] As preferably understood herein, if A is -O-, then Z is H or -CnH2n+1 (where n = 1 - 8), and if A is -NH-, then Z is -CnH2n+1 (where n = 1 - 8). Alternatively, Z can also preferably be defined as Z is H or -CnH2n+1 (where n = 1 - 8). Preferably, if A is -O-, then Z is preferably H.

[0333] 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 (where n = 1 - 8), or Z comprises a second active agent molecule.

[0334] Further preferably, the polymeric carriers as described herein include additional repeating units obtainable by polymerizing: N,N-dimethylacrylamide, N-isobutylacrylamide, N-tert-butylacrylamide, N-hydroxyethylacrylamide, 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 obtainable by polymerizing: 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.

[0335] Further preferably, in the polymeric carriers as described herein, where there are no repeating units of formula (R2) and (R3), the average number of repeating units according to formula (R1) per molecule of copolymer is from 2 to 12, preferably from 2 to 8, more preferably from 2 to 6.

[0336] More preferably, in the polymer carriers as described herein, wherein the repeating units of formula (R2) or (R3) are not functionalized, as defined herein, the average number of repeating units according to formula (R1), (R2) or (R3) per molecule of copolymer is from 10 to 50, preferably from 10 to 40, more preferably from 10 to 30.

[0337] More preferably, in the polymer carriers as described herein, wherein the repeating units of formula (R2) or (R3) are functionalized with a second payload molecule, the average number of repeating units according to formula (R1), (R2) or (R3) per molecule of copolymer is from 4 to 20, preferably from 4 to 15, more preferably from 4 to 10.

[0338] Particularly suitable examples of the polymer carriers for use in the immunoconjugates of the present invention include carriers derived from compounds selected from:

[0339]

[0340] wherein each X is independently -NH(CH2)4- or -NH-C6H4-CH2-; n is from 40 to 120; m is from 4 to 16; o is from 0 to 16; f is -H or -I (it should be understood that I includes both the cold isotopes of iodine and the hot isotopes of iodine), and k and q are designated as the active payload or may be H.

[0341] The terminal group e may be H or may be a modified terminal group, as discussed and illustrated below.

[0342] Regarding terminal group modification, the terminal group e can be introduced via a thiol-reactive group. Suitable reagents include, but are not limited to:

[0343] MC-DBCO

[0344] N-[3-(11,12-Didehydrodibenz[b,f]azocin-5(6H)-yl)-3-oxopropyl]-2,5-dihydro-2,5-dioxo-1H-pyrrole-1-propamide

[0345] Methyltetrazine-PEG4-maleimide

[0346] 3-(2,5-Dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethyl]propamide

[0347] Cas 1802908-02-6

[0348] It should be understood that the payload molecules k and q can be introduced via amino-reactive groups. Suitable reagents for introducing the payload molecules are selected from:

[0349] p-SCN-Bn-CHX-A″-DTPA: [(R)-2-Amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid;

[0350] p-SCN-Bn-DTPA: S-2-(4-Isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid;

[0351] 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;

[0352] p-SCN-Bn-DOTA: S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid;

[0353] NHSDOTA: 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester;

[0354] p-SCN-Bn-NOTA: 2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid;

[0355] NOTA-NHS ester: 2,2'-(7-(2-((2,5-Dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7-triazanonane-1,4-diyl)diacetic acid;

[0356] NODA-GA-NHS ester: 2,2'-(7-(1-Carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazanonane-1,4-diyl)diacetic acid;

[0357] 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-methanoazuleno[3,4]benzo[1,2-b]oxet-9-yl)oxy)-3-oxo-1-phenylpropan-2-yl (2,5-dioxopyrrolidin-1-yl) succinate;

[0358] 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-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate;

[0359] 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;

[0360] DM1-SMCC (maytansine-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;

[0361] SC-VC-PAB-DM1 (CAS: 2259318-47-1);

[0362] 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-naphthacenedione;

[0363] Reagents suitable for introducing kinase inhibitorsPreferably selected from fasudil, sirolimus, imatinib, gefitinib, erlotinib, sorafenib, sunitinib, dasatinib, lapatinib, nilotinib, temsirolimus, everolimus, pazopanib, ruxolitinib, vandetanib, vemurafenib, crizotinib, icotinib, axitinib, tofacitinib, bosutinib, cabozantinib, ponatinib, regorafenib, afatinib, dabrafenib, trametinib, ibrutinib, nintedanib, idelalisib, ceritinib, apatinib, lipfasudil, alectinib, cobimetinib, lenvatinib, palbociclib, ladostigil, osimertinib, omoertinib, neratinib, ribociclib, copanlisib, abemaciclib, acalabrutinib, midostaurin, brigatinib, baricitinib, natasudil, tivozanib, simotinib, fostamatinib, conafenib, binimetinib, Catequentinib, duvelisib, dacomitinib, lorlatinib, larotrectinib, gilteritinib, pyrrotinib, fruquintinib, erdafitinib, alpelisib, ulixertinib, leniolisib, pexidartinib, entrectinib, fedratinib, zanubrutinib, fluoratinib, peficitinib, dergocitinib, alvelestat, selumetinib, tucatinib, pemigatinib, capmatinib, selpercatinib, ripretinib, tilavonib, amitinib, pralsetinib, figitinib, tibamineurin, obutinib, tepotinib, and trilaciclib.

[0364] A reagent suitable for introducing doxorubicin or a derivative thereof. Preferably, the derivative of doxorubicin should be understood as a compound comprising the following moieties:

[0365]

[0366] Preferably, the doxorubicin derivative comprises a DBCO moiety.

[0367] PNU-159682;

[0368] Alternatively, the payload molecules k and q can be introduced via a click reaction in a two-step process. In this case, the amine-reactive reagent is the NHS ester of an azide-functionalized carboxylic acid, and the payload molecules are but not limited to:

[0369] DBCO-DOTA

[0370] 4,7,10-Tetraazacyclododecane-1,4,7-tri(acetic acid)-10-[3-oxo-3-(5-azidodibenzocyclooctyne)acetamide]

[0371] DBCO-PEG4-Val-Cit-PAB-MMAE

[0372] [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadec-1(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]propionylamino]-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-oxohept-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-N-methylcarbamate

[0373] DBCO-Val-Cit-PAB-MMAE

[0374] DBCO-Val-Cit-PAB-MMAF

[0375] DBCO-PEG4-MMAF (CAS: 2360411-65-8)

[0376] DM1-PEG4-DBCO

[0377] DBCO-PEG4-Ahx-DM1

[0378] The reagents suitable for introducing kinase inhibitors are preferably selected from fasudil, sirolimus, imatinib, gefitinib, erlotinib, sorafenib, sunitinib, dasatinib, lapatinib, nilotinib, temsirolimus, everolimus, pazopanib, ruxolitinib, vandetanib, vemurafenib, crizotinib, icotinib, axitinib, tofacitinib, bosutinib, cabozantinib, ponatinib, regorafenib, afatinib, dabrafenib, trametinib, ibrutinib, nintedanib, idelalisib, ceritinib, apatinib, lip fasudil, alectinib, cobimetinib, lenvatinib, palbociclib, radotinib, osimertinib, omoertinib, neratinib, ribociclib, copanlisib, abemaciclib, acalabrutinib, midostaurin, brigatinib, baricitinib, nai fasudil, tivozanib, simotinib, fostamatinib, conafenib, binimetinib, Catequentinib, duvelisib, dacomitinib, lorlatinib, larotrectinib, gilteritinib, pyrrotinib, fruquintinib, erdafitinib, alpelisib, ulixertinib, leniolisib, pemigatinib, camatini, selpercatinib, ripretinib, telarutinib, amitinib, pralsetinib, figitinib, tibatinib, obutinib, tepotinib and trilaciclib.

[0379] Reagents suitable for introducing doxorubicin or its derivatives. Preferably, derivatives of doxorubicin should be understood as compounds containing the following moieties:

[0380]

[0381] Preferably, the doxorubicin derivative contains a DBCO moiety.

[0382] DBCO-PEG4-VC-PAB-DMEA-PNU-159682 (CAS: 2259318-56-2)

[0383] Reagents suitable for introducing AZD7648:

[0384]

[0385] Those skilled in the art should understand that the term "derived" as used herein preferably refers to the attachment of any of these compounds to an antibody or its fragment, for example, by a condensation reaction involving a free carboxylic acid group. It is preferably understood that these formulas do not represent block copolymers, but rather the statistical composition of the polymers obtained. However, it should be understood that block copolymers or gradient copolymers may also be encompassed within the definitions provided herein, as their formation may also be feasible, depending on the specific conditions in the reactor.

[0386] The drug moiety (D) of an antibody-drug conjugate (ADC) can include any compound, moiety, or group having cytotoxic or cytostatic effects. The drug moieties can exert their cytotoxic and cytostatic effects through 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 maytansine, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, deruxtecan, exatecan, elinafide, and their stereoisomers, bioisosteres, analogs, and derivatives having cytotoxic activity. Non-limiting examples of such immunoconjugates are discussed in further detail below. In a preferred embodiment, the drug moiety (D) is a topoisomerase inhibitor, i.e., an inhibitor of topoisomerase I and / or II, such as a derivative of camptothecin, preferably selected from exatecan, deruxtecan, topotecan, irinotecan, SN38, and belotecan. In one embodiment, the active agent (i.e., the drug moiety (D)) is selected from maytansine, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, exatecan, deruxtecan, topotecan, irinotecan, SN38, and belotecan.

[0387] One skilled in the art can attach the drug moiety to the antibody. For example, exatecan can be attached as shown in the following scheme, where R generally represents the antibody or its antigen-binding fragment, including the remainder of the bond between the antibody or its fragment and the drug moiety (D):

[0388]

[0389]

[0390] Another suitable example of a moiety that can be incorporated into the antibody-drug conjugates of the present invention is a moiety derived from a compound according to the following formula:

[0391]

[0392] It should be understood that an addition reaction or a click chemical reaction would be preferred for the attachment of the compounds as shown herein to the antibody or its antibody-binding fragment.

[0393] Another suitable example of a drug moiety incorporated into the antibody-drug conjugates of the present invention is maytansine.

[0394] Exemplary embodiments of antibody-drug conjugate (ADC) compounds include an antibody (Ab) that targets tumor cells, a drug moiety (D), and a linker moiety (L) that attaches the Ab to the 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.

[0395] In one embodiment, the linker moiety is the polymeric carrier as described above.

[0396] In one embodiment, more than one linker moiety is attached to the antibody or an antigen-binding fragment thereof. Thus, in such embodiments, the immunoconjugate can be described according to the formula A-(L-D) i where i is an integer and i > 1.

[0397] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 、I 131 I 125 、Y 90 、Tb 161 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof such as nucleases; antibiotics; toxins such as 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).

[0398] The term "radioisotope / radionuclide" is preferably used as a synonym within the scope of the present invention and preferably represents an atom having an excess of nuclear energy that results in instability. This excess energy can be used in one of three ways: emitted from the atomic nucleus in the form of gamma radiation; transferred to one of its electrons and released as a conversion electron; or used to produce and emit new particles (alpha particles or beta particles) from the atomic nucleus. Radioisotopes / radionuclides are preferably defined herein as isotopes having a half-life of less than 10 19 years.

[0399] Thus, in one embodiment, the present invention relates to one or more immunoconjugates, wherein the immunoconjugate comprises a radioisotope as an active agent. Such conjugates may also be referred to as antibody-radionuclide conjugates.

[0400] The term "antibody-radionuclide conjugate" (ARC) is preferably defined as a variant of such an ADC: wherein the "drug molecule or active molecule" represents a radionuclide / radioisotope covalently bound to an antibody-polymer-conjugate, for example in the case of radioactive iodine, or via a chelate containing, for example, radioactive lutetium, actinium or terbium. The ARC thus formed is capable of delivering a large amount of radiation to tumor tissue, thereby killing tumor cells by disrupting DNA, essential enzymes, etc. It will be apparent to those skilled in the art that the term ARC also encompasses antibody-radionuclide conjugates that do not contain a polymeric carrier but contain a PEG or peptide linker. A suitable example of such a peptide linker is (Ac)-Lys-Ala-Tyr-Ala-Lys(azide)-NH2 [SEQ ID NO.:54] which can be used for subsequent functionalization with DBCO-DOTA.

[0401] As is well known to those skilled in the art, certain antibody-radionuclide conjugates can also be used for diagnostic purposes. Certain radionuclides disclosed herein are monitorable, for example terbium-161 can be visualized with a gamma camera due to its gamma emission and can thus be used to detect cancerous tissues or cell types targeted by the antibody. Terbium-149, which can be used for targeted alpha therapy, is visible in a PET scan and is thus monitorable. 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, terbium-155 are particularly useful in the diagnostic applications described herein and may be referred to as diagnostic radionuclides. As is known to those skilled in the art, these diagnostic 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).

[0402] Those skilled in the art will understand that immunoconjugates in which the active agent includes a radionuclide useful for therapeutic applications (e.g., selected from copper-67, strontium-89, yttrium-90, iodine-131, samarium-153, lutetium-177, radium-223, and actinium-225 (such radionuclides may be referred to as therapeutic radionuclides)) will have a biodistribution substantially the same as that of 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 a therapeutic radionuclide active agent can be supplemented, and for this purpose, immunoconjugates in which the active agent includes a therapeutic radionuclide (as defined herein) can preferably be supplemented at less than 10% by weight. Further preferably, the immunoconjugates of the present invention for combined therapeutic and diagnostic applications can contain two radionuclides, one therapeutic radionuclide and one diagnostic radionuclide, for example attached to a single polymeric carrier, or attached to different repeating units separately. Preferred is such a combination: in which the therapeutic radionuclide and the diagnostic 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 contain isotopes of two different elements, such as indium-111 and lutetium-177, and indium-111 and terbium-161.

[0403] The pharmaceutical formulations of the inventive antibodies or inventive immunoconjugates as described herein are prepared by mixing such antibodies or immunoconjugates of 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 an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as cetalkonium chloride; chlorhexidine digluconate; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); proteins such as serum albumin, 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, sorbitol; osmoprotectants such as ectoine; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g. human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20( Baxter International, Inc.). Certain exemplary sHASEGP (including rHuPH20) and methods of using the same are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0404] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a formulation that is in a form in which the biological activity of the active ingredient contained therein can be effectively exerted and that contains no additional components that are unacceptably toxic to the subject to which the formulation is to be administered.

[0405] "Pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical formulation that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0406] 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 W02006 / 044908, the latter formulation containing a histidine-acetate buffer.

[0407] The formulations of the present invention may also contain more than one active ingredient, preferably those having complementary activities and not adversely affecting each other, depending on the actual needs of the particular indication being treated.

[0408] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules used, respectively, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in coarse emulsions. Such techniques are disclosed in "Remington's Pharmaceutical Sciences", 16th Edition, Osol, A. Ed. (1980).

[0409] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, such as membranes or microcapsules.

[0410] Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0411] 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 treatment. It should be understood that the antibody, immunoconjugate, or pharmaceutical composition of the present invention can be used to treat a disease or disorder.

[0412] As used herein, the term "treatment" (and its grammatical variants such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of an individual being treated and can be used for prevention or during a clinical pathological process. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis. In some embodiments, the antibodies of the present invention are used to delay the development or slow the progression of a disease.

[0413] The antibodies or immunoconjugates of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intralung, and intranasal administration, and, in cases where local treatment is desired, can be administered intracelesionally, intrauterinely, or intravesically. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example by injection, such as intravenous injection or subcutaneous injection, depending in part on whether the administration is to be short-term or long-term. A variety of dosing regimens are contemplated herein, including but not limited to single or multiple administrations, bolus administration, and pulse infusion at different time points.

[0414] The antibodies or immunoconjugates of the present invention will be formulated, dosed, and administered in a manner that is in keeping 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 to which the agent is to be delivered, the method of administration, the timing of administration, and other factors known to the medical practitioner. The antibodies or immunoconjugates need not (but optionally can) be formulated with one or more agents currently used to prevent or treat the disorder being considered. The effective amount of such other agents depends on the amount of antibody or immunoconjugate present in the formulation, the type of disorder or treatment, and the other factors discussed above. These agents are typically used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and by any route determined to be appropriate by empirical / clinical means.

[0415] For the prevention or treatment of a disease, the appropriate dosage of an antibody or immunoconjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend 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 being administered for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody or immunoconjugate, and the judgment of the attending physician. The antibody or immunoconjugate is suitable for administration to a patient either as a single dose or in a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage of the antibody or immunoconjugate of from about 1 μg / kg to 15 mg / kg (e.g., from 0.1 mg / kg to 10 mg / kg) may be administered to the patient, either, for example, by one or more separate administrations or by continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg or higher, depending on the above factors. For repeated administration over several days or longer, treatment will generally continue according to the condition until a desired suppression of disease symptoms is achieved. An exemplary dosage of the antibody or immunoconjugate will be in the range of from about 0.05 mg / kg to about 10 mg / kg. Thus, a single dose or multiple doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered at intervals, for example, weekly or every three weeks (e.g., such that the patient receives from about two doses to about twenty doses, or, for example, about six doses of the antibody). Higher initial loading doses may be administered, followed by one or more lower doses. However, other dosage regimens are also useful. The progress of the therapy can be readily monitored by conventional techniques and assays.

[0416] In one embodiment, the invention 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 the treatment of L1-CAM (CD171)-associated cancer.

[0417] As preferably understood herein, the L1-CAM (CD171)-related cancers are selected from 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 tumor, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, 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 granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular adenocarcinoma, prostate cancer, and head and neck squamous cell carcinoma.

[0418] The antibody or immunoconjugate of the present invention can be used alone or in combination with other agents in treatment. For example, the antibody or immunoconjugate of the present invention can be co-administered with at least one additional therapeutic agent.

[0419] Such combination therapies as described above include co-administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administration, in which case the administration of the antibody or immunoconjugate of the present invention can be carried out before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The antibody or immunoconjugate of the present invention can also be used in combination with radiotherapy.

[0420] Preferably, the antibody or its antigen-binding fragment or immunoconjugate is administered to a subject together with an additional therapeutic agent selected from alkylating agents, platinum-based agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian function inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormone agents, immune checkpoint inhibitors, and bisphosphonate therapeutic agents.

[0421] Preferably, the antibody or its antigen-binding fragment or the immunoconjugate of the present invention is administered to a subject together with an additional therapeutic agent, wherein the additional therapeutic agent can be an agent that sensitizes cells to radiotherapy, such as those selected from protein kinase inhibitors and DNA intercalating agents. Preferably, the protein kinase inhibitor is selected from 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, ruxolitinib, 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 intercalating agent is selected from doxorubicin and nemorubicin.

[0422] The agent that sensitizes cells to radiotherapy can also be AZD7648:

[0423]

[0424] Based on the above disclosure, it should be clear to those skilled in the art that the present invention also relates to the antibody or its antigen-binding fragment of the present invention, the immunoconjugate of the present invention or the pharmaceutical composition of the present invention for manufacturing a drug for treating L1-CAM-related cancers.

[0425] Based on the above disclosure, it should be clear to those skilled in the art that the present invention also relates to a method for treating L1-CAM-related cancers, which method comprises administering to an individual in need thereof the antibody or its antigen-binding fragment of the present invention, the immunoconjugate of the present invention or the pharmaceutical composition of the present invention. It should be understood that the antibody or its antigen-binding fragment of the present invention, or the immunoconjugate of the present invention, or the pharmaceutical composition of the present invention is administered in a therapeutically effective amount.

[0426] "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.

[0427] An "effective amount" of a medicament (e.g., a pharmaceutical formulation) is an amount effective to achieve the desired therapeutic or prophylactic result at a required dosage and for a required period of time.

[0428] The present invention also relates to the antibodies of the present invention, or antigen-binding fragments thereof, or immunoconjugates of the present invention for use in diagnosis. The antibodies, antigen-binding fragments and immunoconjugates of the present invention are particularly useful in the diagnosis of L1-CAM (CD171)-related cancers as described herein.

[0429] Preferably, the L1-CAM (CD171)-related cancer is selected from 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 tumor, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, 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 granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular adenocarcinoma, prostate cancer and head and neck squamous cell carcinoma.

[0430] As is well known to those skilled in the art, an antibody or an antigen-binding fragment thereof can be used in a diagnostic method, particularly an in vitro diagnostic method, wherein the antibody or the fragment is used to detect L1-CAM-positive cells in a tissue, and wherein the antibody or the fragment is subsequently detected by a secondary antibody (preferably a monoclonal antibody).

[0431] As covered by the present invention, a diagnostic immunoconjugate comprises an active agent, wherein the active agent is a moiety capable of detecting and localizing the immunoconjugate. Accordingly, the active agent is not particularly limited as long as it is suitable for diagnostic purposes, and is preferably selected from radionuclides, MRI-active compounds, ultrasound contrast agents, fluorophores (preferably fluorophores in the far red / near infrared spectral region), labels for PET and SPECT, and Gd-based and Fe-based particulate MRI contrast agents. Suitable imaging methods allowing for diagnostic applications are known to those skilled in the art. Preferably, the active agent is a radionuclide. Preferably, the radionuclide is a diagnostic radionuclide, preferably 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.

[0432] Particularly useful in diagnosis are the antibody-radionuclide conjugates of the present invention, wherein the radionuclide is a diagnostic radionuclide, preferably 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, as described above.

[0433] Other embodiments and / or implementations of the present invention are disclosed in the following numbered entries.

[0434] 1. An antibody or an antigen-binding fragment thereof that specifically binds to L1-CAM (CD171), the antibody or the antigen-binding fragment thereof comprising:

[0435] A variable heavy chain region, the variable heavy chain region comprising:

[0436] CDR-H1 characterized by a sequence selected from:

[0437] The sequence according to SEQ ID NO.: 1 (GYWMH),

[0438] The sequence according to SEQ ID NO.: 2 (GYYMH),

[0439] The sequence according to SEQ ID NO.: 3 (GYFMH), and

[0440] The sequence according to SEQ ID NO.: 4 (GYLMH); and

[0441] CDR-H2 characterized by a sequence selected from:

[0442] The sequence according to SEQ ID NO.: 5 (EINPSNGRTNYNERFQG),

[0443] The sequence according to SEQ ID NO.: 6 (EINPSNGRTNYNEKFQG),

[0444] The sequence according to SEQ ID NO.: 7 (EINPSNGRTNYNERFKS),

[0445] The sequence according to SEQ ID NO.: 8 (EINPSNGRTNYNERLKS),

[0446] The sequence according to SEQ ID NO.: 9 (EINPSNARTNYNERFQG),

[0447] The sequence according to SEQ ID NO.: 10 (EINPSNARTNYNEKFQG),

[0448] The sequence according to SEQ ID NO.: 11 (EINPSNARTNYNERFKS) and

[0449] The sequence according to SEQ ID NO.: 12 (EINPSNARTNYNERLKS); and

[0450] A CDR-H3 characterized by the sequence according to SEQ ID NO.: 13 (DYYGTSYNFDY); and / or

[0451] A variable light chain region comprising:

[0452] A CDR-L1 characterized by a sequence selected from:

[0453] The sequence according to SEQ ID NO.: 14 (RANEDINNRLA),

[0454] The sequence according to SEQ ID NO.: 15 (KANEDINNRLA),

[0455] The sequence according to SEQ ID NO.: 16 (QANEDINNRLA),

[0456] The sequence according to SEQ ID NO.: 17 (RANEDINARLA),

[0457] The sequence according to SEQ ID NO.: 18 (KANEDINARLA),

[0458] The sequence according to SEQ ID NO.: 19 (QANEDINARLA),

[0459] The sequence according to SEQ ID NO.: 20 (RANEDINLRLA),

[0460] The sequence according to SEQ ID NO.:21 (KANEDINLRLA), and

[0461] The sequence according to SEQ ID NO.:22 (QANEDINLRLA); and

[0462] CDR-L2 characterized by a sequence selected from the following:

[0463] The sequence according to SEQ ID NO.:23 (GATNLVT) and

[0464] The sequence according to SEQ ID NO.:24 (GASNLVS); and

[0465] CDR-L3 characterized by a sequence selected from the following:

[0466] The sequence according to SEQ ID NO.:25 (QQYWSTPFT),

[0467] The sequence according to SEQ ID NO.:26 (QQYYSTPFT) and

[0468] The sequence according to SEQ ID NO.:27 (QQYFSTPFT).

[0469] 2. The antibody or antigen-binding fragment thereof according to item 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, chimeric antibody, recombinant antibody, antigen-binding fragment of a recombinant antibody, single-chain antibody, humanized antibody, bispecific antibody, multispecific antibody, or an antibody displayed on the surface of a phage or an antibody displayed on the surface of a chimeric antigen receptor (CAR) T cell, preferably, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, preferably an IgG1 antibody.

[0470] 3. The antibody or antigen-binding fragment thereof according to item 1 or 2, wherein the variable heavy chain region comprises CDR-H1 characterized by the sequence according to SEQ ID NO.:1.

[0471] 4. The antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the variable heavy chain region comprises CDR-H2 characterized by the sequence according to SEQ ID NO.:5, 6, 9 or 10, preferably characterized by the sequence according to SEQ ID NO.:5 or 6.

[0472] 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 according to SEQ ID NO.: 14, 15, 17, or 18 or 21, preferably characterized by the sequence according to SEQ ID NO.: 14 or 15.

[0473] 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 according to SEQ ID NO.: 23.

[0474] 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 according to SEQ ID NO.: 25.

[0475] 8. The antibody or antigen-binding fragment thereof according to item 1 or 2, wherein the variable heavy chain region is characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical, to a sequence selected from:

[0476] The sequence according to SEQ ID NO.: 28 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS),

[0477] The sequence according to SEQ ID NO.: 29 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTVSS),

[0478] The sequence according to SEQ ID NO.: 30 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFTGYWMHWIRQPPGKGLEWIGEINPSNGRTNYNERLKSRVTLSVDKSKNQASLKLSSVTAADTAVYFCARDYYGTSYNFDYWGQGTLVTVSS),

[0479] The sequence according to SEQ ID NO.: 31 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), and

[0480] The sequence according to SEQ ID NO.: 32 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWmGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), preferably characterized by the sequence according to SEQ ID NO.: 28 or the sequence according to SEQ ID NO.: 32.

[0481] 9. The antibody or antigen-binding fragment thereof according to item 1 or 2, wherein the variable light chain region is characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical, to a sequence selected from:

[0482] The sequence according to SEQ ID NO.: 33 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK),

[0483] The sequence according to SEQ ID NO.: 34 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDIATYYCQQYWSTPFTFGQGTKLEIK),

[0484] The sequence according to SEQ ID NO.: 35 (EIVMTQSPATLSVSPGERATLSCRANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEFTLTISSLQSEDFAVYYCQQYWSTPFTFGQGTKLEIK),

[0485] The sequence according to SEQ ID NO.: 36 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), and

[0486] The sequence according to SEQ ID NO.: 37 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGAsNLVsGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), preferably characterized by a sequence selected from: the sequence according to SEQ ID NO.: 33, the sequence according to SEQ ID NO.: 36, and the sequence according to SEQ ID NO.: 37.

[0487] 10. An antibody or antigen-binding fragment thereof according to any one of items 1 to 9,

[0488] wherein the variable heavy chain region is characterized by the sequence according to SEQ ID NO.: 28, and the variable light chain region is characterized by the sequence according to SEQ ID NO.: 33; or

[0489] wherein the variable heavy chain region is characterized by the sequence according to SEQ ID NO.: 32, and the variable light chain region is characterized by the sequence according to SEQ ID NO.: 36; or

[0490] wherein the variable heavy chain region is characterized by the sequence according to SEQ ID NO.: 14, and the variable light chain region is characterized by the sequence according to SEQ ID NO.: 37.

[0491] 11. An antibody or antigen-binding fragment thereof according to item 1 or 2,

[0492] wherein the variable heavy chain region is characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical, to the following sequence:

[0493] FH0-CDR-H1-FH1-CDR-H2-FH2-CDR-H3-FH3

[0494] where CDR-H1, CDR-H2, and CDR-H3 are as defined in claim 1, wherein:

[0495] FH0 is characterized by the sequence according to SEQ ID NO.: 38 (QVQLVQSGAEVKKPGASVKVSCKASGYTFT) or the sequence according to SEQ ID NO.: 39 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFT),

[0496] FH1 is characterized by the sequence according to SEQ ID NO.: 40 (WVRQAPGQGLEWIG) or the sequence according to SEQ ID NO.: 41 (WIRQPPGKGLEWIG)

[0497] FH2 is characterized by the sequence according to SEQ ID NO.: 42 (RVTLTVDKSISTAYMELSRLRSDDTAVYFCAR) or the sequence according to SEQ ID NO.: 43 (RVTLSVDKSKNQASLKLSSVTAADTAVYFCAR), and

[0498] FH3 is characterized by the sequence according to SEQ ID NO.: 44 (WGQGTLVTVSS),

[0499] And / or

[0500] wherein the variable light chain region is characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical, to the following sequence:

[0501] FL0-CDR-L1-FL1-CDR-L2-FL2-CDR-L3-FL3

[0502] where CDR-L1, CDR-L2, and CDR-L3 are as defined in claim 1, wherein:

[0503] FL0 is characterized by the sequence according to SEQ ID NO.: 45 (DIQMTQSPSSLSASVGDRVTITC) or the sequence according to SEQ ID NO.: 46 (EIVMTQSPATLSVSPGERATLSC),

[0504] FL1 is characterized by the sequence according to SEQ ID NO.: 47 (WYQQKPGKAPKLLIS) or the sequence according to SEQ IDNO.: 48 (WYQQKPGQAPRLLIS),

[0505] FL2 is characterized by the sequence according to SEQ ID NO.: 49 (GVPSRFSGSGSGKDYTLTISSLQPEDIATYYC) or the sequence according to SEQ ID NO.: 50 (GIPARFSGSGSGKEFTLTISSLQSEDFAVYYC), and

[0506] FL3 is characterized by the sequence according to SEQ ID NO.: 51 (FGQGTKLEIK).

[0507] 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, and the at least one point mutation affects antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), serum half-life and / or the glycosylation state of the antibody.

[0508] 13. The antibody or antigen-binding fragment thereof according to item 11, 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, preferably, wherein the at least one point mutation is selected from L234A, L235A, P331S and N297A.

[0509] 14. The antibody or antigen-binding fragment thereof according to any one of items 1 to 13, characterized in that the dissociation constant K of the antibody to L1-CAM (CD171) D does not exceed 10 -12 M measured in a Biacore-based assay.

[0510] 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.

[0511] 16. A host cell comprising the polynucleotide according to item 15.

[0512] 17. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 14 and an active agent, preferably, wherein the active agent is a cytotoxic agent or a prodrug thereof.

[0513] 18. The immunoconjugate according to item 17, wherein the antibody or antigen-binding fragment thereof is linked to the active agent through a linker moiety, preferably, wherein the linker moiety comprises a polymeric carrier linked to at least one active agent.

[0514] 19. An immunoconjugate according to item 17 or 18, wherein the active agent is a radionuclide, preferably a radionuclide for therapeutic applications and / or a radionuclide for diagnostic use, and the radionuclide for therapeutic applications is preferably selected from copper-67, strontium-89, yttrium-90, iodine-131, samarium-153, terbium-161, lutetium-177, astatine-211, radium-223, and actinium-225, and the radionuclide for diagnostic use is preferably 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.

[0515] 20. A pharmaceutical composition comprising 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, and a pharmaceutically acceptable carrier.

[0516] 21. 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, which is used as a drug.

[0517] 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, which is used for treating L1-CAM (CD171)-related cancers.

[0518] 23. The antibody or antigen-binding fragment thereof for the use according to item 22 or the immunoconjugate for the use according to item 22, wherein the L1-CAM (CD171)-related cancers are selected from 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 tumor, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, 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 granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular adenocarcinoma, prostate cancer, and head and neck squamous cell carcinoma.

[0519] 24. An antibody or antigen-binding fragment thereof for use in the use according to item 22 or 23, or an immunoconjugate for use in the use, wherein the antibody or antigen-binding fragment thereof or the immunoconjugate is administered to a subject in combination with an additional therapeutic agent selected from the group consisting of alkylating agents, platinum-based agents, taxanes, vinca agents, antiestrogen drugs, aromatase inhibitors, ovarian function inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, immune checkpoint inhibitors, and bisphosphonate therapeutic agents.

[0520] 25. 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 diagnosis.

[0521] 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)-related cancers.

[0522] 27. The antibody or antigen-binding fragment thereof for use in the use according to item 26, or the immunoconjugate for use in the use, wherein the L1-CAM (CD171)-related cancers are 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 tumor, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, 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 granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular adenocarcinoma, prostate cancer, and head and neck squamous cell carcinoma.

[0523] Other embodiments or implementations of the present invention are disclosed in the clauses numbered below.

[0524] 1. An antibody or antigen-binding fragment thereof that specifically binds to L1-CAM (CD171), the antibody or antigen-binding fragment thereof comprising:

[0525] A variable heavy chain region, the variable heavy chain region comprising:

[0526] CDR-H1 characterized by a sequence selected from the following:

[0527] The sequence according to SEQ ID NO.: 1 (GYWMH),

[0528] The sequence according to SEQ ID NO.: 2 (GYYMH),

[0529] The sequence according to SEQ ID NO.: 3 (GYFMH), and

[0530] The sequence according to SEQ ID NO.: 4 (GYLMH); and

[0531] CDR-H2 characterized by a sequence selected from the following:

[0532] The sequence according to SEQ ID NO.: 5 (EINPSNGRTNYNERFQG),

[0533] The sequence according to SEQ ID NO.: 6 (EINPSNGRTNYNEKFQG),

[0534] The sequence according to SEQ ID NO.: 7 (EINPSNGRTNYNERFKS),

[0535] The sequence according to SEQ ID NO.: 8 (EINPSNGRTNYNERLKS),

[0536] The sequence according to SEQ ID NO.: 9 (EINPSNARTNYNERFQG),

[0537] The sequence according to SEQ ID NO.: 10 (EINPSNARTNYNEKFQG),

[0538] The sequence according to SEQ ID NO.: 11 (EINPSNARTNYNERFKS) and

[0539] The sequence according to SEQ ID NO.: 12 (EINPSNARTNYNERLKS); and

[0540] CDR-H3 characterized by the sequence according to SEQ ID NO.: 13 (DYYGTSYNFDY); and / or

[0541] Variable light chain region, the variable light chain region comprising:

[0542] CDR-L1 characterized by a sequence selected from the following:

[0543] The sequence according to SEQ ID NO.: 14 (RANEDINNRLA),

[0544] The sequence according to SEQ ID NO.: 15 (KANEDINNRLA),

[0545] The sequence according to SEQ ID NO.: 16 (QANEDINNRLA),

[0546] The sequence according to SEQ ID NO.: 17 (RANEDINARLA),

[0547] The sequence according to SEQ ID NO.: 18 (KANEDINARLA),

[0548] The sequence according to SEQ ID NO.: 19 (QANEDINARLA),

[0549] The sequence according to SEQ ID NO.: 20 (RANEDINLRLA),

[0550] The sequence according to SEQ ID NO.: 21 (KANEDINLRLA), and

[0551] The sequence according to SEQ ID NO.: 22 (QANEDINLRLA); and

[0552] CDR-L2 characterized by a sequence selected from the following:

[0553] The sequence according to SEQ ID NO.: 23 (GATNLVT) and

[0554] The sequence according to SEQ ID NO.: 24 (GASNLVS); and

[0555] CDR-L3 characterized by a sequence selected from the following:

[0556] The sequence according to SEQ ID NO.: 25 (QQYWSTPFT),

[0557] The sequence according to SEQ ID NO.: 26 (QQYYSTPFT) and

[0558] The sequence according to SEQ ID NO.: 27 (QQYFSTPFT),

[0559] Preferably, the antibody or antigen-binding fragment thereof is a monoclonal antibody, chimeric antibody, recombinant antibody, antigen-binding fragment of a recombinant antibody, single-chain antibody, humanized antibody, bispecific antibody, multispecific antibody, or an antibody displayed on the surface of a phage or an antibody displayed 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.

[0560] 2. The antibody or antigen-binding fragment thereof according to clause 1, wherein the variable heavy chain region comprises a CDR-H1 characterized by the sequence according to SEQ ID NO.: 1, and / or

[0561] wherein the variable heavy chain region comprises a CDR-H2 characterized by the sequence according to SEQ ID NO.: 5, 6, 9 or 10, preferably characterized by the sequence according to SEQ ID NO.: 5 or 6, and / or

[0562] wherein the variable light chain region comprises a CDR-L1 characterized by the sequence according to SEQ ID NO.: 14, 15, 17, 18 or 21, preferably characterized by the sequence according to SEQ ID NO.: 14 or 15, and / or

[0563] wherein the variable light chain comprises a CDR-L2 characterized by the sequence according to SEQ ID NO.: 23, and / or

[0564] wherein the variable light chain comprises a CDR-L3 characterized by the sequence according to SEQ ID NO.: 25.

[0565] 3. The antibody or antigen-binding fragment thereof according to clause 1 or 2, wherein the variable heavy chain region is characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical, to a sequence selected from:

[0566] The sequence according to SEQ ID NO.: 28 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS),

[0567] The sequence according to SEQ ID NO.: 29 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTVSS),

[0568] The sequence according to SEQ ID NO.: 30 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFTGYWMHWIRQPPGKGLEWIGEINPSNGRTNYNERLKSRVTLSVDKSKNQASLKLSSVTAADTAVYFCARDYYGTSYNFDYWGQGTLVTVSS),

[0569] The sequence according to SEQ ID NO.: 31 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), and

[0570] The sequence according to SEQ ID NO.: 32 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWmGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), preferably characterized by the sequence according to SEQ ID NO.: 28 or the sequence according to SEQ ID NO.: 32;

[0571] And / or

[0572] Wherein the variable light chain region is characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical, to a sequence selected from:

[0573] The sequence according to SEQ ID NO.: 33 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK),

[0574] The sequence according to SEQ ID NO.: 34 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDIATYYCQQYWSTPFTFGQGTKLEIK),

[0575] The sequence according to SEQ ID NO.: 35 (EIVMTQSPATLSVSPGERATLSCRANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEFTLTISSLQSEDFAVYYCQQYWSTPFTFGQGTKLEIK),

[0576] The sequence according to SEQ ID NO.: 36 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), and

[0577] The sequence according to SEQ ID NO.: 37 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGAsNLVsGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), preferably characterized by a sequence selected from:

[0578] The sequence according to SEQ ID NO.: 33, the sequence according to SEQ ID NO.: 36 and the sequence according to SEQ ID NO.: 37.

[0579] 4. The antibody or antigen-binding fragment thereof according to any one of clauses 1 to 3,

[0580] wherein the variable heavy chain region is characterized by the sequence according to SEQ ID NO.: 28, and the variable light chain region is characterized by the sequence according to SEQ ID NO.: 33; or

[0581] wherein the variable heavy chain region is characterized by the sequence according to SEQ ID NO.: 32, and the variable light chain region is characterized by the sequence according to SEQ ID NO.: 36; or

[0582] Wherein the variable heavy chain region is characterized by the sequence according to SEQ ID NO.: 14, and the variable light chain region is characterized by the sequence according to SEQ ID NO.: 37.

[0583] 5. The antibody or antigen-binding fragment thereof according to clause 1,

[0584] wherein the variable heavy chain region is characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical, to the following sequence:

[0585] FH0-CDR-H1-FH1-CDR-H2-FH2-CDR-H3-FH3

[0586] where CDR-H1, CDR-H2 and CDR-H3 are as defined in clause 1, wherein:

[0587] FH0 is characterized by the sequence according to SEQ ID NO.: 38 (QVQLVQSGAEVKKPGASVKVSCKASGYTFT) or the sequence according to SEQ ID NO.: 39 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFT),

[0588] FH1 is characterized by the sequence according to SEQ ID NO.: 40 (WVRQAPGQGLEWIG) or the sequence according to SEQ ID NO.: 41 (WIRQPPGKGLEWIG)

[0589] FH2 is characterized by the sequence according to SEQ ID NO.: 42 (RVTLTVDKSISTAYMELSRLRSDDTAVYFCAR) or the sequence according to SEQ ID NO.: 43 (RVTLSVDKSKNQASLKLSSVTAADTAVYFCAR), and

[0590] FH3 is characterized by the sequence according to SEQ ID NO.: 44 (WGQGTLVTVSS),

[0591] and / or

[0592] wherein the variable light chain region is characterized by a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical, to the following sequence:

[0593] FL0-CDR-L1-FL1-CDR-L2-FL2-CDR-L3-FL3

[0594] where CDR-L1, CDR-L2 and CDR-L3 are as defined in claim 1, wherein:

[0595] FL0 is characterized by the sequence according to SEQ ID NO.: 45 (DIQMTQSPSSLSASVGDRVTITC) or the sequence according to SEQ ID NO.: 46 (EIVMTQSPATLSVSPGERATLSC),

[0596] FL1 is characterized by the sequence according to SEQ ID NO.: 47 (WYQQKPGKAPKLLIS) or the sequence according to SEQ ID NO.: 48 (WYQQKPGQAPRLLIS),

[0597] FL2 is characterized by the sequence according to SEQ ID NO.: 49 (GVPSRFSGSGSGKDYTLTISSLQPEDIATYYC) or the sequence according to SEQ ID NO.: 50 (GIPARFSGSGSGKEFTLTISSLQSEDFAVYYC), and

[0598] FL3 is characterized by the sequence according to SEQ ID NO.: 51 (FGQGTKLEIK).

[0599] 6. The antibody or antigen-binding fragment thereof according to any one of clauses 1 to 5, wherein the heavy chain further comprises at least one point mutation in the Fc portion, and the at least one point mutation affects antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), serum half-life and / or the glycosylation state of the antibody. Preferably, 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, and more preferably, the at least one point mutation is selected from L234A, L235A, P331S and N297A.

[0600] 7. The antibody or antigen-binding fragment thereof according to any one of clauses 1 to 6, characterized in that the dissociation constant K of the antibody for L1-CAM (CD171) D does not exceed 10 -12 M measured in a Biacore-based assay.

[0601] 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.

[0602] 9. A host cell comprising the polynucleotide according to clause 8.

[0603] 10. An immunoconjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of clauses 1 to 7 and an active agent, preferably, wherein the active agent is a cytotoxic agent or a prodrug thereof,

[0604] Preferably, wherein the antibody or an antigen-binding fragment thereof is linked to the active agent through a linker moiety, preferably, wherein the linker moiety comprises a polymeric carrier linked to at least one active agent,

[0605] and / or

[0606] Preferably, wherein the active agent is a radionuclide, preferably a radionuclide for therapeutic applications and / or a radionuclide for diagnostic use, the radionuclide for therapeutic applications being preferably selected from copper-67, strontium-89, yttrium-90, iodine-131, samarium-153, terbium-161, lutetium-177, astatine-211, radium-223 and actinium-225, and the radionuclide for diagnostic use being preferably 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.

[0607] 11. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of clauses 1 to 7 or the immunoconjugate as described in clause 10, and a pharmaceutically acceptable carrier.

[0608] 12. An antibody or an antigen-binding fragment thereof as described in any one of clauses 1 to 7 or the immunoconjugate as described in clause 10, for use as a medicament.

[0609] 13. An antibody or an antigen-binding fragment thereof as described in any one of clauses 1 to 7 or the immunoconjugate as described in clause 10, for use in the treatment of L1-CAM (CD171)-related cancers,

[0610] Preferably, the L1-CAM (CD171)-related cancers are selected from 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 tumor, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, 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 granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular adenocarcinoma, prostate cancer, and head and neck squamous cell carcinoma,

[0611] and / or

[0612] Preferably, the antibody or its antigen-binding fragment or the immunoconjugate is administered to a subject in combination with an additional therapeutic agent selected from alkylating agents, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian function inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormone agents, immune checkpoint inhibitors, and bisphosphonate therapeutic agents.

[0613] 14. The antibody or its antigen-binding fragment according to any one of clauses 1 to 7 or the immunoconjugate according to clause 10, for use in diagnosis.

[0614] 15. An antibody or antigen-binding fragment thereof as described in any one of clauses 1 to 7, or an immunoconjugate as described in clause 10, for use in diagnosing L1-CAM (CD171)-associated cancer, preferably, wherein the L1-CAM (CD171)-associated cancer is selected from 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 tumor, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, 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 granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular adenocarcinoma, prostate cancer, and head and neck squamous cell carcinoma.

[0615] The present invention will be illustrated in the following examples, which, however, are not to be construed as limiting. It should be noted that, unless otherwise expressly indicated to the contrary, the present invention may relate to each antibody or antigen-binding fragment thereof (especially when defined by amino acid sequence), and / or each polynucleotide disclosed in the examples.

[0616] Example

[0617] Example 1: Humanization of the CE7 mouse monoclonal antibody

[0618] The CE7 murine antibody was humanized by transplanting the three CDRs defined according to the Kabat nomenclature from the light chain variable region (VL) into a human germline VL that is as homologous as possible to the murine antibody VL. Similarly, the three CDRs from the heavy chain variable region (VH) were transplanted into a human germline VH that is as homologous as possible to the murine antibody VH. In addition, several amino acid residues in the framework regions of the selected human germline variable regions were replaced with the amino acid residues present in the murine variable regions (so-called back mutations). Based on the information on the structure of immunoglobulin variable regions and guided by the homology molecular model of the Fv of the CE7 murine monoclonal antibody, these few residues in the framework regions were determined to be crucial in maintaining the CDRs in the correct conformation or in VH / VL packing, and they were thus retained in the humanized version A or, if possible, replaced by their germline counterparts in subsequent humanized versions. Guided by the homology molecular model, in the subsequent version B, where judged feasible, the CDR residues defined according to Kabat were also replaced by their germline counterparts (so-called germlining) in order to increase the degree of humanization (i.e., the percentage sequence identity of both VH and VL between the humanized version and the closest human germline used as the recipient sequence for CDR grafting).

[0619] The added value of combining structural models with pure sequence analysis lies in the potential to distinguish residues facing the epitope from non-epitope residues in the CDR regions. The purpose of the structural model is to allow the extension of the limitations of the humanization process beyond just CDR grafting. Moreover, the structural model allows for a more informed choice of back mutations based on the specific germline involved. It should be noted that the structure defined by the Kabat CDR is not as strict as those defined by other systems; thus, for some germlines, the Kabat definition is too broad. For both the heavy and light chains, we can generally be quite confident that the division of residues from CDR1 and CDR2 into epitope and non-epitope based on the structural model is correct. Similarly, the light chain CDR3 is usually well-described with a high probability. The difficult case is always the heavy chain CDR3.

[0620] The humanized versions A of VH and VL provided herein are conservative versions that clearly minimize / avoid CDR residue alterations and are expected to exhibit similar binding / potency activities to the chimeric antibodies (CE7 murine VH and VL fused to human constant regions) used as reference molecules. The subsequent humanized versions B of VH and VL were designed to achieve a higher percentage sequence identity with the closest human germline (up to as close as 85%). This can be achieved by germlining the framework and / or Kabat CDR amino acid residues (replacing murine residues with their corresponding germline residues).

[0621] For the design of the CDR-grafted version of CE7 murine VH, two human germlines were selected, namely IGHV1-2*06 and IGHV4-34*01. The sequence identities of these two human germlines over the entire V gene were 66.3% and 53.1%, respectively.

[0622] For the design of the CDR-grafted version of CE7 murine VL, three human germlines were selected; IGKV1-NL1*01, IGKV1-33*01 and IGKV3-15*01 were selected. The sequence identities of these three human germlines over the entire V gene with CE7 murine VL were 73.7%, 71.6% and 60.0%, respectively.

[0623] The first step in the humanization process should be to select the best heavy and light chain combinations between the humanized VH and humanized VL versions. For VH, we have humanized versions based on two different human germlines: IGHV1-2*06 with 4 versions (A to D) and IGHV4-34*01 with 3 versions (A to C). This results in a total of 7 different humanized VH. For VL, we have humanized versions based on three human germlines, each of which has 4 versions (A to D). This results in a total of 12 different humanized VL. To obtain all possible combinations between humanized VH and VL, a total of 7 (VH) × 12 (VL) = 84 humanized variants must be generated and purified.

[0624] By testing only version A and version B of the VH humanized versions based on the human germline IGHV1-2*06, and only the combinations of version A of the humanized versions based on the human germline IGHV4-34*01 with version A and version B of the VL humanized versions based on the human germlines IGKV1-NL1*01, IGKV1-33*01 and IGKV3-15*01, this number can be reduced to 18; we will have 3 VH × 6 VL = 18 combinations.

[0625] Based on the results obtained with these 18 humanized mAbs, the best one or two VH / VL combinations will be selected, and then the subsequent versions B, C and D can be tested.

[0626] Table E1 .

[0627]

[0628]

[0629] The best heavy and light chain combinations between the 18 variants should be selected by evaluating the following criteria:

[0630] · The transient expression level (compared to the chimeric version) of the humanized version (such as human IgG1 / κ) produced in mammalian cells (HEK 293 or preferably CHO). Purification is carried out by ELISA or Octet protein A measurement using the TC supernatant of transfected cells before harvest.

[0631] · Binding ability compared to the CE7 chimeric human IgG1 / κ version (EC50 measured by ELISA or FACS; or preferably Kd measured by Biacore or Octet) (this chimeric means the combination of parental murine VH and VL fused to the human constant region).

[0632] · The biological activity of the humanized version in relevant in vitro cell assays compared to the reference CE7 chimeric antibody.

[0633] · Cross-reactivity (in vitro binding activity) with relevant orthologous species (if relevant)

[0634] · Determination of the biophysical properties of the humanized version compared to CE7 chimeric:

[0635] ο SEC-HPLC curve to determine the high molecular weight soluble aggregate level,

[0636] ο SDS-PAGE under non-reducing and reducing conditions,

[0637] ο Differential scanning calorimetry (DSC) analysis using a Microcal VP-capillary DSC system to determine the Tm of Fab, CH2, and CH3.

[0638] Once the best 2 humanized VH and VL pairings are determined, the second round of humanization can be initiated, in which the results of sequence susceptibility variants (see "Sequence Susceptibility" section) will be considered. The positive CDR mutations identified in the sequence susceptibility variants will be introduced into the CDRs of the best 2 humanized VH / VL combinations to obtain the final humanized candidates. The VH and VL humanized versions (C and D of IGHV1-2*01; B and C of IGHV4-34*01) and versions C and D of all light chains can also be tested. Evaluation will be carried out using the same criteria as those used for the first-round candidates above to select the best final candidate.

[0639] Humanization of murine CE7 monoclonal antibody using standard CDR grafting techniques. The principle of this method is to engineer a human antibody that contains only the complementarity-determining regions (CDRs) from the murine monoclonal antibody, with the aim of reducing its immunogenicity when used as a human therapeutic agent. Humanization by CDR grafting requires the antigen-binding residues from the murine antibody to be retained in the humanized antibody; thus, the identification of these residues clearly plays an important role in the protocol. To guide the humanization process and help decide whether to retain the parental murine residues or replace them with their human germline counterparts, a homology molecular model of the Fv of the CE7 murine monoclonal antibody was constructed.

[0640] The CDR grafting protocol used is a modernized version of the method pioneered by Greg Winter and his colleagues at the Medical Research Council, Cambridge, UK. The definition of CDRs is based on the Kabat nomenclature. Selection of the human framework acceptor regions grafted with the CE7 murine CDR regions was accomplished by using the CE7 murine variable region sequences as input to search the IMGT murine and human V gene databases with IgBLAST (http: / / www.ncbi.nlm.nih.gov / igblast / ), which was developed by NCBI for analyzing immunoglobulin V region sequences. The strategy applied was to use human germline sequences that are natural human sequences without the specific somatic mutations found in individual human antibody sequences.

[0641] Sequence susceptibility

[0642] During the past decade, drug developers have become increasingly proactive in early identification and risk assessment to reduce the drug attrition rate. One strategy commonly used to achieve drug-like properties and avoid late-stage failures is the so-called developability or molecular evaluation approach, which aims to optimize the biophysical and chemical properties of molecules before product development. In addition to good biophysical properties (Jain et al., 2017 PNAS; 114:944-949), mAbs also need to have sufficient chemical stability to meet strict process development parameters. The main chemical modifications of interest for mAbs are oxidation (Met, M and Trp, W), deamidation (Asn, N) and isomerization (Asp, D). Therefore, determining the susceptibility of degradation sequences for re-engineering before investing resources in process and product development is expected to reduce complex problems in formulation development and increase the likelihood of successfully developing liquid formulations. Based on a recent study on the deamidation and isomerization susceptibilities of 131 clinical-stage antibodies published in MABS (Lu et al., 2019 MABS, 11:45-57), we analyzed the deamidation and isomerization motifs reported to be modified in the CDRs of CE7 VH and VL. The sequences of the CDRs defined according to Kabat nomenclature are underlined. Within the CDRs, potential sequence susceptibilities are shown in bold. Only potential sequence susceptibilities considered to be true sequence susceptibilities are discussed.

[0643]

[0644]

[0645] In CDR1, W is located at Kabat position Kabat H33. Examination of the molecular model showed that this residue is buried, with its side chain flush with the paratope surface and stacked on VH-CDR3. The indole nitrogen may contact the antigen, and its side chain contacts VH-CDR2 residues E(H55), I(H56)(mc), N(H57), R(H64), VH-CDR3 residue Y(H109); substitution of the lowest energy rotamer of W(H33)Y induced excessive strain (30.5) and altered the contact structure; W(H33)F was similarly negative; W(H33)L induced moderate strain (17.6), but lost contact at R(H64) (which may be a way to improve oxidation susceptibility). Therefore, substitutions of F, Y, and L can be attempted, with L most likely to be effective. See the sequences of the testable WH33F, WH33Y, and WH33L variants below.

[0646] In CDR2, the NG motif is located at Kabat positions H54 and H55. Examination of the molecular model revealed that N H54 is surface-exposed, located at the edge of the paratope surface, with its side chain able to contact the antigen. G H55 is surface-exposed, with its side chain on the non-antigen-facing surface of VH-CDR2, and its alpha-carbon does not contact the vernier or CDRs. Substituting A with G H55 seems to be the best option for improving "NG" susceptibility. Therefore, an A substitution at H55 should be attempted. See the sequence of the GH55A variant tested below.

[0647] In CDR3, the NF motif is located at Kabat positions H100B and H100C. Examination of the molecular model revealed that both of these residues are buried, and thus the risk of deamidation is considered low. CE7-VH-chimeric-WH33Y

[0648] QVQLQQPGAELVKPGASVKLSCKASGYTFTGYYMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSS[SEQ ID NO.:56]

[0649] CE7-VH-chimeric-WH33F

[0650] QVQLQQPGAELVKPGASVKLSCKASGYTFTGYFMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSS[SEQ ID NO.:57]

[0651] CE7-VH-chimeric-WH33L

[0652] QVQLQQPGAELVKPGASVKLSCKASGYTFTGYLMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSS[SEQ ID NO.:58]

[0653] CE7-VH-chimeric-GH55A

[0654] QVQLQQPGAELVKPGASVKLSCKASGYTFTGYWMHWVKQRPGHGLEWIGEINPSNARTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSS [SEQ ID NO.:59]

[0655] CE7 VL:

[0656]

[0657] In CDR1, the motif NN is located at Kabat positions L30 and L31. Examination of the molecular model reveals that N L30 is surface-exposed, with its side chain adjacent to VL CDR3 at the edge of the paratope surface and accessible to the antigen. N L31 is surface-exposed, with its side chain at the edge of the paratope surface, away from its center, and its side chain contacts the regulatory region residues G(L80), Y(L87). Substituting N L31 with L or A seems to be the best choice for NN susceptibility. See the sequences of the NL31L and NL31A variants to be tested below.

[0658] In CDR3, W is located at Kabat position L92. Examination of the molecular model reveals that W L92 is surface-exposed, with its side chain near the center of the paratope surface and having a high probability of contacting the antigen. The best solution to address the oxidative susceptibility may be to substitute W with Y (the phenolic ring overlaps with the indole ring, but the binding will remain unchanged) or F. See the sequences of the WL92Y and WL92F variants tested below.

[0659] CE7-VL-chimeric-NL31L

[0660] DIQMTQSSSSFSVSLGDRVTITCKANEDINLRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYWSTPFTFGSGTELEIK [SEQ ID NO.:61]

[0661] CE7-VL-chimeric-NL31A

[0662] DIQMTQSSSSFSVSLGDRVTITCKANEDINARLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYWSTPFTFGSGTELEIK [SEQ ID NO.:62]

[0663] CE7-VL Chimeric-WL92Y

[0664] DIQMTQSSSSFSVSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYYSTPFTFGSGTELEIK[SEQ ID NO.:63]

[0665] CE7-VL Chimeric-WL92F

[0666] DIQMTQSSSSFSVSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYFSTPFTFGSGTELEIK[SEQ ID NO.:64]

[0667] Heavy chain design

[0668] Amino acid differences with the highest homology to murine germline IGHV1S81*02

[0669] Amino Acid Sequence of VH of Mouse CE7 Hybridoma

[0670]

[0671] GTTLTVSS [The shown sequence is the CE7 VH sequence according to SEQ ID NO.:55]

[0672] The identity between the CE7 murine heavy chain variable (VH) region and the murine germline immunoglobulin VH 1S81*02 (IGHV1S81*02) is 91.8% (90 out of a total of 98 residues in the V gene are identical). The 8 residues that differ between the CE7 VH and the murine germline IGHV1S81*02 are underlined in the CE7 VH.

[0673]

[0674] The residues marked in bold are part of the classification of immunoglobulin VH domain frameworks as defined by Honegger and Plückthun (J. Mol. Biol., 2001, 309:687-699). The authors reported that immunoglobulin VH frameworks can be classified into four different types (I to IV) according to the backbone conformation of framework 1 (especially the conformation of Kabat residues 6, 7, 8, and 9). Mouse CE7 VH has Gln at Kabat position H6 and Pro at position H7, which is typical of Honneger type IV. The human germline IGHV1-2*06 and IGHV4-34*01, which will be used as the acceptor sequences for CDR grafting, are both of Honnerger type III (Gln at Kabat position H6 instead of Pro at position H7). See columns N and O of worksheet VH-IGHV1-2, and columns L and M of VH-IGHV4-34.

[0675] Selection of the human framework receptor VH region

[0676] The selection of the human framework acceptor VH region grafted with the CE7 mouse CDR region was completed by using the mouse CE7 VH amino acid sequence as input and retrieving the IMGT human VH gene database with IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / igblast.cgi). Based on the sequence alignment of the parental antibody with the human germline, the most closely matching entries were determined. The best human germlines used as acceptors were identified based on the following ordered criteria: sequence identity within the framework defined by Kabat, and the identity and / or compatibility of the inter-chain interface residues and the supporting loops with the canonical conformation of the parental CDR.

[0677] One of the important criteria for selecting suitable human germline candidates is to ensure that there is nearly 85% sequence identity between the specific germline and the designed humanized version. We analyzed a large number of human germline sequences and selected two human germlines as the best candidates; one belongs to human family 1, namely IGHV1-2*06, and the other belongs to human family 4, namely IGHV4-34*01. The human germline IGHV1-2*06 has a high sequence identity of 66.3% with mouse CE7 VH (65 out of a total of 98 amino acid residues are identical), while the human germline IGHV4-34*01 has a sequence identity of 53.1% (52 out of a total of 98 amino acid residues are identical).

[0678] In framework 4, the murine CE7 VH gene segment corresponding to the J gene was identified as having the highest homology to the murine germline J2 gene (see columns E, F, and G of VH in Table 1, worksheets VH-IGHV1-2 and VH-IGHV4-34). Comparison of the murine J2 segment gene with the human J segment gene in CDR3 and FR4 revealed the highest homology to the human J4*01 segment.

[0679] Design using the IGHV1-2*06 human germline as the framework receptor region

[0680] Sequence alignment between murine CE7 VH and human germline IGHV1-2*06

[0681] The identity between the CE7 murine heavy chain variable (VH) region and the human germline immunoglobulin VH 1-2*06 (IGHV1-2*06) was 66.3% (65 out of a total of 98 residues in the V gene were identical).

[0682] CDR residues defined according to Kabat nomenclature are highlighted in yellow. Residues that differ between murine CE7 VH and human 13 germline IGHV1-2*06 in the framework regions and CDRs are marked in bold and underlined, respectively.

[0683]

[0684] Humanized version A

[0685] Murine CDRs defined according to Kabat nomenclature were transplanted into IGHV1-2*06 to obtain the sequences detailed below. Residues highlighted in green are framework murine residues (non-CDR residues) retained from the parental murine CE7 VH sequence; they were retained because they may be structurally important for maintaining the full activity of the antibody.

[0686] Version A (VHA)

[0687]

[0688] The identity of the humanized version A (CE7-1-2-VHA) with the IGHV1-2*06 human germline was 84.7% (83 out of a total of 98 residues in the V gene were identical). Residues that differ between the humanized version A and the IGHV1-2*06 human germline in the framework regions and CDRs are marked in bold and underlined, respectively.

[0689]

[0690]

[0691] Details were obtained from the full-length amino acid sequence of Version A in Figure 1, the annotations for each relevant amino acid position in the CE7 VH design of Table 3 when detecting the homology molecular model, and the sequence comparison in VH of Table 1 (Columns E, G, and I of VH-IGHV1-2, corresponding to murine CE7 VH, IGHV1-2*06 germline, and humanized Version A, respectively).

[0692] Humanized version B

[0693] Compared with Version A, three amino acid residues were germline-ized in Version B (i.e., murine residues were replaced by their corresponding IGHV1-2*06 germline residues). In CDR2, Kabat Lys (K) H64 and Kabat Ser (S) H65 were replaced by their corresponding germline IGHV1-2*06 residues (i.e., Gln (Q) and Gly (G)), respectively; and in FR3, Kabat Phe (F) H91 was replaced by the corresponding germline IGHV1-2*06 residue (i.e., Tyr (Y)).

[0694] The different amino acid residues between humanized Versions A and B are underlined.

[0695] Version B (VHB)

[0696]

[0697] The identity of humanized Version B (CE7-1-2-VHB) with the IGHV1-2*06 germline is 87.8% (86 out of a total of 98 residues in the V gene are identical). The different residues between humanized Versions A and B are underlined.

[0698]

[0699] Humanized version C

[0700] Compared with Version B, one amino acid residue was germline-ized in Version C (i.e., murine residue was replaced by the corresponding IGHV1-2*06 germline residue). In CDR2, Kabat Arg (R) H62 was replaced by its corresponding germline IGHV1-2*06 residue (i.e., Lys (K)).

[0701] The different amino acid residues between humanized Versions B and C are underlined.

[0702] Version C (VHC)

[0703]

[0704] The identity of the humanized version C (CE7-1-2-VHC) with the IGHV1-2*06 germline is 88.8% (87 out of a total of 98 residues in the V gene are identical). The residues that differ between humanized versions B and C are underlined.

[0705]

[0706] Humanized version D

[0707] Compared to version C, one amino acid residue was germlined in version D (i.e., the murine residue was replaced by the corresponding IGHV1-2*06 germline residue). In FR2, Kabat Ile (I) H48 was replaced by its corresponding germline IGHV1-2*06 residue (i.e., Met (M)).

[0708] The amino acid residues that differ between humanized versions D and E are underlined.

[0709] Version D (VHD)

[0710]

[0711] The identity of the humanized version D (CE7-1-2-VHD) with the IGHV1-2*06 germline is 89.8% (88 out of a total of 98 residues in the V gene are identical). The residues that differ between humanized versions C and D are underlined.

[0712]

[0713]

[0714] Design using the IGHV4-34*01 human germline as the framework receptor region

[0715] Sequence alignment between murine CE7 VH and human germline IGHV4-34*01

[0716] The sequence identity between the CE7 murine heavy chain variable (VH) region and the germline immunoglobulin VH 4-34*01 (IGHV4-34*01) is 53.1% (52 out of a total of 98 residues in the V gene are identical). The residues that differ between murine CE7 VH and germline IGHV4-34*01 are shown in bold and underlined in the framework and CDR, respectively.

[0717]

[0718] Humanized version A

[0719] The murine CDRs defined according to the Kabat nomenclature (highlighted in yellow) were grafted into IGHV4-34*01 to obtain the sequences detailed below. The framework murine residues (non-CDR residues) were retained from the parental murine CE7 VH sequence; they were retained because they may be structurally important for maintaining the full activity of the antibody.

[0720] Version A

[0721]

[0722] The identity of the humanized version A (CE7-4-34-VHA) with the IGHV4-34*01 human germline is 83.7% (82 out of a total of 98 residues in the V gene are identical). The residues that differ between CE7-434-VHA and the human germline IGHV4-34*01 are marked in bold and underlined in the framework region and CDR, respectively.

[0723]

[0724]

[0725] Humanized version B

[0726] Compared with version A, in version B, three amino acid residues were germlined (i.e., murine residues were replaced by the corresponding IGHV4-34*01 human germline residues); in framework 3 (FR3), Kabat Thr (T) H28 and Kabat Thr (T) H30 were both replaced by Ser (S), and in framework 3 (FR3), Kabat Phe (F) H91 was replaced by Tyr (Y).

[0727] Version B

[0728]

[0729] The identity of the humanized version B (CE7-434-VHB) with the IGHV4-34*01 human germline is 86.7% (85 out of a total of 98 residues in the V gene are identical). Compared with the humanized version A, the germlined amino acid residues in the humanized version B are underlined.

[0730]

[0731] Humanized version C

[0732] Compared with version B, in version C, two amino acid residues were germlineized (i.e., murine residues were replaced by corresponding IGHV4-34*01 human germline residues); in CDR2, Kabat Glu (E) H61 and Kabat Arg (R) H62 were replaced by Pro (P) and Ser (S), respectively.

[0733] Version C

[0734]

[0735]

[0736] The identity of the humanized version C (CE7-434-VHC) with the IGHV4-34*01 human germline is 88.8% (87 out of a total of 98 residues in the V gene are identical). Compared with the humanized version B, the germlineized amino acid residues in the humanized version C are underlined.

[0737]

[0738] Light chain design

[0739] Amino acid differences with the highest homology to murine germline IGKV13-84*01

[0740] Amino acid sequence of the VL of the murine CE7 hybridoma (CDR regions defined according to the Kabat nomenclature are underlined)

[0741]

[0742] The identity between the CE7 murine light chain variable (VL) region and the murine germline immunoglobulin IGKV13-84*01 is 90.5% (86 out of a total of 95 residues in the V gene are identical). The residues different between CE7 VL and the murine germline IGKV13-84*01 are underlined.

[0743]

[0744] Selection of the human framework receptor VL region

[0745] The selection of the human framework receptor VL region transplanted with the CE7 murine CDR region was accomplished by using the murine VL region amino acid sequence as an input and retrieving the IMGT human VL gene database with IgBLAST. Based on the sequence alignment of the parental antibody with the human germline, the most closely matched entries were identified. The best human germlines to be used as receptors were identified based on the following ordered criteria: sequence identity within the framework defined by Kabat, and the identity and / or compatibility of the inter-chain interface residues and the supporting loops with the canonical conformation of the parental CDR. One of the important criteria for selecting a suitable human germline candidate was to ensure an approximate 85% sequence identity between that particular germline and the designed humanized version. We analyzed a large number of human germlines and selected three human germlines (i.e., IGKV1-NL1*01, IGKV1-33*01, and IGKV3-15*01) for designing the humanized versions of CDR transplantation.

[0746] The gene segment of murine CE7 VL corresponding to the J gene was identified as having the highest homology with the murine germline J4 gene (IGKJ4*01). Comparing the murine J4 gene segment with the human J segment genes at CDR3 and FR4, it was found that the human J segment IGKJ2 (IGKJ2*01) had the highest overall homology.

[0747] Design using the IGKV1-NL1*01 human germline as the framework receptor region

[0748] Sequence alignment between murine CE7 VH and human germline IGKV1-NL1*01

[0749] The identity between the CE7 murine light chain variable (VL) region and the human germline immunoglobulin VL 1-NL1*01 (IGKV1-NL1*01) was 73.7% (70 out of a total of 95 residues in the V gene were identical). The CDR residues defined according to Kabat nomenclature are highlighted in yellow. The residues different between murine CE7 VL and the human germline IGKV1-NL1*01 are marked in bold and underlined in the framework region and CDR, respectively.

[0750]

[0751] Humanized version A

[0752] The murine CDRs defined according to Kabat numbering were transplanted into IGKV1-NL1*01 to obtain the sequences detailed below.

[0753] Version A

[0754]

[0755]

[0756] The identity of the humanized version A (CE7-1-NL1-VLA) with IGKV1-NL1*01 is 84.2% (80 out of a total of 95 residues in the V gene are identical). The murine framework residues (non-CDR residues marked in bold) are retained from the parental murine CE7 VL sequence; they are retained because they may be structurally important for maintaining the full activity of the antibody. The positions of the residues that differ between the humanized version A and the human germline IGKV1-NL1*01 are underlined within the CDRs.

[0757]

[0758] Humanized version B

[0759] Compared to version A, in version B one amino acid residue was germline-ized (i.e., replaced by the corresponding human germline residue). In CDR1, Lys (K) Kabat L24 was germline-ized and mutated to Arg (R). Examination of the molecular model indicated that this residue is not likely to contact the antigen directly and it does not play a key structural role; thus, it can replace its corresponding human germline IGKV1-NL1*01 counterpart. The main reason for germline-izing this residue within Kabat CDR L1 is that this change increases the percentage identity of the humanized version B with respect to the human germline IGKV1-NL1*01 to 85.3%.

[0760] Version B

[0761]

[0762] The identity of the humanized version B (CE7-1-NL1-VLB) with the human κ light chain germline of IGKV1-NL1*01 is 85.3% (81 out of a total of 95 residues in the V gene are identical). Compared to the humanized version A, the amino acid residues that were germline-ized in the humanized version B are underlined.

[0763]

[0764] Humanized version C

[0765] Compared to version B, in version C, two amino acid residues were germline-ized (i.e., replaced by their corresponding human germline residues). In CDR2, Thr (T) Kabat L52 and Thr (T) Kabat L56 were germline-ized and mutated to Ser (S). Examination of the molecular model indicated that these residues are not likely to directly contact the antigen and they do not play a key structural role; thus, they can replace their corresponding human germline IGKV1-NL1*01 counterparts. The main reason for germline-izing these residues within the Kabat CDRs is that these changes increase the percent identity of the humanized version C relative to the human germline IGKV1-NL1*01 to 87.4%. 24

[0766] Version C

[0767]

[0768] The humanized version C (CE7-1-NL1-VLC) has 87.4% identity with the IGKV1-NL1*01 human κ light chain germline (83 out of a total of 95 residues in the V gene are identical). Compared to the humanized version B, the germline-ized amino acid residues in the humanized version C are underlined.

[0769]

[0770] Humanized version D

[0771] Compared to version C, in version D, three amino acid residues were germline-ized (i.e., replaced by their corresponding human germline residues). In CDR1, T Asn (N) Kabat L26, Glu (E) L27, and Asp (D) Kabat L28 were germline-ized and mutated to Ser (S), Gln (Q), and Gly (G), respectively. Examination of the molecular model indicated that these residues are not likely to directly contact the antigen and they do not play a key structural role; thus, they can replace their corresponding human germline IGKV1-NL1*01 counterparts. The main reason for germline-izing these residues within the Kabat CDRs is that these changes increase the percent identity of the humanized version D relative to the human germline IGKV1-NL1*01 to 90.5%.

[0772] Version D

[0773]

[0774] The identity of the humanized version D (CE7-1-NL1-VLD) with the human germline IGKV1-NL1*01 kappa light chain is 90.5% (86 out of a total of 95 residues in the V gene are identical). Compared with the humanized version C, the germline-processed amino acid residues in the humanized version D are underlined.

[0775]

[0776] Design using the IGKV1-33*01 human germline as the framework receptor region

[0777] Sequence alignment between murine CE7 VL and human germline IGKV1-33*01

[0778] The identity between the CE7 murine light chain variable (VL) region and the human germline immunoglobulin VL 1-33*01 (IGKV1-33*01) is 71.6% (68 out of a total of 95 residues in the V gene are identical). The CDR residues defined according to Kabat nomenclature are highlighted in yellow. The residues that differ between murine CE7 VL and the human germline IGKV1-33*01 are bolded and underlined in the framework region and CDR, respectively.

[0779]

[0780] Humanized version A

[0781] The murine CDRs defined according to Kabat numbering were transplanted into IGKV1-33*01 to obtain the sequences detailed below.

[0782] Version A

[0783]

[0784] The identity of the humanized version A (CE7-1-33-VLA) with IGKV1-33*01 is 83.2% (79 out of a total of 95 residues in the V gene are identical). The residues in bold are murine framework residues (non-CDR residues) retained from the parental murine CE7 VL sequence; they are retained because they may be structurally important for maintaining the full activity of the antibody. The positions of the residues that differ between the humanized version A and the human germline IGKV1-33*01 are underlined within the CDR.

[0785]

[0786] Humanized version B

[0787] In version B, two amino acid residues were germline-ized (i.e., replaced by their corresponding human germline residues) compared to version A. In CDR1, Lys (K) Kabat L24 was germline-ized and mutated to Gln (Q). In framework 3 (FR3), Leu (L) Kabat L73 was germline-ized and mutated to Phe (F). Examination of the molecular model indicated that these residues were unlikely to directly contact the antigen and that they did not play a key structural role; thus, they could replace their corresponding human germline IGKV1-33*01 counterparts. The main reason for germline-izing these residues within the Kabat CDRs was that these alterations would increase the percent identity of the humanized version B relative to the germline IGKV1-33*01 to 85.3%.

[0788] Version B

[0789]

[0790] The humanized version B (CE7-1-33-VLB) has 85.3% identity with the germline human IGKV1-33*01 kappa light chain (81 out of a total of 95 residues in the V gene are identical). Compared to the humanized version A, the germline-ized amino acid residues in the humanized version B are underlined.

[0791]

[0792] Humanized version C

[0793] In version C, two amino acid residues were germline-ized (i.e., replaced by their corresponding human germline residues) compared to version B. In CDR2, Thr (T) Kabat L52 was germline-ized and mutated to Ser (S). In framework 3 (FR3), Tyr (Y) Kabat L71 was germline-ized and mutated to Phe (F). Examination of the molecular model indicated that these residues were unlikely to directly contact the antigen and that they did not play a key structural role; thus, they could replace their corresponding human germline IGKV1-33*01 counterparts. The main reason for germline-izing these residues within the Kabat CDRs was that these alterations would increase the percent identity of the humanized version C relative to the germline IGKV1-33*01 to 87.4%.

[0794] Version C

[0795]

[0796]

[0797] The identity of the humanized version C (CE7-1-33-VLC) with the IGKV1-33*01 human κ light chain germline is 87.4% (83 out of a total of 95 residues in the V gene are identical). Compared with the humanized version B, the amino acid residues that have been germline-treated in the humanized version C are underlined.

[0798]

[0799] Humanized version D

[0800] Compared with version C, in version D, two amino acid residues were germline-treated (i.e., replaced by their corresponding human germline residues). In CDR1, Asn (N) Kabat L26 and Glu (E) Kabat L27 were germline-treated and mutated to Ser (S) and Gln (Q), respectively. Examination of the molecular model indicates that these residues are unlikely to directly contact the antigen and they do not play a key structural role; thus, they can replace their corresponding human germline IGKV1-33*01 counterparts. The main reason for germline-treating these residues within the Kabat CDR is that these changes will increase the identity percentage of the humanized version C relative to the human germline IGKV1-33*01 to 89.5%.

[0801] Version D

[0802]

[0803] The identity of the humanized version D (CE7-1-33-VLD) with the IGKV1-33*01 human κ light chain germline is 89.5% (85 out of a total of 95 residues in the V gene are identical). Compared with the humanized version C, the amino acid residues that have been germline-treated in the humanized version D are underlined.

[0804]

[0805]

[0806] Design using the IGKV3-15*01 human germline as the framework receptor region

[0807] Sequence alignment between murine CE7 VL and human germline IGKV3-15*01

[0808] The identity between the CE7 murine light chain variable (VL) region and the germline immunoglobulin VL 3-15*01 (IGKV3-15*01) is 60.0% (57 out of a total of 95 residues in the V gene are identical). CDR residues defined according to Kabat nomenclature are labeled. Residues that differ between murine CE7 VL and germline IGKV3-15*01 are labeled in bold and underlined in the framework regions and CDRs, respectively.

[0809]

[0810] Humanized version A

[0811] Murine CDRs defined according to Kabat numbering were transplanted into IGKV3-15*01 to obtain the sequences detailed below.

[0812] Version A

[0813]

[0814] The humanized version A (CE7-3-15-VLA) has an identity of 81.1% with IGKV3-15*01 (77 out of a total of 95 residues in the V gene are identical). Residues labeled in bold are murine framework residues (non-CDR residues) retained from the parental murine CE7 VL sequence; they are retained because they may be structurally important for maintaining the full activity of the antibody. Residues that differ between humanized version A and germline IGKV3-15*01 are underlined at their positions within the CDRs.

[0815]

[0816] Humanized version B

[0817] Compared to version A, two amino acid residues were germlined (i.e., replaced by their corresponding germline residues) in version B. 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 the molecular model indicated that these residues are not likely to contact the antigen directly and that they do not play a critical structural role; thus, they can replace their corresponding germline IGKV3-15*01 counterparts. The main reason for germlining these residues within the Kabat CDRs is that these changes increase the percentage identity of humanized version B relative to germline IGKV3-15*01 to 83.2%.

[0818] Version B

[0819]

[0820] The identity of the humanized version B (CE7-3-15-VLB) with IGKV3-15*01 is 83.2% (79 out of a total of 95 residues in the V gene are identical). The residues marked in bold are murine framework residues (non-CDR residues) retained from the parental murine CE7 VL sequence; they are retained because they may be structurally important for maintaining the full activity of the antibody. Compared with the humanized version A, the germline-processed amino acid residues in the humanized version B are underlined.

[0821]

[0822]

[0823] Humanized version C

[0824] Compared with version B, in version C, two amino acid residues were germline-processed (i.e., replaced by their corresponding human germline residues). In CDR1, Asn (N) Kabat L24 was germline-processed and mutated to Ser (S). In CDR2, Thr (T) Kabat L52 was germline-processed and mutated to Ser (S). Examination of the molecular model indicates that these residues are unlikely to directly contact the antigen and they do not play a key structural role; thus, they can replace their corresponding human germline IGKV3-15*01 counterparts. The main reason for germline-processing these residues within the Kabat CDRs is that these changes increase the identity percentage of the humanized version C relative to the human germline IGKV3-15*01 to 85.3%.

[0825] Version C

[0826]

[0827] The identity of the humanized version B (CE7-3-15-VLC) with IGKV3-15*01 is 85.3% (81 out of a total of 95 residues in the V gene are identical). Compared with the humanized version B, the germline-processed amino acid residues in the humanized version C are underlined.

[0828]

[0829] Humanized version D

[0830] Compared with version C, in version D, two amino acid residues were germline-ized (i.e., replaced by their corresponding human germline residues). In CDR1, Glu (E) Kabat L27 and Asp (D) Kabat L28 were germline-ized and mutated to Gln (Q) and Ser (S), respectively. Examination of the molecular model indicated that these residues are not likely to be in direct contact with the antigen and they do not play a critical structural role; thus, they can replace their corresponding human germline IGKV3-15*01 counterparts. The main reason for germline-izing these residues within the Kabat CDR is that these changes increase the percentage identity of the humanized version C relative to the human germline IGKV3-15*01 to 87.4%.

[0831] Version D

[0832]

[0833] The humanized version D (CE7-3-15-VLD) has 87.4% identity with IGKV3-15*01 (83 out of a total of 95 residues in the V gene are identical). Compared with the humanized version C, the germline-ized amino acid residues in the humanized version D are underlined.

[0834]

[0835] Example 2 - Recombinant antibody production - Variants 1 to 18.

[0836] Genes and expression vectors

[0837] Genes encoding rAb

[0838] Starting from the sequences of the heavy chain (HC) and light chain (LC) variable regions of 18 humanized variant antibodies generated by the computer-aided humanization step, full-length human IgG1κ antibodies carrying the mutations L234A, L235A, and P331S were designed.

[0839] cDNAs encoding the HC and LC variable regions were chemically synthesized and optimized for expression in CHO cells, and then subcloned into a ProteoGenix proprietary mammalian cell expression vector containing the framework of the human IgG1 heavy chain constant region and the human κ 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.

[0840] >CE7-1-2-VHA(L234A L235A+P331S)[SEQ ID NO.:85]

[0841]

[0842] >CE7-1-2-VHB(L234A L235A+P331S)[SEQ ID NO.:86]

[0843]

[0844] >CE7-4-34-VHA(L234A L235A+P331S)[SEQ ID NO.:87]

[0845]

[0846] >CE7-1-NL1-VLA[SEQ ID NO.:88]

[0847] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCTAGCTCCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACCTGCAAGGCCAATGAGGATATCAACAACCGGCTGGCTTGGTATCAGCAGAAGCCCGGCAAGGCTCCCAAGCTGCTGATCTCCGGCGCTACCAATCTGGTGACCGGCGTGCCCTCCAGGTTTAGCGGCAGCGGCTCCGGCAAGGATTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCTACCTACTATTGCCAGCAGTACTGGAGCACCCCTTTTACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0848] >CE7-1-NL1-VLB[SEQ ID NO.:89]

[0849] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGATATCCAGATGACCCAGAGCCCTAGCAGCCTGTCCGCCAGCGTGGGCGATAGGGTGACCATCACCTGCAGGGCTAACGAGGACATCAATAACAGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCAGCGGCGCCACCAATCTGGTGACCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGAAGCGGCAAGGACTACACCCTGACCATCTCCTCCCTGCAGCCTGAGGACTTTGCCACCTATTATTGCCAGCAGTACTGGAGCACCCCTTTCACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0850] >CE7-1-33-VLA[SEQ ID NO.:90]

[0851] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGTAAGGCTAATGAGGACATCAATAACCGGCTGGCTTGGTACCAGCAGAAGCCCGGCAAGGCTCCCAAGCTGCTGATCTCCGGCGCTACCAACCTGGTGACCGGCGTGCCTAGCAGGTTTTCCGGCTCCGGCAGCGGCAAGGATTATACCCTGACCATCTCCTCCCTGCAGCCCGAGGATATCGCTACCTACTACTGCCAGCAGTATTGGTCCACCCCTTTTACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0852] >CE7-1-33-VLB[SEQ ID NO.:91]

[0853] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGATGACCCAGAGCCCCTCCAGCCTGTCCGCTTCCGTGGGCGATAGGGTGACCATCACCTGTCAGGCTAATGAGGACATCAACAACAGGCTGGCTTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCAGCGGCGCCACCAACCTGGTGACCGGCGTGCCAAGCAGGTTCAGCGGCAGCGGCAGCGGAAAGGACTATACCTTCACCATCTCCTCCCTGCAGCCTGAGGACATCGCTACCTATTACTGTCAGCAGTACTGGTCCACCCCCTTCACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0854] >CE7-3-15-VLA[SEQ ID NO.:92]

[0855] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCAGCCTGCTGCTGTGGATCAGCGGCGCCTACGGCGAGATCGTGATGACCCAGTCCCCTGCCACCCTGTCCGTGTCCCCTGGCGAGAGGGCCACCCTGTCTTGCAAGGCTAATGAGGACATCAACAATAGGCTGGCCTGGTATCAGCAGAAGCCTGGCCAGGCTCCTCGGCTGCTGATCAGCGGCGCTACCAACCTGGTGACCGGCATCCCTGCCCGGTTCTCCGGCTCCGGAAGCGGAAAGGAGTATACCCTGACCATCAGCAGCCTGCAGAGCGAGGATTTTGCCGTGTACTATTGCCAGCAGTACTGGTCCACCCCCTTTACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0856] >CE7-3-15-VLB[SEQ ID NO.:93]

[0857] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCAGCGGCGCCTACGGCGAGATCGTGATGACCCAGAGCCCTGCCACCCTGAGCGTGTCCCCTGGCGAGAGAGCCACCCTGTCCTGCCGGGCTAACGAGGACATCAACAACCGGCTGGCTTGGTATCAGCAGAAGCCTGGCCAGGCTCCTAGGCTGCTGATCTCCGGCGCTACCAACCTGGTGACCGGCATCCCTGCCAGGTTTAGCGGCAGCGGCTCCGGCAAGGAGTTCACCCTGACCATCAGCAGCCTGCAGTCCGAGGATTTCGCCGTGTATTATTGTCAGCAGTACTGGTCCACCCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCAGCGGCGCCTACGGCGAGATCGTGATGACCCAGAGCCCTGCCACCCTGAGCGTGTCCCCTGGCGAGAGAGCCACCCTGTCCTGCCGGGCTAACGAGGACATCAACAACCGGCTGGCTTGGTATCAGCAGAAGCCTGGCCAGGCTCCTAGGCTGCTGATCTCCGGCGCTACCAACCTGGTGACCGGCATCCCTGCCAGGTTTAGCGGCAGCGGCTCCGGCAAGGAGTTCACCCTGACCATCAGCAGCCTGCAGTCCGAGGATTTCGCCGTGTATTATTGTCAGCAGTACTGGTCCACCCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0858] Sequences of expressed proteins

[0859] >CE7-1-2-VHA(L234A L235A+P331S)[SEQ ID NO.:94]

[0860] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0861] >CE7-1-2-VHB(L234A L235A+P331S)[SEQ ID NO.:95]

[0862] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0863] >CE7-4-34-VHA(L234A L235A+P331S)[SEQ ID NO.:96]

[0864] MKHLWFFLLLVAAPRWVLSQVQLQQWGAGLLKPSETLSLTCAAYGYTFTGYWMHWIRQPPGKGLEWIGEINPSNGRTNYNERLKSRVTLSVDKSKNQASLKLSSVTAADTAVYFCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0865] >CE7-1-NL1-VLA[SEQ ID NO.:97]

[0866] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0867] >CE7-1-NL1-VLB[SEQ ID NO.:98]

[0868] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0869] >CE7-1-33-VLA[SEQ ID NO.:99]

[0870] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDIATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0871] >CE7-1-33-VLB[SEQ ID NO.:100]

[0872] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCQANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTFTISSLQPEDIATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0873] >CE7-3-15-VLA[SEQ ID NO.:101]

[0874] MVLQTQVFISLLLWISGAYGEIVMTQSPATLSVSPGERATLSCKANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEYTLTISSLQSEDFAVYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0875] >CE7-3-15-VLB[SEQ ID NO.:102]

[0876] MVLQTQVFISLLLWISGAYGEIVMTQSPATLSVSPGERATLSCRANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEFTLTISSLQSEDFAVYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0877] Scale-up production and purification tests

[0878] Short protocol description

[0879] Endotoxin-free DNA preparation was performed on the obtained constructs. As shown in Table E1, eighteen antibodies were expressed by combining HC and LC. Using a proprietary Xten transfection protocol, the plasmids were transiently co-transfected into our proprietary Xten CHO cells. When the viability dropped below 50% (14 days after transfection), cell culture medium samples were collected and then the rAbs were purified on Protein A resin using standard methods:

[0880] - Clarify by 0.22 μm filtration

[0881] - Equilibrate, bind, and wash with PBS pH 7.5

[0882] - Elute by changing the pH with citric acid

[0883] - Neutralize with 1 M Tris-HCl pH 9.0

[0884] - Analyze by PAGE and combine fractions of interest.

[0885] - Perform final QC by PAGE: Qualitative and quantitative analysis by SDS - PAGE

[0886] Combine the eluted fractions and change the buffer to PBS, pH 7.5 by dialysis. Filter the final sample through a 0.22 μm microfilter. The purification profile and final QC are shown in Figure 1 and In Figure 2 . The resulting yield and purity are summarized in Table E2.

[0887] Table E2. Yields and purities obtained for pilot-produced rAb .

[0888] Antibody Concentration Yield*(mg / 30ml) Purity** CE7 variant 1 1.00mg / ml 3.8mg >90％ CE7 variant 2 1.00mg / ml 3.52mg >90％ CE7 variant 3 1.00mg / ml 3.4mg >90％ CE7 variant 4 1.00mg / ml 2.8mg >90％ CE7 variant 5 1.00mg / ml 0.8mg >90％ CE7 variant 6 1.00mg / ml 5.36mg >90％ CE7 variant 7 1.00mg / ml 3.86mg >90％ CE7 variant 8 1.00mg / ml 4.5mg >90％ CE7 variant 9 1.00mg / ml 1.83mg >90％ CE7 variant 10 1.00mg / ml 5.1mg >90％ CE7 variant 11 1.00mg / ml 0.8mg >90％ CE7 variant 12 1.00mg / ml 6.28mg >90％ CE7 variant 13 1.00mg / ml 8.45mg >90％ CE7 variant 14 1.00mg / ml 3.1mg >90％ CE7 variant 15 1.00mg / ml 1.80mg >90％ CE7 variant 16 1.00mg / ml 2.65mg >90％ CE7 variant 17 1.00mg / ml 1.33mg >90％ CE7 variant 18 1.00mg / ml 4.7mg >90％

[0889] * Obtained after purification of a 30 ml test culture.

[0890] ** Based on the full - length antibody observed on non - reducing PAGE

[0891] The analysis results are as As shown in Figure 2 shown.

[0892] Example 3 - Recombinant antibody production - Variants 19 to 28.

[0893] Genes and expression vectors

[0894] Genes encoding rAb

[0895] Starting from the sequences of the heavy chain (HC) and light chain (LC) variable regions of an additional 12 humanized variant antibodies generated by the computer - based humanization step, design full - length human IgG1κ antibodies carrying the mutations L234A, L235A, P331S, and N297A.

[0896] Chemically synthesize the cDNAs encoding the HC and LC variable regions, optimize their expression in CHO cells, and then sub - clone them into a ProteoGenix proprietary mammalian cell expression vector that contains the scaffolds of the human IgG1 heavy chain constant region and the human κ light chain constant region (https: / / www.proteogenix.science / product / xtencho - starter - kit / ). Add a sequence encoding a signal peptide at the 5' / Nter position. The sequences are shown below.

[0897]

[0898] >CE7-1-2-VHB(L234A L235A+P331S N297A)[SEQ ID NO.:103]

[0899]

[0900] >CE7-1-2-VHC(L234A L235A+P331S N297A)[SEQ ID NO.:104]

[0901]

[0902] >CE7-1-2-VHD(L234A L235A+P331S N297A)[SEQ ID NO.:105]

[0903]

[0904] >CE7-1-NL1-VLA[SEQ ID NO.:106]

[0905] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGACATCCAGATGACCCAGAGCCCTTCCTCCCTGTCCGCCAGCGTGGGCGACAGGGTGACCATCACCTGTAAGGCCAATGAGGATATCAATAATCGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTCCGGCGCTACCAATCTGGTGACCGGCGTGCCTTCCCGGTTTTCCGGCTCCGGCAGCGGCAAGGATTATACCCTGACCATCAGCAGCCTGCAGCCCGAGGATTTTGCTACCTATTATTGCCAGCAGTATTGGTCCACCCCCTTCACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0906] >CE7-1-NL1-VLB[SEQ ID NO.:107]

[0907] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCCAGATGACCCAGTCCCCCAGCTCCCTGTCCGCTAGCGTGGGCGACAGGGTGACCATCACCTGTAGGGCCAATGAGGACATCAACAACAGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCTCCTAAGCTGCTGATCTCCGGCGCTACCAATCTGGTGACCGGCGTGCCTTCCAGGTTCTCCGGCTCCGGCTCCGGAAAGGATTACACCCTGACCATCAGCTCCCTGCAGCCTGAGGACTTTGCTACCTATTACTGCCAGCAGTACTGGTCCACCCCCTTTACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0908] >CE7-1-NL1-VLC[SEQ ID NO.:108]

[0909] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGATATCCAGATGACCCAGAGCCCCTCCAGCCTGAGCGCCTCCGTGGGAGACAGGGTGACCATCACCTGTAGGGCCAACGAGGATATCAACAACAGGCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCTCCTAAGCTGCTGATCAGCGGCGCTTCCAACCTGGTGAGCGGCGTGCCCTCCAGGTTTAGCGGCAGCGGCAGCGGAAAGGATTACACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACTATTGCCAGCAGTACTGGAGCACCCCTTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0910] >CE7-1-NL1-VLD[SEQ ID NO.:109]

[0911] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGACATCCAGATGACCCAGTCCCCTAGCTCCCTGAGCGCCTCCGTGGGCGACAGGGTGACCATCACCTGCCGGGCCAGCCAGGGCATCAATAATCGGCTGGCTTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCAGCGGCGCCTCCAACCTGGTGAGCGGCGTGCCTTCCAGGTTCTCCGGCAGCGGCAGCGGCAAGGACTATACCCTGACCATCAGCAGCCTGCAGCCTGAGGACTTTGCCACCTACTATTGCCAGCAGTATTGGTCCACCCCCTTTACCTTTGGCCAGGGCACCAAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0912] The sequence of the expected protein produced is shown below.

[0913] >CE7-1-2-VHB(L234A L235A+P331S)(14047)[SEQ ID NO.:94], as shown above MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0914] >CE7-1-2-VHB(L234A L235A+P331S N297A)[SEQ ID NO.:110]

[0915] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0916] >CE7-1-2-VHC(L234A L235A+P331S N297A)[SEQ ID NO.:111]

[0917] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNEKFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0918] >CE7-1-2-VHD(L234A L235A+P331S N297A)[SEQ ID NO.:112]

[0919] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWMGEINPSNGRTNYNEKFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0920] >CE7-1-NL1-VLA[SEQ ID NO.:97]

[0921] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0922] >CE7-1-NL1-VLB[SEQ ID NO.:98]

[0923] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0924] >CE7-1-NL1-VLC[SEQ ID NO.:113]

[0925] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGASNLVSGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0926] >CE7-1-NL1-VLD[SEQ ID NO.:114]

[0927] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCRASQGINNRLAWYQQKPGKAPKLLISGASNLVSGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0928] Scaled-up production and purification testing

[0929] Short protocol description

[0930] Endotoxin-free DNA preparation of the obtained constructs was performed as described herein. Twelve antibodies were expressed by combining HC and LC as follows:

[0931] Table E3 .

[0932]

[0933]

[0934] Small-scale antibody production was carried out as described in Example 2. The results are shown in In Figures 3 and In Figure 4 . The resulting yields and purities are summarized in Table E4.

[0935] Table E4. Yields and purities obtained for pilot-produced rAb .

[0936] Antibody Concentration Specification Quantity Yield* Purity** CE7 variant 7 0.90mg / ml 1.70ml / vial 2 vials 3.06mg / 30ml >90％ CE7 variant 19 0.77mg / ml 1.83ml / vial 2 vials 2.82mg / 30ml >90％ CE7 variant 20 0.72mg / ml 1.50ml / vial 2 vials 2.16mg / 30ml >90％ CE7 variant 21 0.65mg / ml 1.52ml / vial 2 vials 1.98mg / 30ml >90％ CE7 variant 22 1.17mg / ml 1.86ml / vial 1 vial 2.18mg / 30ml >90％ CE7 variant 23 0.70mg / ml 1.75ml / vial 1 vial 1.23mg / 30ml >90％ CE7 variant 24 0.67mg / ml 1.60ml / vial 2 vials 2.14mg / 30ml >90％ CE7 variant 25 0.82mg / ml 1.41ml / vial 2 vials 2.31mg / 30ml >90％ CE7 variant 26 0.67mg / ml 1.60ml / vial 2 vials 2.14mg / 30ml >90％ CE7 variant 27 0.94mg / ml 1.60ml / vial 1 vial 1.50mg / 30ml >90％ CE7 variant 28 0.97mg / ml 1.70ml / vial 1 vial 1.65mg / 30ml >90％ CE7 variant 7-AG 1.66mg / ml 1.58ml / vial 6 vials 15.74mg / 200ml >90％

[0937] *Obtained after purification of 30 ml test culture.

[0938] **Based on full-length antibodies observed on non-reducing PAGE

[0939] The analysis results are as As shown in Figure 2 shown.

[0940] Final buffer: PBS pH 7.5.

[0941] Expression and purification results of non-glycosylated forms of variants 4, 7, 15, 17, and 18 .

[0942] Starting from the sequences of the heavy chain (HC) and light chain (LC) variable regions of 18 humanized variant antibodies generated by the computer humanization step, full-length human IgG1κ antibodies carrying the mutations L234A, L235A, P331S and N297A were designed. The cDNAs encoding HC and LC were chemically synthesized and optimized for their expression in CHO cells, and then subcloned into a proprietary mammalian cell expression vector of ProteoGenix, which contains the framework of the human IgG1 heavy chain constant region and the human κ 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.

[0943] >CE7-1-2-VHA(L234A L235A+P331S+N297A)[SEQ ID NO.:115]

[0944]

[0945] The following sequences are the sequences mentioned above:

[0946] >CE7-1-2-VHB(L234A L235A+P331S+N297A)[SEQ ID NO.:103]

[0947] >CE7-4-34-VHA(L234A L235A+P331S+N297A)[SEQ ID NO.:87]

[0948] >CE7-1-NL1-VLA[SEQ ID NO.:88]

[0949] >CE7-1-33-VLA[SEQ ID NO.:90]

[0950] >CE7-1-33-VLB[SEQ ID NO.:91]

[0951] >CE7-3-15-VLA[SEQ ID NO.:92]

[0952] >CE7-3-15-VLB[SEQ ID NO.:93]

[0953] Based on the following sequences, the expected protein products are defined as follows or as defined above:

[0954] >CE7-1-2-VHA(L234A L235A+P331S+N297A)[SEQ ID NO.:116]

[0955] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0956] Scale-up production and purification testing

[0957] Short protocol description

[0958] As described herein, endotoxin-free DNA preparation of the obtained construct was performed. Five antibodies were expressed by combining HC and LC as follows:

[0959] Table E5

[0960]

[0961] The results of small-scale production are summarized in Figure 5 and in Figure 6 . The yield and purity data are summarized in Table E6.

[0962] Table E6. Yield and purity obtained for pilot production of rAb .

[0963] Antibody Concentration Yield*(mg / 80ml) Purity** CE7 variant 4_AG 2.54mg / ml 11.43mg >90％ CE7 variant 7_AG 3.26mg / ml 11.41mg >90％ CE7 variant 15_AG 1.94mg / ml 10.36mg >90％ CE7 variant 17_AG 1.09mg / ml 3.73mg >90％ CE7 variant 18_AG 1.99mg / ml 9.67mg >90％

[0964] * Obtained after purification of 30 ml test culture.

[0965] ** Based on full-length antibodies observed on non-reducing PAGE

[0966] The analysis results are as as shown in Figure 6 shown.

[0967] Example 5 - SEC HPLC analysis

[0968] Starting from the samples of each protein obtained after the final adjustment and aliquoting as described in Example 4, SEC-HPLC analysis was performed to evaluate the aggregation levels of the first three candidates: CE7 variant 4_AG, CE7 variant 7_AG, and CE7 variant 17_AG. The analysis was carried out using an HPLC Waters 2695 apparatus equipped with a photodiode array detector (2996).

[0969] Buffer preparation

[0970] - Mobile phase: 100 mM sodium sulfate, 100 mM phosphate, pH 6.7 buffer (14.2 g / L of Na2SO4, 15.6 g / L of Na2HPO4·12H2O, 6.8 g / L of NaH2P04),

[0971] Remove air bubbles from all buffers.

[0972] Sample preparation

[0973] - Protein preparation: Starting from an additional aliquot of each final sample stored at -80 °C overnight, the protein sample was diluted to 1 mg / ml with DD water, centrifuged at 12000 g for 10 minutes, and the supernatant was collected

[0974] Analysis conditions

[0975] - Blank control: Dilute the sample (5x) with DD water

[0976] - Column: G3000SWXL, TOSOH, 7.8 × 300 mm

[0977] - HPLC: Waters 2695

[0978] - Elution gradient: Isocratic elution with the mobile phase

[0979] - Flow rate: 0.8 ml / min

[0980] - Temperature: 25 °C

[0981] - Injection volume: 30 μl

[0982] - Detection wavelength: 280 nm

[0983] - Collection date and time: 25 minutes

[0984] - Equilibrate with 100% mobile phase for 5 minutes

[0985] - Running samples: Inject a blank control, then inject the samples

[0986] - Data analysis

[0987] The results are shown in Table E7.

[0988] Table E7. SEC HPLC data

[0989]

[0990] * Purity of intact antibody monomers detected in the sample.

[0991] When several peaks are detected, the data corresponding to the peak of intact antibody monomers are underlined. When a single peak is detected, an estimated value of the concentration (purity) is given.

[0992] The SEC-HPLC chromatograms of the obtained samples are also shown in Figure 7. SEC-HPLC analysis shows that all three antibody samples exhibit an ideal heterotetrameric antibody composed of two heavy chains and two light chains at the optimal concentration and very few aggregates, i.e., <5%.

[0993] Example 6 - Determination of the KD of antibody / antigen interaction by surface plasmon resonance (SPR) technology (Biacore 8K) KD

[0994] In this study, the Kd of 3 humanized antibodies against their soluble antigens has been determined by Biacore 8K. The L1-CAM recombinant protein (His-tag) has been used, which was obtained from Sinobiological with the catalog number 10140-H08H ( https: / / www.sinobiological.com / recombinant-proteins / human-l1cam-10140-h08h , as accessed on September 2, 2022). In the SPR experiment, the antigen was immobilized on the sensor chip, and the solution containing different concentrations of antibody was flowed over the antigen. The kinetic parameters, including their on / off rates (ka and kd), were measured in real time. Based on this, the binding affinity constant KD was determined. The materials used are summarized in Table E8.

[0995] Table E8. Material information

[0996] Product Type Concentration Purity MW (kDa) L1-CAM Ligand 1.00mg / ml >90％ 143 CE7 variant 4-AG Analyte 2.54mg / ml >90％ 150 CE7 variant 7-AG Analyte 3.26mg / ml >90％ 150 CE7 variant 18-AG Analyte 1.99mg / ml >90％ 150 Chimeric cHCE7 Analyte 2.10mg / ml >90％ 150 Deglycosylated cHCE7 Analyte 3.40mg / ml >90％ 150

[0997] The following buffers were used in the experiment:

[0998] Antigen dilution buffer: NaAc pH 4.5 (20 mM NaAc pH 4.5)

[0999] Running buffer / antibody dilution buffer: HBS-EP+ (0.01 M Hepes pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.01% surfactant P20)

[1000] Regeneration buffer: glycine pH 1.5

[1001] The CM5 sensor chip is used, which is a glass slide coated with a thin layer of gold and covalently linked to a carboxymethyl dextran matrix. Gold is necessary to generate the SPR response, and the dextran matrix allows the use of maleimide chemistry to covalently immobilize biomolecules.

[1002] Method

[1003] Channel 1: No coupling, used as a negative control.

[1004] Channel 2: L1-CAM (10 μg / ml) was immobilized on the 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. Therefore, the response is proportional to the number of antibody molecules interacting with the antigen.

[1005] Antibodies of determined concentrations were passed over the CM5 chip, and the response was captured over time, showing the course of the interaction and binding / dissociation cycles. Then, regeneration was performed to remove all remaining bound antibodies from the chip, and new concentrations were tested as described herein. After continuously testing different concentrations, kinetic parameters and affinities were calculated using BIA evaluation software.

[1006] The following concentrations were tested: 1.56 nM, 3.125 nM, 6.25 nM, 32 nM, 12.5 nM, and 25 nM. The sensorgrams are shown in Figure 8, and the results are summarized in Table E9.

[1007] Table E9 .

[1008] Ka (1 / Ms) Kd (1 / s) KD (M) CE7 variant 4-AG <![CDATA[1.23×10 6 > <![CDATA[8.18×10 -7 > <![CDATA[6.64×10 -13 > CE7 variant 7-AG <![CDATA[1.03×10 6 > <![CDATA[3.73×10 -8 > <![CDATA[3.62×10 -14 > CE7 variant 18-AG <![CDATA[1.09×10 6 > <![CDATA[1.35×10 -6 > <![CDATA[1.24×10 -12 <!-- 82 -->]]> Chimeric cHCE7 <![CDATA[8.45×10 5 > <![CDATA[4.39×10 -5 > <![CDATA[5.19×10 -11 > Deglycosylated cHCE7 <![CDATA[8.06×10 5 > <![CDATA[9.59×10 -5 > <![CDATA[1.2×10 -10 >

[1009] Three humanized non-glycosylated antibodies against L1-CAM and two control antibodies have KD values in the range of 10 -10 to 10 -13 , indicating very high affinity for the antigen, for the following reasons:

[1010] - A higher KD value in the range of 10 -13 indicates a very strong interaction,

[1011] - More favorable kinetic parameters, indicating rapid association and no dissociation,

[1012] - The shape of the sensorgram shows a clear concentration-response relationship.

[1013] Example 7 - Determination of the KD of antibody / antigen interaction by surface plasmon resonance (SPR) technology (Biacore 8K) KD

[1014] The KD of four monoclonal antibodies against their soluble antigens was determined using a Biacore 8K as described in Example 6.

[1015] Table E10 summarizes the materials used in this study.

[1016] Table E10. Material information

[1017] Product Type Concentration Purity MW (kDa) L1-CAM Ligand 1.00mg / ml >90％ 143 CE7 variant 7 Analyte 0.90mg / ml >90％ 150 CE7 variant 26 Analyte 0.67mg / ml >90％ 150 CE7 variant 27 Analyte 0.94mg / ml >90％ 150 CE7 variant 28 Analyte 0.97mg / ml >90％ 150

[1018] It should be noted that variants 26, 27, and 28 include the N297A mutation and are thus non-glycosylated. Variant 7 as used herein does not include this mutation and is expected to be glycosylated.

[1019] For a description of the experimental conditions, see Example 6.

[1020] The results are summarized in Figure 9 and Table E11.

[1021] Table E11. Kinetic parameters and affinity of antibody / antigen interaction

[1022] Ka (1 / Ms) Kd (1 / s) KD (M) CE7 variant 7 <![CDATA[6.15×10 5 > <![CDATA[5.51×10 -7 > <![CDATA[8.96×10 -13 > CE7 variant 26 8.91×105 <![CDATA[1.65×10 -7 > <![CDATA[1.86×10 -13 > CE7 variant 27 <![CDATA[7.16×10 5 > <![CDATA[1.02×10 -7 > <![CDATA[1.43×10 -13 > CE7 variant 28 <![CDATA[7.87×10 5 > <![CDATA[3.62×10 -5 > <![CDATA[4.6×10 -11 >

[1023] Example 8 - Production-susceptible variants 1 to 8 。

[1024] Starting from the sequences of the heavy chain (HC) and light chain (LC) variable regions of eight additional susceptibility variant antibodies generated by the computer-aided humanization step, full-length human IgG1κ antibodies carrying the mutations L234A, L235A, P331S, and N297A were designed. It should be noted that these susceptibility variants include the mutations that have been made on the chimeric CE7 antibody framework.

[1025] The cDNAs encoding the HC and LC variable regions were chemically synthesized and optimized for expression in CHO cells, and then subcloned into a ProteoGenix proprietary mammalian cell expression vector that contains the scaffolds of the human IgG1 heavy chain constant region and the human κ 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.

[1026] >CE7-VH-chimeric-WH33Y(L234A L235A+P331S N297A)[SEQ ID NO.:117]

[1027]

[1028] >CE7-VH Chimeric-WH33F (L234A L235A+P331S N297A) [SEQ ID NO.:118]

[1029]

[1030] >CE7-VH-chimeric-WH33L (L234A L235A+P331S N297A) [SEQ ID NO.:119]

[1031]

[1032] >CE7-VH-Chimeric-GH55A(L234A L235A+P331S N297A)[SEQ ID NO.:120]

[1033]

[1034] >CE7-VH chimeric (L234A L235A+P331S N297A) [SEQ ID NO.:121]

[1035]

[1036] >CE7-VL chimeric [SEQ ID NO.:122]

[1037] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCAGCGGCGCCTACGGCGATATCCAGATGACCCAGAGCTCCAGCTCCTTTAGCGTGAGCCTGGGCGACCGGGTGACCATCACCTGTAAGGCTAATGAGGACATCAATAACAGGCTGGCTTGGTACCAGCAGACCCCCGGCAATAGCCCTAGGCTGCTGATCAGCGGCGCTACCAATCTGGTGACCGGCGTGCCTAGCAGGTTTAGCGGCTCCGGCTCCGGCAAGGATTACACCCTGACCATCACCTCCCTGCAGGCCGAGGATTTTGCTACCTACTATTGTCAGCAGTACTGGAGCACCCCTTTTACCTTTGGCTCCGGCACCGAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[1038] >CE7-VL chimeric-NL31L [SEQ ID NO.:123]

[1039] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCAGCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGACATCCAGATGACCCAGAGCTCCTCCTCCTTTTCCGTGTCCCTGGGCGATCGGGTGACCATCACCTGTAAGGCCAATGAGGATATCAACCTGAGGCTGGCCTGGTACCAGCAGACCCCTGGCAACAGCCCTAGGCTGCTGATCTCCGGCGCTACCAATCTGGTGACCGGCGTGCCTAGCCGGTTTAGCGGCTCCGGCAGCGGCAAGGACTATACCCTGACCATCACCAGCCTGCAGGCCGAGGACTTTGCTACCTATTATTGTCAGCAGTACTGGAGCACCCCCTTTACCTTTGGCTCCGGCACCGAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[1040] >CE7-VL-chimeric-NL31A [SEQ ID NO.:124]

[1041] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCTCCGGCGCTTATGGCGATATCCAGATGACCCAGAGCTCCAGCAGCTTCTCCGTGAGCCTGGGCGATAGGGTGACCATCACCTGTAAGGCCAACGAGGATATCAATGCTAGGCTGGCTTGGTATCAGCAGACCCCTGGCAATAGCCCCCGGCTGCTGATCTCCGGCGCCACAAACCTGGTGACCGGCGTGCCTTCCAGGTTCTCCGGCAGCGGCTCCGGCAAGGATTATACCCTGACCATCACCAGCCTGCAGGCTGAGGATTTTGCTACCTACTACTGTCAGCAGTATTGGAGCACCCCCTTTACCTTTGGCTCCGGCACCGAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[1042] >CE7-VL-chimeric-WL92Y [SEQ ID NO.:125]

[1043] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCAGCCTGCTGCTGTGGATCTCCGGCGCTTATGGCGATATCCAGATGACCCAGAGCTCCTCCTCCTTTAGCGTGAGCCTGGGCGATCGGGTGACCATCACCTGTAAGGCCAACGAGGACATCAATAACAGGCTGGCTTGGTACCAGCAGACCCCTGGCAACAGCCCTCGGCTGCTGATCAGCGGCGCCACCAATCTGGTGACCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGATCCGGCAAGGACTACACCCTGACCATCACCAGCCTGCAGGCTGAGGACTTTGCCACCTATTACTGTCAGCAGTACTATTCCACCCCTTTCACCTTCGGCTCCGGCACCGAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[1044] >CE7-VL-Chimeric-WL92F [SEQ ID NO.:126]

[1045] GAATTCgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCAGCGGCGCTTATGGCGACATCCAGATGACCCAGAGCAGCTCCAGCTTCTCCGTGTCCCTGGGCGACCGGGTGACCATCACCTGTAAGGCCAATGAGGATATCAACAACCGGCTGGCCTGGTATCAGCAGACCCCTGGCAACAGCCCCAGGCTGCTGATCTCCGGCGCCACCAATCTGGTGACCGGCGTGCCCTCCCGGTTTTCCGGCAGCGGAAGCGGCAAGGATTACACCCTGACCATCACCTCCCTGCAGGCTGAGGATTTCGCTACCTACTATTGCCAGCAGTACTTTTCCACCCCCTTTACCTTTGGCTCCGGCACCGAGCTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[1046] The sequence of the expected protein produced is shown below.

[1047] >CE7-VH-chimeric-WH33Y(L234A L235A+P331S N297A)[SEQ ID NO.:127]

[1048] MKHLWFFLLLVAAPRWVLSQVQLQQPGAELVKPGASVKLSCKASGYTFTGYYMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1049] >CE7-VH-Chimeric-WH33F (L234A L235A+P331S N297A) [SEQ ID NO.:128]

[1050] MKHLWFFLLLVAAPRWVLSQVQLQQPGAELVKPGASVKLSCKASGYTFTGYFMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1051] >CE7-VH-Chimeric-WH33L (L234A L235A+P331S N297A) [SEQ ID NO.:129]

[1052] MKHLWFFLLLVAAPRWVLSQVQLQQPGAELVKPGASVKLSCKASGYTFTGYLMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1053] >CE7-VH-Chimeric-GH55A(L234A L235A+P331S N297A)[SEQ ID NO.:130]

[1054] MKHLWFFLLLVAAPRWVLSQVQLQQPGAELVKPGASVKLSCKASGYTFTGYWMHWVKQRPGHGLEWIGEINPSNARTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1055] >CE7-VH Chimeric (L234A L235A+P331S N297A) [SEQ ID NO.:131]

[1056] MKHLWFFLLLVAAPRWVLSQVQLQQPGAELVKPGASVKLSCKASGYTFTGYWMHWVKQRPGHGLEWIGEINPSNGRTNYNERFKSKATLTVDKSSTTAFMQLSGLTSEDSAVYFCARDYYGTSYNFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1057] >CE7-VL Chimeric [SEQ ID NO.:132]

[1058] MVLQTQVFISLLLWISGAYGDIQMTQSSSSFSVSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYWSTPFTFGSGTELEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1059] >CE7-VL Chimeric-NL31L [SEQ ID NO.:133]

[1060] MVLQTQVFISLLLWISGAYGDIQMTQSSSSFSVSLGDRVTITCKANEDINLRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYWSTPFTFGSGTELEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1061] >CE7-VL-Chimeric-NL31A [SEQ ID NO.:134]

[1062] MVLQTQVFISLLLWISGAYGDIQMTQSSSSFSVSLGDRVTITCKANEDINARLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYWSTPFTFGSGTELEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1063] >CE7-VL-Chimeric-WL92Y [SEQ ID NO.:135]

[1064] MVLQTQVFISLLLWISGAYGDIQMTQSSSSFSVSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYYSTPFTFGSGTELEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1065] >CE7-VL-Chimeric-WL92F [SEQ ID NO.:136]

[1066] MVLQTQVFISLLLWISGAYGDIQMTQSSSSFSVSLGDRVTITCKANEDINNRLAWYQQTPGNSPRLLISGATNLVTGVPSRFSGSGSGKDYTLTITSLQAEDFATYYCQQYFSTPFTFGSGTELEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1067] As described above, endotoxin-free DNA preparation of the obtained constructs was carried out. Eight antibodies were expressed by combining HC and LC as follows:

[1068] Table E12

[1069] Antibody ID Heavy chain Light chain LV1 pTXs1-CE7-VH-chimeric-WH33Y pTXs1-CE7-VL-chimeric LV2 pTXs1-CE7-VH-chimeric-WH33F pTXs1-CE7-VL chimera LV3 pTXs1-CE7-VH chimera-WH33L pTXs1-CE7-VL chimera LV4 pTXs1-CE7-VH chimera-GH55A pTXs1-CE7-VL chimera LV5 pTXs1-CE7-VH chimera pTXs1-CE7-VL chimera-NL31L LV6 pTXs1-CE7-VH chimera pTXs1-CE7-VL chimera-NL31A LV7 pTXs1-CE7-VH chimera pTXs1-CE7-VL chimera-WL92Y LV8 pTXs1-CE7-VH chimera pTXs1-CE7-VL chimera-WL92F

[1070] Using the method described in Example 2 or Example 3. The purification profiles and final QC are shown in Figure 10 and in Figure 11 . The yields and purities of the obtained antibodies are summarized in Table E13.

[1071] Table E13. Yield and purity obtained for pilot-scale rAb

[1072] Antibody Concentration Specification Quantity Yield* Purity** LV1 0.59 mg / ml 1.30 ml / vial 2 vials 1.53 mg / 30 ml >90％ LV2 1.73 mg / ml 0.70 ml / vial 1 vial 1.21 mg / 30 ml >90％ LV3 0.37 mg / ml 1.40 ml / vial 2 vials 1.04 mg / 30 ml >90％ LV4 0.20 mg / ml 1.60 ml / vial 4 vials 1.28 mg / 30 ml >90％ LV5 0.51 mg / ml 1.58 ml / vial 3 vials 2.42 mg / 30 ml >90％ LV6 0.54 mg / ml 1.50 ml / vial 3 vials 2.43 mg / 30 ml >90％ LV7 0.40 mg / ml 1.50 ml / vial 3 vials 1.80 mg / 30 ml >90％ LV8 0.48 mg / ml 1.57 ml / vial 3 vials 2.26 mg / 30 ml >90％

[1073] * Obtained after purification of 30 ml test culture.

[1074] ** Based on full-length antibodies observed on non-reducing PAGE

[1075] The analysis results are as shown in as shown in Figure 2 shown.

[1076] Final buffer: PBS pH 7.5.

[1077] Example 9 - SEC-HPLC Analysis of Susceptibility Variants

[1078] Short protocol description

[1079] SEC-HPLC analysis was performed to evaluate the purity level and quantify the proportion of soluble aggregates in the susceptible antibody samples. The analysis was carried out using an HPLC Waters 2695 device equipped with a photodiode array detector (2996).

[1080] Buffer preparation

[1081] - Mobile phase: 100 mM sodium sulfate, 100 mM phosphate, pH 6.7 buffer (14.2 g / L of Na2SO4, 15.6 g / L of Na2HPO4·12H2O, 6.8 g / L of NaH2PO4)

[1082] Remove air bubbles from all buffers.

[1083] Sample preparation

[1084] - Protein preparation: Start with an additional aliquot of each final sample stored at -80 °C overnight and dilute the protein sample to 1 mg / ml with DD water

[1085] - Centrifuge at 12,000 g for 10 minutes and collect the supernatant

[1086] Analysis conditions

[1087] - Blank control: Dilute the sample with DD water (5x)

[1088] - Column: G3000SWXL, TOSOH, 7.8 × 300 mm

[1089] - HPLC: Waters 2695

[1090] - Elution gradient: Isocratic elution with the mobile phase

[1091] - Flow rate: 0.8 ml / minute

[1092] - Temperature: 25 °C

[1093] - Injection volume: 30 μl

[1094] - Detection wavelength: 280 nm

[1095] - Collection date and time: 25 minutes

[1096] - Equilibrate with 100% mobile phase for 5 minutes

[1097] - Run the sample: Inject a blank control and then inject the sample

[1098] - Data analysis

[1099] The results are summarized in Table E14.

[1100] Table E14. SEC-HPLC data

[1101]

[1102] When several peaks are detected, the data for the peak corresponding to the intact antibody monomer are presented in bold.

[1103] When a single peak is detected, an estimated value of the concentration (purity) is given.

[1104] Example 10. Differential Scanning Fluorimetry Analysis

[1105] Short protocol description

[1106] DSF analysis was performed to evaluate the thermal stability (unfolding) of 18 humanized variant antibodies. The DSF work was carried out using a Nanotemper-nanoDSF system and standard methods. The mAb was heated from 40 °C to 90 °C at a linear heating rate of 1 °C / min. Tryptophan fluorescence at 350 nm and 330 nm was collected at a rate of 10 data points per minute. The midpoint of the unfolding transition was automatically determined based on the second derivative of the fluorescence ratio (F350 / F330). The samples are summarized in Table E15.

[1107] Table E15. DSF samples 。

[1108] Sample Quantity Storage buffer CE7 variant 1 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 variant 2 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 variant 3 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 variant 4 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 variant 5 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 variant 6 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 variant 7 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 variant 8 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 variant 9 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 variant 10 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 11 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 12 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 13 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 14 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 15 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 16 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 17 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 18 1.00 mg / ml, 50 μl / vial PBS, pH 7.5

[1109] The analysis results are shown in Table E16.

[1110] Table E16. Tm Values of Each Antibody 。

[1111]

[1112]

[1113] All antibodies showed two unfolding transitions in the plot of fluorescence ratio (F330 / F350) versus temperature. The different unfolding events can be attributed to the different thermal stabilities of the Fab and Fc domains of the antibody. DSF analysis showed that variants 5, 7, 8, 9, 10, and 11 had similar thermal stabilities, with Tm1 being approximately 68 °C and Tm2 being approximately 80 °C. Variants 13, 14, 15, 16, 17, and 18 all showed a slight decrease in Tm2 (approximately 74 °C).

[1114] Example 11. Differential Scanning Fluorimetry Analysis

[1115] The samples summarized in Table E17 were further analyzed as described in Example 10.

[1116] Table E17. DSF Samples

[1117] CE7 Variant 4AG 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 7AG 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 18AG 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 Chimeric cHCE7 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 Deglycosylated cHCE7 1.00 mg / ml, 50 μl / vial PBS, pH 7.5

[1118] The analysis results are shown in Table E18.

[1119] Table E18. Tm Values of Test Antibodies 。

[1120] Sample Tm 1 (°C) Tm 2 (°C) CE7 Variant 4AG 58.7℃ 80.0℃ CE7 Variant 7AG 58.3℃ 80.2℃ CE7 Variant 18AG 58.4℃ 74.3℃ Chimeric cHCE7 66.0℃ 73.8℃ Deglycosylated cHCE7 60.2℃ 73.6℃

[1121] The DSF analysis results were similar to those observed in Example 10, showing two unfolding events. The Tm1 and Tm2 of the chimeric antibody were comparable to Tm1 of the non-glycosylated variant. The non-glycosylated chimeric antibody had a Tm1 value similar to that of three humanized variants, but lower than Tm1 of the chimeric antibody, which may correspond to the unfolding event of the Fc domain. The Tm2 values of these three humanized non-glycosylated variants were higher and very similar to the Tm2 values obtained from the same non-glycosylated version. This indicates that Tm2 may correspond to the unfolding of the Fab domain.

[1122] Example 12. Differential Scanning Fluorimetry Analysis

[1123] The samples summarized in Table E19 were further analyzed as described in Example 10. It should be noted that all variants described therein (except for CE7 variant 7) were non-glycosylated (i.e., containing the mutation N297A in the heavy chain).

[1124] Table E19. DSF Samples

[1125] CE7 Variant 7 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 7-AG 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 19 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 20 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 21 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 22 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 23 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 24 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 25 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 26 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 27 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 CE7 Variant 28 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 LV1 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 LV2 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 LV3 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 LV4 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 LV5 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 LV6 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 LV7 1.00 mg / ml, 50 μl / vial PBS, pH 7.5 LV8 1.00 mg / ml, 50 μl / vial PBS, pH 7.5

[1126] The analysis results are shown in Table E20.

[1127] Table E20. Tm Values of Test Antibodies 。

[1128]

[1129]

[1130] All antibodies showed two unfolding transitions in the plot of fluorescence ratio (F330 / F350) versus temperature (Figure 1). The different unfolding events can be attributed to the different thermal stabilities of the Fab and Fc domains of the antibody. DSF analysis showed that additional variants 19 to 28 had similar thermal stabilities, with Tm1 of approximately 57 °C and Tm2 of approximately 80 °C, which were very similar to variant 7-AG. The susceptible variants LV1 to LV8 all showed a slight decrease in Tm2 (approximately 72 °C).

[1131] Example 13—Affinity of Antibody for L1-CAM—ELISA Study

[1132] The affinity of the antibody for L1-CAM was determined using the ELISA method according to the following protocol:

[1133] 1. Antigen coating: Human L1-CAM, 2 μg / ml, 100 μl / well, 37 °C, 2 hours

[1134] 2. Blocking: 3% BSA-PBS, 300 μl / well, 37°C, 1.5 h

[1135] 3. Washing: PBST, 300 μl / well, 2 times

[1136] 4. Primary antibody (humanized variant), 100 μl / well, 37°C, 30 min

[1137] 5. Washing: PBST, 300 μl / well, 3 times

[1138] 6. Secondary antibody, 100 μl / well, 37°C, 30 min

[1139] 7. Washing: PBST, 300 μl / well, 3 times

[1140] 8. TMB substrate solution: 100 μl / well, 37°C, 5 min to 10 min

[1141] 9. Termination: 2M HCl, 50 μl / well

[1142] 10. Reading: OD450 - OD630

[1143] Table E21

[1144]

[1145]

[1146] Example 14—GCI Study of Antibody Binding

[1147] In this study, the binding of certain antibodies of the present invention to L1-CAM was examined.

[1148] Basic principle:

[1149] · This experiment should study the binding of L1-CAM to antibodies captured by Protein A / G in a multi-cycle kinetic experiment. See FAQ.

[1150] · The ligand will be re-captured in each analysis cycle.

[1151] · The analyte will be injected onto the ligand and reference surfaces at increasing concentrations.

[1152] · After each analysis cycle, the ligand-analyte complex will be removed from the capture surface by injecting a regeneration solution.

[1153] · A DMSO solvent correction curve for solvent correction will be established. See FAQ.

[1154] · The non-specific and ligand-specific analyte binding of the original sensorgram will be detected.

[1155] ·Finally, the data will be double-referenced and, where applicable, fitted to an appropriate kinetic model.

[1156] Table E22. Details of GCI Setup 。

[1157]

[1158]

[1159] Figure 13A A double-referenced, solvent-corrected sensorgram showing the interaction between variant 7AG and L1-CAM is presented, and the measured response values are shown on the graph. Dose-responsive binding was detected. The data were fitted to a 1:1 interaction model, where the fitted curve is shown in black, giving a calculated affinity ≈4 nM. The ligand activity was calculated to be 28%. Lower concentrations were excluded to improve the fit quality.

[1160] Figure 13B A double-referenced, solvent-corrected sensorgram showing the interaction between deglycosylated cHCE7 and L1-CAM is presented, and the measured response values are shown on the graph. Dose-responsive binding was detected. The data were fitted to a 1:1 interaction model, where the fitted curve is shown in black, giving a calculated affinity ≈4 nM. The ligand activity was calculated to be 28%. Lower concentrations were excluded to improve the fit quality.

[1161] Thus, the study confirmed that the affinities of the humanized antibody and the chimeric antibody are comparable.

[1162] Example 15—Functionalization of the Antibodies of the Invention with an Azide Linker Using MTG

[1163] The humanized antibody variant (PBS solution at 5 mg / L, pH 7.5) was deglycosylated by mixing the humanized antibody variant with N-glycosidase (5.45 U / mg antibody) (N-glycosidase F, 1 U / μL, REF: 11365177001, Sigma Aldrich, Germany) in a 1.5 mL reaction tube and incubating at 37 °C for 24 h with gentle shaking. The completion of the reaction was analyzed by LC-MS. Subsequently, the buffer of the deglycosylated antibody was changed to Trizma buffer (50 mM pH 7.5) using a PD-10 desalting column. After that, the deglycosylated antibody (4 mg / ml) was incubated in a 5 ml low protein-binding reaction tube with a mixture of the following: microbial transglutaminase (MTG) (2.5 U / mg antibody [Andracon TM, Catalog number Log.T-153, Supplier Zedira, Germany], 80 equivalents / antibody of amino-PEG4-azide [Supplier Broadpharm, Catalog number Log.BP-21615, USA], in order to modify residue Q295 in the heavy chain (HC) of the antibody using the click-reactive azide linker. The conjugation reaction was carried out by incubating at 37 °C for 24 hours. The completion of the reaction was analyzed by LC-MS (HPLC 2795 Waters, ESI-TOF LCT Premier). The conjugation efficiency was calculated to be >90% by comparing the AUC of HC (unmodified) with HC (linker-modified), and the mass difference of the linker 245 Da was used to identify the peaks. After that, using the manufacturer's protocol, the residual enzyme and excess PEG linker were removed by protein A affinity chromatography [Protein Sepharose 4 Fast Flow, Supplier GE Healthcare USA, Catalog number Log.17.5280-20, where the column is SPE Catalog number Log.120.1362, Biotage, Sweden] The purified antibody-linker-azide conjugate was buffer-exchanged (5 times) to PBS pH 7.5 using an ultracentrifugation Ultra-15 centrifugal filter with a molecular weight cut-off of 30 kDa MWCO, Catalog number Log.UFC903008, Supplier Sigma Aldrich, Germany]. The antibody-linker-azide conjugate was stored at -20 °C for later use.

[1164] Example 16—Functionalization of the Nonglycosylated Antibodies of the Invention with an Azide Linker Using MTG

[1165] The non-glycosylated version of the humanized antibody of the present invention employed the same conjugation protocol as provided in Example 3, except that the enzymatic deglycosylation step was omitted. The conjugation efficiency with MTG was comparable to that described in Example 15.

[1166] Example 17—Functionalization of Variant 7AG-Q295-NH-PEG4-Azide with a Metal Chelator (DBCODOTA) Functionalization

[1167] The azide-modified antibody-linker conjugate was functionalized with a metal chelator to generate a model antibody-radionuclide conjugate (ARC) containing 2 DOTA chelators per mAb. Briefly, 23.2 μM variant 7AG-[Q295-NH-PEG4-azide]2 (synthesized according to the protocol provided in Example 4) was dissolved in PBS pH 7.5, 1 mM ammonium acetate (pH 5.5) was added, and then incubated with a 10-fold molar excess of click-reactive DBCO-DOTA (supplier Macrocyclics, Texas (USA), catalog number Log.B-283) at room temperature for 16 h. Excess DBCO-DOTA was then removed by ultrafiltration [Vivaspin 6, 30 kDa MWCO, catalog number Log.VS0621, Bio-rad, Germany].

[1168] Example 18—Mouse Biodistribution Study of Variant 7AG-[Q295-NH-PEG4-Click-(DOTA)]2

[1169] huCE7 variant 7AG-[Q295-NH-PEG4-click-(DOTA)]2 was obtained from Example 5, then radiolabeled with lutetium-177 and used in murine biodistribution studies to characterize tumor uptake.

[1170] Table E23 .

[1171]

[1172] The reaction was carried out in a 1.5 mL reaction tube with gentle stirring at 37 °C for 1 h. 1 μL of the 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 equipped with a γ-counting detector to monitor the labeling efficiency.

[1173] Cells for the tumor model:

[1174] 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 amphotericin (BioConcept, Allschwil, Switzerland). The cell line was cultured in a humidified environment containing 5% CO2. The cells were then injected into mice (5 million cells per animal) to generate a Her2 and L1-CAM-positive ovarian cancer tumor model.

[1175] Biodistribution of the murine tumor model:

[1176] A total of 32 mice bearing SKOV 3ip tumors were injected with 100 μL of an injection solution of 150 kBq huCE7 variant 7AG-[PEG4-DOTA(Lu-177)]2 (16 mice). In this study, cHCE7-[PEG4-DOTA(Lu-177)]2 (16 mice) was used as a control for tumor uptake. Four mice in each group were sacrificed at 24 hours, 48 hours, 72 hours, and 96 hours after injection, and the organs / tissues were dissected, weighed, and the radioactivity intensity was measured. The % i.A. / g was calculated and shown in In Figure 18 .

[1177] Within the monitoring time range from 24 hours to 96 hours, the tumor uptake between the humanized antibody huCE7 variant 7AG and the chimeric cHCE7 was comparable, at approximately 68% + / - 7.7% i.A. / g; the maximum activity reached 77% + / - 7.2% i.A. / g per gram of tissue at 96 hours, indicating that the humanized framework did not interfere with tumor uptake.

[1178] The organ distribution of the humanized antibody huCE7 variant 7AG was within the literature values of typical ARC ( Figure 18) .

[1179] Example 19—Functionalization of huCE7 Variant 7AG-[Q295-NH-PEG4-Azide] with a Toxin (MMAE) Functionalization

[1180] The azide-modified antibody-linker conjugate was functionalized with the toxin monomethyl auristatin E (MMAE) to generate a model containing 4 toxins / mAb, namely the antibody-drug conjugate. Briefly, variant 7-[Q295-NH-PEG4-azide]2 synthesized according to the protocol provided in Example 4 was incubated with a 10-fold molar excess of click-reactive DBCO-(PEG2-vc-PAB-MMAE)2 (supplied by SyntaBio, USA) in PBS pH 7.5 supplemented with 10% (m / v) dimethylacetamide at room temperature for 16 hours. Subsequently, the ADC was purified into PBS pH 7.5 through a Sephadex G25 column, and then the residual toxin linker was removed with activated carbon. The resulting ADC [huCE7-V7AG-(MMAE)4] was concentrated to approximately 3 mg / mL and then subjected to a final 0.2 μm filtration.

[1181] DAR analysis of the ADC:

[1182] As determined by SEC, the monomer accounted for 96.8% of the sample. The total concentration determined by SEC was 2.8 mg / mL. The drug-to-antibody ratio determined by MS was 3.9, and the drug-to-antibody ratio determined by HIC was 3.6. A total of 3.3 mg of huCE7-V7AG-(MMAE2)2 was produced.

[1183] Example 20—Zebrafish Toxicity and Tumor Efficacy Study with huCE7 Variant 7-[Q295-NH-PEG4-Click-Branched PEG-(MMAE)2]2 Part 1: Toxicity Assessment of the huCE7-V7AG Antibody

[1184] This study aimed to evaluate the toxicity and anti-tumor effects of 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. Three days after implantation, the anti-cancer efficacy of the antibodies was determined by changes in primary tumor size (i.e., tumor growth or shrinkage) and the number of tumor cells that spread to the distal caudal vein plexus (CVP), as described below.

[1185] The study design was divided into three parts and conducted at BioReperia AB (Wahlbecksgatan 25, 582 13 )

[1186] Part 1: Toxicity assessment of huCE7-V7AG antibody (reduced toxicity)

[1187] Part 2: Toxicity assessment of HUCE7-V7AG-(MMAE)4 antibody (comprehensive toxicity assessment)

[1188] Part 3: Efficacy assessment using the ZTX-ONCOLEADS model (BioReperia AB, Sweden)

[1189] Table E24: Grouping Description of the Toxicological Analysis of the huCE7-V7AG Antibody

[1190] A reduced toxicity study of huCE7-V7AG antibody and vehicle control at more than 5 concentrations (Table E24). The antibody was administered intravenously. Each group consisted of 20 zebrafish larvae at 48 hours post-fertilization and was incubated at 35.5 °C for 3 days after injection. Assessment was performed 72 hours after treatment, and terminal assessment was based on the following parameters: live / dead status and LD50 concentration.

[1191] Figure 17)

[1192]

[1193] Results:

[1194] Toxicity assessment was performed on different concentrations of "naked" huCE7-V7AG. The results showed that these embryos were very tolerant to the antibody, and only one embryo died in all experimental groups (at a concentration of 0.1 mg / mL) (Part 2: Toxicity Assessment of the HUCE7-V7AG-(MMAE)4 Antibody Since the mortality rate of all experimental groups did not exceed 50%, the LD50 linear fitting readings could not be directly adopted.

[1195] Table E25: Grouping Description of the Toxicological Analysis of the HUCE7-V7AG-(MMAE)4 Antibody

[1196] After obtaining the toxicity results of the huCE7-V7AG antibody, it was decided to conduct a comprehensive toxicity assessment of the HUCE7-V7AG-(MMAE)4 antibody. A comprehensive toxicity study of the HUCE7-V7AG-(MMAE)4 antibody at more than 5 concentrations and a vehicle control (Table E25). The antibody was administered intravenously. Each group would consist of 20 zebrafish larvae 48 hours after fertilization and be continuously incubated at 35.5 °C for 3 days after injection. Assessments were made at 24 hours, 48 hours, and 72 hours after treatment, and terminal assessments were made based on the following parameters: live / dead status, LT50, LD50, and non-lethal toxicity such as pericardial edema, malformation, necrosis, teratogenesis, etc. Representative images of the embryos were obtained at each time point.

[1197] Table E25.2—Percentage of Embryo Survival

[1198]

[1199] Results: Embryo survival after exposure to intravenously injected HuCE7-V7Ag(MMAE)4

[1200] Table E25.3—Percentage of Embryos Exhibiting Non-Lethal Toxicity after 72 Hours of Treatment

[1201]

[1202] Part 3: Efficacy Assessment Using ZTX-ONCOLEADS .

[1203]

[1204] The MMAE-loaded ADC showed good compatibility with the non-tumor zebrafish model. Since the 50% lethality rate was not reached (see Tables E25.2 and E25.3), the LT50 or LD50 concentrations could not be determined; additionally, all experimental groups were within the background mortality rate range of intravenously injected embryos. Regarding non-lethal toxicity, the 2.8 mg / mL concentration showed the highest percentage of embryos with partial toxicity, which was consistent with expectations but was still applicable to the efficacy study. However, the dose range for the efficacy study (Part 3) was set between 0.1 mg / ml and 0.9 mg / ml.

[1205] Table E26. Experimental Group Description of the Efficacy Analysis of huCE7-V7AG and HUCE7-V7AG-

[1206] The MAXFTN-401 cancer cells were used to evaluate the anti-tumor efficacy of huCE7-V7AG and HUCE7-V7AG-(MMAE)4 antibodies. The study consisted of 5 experimental groups (Table E26). The concentration of the antibody to be tested was determined based on the results of toxicity assessment. The tumor cells were implanted subcutaneously, and antibody treatment was administered intravenously after tumor implantation. Each experimental group included 20 tumor-bearing embryos and was incubated continuously at 35.5 °C for 3 days. Three days after implantation, the anti-tumor efficacy of the antibody was determined by the change in the size of the primary tumor (i.e., tumor growth or shrinkage) and the number of tumor cells that spread to the CVP.

[1207] Antibodies Using ZTX-ONCOLEADS (MMAE)4 Analysis Part 4: Experimental Group Description of the Efficacy Analysis of huCE7-V7AG and HUCE7-V7AG-

[1208]

[1209] The detailed results and descriptions of this study are shown in Example 28.

[1210] Antibodies Using ZTX-ONCOLEADS

[1211] The cDNA encoding the HC and LC sequences was chemically synthesized and optimized for expression in CHO cells, and then subcloned into a ProteoGenix proprietary mammalian cell expression vector. The sequences are shown below:

[1212] >CE7-1-2-VHB-WH33Y-GH55A[SEQ ID NO.:137]

[1213]

[1214] >CE7-1-2-VHB-WH33F-GH55A[SEQ ID NO:138]

[1215]

[1216] >CE7-1-NL1-VLA-NL31A-WL92Y[SEQ ID NO.:139]

[1217] GAATTCGCCGCCACCATGGTGCTGCAGACCCAGGTGTTTATCAGCCTGCTGCTGTGGATCTCCGGCGCTTATGGCGATATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCTTCTGTGGGAGATAGGGTGACCATCACCTGTAAGGCTAATGAGGATATCAACGCTCGGCTGGCCTGGTACCAGCAGAAGCCTGGAAAGGCTCCTAAGCTGCTGATCTCCGGCGCCACAAACCTGGTGACCGGAGTTCCTTCCCGGTTCTCCGGAAGCGGATCCGGAAAGGACTATACCCTGACCATCTCCAGCCTGCAGCCCGAAGATTTCGCTACCTATTACTGCCAGCAGTATTATAGCACCCCCTTCACCTTCGGCCAGGGCACCAAACTGGAGATCAAGCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[1218] >CE7-1-NL1-VLA-NL31A-WL92F[SEQ ID NO:140]

[1219] GAATTCGCCGCCACCATGGTGCTGCAGACCCAGGTGTTTATCAGCCTGCTGCTGTGGATCTCCGGCGCTTATGGCGATATCCAGATGACCCAGTCCCCCTCCTCCCTGTCTGCTTCTGTGGGAGATAGGGTGACCATCACCTGTAAGGCTAATGAGGATATCAACGCTCGGCTGGCCTGGTACCAGCAGAAGCCTGGAAAGGCTCCTAAGCTGCTGATCTCCGGCGCCACAAACCTGGTGACCGGAGTTCCTTCCCGGTTCTCCGGAAGCGGATCCGGAAAGGACTATACCCTGACCATCTCCAGCCTGCAGCCCGAAGATTTCGCTACCTATTACTGCCAGCAGTATTTCAGCACCCCCTTCACCTTCGGCCAGGGCACCAAACTGGAGATCAAGCGTACGGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[1220] The sequence of the expected protein produced is shown below.

[1221] >CE7-1-2-VHB-WH33Y-GH55A(L234A L235A+P331S+N297A)[SEQ ID NO.:141]

[1222] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWIGEINPSNARTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1223] >CE7-1-2-VHB-WH33F-GH55A(L234A L235A+P331S+N297A)[SEQ ID NO.:142]

[1224] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYFMHWVRQAPGQGLEWIGEINPSNARTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1225] >CE7-1-NL1-VLA-NL31A-WL92Y[SEQ ID NO.:143]

[1226] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKANEDINARLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYYSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1227] >CE7-1-NL1-VLA-NL31A-WL92F[SEQ ID NO.:144]

[1228] MVLQTQVFISLLLWISGAYGDIQMTQSPSSLSASVGDRVTITCKANEDINARLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYFSTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1229] Example 21—Expression and Purification of Susceptibility Variant LV29-32

[1230] Production and Purification

[1231] As described above, endotoxin-free DNA preparation was performed on the obtained constructs.

[1232] Four antibodies were expressed by combining HC and LC as follows:

[1233] Short Protocol Description .

[1234]

[1235] Using a proprietary Xten transfection protocol, the plasmids were transiently co-transfected into our proprietary XtenCHO cells. When the viability dropped below 50% (14 days after transfection), the culture medium was collected and purified as follows.

[1236] - Clarify by 0.22 μm filtration

[1237] - Equilibrate, bind, and wash with PBS pH 7.5

[1238] - Elute by changing the pH with citric acid

[1239] - Neutralize with 1 M Tris-HCl pH 9.0

[1240] - Analyze by PAGE and pool the fractions of interest.

[1241] - Perform final QC by PAGE: Qualitative and quantitative by SDS-PAGE

[1242] Combine the elution fractions and change the buffer to PBS, pH 7.5, by dialysis. Filter the final sample through a 0.22 μm microporous filter and determine the endotoxin level using a chromogenic LAL endotoxin assay kit according to the IFU (Genscript Toxinsensor kit #L00350). The purification test results and QC are as Table E27 Figure 14 and shown.

[1243] The resulting yield and purity are summarized in Table E28.

[1244] Figure 15 .

[1245] Table E28 Antibody Concentration Volume Number of Vials Yield* Purity (%)** LV29 2.44 mg / ml 1.50 ml / vial 2 vials >90％ 7.32 mg / 30 ml LV30 2.24 mg / ml 1.55 ml / vial 2 vials 6.94 mg / 30 ml >90％ LV31 2.36 mg / ml 1.62 ml / vial 2 vials 7.65 mg / 30 ml >90％ LV32 1.92 mg / ml 1.74 ml / vial 2 vials 6.68 mg / 30 ml >90％

[1246] Example 22 - SEC-HPLC Study of Susceptibility Variants

[1247] Buffer Preparation

[1248] - Mobile phase: 100 mM sodium sulfate, 100 mM phosphate, pH 6.7 buffer (14.2 g / L of Na2SO4, 15.6 g / L of Na2HPO4·12H2O, 6.8 g / L of NaH2PO4). Remove air bubbles from all buffers.

[1249] Sample Preparation

[1250] - Protein preparation: Start with an additional aliquot of each final sample stored at -80 °C overnight and dilute the protein sample to 1 mg / ml with DD water

[1251] - Centrifuge at 12,000 g for 10 minutes and collect the supernatant

[1252] Analysis Conditions

[1253] - Blank control: Dilute the sample (5x) with DD water

[1254] - Column: G3000SWXL, TOSOH, 7.8 × 300 mm

[1255] - HPLC: Waters 2695

[1256] - Elution gradient: Isocratic elution with the mobile phase

[1257] - Flow rate: 1.0 ml / minute

[1258] - Temperature: 25 °C

[1259] - Injection volume: 30 μl

[1260] - Detection wavelength: 280 nm

[1261] - Collection date and time: 25 minutes

[1262] - Equilibrate with 100% mobile phase for 5 minutes

[1263] - Run the sample: Inject a blank control, then inject the sample

[1264] - Data analysis

[1265] The results are shown in Table E29 and In Figure 16 。

[1266] ...

Claims

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

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the variable heavy chain region comprises CDR-H2 characterized by the sequence according to 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 CDR-L1 characterized by the sequence according to SEQ ID NO.: 14, 15, 17, 18 or 21.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the variable light chain region comprises CDR-L1 characterized by the sequence according to 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 CDR-L2 characterized by the sequence according to 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 CDR-L3 characterized by the sequence according to SEQ ID NO.:

25.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the variable heavy chain region is 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 according to SEQ ID NO.: 28 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), The sequence according to SEQ ID NO.: 29 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNERFKSRVTLTVDKSISTAYMELSRLRSDDTAVYFCARDYYGTSYNFDYWGQGTLVTVSS), The sequence according to SEQ ID NO.: 30 (QVQLQQWGAGLLKPSETLSLTCAAYGYTFTGYWMHWIRQPPGKGLEWIGEINPSNGRTNYNERLKSRVTLSVDKSKNQASLKLSSVTAADTAVYFCARDYYGTSYNFDYWGQGTLVTVSS), The sequence according to SEQ ID NO.: 31 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWIGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS), and The sequence according to SEQ ID NO.: 32 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWMHWVRQAPGQGLEWmGEINPSNGRTNYNEkFQGRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDYYGTSYNFDYWGQGTLVTVSS) 13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the variable heavy chain region is characterized by the sequence according to SEQ ID NO.: 28 or the sequence according to SEQ ID NO.:

32.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the variable light chain region is 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 according to SEQ ID NO.: 33 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), The sequence according to SEQ ID NO.: 34 (DIQMTQSPSSLSASVGDRVTITCKANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDIATYYCQQYWSTPFTFGQGTKLEIK) The sequence according to SEQ ID NO.: 35 (EIVMTQSPATLSVSPGERATLSCRANEDINNRLAWYQQKPGQAPRLLISGATNLVTGIPARFSGSGSGKEFTLTISSLQSEDFAVYYCQQYWSTPFTFGQGTKLEIK), The sequence according to SEQ ID NO.: 36 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGATNLVTGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK), and The sequence according to SEQ ID NO.: 37 (DIQMTQSPSSLSASVGDRVTITCRANEDINNRLAWYQQKPGKAPKLLISGAsNLVsGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKLEIK).

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

37.

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

37.

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

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy 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, still more preferably identical, to a sequence selected from the sequences according to SEQ ID NOs.: 110, 142, 155 to 163, preferably identical to the sequence according to SEQ ID NOs.: 110, 142, 155, 160 or 162, and 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, still more preferably identical to the sequence according to SEQ ID NO.: 97 or 143.

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

143.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain 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, still more preferably identical to the sequences according to SEQ ID NO.: 110, 142, 155 to 163, and wherein the light chain 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, still more preferably identical to the sequence according to SEQ ID NO.: 97 or 143.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain 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, still more preferably identical to a sequence selected from: the sequences according to SEQ ID NO.: 95 or 164 to 172, and wherein the light chain 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, still more preferably identical to the sequence according to SEQ ID NO.: 97 or 143.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain 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, still more preferably identical to a sequence selected from: the sequences according to SEQ ID NO.: 173 to 183, and wherein the light chain 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, still more preferably identical to the sequence according to SEQ ID NO.:

193.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain 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, still more preferably identical to a sequence selected from: the sequences according to SEQ ID NO.: 173 to 183, and wherein the light chain 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, still more preferably identical to the sequence according to SEQ ID NO.:

194.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain 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, still more preferably identical to a sequence selected from: the sequences according to SEQ ID NO.: 184 to 192, and wherein the light chain 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, still more preferably identical to the sequence according to SEQ ID NO.:

193.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain 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, still more preferably identical to a sequence selected from: the sequences according to SEQ ID NO.: 184 to 192, and wherein the light chain 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, still more preferably identical to the sequence according to SEQ ID NO.:

194.

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

194.

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

28. The antibody or antigen-binding fragment thereof according to 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, and more preferably, wherein the at least one point mutation is selected from L234A, L235A, P331S and N297A.

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

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 according to claim 30.

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

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

34. The immunoconjugate according to claim 32 or 33, wherein the active agent is a radionuclide, preferably a radionuclide for therapeutic applications and / or a radionuclide for diagnostic use, and the radionuclide for therapeutic applications is preferably selected from copper-67, strontium-89, yttrium-90, iodine-131, samarium-153, terbium-161, lutetium-177, astatine-211, radium-223 and actinium-225, and the radionuclide for diagnostic use is preferably 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. The immunoconjugate according to claim 32 or 33, wherein the active agent is selected from maytansine, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU-159682, anthracyclomycin, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, irinotecan, deruxtecan, topotecan, irinotecan, SN38 and belotecan.

36. A pharmaceutical composition comprising 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, and a pharmaceutically acceptable carrier.

37. 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, which is used as a drug.

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. An antibody or antigen-binding fragment thereof for use according to claim 38 or an immunoconjugate for use according to claim 38, wherein the L1-CAM (CD171)-associated cancer is selected from 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 tumor, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, 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 granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular adenocarcinoma, prostate cancer, and head and neck squamous cell carcinoma.

40. An antibody or antigen-binding fragment thereof for use according to claim 38 or 39 or an immunoconjugate for use according to claim 38 or 39, wherein the antibody or antigen-binding fragment thereof or the immunoconjugate is administered to a subject in combination with an additional therapeutic agent selected from alkylating agents, platinum-based agents, taxanes, vinca agents, antiestrogen drugs, aromatase inhibitors, ovarian function inhibitors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormone agents, immune checkpoint inhibitors, and bisphosphonate therapeutic agents.

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 the diagnosis of L1-CAM (CD171)-associated cancer.

43. An antibody or antigen-binding fragment thereof for use in the use according to claim 42, or an immunoconjugate for use in the use according to claim 42, wherein the L1-CAM (CD171)-related cancer is selected from 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 tumor, neuroendocrine tumor, gastrointestinal stromal tumor (GIST), pheochromocytoma, glioma, 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 granulosa cell tumor, schwannoma, primitive neuroectodermal tumor (PNET), epithelioid sarcoma, olfactory neuroblastoma, medulloblastoma, capillary hemangioma, Kaposi's sarcoma, rhabdomyosarcoma, submandibular adenocarcinoma, prostate cancer, and head and neck squamous cell carcinoma.

Images