Anti-CEACAM5 antibodies and conjugates and uses thereof

Abstract

The present invention provides an antibody that binds to human CEACAM5 protein, as well as an isolated nucleic acid and a host cell that contain a sequence encoding the antibody.The present invention also provides an immunoconjugate that contains the antibody linked to a growth inhibitory drug, and a pharmaceutical composition that contains the antibody or immunoconjugate of the present invention.The present invention also provides the use of the antibody, immunoconjugate, and pharmaceutical composition of the present invention for the treatment of cancer or for diagnostic purposes.

Description

[Technical field]

[0001] The present invention relates to an antibody that binds to human CEACAM5 protein, and an isolated nucleic acid and host cell that contain a sequence that codes for said antibody.The present invention also relates to an immunoconjugate that contains said antibody linked to a growth inhibitory drug, and to a pharmaceutical composition that contains the antibody or immunoconjugate of the present invention.The present invention also relates to the use of the antibody, immunoconjugate, and pharmaceutical composition of the present invention for the treatment of cancer or for diagnostic purposes. [Background technology]

[0002] Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion. CEA was first identified in 1965 as a protein normally expressed by fetal intestine during the first 6 months of pregnancy (Gold and Freedman, J Exp Med, 121, 439, 1965) and has been found in many cancers, such as colorectal or pancreatic cancer. The CEA family belongs to the immunoglobulin superfamily. The CEA family, consisting of 18 genes, is subdivided into two subgroups of proteins: the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) subgroup and the pregnancy-specific glycoprotein subgroup (Kammerer & Zimmermann, BMC Biology 2010, 8:12).

[0003] In humans, the CEACAM subgroup consists of seven members: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, and CEACAM8. CEACAM5, which is identical to the originally identified CEA, has been reported to be highly expressed on the surface of cancer cells, such as colon, stomach, lung, and pancreatic tumor cells, and its expression in normal tissues is restricted to a small number of normal epithelial cells, such as colon and esophageal epithelial cells. Therefore, CEACAM5 may constitute a suitable therapeutic target for tumor-specific targeting approaches, such as immunoconjugates.

[0004] The extracellular domain of CEACAM family members is composed of repeated immunoglobulin-like (Ig-like) domains, which are classified into three types A, B, and N according to sequence homology. CEACAM5 contains seven such domains, namely N, A1, B1, A2, B2, A3, and B3. The CEACAM5 A1, A2, and A3 domains on the one hand, and the B1, B2, and B3 domains on the other hand, show high sequence homology, with the A domain of human CEACAM5 exhibiting 84-87% pairwise sequence similarity, and the B domain 69-80%. Furthermore, other human CEACAM members, namely CEACAM1, CEACAM6, CEACAM7, and CEACAM8, which display their structural A and / or B domains, show homology with human CEACAM5. In particular, the A and B domains of the human CEACAM6 protein show sequence homology with either the A1 and A3 domains or the B1 to B3 domains of human CEACAM5, respectively, which is even higher than that observed between the A and B domains of human CEACAM5.

[0005] Anti-CEA antibodies have been produced for diagnostic or therapeutic purposes targeting CEA. Specificity for related antigens has always been mentioned as a concern in this technical field, for example by Sharkey et al (1990, Cancer Research 50, 2823). Due to the above-mentioned homology, some of the previously described antibodies demonstrate binding to the repeated epitopes of CEACAM5 present on different immunoglobulin domains, and show cross-reactivity to other CEACAM family members such as CEACAM1, CEACAM6, CEACAM7, or CEACAM8, and therefore may lack specificity for CEACAM5. However, for CEA targeting therapy, it is desirable for anti-CEACAM5 antibodies to have specificity that binds to human CEACAM5-expressing tumor cells but not to certain normal tissues expressing other CEACAM family members. It is noteworthy that CEACAM1, CEACAM6 and CEACAM8 have been described to be expressed by neutrophils of humans and non-human primates (Ebrahimmnejad et al, 2000, Exp Cell Res, 260, 365; Zhao et al, 2004, J Immunol Methods 293, 207; Strickland et al, 2009 J Pathol, 218, 380), where they have been shown to regulate granulopoiesis and play a role in immune responses. For therapeutic purposes, cross-reactivity of anti-CEACAM5 antibodies with CEACAM1, CEACAM6, CEACAM7, or CEACAM8 may therefore reduce the therapeutic index of the compound due to increased toxicity in normal tissues. Thus, there is a need for antibodies specifically directed against CEACAM5 that do not cross-react with other molecules of the CEACAM family, for example for use as part of an antibody-drug conjugate (ADC) or for use in any other manner that results in killing of target cells.

[0006] Moreover, since CEACAM5 has been described to be expressed in several normal cell tissues, it is desirable to develop anti-CEACAM5 antibodies capable of binding to human CEACAM5 and cynomolgus monkey (Macaca fascicularis) CEACAM5, because such antibodies can be readily tested in preclinical toxicology studies in cynomolgus monkeys to assess their safety profile.

[0007] The combination of a) the need for species cross-reactivity and b) specificity for human and Macaca fascicularis CEACAM5, i.e., no cross-reactivity with other Macaca fascicularis and human CEACAM family members, adds an additional layer of complexity to the development of novel anti-CEACAM5 antibodies, especially in light of the overall sequence homology between the human and Macaca fascicularis CEACAM proteins.

[0008] CEACAM5 has also been described in the literature as a surface protein that internalizes poorly (reviewed in Schmidt et al, 2008, Cancer Immunol. Immunother. 57, 1879), presenting an additional challenge for antibody-drug conjugates directed against this target protein.

[0009] Known anti-CEACAM5 antibodies include Immunomedics' labetuzumab (also known as hMN14; Sharkey et al, 1995, Cancer Research 55, 5935). This antibody has been shown not to bind to related antigens, but is also not cross-reactive with CEACAM5 from Macaca fascicularis. Labetuzumab has also been used as part of an antibody-drug conjugate (ADC), namely labetuzumab govitecan. Labetuzumab govitecan is an ADC composed of the cytotoxic drug SN38 conjugated to the anti-CEACAM5 antibody labetuzumab via a linker (called CL2A) containing a pH-sensitive carbonate and a short polyethylene glycol (PEG) chain. Labetuzumab govitecan is characterized by significant instability of the linker structure used, leading to early systemic loss of the cytotoxic payload after parenteral application. This degradation process may limit antitumor activity and increase the risk of side effects. Another known anti-CEACAM5 ADC is Sanofi's SAR408701 (Tsamitamab Rabtansine), which comprises the anti-CEACAM5 antibody SAR408377 (Tsamitamab, also referred to as huMab2-3) covalently linked to the potent microtubule-destabilizing maytansinoid cytotoxic agent DM4 via an N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB) linker. SAR408701 is associated with toxic side effects (including keratitis and keratopathy) on several organs and tissues, including the cornea of ​​the eye. Also, the efficacy of microtubule inhibitor-based ADCs may be limited in certain cancer indications, such as colorectal cancer. To date, no anti-CEACAM5 antibody or ADC has been approved for any therapeutic use in the clinic. In general, only a few ADCs have been approved for the treatment of solid tumors.

[0010] Some ADCs are dose-limiting in patients due to side effects of the released payload due to cellular catabolism, which can result in toxicity in bone marrow and circulating blood cells (e.g., neutropenia, reticulocytopenia, lymphopenia).

[0011] Due to the drug coupling to antibody, ADCs often have suboptimal half-life in humans, only a few days in circulation.This is significantly lower than the half-life of corresponding unconjugated antibodies.The relatively high clearance of these ADCs is related to cellular degradation after target-independent uptake, leading to substantial release of toxic drug payloads that can induce side effects.Therefore, it is the objective of the ADCs disclosed herein to improve their half-life in order to reduce unwanted side effects.

[0012] In view of the above, there remains a need for new and improved therapeutic agents for the treatment of cancer, e.g., for different solid tumor indications including, e.g., CRC, pancreatic cancer, gastric cancer, NSCLC, esophageal cancer, and prostate cancer. Summary of the Invention

[0013] The present invention addresses this and other needs in the art, in particular by providing monoclonal antibodies directed against CEACAM5 (reactive with both human and Macaca fascicularis proteins) and by providing immunoconjugates (also referred to herein as antibody-drug conjugates (ADCs)) comprising said antibodies. These immunoconjugates have a cytotoxic effect, killing tumor cells in vitro and inhibiting tumor growth in vivo. The present invention relates to the embodiments described in the claims and further described herein below.

[0014] In an attempt to generate new antibodies against CEACAM5 with optimal properties for therapeutic purposes, particularly in the format of an immunoconjugate, the inventors have carried out extensive research and development to select antibodies with advantageous profiles and to develop immunoconjugates based thereon.

[0015] The present inventors have been able to select and produce optimized IgGs that unexpectedly contain several desirable features. These antibodies bind with high affinity to the A2-B2 domain of human CEACAM5 and do not recognize human CEACAM1, CEACAM6, CEACAM7, and CEACAM8 proteins. In the cellular context, these antibodies show high affinity to CEACAM5-expressing tumor cells and are internalized. Moreover, these antibodies also bind to Macaca fascicularis CEACAM5 protein, with affinities for monkey and human proteins within 10-fold of each other. Although the antibodies of the present invention bind to the A2-B2 domain of Macaca fascicularis CEACAM5, they do not recognize another Macaca fascicularis CEACAM protein, CEACAM6.

[0016] The inventors have also shown that the antibodies they have generated can induce cytotoxic effects against tumor cells in vitro when combined with cytotoxic drugs as immunoconjugates. The antibodies conjugated to cytotoxic drugs (i.e., the immunoconjugates of the present invention) can also significantly inhibit tumor growth in mice bearing CEACAM5-expressing tumors. The linker connecting the drug and the antibody was designed to maximize systemic stability after parenteral application. The release of exatecan from the immunoconjugates of the present invention within target cells leads to very high titers and a significant bystander effect. The strong bystander effect may be beneficial for the treatment of patients with heterogeneous target expression.

[0017] Generally, high systemic exposure is desired, which ultimately leads to effective tumor targeting and improved cytotoxic payload deployment to tumor tissues and cells in enhanced tumor cell killing compared to compounds with lower systemic exposure.

[0018] Furthermore, the antibody-drug-conjugates of the invention are improved by the inclusion of molecular modifications to reduce target-independent cellular degradation leading to molecules with lower clearance, higher systemic exposure and reduced payload release.

[0019] The present invention relates to antibody modification and payload conjugation strategies that significantly reduce off-target cellular catabolism of such ADCs, thereby reducing the levels of released payload while improving efficacy driven by higher ADC exposure. These modifications thus provide drugs with improved therapeutic windows with reduced side effects and increased antitumor activity. As described herein, the exposure and half-life of ADCs in accordance with the invention can be determined, for example, by: (1) by modifying the antibody heavy chain by including a YTE mutation and linking it to a drug using interchain cysteine ​​conjugation; or (2) It will be improved by modification of the antibody heavy chain with YTE mutations and by using an innovative enzymatic conjugation method that uses the microbial transglutaminase (also referred to herein as "TGase" and "mTG") enzyme to attach drugs to antibodies.

[0020] Surprisingly, high exposure leading to low clearance values ​​in human predictive animal models was obtained by the above approach (2). Such modifications improved the exposure of the ADC by a factor of about 10x compared to variants that did not contain these modifications. This resulted in a significant reduction in the released payload levels and a significant increase in the concentration of antitumor active ADC in circulation. The improved ADC resembled the PK profile of the original YTE antibody without the drug linker and showed improved half-life over prior art ADCs.

[0021] These results represent reduced toxicity and improved efficacy in clinical cancer therapy compared to prior art molecules. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 Binding of mAb1 to recombinant human (rh) CEACAM5 ECD or its domains N-A1-B1, A2-B2, A3-B3, or to recombinant Macaca fascicularis (mf) CEACAM5 ECD in an ELISA assay. [Diagram 2] Figure 2. EC50 of anti-CEACAM5 antibodies binding to MKN-45 cells. Cell binding of mAb1 compared to antibodies huMab2-3 and hmn-14 to the MKN45 cell line expressing CEACAM5. [Diagram 3] FIG. 3. Internalization of pHrodo-labeled antibodies into late endosomes and lysosomes of cells (total fluorescence intensity per cell, average of triplicates). [Figure 4] Figure 4. Fluorescence intensity per cell from 700 to 1200 min, which is the linear part of the curve. The slope of the line was measured and compared between samples (see Example 1.6.5). [Diagram 5] Figure 5. IHC staining with antibody rb8G4 in FFPE cancer cell lines. [Figure 6] Figure 6 Correlation of CEACAM5 mRNA expression and IHC staining for 104 cancer cell lines. [Figure 7] FIG. 7 IHC staining with antibody rb8G4 in normal human tissues. [Figure 8] FIG. 8 CEACAM5 mRNA expression in normal human tissues. [Figure 9] Figure 9. IHC staining of human colon cancer tissue using antibody rb8G4. [Figure 10] Figure 10 IHC staining of human gastric cancer tissues using antibody rb8G4. [Figure 11] Figure 11. IHC staining of human esophageal cancer tissue using antibody rb8G4. [Figure 12] Figure 12 IHC staining of human non-small cell lung cancer tissues using antibody rb8G4. [Figure 13-1]FIG. 13 Binding of mAb1 (FIG. 13A) and rb8G4 (FIG. 13B) to CEACAM5 in cancer cell line lysates examined by Western blotting. [Figure 13-2] FIG. 13 Binding of mAb1 (FIG. 13A) and rb8G4 (FIG. 13B) to CEACAM5 in cancer cell line lysates examined by Western blotting. [Figure 14] FIG. 14. Typical SEC chromatograms showing the purity of the stock mAb, the conjugate after UF, and the final bulk drug substance (BDS). [Figure 15] Figure 15. Typical RP-HPLC chromatogram showing separation of light and heavy chains. The chromatogram shows an overlay of the stock mAb, crude ADC, and final BDS. [Figure 16] FIG. 16 is a typical chromatogram showing free drug levels of the NAC standard and the final BDS. [Figure 17] FIG. 17. Typical SEC chromatograms showing the purity of the stock mAb and the final BDS. [Figure 18] Figure 18. Typical RP-HPLC chromatogram showing separation of light and heavy chains. The chromatogram shows an overlay of the stock mAb and the final BDS. [Figure 19] FIG. 19 is a typical chromatogram showing free drug levels of the NAC standard and the final BDS. [Figure 20] Figure 20. ADC stability for human, mouse, and cynomolgus serum. Conjugated exatecan concentrations were calculated using free exatecan (initial dose approximately 10 μM) (normalized data). [Figure 21] Figure 21. ADC3 control stability for mouse serum and buffer. Conjugated SN38 concentration was calculated using free SN38 (ADC protein concentration at initial dose of 50 μg / mL) (not normalized). [Figure 22]Figure 22 Payload release profile of ADC1 and ADC2 in human liver lysosomes (pH 5.0). Conjugated drug concentrations were calculated using, for example, free exatecan (initial concentration approximately 10 μM exatecan). Normalized data. [Diagram 23] FIG. 23 is ADC catabolite profiling confirming free exatecan as a lysosomal release product. [Figure 24-1] Figure 24. In vitro potency of ADC1, ADC2, and free payload against antigen-positive SK-CO-1 (Figure 24A) and SNU-16 (Figure 24B) cell lines compared to antigen-negative MDA-MB-231 (Figure 24C) cell line. One representative experiment is shown. Mean ± SD of triplicates. The legend assigning the three different series of data points to ADC1, ADC2, and payload, respectively, shown in Figure 24C also applies to Figures 24A and 24B. [Figure 24-2] Figure 24. In vitro potency of ADC1, ADC2, and free payload against antigen-positive SK-CO-1 (Figure 24A) and SNU-16 (Figure 24B) cell lines compared to antigen-negative MDA-MB-231 (Figure 24C) cell line. One representative experiment is shown. Mean ± SD of triplicates. The legend assigning the three different series of data points to ADC1, ADC2, and payload, respectively, shown in Figure 24C also applies to Figures 24A and 24B. [Diagram 25] Figure 25. Comparison of ADC1 and ADC2 with their respective isotype controls in SK-CO-1 cell line. One representative experiment is shown. Mean ± SD of triplicates. [Figure 26] Figure 26. In vitro potency of ADC1, ADC2, ADC SAR DM4, ADC mAb1 DM4, and free payload against antigen-positive SK-CO-1 (Figure 26A) compared to antigen-negative MDA-MB-231 (Figure 26B) cell lines. One representative experiment (mean ± SD of triplicates) is shown; the legend shown in Figure 26B also applies to Figure 26A. [Figure 27]Figure 27. Strong bystander effect of ADC1 and ADC2 on antigen-negative MDA-MB-231 cells in co-culture with antigen-positive SK-CO-1 cells (Figure 27A). There is no non-specific effect of ADC1 or ADC2 on MDA-MB-231 cells alone (Figure 27B). One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 28-1] FIG. 28. The bystander effect of ADC1 and ADC2 on antigen-negative MDA-MB-231 cells in co-culture with antigen-positive SK-CO-1 cells is stronger than that of ADC SAR DM4 (FIG. 28A and FIG. 28B). [Figure 28-2] There is no non-specific effect of the tested ADCs on MDA-MB-231 cells alone (Figure 28C). One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 29] FIG. 29. Efficacy of ADC1 and ADC2 in a CRC PDX model (COPF217) after a single treatment. [Diagram 30] FIG. 30. Efficacy of ADC1 in NSCLC PDX models (LUPF160151) after a single treatment. [Diagram 31] FIG. 31 Efficacy of ADC1 in a gastric cancer PDX model (GAX066) after a single treatment. [Diagram 32] FIG. 32 Efficacy of ADC1 compared to ADC3 in the pancreatic xenograft model (HPAF-II). [Diagram 33] FIG. 33. Efficacy of ADC1 compared to ADC SAR DM4 in CRC PDX model (COPF230). [Diagram 34] FIG. 34. Efficacy of ADC1 compared to ADC SAR DM4 in CRC PDX model (REPF210). [Diagram 35] FIG. 35 Efficacy of ADC1 compared to ADC SAR DM4 in gastric PDX model GAPF313 (interim analysis of ongoing experiment). [Diagram 36]FIG. 36. Typical SEC chromatograms showing the purity of input mAb and final BDS for mAb1-M / ADC1-M. [Figure 37] FIG. 37 Typical RP-HPLC chromatograms illustrating the DAR determination of the final BDS for mAb1-M / ADC1-M. [Figure 38] FIG. 38. Representative SEC chromatograms showing the purity of input mAb and final BDS for ADC7-M, ADC2-M, and ADC5-M. [Figure 39] FIG. 39 Typical RP-HPLC chromatograms illustrating the DAR determination of the final BDS for ADC7-M, ADC2-M, and ADC5-M. [Figure 40-1] Figure 40. In vitro potency of ADC1-M, ADC4-M, and free payload against antigen-positive SK-CO-1 (Figure 40a), SNU-16 (Figure 40b), MKN-45 (Figure 40c), and LS174T (Figure 40d) cell lines compared to antigen-negative MDA-MB-231 (Figure 40e) cell line. One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 40-2] Figure 40. In vitro potency of ADC1-M, ADC4-M, and free payload against antigen-positive SK-CO-1 (Figure 40a), SNU-16 (Figure 40b), MKN-45 (Figure 40c), and LS174T (Figure 40d) cell lines compared to antigen-negative MDA-MB-231 (Figure 40e) cell line. One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 40-3] Figure 40. In vitro potency of ADC1-M, ADC4-M, and free payload against antigen-positive SK-CO-1 (Figure 40a), SNU-16 (Figure 40b), MKN-45 (Figure 40c), and LS174T (Figure 40d) cell lines compared to antigen-negative MDA-MB-231 (Figure 40e) cell line. One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 41-1]Figure 41. In vitro potency of ADC2-M, ADC5-M, and free payload against antigen-positive SK-CO-1 (Figure 41a), SNU-16 (Figure 41b), MKN-45 (Figure 41c), and LS174T (Figure 41d) cell lines compared to antigen-negative MDA-MB-231 (Figure 41e) cell line. One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 41-2] Figure 41. In vitro potency of ADC2-M, ADC5-M, and free payload against antigen-positive SK-CO-1 (Figure 41a), SNU-16 (Figure 41b), MKN-45 (Figure 41c), and LS174T (Figure 41d) cell lines compared to antigen-negative MDA-MB-231 (Figure 41e) cell line. One representative experiment (mean ± SD of duplicate experiments) is shown. [Figure 41-3] Figure 41. In vitro potency of ADC2-M, ADC5-M, and free payload against antigen-positive SK-CO-1 (Figure 41a), SNU-16 (Figure 41b), MKN-45 (Figure 41c), and LS174T (Figure 41d) cell lines compared to antigen-negative MDA-MB-231 (Figure 41e) cell line. One representative experiment (mean ± SD of duplicate experiments) is shown. [Diagram 42] FIG. 42 Pharmacokinetic profiles in huFcRn Tg276 mice (total antibody) for ADC1, ADC1-M, ADC2-M, ADC6-M, and ADC7-M. [Diagram 43] FIG. 43. Tumor volume change following treatment with ADC1-M and ADC2-M versus vehicle control. [Diagram 44] Figure 44. ADC SAR Strong bystander effect of ADC1-M, ADC2-M, ADC6-M, and ADC7-M on antigen-negative MDA-MB-231 cells in co-culture with antigen-positive SK-CO-1 cells compared to DM4. One representative experiment (mean ± SD of duplicate experiments) is shown. [Diagram 45] FIG. 45. Efficacy of ADC1-M and ADC3-M compared to ADC8 in the HPAF-II xenograft model. [Figure 46]Figure 46. Efficacy of ADC1-M, ADC3-M compared to ADC SAR DM4 in a CRC PDX model (COPF230) after a single dose. [Figure 47] FIG. 47. Efficacy of ADC1-M, ADC3-M compared to ADC SAR DM4 in a GC PDX model (GAPF313) after multiple treatments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] definition "CEACAM5" as used herein refers to "carcinoembryonic antigen-related cell adhesion molecule 5", also known as "CD66e" (cluster of differentiation 66e). CEACAM5 is a glycoprotein involved in cell adhesion. CEACAM5 is highly expressed on the surface of, for example, colon cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer, and other solid tumors, among others. The reference sequence of full-length human CEACAM5, including the signal peptide (positions 1-34) and propeptide (positions 686-702), is available from the GenBank database under accession number AAA51967.1. The amino acid sequence reads as follows: (SEQ ID NO: 1).Five nonsynonymous SNPs have been identified with frequencies higher than 2% in the Caucasian population. Four of them are located in the N domain of human CEACAM5 (positions 80, 83, 112, 113) and the last one in the A2 domain (position 398). GenBank AAA51967.1 contains the major haplotypes (I80, V83, I112, I113, and E398).

[0024] A "domain" or "region" is any region of a protein that is generally defined based on sequence homology and often relates to a particular structural or functional entity. CEACAM family members are known to be composed of Ig-like domains. The term "domain" is used in this document either to refer to individual Ig-like domains, such as the "N domain," or to groups of contiguous domains, such as the "A2-B2 domain."

[0025] The domain organization of human CEACAM5 is as follows (based on the GenBank AAA51967.1 sequence; SEQ ID NO:1): [Table A]

[0026] Thus, the A2-B2 domain of human CEACAM5 consists of amino acids 321-498 of SEQ ID NO:1.

[0027] The reference sequence for the Macaca fascicularis CEACAM5 protein is available (NCBI reference sequence XP_005589491.1) and this amino acid sequence reads as follows:

number

[0028] A sequence that "encodes" an expression product such as a polypeptide, protein, or enzyme, or a "coding sequence" is a nucleotide sequence that, when expressed, results in the production of that polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. A protein coding sequence includes a start codon (usually ATG) and a stop codon.

[0029] The reference of a particular protein (e.g., an antibody) as used herein can include polypeptides having a native amino acid sequence, as well as mutants and modified forms, regardless of their origin or mode of preparation. A protein having a native amino acid sequence is a protein having the same amino acid sequence as that obtained from nature. Such native sequence proteins can be isolated from nature or prepared using standard recombinant and / or synthetic methods. Native sequence proteins specifically encompass naturally occurring truncated or soluble forms, naturally occurring mutant forms (e.g., alternatively spliced ​​forms), naturally occurring allelic variants, and forms that include post-translational modifications. Native sequence proteins include proteins that carry post-translational modifications, such as glycosylation or phosphorylation, or other modifications of some amino acid residues.

[0030] The term "gene" refers to a DNA sequence that codes for or corresponds to a specific sequence of amino acids, including all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, that determine the conditions under which the gene is expressed. Some genes that are not structural genes are transcribed from DNA into RNA but are not translated into amino acid sequences. Other genes function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene contemplates genomic sequences that code for proteins, i.e., sequences that include regulators, promoter, intron, and exon sequences.

[0031] As used herein, a sequence that is "at least 85% identical" to a reference sequence is a sequence that has 85% or more, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of the reference sequence. Thus, the percentage of "sequence identity" can be determined by comparing two such sequences over their entire length by global pairwise alignment using the algorithm of Needleman and Wunsch (J.Mol.Biol.48:443 (1970)). For example, the program Needle (EMBOSS) is used with the BLOSUM62 matrix and the following parameters: gap open=10, gap extension=0.5, end gap penalty=not applicable, end gap open=10, end gap extension=0.5 (these are standard settings).

[0032] A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain with similar chemical properties (e.g., charge, size, or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. Examples of groups of amino acids with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitutions can also be defined based on amino acid size.

[0033] An "antibody" (also referred to as an "immunoglobulin") is, for example, a natural or conventional type of antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (I) and kappa (k). There are five main heavy chain classes (or isotypes) that determine the functional activity aspects of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each antibody chain contains distinct sequence domains (or regions). The light chain of a typical IgG antibody contains two regions, the variable region (VL) and the constant region (CL). The heavy chain of a typical IgG antibody contains four regions, the variable region (VH) and the constant region (CH), and the remaining three constant domains (CH1, CH2, and CH3). The variable regions of both the light and heavy chains determine the binding and specificity to the antigen. The constant regions of the light and heavy chains can confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the Fab fragment of an antibody and consists of the variable portions of one light chain and one heavy chain.

[0034] The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is mainly made up of residues that are from the so-called hypervariable or complementarity determining regions (CDRs). Complementarity determining regions (CDRs) therefore refer to amino acid sequences that together define the binding affinity and specificity of the Fv region of an antibody. The light (L) and heavy (H) chains of an antibody have three CDRs, designated CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, the antigen binding site of a conventional antibody encompasses six CDRs, including a set of CDRs from each of the heavy and light chain variable regions.

[0035] "Framework region" (FR) refers to the amino acid sequence inserted between the CDRs, i.e., the portion of the immunoglobulin light and heavy chain variable regions that is relatively conserved among different immunoglobulins of a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. As used herein, a "human framework region" is a framework region that is substantially (about 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the framework region of a naturally occurring human antibody.

[0036] In the context of the present invention, the definition of CDR / FR in an immunoglobulin light or heavy chain is determined based on the definition of IMGT (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

[0037] As used herein, the term "antibody" includes conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, such as the variable heavy chains of single domain antibodies. As used herein, the term "antibody" also includes chimeric, humanized, bispecific or multispecific antibodies, as well as other types of engineered antibodies. The term "antibody" includes monoclonal antibodies.

[0038] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single amino acid sequence directed against a specific antigen and should not be construed as requiring production of the antibody by any particular method. A monoclonal antibody can be produced, for example, by a single clone of a B cell or hybridoma, but can also be produced by methods including recombinant, e.g., genetic or protein engineering.

[0039] The term "chimeric antibody" in its broadest sense refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In some embodiments, chimeric antibodies contain the VH and VL of an antibody derived from a non-human animal, together with the CH and CL of another antibody, which in some embodiments is a human antibody. The non-human animal may be any animal, such as mouse, rat, hamster, rabbit, etc. Chimeric antibodies also refer to multispecific antibodies that have specificity for at least two different antigens.

[0040] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and has been modified, for example, by replacing certain amino acids in the framework regions of VH and VL to avoid or minimize immune responses in humans. The constant regions of a humanized antibody are typically human CH and CL regions.

[0041] A "fragment" of an antibody (e.g., of a conventional antibody) comprises a portion of an intact antibody, such as an IgG, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody can also be a heavy chain antibody or a single domain antibody, such as a VHH.

[0042] The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 Da and antigen-binding activity, in which about half of the N-terminal heavy chain and the entire light chain are linked via disulfide bonds. It is usually obtained from the fragment by treating IgG with the protease papain.

[0043] The term "F(ab')2" refers to an antibody fragment with a molecular weight of about 100,000 Da and antigen-binding activity, which is slightly larger than two identical Fab fragments linked via disulfide bonds in the hinge region. It is usually obtained from among the fragments by treating IgG with the protease pepsin.

[0044] The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 Da and antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0045] Single chain Fvs ("scFvs") are covalently linked VH::VL heterodimers, which are usually expressed from gene fusions that include genes encoding VH and VL linked by a peptide-encoding linker. The human scFv fragments of the invention include CDRs that are held in the proper configuration, for example by using recombinant gene technology. Bivalent and multivalent antibody fragments form spontaneously by association of monovalent scFvs, or are generated by coupling monovalent scFvs by a peptide linker, such as bivalent sc(Fv)2. A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" refers to two dsFvs linked by a peptide linker.

[0046] The term "bispecific antibody" or "BsAb" refers to an antibody that contains two different antigen binding sites. Thus, BsAbs are capable of binding, for example, to two different antigens simultaneously. Genetic engineering is frequently used to design, modify, and generate antibodies or antibody derivatives with a desired set of binding properties and effector functions, as described, for example, in EP 2 050 764 A1.

[0047] The term "multispecific antibody" refers to an antibody that contains two or more different antigen-binding sites.

[0048] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites.

[0049] The term "hybridoma" refers to cells obtained by subjecting B cells prepared by immunizing a non-human mammal with an antigen to cell fusion with myeloma cells derived from a mouse or the like, which produce the desired monoclonal antibody having antigen specificity.

[0050] "Purified" or "isolated," when referring to a polypeptide (e.g., an antibody) or nucleotide sequence, means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" means that at least 75%, 85%, 95%, 96%, 97%, or 98% by weight of biological macromolecules of the same type are present. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide of interest; however, the molecule may include some additional bases or moieties that do not adversely affect the basic characteristics of the composition.

[0051] As used herein, the term "subject" refers to a mammal, such as a rodent, cat, dog, primate, or human. In an embodiment of the invention, the subject (or patient) is a human.

[0052] Antibodies of the Invention The present inventors have succeeded in generating, screening and selecting specific anti-CEACAM5 antibodies, which surprisingly exhibit a combination of several properties that make them ideally suited for use in cancer therapy, especially as part of an immunoconjugate (antibody-drug conjugate).For example, the antibodies of the present invention exhibit high affinity for both human and Macaca fascicularis CEACAM5 proteins, and they do not significantly cross-react with either human CEACAM1, CEACAM6, CEACAM7 and CEACAM8 proteins, or Macaca fascicularis CEACAM6 protein.The present inventors have determined the amino acid sequence of such monoclonal antibodies according to the present invention. The present invention provides an isolated antibody that binds to human CEACAM5 protein; The isolated antibody comprises: (i) at least one light chain constant region (CL) comprising a sequence selected from the group consisting of GGTLQSPP, LLQGA, GGLLQGPP, TLQSG, TLQSPP and TLQSA (preferably GGTLQSPP), preferably comprising this sequence at the C-terminus of the light chain constant region; and / or

[0053] (ii) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions: (a) L234A and L235A (LALA mutations); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y and S254T and T256E (YTE mutation); (e) K222R; Eu numbering is used for the amino acid substitutions. Here, the isolated antibody comprises a CDR1-H consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2-H consisting of the amino acid sequence of SEQ ID NO: 4, a CDR3-H consisting of the amino acid sequence of SEQ ID NO: 5, a CDR1-L consisting of the amino acid sequence of SEQ ID NO: 6, a CDR2-L consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR3-L consisting of the amino acid sequence of SEQ ID NO: 8. The present invention further provides an isolated antibody that binds to a human CEACAM5 protein; wherein the isolated antibody comprises: (i) at least one light chain constant region (CL) comprising a sequence selected from the group consisting of GGTLQSPP, LLQGA, GGLLQGPP, TLQSG, TLQSPP and TLQSA (preferably GGTLQSPP), preferably comprising this sequence at the C-terminus of the light chain constant region; And (ii) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions: (a) L234A and L235A (LALA mutations); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y and S254T and T256E (YTE mutation); (e) K222R; Eu numbering is used for the amino acid substitutions. Here, the isolated antibody comprises a CDR1-H consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2-H consisting of the amino acid sequence of SEQ ID NO: 4, a CDR3-H consisting of the amino acid sequence of SEQ ID NO: 5, a CDR1-L consisting of the amino acid sequence of SEQ ID NO: 6, a CDR2-L consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR3-L consisting of the amino acid sequence of SEQ ID NO: 8.

[0054] Preferably, the isolated antibody of the invention comprises framework regions FR1, FR2, FR3, FR4, FR5, FR6, FR7, and FR8 having the structures FR1-CDR1-H-FR2-CDR2-H-FR3-CDR3-H-FR4, and FR5-CDR1-L-FR6-CDR2-L-FR7-CDR3-L-FR8; where FR1 consists of SEQ ID NO: 54, FR2 consists of SEQ ID NO: 55, FR3 consists of SEQ ID NO: 56, FR4 consists of SEQ ID NO: 57, FR5 consists of SEQ ID NO: 58, FR6 consists of SEQ ID NO: 59, FR7 consists of SEQ ID NO: 60, and FR8 consists of SEQ ID NO: 61. The present invention further provides an isolated antibody that binds to human CEACAM5 protein, wherein the isolated antibody preferably comprises a CDR1-H consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2-H consisting of the amino acid sequence of SEQ ID NO: 4, a CDR3-H consisting of the amino acid sequence of SEQ ID NO: 5, a CDR1-L consisting of the amino acid sequence of SEQ ID NO: 6, a CDR2-L consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR3-L consisting of the amino acid sequence of SEQ ID NO: 8: wherein said isolated antibody comprises framework regions FR1, FR2, FR3, FR4, FR5, FR6, FR7, and FR8 having the structures FR1-CDR1-H-FR2-CDR2-H-FR3-CDR3-H-FR4, and FR5-CDR1-L-FR6-CDR2-L-FR7-CDR3-L-FR8, wherein FR1 consists of SEQ ID NO: 54 (EVQLQESGPGLVKPSQTLSLTCTVS), FR2 consists of SEQ ID NO: 55 (LTWIRQHPGKGLEWIGY), and FR3 consists of SEQ ID NO: 56 (YFNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYC), FR4 consists of SEQ ID NO: 57 (WGQGTLVTVSS), FR5 consists of SEQ ID NO: 58 (EIVLTQSPATLSVSPGERATLSCRTS), FR6 consists of SEQ ID NO: 59 (LAWYQQKPGQAPRLLIY), FR7 consists of SEQ ID NO: 60 (TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC), and FR8 consists of SEQ ID NO: 61 (FGPGTKVDIK). The present invention also provides an isolated antibody that binds to a human CEACAM5 protein, wherein the isolated antibody comprises: (i) at least one light chain constant region (CL) comprising a sequence selected from the group consisting of GGTLQSPP, LLQGA, GGLLQGPP, TLQSG, TLQSPP, and TLQSA, most preferably the sequence GGTLQSPP, preferably comprising this sequence at the C-terminus of the light chain constant region; and / or (ii) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions: (a) L234A and L235A (LALA mutations); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y and S254T and T256E (YTE mutation); (e) K222R; wherein Eu numbering is used for said amino acid substitutions; Preferably, the isolated antibody comprises a CDR1-H consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2-H consisting of the amino acid sequence of SEQ ID NO: 4, a CDR3-H consisting of the amino acid sequence of SEQ ID NO: 5, a CDR1-L consisting of the amino acid sequence of SEQ ID NO: 6, a CDR2-L consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR3-L consisting of the amino acid sequence of SEQ ID NO: 8: Preferably, said isolated antibody herein comprises framework regions FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 having the structures FR1-CDR1-H-FR2-CDR2-H-FR3-CDR3-H-FR4, and FR5-CDR1-L-FR6-CDR2-L-FR7-CDR3-L-FR8, where FR1 consists of SEQ ID NO: 54, FR2 consists of SEQ ID NO: 55, FR3 consists of SEQ ID NO: 56, FR4 consists of SEQ ID NO: 57, FR5 consists of SEQ ID NO: 58, FR6 consists of SEQ ID NO: 59, FR7 consists of SEQ ID NO: 60 and FR8 consists of SEQ ID NO: 61. The present invention also provides an isolated antibody, which binds to human CEACAM5 protein, which comprises a CDR1-H consisting of the amino acid sequence DGSVSRGGYY (SEQ ID NO:3), a CDR2-H consisting of the amino acid sequence IYYSGST (SEQ ID NO:4), a CDR3-H consisting of the amino acid sequence ARGIAVAPFDY (SEQ ID NO:5), a CDR1-L consisting of the amino acid sequence QSVRSN (SEQ ID NO:6), a CDR2-L consisting of the amino acid sequence AAS (SEQ ID NO:7), and a CDR3-L consisting of the amino acid sequence QQYTNWPFT (SEQ ID NO:8), wherein the isolated antibody comprises: (i) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions: (a) L234A and L235A (LALA mutations); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y and S254T and T256E (YTE mutation); (e) K222R; and / or (ii) at least one light chain constant region (CL) comprising the sequence GGTLQSPP, preferably at its C-terminus. Eu numbering is used for the amino acid substitution. The Eu numbering system is well known (see Edelman et al., Proc. Natl. Acad. Sci. USA 1969, 63, 78-85 and Kabat, EA et al., National Institutes of Health (US) Office of the Director. Sequences of Proteins of Immunological Interest, 5th ed.; DIANE Publishing: Collingdale, PA, USA, 1991), and the position of the indicated amino acid substitution follows this numbering system. Amino acid substitution is specified using one-letter amino acid code. Also, GGTLQSPP can be included multiple times in the light chain constant region (CL), and alternatively or additionally, can also be included in the heavy chain constant region (CH). Preferably, GGTLQSPP is included once per light chain constant region (CL) in both light chain constant regions (CL) of the antibody of the present invention.

[0055] The antibodies of the present invention may preferably also bind to Macaca fascicularis CEACAM5 protein.

[0056] In certain embodiments of the isolated antibodies of the invention, both heavy chain constant regions (CH) comprise one or more of the amino acid substitutions (a) to (e) above, and / or wherein both light chain constant regions comprise the sequence GGTLQSPP. Preferred combinations of CL and CH chain modifications are outlined in Table 4 below, which shows the modification combinations for antibodies mAb1-M, mAb2-M, mAb3-M, mAb6-M, and mAb7-M. Preferably, the antibodies of the invention comprise any of the following heavy chain constant region (CH) and light chain constant region (CL) modifications: (a) the C H contains the amino acid substitutions L234A, L235A (a LALA mutation) and M252Y, S254T, and T256E (a YTE mutation); or (b) the CH comprises the amino acid substitutions L234A, L235A (LALA mutations) and M252Y, S254T, and T256E (YTE mutations), and a light chain constant region (CL) comprising (preferably at the C-terminus) the sequence GGTLQSPP; or (c) the CH comprises the amino acid substitutions L234A, L235A (LALA mutation) and M252Y, S254T, and T256E (YTE mutation) and K222R, and a light chain constant region (CL) comprising (preferably at the C-terminus) the sequence GGTLQSPP; or (d) C-H contains the amino acid substitutions L234A, L235A (LALA mutation); or (e) CH comprises the amino acid substitutions L234A, L235A (LALA mutation), and a light chain constant region (CL) comprising (preferably at the C-terminus) the sequence GGTLQSPP.

[0057] Preferably, both CL and both CH regions of an antibody of the invention contain the modifications outlined in (a) to (e) above.

[0058] In a further embodiment of the isolated antibody of the invention, at least one heavy chain constant region (CH) has the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31), or ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDRTHTCPPCPPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32), and / or at least one of the light chain constant regions (CL) comprises the amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGTLQSPP (SEQ ID NO: 33).

[0059] In yet further embodiments of the isolated antibody of the present invention, said heavy chain constant region (CH) and light chain constant region (CL) have any of the following sequence combinations: (1) both CHs comprise a sequence of SEQ ID NO:31 and both CLs comprise a sequence of SEQ ID NO:12; or (2) both CHs comprise a sequence of SEQ ID NO:31 and both CLs comprise a sequence of SEQ ID NO:33; or (3) both CHs comprise a sequence of SEQ ID NO:32 and both CLs comprise a sequence of SEQ ID NO:33; or (4) both CHs comprise the sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:50) and both CLs comprise the sequence of SEQ ID NO:12; or (5) both CHs comprise a sequence of SEQ ID NO:50 and both CLs comprise a sequence of SEQ ID NO:33; or (6) at least one CH comprises the sequence of SEQ ID NO:31 and one CL comprises the sequence of SEQ ID NO:12; or (7) at least one CH comprises the sequence of SEQ ID NO: 31 and one CL comprises the sequence of SEQ ID NO: 33; or (8) at least one CH comprises the sequence of SEQ ID NO: 32 and one CL comprises the sequence of SEQ ID NO: 33; or (9) at least one CH comprises the sequence of SEQ ID NO:50 and one CL comprises the sequence of SEQ ID NO:12; or 10) At least one CH comprises the sequence of SEQ ID NO:50 and one CL comprises the sequence of SEQ ID NO:33.

[0060] In an embodiment of the invention, an antibody having the six CDR sequences mentioned above comprises the amino acid sequence

number

number

[0061] In an embodiment of the invention, the antibody having the six CDR sequences referred to above comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:9 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:10.

[0062] In an embodiment of the invention, the antibody further comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 11), and a light chain constant region (CL) comprising an amino acid sequence at least 85% identical to the amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

[0063] In an embodiment of the invention, the antibody comprises a heavy chain constant region (CH) comprising the amino acid sequence of SEQ ID NO:11 and a light chain constant region (CL) comprising the amino acid sequence of SEQ ID NO:12.

[0064] It is known that the heavy chain constant region (CH) of an antibody can contain a C-terminal lysine (K) without losing any binding functionality. Thus, in the sequences described herein for the antibodies of the present invention, the heavy chain constant region (CH) can optionally contain an additional lysine (K) at the C-terminus. A heavy chain constant region (CH) without lysine is preferred for the antibody-drug conjugates disclosed herein. In a more specific embodiment, the antibody of the invention is an isolated antibody that binds to human CEACAM5 protein and has the amino acid sequence EVQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQ The heavy chain (HC) comprises an amino acid sequence at least 85% identical to PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 13), and a light chain (LC) comprises an amino acid sequence at least 85% identical to the amino acid sequence EIVLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14).In an even more specific embodiment of the invention, the antibody comprises: (i) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 34 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 14; or (ii) the amino acid sequence EVQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI A heavy chain (HC) comprising the amino acid sequence (SEQ ID NO: 34) of CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG; or (iii) Amino acid sequence EVQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDRTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL a heavy chain (HC) comprising PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35) and a light chain (LC) comprising the amino acid sequence EIVLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGTLQSPP (SEQ ID NO: 36); or (iv) Amino acid sequence EVQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSV A heavy chain (HC) comprising FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:51) and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:14; or (v) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 51 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 36.

[0065] In yet more specific embodiments of the present invention, the antibody consists of: (i) two identical heavy chains (HC) comprising the amino acid sequence of SEQ ID NO: 34 and two identical light chains (LC) comprising the amino acid sequence of SEQ ID NO: 14; or (ii) two identical heavy chains (HC) comprising the amino acid sequence of SEQ ID NO: 34 and two identical light chains (LC) comprising the amino acid sequence of SEQ ID NO: 36; or (iii) two identical heavy chains (HC) comprising the amino acid sequence of SEQ ID NO: 35 and two identical light chains (LC) comprising the amino acid sequence of SEQ ID NO: 36; or (iv) two identical heavy chains (HC) comprising the amino acid sequence of SEQ ID NO: 51 and two identical light chains (LC) comprising the amino acid sequence of SEQ ID NO: 14; or (v) two identical heavy chains (HC) comprising the amino acid sequence of SEQ ID NO: 51 and two identical light chains (LC) comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, one or more individual amino acids of the antibody of the present invention may be modified by substitution, in particular by conservative substitution, in one or more of the above-mentioned sequences, including the CDR sequences. Such modifications may be intended, for example, to remove glycosylation or deamidation sites, for example in connection with humanization of the antibody.

[0066] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and that is composed of two identical heavy chains (HC) consisting of the amino acid sequence of SEQ ID NO: 13 and two identical light chains (LC) consisting of the amino acid sequence of SEQ ID NO: 14. This particular antibody is also referred to herein as "mAb1."

[0067] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and that is composed of two identical heavy chains (HC) consisting of the amino acid sequence of SEQ ID NO: 34 and two identical light chains (LC) consisting of the amino acid sequence of SEQ ID NO: 14. This particular antibody is also referred to herein as "mAb1-M."

[0068] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and that is composed of two identical heavy chains (HC) consisting of the amino acid sequence of SEQ ID NO: 34 and two identical light chains (LC) consisting of the amino acid sequence of SEQ ID NO: 36. This particular antibody is also referred to herein as "mAb2-M."

[0069] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and that is composed of two identical heavy chains (HC) consisting of the amino acid sequence of SEQ ID NO: 35 and two identical light chains (LC) consisting of the amino acid sequence of SEQ ID NO: 36. This particular antibody is also referred to herein as "mAb3-M."

[0070] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and that is composed of two identical heavy chains (HC) consisting of the amino acid sequence of SEQ ID NO: 51 and two identical light chains (LC) consisting of the amino acid sequence of SEQ ID NO: 14. This particular antibody is also referred to herein as "mAb6-M."

[0071] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and that is composed of two identical heavy chains (HC) consisting of the amino acid sequence of SEQ ID NO: 51 and two identical light chains (LC) consisting of the amino acid sequence of SEQ ID NO: 36. This particular antibody is also referred to herein as "mAb7-M."

[0072] In some embodiments, the antibodies of the invention bind to the A2-B2 domain of human and Macaca fascicularis CEACAM5. The invention also provides antibodies that compete for binding to the A2-B2 domain of human and / or Macaca fascicularis CEACAM5 protein with an antibody comprising the heavy and light chain variable regions of mAb1 (i.e., VH and VL corresponding to SEQ ID NOs: 9 and 10, respectively) and the heavy chain constant region (CH) and light chain constant region (CL) from any of mAb1-M, mAb2-M, mAb3-M, mAb6-M, or mAb7-M.

[0073] The ability of a candidate antibody to compete with an antibody comprising the VH and VL of mAb1 (hereinafter referred to as the "reference" antibody in the context of competition with the candidate antibody) for binding to the A2-B2 domain of human and / or Macaca fascicularis CEACAM5 protein can be readily assayed, for example, by competitive ELISA. Here, an antigen (i.e., a polypeptide comprising or consisting of the A2-B2 domain of human or Macaca fascicularis CEACAM5, or a fragment of human or Macaca fascicularis CEACAM5 encompassing the A2-B2 domain, in particular the extracellular domain of human or Macaca fascicularis CEACAM5) is bound to a solid support, two solutions containing the candidate antibody and the reference antibody, respectively, are added, and the antibodies are allowed to compete for binding to the antigen. The amount of the reference antibody bound to the antigen can then be measured and compared to the amount of the reference antibody bound to the antigen when measured against a negative control (e.g., a solution containing no antibody). A reduced amount of bound reference antibody in the presence of the candidate antibody compared to the amount of bound reference antibody in the presence of the negative control indicates that the candidate antibody competes with the reference antibody. Conveniently, the reference antibody can be labeled (e.g., fluorescent) to facilitate detection of bound reference antibody. Repeated measurements can be performed with serial dilutions of the candidate and / or reference antibodies.

[0074] In some embodiments, the antibodies of the present invention do not bind to or significantly cross-react with any of the human CEACAM1, human CEACAM6, human CEACAM7, human CEACAM8, Macaca fascicularis CEACAM6 proteins. In some embodiments, the antibodies do not bind to or significantly cross-react with the extracellular domains of the above-mentioned human and Macaca fascicularis CEACAM proteins other than CEACAM5.

[0075] "Affinity" is theoretically defined by the equilibrium association between the whole antibody and the antigen. It can be measured by various known methods, for example, by measuring the association and dissociation rates by surface plasmon resonance or by measuring the EC in immunochemical assays (ELISA, FACS). 50 (or apparent K D In these assays, EC 50 is the concentration of antibody that induces a response halfway between baseline and maximum by ELISA (enzyme-linked immunosorbent assay) in a given concentration of antigen or by FACS (fluorescence-activated cell sorting) in cells expressing the antigen after some defined period of exposure.

[0076] Monoclonal antibodies that bind to antigen 1 (Ag1) are known to be effective in treating EC 50 A monoclonal antibody that binds Ag1 is "cross-reactive" to antigen 2 (Ag2) if its affinity for Ag1 is in a similar range for both antigens. In this application, a monoclonal antibody that binds Ag1 is cross-reactive to Ag2 if its affinity for Ag2 is within 10-fold (e.g., within 5-fold) of its affinity for Ag1. Affinity is measured in the same way for both antigens.

[0077] A monoclonal antibody that binds to Ag1 is "not significantly cross-reactive" to Ag2 when the affinities for the two antigens are very different. If the binding response is too low, the affinity for Ag2 may not be measurable. In this application, a monoclonal antibody that binds to Ag1 is not significantly cross-reactive to Ag2 when the binding response of the monoclonal antibody to Ag2 is less than 5% of the binding response of the same monoclonal antibody to Ag1 in the same experimental setting and at the same antibody concentration. In practice, the antibody concentration used is chosen to be within the range of the EC 50 Or it may be the concentration required to reach a saturation plateau. A monoclonal antibody "specifically binds" (or is "specific for") Ag1 when it is not significantly cross-reactive to Ag2.

[0078] In some embodiments, an antibody according to the invention has an affinity for Macaca fascicularis CEACAM5 that is within 10-fold (e.g., within 5-fold) of its affinity for human CEACAM5. Thus, an antibody according to the invention can be used in toxicology studies conducted in monkeys, since the toxicity profile observed in monkeys will be relevant for predicting potential adverse effects in humans.

[0079] In some embodiments, the antibodies of the present invention have an affinity for human CEACAM5 or Macaca fascicularis CEACAM5, or both, that is ≦10 nM. For example, the antibodies of the present invention may have an affinity for human CEACAM5 that is between 1 nM and 10 nM, for example, an affinity for human CEACAM5 of about 6 nM.

[0080] Affinity for human CEACAM5 or Macaca fascicularis CEACAM5 can be determined, for example, as an EC50 value in an ELISA using soluble recombinant CEACAM5 as the capture antigen.

[0081] Alternatively, for antibodies of the invention, the apparent dissociation constant (apparent KD) can be determined by FACS analysis, for example with the tumor cell line MKN45 (DSMZ, ACC409).

[0082] Furthermore, it has been shown that antibodies according to the invention are capable of detecting CEACAM5 expression by immunohistochemistry, for example from frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections.

[0083] Any combination of the above and below aspects herein forms part of the present invention.

[0084] In some embodiments, the antibody according to the invention is a conventional antibody, such as a conventional monoclonal antibody, or an antibody fragment, a bispecific or multispecific antibody.

[0085] In some embodiments, the antibody according to the invention comprises or consists of an IgG or a fragment thereof.

[0086] In some embodiments, the antibody of the present invention may be, for example, a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. Numerous methods for humanization of antibody sequences are known in the art. See, for example, the review by Almagro & Fransson (2008) Front Biosci.13:1619-1633. One commonly used method is CDR grafting or antibody reshaping, which involves grafting the CDR sequences of a donor antibody, typically a murine antibody, onto the framework scaffold of a human antibody of different specificity. Since CDR grafting reduces the binding specificity and affinity, and therefore the biological activity, of the CDR-grafted non-human antibody, back mutations are introduced at selected positions of the CDR-grafted antibody to retain the binding specificity and affinity of the parent antibody. Identification of possible back mutation positions can be performed using information available from the literature and antibody databases. Amino acid residues that are candidates for back mutations are typically those located on the surface of the antibody molecule, and buried or low surface-exposed residues will usually not be altered. Amino acid residues that are candidates for backmutation are typically those located on the surface of the antibody molecule; buried or less surface-exposed residues usually will not be altered. Another alternative technique is known as "guided selection" (Jespers et al. (1994) Biotechnology 12, 899), which can be used to derive fully human antibodies from murine antibodies that preserve the epitopes and binding characteristics of the parent antibody.

[0087] For chimeric antibodies, humanization typically involves altering the framework regions of the variable region sequences.

[0088] Although amino acid residues that are part of the CDRs are typically not changed in connection with humanization, in certain cases it may be desirable to change individual CDR amino acid residues, for example to remove glycosylation sites, deamidation sites, or unwanted cysteine ​​residues. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X is any amino acid except Pro. Removal of an N-glycosylation site is achieved, for example, by mutating either the Asn or Ser / Thr residue to a different residue by conservative substitution. Deamidation of asparagine and glutamine residues may occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present primarily in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. Thus, when such a deamidation site, e.g. Asn-Gly, is present in a CDR sequence, it is desirable to remove the site, typically by conservative substitution to remove one of the involved residues. Substitutions on the CDR sequences to remove one of the involved residues are also intended to be encompassed by the invention.

[0089] In a humanized antibody or fragment thereof, the heavy and light chain variable domains comprise human receptor framework regions. The humanized antibody further comprises human heavy and light chain constant domains, where present.

[0090] In some embodiments, an antibody in accordance with the invention may be an antibody fragment (e.g., a humanized antibody fragment) selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and a diabody.

[0091] In some embodiments, an antibody according to the invention may be a bispecific or multispecific antibody formed from antibody fragments, at least one antibody fragment being a fragment of an antibody according to the invention. Multispecific antibodies are multivalent protein complexes, e.g. as described in EP 2 050 764 A1 or US 2005 / 0003403 A1.

[0092] The bispecific or multispecific antibody according to the present invention may have specificity for (a) human and Macaca fascicularis CEACAM5 protein and (b) at least one other antigen. In some embodiments, the at least one other antigen is not a human or Macaca fascicularis CEACAM family member. In other embodiments, the at least one other antigen may be an epitope on human or Macaca fascicularis CEACAM5 other than the epitope targeted by mAb1.

[0093] The antibodies of the invention may be produced by any technique known in the art. Antibodies according to the invention may, for example, be used in isolated (e.g., purified) form or may be contained in a vector, such as a membrane or lipid vesicle (e.g., liposome).

[0094] Nucleic acids and host cells of the invention A further aspect of the present invention relates to an isolated nucleic acid comprising or consisting of a nucleic acid sequence encoding an antibody of the invention as defined above.

[0095] Typically, the nucleic acid is a DNA or RNA molecule, which is comprised on any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0096] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle for introducing DNA or RNA sequences (e.g., foreign genes) into a host cell to transform the host and promote expression (e.g., transcription and translation) of the introduced sequences.

[0097] Therefore, a further aspect of the present invention relates to a vector comprising a nucleic acid of the invention as defined above.

[0098] Such vectors contain control elements, such as promoters, enhancers, terminators, etc., for inducing or directing expression of the polypeptide upon administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), immunoglobulin H chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983), etc.

[0099] Any expression vector for animal cells can be used as long as the gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1 beta d2-4- (Miyaji H et al. 1990), etc.

[0100] Other examples of plasmids include replicative plasmids that contain an origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR, and the like.

[0101] Other examples of viral vectors include adenovirus, retrovirus, herpes virus and AAV vectors. Such recombinant viruses are produced by techniques known in the art, for example by transfecting packaging cells or by transient transfection with helper plasmid or virus. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0102] A further object of the present invention relates to a host cell transfected, infected or transformed with a nucleic acid and / or a vector according to the invention.

[0103] The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous) gene, DNA or RNA sequence into a host cell, where the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme, encoded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed."

[0104] The nucleic acids of the invention can be used to produce the antibodies of the invention in a suitable expression system. The term "expression system" means a host cell and a compatible vector under suitable conditions for the expression of a protein encoded by, for example, foreign DNA carried by the vector and introduced into the host cell.

[0105] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., generated from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. In some embodiments, YB2 / 0 cells are used because the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in YB2 / 0 cells.

[0106] For the expression of humanized antibodies, the expression vector is either a type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are present on separate vectors, or a type in which both genes are present on the same vector (tandem type). From the viewpoints of ease of construction of the humanized antibody expression vector, ease of introduction into animal cells, and balance of the expression levels of antibody H and L chains in animal cells, the humanized antibody expression vector is a tandem type. Shitara K et al. J Immunol Methods. 1994 Jan 3; 167 (1-2): 271-8). Examples of tandem type humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354), pEE18, etc.

[0107] The present invention also relates to a method for producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of: (i) introducing into a competent host cell, in vitro or ex vivo, a recombinant nucleic acid or vector as described above, (ii) culturing in vitro or ex vivo the obtained recombinant host cell, and (iii) optionally selecting cells which express and / or secrete said antibody.

[0108] Such recombinant host cells can be used for the production of the antibodies of the invention.

[0109] Methods for Producing Antibodies of the Invention The antibodies of the present invention may be produced by any technique known in the art, including, without limitation, chemical, biological, genetic or enzymatic techniques, either alone or in combination.

[0110] Knowing the amino acid sequence of the desired antibody, one of skill in the art can readily produce said antibody or immunoglobulin chains using standard techniques for the production of polypeptides. For example, they can be synthesized using a commercially available peptide synthesizer (such as those produced by Applied Biosystems, Foster City, California) using the well-known solid-phase method, following the manufacturer's instructions. Alternatively, the antibodies and immunoglobulin chains of the invention can be produced by recombinant DNA techniques known in the art. For example, these polypeptides (e.g., antibodies) are obtained as DNA expression products following incorporation of a DNA sequence encoding the desired polypeptide onto an expression vector and introduction of such vector into a suitable eukaryotic or prokaryotic host that will express the desired polypeptide, from which they can then be isolated using well-known techniques.

[0111] For example, the invention provides the following DNA sequence encoding the antibody mAb1: mAb1 heavy chain nucleotide sequence Here, the mVk signal peptide is Underlined , Start and stop codons are in italics; VH region sequences in boldface; The CDRs are indicated by double underlining:

number

number

[0112] The present invention also provides the following DNA sequences encoding the antibodies mAb1-M, mAb2-M, and mAb3-M, mAb6-M, mAb7-M: ------- SEQ ID NO: 37: HC mAb1-M and mAb2-M (HC-LALA-YTE) (nucleotides) The signal peptide Underlined , Start and stop codons are in italics; VH region sequences in boldface; The CDRs are indicated by double underlining:

number

number

number

number

number

number

number

number

number

[0113] The present invention further relates to a method for producing an antibody of the present invention, comprising the steps of: (i) culturing a transformed host cell according to the present invention; (ii) expressing the antibody; and (iii) recovering the expressed antibody.

[0114] The antibodies of the invention can be suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0115] In some embodiments, the humanized chimeric antibodies of the present invention can be produced by obtaining nucleic acid sequences encoding the humanized VL and VH regions as described above, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cells harboring genes encoding a human antibody CH and a human antibody CL, and expressing the coding sequences by introducing the expression vector into the animal cells.

[0116] As the CH domain of the human chimeric antibody, any region belonging to the human immunoglobulin heavy chain is used. For example, IgG class is preferable, and any one of the subclasses belonging to the IgG class, such as IgG1, IgG2, IgG3, and IgG4, may be used. As the CL of the human chimeric antibody, any region belonging to the human immunoglobulin light chain is used, and those of the kappa class or lambda class may be used.

[0117] Methods for producing humanized or chimeric antibodies can involve conventional recombinant DNA and gene transfection techniques and are known in the art (see, for example, Morrison SL. et al. (1984) and patent documents US 5,202,238; and US 5,204,244).

[0118] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using various techniques known in the art, such as the techniques disclosed in application WO2009 / 032661, CDR grafting (EP 239,400; PCT Publication WO91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. al. (1994), and chain shuffling (U.S. Patent No. 5,565,332). General recombinant DNA techniques for the preparation of such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0119] The Fab of the present invention can be obtained by treating an antibody (e.g., IgG) of the present invention with a protease such as papain. Alternatively, the Fab can be produced by inserting DNA sequences encoding both chains of the Fab of the antibody into a vector for prokaryotic or eukaryotic expression and introducing the vector into a prokaryotic or eukaryotic cell (as appropriate) to express the Fab.

[0120] The F(ab')2 of the present invention can be obtained by treating the antibody (e.g., IgG) of the present invention with the protease pepsin. F(ab')2 can also be produced by linking the Fab' described below via a thioether bond or disulfide bond.

[0121] The Fab' of the present invention can be obtained by treating the F(ab')2 of the present invention with a reducing agent such as dithiothreitol. Alternatively, the Fab' can be produced by inserting a DNA sequence encoding the Fab' chain of the antibody into a vector for prokaryotic expression or a vector for eukaryotic expression and introducing the vector into a prokaryotic or eukaryotic cell (as appropriate) to effect expression thereof.

[0122] The scFv of the present invention can be produced by taking the sequences of the CDRs or VH and VL domains as previously described for the antibodies of the present invention, constructing DNA encoding the scFv fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic cell (as appropriate) to express the scFv. To generate humanized scFv fragments, a well-known technique called CDR grafting is used. This involves selecting the complementarity determining regions (CDRs) according to the present invention and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, for example, WO98 / 45322; WO 87 / 02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

[0123] Modification of the antibodies of the present invention Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, modifications may be desired to improve the binding affinity and / or other biological properties of the antibody.

[0124] Modifications and changes can be made to the structure of the antibodies of the invention and the DNA sequences encoding them, resulting in functional antibodies or polypeptides possessing desired properties.

[0125] When making modifications to the amino acid sequence of a polypeptide, the hydropathic index of the amino acids is taken into consideration. The importance of the hydropathic amino acid index to the interactive biological function of a protein is generally understood in the art. It is accepted that the relative hydropathic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); aspartic acid (-3.5); lysine (-3.9); and arginine (-4.5).

[0126] Further aspects of the invention also encompass function-conservative variants of the polypeptides of the invention.

[0127] For example, certain amino acids can be substituted by other amino acids in a protein structure without appreciable loss of activity. Because the interactive capacity and properties of a protein determine its biological functional activity, certain amino acid substitutions can be made in a protein sequence and, of course, in its coding DNA sequence, while still obtaining a protein with similar properties. It is therefore contemplated that various modifications can be made in the antibody sequences of the present invention, or in the corresponding DNA sequences encoding said polypeptides, without appreciable loss of their biological activity.

[0128] It is known in the art that certain amino acids can be substituted by other amino acids with similar hydropathic index or score to result in proteins with similar biological activity, i.e., biologically functional equivalent proteins. It is also possible to use well-established techniques such as alanine scanning approach to identify all amino acids that can be substituted in the antibody or polypeptide of the present invention without significant loss of binding to the antigen. Such residues are considered neutral, since they are not involved in antigen binding or in maintaining the structure of the antibody. One or more of these neutral positions can be substituted by alanine or another amino acid without changing the main properties of the antibody or polypeptide of the present invention.

[0129] Neutral positions can be seen as positions where any amino acid substitution can be incorporated. In fact, in the principle of alanine scanning, alanine is chosen because this residue has no particular structural or chemical features. It is generally accepted that if alanine can be substituted for a particular amino acid without changing the properties of a protein, it is highly likely that many, if not all, other amino acid substitutions will also be neutral. In the opposite case, where alanine is the wild-type amino acid, if a particular substitution can be shown as neutral, it is highly likely that other substitutions will also be neutral.

[0130] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account any of the foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0131] It may also be desirable to modify the antibody of the present invention for effector function, for example, to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody, or to alter, for example, binding to Fc receptors. This is accomplished by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or in addition, a cysteine ​​residue(s) is / are introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated has improved internalization capacity and / or increased complement-mediated cell killing and / or antibody-dependent cellular cytotoxicity (ADCC) (Caron PC.et al.1992; and Shopes B.1992). In some embodiments, the antibody of the present invention may be an antibody with a modified amino acid sequence that results in reduced or abolished binding to most Fcγ receptors. This may reduce uptake and toxicity in normal cells and tissues expressing such receptors, such as macrophages, hepatic sinusoidal cells, etc. An example of such an antibody is one that contains two leucine (L) residues at positions 234 and 235 substituted with alanine (A) (i.e., LALA). This double substitution has been demonstrated to reduce Fc binding to FcγR and, consequently, also reduces ADCC and reduces complement binding / activation. Another example of such an antibody is one that contains the substitution P329G in addition to the LALA double substitution (i.e., PG-LALA; see, e.g., Schlothauer et al., Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions, Protein Engineering, Design and Selection, Volume 29, Issue 10, October 2016, p. 457-466).In some embodiments, an antibody of the present invention is therefore an antibody that (i) contains, for example, a LALA or PG-LALA set of substitutions and (ii) has an amino acid sequence that is otherwise identical to the amino acid sequence of one of the antibodies of the present invention described herein above with reference to the respective SEQ ID NOs.

[0132] Another type of amino acid modification of the antibodies of the invention is useful for altering the original glycosylation pattern of the antibody, i.e., by deleting one or more carbohydrate moieties found on the antibody and / or adding one or more glycosylation sites that are not present on the antibody. The presence of either of the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, creates a potential glycosylation site. Addition or deletion of glycosylation sites of the antibody can conveniently be accomplished by altering the amino acid sequence such that it contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites).

[0133] Another type of modification involves the removal of sequences identified in silico or experimentally that potentially result in degradation products or heterogeneity of antibody preparations. As an example, deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present primarily in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. Thus, when such deamidation sites, particularly Asn-Gly, are present in antibodies or polypeptides, it is considered to remove the site, typically by conservative substitution to remove one of the involved residues. Such substitutions on the sequence to remove one or more of the involved residues are also intended to be encompassed by the present invention.

[0134] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell with glycosylation capabilities for N- or O-linked glycosylation. Depending on the mode of coupling used, the sugar(s) are attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as on cysteine, (d) free hydroxyl groups, such as on serine, threonine, or hydroxyproline, (e) aromatic residues, such as on phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO87 / 05330.

[0135] Removal of carbohydrate moieties present on antibodies is accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described in Sojahr H. et al. (1987) and Edge, A. S. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exoglycosidases as described in Thotakura, N. R. et al. (1987).

[0136] Another type of covalent modification of the antibody involves linking it to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, e.g., in the manner described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337.

[0137] Other amino acid sequence modifications known in the art may also be applied to the antibodies of the invention.

[0138] Immunoconjugates of the Invention The present invention provides immunoconjugates, also referred to herein as antibody-drug conjugates, or more simply as conjugates.All these terms used herein have the same meaning and can be interchanged.Suitable methods for preparing immunoconjugates are known in the art.The immunoconjugates of the present invention can be prepared, for example, by in vitro methods described herein.

[0139] The invention provides immunoconjugates comprising an antibody of the invention (e.g., mAb1, or an antibody having the same six CDRs as mAb1) covalently linked via a linker to at least one growth inhibitory agent.

[0140] The term "growth inhibitory agent" (also referred to as "anti-proliferative agent") refers to a molecule or compound or composition that inhibits the growth of cells, such as tumor cells, in vitro and / or in vivo.

[0141] In some embodiments, the growth inhibitory agent is a cytotoxic drug (also referred to as a cytotoxic agent). In some embodiments, the growth inhibitory agent is a radioactive moiety.

[0142] The term "cytotoxic drug" as used herein refers to a substance that directly or indirectly inhibits or interferes with the function of a cell and / or causes the destruction of a cell. The term "cytotoxic drug" includes, for example, chemotherapeutic agents, enzymes, antibiotics, toxins, such as small molecule toxins or enzymatically active toxins, toxoids, vincas, taxanes, maytansinoids or maytansinoid analogs, tomaymycin or pyrrolobenzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatins or dolastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents, CC-1065, and CC-1065 analogs.

[0143] Topoisomerase I inhibitors are molecules or compounds that inhibit the human enzyme topoisomerase I, which is involved in changing DNA topology by catalyzing the transient breakage and religation of one strand of DNA.Topoisomerase I inhibitors are highly toxic, for example, to mammalian cell division.Examples of suitable topoisomerase I inhibitors include camptothecin (CPT) and its analogs, such as topotecan, irinotecan, ciratecan, cositecan, exatecan, lutotecan, gimatecan, belotecan, and rubitecan.

[0144] In some embodiments, the immunoconjugate of the present invention comprises the cytotoxic drug exatecan as a growth inhibitory agent. Exatecan has the chemical name (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo(de)pyrano(3',4':6,7)indolizino(1,2-b)quinoline-10,13-dione. Exatecan has the following structural formula (I):

[0145] [ka] It is represented by:

[0146] In further embodiments of the present invention, other CPT analogs and other cytotoxic drugs are used, for example as listed above. Examples of some cytotoxic drugs and methods of conjugation are further described in application WO2008 / 010101, which is incorporated by reference.

[0147] The term "radioactive moiety" refers to At 211 , Bi 212 , Er 169 , I 131 , I 125 , Y 90 , In 111 , P 32 , Re 186 , Re 188 , Sm 153 , Sr 89Radioisotopes generally emit mainly beta radiation. In some embodiments, the radioisotope is an alpha-emitting isotope, such as thorium-227, which emits alpha radiation. The immunoconjugate can be prepared, for example, as described in application WO2004 / 091668.

[0148] In the immunoconjugates of the present invention, the antibodies of the present invention are covalently linked to at least one growth inhibitory agent via a linker. As used herein, "linker" refers to a chemical moiety that includes a covalent bond and / or any chain of atoms that covalently attaches a growth inhibitory agent to an antibody. Linkers are known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Conjugation of the antibodies of the invention with cytotoxic drugs or other growth inhibitory agents can be accomplished using a variety of bifunctional protein coupling agents, including, for example, but not limited to, N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl(2-pyridyldithio)propionate (SPDP), succinimidyl(N-maleimidomethyl) Cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies (WO 94 / 11026).

[0149] In an embodiment of the present invention, the linker is a "cleavable linker". This facilitates the release of cytotoxic drugs or other growth-inhibitory agents in or near cells, such as tumor cells. In some embodiments, the linker is a linker that can be cleaved in the endosome of mammalian cells. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase-labile linker, a photolabile linker, or a disulfide-containing linker is used (see, for example, US Patent No. 5,208,020).

[0150] The following nomenclature is also used herein when referring to structural formulas representing immunoconjugates, where a combination of a growth inhibitory drug and a linker is also referred to as a [(linker)-(growth inhibitory drug)] moiety, e.g., a combination of an exatecan molecule and a linker is also referred to as a [(linker)-(exatecan)] moiety.

[0151] In some particular embodiments of the invention, the linker is a linker cleavable by the human enzyme glucuronidase. Thus, for example, the immunoconjugates of the invention include a linker cleavable by glucuronidase, as represented by the following formula (II) or (IIA): [ka] [ka] wherein the antibody is an antibody of the invention, where S is a sulfur atom of the antibody, and n is the number of [(linker)-(growth inhibitory agent)] moieties covalently linked to the antibody. The number n can be, for example, between 1 and 10. In more specific embodiments including the above formula (II), n is between 7 and 8; in even more specific embodiments using the above formula (II), n is between 7.5 and 8.0 (i.e., about 8). In embodiments using the above formula IIA, n is preferably between 3 and 4, most preferably between 3.5 and 4.0 (i.e., about 4). In some embodiments, S is a sulfur atom of a cysteine ​​of the antibody. In some embodiments, the antibody is mAb1-M, mAb2-M, mAb3-M, mAb6-M, or mAb7-M.

[0152] The number n is also referred to as the "drug-to-antibody ratio" (or "DAR"). This number n should always be understood as the average number for any given (preparation of) immunoconjugate.

[0153] In the above formula (II), the chemical structure between the sulfur atom of the antibody and the growth inhibitory drug is a linker. One of these linkers is also contained in each of the formulas (IV) to (VIIIA) further shown below.

[0154] In any one of the embodiments having a glucuronidase-cleavable linker, as described above, the growth inhibitory drug is, for example, exetecan.

[0155] Thus, in some embodiments, the present invention provides an immunoconjugate comprising an antibody according to the present invention covalently linked via a linker to exatecan, wherein the conjugate has the following formula (IV) or formula (IVA):

[0156] [ka] where S is a sulfur atom of the antibody, and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, between 1 and 10. In more specific embodiments involving formula IV, n is between 7 and 8; in even more specific embodiments based on formula IV, n is between 7.5 and 8.0 (i.e., about 8). In embodiments using formula IVA above, n is preferably between 3 and 4, most preferably between 3.5 and 4.0 (i.e., about 4). In some embodiments, the antibody is mAb1-M, mAb2-M, mAb3-M, mAb6-M, or mAb7-M.

[0157] In some embodiments, in the immunoconjugates of the present invention, such as the exatecan conjugates with glucuronidase cleavable linkers described above, the linker is covalently attached to the antibody at the sulfur atom of a cysteine ​​residue of the antibody. For example, this cysteine ​​residue of the antibody can be one of the cysteine ​​residues that can form an interchain disulfide bond (also referred to herein as an interchain disulfide bridge). Since there are four interchain disulfide bonds on an IgG1 antibody involving a total of eight cysteine ​​residues, the attachment of the linker to the antibody at the sulfur atom of such a cysteine ​​residue means that the DAR can be up to 8. In such cases, the DAR is typically between 7 and 8, for example between 7.5 and 8.0 (i.e., about 8), provided that the antibody is an IgG1 or has the same number of interchain disulfide bonds as an IgG1.

[0158] Thus, in some embodiments, the present invention provides an immunoconjugate comprising an antibody according to the invention covalently linked to an execan via a linker, wherein the conjugate has the following formula (VI) or formula (VIA):

[0159] [ka] where S is the sulfur atom of the cysteine ​​of the antibody, and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also referred to as DAR) can be, for example, between 1 and 10. In a more specific embodiment based on the above formula VI, n is between 7 and 8; in an even more specific embodiment using formula VI, n is between 7.5 and 8.5 (preferably 8). In an embodiment using the above formula VIA, n is preferably between 3 and 5, more preferably between 3.5 and 4.5, and most preferably 4.

[0160] In any of the immunoconjugates described above, any of the antibodies of the present invention (as described herein above and below) may be used. In some embodiments, the immunoconjugates of the present invention comprise mAb1-M, mAb2-M, mAb3-M, mAb6-M or mAb7-M as the antibody.

[0161] Thus, in some embodiments, the present invention provides an immunoconjugate comprising an antibody according to the present invention (preferably selected from the group consisting of mAb1-M, mAb2-M, mAb3-M, mAb6-M, and mAb7-M) covalently linked to an execan via a linker, wherein the conjugate has the following formula (VIII) or formula (VIIIA):

[0162] [ka] where S is a sulfur atom of a cysteine ​​of an antibody (preferably, mAb1-M, mAb2-M, mAb3-M, mAb6-M or mAb7-M), and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody (preferably, mAb1-M, mAb2-M, mAb3-M, mAb6-M or mAb7-M). The number n (also referred to as DAR) can be, for example, between 1 and 10. In more specific embodiments, n is between 7 and 8; in even more specific embodiments, n is between 7.5 and 8.0 (i.e., about 8). In some embodiments, S is a sulfur atom of a cysteine ​​of an antibody (preferably, mAb1-M, mAb2-M, mAb3-M, mAb6-M or mAb7-M) capable of forming an interchain disulfide bridge, and the DAR is about 8. An example of such an immunoconjugate (i.e., "ADC1") is further described in the Examples. Preferred immunoconjugates of the invention are listed below: [Table B]

[0163] In a further preferred embodiment, the present invention provides an antibody-drug conjugate, wherein the drug is exatecan, and wherein the antibody-drug conjugate comprises any one of the following: (i) to (v): (i) an anti-CEACAM5 monoclonal antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 14 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34, wherein exatecan is linked to said antibody by the linker type and conjugate type shown for ADC1-M in Table 4; or (ii) an anti-CEACAM5 monoclonal antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 36 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34, wherein exatecan is linked to the antibody by the linker type and conjugate type shown for ADC2-M in Table 4; or (iii) an anti-CEACAM5 monoclonal antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 36 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 35, wherein exatecan is linked to the antibody by the linker type and conjugate type shown for ADC3-M in Table 4; or (iv) an anti-CEACAM5 monoclonal antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 14 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 51, wherein exatecan is linked to the antibody by the linker type and conjugate type shown for ADC6-M in Table 4; or (v) an anti-CEACAM5 monoclonal antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 36 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 51, wherein exatecan is linked to the antibody by the linker type and conjugate type shown for ADC7-M in Table 4.

[0164] In a further preferred embodiment, the present invention provides an antibody-drug conjugate selected from ADC1-M, ADC2-M, ADC3-M, ADC6-M, and ADC7-M, having all of the properties of the respective ADCs outlined in Table 4, including the respective DARs shown in Table 4.

[0165] In other embodiments of the invention, the linker may be a "non-cleavable linker" (e.g., an SMCC linker). Release of the growth inhibitory drug from the antibody may occur by lysosomal degradation of the antibody.

[0166] In another embodiment of the invention, the immunoconjugate is a fusion protein comprising an antibody of the invention and a cytotoxic or growth inhibitory polypeptide (as a growth inhibitory agent). Such fusion proteins are produced by recombinant techniques or peptide synthesis, methods known in the art. The coding DNA molecule contains respective regions encoding the two parts of the conjugate (the antibody and the cytotoxic or growth inhibitory polypeptide, respectively), either adjacent to each other or separated by a region encoding a linker peptide.

[0167] The antibodies of the invention may also be used in directed enzyme prodrug therapy, such as antibody-directed enzyme prodrug therapy, by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapy drug, see WO 81 / 01145) into an active, cytotoxic drug (see, e.g., WO 88 / 07378 and U.S. Pat. No. 4,975,278). The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug to convert it into its more active, cytotoxic form. Enzymes that are useful in this context include, but are not limited to: alkaline phosphatases, which are useful for converting phosphate-containing prodrugs to free drugs; arylsulfatases, which are useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminases, which are useful for converting the non-toxic fluorocytosine to the anti-cancer drug 5-fluorouracil; proteases such as Serratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L), which are useful for converting peptide-containing prodrugs to free drugs. carbohydrate cleaving enzymes such as O-galactosidase and neuraminidase, useful for converting glycosylated prodrugs to free drugs; P-lactamases, useful for converting P-lactam derivatized drugs to free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogen with phenoxyacetyl or phenylacetyl groups, respectively, to free drugs. Enzymes may be covalently attached to the antibodies of the invention by techniques known in the art, such as the use of the linkers discussed above.

[0168] Suitable methods for preparing the immunoconjugates of the present invention are known in the art (see, for example, Hermanson GT, Bioconjugate Techniques, Third Edition, 2013, Academic Press). For example, methods for conjugating cytotoxic drugs to antibodies via linkers that are covalently attached to cysteine ​​residues of the interchain disulfide bridges of the antibody are well known.

[0169] In general, the immunoconjugates of the invention can be obtained, for example, by a process comprising the following steps: (i) preparing a compound comprising a linker and a growth inhibitory drug (e.g., a cytotoxic drug), also referred to herein as a "drug-linker compound"; (ii) contacting an optionally buffered aqueous solution of an antibody according to the invention with a solution of a Drug-Linker Compound; (iii) optionally then separating the conjugate formed in (ii) from unreacted antibody and / or drug-linker compound.

[0170] Aqueous solutions of antibodies can be buffered with buffers such as histidine, potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer is selected depending on the nature of the antibody. The drug-linker compound can be dissolved in an organic polar solvent such as dimethylsulfoxide (DMSO) or dimethylacetamide (DMA).

[0171] For conjugation to cysteine ​​residues of the antibody, the antibody is subjected to reduction (e.g. using TCEP) prior to step (ii). Suitable reducing conditions for reducing only interchain disulfide bonds are known in the art.

[0172] The reaction temperature for conjugation is usually between 20° C. and 40° C. The reaction time can vary, typically from 1 to 24 hours. The reaction between the antibody and the drug-linker compound can be monitored by refractive index measurement and / or size exclusion chromatography (SEC) with a UV detector. If the conjugate yield is too low, the reaction time can be extended.

[0173] A number of different chromatographic methods can be used by those skilled in the art to carry out the separation of step (iii). The conjugate can be purified, for example, by SEC, adsorption chromatography (e.g., ion exchange chromatography IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed support chromatography such as hydroxyapatite chromatography, or high performance liquid chromatography (HPLC) such as reverse phase HPLC. Purification by dialysis or filtration or diafiltration can also be used.

[0174] After steps (ii) and / or (iii), the conjugate-containing solution may be subjected to an additional step (iv) of purification, for example by chromatography, ultrafiltration and / or diafiltration. Such additional steps of purification, for example by chromatography, ultrafiltration and / or diafiltration, may also be performed on the antibody-containing solution after the reduction reaction, in cases where reduction is performed prior to conjugation.

[0175] The conjugate is recovered in aqueous solution at the end of such process. Drug-to-antibody ratio (DAR) is a number that can vary depending on the nature of the antibody and drug-linker compound used, as well as the experimental conditions used for conjugation (e.g., ratio (drug-linker compound) / (antibody), reaction time, solvent, and the nature of cosolvents, if any). Thus, contact between antibody and drug-linker compound can lead to a mixture that contains several conjugates that differ from each other by different drug-to-antibody ratios. Thus, the DAR determined is an average value.

[0176] Conjugation at the cysteine ​​residues of the interchain disulfide bridges using an antibody having four interchain disulfide bridges (e.g., mAb1 or any IgG1 antibody) is a method known in the art and offers the advantage that by choosing reaction conditions that allow the conjugation to proceed to completion (or at least close to completion), a relatively uniform DAR of about 8 can be achieved.

[0177] An exemplary method that can be used to determine the DAR consists of spectrophotometrically measuring the ratio of the absorbance of a solution of the purified conjugate at λD and 280 nm. 280 nm is a wavelength commonly used to measure protein concentrations, such as antibody concentrations. The wavelength λD is selected to allow the drug to be distinguished from the antibody, i.e., as is readily known to those skilled in the art, λD is the wavelength at which the drug has high absorbance, and λD is sufficiently far from 280 nm to avoid substantial overlap of the absorbance peaks of the drug and the antibody. For example, λD can be selected as 370 nm for exatecan (or camptothecin or other camptothecin analogues), or 252 nm for maytansinoid molecules.

[0178] The method of DAR calculation is derived, for example, from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science. The absorbance of the conjugate at λD (AλD) and 280 nm (A280) is measured at the monomer peak of a size exclusion chromatography (SEC) analysis (calculating the "DAR(SEC)" parameter) or using a classical spectrophotometer instrument (calculating the "DAR(UV)" parameter). The absorbance can be expressed as follows: A λD =(C D ×ε DλD )+(C A ×ε AλD ) A 280 =(C D ×ε D280 )+(CA ×ε A280 ) During the ceremony: C D and C A are the concentrations of drug and antibody in solution, respectively, ε DλD and ε D280 are λ D and the molar extinction coefficient of the drug at 280 nm, ε AλD and ε A280 are λ D and the molar extinction coefficient of the antibody at 280 nm. Solving these two equations with two unknowns leads to the following equation: C D =[(ε A280 ×A λD )-(ε AλD ×A 280 )] / [(ε DλD ×ε A280 )-(ε AλD ×ε D280 )] C A =[A 280 -(C D ×ε D280 )] / ε A280

[0179] The average DAR is then calculated from the ratio of the drug concentration to that of the antibody: DAR=C D / C A .

[0180] An alternative method for preparing an immunoconjugate of the invention is now described. This method may be used, inter alia, for an antibody comprising an amino acid sequence selected from the group consisting of GGTLQSPP, LLQGA, GGLLQGPP, TLQSG, TLQSPP, and TLQSA in at least one of its light chain constant regions (CL) and / or at least one of its heavy chain constant regions (CH). Thus, a further aspect of the invention relates to a method for producing an antibody-linker conjugate comprising the steps of: (1) providing an antibody comprising in at least one, preferably both, of its light chain constant regions (CL) and / or in at least one, preferably both, of its heavy chain constant regions (CH), an amino acid sequence selected from the group consisting of GGTLQSPP, LLQGA, GGLLQGPP, TLQSG, TLQSPP, and TLQSA, more preferably the amino acid sequence TLQSPP or GGTLQSPP (most preferably the sequence GGTLQSPP): (2) Mixing together at least the following components in a reaction buffer: (a) the antibody provided in step (1); (b) a microbial transglutaminase, preferably a transglutaminase comprising the following amino acid sequence:

number

[0181] In one embodiment, the transglutaminase is

number

[0182] In a preferred embodiment of the method, the reaction buffer is 7% DMSO, 24 mM HEPES, pH 7.0.

[0183] Preferably, the antibody used in the methods of the invention further comprises the sequences LLQGA and / or GGLLQGPP in at least one of its light chain constant regions (CL) and / or at least one of its heavy chain constant regions (CH). In the methods of the present invention, the linker has the following formula: [ka] where R is the remainder of the linker, which may also optionally include a drug. Accordingly, the drug is preferably exatecan. In a more preferred embodiment of the method of the present invention, the linker is NH2-GGG-beta-glucuronide.

[0184] In a preferred embodiment of the method of the present invention, in step (2), the mixture comprises the following drug-linker: [ka] Includes.

[0185] In a further embodiment of the method of the present invention, the mixture of step (2) comprises 5 molar equivalents of linker or drug-linker per conjugation site, respectively, wherein the conjugation site is the sequence LLQGA, GGLLQGPP, GGTLQSPP, TLQSG, TLQSPP or TLQSA contained in the light chain constant region (CL) and / or heavy chain constant region (CH) of said antibody.

[0186] In further embodiments of the methods of the invention, the antibody is an anti-CEACAM5 antibody of the invention as described herein, and / or the drug is a growth inhibitory drug as defined herein.

[0187] A further aspect of the invention relates to antibody-linker conjugates producible according to the methods of the invention, where the linker is preferably a linker or drug linker as described herein in the context of the ADCs of the invention.

[0188] Exemplary methods for preparing the immunoconjugates of the invention are described in the Examples.

[0189] Drug-Linker Compounds The present invention also provides compounds comprising a linker and a growth inhibitory drug (e.g., a cytotoxic drug), also referred to herein as a "drug-linker compound." For example, the present invention provides compounds of the following formula (X) or formula (XA):

[0190] [ka] [ka] or a physiologically acceptable salt thereof. A compound according to formula X is also referred to herein as "Drug-Linker Compound 1," "Compound DL1," or "DL1," and a compound according to formula XA is also referred to herein as "Drug-Linker Compound 1-M," "Compound DL1-M," or "DL1-M."

[0191] These Drug-Linker compounds can be used to prepare the immunoconjugates of the present invention as described herein above and below.

[0192] Drug-Linker Compounds of the invention (eg, those of formula (X) or (XA) depicted above) can be prepared by chemical synthesis, for example, as further described in the Examples below.

[0193] Pharmaceutical Compositions The antibodies or immunoconjugates of the invention are combined with pharma- ceutically acceptable carriers, diluents and / or excipients, optionally sustained release matrices of classes including, but not limited to, biodegradable polymers, non-biodegradable polymers, lipids or sugars, to form a pharmaceutical composition.

[0194] Accordingly, another aspect of the present invention relates to a pharmaceutical composition comprising an antibody or immunoconjugate of the invention and a pharma- ceutically acceptable carrier, diluent and / or excipient.

[0195] The terms "pharmaceutical" or "pharmaceutical acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other undesired reactions when administered to a mammal, particularly a human, as appropriate. A pharmaceutically acceptable carrier, diluent, or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0196] The term "pharmaceutical acceptable carrier" as used herein includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, etc. Examples of suitable carriers, diluents and / or excipients include, but are not limited to, water, amino acids, saline, phosphate buffered saline, buffer phosphate, acetate, citric acid, succinic acid; amino acids and derivatives such as histidine, arginine, glycine, proline, glycylglycine, etc.; inorganic salts such as NaCl or calcium chloride; sugars or polyalcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as polysorbate 80, polysorbate 20, poloxamer 188; and the like, and one or more of these combinations. In many cases, it will be useful to include an isotonicity agent such as sugar, polyalcohol, or sodium chloride in the pharmaceutical composition. The formulation may also contain an antioxidant such as tryptamine and / or a stabilizer such as Tween 20.

[0197] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will of course depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, etc.

[0198] Pharmaceutical compositions of the present invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, and the like.

[0199] In some embodiments, the pharmaceutical composition contains a pharma- ceutically acceptable base for the formulation for injection. These can be isotonic sterile saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or mixtures of such salts), or dry, especially lyophilized, compositions. This allows the constitution of an injectable solution by the addition of sterile water or saline, depending on the case.

[0200] The pharmaceutical composition may be administered from a drug combination device.

[0201] The dose used for administration can be adapted as a function of various parameters, for example as a function of the administration mode used, the pathology involved, or alternatively the desired duration of treatment.

[0202] To prepare a pharmaceutical composition, an effective amount of the antibody or immunoconjugate of the invention may be dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium.

[0203] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all such cases, the form must be sterile and injectable by a suitable device or system for delivery without degradation, it must be stable under the conditions of manufacture and storage, and it must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0204] The solution of the active compound as free base or pharmacologically acceptable salt can be prepared in the water suitably mixed with surfactant.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, and their mixture, and in oil.Under normal conditions of storage and use, these preparations can contain preservatives to prevent the growth of microorganisms.

[0205] The antibody or immunoconjugate of the present invention can be formulated into pharmaceutical compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, glycine, histidine, procaine, etc.

[0206] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some cases, it may be desirable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by the use of agents that delay absorption, for example, aluminum monostearate and gelatin, in the compositions.

[0207] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with any of the other ingredients listed above, followed by filtration sterilization. In general, dispersions can be prepared by incorporating various sterilized active ingredients into a sterile base containing a basic dispersion medium and the other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying technology. These produce a powder of the active ingredient plus any additional desired ingredients from its solution that has previously been sterile-filtered.

[0208] The preparation of more or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as a solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0209] Depending on the formulation, solutions may be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the injectable solutions described above, although drug release capsules and the like may also be employed.

[0210] For parenteral administration by aqueous solution, for example, the solution may be suitably buffered if necessary, and the liquid diluent may first be rendered isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous medium that may be employed will be known to those skilled in the art in light of this disclosure. For example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous infusion or injected into the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, p.1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject to be treated. The person administering will, in any event, determine the appropriate dose for the individual subject.

[0211] The antibodies or immunoconjugates of the invention can be formulated in therapeutic mixtures to contain, for example, about 0.01 to 100 milligrams per dose.

[0212] In addition to antibodies or immunoconjugates formulated for parenteral administration, such as intravenous or intramuscular injection, other pharma- ceutically acceptable forms include, for example, tablets or other solids for oral administration, time-release capsules, and any other forms currently in use.

[0213] In some embodiments, the use of liposomes and / or nanoparticles is contemplated for the introduction of polypeptides into host cells. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0214] Nanocapsules can generally entrap compounds in a stable and reproducible manner. To avoid side effects caused by intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles or biodegradable polylactide or polylactide-co-glycolide nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be easily made by those skilled in the art.

[0215] Liposomes can be formed from phospholipids that are dispersed in aqueous media and spontaneously form multilamellar concentric bilayer vesicles, also called multilamellar vesicles (MLVs). MLVs generally have diameters ranging from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 A that contain aqueous solution in their cores. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0216] Besides the examples mentioned above, further pharmaceutical forms such as nanoparticles, microparticles and capsules, implants (eg lipid implants), or self-solidifying or self-emulsifying systems are also contemplated.

[0217] Therapeutic Methods and Uses The inventors found that the antibodies of the invention (e.g., mAb1) can be internalized as part of a CEACAM5-antibody complex after binding. Moreover, they showed that such antibodies conjugated to a cytotoxic drug (exatecan) mediate cytotoxic effects against tumor cells in vitro. The inventors also showed that these immunoconjugates of the invention induce significant antitumor activity in vivo with a single injection when used at a dose of 10 mg / kg, e.g., in a mouse xenograft model of patient-derived human colon cancer. In fact, the immunoconjugates of the invention show broad activity in a large set of in vitro and in vivo models. The cytotoxic potency correlates well with the target (CEACAM5) expression and is much lower in target-negative cells. Very good antitumor activity was demonstrated in several cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models of different cancer types. The immunoconjugate was well tolerated in a dose-ranging study in non-human primates and had a side effect profile typical of topoisomerase-I inhibitor chemotherapy. Preclinical data indicates a good therapeutic window for subsequent clinical trials. Thus, the antibodies, immunoconjugates and pharmaceutical compositions of the present invention may be useful in treating cancer.

[0218] Thus, the present invention provides an antibody, immunoconjugate or pharmaceutical composition of the present invention for use as a medicament. For example, the present invention provides an antibody, immunoconjugate or pharmaceutical composition of the present invention for use in treating cancer. The present invention further provides a method of treating cancer, comprising administering an antibody, immunoconjugate or pharmaceutical composition of the present invention to a subject in need thereof.

[0219] The cancer to be treated by the antibody, immunoconjugate, or pharmaceutical composition of the present invention is preferably a cancer that expresses CEACAM5, more preferably a cancer that overexpresses CEACAM5 compared to normal (i.e., non-tumor) cells of the same tissue origin. Expression of CEACAM5 by cells can be readily assayed, for example, by immunohistochemical methods, for example, by using an antibody according to the present invention (or a commercially available anti-CEACAM5 antibody), for example, as described in the next section "Diagnostic Uses."

[0220] In some embodiments, the cancer to be treated with the antibody, immunoconjugate, or pharmaceutical composition of the present invention is colon cancer, non-small cell lung cancer, pancreatic cancer, gastric cancer, cervical cancer, esophageal cancer (e.g., esophageal adenocarcinoma), bile duct cancer, breast cancer, prostate cancer, ovarian cancer, urothelial cancer, bladder cancer, or gastric, uterine, endometrial, thyroid, or skin cancer. In some specific embodiments, the cancer to be treated with the antibody, immunoconjugate, or pharmaceutical composition of the present invention is colon cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, or prostate cancer.

[0221] The antibodies or immunoconjugates of the invention may be used in cancer therapy, either alone or in combination with any suitable growth inhibitory agent.

[0222] The antibodies of the present invention can be conjugated (linked) to growth inhibitory drugs as described above. Thus, the antibodies of the present invention can be useful for targeting the growth inhibitory drugs to cancerous cells that express or overexpress CEACAM5 on their surface.

[0223] It is also well known that therapeutic monoclonal antibodies can lead to the depletion of cells bearing the antigen specifically recognized by the antibody. This depletion can be mediated by at least three mechanisms: antibody-mediated cytotoxicity (ADCC), complement-dependent lysis, and direct inhibition of tumor growth via signals mediated by the antigen targeted by the antibody.

[0224] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to and subsequently kill antigen-bearing target cells. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as those described in US Patent Nos. 5,500,362 or 5,821,337, can be performed.

[0225] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system to antibodies that are bound to their corresponding antigens. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al. (Journal of Immunological Methods. 1997 Mar;202(2):163-171).

[0226] In some embodiments, the antibodies of the present invention may have altered amino acid sequences that result in reduced or abolished binding to most Fcγ receptors, which may reduce uptake and toxicity in normal cells and tissues that express such receptors, e.g., macrophages, hepatic sinusoidal cells, etc.

[0227] Certain aspects of the invention relate to methods of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an antibody, immunoconjugate or pharmaceutical composition of the invention.

[0228] In the context of the present invention, the term "treating" or "treatment" as used herein means to reverse, alleviate, inhibit the progression of, or prevent the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treating cancer" as used herein means to inhibit the growth of malignant cells of a tumor and / or the progression of metastases from said tumor. Such treatment may also lead to the regression of tumor growth, i.e., a reduction in the size of a measurable tumor. For example, such treatment may lead to the complete regression of tumors or metastases.

[0229] In the context of the therapeutic application of the present invention, the term "subject" or "patient" or "subject in need thereof" or "patient in need thereof" refers to a subject (e.g., a human or non-human mammal) that is suffering from or likely to be suffering from a tumor. For example, said patient may be a patient that has been determined to be sensitive to a therapeutic agent that targets CEACAM5, in particular an antibody or immunoconjugate according to the present invention, for example according to the method described herein below.

[0230] "Therapeutically effective amount" means an amount sufficient to treat said cancer disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily usage of the antibodies, immunoconjugates and pharmaceutical compositions of the present invention (collectively referred to as "therapeutic agents") will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder to be treated and the severity of the disorder; the activity of the specific therapeutic agent employed; the age, weight, general health, sex and diet of the patient; the administration time, route of administration and excretion rate of the specific therapeutic agent employed; the duration of treatment; drugs used in combination or simultaneously with the specific therapeutic agent employed; and similar factors well known in the medical art. For example, it is well within the skill of the art to start the dose of a compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0231] The antibodies, immunoconjugates or pharmaceutical compositions of the invention may also be used to inhibit the progression of cancer metastasis.

[0232] The antibodies, immunoconjugates or pharmaceutical compositions of the invention may also be used in combination with any other therapeutic intervention for treating cancer (e.g., adjuvant therapy) and / or reducing the growth of metastatic cancer. For example, the other therapeutic intervention for such combination may be a standard of care (SOC) therapeutic agent for the cancer being treated.

[0233] The efficacy of treatment with an antibody or immunoconjugate or pharmaceutical composition according to the invention can be readily analyzed in vivo, e.g. in a mouse model of cancer, by measuring, e.g., the change in tumor volume, % tumor regression, partial regression or complete regression between treated and control groups.

[0234] Diagnostic Use CEACAM5 has been reported to be highly expressed on the surface of cancer cells, e.g., colon, gastric, lung, and pancreatic tumor cells, while expression in normal tissues is restricted to a small number of normal epithelial cells, such as colonic and esophageal epithelial cells.

[0235] CEACAM5 therefore constitutes a cancer marker and has the potential to be used, for example, to indicate the effectiveness of anti-cancer treatment or to detect disease recurrence.

[0236] In some embodiments, the antibody of the present invention can be used as a component of an assay in the context of a therapy targeting CEACAM5-expressing tumors to determine the sensitivity of a patient to a therapeutic agent, to monitor the effectiveness of an anti-cancer therapy, or to detect disease recurrence after treatment.In some embodiments, the same antibody of the present invention can be used both as a component of a therapeutic agent and as a component of a diagnostic assay.

[0237] Therefore, a further aspect of the present invention relates to the use of the antibody according to the present invention for detecting CEACAM5 expression ex vivo from a biological sample from a subject. Another aspect of the present invention relates to the use of the antibody according to the present invention for detecting CEACAM5 expression in a subject in vivo. When used for detecting CEACAM5, the antibody can be labeled with a detectable molecule, such as a fluorophore or an enzyme.

[0238] The detection of CEACAM5 can be intended, for example, to: a) diagnosing the presence of cancer in a subject; or b) determining the sensitivity of a patient with cancer to a therapeutic agent targeting CEACAM5, in particular an antibody or immunoconjugate according to the invention; or c) monitoring the efficacy of an anti-CEACAM5 cancer treatment or detecting cancer relapse after an anti-CEACAM5 cancer treatment, in particular, wherein said treatment is a treatment with an antibody or immunoconjugate according to the invention: By detecting the expression of the surface protein CEACAM5 on tumor cells.

[0239] In an embodiment, the antibody is intended for in vitro or ex vivo diagnostic use.For example, CEACAM5 can be detected from a biological sample obtained from a subject in vitro or ex vivo using the antibody of the present invention.The use according to the present invention can also be in vivo use.For example, the antibody according to the present invention can be administered to a subject, and antibody-cell complexes can be detected and / or quantified.The detection of said complexes is an indication of cancer.

[0240] The present invention further relates to an in vitro or ex vivo method of detecting the presence of cancer in a subject comprising the steps of: (a) contacting a biological sample from a subject with an antibody according to the invention, in particular under suitable conditions for the antibody to form a complex with said biological sample; (b) determining the level of antibody bound to the biological sample; and (c) detecting the presence of cancer by comparing the measured level of bound antibody to a control, where an increased level of bound antibody compared to the control is indicative of cancer.

[0241] The present invention also relates to an in vitro or ex vivo method for determining the sensitivity of a patient suffering from cancer to a therapeutic agent targeting CEACAM5, in particular to an antibody or immunoconjugate according to the invention, which method comprises the steps of: (a) contacting a biological sample from a patient with cancer with an antibody according to the invention, in particular under suitable conditions for the antibody to form a complex with said biological sample; (b) determining the level of antibody bound to the biological sample; and (c) comparing the measured level of bound antibody to the biological sample with the level of bound antibody to a control: wherein an increased level of bound antibody to said biological sample, relative to a control, is indicative of a patient susceptible to a therapeutic agent that targets CEACAM5.

[0242] In the above methods, the control can be a normal, non-cancerous biological sample of the same type, or a reference value determined as representative of antibody binding levels in a normal biological sample of the same type.

[0243] In certain embodiments, the antibodies of the present invention are useful for diagnosing CEACAM5-expressing cancers, such as colon cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer, or other solid tumors that express CEACAM5.

[0244] The present invention further relates to an in vitro or ex vivo method of monitoring the efficacy of an anti-CEACAM5 cancer treatment comprising the steps of: (a) contacting a biological sample from a subject undergoing anti-CEACAM5 cancer treatment with an antibody according to the invention, in particular under conditions suitable for the antibody to form a complex with said biological sample; (b) determining the level of antibody bound to the biological sample; and (c) comparing the measured level of bound antibody to the level of bound antibody in a control: Wherein a decreased level of bound antibody to the biological sample compared to the control is indicative of the efficacy of the anti-CEACAM5 cancer treatment. In the method, an increased level of bound antibody to the biological sample compared to the control is indicative of the ineffectiveness of the anti-CEACAM5 cancer treatment. In one embodiment of this method of monitoring efficacy, the control is a biological sample of the same type as the biological sample subjected to analysis, but obtained from the subject at an earlier time point during the course of the anti-CEACAM5 cancer treatment.

[0245] The present invention further relates to an in vitro or ex vivo method for detecting cancer relapse following anti-CEACAM5 cancer treatment comprising the steps of: (a) contacting a biological sample from a subject with an antibody according to the invention, in particular under suitable conditions for the antibody to form a complex with said biological sample, and the subject has completed an anti-CEACAM5 cancer treatment; (b) determining the level of antibody bound to the biological sample; and (c) comparing the measured level of bound antibody to the level of bound antibody in a control: Wherein an increased level of bound antibody to said biological sample compared to a control is indicative of cancer relapse after anti-CEACAM5 cancer treatment. Said control may in particular be a biological sample of the same type as the biological sample subjected to analysis, but obtained from the subject before, i.e., upon or after completion of anti-CEACAM5 cancer treatment.

[0246] The anti-CEACAM5 cancer treatment is, for example, a treatment using an antibody or immunoconjugate according to the present invention. The anti-CEACAM5 cancer treatment targets CEACAM5-expressing cancer, such as colon cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer, or other solid tumors that express CEACAM5.

[0247] In some embodiments, the antibodies of the present invention may be labeled with a detectable molecule or substance, such as a fluorescent molecule or fluorophore, a radioactive molecule, an enzyme, or any other label known in the art that provides (either directly or indirectly) a signal.

[0248] With respect to the antibodies according to the invention, the term "labeled", as used herein, is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)) to the polypeptide, as well as indirect labeling of the polypeptide by reactivity with a detectable substance.

[0249] The antibodies of the present invention can be labeled with a radioactive molecule by any method known in the art. For example, the radioactive molecule can be I 123 , I 124 , In 111 , Re 186 , Re188 , Tc 99 Examples of radioactive atoms include, but are not limited to, radioactive atoms for scintigraphic studies such as, for example, iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0250] "Biological sample" encompasses various sample types obtained from a subject that can be used for diagnostic or monitoring assays. Biological samples include, but are not limited to, blood and other liquid samples of biological origin, solid tissue samples, such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny. Thus, biological samples encompass clinical samples, cells from culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples such as tumor samples.

[0251] In some embodiments, the biological sample may be a formalin-fixed paraffin-embedded (FFPE) tissue sample.

[0252] The present invention also relates to an in vivo method of detecting the presence of cancer in a subject comprising the steps of: a) administering to a patient an antibody according to the invention, wherein the antibody is labeled with a detectable molecule: b) detecting the localization of said antibody in the patient by imaging, for example by detecting a detectable molecule.

[0253] In the above methods, the cancer can be a CEACAM5-expressing cancer, such as colon cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer or other solid tumors that express CEACAM5.

[0254] The antibodies of the invention may also be useful for staging cancer (e.g., by radioimaging). They may be used alone or in combination with other cancer markers.

[0255] As used herein, the term "detection" or "detected" encompasses qualitative and / or quantitative detection (ie, measuring the levels), with or without reference to a control.

[0256] In the context of the present invention, the term "diagnosing" as used herein means the determination of the nature of a medical condition and is intended to identify the pathology from which a subject suffers based on a number of collected data.

[0257] kit Finally, the present invention also provides a kit comprising at least one antibody or immunoconjugate of the present invention. The kit containing the antibody of the present invention can find use for detecting surface protein CEACAM5 or for therapeutic or diagnostic assays. The kit of the present invention can contain the antibody coupled to a solid support, such as a tissue culture plate or beads (e.g., sepharose beads). The kit containing the antibody for detecting and quantifying surface protein CEACAM5 in vitro, for example by ELISA or Western blot, can be provided. Such antibodies useful for detection can be provided with a label, such as a fluorescent or radioactive label.

[0258] A brief description of the sequence Amino acid sequence: SEQ ID NO: 1 Human CEACAM5 protein sequence according to GenBank accession number AAA51967.1 SEQ ID NO:2 Macaca fascicularis CEACAM5 protein sequence (NCBI reference sequence XP_005589491.1) SEQ ID NO: 3 CDR1-H of mAb1 SEQ ID NO: 4 CDR2-H of mAb1 SEQ ID NO: 5 CDR3-H of mAb1 SEQ ID NO: 6 CDR1-L of mAb1 SEQ ID NO: 7 CDR2-L of mAb1 SEQ ID NO: 8 CDR3-L of mAb1 SEQ ID NO: 9 VH of mAb1 SEQ ID NO: 10 VL of mAb1 SEQ ID NO: 11 CH of mAb1 SEQ ID NO: 12 CL of mAb1, mAb1-M and mAb6-M SEQ ID NO: 13 HC of mAb1 SEQ ID NO: 14 LC of mAb1, mAb1-M and mAb6-M

[0259] Nucleic acid sequence: SEQ ID NO: 15 DNA sequence encoding the HC of mAb1 SEQ ID NO: 16 DNA sequence encoding the LC of mAb1

[0260] Amino acid sequence: SEQ ID NO: 17 HC of antibody hu8G4 SEQ ID NO: 18 LC of antibody hu8G4 SEQ ID NO: 19 Optimized antibody variant 1 HC SEQ ID NO: 20 LC of optimized antibody variant 1 SEQ ID NO: 21 LC of optimized antibody variant 2 SEQ ID NO: 22 LC of optimized antibody variant 4 SEQ ID NO: 23 LC of optimized antibody variant 5 SEQ ID NO: 24 Optimized antibody variant 6 HC SEQ ID NO: 25 HC of huMab2-3 (allotype) SEQ ID NO: 26 LC of huMab2-3 SEQ ID NO:27 hmn-14 HC SEQ ID NO:28 LC of hmn-14 SEQ ID NO:29 rb8G4HC SEQ ID NO: 30 LC of rb8G4 SEQ ID NO: 31 CH(CH-LALA-YTE) of mAb1-M and mAb2-M SEQ ID NO: 32 CH of mAb3-M (CH-K222R-LALA-YTE) SEQ ID NO: 33 CL (CL-tag) of mAb2-M, mAb3-M, and mAb7-M SEQ ID NO: 34 HC of mAb1-M and mAb2-M (HC-LALA-YTE) SEQ ID NO: 35 HC of mAb3-M (HC-K222R-LALA-YTE) SEQ ID NO: 36 LC (LC-tag) of mAb2-M, mAb3-M, and mAb7-M SEQ ID NO: 40 HC of mAb5-M (anti-CD20-HC) SEQ ID NO: 41 LC of mAb5-M (anti-CD20-LC) SEQ ID NO: 44 Transglutaminase SEQ ID NO: 45 Transglutaminase (activated, version 1) SEQ ID NO: 47 Transglutaminase (activated, version 2) SEQ ID NO: 48 Transglutaminase (activated, version 3) SEQ ID NO: 49 Transglutaminase (activated, version 4) SEQ ID NO: 50 CH(CH-LALA) of mAb6-M and mAb7-M SEQ ID NO: 51 HC of mAb6-M and mAb7-M (HC-LALA) SEQ ID NO: 54 Framework region FR1 SEQ ID NO: 55 Framework region FR2 SEQ ID NO: 56 Framework region FR3 SEQ ID NO: 57 Framework region FR4 SEQ ID NO: 58 Framework region FR5 SEQ ID NO: 59 Framework region FR6 SEQ ID NO: 60 Framework region FR7 SEQ ID NO: 61 Framework region FR8

[0261] Nucleic acid sequence: SEQ ID NO: 37 DNA sequence encoding the HC (HC-LALA-YTE) of mAb1-M and mAb2-M SEQ ID NO: 38 DNA sequence encoding HC (LC-K222R-LALA-YTE) of mAb3-M SEQ ID NO: 39 DNA sequence encoding the LC (LC-tag) of mAb2-M, mAb3-M, and mAb7-M SEQ ID NO: 42 DNA sequence encoding the HC of mAb5-M (anti-CD20-HC) SEQ ID NO: 43 DNA sequence encoding the LC of mAb5-M (anti-CD20-LC) SEQ ID NO: 46 DNA sequence encoding transglutaminase SEQ ID NO: 52 DNA sequence encoding the HC (HC-LALA) of mAb6-M and mAb7-M SEQ ID NO: 53 DNA sequence encoding the LC of mAb1-M and mAb6-M

[0262] Amino acid sequence: SEQ ID NO: 62 Labetuzumab heavy chain SEQ ID NO:63 Labetuzumab light chain SEQ ID NO: 64 Rituximab HC SEQ ID NO: 65 Rituximab LC

[0263] example Example 1: Anti-CEACAM5 antibody 1.1 Immunization of transgenic rats and isolation of hybridomas To generate monoclonal antibodies against human CEACAM5 protein (carcinoembryonic antigen-related cell adhesion molecule 5; CD66e), human immunoglobulin gene transgenic rats (OmniRat™) were obtained from CHARLES RIVER LABORATORIES INTERNATIONAL INC. (Wilmington, Mass.). Five rats were immunized four times with CEACAM5 cDNA (encoding amino acids 35-675 of the human CEACAM5 protein sequence with UniProt ID no. P06731, the sequence of which is identical to SEQ ID NO:1 except for the replacement of E398 of SEQ ID NO:1 by K398) cloned into Aldevron's proprietary immunization vector (pB8-CEA-hum-MC) and transiently transfected into OMT rat cells using a gene gun.

[0264] Anti-CEACAM5 titers were assessed by cell-based ELISA (CELISA) assay using cells expressing CEACAM5 on their cell membrane (titer results are presented below). Serum of immunized animals was taken on day 31 of the immunization protocol after four rounds of genetic material immunization (IS31d-4). Serum diluted in PBS+3% FBS was tested by flow cytometry against mammalian cells transiently transfected with CEACAM5 cDNA cloned into Aldevron's proprietary expression vector (pB1-CEA-hum-MC). Goat anti-rat IgG R-phycoerythrin conjugate (Southern Biotech, #3030-09) was used as the secondary antibody at 10 μg / ml.

[0265] All animals were sacrificed and lymphocytes from lymph nodes were pooled and cryopreserved for future use. The cells were fused with the Ag8 mouse myeloma cell line to generate viable hybridomas. Hybridoma cells from this fusion were then transferred to ten 96-well plates.

[0266] 1.2 CEACAM5 specificity Hybridoma supernatants were screened using a cell-based ELISA (CELISA) assay for detection of anti-CEACAM5 antibodies that did not bind to CEACAM1 (BGP), CEACAM3 (CGM1a), CEACAM4 (CGM7), CEACAM6 (NCA), and CEACAM8 (NCA-95). Goat anti-rat IgG R-phycoerythrin conjugate (Southern Biotech, #3030-09) was used as the secondary antibody at 10 μg / ml.

[0267] Clones showing specificity for human CEACAM5, but not for its related proteins, were transferred to one 96-well plate and hybridoma supernatants were evaluated for specificity and cross-reactivity in an ELISA assay. In this analysis, the 8G4 hybridoma clone and its subclones showed specificity for human CEACAM5 and cross-reactivity for Macaca fascicularis CEACAM5.

[0268] 1.3 Detection and cloning of antibody sequences Total RNA was prepared from each hybridoma clone according to the RNeasy 96 protocol, Qiagen. Total RNA was then transcribed into cDNA using random hexamers and SuperScript® III.

[0269] The resulting cDNA was quality controlled by qPCR, and VH and Vk were amplified by PCR. The PCR products were purified using the AMpure XP PCR cleanup kit in combination with a KingFisher instrument.

[0270] The VH and Vk genes of the 8G4 subclone were cloned into the destination vectors hi00_pTT5_VH_ccdB and hh00_pTT5_Vk_ccdB, respectively, using a homologous recombination procedure (so-called "Lucigen cloning"). The reaction mix was transformed into One Shot® Mach1™ T1R chemically competent E. coli. Correctly recombined clones were confirmed by Sanger sequencing.

[0271] 1.4 Humanization and biochemical characterization of hits and candidate selection 8G4 and other clones were reformatted and expressed as human IgG1 molecules. They were evaluated by SDS-PAGE, size-exclusion chromatography (SEC), selectivity, affinity, cell binding and potency. Based on the results, one humanized candidate antibody, designated hu8G4, was selected for amino acid sequence optimization to improve manufacturability and affinity.

[0272] The amino acid sequence of the humanized candidate antibody hu8G4 is as follows: Heavy Chain: EVQLVESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRLTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17)

[0273] Light chain: ETTLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTIGSLQSEDFAVYFCQQYTNWPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18)

[0274] 1.5 Biophysical improvement strategies especially for hu8G4 leading to mAb1 Evaluation of the variable region sequences of hu8G4 identified six non-germline amino acid residues on the light chain framework and two non-germline amino acid residues on the heavy chain framework. Evaluation of amino acids and sequence motifs potentially prone to post-translational modification, such as deamidation motifs, surface-accessible methionines, and free cysteines, did not identify any amino acid residues with increased propensity. Several designed antibody sequences were generated in which certain amino acids were replaced by the germline-associated amino acid at that position. The different VH and VL optimized designs were then co-expressed in HEK293 6E cells as Fab and full-length IgG1 molecules, purified, and tested (see, for example, optimized variants 1-10 below).

[0275] The amino acid sequences of the 10 optimized antibody variants in full-length IgG1 format were as follows: Variant 1 (VH1.00 / VL1.00)

[0276] HC: EVQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRLTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19)

[0277] LC: ETTLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTIGSLQSEDFAVYFCQQYTNWPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 20) Variant 2 (VH1.00 / VL1.01) HC: SEQ ID NO: 19

[0278] LC: EIVLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYTNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 21) Variant 3 (VH1.00 / VL1.02) HC: SEQ ID NO: 19 LC: SEQ ID NO: 14 Variant 4 (VH1.00 / VL1.03) HC: SEQ ID NO: 19

[0279] LC: EIVMTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYTNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22) Variant 5 (VH1.00 / VL1.04) HC: SEQ ID NO: 19

[0280] LC: EIVMTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23) Variant 6 (VH1.02 / VL1.00)

[0281] HC: EVQLQESGPG LVKPSQTLSL TCTVSDGSVS RGGYYLTWIR QHPGKGLEWI GYIYYSGSTY FNPSLRSRVT MSVDTSKNQF SLKLSSVTAA DTAVYYCARG IAVAPFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK (SEQ ID NO: 24) LC: SEQ ID NO:20 Variant 7 (VH1.02 / VL1.01) HC: SEQ ID NO:24 LC: SEQ ID NO:21 Variant 8 (VH1.02 / VL1.02) HC: SEQ ID NO:24 LC: SEQ ID NO: 14 Variant 9 (VH1.02 / VL1.03) HC: SEQ ID NO:24 LC: SEQ ID NO:22 Variant 10 (VH1.02 / VL1.04) HC: SEQ ID NO:24 LC: SEQ ID NO:23

[0282] All of the optimized variants 1 to 10 performed similarly well in terms of maintaining quality as assayed by percent aggregates by size exclusion chromatography, maintaining stability based on thermal unfolding monitored by fluorescence (FMTU), retaining binding to the MKN-45 cancer cell line, and maintaining selectivity for the target. Variant 8 (i.e., the variant encompassing VH1.02 and VL1.02) was selected for further development as an optimized variant with particularly similar sequence to the germline.

[0283] Further sequence optimization of the selected variant 8 was then performed, particularly to reduce IgG Fc effector function. Compared to the parent clone, the resulting final sequence-optimized (so) clone, designated so8G4 (also referred to herein as mAb1), exhibits improved affinity and improved manufacturability, and exhibits no or reduced binding to FcγRI, FcγRIIa, FcγRIIIa, FcγRIIIa / complex, C1q, FcγRIIb, and FcγRIIIb, while maintaining affinity to CEACAM5 and FcRn. The amino acid sequence of this final sequence-optimized antibody so8G4 (also referred to herein as mAb1) is as follows: Heavy Chain (HC): SEQ ID NO: 13 (as defined herein above) Light chain (LC): SEQ ID NO: 14 (as defined herein above)

[0284] 1.6 In vitro characterization of mAb1 Antibody mAb1 was characterized by in vitro assays for several properties, including binding affinity, selectivity, and internalization.

[0285] 1.6.1 Binding affinity To determine the binding affinity of soluble antibody analytes to the captured target protein CEACAM5 (human or cynomolgus monkey Macaca fascicularis). The following experimental conditions were used with the Octet Red instrument: Streptavidin-coated biosensors. Biotinylated target protein concentration (here ECD stands for extracellular domain): · R&D Systems human_CEACAM5_ECD-his-biotin (biotinylated using standard methods) was captured at 2.5μg / ml for 900s at 1000rpm. Recombinant Macaca fascicularis CEACAM5_ECD-His-biotin obtained from Syngene (biotinylated using standard methods) was captured at 5 μg / ml for 900 s at 1000 rpm. Analyte antibody concentrations: 200, 100, 50, 25, 12.5, 6.25, 0 nM. Binding affinity KD (equilibrium dissociation constant) values ​​were determined from the measured binding kinetic association (ka) and dissociation (kd) rate constants using Octet evaluation software, where KD=kd / ka. The antibody was used in Fab format. Results for Fab generated from mAb1: The binding affinity KD for human CEACAM5 was 6.3±1.98 nM. The binding affinity KD for cynomolgus monkey CEACAM5 was 14.1±2.53 nM.

[0286] 1.6.2. Selectivity a) Species and domains The selectivity of mAb1 was determined in an ELISA assay by titrating the antibody from 4 nM to 0.25 pM and applying it to 1 μg / ml of bound recombinant human (rh) CEACAM5 ECD or its domains N-A1-B1, A2-B2, A3-B3 or bound recombinant Macaca fascicularis (mf) CEACAM5 ECD, all from Syngene. The results are shown in Figure 1 and summarized below: The binding EC50 for rhCEACAM5 is 153.4 pM. The binding EC50 for the rhA2-B2 domain is 166.9 pM. The binding EC50 for mfCEACAM5 is 324.3 pM. No binding was detected to rhN-A1-B1 or rhA3-B3 or to BSA (bovine serum albumin, served as a negative control).

[0287] b) Different CEACAM proteins The selectivity of mAb1 Fab against human CEACAM5 and other human CEACAM family members was determined by ELISA assay. Proteins were coated onto 96-well assay plates: huCEACAM5-His6 (R&D Systems #4128-CM), huCEACAM6-His6 (3934-CM R&D Systems, and recombinant protein obtained from Syngene), huCEACAM1-His6 (2244-CM R&D Systems), huCEACAM3-His6 (C449 Novoprotein), huCEACAM7-His6 (C926 Novoprotein), huCEACAM8-His6 (C583 Novoprotein), huPSG1-His6 (CC66 Novoprotein). Each protein was coated onto the plate at a concentration of 12 nM.

[0288] result: The Fab of mAb1 bound to human CEACAM5 (EC50 of 3.04 nM) but did not bind to other human CEACAM family members in ELISA assays, even when 1000 nM of the Fab of mAb1 was used, a concentration more than 300-fold higher than the EC50 for binding to human CEACAM5.

[0289] The Fab of mAb1 also did not bind to an unrelated protein (BSA) in an ELISA assay at all concentrations tested.

[0290] 1.6.3 Cell Binding of mAb1 The ability of an antibody to bind its target protein on cells was determined by titrating the antibody against cells expressing the target (e.g., human CEACAM5) and measuring the fluorescent MFI of the cells. Model cells for antibody binding comparison were the MKN45 cell line expressing human CEACAM5 and the CHO cell line expressing mfCEACAM5. Titration was 10-point x 4 dilutions with assay buffer (PBS x 1 containing 1% BSA), with a curve starting concentration of 2000 nM.

[0291] Example data: mAb1 bound to human CEACAM5-expressing MKN-45 cell line with an EC50 of 10.62 ± 1.6 nM. mAb1 bound to the mfCEACAM5-expressing CHO cell line with an EC50 of 4.8 ± 0.6 nM.

[0292] 1.6.4 Cell binding comparison with known antibodies The cell binding of our lead antibody mAb1 was compared to the ADC-related known antibodies huMab2-3 (as is from the known ADC SAR408701) and hMN14 (also referred to herein as hmn-14) (as is from the known ADC labetuzumab govitecan or IMMU-130) on the CEACAM5-expressing MKN45 cell line.

[0293] The following amino acid sequences were used for the known antibodies referred to above in the experiments described herein below:

[0294] huMab2-3: HC(Allotype): EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25) LC: DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26)

[0295] hmn-14: HC: EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINY APSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27) LC: DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPS RFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 28)

[0296] Rituximab was included in the comparison as a control. The results for antibody binding to cells are shown in Figure 2 and summarized below: mAb1(so8G4)=8.3nM huMab2-3=6nM hmn-14=11.8nM

[0297] Average of several experiments (EC50): mAb1 (so8G4)=10.4nM±1.6nM(n=12) huMab2-3 = 4.9 nM ± 1.6 nM (n = 3) hmn-14=16nM(n=2)

[0298] The cell binding of anti-CEACAM5 antibodies to the MKN45 cell line shows that the binding of mAb1 and the known antibodies is similar.

[0299] 1.6.5 Internalization Assays using Cell Discoverer A relevant property of an ADC is its internalization into target-expressing cells and lysosomes. Therefore, internalization is a relevant property of an antibody to be used as part of an ADC. The rate of antibody internalization into late endosomes and lysosomes (low pH vesicles) can be monitored by directly labeling the antibody with a pH-sensitive dye (pHrodo), which upon excitation emits strong fluorescence at a pH below 6.0. This fluorescence can be imaged with a Cell-Discoverer7 (Zeiss) and the internalization rate can be calculated.

[0300] To analyze the internalization kinetics of several antibodies, MKN45 cells were seeded at 25,000 cells / well in 96-well dark clear flat-bottom plates (Cellvis). Cells were cultured overnight in 100 μl / well of RMPI-1640+10% FBS (Thermo). The cell medium was removed and cells were stained with 100 μl of 10 μg / ml Hoechst dye diluted in PBS×1 for 15 min at room temperature (RT) in the dark. Then, cells were washed twice with PBS×1.

[0301] Anti-CEACAM5 human IgG antibodies (so8G4 (i.e. mAb1), humab2-3, hmn-14) and anti-MerTK antibodies (Merck) were directly labeled with pHrodo, diluted to a concentration of 100 nM in warmed RPMI1640 + 10% FBS without phenol red, and added to their respective wells. The plates were incubated at 37°C, 5% CO in a Cell Discoverer. 2The cells were incubated at RT for 20 h with images taken every 20 min as further described below.

[0302] Internalization of pHrodo-labeled antibodies into late endosomes and lysosomes of cells was imaged by Cell-Discoverer7 (Zeiss) using fluorescence at 567 nm excitation and 592 / 25 nm emission detector. Cell nuclei were labeled with Hoechst and imaged at 385 nm excitation and 425 / 30 nm emission detector.

[0303] The fluorescence of each well was recorded every 20 min for 20 h. Analysis of the total fluorescence intensity (SFI) per cell was calculated using Zen software (ZEN3.1) and Excel analysis of linear regression.

[0304] The results are shown in Figures 3 and 4, and the slopes of the linear parts of the curves (see Figure 4) are also summarized in the table below: [Table C]

[0305] Conclusion: 1. so8G4 (mAb1) has a higher average binding rate (28958±766) than humab2-3 (18917±1416) and hmn-14 (22268±3060). 2. so8G4 (mAb1) also has high internalization potency compared to humab2-3 and hmn-14. 3. mAb1-M, mAb2-M, mAb3-M, mAb6-M, and mAb7-M are expected to exhibit internalization properties corresponding to mAb1.

[0306] 1.7 Exemplary Methods for Producing mAb1, e.g., for Use in Conjugation to Drug-Linker Compounds Anti-CEACAM5 antibody mAb1 was produced by recombinant CHO-K1Sv cell line. Cell culture was performed in batch mode in 200 l single-use bioreactors. Cells were grown in proprietary CHO fed-batch growth medium supplemented with glucose at 37°C. At days 3, 5, 7, and 10 after inoculation, the culture was fed with a proprietary mixture of feed components.

[0307] Crude conditioned medium from the bioreactor runs was transferred to a 3 x 1.1 m 2 Millistak+Pod DOHC (Millipore MD0HC10FS1) and 1.1m 2 The solution was clarified using a Millistak+Pod XOHC (Millipore #MX0HC01 FS1) filter, followed by a final filtration through a Millipore Opticap XL3 0.5 / 0.2 μm filter (Millipore #KHGES03HH3).

[0308] After clarification, the antibody mAb1 was purified using a standard antibody purification process consisting of a Protein A capture step and an ion exchange chromatography step. The anti-CEACAM5 antibody mAb1 served as an intermediate for the production of ADC molecules.

[0309] 1.8 Expression and purification of a human / rabbit chimeric variant of mAb1 and its use for immunohistochemistry (IHC) in formaldehyde-fixed paraffin-embedded cell lines and human tumor tissues A human / rabbit chimeric variant of mAb1 was generated by conventional recombinant methods. The human / rabbit chimeric variant of mAb1 (also referred to herein as "rb8G4") had the following amino acid sequence: Heavy chain VQLQESGPGLVKPSQTLSLTCTVSDGSVSRGGYYLTWIRQHPGKGLEWIGYIYYSGSTYFNPSLRSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARGIAVAPFDYWGQGT LVTVSSQPKAPSVFPLAPCCGDTPSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSEDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHEDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO: 29) Light chain EIVLTQSPATLSVSPGERATLSCRTSQSVRSNLAWYQQKPGQAPRLLIYAASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTNWPFTFGPGTKVDIKRDPVAPSVLLFPPSKEELTTGTATIVCVANKFYPSDITVTWKVDGTTQQSGIENSKTPQSPEDNTYSLSSTLSLTSAQYNSHSVYTCEVVQGSASPIVQSFNRGDC (SEQ ID NO: 30)

[0310] Antibody rb8G4 was expressed in HEK cells (Expi293 suspension cells) by transient transfection and purified using MabSelect SuRe and citrate buffer. rb8G4 was then used for IHC of formaldehyde-fixed paraffin-embedded cell lines and human tumor tissues.

[0311] material and method Cell lines and tissues Human cancer cell lines were cultured from Merck cell banks, fixed in 4% buffered formaldehyde, and paraffin-embedded (FFPE). Paraffin-embedded cell lines were arranged on cell line microarrays (CMA) (Zytomed). FFPE tissue sections from human organ tissue microarrays (TMA) were obtained from amsbio (FDA standard tissue array, T8234701). FFPE human tumor samples were provided by BioIVT and Indivumed GmbH.

[0312] method For IHC staining with anti-CEACAM-5 antibody rb8G4, 4 μm sections from formaldehyde-fixed paraffin-embedded (FFPE) cancer cell line microarrays (CMA) and human tumor tissues were mounted on charged slides (SuperFrost Ultra Plus, Thermo Fisher Scientific or TOMO, Matsunami). The staining procedure was performed using the Discovery XT (Roche Diagnostics) staining platform. After deparaffinization, sections were heated for antigen retrieval in Tris-EDTA buffer pH 8 (CC1, Roche Diagnostics). Sections were incubated with the primary monoclonal antibody rb8G4 diluted to 0.5 or 0.7 μg / ml in phosphate-buffered saline (PBS) or antibody dilution buffer (DCS). Clone DA1E (rabbit monoclonal IgG, NEB) served as an isotype control antibody. Next to the primary antibody was the HQ anti-rabbit IgG detection kit (Roche Diagnostics). Slides were counterstained with hematoxylin, washed in tap water, dehydrated, and coverslip mounted with Entellan Neu (VWR) permanent mounting media. TMAs with CMA and human organ tissues were stained and scanned with a NanoZoomer (Hamamatsu) at a resolution of 0.46 μm / pixel. Human tumor sections were stained and scanned using an AxioScan.Z1 (Zeiss) instrument at a resolution of 0.44 μm / pixel. CMA scans were analyzed by the image analysis software HALO (Indica Labs, USA). For determination of the amount of antigen present, the brown staining positive area was calculated as a percent area of ​​the viable tissue area. Staining (arbitrary units) is calculated as follows: Antibody staining (AU) = % area of ​​positive tissue * average optical density of brown (OD ranges from 0 to 1)

[0313] Maximum antibody staining is 100 = 100% of the tissue area is black (greyscale OD value is 1).

[0314] CEACAM-5 mRNA data for cancer cell lines was obtained from the Cancer Cell Line Encyclopedia (CCLE; Broad Institute of MIT & Harvard).

[0315] result Validation in cancer cell lines and human normal tissues Antibody rb8G4 showed signal in the cytoplasm and plasma membrane in FFPE cancer cell lines (Figure 5).

[0316] The specificity of antibody rb8G4 for FFPE tissues / cells was shown by comparing the staining signal of 104 cancer cell lines with the mRNA expression of these cell lines (CCLE dataset). The resulting Pearson correlation coefficient of r=0.88 supports the conclusion that antibody rb8G4 (also referred to as SO8G4AB323) detects the CEACAM-5 epitope from FFPE tissues / cells (Figure 6). The cancer cell line microarray and individually selected positive and negative cell lines served as control matrices in staining runs with human normal and tumor tissues.

[0317] Staining with antibody rb8G4 in normal human tissues (Figure 7) was consistent with CEACAM-5 mRNA expression (Figure 8) (source: http: / / www.proteinatlas.org / ENSG00000105388-CEACAM5 / tissue), further supporting the specificity of the antibody for CEACAM-5.

[0318] Human tumor tissue Antibody rb8G4 stained positive in several human tumor indications, as shown in colon cancer (Figure 9), gastric cancer (Figure 10), esophageal cancer (Figure 11), and non-small cell lung cancer (Figure 12). The signal is localized to the cytoplasm and plasma membrane.

[0319] 1.9 Flow cytometry and Western blot using mAb1 and rb8G4 The binding of mAb1, rb8G4 and a commercial anti-CEACAM5 antibody to CEACAM5 positive and negative cell lines was compared.

[0320] Methods used: 5E5 to 1E6 cells were used for flow cytometry analysis using a BD FACSCanto II (BD Biosciences) in 5mL polystyrene tubes. Staining with 10μg / mL of primary antibody (mAb1, rb8G4, mouse monoclonal Agilent Dako #M7072 clone #IL7) and respective fluorescently labeled secondary antibodies (donkey anti-human IgG, Jackson-Dianova #709-116-149; donkey anti-mouse IgG, Jackson ImmunoResearch #715-116-150, donkey anti-rabbit IgG, Jackson-Dianova #711-116-152) was performed in 50μL of 1% PBS / BSA at 4℃ for 20 to 30min. Between and after the staining steps, cells were washed three times with 1% PBS / BSA and resuspended in 500 μL of 1% PBS / BSA (including 0.2 μg / mL DAPI for live cell gating) for flow cytometry analysis. Data evaluation was performed using FlowJo software (BD Biosciences).

[0321] Results: mAb1 and rb8G4 showed binding corresponding to the mRNA expression level data only in CEACAM5 positive cell lines (Table 1 below; MKN-45, NCI-H441). In contrast, with the commercial antibody, binding was weaker and limited to high CEACAM5 cell lines (Table 1 below; MKN-45). In conclusion, mAb1 and rb8G4 specifically detect CEACAM5 positive cancer cells and can be utilized as detection agents. [Table 1]

[0322] Table 1. Binding of different antibodies to CEACAM5-positive and CEACAM5-negative cell lines. The quotient of the median fluorescence intensity (MFI) of each antibody divided by the MFI of the secondary antibody only control is listed per cell line.

[0323] The binding of human mAb1 and rb8G4 to CEACAM5 positive and negative cell line lysates was also examined by Western blot.

[0324] Methods used: Western blots were performed according to standard protocols (Sambrook, J. & Russell, DW, 2001. Molecular Cloning: A Laboratory Manual, Volume 1, CSHL Press). For SDS-PAGE followed by wet blotting of membranes, 15 μg of total protein of RIPA cell lysates quantified by BCA kit (Thermo Scientific, #23227) was loaded per lane. Criterion XT 4-12% gels (Bio-Rad, #3450125) using MOPS running buffer (Bio-Rad, #1610788) were used in a Criterion electrophoresis cell (Bio-Rad, #1656001). Protein transfer was confirmed by Ponceau staining. Membranes were washed before and between staining with primary (mAb1 or rb8G4) and secondary antibodies (anti-human IgG, Jackson ImmunoResearch, #109-035-098 or anti-rabbit IgG, CellSignaling, #7074) at 0.5 μg / mL to 1 μg / mL. Stained membranes were visualized with ECL detection reagents using a Fusion FX imaging system (Vilber).

[0325] The results are shown in Figures 13A and 13B: both antibodies bound in a similar pattern corresponding to the expected migration rate of highly glycosylated CEACAM5. CEACAM5 detection by mAb1 (Figure 13A) and rb8G4 (Figure 13B) was specific to CEACAM5-positive cell lines and the intensity correlated with the mRNA expression level. A secondary band observed at lower intensity corresponds to a potential second isoform previously described (Hatakeyama et al.: Novel protein isoforms of carcinoembryonic antigen are secreted from pancreatic, gastric and colorectal cancer cells. BMC Research Notes 2013 6:381).

[0326] Example 2: Synthesis of Drug-Linker Compounds with Glucuronide-Based Linkers: Drug-Linker Compound 1 (DL1) and Drug-Linker Compound 1-M (DL1-M) [ka]

[0327] Synthetic Route to Compound 9 (also referred to herein as Drug-Linker Compound 1 (DL1)). [ka] Synthetic Route to Compound 11 (also referred to herein as Drug-Linker Compound 1-M (DL1-M) Protocol for the chemical preparation of compound 9 Step 1: Compound 1 [ka]

[0328] To a stirred solution of (2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-bromo-tetrahydro-pyran-2-carboxylic acid methyl ester (8.30 g; 20.90 mmol; 1.00 eq.) and 4-hydroxy-3-nitro-benzaldehyde (5.24 g; 31.35 mmol; 1.50 eq.) in acetonitrile (83.00 ml; 10.00 V) was added silver(I) oxide (9.69 g; 41.80 mmol; 2.00 eq.). The reaction mixture was stirred at RT for 16 h. The reaction mixture was filtered through Celite. The filtrate was concentrated under vacuum to give a solid. The solid was dissolved in EtOAc and washed with 10% aqueous NaHCO3 to remove excess 4-hydroxy-3-nitro-benzaldehyde. The organic layer was concentrated under vacuum to give compound 1 as a sand-colored solid. Yield: 9.0g Yield: 89.1%

[0329] Analysis data : NMR: 1H-NMR (400 MHz,DMSO-d6): 9.98 (s,1H),8.46 (s,1H),8.25-8.21 (m,1H),7.64 (d,J = 11.60Hz,1H),5.94 (d,J= 10.00 Hz,1H),5.51-5.44 (m,1H),5.20-5.09 (m,2H),4.80 (d,J = 13.20 Hz,1H),3.64 (s,3H),2.09 (s,9H). Step 2: Compound 2 [ka]

[0330] To a stirred solution of compound 1 (9.00 g; 18.62 mmol; 1.00 eq.) in propan-2-ol (33.00 ml; 3.67 V) and CHCl3 (167.00 ml; 18.56 V) was added silica gel 60-120 (3.60 g; 112.09 mmol; 6.02 eq.) followed by sodium borohydride (1.80 g; 46.55 mmol; 2.50 eq.). The reaction mixture was stirred at RT for 1 h. After completion, the reaction mixture was cooled and H 2 The mixture was quenched with O and filtered through Celite. The filtrate was extracted with dichloromethane and 2 Drying over SO4 The solvent was concentrated to give compound 2 as an off-white powder. Yield: 8.70g Yield: 92.4% Analysis data LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm, mobile phase A: 0.1% HCOOH, in H2O: ACN (95:5), B: ACN RT(min):2.05;M+H:503.2, Purity:96.6% Step 3: Compound 3 [ka]

[0331] To a stirred solution of compound 2 (8.70 g; 17.21 mmol; 1.00 eq.) in ethyl acetate (100.00 ml; 11.49 V) and THF (100.00 ml; 11.49 V) was added palladium on carbon (10% w / w) (2.50 g; 2.35 mmol; 0.14 eq.). The reaction mixture was stirred under hydrogen atmosphere at RT for 3 h. After completion, the reaction mixture was filtered through Celite. The solvent was concentrated under vacuum to give compound 3 as an off-white solid. Yield: 8.5g Yield: 100%

[0332] Analysis data : LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm, mobile phase A: 0.1% HCOOH, in H2O: ACN (95:5), B: ACN RT(min):1.73;M+H:456.10, Purity:95.1% Step 4: Compound 4 [ka]

[0333] To a stirred solution of compound 3 (10.00 g; 20.89 mmol; 1.00 eq.) and (9H-fluoren-9-ylmethoxycarbonylamino)-acetic acid (7.60 g; 25.06 mmol; 1.20 eq.) in DCM (250.00 ml; 25.00 V), 2-ethoxy-2H-quinoline-1-carboxylic acid ethyl ester (15.65 g; 62.66 mmol; 3.00 eq.) was added at 0° C. The reaction mixture was stirred at RT for 16 h. After completion, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (56% EtOAc:Petroleum Ether) to obtain the compound with 80% purity. The compound was further purified by washing with 30% EtOAc and Pet Ether to obtain compound 4 as a white solid. Yield: 8.5g Yield: 50.7%

[0334] Analysis data : LCMS: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 μm, Mobile phase A: 0.1% HCOOH, H 2 O: ACN (95:5), B: ACN RT(min):3.03;M+H:735.2, Purity:81.9% Step 5: Compound 5 [ka]

[0335] To a stirred solution of compound 4 (2.00 g; 2.49 mmol; 1.00 eq.) in THF (40.00 ml; 20.00 V) at 0° C. was added carbonic acid bis-(4-nitro-phenyl) ester (3.06 g; 9.97 mmol; 4.00 eq.) and DIPEA (4.40 ml; 24.92 mmol; 10.00 eq.). The reaction mixture was stirred at RT for 12 h. After completion of the reaction, the reaction mixture was concentrated under vacuum. The crude product was purified by column chromatography using silica gel (230-400) and Pet ether / ethyl acetate as eluent to give compound 5 as a pale yellow solid. Yield: 2.0g Yield: 84.6%

[0336] Analysis data : LCMS: Column: X-Bridge C8 (50 × 4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA in Milli-Q water; B: ACN RT(min):3.24;M+H:900.20, Purity:94.9% Step 6: Compound 6 [ka]

[0337] Compound 5 (1,369 g; 1,00 eq.) was dissolved in N,N-dimethylformamide (15,00 ml) and exatecan mesylate (679,7 mg; 1,00 eq.), 4-methylmorpholine for synthesis (0,422 ml; 3,00 eq.) and 1-hydroxybenzotriazole (172,8 mg; 1,00 eq.) were added. The reaction mixture was stirred overnight at room temperature. After the stirring time, the reaction suspension turned into a brown solution. The reaction was monitored by LC-MS, which indicated complete conversion of the starting material. The reaction mixture was purified by RP flash chromatography. The product-containing fractions were combined, concentrated under vacuum and lyophilized overnight to give compound 6 as a yellow solid. Yield: 1.59g Yield: 87.5%

[0338] Analysis data : LCMS: Column: Chromolis HR RP-18e (50-4, 6 mm); Mobile phase A: 0.05% HCOOH, H 2 in O; B: 0.04% HCOOH and 1% H 2 O in ACN; T: 40°C; Flow rate: 3.3 ml / min; MS: 100-2000, amu positive; 1% → 100%B: 0 → 2.0 min; 100%B: 2.0 → 2.5 min

[0339] RT(min):1.95;M+H:1196.40, Purity:84.4%. Step 7: Compound 7 [ka]

[0340] Compound 6 (1,586 g; 1,00 eq.) was dissolved in tetrahydrofuran (50,00 ml) and a solution of LiOH (0.1 M) (containing lithium hydroxide hydrate (281,77 mg; 6,00 eq.) in water (67,100 ml)) was added dropwise at 0° C. The pH value was checked during the addition. The pH should not exceed 10. The addition of the LiOH solution was complete after 1.5 h. The reaction was monitored by LC-MS, which showed complete conversion of the starting material. The reaction was quenched with citric acid solution and the pH was adjusted to 5. The reaction mixture was concentrated under reduced pressure. The crude was purified by preparative HPLC. The product-containing fractions were combined and lyophilized to give compound 7 as a dark yellow solid. Yield: 728mg Yield: 54.8%

[0341] Analysis data : LCMS: Column: Chromolis HR RP-18e (50-4, 6 mm); Mobile phase A: 0.05% HCOOH, H 2 in O; B: 0.04% HCOOH and 1% H 2 O in ACN; T: 40°C; Flow rate: 3.3 ml / min; MS: 100-2000, amu positive; 1% → 100%B: 0 → 2.0 min; 100%B: 2.0 → 2.5 min

[0342] RT(min):1.68;M+H:1056.30, Purity:98.5%. Step 8: Compound 8 [ka]

[0343] Compound 7 (728,000 mg; 1,00 eq.) was dissolved in N,N-dimethylformamide (20,00 ml). Piperidine (136,513 μl; 2,00 eq.) was added and the solution was stirred at RT for a total of 4 h. The reaction was monitored by LC-MS, which showed complete conversion of the starting material. The reaction mixture was concentrated under reduced pressure and the crude product was purified by RP flash chromatography. The product-containing fractions were combined, the solvent was partially removed and it was lyophilized overnight to give compound 8 as a yellow solid. Yield: 706mg Yield: 100%

[0344] Analysis data : LCMS: Column: Chromolis HR RP-18e (50-4, 6 mm); Mobile phase A: 0.05% HCOOH, H 2 in O; B: 0.04% HCOOH and 1% H 2 O in ACN; T: 40°C; Flow rate: 3.3 ml / min; MS: 100-2000, amu positive; 1% → 100%B: 0 → 2.0 min; 100%B: 2.0 → 2.5 min

[0345] RT(min):1.22;M+H:834.30, Purity:97.6%. Step 9: Compound 9 [ka]

[0346] To a solution of compound 8 (854 mg; 1,00 eq.) in dimethylformamide (30,00 ml) was added N-ethyldiisopropylamine (149,234 μl; 1,00 eq.) and 3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (233,61 mg; 1,00 eq.). The reaction mixture was stirred at RT for 3 h. The reaction was monitored by LC-MS, which showed complete conversion of the starting material. The reaction mixture was concentrated under reduced pressure and the crude product was purified by RP flash chromatography. The product-containing fractions were combined, concentrated and lyophilized to give the desired product in 91% purity. This material was again purified by RP chromatography to give compound 9 as a yellow solid. Yield: 580mg Yield: 60.1%

[0347] Analysis data : LCMS: Column: Chromolis HR RP-18e (50-4, 6 mm); Mobile phase A: 0.05% HCOOH, H 2 in O; B: 0.04% HCOOH and 1% H 2 O in ACN; T: 40°C; Flow rate: 3.3 ml / min; MS: 100-2000, amu positive; 1% → 100%B: 0 → 2.0 min; 100%B: 2.0 → 2.5 min RT(min): 1.38; M+H: 985.30, purity: 90% (the other 10% of the isomer can be removed by HPLC)

[0348] 1 H NMR (500 MHz, DMSO-d 6)δ 13.10 - 12.44 (m,1H),9.08 (s,1H),8.32 (t,J = 5.8 Hz,1H),8.16 (s,1H),8.02 (d,J = 8.8 Hz,1H),7.76 (d,J = 10.9 Hz,1H),7.31 (s,1H),7.15 - 7.09 (m,2H),6.98 (s,2H),5.48 - 5.38 (m,2H),5.32 - 5.22 (m,3H),5.11 - 5.01 (m,2H),4.87 (d,J = 7.6 Hz,1H),3.92 - 3.88 (m,1H),3.89 - 3.84 (m,2H),3.65 - 3.61 (m,2H),3.46 - 3.41 (m,1H),3.42 - 3.37 (m,1H),3.38 - 3.31 (m,1H),3.28 - 3.20 (m,1H),3.15 - 3.07 (m,1H),2.48 - 2.44 (m,2H),2.38 (s,3H),2.24 - 2.13 (m,2H),1.94 - 1.80 (m,2H),0.88 (t,J = 7.3 Hz,3H).

[0349] Chemical preparation protocol for compound 11 Step 1: Compound 10 [ka] To a solution of compound 8 (289.00 mg; 1.00 eq) in dimethylformamide (10.00 ml) was added N-ethyldiisopropylamino (0.104 ml, 2.00 eq) and 2,5-dioxopyrrolidin-1-yl(tert-butoxycarbonyl)glycylglycinate (105.70 mg; 1.00 eq). The reaction mixture was stirred at room temperature for 1 hour and the reaction was reflected by LC-MS. After completion, the solvent was removed under vacuum and the crude product was purified by preparative HPLC on a Sunfire column. The fractions containing the product were combined and lyophilized to give the trifluoroacetate salt of compound 10 as a yellow solid.

[0350] Yield: 162.00 mg, 0.14 mmol. Yield: 44.8%

[0351] Analysis data : LCMS: Column: Chromolis HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in HO; B: 0.04% HCOOH and 1% HO in ACN; T: 40 °C; Flow rate: 3,3 ml / min; MS: 100-2000, amu positive; 1% → 100% B: 0 → 2,0 min; 100% B: 2,0 → 2,5 min RT(min):1.41;M+H:1048.1, Purity:97.9%

[0352] Step 2: Compound 11 [ka] To a suspension of compound 10 (162.00 mg; 1.00 eq) in dichloromethane (5.00 ml) was added 4.0 M hydrogen chloride solution in 1,4-dioxane (682.43 μl, 20.00 eq), which was stirred at room temperature for 3.5 h and the reaction was monitored by LC-MS. After completion, the reaction mixture was concentrated under vacuum and the crude product was purified by preparative HPLC. The fractions containing the product were combined and lyophilized to give the trifluoroacetate salt of compound 11 as a yellow solid.

[0353] Yield: 113 mg; 0.11 mmol. Yield: 77.0%

[0354] Analysis data : LCMS: Column: Chromolis HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in HO; B: 0.04% HCOOH and 1% HO in ACN; T: 40 °C; Flow rate: 3,3 ml / min; MS: 100-2000, amu positive; 1% → 100% B: 0 → 2,0 min; 100% B: 2,0 → 2,5 min RT(min):1.22;M+H:948.0, M+2H:474.8;Purity:98.8%

[0355] 1 1H NMR (700 MHz, DMSO-d6) δ 12.93 - 12.79 (m, 1H), 9.14 (s, 1H), 8.61 (t, J = 5.8 Hz, 1H), 8.41 (t, J = 5.9 Hz, 1H), 8.14 (s, 1H), 8.05 (d, J = 8.8 Hz, 1H), 8.03 - 7.93 (m, 3H), 7.79 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 7.13 (s, 2H), 6.58 - 6.47 (m, 1H), 5.80 - 5.61 (m, 1H), 5.48 - 5.40 (m, 2H), 5.31 - 5.24 (m, 3H), 5.08 (d, J = 12.2 Hz, 1H), 5.03 (d, J = 12.3 Hz, 1H), 4.91 (d, J = 7.7 Hz, 1H), 3.96 (d, J = 5.8 Hz, 2H), 3.95 - 3.86 (m, 3H), 3.62 (q, J = 5.8 Hz, 2H), 3.43 (t, J = 9.3 Hz, 1H), 3.41 (t, J = 8.5 Hz, 1H), 3.35 (t, J = 9.0 Hz, 1H), 3.27 - 3.20 (m, 1H), 3.15 - 3.09 (m, 1H), 2.38 (s, 3H), 2.23 - 2.13 (m, 2H), 1.93 - 1.81 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0356] Example 3: Synthesis of a drug-linker compound with a linker cleavable by legumain: Drug-linker compound 2 (DL2)

Chem.

[0357] Preparative HPLC: Column: Sunfire Prep C18 OBD - 75.0g (250bar) Solvent A: Water, 0.1% TFA, Solvent C: Solvent B: acetonitrile, 0.1% TFA [ka]

[0358] Step 2 (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-4-amino-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1,4-dioxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (365 mg; 0,34 mmol; 1,00 eq.) was dissolved in N,N-(4,00 ml). Additional piperidine for synthesis (0.07 ml; 0.69 mmol; 2.00 eq.) was added and the reaction solution was stirred at rt for 1 h.

[0359] The reaction mixture was purified by preparative HPLC to yield 300 mg (0.314 mmol) of 4-((S)-4-amino-2-((S)-2-((S)-2-aminopropanamido)propanamido)-4-oxobutanamido)benzyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate. LC / MS:[M+H]:841.3

[0360] Preparative HPLC for purification: RediSep column: C18 130g SN:E0410A0D24BE1Lot:262118923W Flow rate: 75ml / min Conditions - Volume: 390.0ml Eluent: A1 water, 0.1% TFA Eluent: B1 acetonitrile, 0.1% TFA [ka]

[0361] Step 3 4-((S)-4-amino-2-((S)-2-((S)-2-aminopropanamido)propanamido)-4-oxobutanamido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]phenyl)-2-((S)-4-amino ...4-aminopropanamido)propanamido)-4-oxobutanamido) To a solution of ]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (571 mg; 0.60 mmol; 1.00 eq.) was added N-ethyldiisopropylamine (203 μl; 1.20 mmol; 2.00 eq.) and N-succinimidyl 3-maleimidopropionate (162 mg; 0.60 mmol; 1.00 eq.). The reaction mixture was then stirred for 10 min and monitored by LC / MS.

[0362] The reaction mixture was purified by preparative HPLC to yield 378 mg (0.35 mmol) of DL2. LC / MS:[M+H]:992.4 Preparative HPLC for purification: RediSep column: C18 86g SN:E0410A8B46130Lot:281729189W Flow rate: 60ml / min Conditions - Volume: 264,0ml Eluent 1: A1 Water, 0.1% TFA Eluent 2: B1 acetonitrile, 0.1% TFA

[0363] Sequence Analytics: Method information: A: H2O+0.05%HCOOH | B: MeCN+0.04%HCOOH+1%H2O T: 40℃ | Flow rate: 3.3ml / min | MS: 100-2000amu positive Column: Chromolys HR RP-18e 50-4,6mm 0%→100%B:0→2.0min|100%B:2,0→2,5min

[0364] Example 4: Preparation of Immunoconjugates Glucuronide-based conjugates of mAb1 (referred to as ADC1) and mAb1-M (the resulting immunoconjugate is referred to as ADC1-M), mAb4-M (the resulting immunoconjugate is referred to as ADC4-M), and mAb6-M (the resulting immunoconjugate is referred to as ADC6-M). The following method is described in the context of mAb1 (the resulting immunoconjugate is referred to as ADC1), however the same method may also be used for mAb1-M (the resulting immunoconjugate is referred to as ADC1-M), mAb4-M (the resulting immunoconjugate is referred to as ADC4-M), and mAb6-M (the resulting immunoconjugate is referred to as ADC6-M).

[0365] 4.1 Conjugation Process Antibody preparation Antibody mAb1 (as defined herein above) was thawed at 2-8°C up to 3 days prior to conjugation and stored at 2-8°C until use. mAb (>10 g) was equilibrated at room temperature on the day of conjugation prior to use. mAb (9.6 mg / mL) was aliquoted (10.0 g, 1041.7 mL) and diluted to 5.59 mg / mL using conjugation buffer (200 mM histidine, pH 6.5). The mAb solution was added to a 3 L Chemglass jacketed reactor and set to 25±2°C with stirring at 50 rpm.

[0366] Antibody reduction 7.0 mol eq. (9.7 mL) of 50 mM TCEP solution (50 mM TCEP in conjugation buffer) was added to the mAb solution vial and the reaction was allowed to proceed at 25±2° C. for 3 hours.

[0367] Conjugation Drug-linker compound 1 (DL1) of formula (X) was weighed out and dissolved in DMSO to prepare a 20 mM solution. 90% (148.6 mL) of the required DMSO was added to the reactor. Immediately after the DMSO addition, 10.0 molar equivalents (38.2 mL) of the 20 mM drug-linker solution was added to the reactor. Then, 10% (18.2 mL) of the remaining required DMSO was used to rinse the drug-linker vial to ensure complete transfer. After the final addition, the reaction was allowed to proceed for 1 hour at 25±2° C. The total volume during conjugation was 1997.0 mL.

[0368] NOTE: The overall DMSO concentration in the reaction was 10% (v / v) (DMSO + drug linker solution).

[0369] Quench 35 mol eq. (48.5 mL) of 50 mM NAC was added to the reactor and the reaction was allowed to proceed at 25±2° C. for 30 min.

[0370] filtration The filtered crude conjugate solution was transferred from the reaction vessel and then filtered using a Millipak Gamma Gold 60 (MPGL06GH2) to give 1993.6 mL (filter loading: 324.7 g / m2 [protein], 66.5 L / m2 [solution]) of filtered crude conjugate.

[0371] Diafiltration The filtered crude conjugate solution was buffer exchanged (DV=1993.6 mL) through a Pellicon 3 (30 kDa) Biomax membrane (1×0.11 m2, 300 LMH (550 mL / min), 16 psi TMP, actual loading 88.5 g / m2). The crude conjugate was buffer exchanged for 16 diavolumes using diafiltration buffer (10 mM histidine, pH 5.5). After buffer exchange, the solution was concentrated to ≧25 mg / mL, transferred to a bottle, and the membrane was flushed with diafiltration buffer. The total volume recovered from the UF / DF was 361.5 mL.

[0372] Formulation The concentrated ADC (i.e., ADC1) was diluted to 20.0 mg / mL with 112.1 mL of diafiltration buffer (10 mM histidine, pH 5.5). The resulting solution was diluted to 15.0 mg / mL with 157.6 mL of 4× formulation buffer (10 mM histidine, 12% (w / v) trehalose dihydrate, 400 mM NaCl, pH 5.5) to give a final target bulk drug substance (BDS) concentration of 15.0 mg / mL.

[0373] filtration The final formulated ADC was filtered using a 0.2 μm Millipak Gamma Gold 40 (MPGL04GH2) filter to yield 619.6 mL (filter load: 464.6 g / m2 [protein], 31.0 L / m2 [solution]) of ADC1 BDS. The material was packaged in HDPE bottles and stored at ≦−65° C.

[0374] 4.2 Methods: Characterization of Drug Substance: ADC1 Size Exclusion Chromatography (SEC) SEC Method Parameters Wavelength 280nm Column: Tosoh TSKgel 7.8mm x 300mm, 5μm (P / N0008541) Mobile phase 0.14M potassium phosphate monobasic 50mM Sodium Phosphate Monobasic 0.06M Dibasic Potassium Phosphate 0.25M Potassium Chloride 5% IPA Injection volume 20μL Temperature 25℃ Flow rate 0.5mL / min Run time 30min Typical SEC chromatograms showing the purity of stock mAb, conjugate after UF, and final BDS: FIG. 14.

[0375] For the BDS material presented above, a HMWS of 1.7% and a monomer purity of 96.9% were reported. Reverse Phase HPLC (RP-HPLC) Method RP-HPLC method parameters Wavelength 280nm Column: PLRP-S 1000Å (50×2.1mm, 8μm) column, Agilent (P / N PL1912-1802) Mobile phase A: 0.1% formic acid, 0.01% TFA in water Mobile phase B 0.1% formic acid, 0.01% TFA in ACN gradient [Table D] Injection volume 10μL Column temperature: 80℃ Flow rate 1.0mL / min Run time 30min

[0376] Sample Preparation Dilute sample to 2 mg / mL and add 40 μL to a microcentrifuge tube. Add 60 μL of approximately 8 M Guanidine HCl, approximately 130 mM Tris, approximately 1 mM EDTA, pH 7.6 buffer. Add 2 μL of 500 mM DTT and vortex to mix. Incubate sample at 37 ± 2°C for 30 ± 2 min.

[0377] Typical RP-HPLC chromatogram showing separation of light and heavy chains: Figure 15. The chromatogram shows an overlay of the stock mAb, crude ADC, and final BDS.

[0378] For the ADC1 BDS material above, a DAR of 7.9 was reported. Free drug method. Free drug method parameters Wavelength 254nm Column: Phenomenex Gemini, C18, 2 x 150 mm, 3 μm (P / N00F-4439-B0) Mobile phase A: 0.1% formic acid in water Mobile phase B 0.1% formic acid in acetonitrile gradient [Table E] Injection volume 10.00μL Column temperature: 50℃ Flow rate 0.75mL / min Sample preparation Protein drop: 100 μL drug substance + 250 μL cold MeOH + 50 μL 3M MgCl 2 Spin at 20,000 rpm for 10 minutes.

[0379] Standard preparation: Mix 20 μL 20 mM DL1 (drug-linker compound 1 in DMSO) + 20 μL DMSO + 40 μL MeOH + 20 μL 200 mM NAC in diafiltration buffer. Incubate overnight to give 4 mM DL-NAC. Dilute 4 mM DL-NAC with MeOH to give 4 μM DL-NAC standard.

[0380] Typical chromatograms showing free drug levels of NAC standard and final BDS: FIG.

[0381] Residual free drug levels below 2.4% (molar ratio) were reported for the ADC1 BDS material shown above.

[0382] Example 5: Preparation of immunoconjugate: peptide-based conjugate of mAb1 (referred to as ADC2) 5.1 Conjugation Process Antibody preparation Antibody mAb1 (as defined herein above) was thawed at 2-8°C up to 3 days prior to conjugation and stored at 2-8°C until use. mAb (>9.5g) was equilibrated at room temperature on the day of conjugation prior to use. mAb (9.6mg / mL) was aliquoted (9.5g, 989.6mL) and diluted to 5.59mg / mL using conjugation buffer (200mM histidine, pH 6.5). The mAb solution was added to a 3L Chemglass jacketed reactor and set to 25±2°C with stirring at 50 rpm.

[0383] Antibody reduction 8.0 mol eq. (10.5 mL) of 50 mM TCEP solution (50 mM TCEP in conjugation buffer) was added to the mAb solution vial and the reaction was allowed to proceed at 25±2° C. for 3 hours.

[0384] Diafiltration The reduced mAb solution was buffer exchanged to 6 DV (DV = 1706.9 mL). A Pellicon 3 (30 kDa) Biomax membrane was used (1 × 0.11 m2, 300 LMH (550 mL / min), TMP at 16 psi, actual loading 86.3 g / m2. The reduced antibody was buffer exchanged using conjugation buffer (200 mM histidine, pH 6.5). After buffer exchange, the reduced mAb solution was collected back into the reactor and the membrane was flushed with conjugation buffer. Conjugation Drug-linker compound 2 (DL2) was weighed out and dissolved in DMSO to prepare a 20 mM drug-linker solution. 90% (142.6 mL) of the required DMSO was added to the reactor. Immediately following the DMSO addition, 9.5 molar equivalents (31.2 mL) of the 20 mM drug-linker solution was added to the reactor. Then, 10% (15.8 mL) of the remaining required DMSO was used to rinse the drug-linker vial to ensure complete transfer. After the final addition, the reaction was allowed to proceed for 2 hours at 25±2°C. The total volume during the conjugation reaction was 1894.5 mL.

[0385] Quench 35 mol eq. (46 mL) of 50 mM NAC was added to the reactor and the reaction was allowed to proceed at 25±2° C. for 45 min.

[0386] filtration The crude conjugate solution was transferred from the reaction vessel and filtered using a Millipak Gamma Gold 60 (MPGL06GH2) to yield 1897.3 mL (filter load: 308.9 g / m2 [protein], 63.2 L / m2 [solution]) of filtered crude conjugate.

[0387] Diafiltration The filtered crude conjugate solution was buffer exchanged (DV=1897.3 mL) through a Pellicon 3 (30 kDa) Biomax membrane (1×0.11 m2, 300 LMH (550 mL / min), 16 psi TMP, actual loading 84.2 g / m2). The initial 12 DV were performed using conjugation buffer (200 mM histidine, pH 6.5), then switched to standard diafiltration buffer (10 mM histidine, pH 5.5) for an additional 8 DV. After the buffer completed the buffer exchange, the solution was concentrated to ≥25 mg / mL, transferred to a bottle, and the membrane was flushed with diafiltration buffer. The total pool volume recovered from the UF / DF was 335.7 mL.

[0388] Formulation The concentrated ADC (i.e., ADC2) was diluted to 20.0 mg / mL with 84.7 mL of diafiltration buffer (10 mM histidine, pH 5.5). The resulting solution was diluted with 138.6 mL of 4× formulation buffer (10 mM histidine, 12% (w / v) trehalose dihydrate, 400 mM NaCl, pH 5.5) to a final target BDS concentration of 15.0 mg / mL.

[0389] filtration The final formulated ADC was sterile filtered using a Millipak Gamma Gold 60 (MPGL06GH2) to yield 549.3 mL (filter load: 411.4 g / m2 [protein], 27.5 L / m2 [solution]) of ADC2 BDS. The material was packaged in HDPE bottles and stored at ≦−65° C. 5.2 Methods: Characterization of Drug Substance: ADC2 Size Exclusion Chromatography (SEC) SEC Method Parameters Wavelength 280nm Column: Tosoh TSKgel 7.8mm x 300mm, 5μm (P / N0008541) Mobile phase 50mM monobasic sodium phosphate 0.4M Sodium Perchlorate pH 6.3 Injection volume 1μL Column temperature: 25℃ Flow rate 0.5mL / min Run time 30min

[0390] Typical SEC chromatogram showing the purity of the stock mAb and the final BDS: FIG.

[0391] For the ADC2 BDS material presented above, a HMWS of 4.2% and a monomer purity of 95.8% were reported. Reverse Phase HPLC (RP-HPLC) Method RP-HPLC method parameters Wavelength 280nm Column: PLRP-S 1000Å (50×2.1mm, 8μm) column, Agilent (P / N PL1912-1802) Mobile phase A: 0.1% formic acid, 0.01% TFA in water Mobile phase B 0.1% formic acid, 0.01% TFA in ACN gradient [Table F] Injection volume 10μL Column temperature: 80℃ Flow rate 1.0mL / min Run time 30min

[0392] Sample Preparation Dilute sample to 2 mg / mL and add 40 μL to a microcentrifuge tube. Add 60 μL of approximately 8 M Guanidine HCl, approximately 130 mM Tris, approximately 1 mM EDTA, pH 7.6 buffer. Add 2 μL of 500 mM DTT and vortex to mix. Incubate sample at 37 ± 2°C for 30 ± 2 min.

[0393] Typical RP-HPLC chromatogram showing separation of light and heavy chains: Figure 18. The chromatogram shows an overlay of the stock mAb and the final BDS.

[0394] For the ADC2 BDS material above, a DAR of 7.6 was reported. Free Drug Method Free drug method parameters Wavelength 254nm Column: Phenomenex Gemini, C18, 2 x 150 mm, 3 μm (P / N00F-4439-B0) Mobile phase A: 0.1% formic acid in water Mobile phase B 0.1% formic acid in acetonitrile gradient [Table G] Injection volume 10.00μL Column temperature: 50℃ Flow rate 0.75mL / min Sample preparation Protein drop: 100 μL drug substance + 250 μL cold MeOH + 50 μL 3M MgCl 2 Spin at 20,000 rpm for 10 minutes.

[0395] Standard preparation: Mix 20 μL 20 mM DL2 (drug-linker compound 2 in DMSO) + 20 μL DMSO + 40 μL MeOH + 20 μL 200 mM NAC in diafiltration buffer. Incubate overnight to give 4 mM DL-NAC. Dilute 4 mM DL-NAC with MeOH to give 4 μM DL-NAC standard.

[0396] Typical chromatograms showing free drug levels of NAC standard and final BDS: FIG.

[0397] For the ADC2 BDS material shown above, residual free drug levels below 1.9% (molar ratio) were reported.

[0398] Example 6: Analogs of ADC SAR408701 6.1 Antibodies For the purposes of further comparative experiments, an analog of Sanofi's anti-CEACAM5 ADC SAR408701 was prepared based on a monoclonal antibody with the following sequence: Heavy chain: SEQ ID NO: 25 Light chain: SEQ ID NO: 26

[0399] 6.2 Drug-Linker Compounds As drug-linker molecule conjugated to the above mentioned antibody, SPDB-DM4 (obtained from Levena Biopharma) was used: Product name: SPDB-DM4 structure: [ka] Expected mass: 994.35 Average observed mass: 995.5 (Ms+H + ) Mass Spectral Analysis: Consistent and showed correct MW HPLC analysis: purity >95% Appearance: White powder

[0400] 6.3 Conjugation Antibodies were thawed at 2-8 °C up to 3 days prior to conjugation and stored at 2-8 °C until use. Antibodies (175 mg) were equilibrated at room temperature on the day of conjugation prior to use. Antibodies (7.9 mg / mL) were diluted to 5 mg / mL using conjugation buffer (PBS, pH 7.4) and a 5 mM DMSO solution of SPDB-DM4 (Levena Biopharma) (8 molar equivalents relative to antibody). The reaction solutions were mixed and incubated at 25 °C for 4 h.

[0401] 6.4 Preparative Size Exclusion Chromatography, Desalting and Filtration The reaction mixture was purified using preparative size-exclusion chromatography. A Superdex 200 pg (50 / 60) column was connected to an Akta Avant 25 system (GE Healthcare) and equilibrated with PBS, pH 7.4 according to the manufacturer's instructions. The reaction mixture was then injected and run through the column at a flow rate of 10 ml / min with PBS, pH 7.4 as running buffer. ADC-containing fractions were determined by UV light absorption at 280 nm, pooled, and concentrated. The ADC material was concentrated using a 15 ml Amicon Ultra 50 kDa cutoff centrifugal device (Merck Millipore) according to the manufacturer's instructions. The concentrated ADC material was transferred to formulation buffer (10 mM histidine, 130 mM glycine, 5% sucrose, pH 5.5) using a HiPrep 26 / 10 desalting column (GE Healthcare) at a flow rate of 10 ml / min by an Akta Avant 25 system (GE Healthcare) according to the manufacturer's instructions. The resulting ADC material was filtered using a 0.2 μm filter (Merck Millipore), aliquoted, and then flash frozen in liquid nitrogen. The final concentration of the ADC material was 5.82 mg / ml, and the material was kept at -80°C until further use. The ADC resulting from this work is also referred to herein as "ADC SAR DM4" or simply "ADC SAR". This ADC is an analog of SAR408701.

[0402] Example 7: ADC based on mAb1 and SPDB-DM4 Another ADC was prepared based on the antibody mAb1 (as described herein above) and the drug-linker compound SPDB-DM4, i.e., the same drug-linker compound as the ADC SAR DM4 described above. The ADC resulting from this work is referred to herein as "ADC mAb1 DM4" and was prepared as follows: 7.1 Materials used: ·Antibody: mAb1, 1mg / mL, in 10mM HEPES, pH5.8 Conjugation buffer: 10mM HEPES, pH 5.8 Drug-linker compound: SPDB-DM4, 2 mg / mL in DMF

[0403] 7.2 Method: Conjugation: Approximately 30-fold molar excess of drug-linker molecule was used for conjugation (75 mL antibody + 7.1 mL SPDB-DM4 drug-linker) and incubated for 5 h at room temperature with slow rocking. Purification: The ADC was buffer exchanged into 20 mM histidine, 150 mM NaCl, pH 6.0 to remove free drug. Formulation buffer: 20mM histidine, 150mM NaCl, pH 6.0

[0404] 7.3 Purified ADC analytical details: Final yield: 40mg ·Concentration: 2.2mg / mL DAR:4.4

[0405] Example 8: Characterization of drug release from ADC1 and ADC2 8.1 Materials and Methods 8.1.1 Test specimen [Table H]

[0406] 8.1.2 Materials All reagents and buffers were stored according to the manufacturer's instructions and used prior to the batch expiration date. [Table I]

[0407] 8.1.3 Equipment [Table J]

[0408] 8.1.4 Procedure 8.1.4.1 Serum sample preparation 2M HEPES solution: 52.1 g HEPES was dissolved in 75 mL Milli-Q water and 15 mL 25% HCl, adjusted to pH 7.55 and added to 100 mL. This solution was mixed with serum at 15% v / v to obtain stabilized serum with pH 7.3-7.4. Human serum from Biowest (lot no. S15594S4200) was thawed. 100 mL serum was mixed with 15 mL 2M HEPES buffer. Mouse serum from Biowest (lot no. S18169S2160) was thawed. 100 mL serum was mixed with 15 mL 2M HEPES buffer. Cynomolgus serum was thawed and 8.5 mL serum was mixed with 1.5 mL 2M HEPES buffer. pH was measured (7.37) and serum was sterile filtered. 2 mL aliquots were frozen at -20 °C.

[0409] Prepared serum was thawed at RT. The desired ADC protein concentration was prepared in triplicate at 180 μg / mL for subsequent free payload analytics by LC-MS. After ADC was added to serum, individual batches were mixed and divided into 20 μL aliquots. In addition, one 96 h sample of 20 μL for each ADC was pipetted and used for workup analysis to measure overall recovery. 0 h samples were frozen directly at -80°C, remaining samples were incubated at 37°C and 5% CO2, and reactions were stopped by storage at -80°C for 2 / 4 / 6 / 24 / 48 / 72, 96 h incubations.

[0410] 8.1.4.2 Human liver lysosome sample preparation The pH was adjusted to either pH 5.0 or pH 4.0 using assay buffer. Lysosome stability preparation: 80 μL human liver lysosomes were prepared for triplicate measurements (n=3) as shown exemplarily for ADC1: 2.76 μL ADC1+6 μL human liver lysosomes+71.2 μL legumain assay buffer. Preparation of MeOH+PIC (1:200): 10 μL PIC III+1990 μL MeOH. The reaction was started by transferring the Eppendorf tube to a Thermomix preheated to 37° C. Subsequently, 10 μL aliquots were withdrawn after 0, 1, 2, 4, 24 and 48 hours and mixed with 40 μL PIC III (1:200).

[0411] 8.2 Results Stability of the ADCs in human, mouse and cynomolgus serum (Figure 20). Conjugated exatecan concentrations were calculated using free exatecan (initial dose approximately 10 μM) (normalized data). Similar profiles were obtained in human, cynomolgus, and mouse serum. Only minor warhead release was observed, most notable in mouse serum for ADC2 (5.9% free of initial conjugated payload at 96 h) and ADC1 (1.4%).

[0412] ADC3 control stability for mouse serum and buffer (Figure 21). Conjugated SN38 concentrations were calculated using free SN38 (ADC protein concentration at initial dose of 50 μg / mL) (not normalized). Significant SN-38 release was observed for both matrices.

[0413] Payload release profiles of ADC1 and ADC2 in human liver lysosomes (pH 5.0) (Figure 22). Conjugated drug concentrations were calculated using, for example, free exatecan (initial concentration approximately 10 μM exatecan). Normalized data. For payload release mediated by ADC1- and ADC2 cleavage, intermediate levels of payload release were observed (both approximately 40% of initial total conjugated payload).

[0414] ADC catabolite profiling confirms free exatecan as a lysosomal release product (FIG. 23). To confirm exatecan as the major release product, an ADC1 catabolite profiling study was performed in human lysosomal extracts, in which comparison of TIC-MS and extracted ion chromatograms at various time points showed that the expected exatecan catabolite was subsequently released from ADC1 during incubation (0 h, 4 h, 24 h). Retention time 9.33 min, detected mass m / z 436.1671 ([M+H] + , C 24 H 23 O 4 N 4 F) and the MS / MS spectrum of the detected catabolites is consistent with that of exatecan.

[0415] Example 9: ADC1 and ADC2 specifically kill cancer cells in vitro with high potency Human cancer cell lines were used to evaluate the potential of ADC1 and ADC2 to kill cancer cells. ADC1 and ADC2 showed sub-nanomolar in vitro potency against different CEACAM5-positive cell lines and minor effects against CEACAM5-negative cell lines (Table 2 below). As shown in the exemplary dose-response curves (Figure 24A / B), ADC1 and ADC2 were highly potent against the CEACAM5-positive cell lines SK-CO-1 and SNU-16. In contrast, the effects of ADC1 and ADC2 against antigen-negative MDA-MB-231 were limited to the highest concentrations tested (Figure 24C).

[0416] Rituximab isotype control ADCs utilizing similar linkers and payloads as ADC1 and ADC2 showed much less efficacy against the SK-CO-1 cell line (Figure 25).

[0417] In conclusion, ADC1 and ADC2 specifically kill CEACAM5-expressing human cancer cell lines in vitro with high potency. [Table 2]

[0418] Table 2. Potency of ADC1, ADC2 and free payload against multiple human cell lines. Maximum effect compared to untreated control at highest tested compound concentration is indicated in brackets. For each cell line, CEACAM5 expression is indicated.

[0419] Methods – Viability Assay: The cytotoxic effect of ADCs on cancer cell lines was measured by cell viability assay. The day before treatment, cells were seeded in 96-well plates in a volume of 90 μL. Test compounds (ADCs or free payload) were formulated in cell culture medium at 10x the starting concentration. Test compounds were serially diluted (1:4) and 10 μL of each dilution was added to the cells in triplicate. Plates were cultured at 37° C. in a CO2 incubator for 6 days. For cell viability measurements, Cell Titer-Glo® reagent (Promega™ Corp, Madison, WI) was added to each well and plates were processed according to the manufacturer's instructions. Luminescence signals were measured using a Varioskan plate reader (Thermo Fisher). Luminescence readings were converted to % viability relative to untreated cells. Data were fitted by nonlinear regression analysis using GraphPad Prism with log(inhibitor) vs. response, variable slope, 4-parameter fit equation. Data are presented as % relative cell viability versus molar compound concentration, and error bars indicate standard deviation (SD) of triplicates. Geometric mean IC50 values ​​from multiple experiments were calculated.

[0420] Using the same method as above, ADC1 and ADC2 were also compared with ADC SAR DM4 in terms of their cytotoxic effects against antigen-positive SK-CO-1 and antigen-negative MDA-MB-231 cell lines. ADC1 and ADC2 showed 2.9- and 2.7-fold higher potency than ADC SAR DM4 against SK-CO-1 cancer cells, respectively (Figure 26A). Non-specific effects against antigen-negative MDA-MB-231 were slightly higher for ADC SAR DM4 compared with ADC1 and ADC2 (Figure 26B). ADC SAR DM4 and ADC mAb1 DM4 showed similar potency against SK-CO-1, with a trend toward slightly higher potency for ADC mAb1 DM4 (Figure 26A).

[0421] Example 10: ADC1 and ADC2 mediate a strong bystander effect on antigen-negative cells in co-culture with antigen-positive cells The potential of ADC1 and ADC2 to mediate bystander effects on antigen-negative cells in close proximity to antigen-positive cells was evaluated in a bystander assay. ADC1 and ADC2 showed a strong bystander effect on CEACAM5-negative MDA-MB-231 cells in the presence of CEACAM5-positive SK-CO-1 (Figure 27A). Coculture experiments were performed at an ADC concentration of 1 nM, which caused maximal inhibition of CEACAM5-positive SK-CO-1 cell viability for ADC1 (Figure 26A), but no effect on CEACAM5-negative MDA-MB-231 cells (Figure 26B). Consistent with these findings, no nonspecific effects of ADC1 or ADC2 were observed in the MDA-MB-231-only control in the bystander assay setup (Figure 27B).

[0422] In conclusion, ADC1 and ADC2 mediate strong bystander effects against antigen-negative cancer cells in coculture with antigen-positive cells. These findings support their potential to effectively target tumors with heterogeneous target expression.

[0423] Given the utilization of the same drug-linker and Fv region, it is expected that ADC1-M, ADC2-M, ADC3-M, as well as ADC6-M and ADC7-M, will mediate similar strong bystander effects as ADC1 and ADC2.

[0424] Compared to ADC SAR, ADC1 and ADC2 mediated a much stronger bystander effect on antigen-negative cells in co-culture with antigen-positive cells (Figure 28A, Figure 28B). ADC mAb1 DM4, which utilizes the same antibody (i.e., mAb1) as ADC1 and ADC2 with the drug-linker molecule (i.e., SPDB-DM4) utilized in ADC SAR DM4, also showed a more pronounced bystander effect than ADC SAR DM4 (Figure 28A, Figure 28B). This indicated that mAb1 contributes to the high bystander effect observed in ADC1 and ADC2 compared to ADC SAR utilizing a different antibody. Therefore, it is expected that ADC1-M, ADC2-M, ADC3-M, ADC6-M, and ADC7-M also mediated a stronger bystander effect than ADC SAR.

[0425] For all ADCs tested, the extent of the bystander effect increased by increasing the number of antigen-positive cells added to a fixed number of antigen-negative cells (compare Figure 28A and Figure 28B). Without wishing to be bound by theory, this may be the result of more ADC being processed by a greater number of antigen-positive cells, releasing more free payload, which is responsible for the bystander effect on the antigen-negative cells.

[0426] In MDA-MB-231 cells alone, no non-specific effects of the tested ADCs were observed (Figure 28C).

[0427] Methods – Bystander Assay The cytotoxic effect of ADCs against antigen-negative cancer cell lines in co-culture with antigen-positive cancer cell lines was measured by bystander assay. 1,000 CEACAM5-negative MDA-MB-231 cells were seeded with 750 or 3000 CEACAM5-positive SK-CO-1 cells per well in co-culture experiments. As a control, 1000 MDA-MB-231 cells alone were seeded in parallel. The day before treatment, cells were seeded in a total volume of 90 μL in 96-well plates. Test compounds were formulated at 10x the final concentration of 1E-9M in cell culture medium and 10 μL was added to the cells in duplicate. Plates were cultured at 37°C in a CO2 incubator for 6 days.

[0428] Prior to immunofluorescence staining, the medium was removed and cells were treated with 100% methanol (-20°C) for 30 min. After methanol removal and one PBS washing step, cells were treated with 2.5% paraformaldehyde (PFA) in 0.2% Triton X-100 in PBS for 15 min at room temperature. After solution removal and one PBS washing step, cells were treated with 1% BSA / 0.1% Tween / 0.1% sodium azide in PBS for at least 1 h at room temperature. Antigen-positive and antigen-negative cells were identified by immunofluorescence staining with 10 μg / mL human anti-CEACAM5 (mAb1) primary antibody and 1:2000 dilution of donkey anti-human IgG fluorescent (phycoerythrin)-labeled secondary antibody (Jackson ImmunoResearch, #709-116-149). Cells were identified by nuclear staining using 1 μg / mL Hoechst33342 (Life technologies, cat#H3570) dye. Staining was performed in 1% BSA / 0.1% sodium azide in PBS for 30 min at room temperature. Secondary antibody staining was combined with Hoechst dye staining. Between and after the staining steps, cells were washed three times with PBS.

[0429] Plates were imaged with a confocal quantitative imaging cytometer CQ1 (Yokogawa® Electric Corporation, Tokyo, Japan). Analysis was adapted from the CQ1 software (Yokogawa) template "Nucleus and pseudo-Cell body" and FCS export files were analyzed using FlowJo (BD). Antigen-positive and antigen-negative cells were differentiated and quantified based on the absence or staining with fluorescently labeled antibodies around the nucleus. Bar graphs show the number of identified antigen-positive and antigen-negative cells per treatment condition.

[0430] Example 11: Efficacy of ADC1 and ADC2 in patient-derived xenograft (PDX) mouse models of colorectal cancer (CRC) In vivo antitumor efficacy was evaluated in a human patient-derived CRC xenograft model, COPF217 (Shanghai LideBiotech CO.,LTD). COPF217 tumor fragments were implanted subcutaneously into the right flank of 6- to 8-week-old immunodeficient female mice (NU-Foxn1nu, Charles River). Tumors reached a mean volume of 165 mm 3 Upon reaching 18 days, 6 mice / group were treated intravenously once with vehicle (saline solution) or ADC1 or ADC2 (each at a dose of 10 mg / kg; day 0). Tumor length (L) and width (W) were measured with calipers and tumor volume was calculated using the formula L x (W^2) / 2.

[0431] A single treatment with ADC1 or ADC2 at a dose of 10 mg / kg led to a significant antitumor effect. The two substances show similar effects leading to tumor stagnation (FIG. 29). Treatment with ADC1 or ADC2 had no significant impact on body weight (data not shown).

[0432] Further experiments with other CRC PDX models: In additional experiments corresponding to those described above for COPF217, a single treatment with ADC1 also resulted in tumor stasis or regression in 12 other CRC PDX models with high CEACAM5 expression.

[0433] Example 12: Efficacy of ADC1 in non-small cell lung cancer (NSCLC) PDX mouse models In vivo antitumor efficacy was evaluated in a human patient-derived NSCLC xenograft model LUPF160151 (Shanghai LideBiotech CO.,LTD). LUPF160151 tumor fragments were subcutaneously implanted into the right flank of 6- to 8-week-old immunodeficient female mice (NU-Foxn1nu, Charles River). When tumors reached 180 mm 3 When the tumor reached an average volume of 1000 mg / kg, 5 mice / group were treated once intravenously with vehicle (saline solution) or ADC1 (6 mg / kg; day 0). The length (L) and width (W) of the tumor were measured with calipers, and tumor volume was calculated using the formula L x (W^2) / 2.

[0434] A single treatment with ADC1 at a dose of 6 mg / kg led to a significant antitumor effect (Figure 30) without impact on body weight (data not shown). The model LUPF160151 showed heterogeneous CEACAM5 expression, with CEACAM5-negative tumor cells adjacent to CEACAM5-positive tumor cells. Thus, the good efficacy of ADC1 in this model indicates a strong bystander effect of ADC.

[0435] Example 13: Efficacy of ADC1 in gastric cancer PDX mouse models In vivo antitumor efficacy was evaluated in a human patient-derived gastric cancer xenograft model, GAX066 (Shanghai ChemPartner Co., Ltd). Tumor fragments were subcutaneously implanted into the right flank of immunodeficient female mice (Nu / Nu mice, Beijing Vital River Lab Animal Technology Co. Ltd, 18-22 g). Tumors reached an average volume of 220 mm 3Upon reaching 18 days, 6 mice / group were treated once intravenously with vehicle (saline solution) or ADC1 (3 or 10 mg / kg; day 0). Tumor length (L) and width (W) were measured with calipers and tumor volume was calculated using the formula L x (W^2) / 2.

[0436] A single treatment with 3 or 10 mg / kg ADC1 caused significant antitumor effects (Figure 31) without impact on body weight (data not shown). In five of six tumors, treatment with 10 mg / kg resulted in complete tumor regression.

[0437] Example 14: Efficacy of ADC1 compared to ADC3 in pancreatic cell line derived tumor models The efficacy of ADC1 compared to ADC3 was evaluated in the human pancreatic cell line-derived xenograft model HPAF-II (ATCC, CRL-1997). 6 HPAF-II cells were injected subcutaneously into the right flank of 6- to 8-week-old immunodeficient female mice (Hsd: Athymic Nude-Foxn1nu, Envigo). Tumors grew to an average volume of 150 mm. 3 Upon reaching 18 days, 10 mice / group were treated intravenously once with vehicle (saline solution) or ADC1 (1 mg / kg or 6 mg / kg; day 0) or ADC3 (1 mg / kg or 6 mg / kg; day 0). Tumor length (L) and width (W) were measured with calipers and tumor volume was calculated using L x (W^2) / 2.

[0438] A single treatment with ADC1 at a dose of 6 mg / kg led to a significant antitumor effect. The effect is dose-dependent, since a single treatment with 1 mg / kg only led to a mild but significant transient antitumor effect. In contrast, a single treatment with the same dose of ADC3 did not show a significant antitumor effect at either dose (Figure 32). All treatments had no significant effect on body weight (data not shown). It is expected that ADC1-M, ADC2-M, ADC3-M, ADC6-M, and ADC7-M, like ADC1, are well-outperformed by ADC3 in this respect.

[0439] Example 15: Efficacy of ADC1 compared to ADC SAR DM4 in two CRC PDX mouse models In vivo antitumor efficacy was evaluated in human patient-derived CRC xenograft models COPF230 and REPF210 (Shanghai LideBiotech CO.,LTD). Tumor fragments were subcutaneously implanted into the right flank of 6- to 8-week-old immunodeficient female mice (NU-Foxn1nu, Charles River). When tumors reached approximately 170 mm 3 When the tumor reached an average volume of 1000 mg / kg, 6 mice / group were treated intravenously once (day 0) with vehicle (saline solution), ADC1 (6 mg / kg) or ADC SAR DM4 (6 mg / kg). The length (L) and width (W) of the tumor were measured with calipers and the tumor volume was calculated using the formula L x (W^2) / 2.

[0440] A single treatment with ADC1 led to a significant antitumor effect in both PDX models COPF230 (Figure 33) and REPF210 (Figure 34). In contrast, a single treatment with the same dose of ADC SAR DM4 showed no antitumor effect in either CRC PDX model (Figure 33, Figure 34). No significant effect on body weight was observed in any of the treatment groups (data not shown). It is expected that ADC1-M, ADC2-M, ADC3-M, ADC6-M, and ADC7-M, like ADC1, will be superior to ADC SAR DM4 in this respect.

[0441] Example 16: Efficacy of ADC1 compared to ADC SAR DM4 in a gastric PDX mouse model (GAPF313) In vivo antitumor efficacy was evaluated in a human patient-derived gastric xenograft model, GAPF313 (Shanghai LideBiotech CO.,LTD). Tumor fragments were implanted subcutaneously into the right flank of 6- to 8-week-old immunodeficient female mice (NU-Foxn1nu, Charles River). Tumors reached an average volume of approximately 180 mm 3Upon reaching 18 days, 6 mice / group were treated intravenously with vehicle (saline solution), ADC1 (4 mg / kg or 7 mg / kg, Q2W x 3) or ADC SAR DM4 (4.7 mg / kg, Q2W x 3) 3 times every other week starting on day 0. Tumor length (L) and width (W) were measured with calipers and tumor volume was calculated using the formula L x (W^2) / 2.

[0442] Interim data analysis of ongoing studies demonstrates clear antitumor efficacy of ADC1 in this model, and no effect of ADC SAR DM4 (Figure 35). All treatments were well tolerated (data not shown). It is expected that ADC1-M, ADC2-M, ADC3-M, ADC6-M, and ADC7-M, as well as ADC1, will outperform ADC SAR DM4 in this respect.

[0443] Example 17: Safety profile of ADC1 – Pilot toxicity study in cynomolgus monkeys To examine the safety profile, ADC1 was administered at doses of 0, 3, 10, and 30 mg / kg by 30 min iv infusion to cynomolgus monkeys three times at 3-week intervals (days 1, 22, and 43), and the animals were sacrificed on day 50 for gross and histopathological examination. As a result, it was found that ADC1 has a relatively favorable safety profile in that it lacks toxicity in certain organs affected by known ADC toxic side effects.

[0444] Example 18: Expression and purification of engineered antibodies and transglutaminase In the following, the expression and purification of the modified antibodies are described. These modified antibodies can be used to generate the respective antibody-drug conjugates. The characteristics of the modified antibodies mAb1-M, mAb2-M, mAb3-M, mAb4-M, mAb5-M, mAb6-M, and mAb7-M are summarized in Table 4 below. [Table 4-1] [Table 4-2] Table 4.

[0445] DNA sequences encoding antibodies mAb1-M, mAb2-M, mAb3-M, mAb5-M, mAb6-M, and mAb7-M were synthesized at GeneArt (Life Technologies) and cloned into pTT5 plasmid for recombinant expression. The resulting plasmids were used for transient transfection and recombinant protein expression in shake flasks. ExpiCHO expression system (Gibco™, Thermo Fisher Scientific Inc.) was used. Seven days after transfection, the supernatant was harvested and the expressed antibodies were purified. A standard stepwise process was used, including Protein A affinity chromatography (HiTrap MabSelect SuRe column, Cytiva) and size exclusion chromatography (HiLoad Superdex 200pg column, Cytiva).

[0446] mAb4-M / rituximab (F. Hoffmann-La Roche Ltd., Basel, Switzerland) was purchased of pharmaceutical grade quality.

[0447] Expression and purification of transglutaminase A DNA sequence (SEQ ID NO: 46) encoding the transglutaminase enzyme mTG was synthesized by GeneArt (Life Technologies) and cloned into the pET30a plasmid for recombinant expression. The Escherichia coli BL21(DE3) strain transformed with the constructed plasmid was grown overnight in a shake flask at 28°C and 130 rpm (50 mm shaker diameter) in lysogen broth medium supplemented with 5 g / l glucose, 10 ml / 100 ml of 10x phosphate buffered saline and 30 mg / l kanamycin. This culture was used to grow 9.5 liters of growth medium (50 g / l yeast extract, 10 g / l peptone, 0.5 g / l MgSO4). 4 x7H 2The fermenter was inoculated at an optical density of 0.00002 containing 0.00002 HO and 2 ml of 50% Desmophen (antifoam by Rhein Chemie Rheinau). The fermenter was run overnight (16 h) at 28 °C, 800 U / min rotation, 5 Nl / min aeration and pH 7.0-7.4. At OD5 the culture was induced with 0.1 mM IPTG until an OD of approximately 30 was reached (5-6 h). In case of foaming or a drop in oxygen concentration below 2 mg / ml, more Desmophen was added or the rotation was increased to 1000 rpm, respectively. The cell mass was harvested by continuous flow-through centrifugation.

[0448] 50 g of cell pellet was resuspended in 250 ml of 50 mM Na-acetate, 1 mM DTT, 5 mM MgCl2, 25 U / ml benzonase (Millipore) pH 5.5 and disrupted using a French press. The supernatant was clarified by centrifugation and filtration (0.8 / 0.2 μm pore size), the pH was adjusted to 5.5, loaded onto a Fractogel® SO3-(M) 85 ml column (Millipore) and then eluted with a 20 CV linear gradient from 0 to 1 M NaCl. Fractions with purified protein of the expected size were identified by SDS-PAGE, pooled and dialyzed overnight at 4°C against 50 mM Tris / HCl, 300 mM NaCl, pH 8.0. The transglutaminase was then proteolyzed at 37°C for 60 min at a final concentration of 2 mg / ml using 0.5 U / ml Dispase® I (Sigma Aldrich) and 2 mM CaCl2, followed by the addition of 5 mM EDTA. The reaction mix was dialyzed overnight at 4°C against 50 mM sodium phosphate buffer pH 6.0, loaded onto a Fractogel® SO3-column (Millipore) and eluted with a 20 CV linear gradient from 0 to 1 M NaCl. Fractions with efficiently cleaved and purified protein were identified by SDS-PAGE, pooled, concentrated, and purified using a HiLoad Superdex 75 pg size-exclusion column (Cytiva) with 24 HEPES, pH 7, 100 mM NaCl as the running buffer. Transglutaminase-containing fractions were pooled, concentrated to >20 mg / ml, flash frozen in liquid nitrogen, and stored at -80°C. Enzyme activity was determined using the ZediXclusive microbial transglutaminase assay (Zedira).

[0449] Example 19: ADC preparation, conjugation and characterization of ADC1-M, ADC4-M and ADC6-M 19.1. Antibody Preparation and Conjugation Monoclonal antibodies (mAbs) formulated in 50 mM histidine, 100 mM NaCl, pH 6.5 were stored at -80°C. Prior to conjugation, the mAbs were thawed at RT and the protein concentration was adjusted to 5 mg / ml by dilution with formulation buffer. The mAbs were then reduced by adding 10-12 molar excess (relative to the mAb) of TCEP and incubated at 37°C for 2-3 h. 16-24 molar equivalents (relative to the mAb) of DL1 (from a 10 mM stock solution in DMSO) were added and incubated at 25°C for 60 min. The reaction mix was quenched by adding 25 molar equivalents (relative to the mAb) of N-acetyl-cysteine ​​(from a 25 mM DMSO stock solution) and incubated at 25°C for 30 min. The ADCs were separated from DL1 and possible high molecular weight species (HMWS) by size exclusion chromatography (SEC). Prior to SEC purification, samples were centrifuged at 4000×g for 2 min to remove possible precipitates. SEC was performed using an Akta LC system (Cytiva) with a flow rate of 2.5 ml / min and a HiLoad Superdex 200 26 / 60 Increase column in combination with 50 mM histidine, 100 mM NaCl, pH 6.5 as running buffer. The fractionated samples containing the ADC material were pooled and concentrated to 6 mg / ml using an Amicon Ultra 15 50K centrifuge (Millipore), followed by a final buffer exchange to 10 mM histidine, 40 mM NaCl, 6% trehalose, 0.05% TWEEN, pH 5.5 using a HiTrap desalting column in combination with an Akta LC system (Cytiva). The final bulk drug substance (BDS) was filtered through a 0.22 μm sterile filter unit (Millipore) and flash frozen in liquid nitrogen until further use.

[0450] 19.2.Quality Attributes ADC1-M [Table K]

[0451] ADC6-M [Table L]

[0452] ADC4-M [Table M]

[0453] 9.3 Drug Substance Characterization Size Exclusion Chromatography (SEC) Monomer content and purity were assessed by size-exclusion chromatography on a Waters Bio Resolve SEC mAb column. Wavelength 214nm Column: Waters Bio Resolve SEC mAb column, 200Å, 2.5μm, 4.6×150mm Mobile phase 50mM monobasic sodium phosphate 0.4M Sodium Perchlorate, pH 6.3 Injection volume 5μL Column temperature: 25℃ Flow rate 0.35mL / min

[0454] A typical SEC chromatogram showing the purity of the input mAb and the final BDS is shown in FIG. Reverse Phase HPLC (RP-HPLC) Method RP-HPLC method parameters Wavelength 214nm Column: PLRP-S 1000Å (50×2.1mm, 8μm) column, Agilent Mobile phase A Lichrosolv H2O+0.1%TFA Mobile phase B 100%AcN+0.1%TFA Gradient 30-45%B(7.5min) Injection volume 10μL Column temperature: 65℃ Flow rate 1.0mL / min

[0455] Sample preparation: 40 μl of sample (2.2 mg / ml) was mixed with 4 μl of 0.5 M TCEP and incubated for 5 min at RT. Then, 40 μl of 0.5 M iodoacetamide was added and incubated for 15 min at 37° C. 10 μl of this reaction was injected.

[0456] A typical RP-HPLC chromatogram illustrating the DAR determination of the final BDS is shown in FIG. Free Drug Method Wavelength 254nm Column: Phenomenex Gemini, C18, 2 x 150 mm, 3 μm (P / N00F-4439-B0) Mobile phase A: 0.1% formic acid in water Mobile phase B 0.1% formic acid in acetonitrile gradient [Table N] Injection volume 10.00μL Column temperature: 50℃ Flow rate 0.75mL / min Sample preparation Protein drop: 100 μL drug substance + 250 μL cold MeOH + 50 μL 3M MgCl2. Spin at 20,000 rpm for 10 min.

[0457] Standard preparation: Mix 20 μL 20 mM DL (MSC2702209A in DMSO) + 20 μL DMSO + 40 μL MeOH + 20 μL 200 mM NAC in diafiltration buffer. Incubate overnight to give 4 mM DL-NAC. Dilute 4 mM DL-NAC with MeOH to give 4 μM DL-NAC standard.

[0458] Endotoxin characterization Endotoxin was determined by kinetic chromogenic LAL assay using the Endosafe PTS endotoxin system (Charles River). Buffers and antibodies were diluted 10-fold with LAL reagent water. ADC was diluted 10-fold with LAL reagent water. All samples were analyzed on a 0.01-1 EU / mL cartridge. EU / mL values ​​were converted to EU / mg by dividing by ADC[P]mg / mL.

[0459] Example 20: ADC preparation, conjugation and characterization of ADC7-M, ADC2-M, ADC5-M and ADC3-M 20.1. Antibody Preparation and Conjugation Monoclonal antibodies (mAbs) were stored at -80°C. Prior to conjugation, mAbs were thawed at RT and buffer exchanged into 24 mM HEPES, pH 7.0 using a HiTrap desalting column in combination with an Akta liquid chromatography (LC) system (Cytiva). Microbial transglutaminase (mTG) was used to couple the drug linker (DL) to the antibody. The reaction setup was as follows: 5 mg / ml mAb, 5 molar equivalents of DL1-M per conjugation site, 20 U / ml mTG, 7% DMSO, 24 mM HEPES, pH 7.0. The reaction was carried out at 37°C for 18 h. ADCs were separated from DL and mTG by size exclusion chromatography (SEC). Prior to SEC purification, the NaCl concentration of the samples was adjusted to 100 mM using a 5 M NaCl stock solution. SEC was performed using a HiLoad Superdex 200 26 / 60 Increase column coupled to an Akta LC system (Cytiva) at a flow rate of 2.5 ml / min and 10 mM histidine, 100 mM NaCl, pH 5.5 as the running buffer. The fractionated samples containing the ADC material were pooled and concentrated to 8 mg / ml using an Amicon Ultra 15 50K centrifuge (Millipore), followed by a final buffer exchange to 10 mM histidine, 40 mM NaCl, 6% trehalose, 0.05% TWEEN, pH 5.5 using a HiTrap desalting column coupled to an Akta LC system (Cytiva). The final bulk drug substance (BDS) was filtered through a 0.22 μm sterile filter unit (Millipore) and flash frozen in liquid nitrogen until further use.

[0460] 20.2 Quality attributes ADC7-M [Table O]

[0461] ADC2-M [Table P]

[0462] ADC3-M [Table Q]

[0463] ADC5-M [Table R]

[0464] 20.3. Methods: Characterization of Drug Substances Size Exclusion Chromatography (SEC) Monomer content and purity were assessed by size exclusion chromatography on a TOSOH TSKgel column. Injection volumes ranged from 1-5 μl (up to 10 μg protein). Wavelength 214nm Column: Tosoh TSKgel 7.8mm x 300mm, 5μm Mobile phase 50 mM sodium phosphate monobasic, 0.4 M sodium perchlorate, pH 6.3. Injection volume: 1-5μL Column temperature: 25℃ Flow rate 0.5mL / min

[0465] A typical SEC chromatogram showing the purity of the input mAb and the final BDS is shown in FIG. Reverse Phase HPLC (RP-HPLC) Method RP-HPLC method parameters. Wavelength 214nm Column: PLRP-S 1000Å (50×2.1mm, 8μm) column, Agilent Mobile phase A Lichrosolv H2O+0.1%TFA Mobile phase B 100%AcN+0.1%TFA Gradient 30-45%B(7.5min) Injection volume 10μL Column temperature: 65℃ Flow rate 1.0mL / min

[0466] Sample preparation: 40 μl of sample (2.2 mg / ml) was mixed with 4 μl of 0.5 M TCEP and incubated for 5 min at RT. Then, 40 μl of 0.5 M iodoacetamide was added and incubated for 15 min at 37° C. 10 μl of this reaction was injected.

[0467] A typical RP-HPLC chromatogram illustrating the DAR determination of the final BDS is shown in FIG. Free drug method. Wavelength 254nm Column: Phenomenex Gemini, C18, 2 x 150 mm, 3 μm (P / N00F-4439-B0) Mobile phase A: 0.1% formic acid in water Mobile phase B 0.1% formic acid in acetonitrile gradient [Table S] Injection volume 10.00μL Column temperature: 50℃ Flow rate 0.75mL / min Sample preparation Protein drop: 100 μL drug substance + 250 μL cold MeOH + 50 μL 3M MgCl2. Spin at 20,000 rpm for 10 min.

[0468] Standard preparation: Mix 20 μL 20 mM DL (MSC2702209A in DMSO) + 20 μL DMSO + 40 μL MeOH + 20 μL 200 mM NAC in diafiltration buffer. Incubate overnight to give 4 mM DL-NAC. Dilute 4 mM DL-NAC with MeOH to give 4 μM DL-NAC standard.

[0469] Endotoxin characterization Endotoxin was determined by kinetic chromogenic LAL assay using the Endosafe PTS endotoxin system (Charles River). Buffers and antibodies were diluted 10-fold with LAL reagent water. ADC was diluted 10-fold with LAL reagent water. All samples were analyzed on a 0.01-1 EU / mL cartridge. EU / mL values ​​were converted to EU / mg by dividing by ADC[P]mg / mL.

[0470] Example 21: ADC1-M and ADC2-M specifically kill cancer cells in vitro with high potency Human cancer cell lines were used to evaluate the potential of ADC1-M and ADC2-M to kill cancer cells. ADC1-M and ADC2-M exhibited subnanomolar and subnanomolar to single-digit nanomolar in vitro potencies, respectively, against different CEACAM5-positive cell lines (Table 3). In contrast, the effects of ADC1-M and ADC2-M were minor against the CEACAM5-negative cell line MDA-MB-231 (Table 3). As shown in the exemplary dose-response curves, ADC1-M and ADC2-M were highly potent against the CEACAM5-positive cell lines SK-CO-1, SNU-16, MKN-45, and LS174T (Figures 40a-d and 41a-d). In contrast, ADC1-M and ADC2-M had only minor effects on antigen-negative MDA-MB-231 cell viability (Figures 40e and 41e). An isotype control ADC utilizing the same linker payload as ADC1-M and ADC2-M showed much less efficacy against the CEACAM5-positive cell lines tested (Figure 40 and Figure 41).

[0471] In conclusion, ADC1-M and ADC2-M specifically kill CEACAM5-expressing human cancer cell lines in vitro with high potency. [Table 3]

[0472] Table 3. Potency of ADC1-M, ADC2-M and free payload against multiple human cell lines. Maximum effect compared to untreated control at highest tested compound concentration is indicated in brackets. Potency cutoff to report -50% effect, otherwise "n / a". CEACAM5 expression is indicated for each cell line.

[0473] Methods – Viability Assay: The cytotoxic effect of ADCs on cancer cell lines was measured by cell viability assay. The day before treatment, cells were seeded in 96-well plates in a volume of 90 μL. Test compounds (ADCs or free payload) were formulated in cell culture medium at 10x the starting concentration. Test compounds were serially diluted (1:4) and 10 μL of each dilution was added to cells in triplicate. Plates were cultured at 37° C. in a CO2 incubator for 6 days. For cell viability measurements, Cell Titer-Glo® reagent (Promega™ Corp, Madison, WI) was added to each well and plates were processed according to the manufacturer's instructions. Luminescence signals were measured using a Varioskan plate reader (Thermo Fisher). Luminescence readings were converted to % viability relative to untreated cells. Data were fitted by nonlinear regression analysis using log(inhibitor) vs. response, variable slope, 4-parameter fit equation using Genedata Screener or GraphPad Prism. Data are presented as % relative cell viability versus compound molar concentration, and error bars indicate standard deviation (SD) of duplicate or triplicate. Geometric mean IC50 values ​​from multiple experiments were calculated.

[0474] ADC1-M and ADC2-M were also compared with ADC SAR DM4 in terms of their cytotoxic effects on antigen-positive SK-CO-1 and antigen-negative MDA-MB-231 cell lines. ADC1-M and ADC2-M showed similar potency to ADC SAR DM4 on SK-CO-1 cancer cells (Fig. 40a and Fig. 41a compared to Fig. 26a). Non-specific effects on antigen-negative MDA-MB-231 were higher for ADC SARDM4 compared to ADC1-M and ADC2-M (Fig. 40e and Fig. 41e compared to Fig. 26b), with the difference being more pronounced for ADC2-M than for ADC1-M.

[0475] ADC1-M and ADC2-M were also generated utilizing an antibody scaffold lacking the YTE mutation, and both of these ADCs (ADC6-M and ADC7-M) showed similar results as their respective ADCs (ADC1-M and ADC2-M) that contained the YTE mutation.

[0476] Example 23: Pharmacokinetic studies of ADCs Pharmacokinetic studies in human FcRn transgenic (276 hemizygous model) mice were performed after a single intravenous dose of 3 mg / kg ADC1, ADC1-M, ADC6-M, ADC7-M, ADC2-M. Samples were taken from 6 animals per treatment group at 1, 24, 48, 72, 144, 168, 240, 336, and 504 h post-dose for ADC1-M, ADC6-M, ADC7-M, and ADC2-M, and from 9 animals per treatment group at 0.08 (approximately 5 min), 4, 24, 48, 72, 168, 240, 336, and 504 h post-dose for ADC1. Data were pooled for PK analysis. See Figure 42 for plasma concentrations per treatment. Back-extrapolation of plasma concentrations at time 0 (C0) yielded concentrations slightly lower than the theoretical maximum for ADC1-M, ADC7-M, and ADC2-M, and close to the theoretical maximum for ADC6-M and ADC1 (assuming dilution by a plasma volume of 40 mL / kg). Plasma concentrations declined with a biphasic profile. A sharp decrease in plasma concentrations was observed up to 24 h, followed by a slower decrease until the last sampling time (504 h) in all molecular groups, with the exception of animals treated with ADC6-M, which were quantifiable up to 336 h.

[0477] The longest terminal half-life (t1 / 2) value was calculated for ADC2-M with Lala-YTE variant with DAR=4 (190h), followed by ADC7-M with Lala variant with DAR=4 (122h), ADC1-M with Lala-YTE variant and DAR=8 (64.5h), and ADC6-M with Lala variant with DAR=8 (33.8h), and finally ADC1 with IgG1.4 with DAR=8 (29.6h). The % of extrapolated AUCinf was lower than 20%, allowing reliable calculation of AUC0-inf and derived parameters (Cl, Vz and Vss). In transgenic mice, the highest AUC0-inf and lowest Cl values ​​were calculated for ADC2-M (Lala-YTE variant, DAR=4), followed by ADC7-M, ADC6-M, ADC1-M, and ADC1. AUC0-inf and Cl values ​​ranged from 1.360000 to 10.200000 h*ng / mL and 0.293 to 1.16 mL / h / kg, respectively. No differences in the relevant volume of distribution (Vss) were observed, with Vss ranging from 49.8 to 113 mL / kg.

[0478] In conclusion, after a single iv dose of 3 mg / kg in transgenic mice, the best PK profile was exhibited by ADC2-M with the highest plasma exposure, lowest Cl and longest t1 / 2. The PK profile was improved by moving from IgG1.4 to the Lala variant, and the strongest effect was observed by further including the YTE variant, reducing the DAR from 8 to 4.

[0479] Table 5: PK parameters of the Ceacam5 backup molecule after iv administration of 3 mg / kg. Antibody-drug conjugates (ADCs) and drug-to-antibody ratios (DARs) are also indicated. [Table 5]

[0480] Example 24: Efficacy of ADC1-M and ADC2-M in pancreatic cell line derived tumor models The efficacy of ADC1-M and ADC2-M was evaluated in the human pancreatic cell line-derived xenograft model BxPC3 (ATCC, CRL-1687). 6 of BxPC3 cells were injected subcutaneously into the right flank of 6- to 8-week-old immunodeficient female mice (Hsd: Athymic Nude-Foxn1nu, Envigo). When tumors reached an average volume of 85 mm3, 10 mice / group were treated intravenously once with (saline solution) or ADC1-M (5 mg / kg; day 0), or with ADC2-M (5 mg / kg or 10 mg / kg; day 0). Tumor length (L) and width (W) were measured with calipers, and tumor volume was calculated using L x (W^2) / 2.

[0481] Single treatment with ADC1-M at a dose of 5 mg / kg and ADC2-M at a dose of 5 mg / kg or 10 mg / kg led to significant antitumor effects. Both ADCs have similar efficacy in this model (Figure 43). Treatment had no significant effect on body weight (data not shown).

[0482] Example 25: Preparation of ADC Labetuzumab Govitecan Analog ADC8 25.1 Antibodies For purposes of further comparative experiments, additional analogs of the ADC labetuzumab govitecan were prepared based on a monoclonal antibody having the following sequence: Heavy Chain: DIQLTQSPSS LSASVGDRVT ITCKASQDVG TSVAWYQQKP GKAPKLLIYW TSTRHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ YSLYRSFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GECEVQLVES GGGVVQPGRS LRLSCSASGF DFTTYWMSWV RQAPGKGLEW IGEIHPDSST INYAPSLKDR FTISRDNAKN TLFLQMDSLR PEDTGVYFCA SLYFGFPWFA YWGQGTPVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK (SEQ ID NO: 62) Light Chain: DIQLTQSPSS LSASVGDRVT ITCKASQDVG TSVAWYQQKP GKAPKLLIYW TSTRHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ YSLYRSFGQG TKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC (SEQ ID NO: 63)

[0483] 25.2 Drug-Linker Compounds As a drug-linker molecule to be conjugated to the above-mentioned antibody, a molecule of the following structure was used:

[0484] [ka]

[0485] This drug-linker molecule was purchased from SyntaBio LLC, 10239 Flanders Ct, San Diego, CA 92121. Lot No. S041070422.

[0486] 25.3 Conjugation Monoclonal antibodies (mAbs) were thawed at 2-8°C up to 3 days prior to conjugation and stored at 2-8°C in PBS, pH 6.8 until further use. On the day of conjugation, the pH of the mAb solution was adjusted to a final concentration of 5% (v / v) by the addition of 0.5 M Tris, 0.025 M EDTA, pH 8.5. After pH adjustment, the mAbs were reduced using 10 molar equivalents of TCEP and incubation at 20°C for 120 min. Subsequently, the mAb solution was diluted 1:1 with 20 mM histidine, 80 mM NaCl, pH 5.5, the DMSO concentration was adjusted to 10% (v / v), and the reaction was initiated by the addition of 16 molar equivalents of the drug-linker mentioned above. The reaction was incubated at 20°C for 60 min and finally quenched by the addition of 100 mM NAC (n-acetyl-cysteine). The conjugated mAb (i.e., ADC) was then subjected to preparative size-exclusion chromatography.

[0487] 25.4 Preparative Size Exclusion Chromatography, Desalting and Filtration The reaction mixture was purified using preparative size-exclusion chromatography. A GE HiLoad 26 / 60 Superdex S200 column was connected to an Akta Avant 25 system (GE Healthcare) and equilibrated with 20 mM histidine, 80 mM NaCl, pH 5.5 according to the manufacturer's instructions. The reaction mixture was then injected and run through the column at a flow rate of 5 ml / min using 20 mM histidine, 80 mM NaCl, pH 5.5 as the running buffer. ADC-containing fractions were determined by UV light absorption at 280 nm, pooled, and concentrated. The ADC material was concentrated using a Vivaspin VS2022 device (Sartorius UK Ltd.) according to the manufacturer's instructions. The concentrated ADC material was transferred to formulation buffer (10 mM histidine, 100 mM NaCl, 3% trehalose, 0.05% (w / v) PS20, pH 5.5). A HiPrep 26 / 10 desalting column (GE Healthcare) was used at a flow rate of 10 ml / min on an Akta Avant 25 system (GE Healthcare) according to the manufacturer's instructions. The final ADC material was filtered using a 0.2 μm filter (0.2 μm PES filter, Merck Millipore), aliquoted, and then flash frozen in liquid nitrogen. The final concentration of the ADC material (drug substance) was 7.7 mg / ml. The material was kept at -80°C until further use. The ADC resulting from this work is referred to herein as "ADC8". This ADC is an analogue of labetuzumab govitecan.

[0488] 25.5 Further Characterization of ADC8 Drug Substance The ADC8 drug substance obtained above was further analyzed (a) by size exclusion chromatography (SEC), which showed a monomeric purity of 99.3%, (b) by reversed phase HPLC (RP-HPLC), which showed a DAR of 7.7, and (c) by a RP HPLC based free drug method, which showed residual free drug levels below 0.02% (by molar ratio).

[0489] Example 26: ADC1-M, ADC2-M, ADC6-M and ADC7-M kill cancer cells with higher specificity than ADC SAR DM4 and ADC8 Using the same method described in Example 21, human cancer cell lines were used to compare the effects of ADC1-M, ADC2-M, ADC6-M, and ADC7-M, as well as the ADC SARs DM4 and ADC8, on cancer cells with CEACAM5 expression relative to their effects on cancer cells lacking CEACAM5 expression. The fold reduction in IC50, defined as the specificity index, was calculated by dividing the IC50 on CEACAM5-negative MDA-MB-231 cells by the IC50 on each CEACAM5-positive cell line (see Table 6). The higher the value of the specificity index, the more specific the tested ADC is. ADC1-M, ADC2-M, ADC6-M, and ADC7-M showed much lower IC50 in CEACAM5-positive SK-CO-1, SNU-16, and MKN-45 cells than in CEACAM5-negative MDA-MB-231 cells. These resulted in specificity indices ranging from 116 to 874. The specificity indices of ADC2-M and ADC7-M are probably underestimated due to the lack of effect on MDA-MB-231 cells in the tested concentration range (as shown for ADC2-M in Table 3 and Figure 41e). For this reason, the specificity indices were calculated only for ADC2-M and ADC7-M, setting the IC50 at the highest tested concentration of 100 nM. In contrast, ADC SAR DM4 and ADC8 showed lower specificity indices ranging from 26 to 73 and 1.8 to 4.8, respectively. In conclusion, a more selective killing of CEACAM5-positive cancer cells by ADC1-M, ADC2-M, ADC6-M, and ADC7-M was shown in vitro compared to ADC SAR DM4 and ADC8. [Table 6]

[0490] Table 6. Relative potency increase (specificity index) of ADCs in CEACAM5-positive cell lines compared to the CEACAM5-negative MDA-MB-231 cell line. The respective specificity index of each ADC was calculated by dividing the IC50 in MDA-MB-231 by the IC50 in each of the three CEACAM5-positive cell lines (theoretical index of 1 for MDA-MB-231 is reported for illustrative purposes only). Calculations were based on the geometric mean IC50 of two to four individual experiments.

[0491] Example 27: ADC1-M, ADC2-M, ADC6-M, ADC7-M mediate stronger bystander effects than ADC SAR DM4 against antigen-negative cells in co-culture with antigen-positive cells Using the same method as in Example 10, the potential of ADC1-M, ADC2-M, ADC6-M, ADC7-M, and ADC SAR DM4 to mediate bystander effects on antigen-negative cells in close proximity to antigen-positive cells was evaluated in a bystander assay. ADC1-M, ADC2-M, ADC6-M, and ADC7-M showed strong bystander effects on CEACAM5-negative MDA-MB-231 cells in the presence of CEACAM5-positive SK-CO-1 (Figure 44). Co-culture experiments were performed at an ADC concentration of 1 nM, which caused maximal inhibition of CEACAM5-positive SK-CO-1 cell viability with ADC1 (Figure 26A), but had no effect on CEACAM5-negative MDA-MB-231 cells alone (Figure 26B). Consistent with these findings, non-specific effects of ADC1-M, ADC2-M, ADC6-M, ADC7-M, and ADC SAR DM4 were not observed in the MDA-MB-231 only control in the bystander assay set-up.

[0492] In conclusion, ADC1-M, ADC2-M, ADC6-M, and ADC7-M mediate strong bystander effects against antigen-negative cancer cells in coculture with antigen-positive cells. These findings support their potential to effectively target tumors with heterogeneous target expression.

[0493] Compared to the ADC SAR DM4, ADC1-M, ADC2-M, ADC6-M, and ADC7-M mediated a much stronger bystander effect on antigen-negative cells in coculture with antigen-positive cells (FIG. 44).

[0494] For all ADCs tested, the extent of the bystander effect increased with increasing numbers of antigen-positive cells added to a fixed number of antigen-negative cells. Without wishing to be bound by theory, this may be the result of more ADC being processed by a greater number of antigen-positive cells, releasing more free payload, which is responsible for the bystander effect on the antigen-negative cells.

[0495] No non-specific effects of the tested ADCs were observed in bystander analyses in MDA-MB-231 cells alone for all tested ADCs at 1E-9M test concentrations.

[0496] Example 28: Efficacy of ADC1-M and ADC3-M compared to ADC8 The efficacy of ADC1-M and ADC3-M compared to ADC8 was evaluated in a human pancreatic adenocarcinoma cell line-derived xenograft model, HPAF-II (ATCC, CRL-1997). Six to eight week old immunodeficient female mice (Hsd: Athymic Nude-Foxn1nu, Envigo) were injected with 5 × 10 6 HPAF-II cells were injected subcutaneously into the right flank. 3 When the tumor reached an average volume of 1000 mg / kg, 10 mice / group were treated once intravenously with ADC1-M (1 mg / kg or 6 mg / kg) or ADC3-M (1 mg / kg or 6 mg / kg) or ADC8 (1 mg / kg or 6 mg / kg). The length (L) and width (W) of the tumor were measured with calipers, and the tumor volume was calculated using L×W^2 / 2.

[0497] Single treatment with ADC1-M or ADC3-M at a dose of 1 or 6 mg / kg led to a significant antitumor effect compared to vehicle control. The effect is dose-dependent, since a single treatment with 1 mg / kg only led to a minor and temporary antitumor effect, whereas 6 mg / kg showed a much stronger antitumor effect. In contrast, a single treatment with ADC8 did not show a significant antitumor effect at either dose (Figure 45). All treatments had no significant effect on body weight (data not shown).

[0498] Example 29: Efficacy of ADC1-M and ADC3-M compared to ADC SAR DM4 in CRC PDX mouse models Antitumor efficacy of ADC1-M and ADC3-M compared to ADC SAR DM4 was determined using the human CRC xenograft model COPF230. The study was conducted at Lide Biotech (Shanghai). Six to eight week old immunodeficient female mice (Crl: NU-Foxn1nu) were subcutaneously implanted with COPF230 tumor fragments in the right flank. Tumors were 165 mm 3 When tumors reached an average volume of 1000 mg / kg, 6 mice / group were treated intravenously once with vehicle (saline solution) or ADC1-M, ADC3-M, or ADC SAR DM4 (6 mg / kg each). Tumor length (L) and width (W) were measured with calipers, and tumor volume was calculated using L×W^2 / 2.

[0499] A single treatment with 6 mg / kg ADC1-M or ADC3-M led to similar and significant antitumor effects compared to vehicle control groups. In contrast, a single treatment with 6 mg / kg ADC SAR DM4 demonstrated only minor antitumor effects (Figure 46). All treatments had no significant impact on body weight (data not shown).

[0500] Example 30: Efficacy of ADC1-M and ADC3-M compared to ADC SAR DM4 in GC PDX mouse models The antitumor efficacy of ADC1-M and ADC3-M compared to ADC SAR DM4 was evaluated in the human GC xenograft model GAPF313. The study was conducted at Lide Biotech (Shanghai). Six- to eight-week-old immunodeficient female mice (Crl: NU-Foxn1nu) were subcutaneously implanted with GAPF313 tumor fragments in the right flank. Tumors reached a mean volume of 176 mm 3 Upon reaching 18 days of age, 6 mice / group were treated intravenously every other week (3 cycles) with vehicle (saline solution) or 4 mg / kg ADC1-M or ADC3-M or 4.7 mg / kg ADC SAR DM4. Tumor length (L) and width (W) were measured with calipers and tumor volume was calculated using L×W^2 / 2.

[0501] Multiple treatments with ADC1-M or ADC3-M led to similar and significant antitumor effects compared to vehicle controls. ADC SAR DM4 was not significantly more effective than ADC1-M and ADC3-M (FIG. 47). All treatments had no significant impact on body weight (data not shown).

Claims

1. An isolated antibody that binds to the human CEACAM5 protein, The antibodies isolated here (i) comprising at least one light chain constant region (CL) containing a sequence selected from the group consisting of GGTLQSPP, LLQGA, GGLLQGPP, TLQSG, TLQSPP, and TLQSA, preferably containing this sequence at the C-terminus of the light chain constant region; and (ii) The following amino acid substitutions are optional: (a) L234A and L235A (LALA mutations); (b) L234A, L235A, and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y, S254T, and T256E (YTE mutations); (e)K222R It includes at least one heavy chain constant region (CH) containing one or more of the following: Furthermore, Eu numbering is used here for the amino acid substitution; Furthermore, the isolated antibody here comprises CDR1-H consisting of the amino acid sequence of SEQ ID NO: 3, CDR2-H consisting of the amino acid sequence of SEQ ID NO: 4, CDR3-H consisting of the amino acid sequence of SEQ ID NO: 5, CDR1-L consisting of the amino acid sequence of SEQ ID NO: 6, CDR2-L consisting of the amino acid sequence of SEQ ID NO: 7, and CDR3-L consisting of the amino acid sequence of SEQ ID NO:

8. The isolated antibody mentioned above.

2. The isolated antibody according to claim 1, comprising framework regions FR1, FR2, FR3, FR4, FR5, FR6, FR7, and FR8 having the structures FR1 - CDR1-H - FR2 - CDR2-H - FR3 - CDR3-H - FR4, and FR5 - CDR1-L - FR6 - CDR2-L - FR7 - CDR3-L - FR8; where FR1 is sequence number 54, FR2 is sequence number 55, FR3 is sequence number 56, FR4 is sequence number 57, FR5 is sequence number 58, FR6 is sequence number 59, FR7 is sequence number 60, and FR8 is sequence number 61.

3. An isolated antibody that binds to the human CEACAM5 protein, Here, the isolated antibody, (i) comprising at least one light chain constant region (CL) containing a sequence selected from the group consisting of GGTLQSPP, TLQSPP, TLQSA, and TLQSG, preferably containing this sequence at the C-terminus of the light chain constant region; and (ii) The following amino acid substitutions are optional: (a) L234A and L235A (LALA mutations); (b) L234A, L235A, and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y, S254T, and T256E (YTE mutations); (e)K222R It includes at least one heavy chain constant region (CH) containing one or more of the following: Furthermore, Eu numbering is used here for the amino acid substitution; Preferably, the isolated antibody comprises CDR1-H consisting of the amino acid sequence of SEQ ID NO: 3, CDR2-H consisting of the amino acid sequence of SEQ ID NO: 4, CDR3-H consisting of the amino acid sequence of SEQ ID NO: 5, CDR1-L consisting of the amino acid sequence of SEQ ID NO: 6, CDR2-L consisting of the amino acid sequence of SEQ ID NO: 7, and CDR3-L consisting of the amino acid sequence of SEQ ID NO: 8; Furthermore, the isolated antibody comprises framework regions FR1, FR2, FR3, FR4, FR5, FR6, FR7, and FR8 having the structures FR1 - CDR1-H - FR2 - CDR2-H - FR3 - CDR3-H - FR4, and FR5 - CDR1-L - FR6 - CDR2-L - FR7 - CDR3-L - FR8; where FR1 is sequence number 54, FR2 is sequence number 55, FR3 is sequence number 56, FR4 is sequence number 57, FR5 is sequence number 58, FR6 is sequence number 59, FR7 is sequence number 60, and FR8 is sequence number 61. The isolated antibody mentioned above.

4. An isolated antibody according to any one of claims 1 to 3, wherein both heavy chain constant regions (CH) contain one or more of the amino acid substitutions (a) to (e), and / or both light chain constant regions contain the sequence GGTLQSPP.

5. The antibody according to any one of claims 1 to 3, comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:

10.

6. (i) A heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 34 and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 14; or (ii) A heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 34 and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 36; or (iii) A heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 35 and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 36; or (iv) A heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 51 and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 14; or (v) Heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 51 and light chain (LC) containing the amino acid sequence of SEQ ID NO: 36 An antibody according to any one of claims 1 to 3, comprising:

7. An isolated nucleic acid comprising a nucleic acid sequence encoding an antibody according to any one of claims 1 to 3.

8. A host cell transformed with the nucleic acid described in claim 7.

9. An immunoconjugate comprising an antibody according to any one of claims 1 to 3, covalently linked to at least one growth inhibitor via a linker.

10. The immunoconjugate according to claim 9, wherein the growth inhibitory agent is exatecan.

11. The immunoconjugate is of the following formula (IV) or formula (IVA): 【Chemistry 1】 An immunoconjugate having the formula, wherein S is a sulfur atom of the antibody, and n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, according to claim 9.

12. The immunoconjugate according to claim 11, wherein the immunoconjugate has formula (IVA), and n is between 1 and 10, preferably 4.

13. A pharmaceutical composition comprising the immunoconjugate according to claim 9, further comprising a pharmaceutically acceptable carrier, diluent, and / or excipient.

14. A pharmaceutical composition according to claim 13 for use as a medicine.

15. A pharmaceutical composition according to claim 13 for use in the treatment of cancer.

16. The pharmaceutical composition for use according to claim 15, wherein the cancer is a CEACAM5-expressing cancer.

17. The pharmaceutical composition for use according to claim 15, wherein the cancer is colorectal cancer, stomach cancer, lung cancer, pancreatic cancer, esophageal cancer, or prostate cancer.

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