Anti-CEACAM5 antibody and conjugate and application thereof

Abstract

The present invention provides antibodies that bind to the human CEACAM5 protein, as well as isolated nucleic acids and host cells comprising sequences encoding the antibodies. The invention also provides immunoconjugates comprising said antibodies linked to a growth inhibitor, and pharmaceutical compositions comprising the antibodies or immunoconjugates of the invention. The invention also provides the use of the antibodies, immunoconjugates and pharmaceutical compositions of the invention for the treatment of cancer or for diagnostic purposes.

Description

Technical Field

[0001] The present invention relates to antibodies that bind to human CEACAM5 protein, and isolated nucleic acids and host cells comprising sequences encoding the antibodies. The present invention also relates to immunoconjugates comprising the antibodies linked to growth inhibitors, and pharmaceutical compositions comprising the antibodies or immunoconjugates of the present invention. The present invention also relates to the use of the antibodies, immunoconjugates and pharmaceutical compositions of the present invention for treating cancer or for diagnostic purposes. Background Art

[0002] Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion. CEA was first identified in 1965 (Gold and Freedman, J Exp Med, 121, 439, 1965) as a protein normally expressed by the fetal intestine during the first six months of pregnancy, and is found in many cancers, such as colorectal cancer or pancreatic cancer. The CEA family belongs to the immunoglobulin superfamily. The CEA family consisting of 18 genes is subdivided into two protein subgroups: 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 7 members: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7 and CEACAM8. CEACAM5, identical to the originally identified CEA, is reported to be highly expressed on the surface of cancer cells such as colorectal, gastric, lung and pancreatic tumor cells, and expression in normal tissues is restricted to some normal epithelial cells, such as colon and esophageal epithelial cells. Thus, CEACAM5 may constitute a therapeutic target suitable for tumor-specific targeting approaches, such as immunoconjugates.

[0004] The extracellular domain of the CEACAM family members consists of repeated immunoglobulin-like (Ig-like) domains, which have been classified into three types, A, B and N, based on 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 presenting 84% to 87% pairwise sequence similarity and the B domain presenting 69% to 80% pairwise sequence similarity. In addition, other human CEACAM members, namely CEACAM1, CEACAM6, CEACAM7 and CEACAM8, which present A and / or B domains in their structures, show homology with human CEACAM5. In particular, the A and B domains of the human CEACAM6 protein show sequence homologies with the A1 and A3 domains and any of the B1 to B3 domains of human CEACAM5, respectively, which are even higher than those observed in the A and B domains of human CEACAM5.

[0005] Anti-CEA antibodies for diagnostic or therapeutic purposes for CEA targeting have been produced. Specificity for related antigens has been mentioned as a concern in this field, such as Sharkey et al. (1990, Cancer Research 50, 2823). Due to the homology mentioned above, some previously described antibodies may show, for example, binding to repeated epitopes of CEACAM5 present in different immunoglobulin domains, and show cross-reactivity with other CEACAM family members, such as CEACAM1, CEACAM6, CEACAM7 or CEACAM8, thereby lacking specificity for CEACAM5. However, for CEA targeted therapy, the specificity of anti-CEACAM5 antibodies is required so that they bind to tumor cells expressing human CEACAM5, but not to certain normal tissues expressing other CEACAM family members. It is noteworthy that CEACAM1, CEACAM6 and CEACAM8 have been described as being 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 granulocyte production 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. Therefore, antibodies specific for CEACAM5 that do not cross-react with other molecules of the CEACAM family are needed, for example, for use as part of an antibody drug conjugate (ADC) or in any other manner that results in killing of target cells.

[0006] Furthermore, since CEACAM5 has been described to be expressed in some normal cell tissues, it is desirable to develop anti-CEACAM5 antibodies that are able to bind to human CEACAM5 as well as cynomolgus monkey (Macaca fascicularis) CEACAM5, as such antibodies can be easily tested in preclinical toxicology studies in cynomolgus monkeys to assess their safety profile.

[0007] The combination of the need for a) species cross-reactivity and b) specificity for human and cynomolgus macaque CEACAM5 (i.e., lack of cross-reactivity with other cynomolgus macaque and human CEACAM family members) further complicates the development of new anti-CEACAM5 antibodies, especially given the overall sequence homology between human and cynomolgus macaque CEACAM proteins.

[0008] Furthermore, CEACAM5 has been described in the literature as a surface protein that is poorly internalized (reviewed in Schmidt et al., 2008, Cancer Immunol. Immunother. 57, 1879), which poses further challenges for antibody drug conjugates against this target protein.

[0009] Known anti-CEACAM5 antibodies include Immunomedics' labetuzumab (also known as hMN14; Sharkey et al., 1995, Cancer Research 55, 5935). It has been shown that the antibody does not bind to related antigens, but also does not cross-react with CEACAM5 from cynomolgus macaques. Labetuzumab has also been used as part of an antibody-drug conjugate (ADC), namely as govitecan-labetuzumab. Govitecan-labetuzumab is an ADC consisting of the cytotoxic drug SN38 conjugated to the anti-CEACAM5 antibody labetuzumab via a linker (called CL2A), which contains a pH-sensitive carbonate and a short polyethylene glycol (PEG) chain. Govitecan-labetuzumab is characterized by significant instability of the linker structure used, which leads to early systemic loss of the cytotoxic payload after parenteral administration. This degradation process can limit anti-tumor activity and increase the risk of side effects. Another known anti-CEACAM5 ADC is Sanofi's SAR408701 (tusamitamab ravtansine), which comprises the anti-CEACAM5 antibody SAR408377 (tusamitamab; also known as huMab2-3) covalently linked to the cytotoxic agent DM4, a potent microtubule-destabilizing maytansinoid, via an N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB) linker. SAR408701 is associated with toxic side effects, including keratitis and corneal disease, to several organs and tissues, including the cornea. In addition, the effectiveness of ADCs based on microtubule inhibitors 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, few ADCs have been approved for the treatment of solid tumors.

[0010] Some ADCs may be dose-limiting in patients due to side effects of the payload being released through cellular catabolism, leading to toxicity in the bone marrow and circulating blood cells (e.g., neutropenia, reticulocytopenia, lymphocytopenia).

[0011] Due to the drug-antibody coupling, ADCs typically have a suboptimal half-life in humans of only a few days in the circulation, which is significantly lower than the half-life of the corresponding unconjugated antibody. The relatively high clearance of these ADCs is associated with cellular degradation after target-independent uptake and leads to a large release of toxic drug payloads, which can induce side effects. Thus, the purpose of the ADCs disclosed herein is to improve their half-life to reduce unwanted side effects.

[0012] In view of the foregoing, there remains a need for new and improved therapeutics for the treatment of cancer, such as for different solid tumor indications including, for example, CRC, pancreatic cancer, gastric cancer, NSCLC, esophageal cancer, and prostate cancer. Summary of the invention

[0013] The present invention addresses this need and other needs in the art by, inter alia, providing monoclonal antibodies against CEACAM5 (reactive with both human and cynomolgus macaque proteins) and by providing immunoconjugates (also referred to herein as antibody-drug conjugates (ADCs)) comprising said antibodies; these immunoconjugates have cytotoxic effects, killing tumor cells in vitro and inhibiting tumor growth in vivo. The present invention relates to the embodiments described in the claims and in the further description below.

[0014] In order to generate new antibodies against CEACAM5 with optimal properties for therapeutic purposes, in particular in the form of immunoconjugates, the inventors have performed extensive research and development to select antibodies with a favorable profile and to develop immunoconjugates based on this.

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

[0016] The inventors have also shown that the antibodies they generated can induce cytotoxic effects on tumor cells in vitro when combined with cytotoxic drugs in immunoconjugates. Antibodies conjugated to cytotoxic drugs (i.e., immunoconjugates of the present invention) can also significantly inhibit tumor growth in mice carrying tumors expressing CEACAM5. The linker connecting the drug to the antibody is designed to maximize systemic stability after parenteral administration. The release of exatecan from the immunoconjugates of the present invention within target cells results in very high efficacy and a prominent bystander effect. A potent bystander effect can be beneficial in treating patients with heterogeneous target expression.

[0017] Generally, high systemic exposure is desirable. Compared to compounds with lower systemic exposure, high systemic exposure of ADC drugs leads to more effective tumor targeting and improved distribution of cytotoxic payload in tumor tissues and cells, and ultimately leads to enhanced tumor cell killing.

[0018] Furthermore, the antibody-drug-conjugates of the invention are improved by including molecular modifications to reduce target-independent cellular degradation, resulting in 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 level of released payload while improving efficacy driven by higher ADC exposure. Therefore, these modifications will provide an improved therapeutic window for drugs by reducing side effects and improving anti-tumor activity.

[0020] As described herein, the exposure and half-life of ADCs according to the present invention will be improved by, for example: (1) modifying the antibody heavy chain by including a YTE mutation and linking it to the drug using interchain cysteine ​​conjugation, or (2) by modifying the antibody heavy chain with a YTE mutation and using an innovative enzymatic conjugation method that uses the microbial transglutaminase (also referred to herein as "TGase" and "mTG") enzyme to attach the drug to the antibody.

[0021] Unexpectedly, high exposures that resulted in low clearance values ​​in human predictive animal models were obtained by the above method (2). With such modifications, the exposure of the ADC was improved to about 10-fold compared to variants not containing these modifications, which therefore resulted in a significant reduction in the level of released payload and a significant increase in the concentration of anti-tumor active ADC in the circulation. The improved ADC had a similar PK profile to the original YTE antibody not containing a drug linker and exhibited an improved half-life that exceeded prior art ADCs.

[0022] These results will reduce toxicity and improve the efficacy of clinical cancer therapies compared to prior art molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Binding of mAb1 to recombinant human (rh) CEACAM5 ECD or its domains N-A1-B1, A2-B2, A3-B3 or to recombinant cynomolgus macaque (mf) CEACAM5 ECD in ELISA assay.

[0024] Figure 2 : EC50 of anti-CEACAM5 antibody binding to MKN-45 cells: Cellular binding of mAb1 on the CEACAM5 expressing MKN45 cell line compared to antibodies huMab2-3 and hmn-14.

[0025] Figure 3 : pHrodo-labeled antibodies were internalized into late endosomes and lysosomes of cells (total fluorescence intensity per cell, average of triplicates).

[0026] Figure 4 : Fluorescence intensity per cell from 700 min to 1200 min, which is the linear portion of the curve. The linear slope was measured and compared between samples (see Example 1.6.5).

[0027] Figure 5 : IHC staining of FFPE cancer cell lines using antibody rb8G4.

[0028] Figure 6 : Correlation between CEACAM5 mRNA expression and IHC staining in 104 cancer cell lines.

[0029] Figure 7 : IHC staining of normal human tissues using antibody rb8G4.

[0030] Figure 8 : CEACAM5 mRNA expression in normal human tissues.

[0031] Fig. 9 : IHC staining of human colorectal cancer tissues using antibody rb8G4.

[0032] Fig.10 : IHC staining of human gastric cancer tissues using antibody rb8G4.

[0033] Fig.11 : IHC staining of human esophageal cancer tissues using antibody rb8G4.

[0034] Fig.12 : IHC staining of human non-small cell lung cancer tissues using antibody rb8G4.

[0035] Figure 13: mAb1 ( Fig.13A ) and rb8G4( Fig. 13B ) binding to CEACAM5.

[0036] Fig.14 : Typical SEC chromatograms showing the purity of stock mAb, conjugate after UF and final drug substance (BDS).

[0037] Fig.15 : A typical RP-HPLC chromatogram showing the separation of light and heavy chains. The chromatogram shows the overlap of the stock mAb, crude ADC and final BDS.

[0038] Fig.16 : Typical chromatograms showing free drug levels of NAC standards and final BDS.

[0039] Fig.17 : Typical SEC chromatograms showing the purity of stock mAb and final BDS.

[0040] Fig.18 : Typical RP-HPLC chromatogram showing separation of light and heavy chains. The chromatogram shows the overlay of the stock mAb and the final BDS.

[0041] Fig.19 : Typical chromatograms showing free drug levels of NAC standards and final BDS.

[0042] Fig. 20 : ADC stability in human, mouse and cynomolgus monkey sera. Conjugated exatecan concentrations (initial dose approximately 10 μM) were calculated using free exatecan (normalized data).

[0043] Fig.21 : ADC3 stability control in mouse serum and buffer. Conjugated SN38 concentration (initial dose 50 μg / mL ADC protein concentration) was calculated using free SN38 (unnormalized).

[0044] Fig. 22 : Payload release curves of ADC1 and ADC2 in human liver lysosomes (pH 5.0). Conjugated drug concentrations were calculated using, for example, free exatecan (initial concentration of approximately 10 μM exatecan) normalized data.

[0045] Fig.23 ADC catabolite analysis confirmed that free exatecan was a lysosomal release product.

[0046] Figure 24: ADC1, ADC2 and free payload against antigen-positive SK-CO-1 ( Fig.24A ) and SNU-16( Fig. 24B ) cell lines and antigen-negative MDA-MB-231 ( Fig.24C ) cell lines. One representative experiment is shown, with mean ± SD of triplicates. Fig.24C The legend for assigning three different series of data points to ADC1, ADC2, and payload also applies to Fig.24A and Fig. 24B .

[0047] Fig.25 : Comparison of ADC1 and ADC2 with corresponding isotype controls on SK-CO-1 cell line. Shown is one representative experiment with mean ± SD of triplicates.

[0048] Figure 26: ADC1, ADC2, ADC SAR DM4, ADC mAb1 DM4 and free payload against antigen-positive SK-CO-1 ( Fig.26A ) and antigen-negative MDA-MB-231( Fig.26B ) cell lines compared to in vitro potency. One representative experiment is shown, with mean ± SD of triplicates; Fig.26B The legend shown in also applies to Fig.26A .

[0049] FIG. 27 : Potent bystander effects of ADC1 and ADC2 on antigen-negative MDA-MB-231 cells co-cultured with antigen-positive SK-CO-1 cells ( Fig.27A ADC1 or ADC2 had no nonspecific effects on MDA-MB-231 cells alone ( Fig.27B ). Shown is one representative experiment with mean ± SD of duplicates.

[0050] FIG28 : ADC1 and ADC2 have a more potent bystander effect on antigen-negative MDA-MB-231 cells co-cultured with antigen-positive SK-CO-1 cells than ADC SAR DM4 ( Fig.28A and Fig.28B The tested ADCs had no nonspecific effects on MDA-MB-231 cells alone ( Fig.28C ). Shown is one representative experiment with mean ± SD of duplicates.

[0051] Fig.29 : Efficacy of ADC1 and ADC2 in a CRC PDX model (COPF217) after a single treatment.

[0052] Fig.30 : Efficacy of ADC1 in a NSCLC PDX model (LUPF160151) after a single treatment.

[0053] Fig.31 : Efficacy of ADC1 in a gastric cancer PDX model (GAX066) after a single treatment.

[0054] Fig.32 : Efficacy of ADC1 compared with ADC3 in a pancreatic xenograft model (HPAF-II).

[0055] Fig.33 : Efficacy of ADC1 compared with ADC SAR DM4 in the CRC PDX model (COPF230).

[0056] Fig.34 : Efficacy of ADC 1 compared with ADC SAR DM4 in the CRC PDX model (REPF210).

[0057] Fig.35 : Efficacy of ADC1 compared to ADC SAR DM4 in the gastric PDX model GAPF313 (interim analysis of ongoing experiments).

[0058] Fig.36 : Typical SEC chromatograms showing the purity of input mAb and final BDS of mAb1-M / ADC1-M.

[0059] Fig.37 : shows a typical RP-HPLC chromatogram of DAR determination of the final BDS of mAb1-M / ADC1-M.

[0060] Fig.38 : Typical SEC chromatograms showing the purity of input mAbs and final BDS for ADC7-M, ADC2-M, and ADC5-M.

[0061] Fig.39 : shows typical RP-HPLC chromatograms of DAR determination of final BDS of ADC7-M, ADC2-M and ADC5-M.

[0062] Fig.40 : ADC1-M, ADC4-M and free payload against antigen-positive SK-CO-1 ( Fig.40 a) SNU-16( Fig.40 b) MKN-45( Fig.40 c) and LS174T( Fig.40 d) Cell lines and antigen-negative MDA-MB-231 ( Fig.40 e) In vitro potency compared to cell lines. One representative experiment is shown, mean ± SD of duplicates.

[0063] Fig.41: ADC2-M, ADC5-M and free payload against antigen-positive SK-CO-1 ( Fig.41 a) SNU-16( Fig.41 b) MKN-45( Fig.41 c) and LS174T( Fig.41 d) Cell lines and antigen-negative MDA-MB-231 ( Fig.41 e) In vitro potency compared to cell lines. One representative experiment is shown, mean ± SD of duplicates.

[0064] Fig.42 : Pharmacokinetic profiles of ADC1, ADC1-M, ADC2-M, ADC6-M and ADC7-M in huFcRn Tg276 mice (total antibody).

[0065] Fig.43 : Changes in tumor volume after treatment with ADC1-M and ADC2-M relative to vehicle control.

[0066] Fig.44 : More potent bystander effect of ADC1-M, ADC2-M, ADC6-M and ADC7-M on antigen-negative MDA-MB-231 cells co-cultured with antigen-positive SK-CO-1 cells compared to ADC SAR DM4. One representative experiment is shown, mean ± SD of duplicates.

[0067] Fig.45 : Efficacy of ADC1-M and ADC3-M compared to ADC8 in the HPAF-II xenograft model.

[0068] Fig.46 : Efficacy of ADC1-M, ADC3-M compared with ADC SAR DM4 in the CRC PDX model (COPF230) after a single treatment.

[0069] Fig.47 : Efficacy of ADC1-M, ADC3-M compared to ADC SAR DM4 in a GC PDX model (GAPF313) after multiple treatments. DETAILED DESCRIPTION definition

[0070] As used herein, "CEACAM5" 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, for example, on the surface of colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer and other solid tumors. The reference sequence of the full-length human CEACAM5, including the signal peptide (positions 1-34) and the propeptide (positions 686-702), can be obtained from the GenBank database under the accession number AAA51967.1; the amino acid sequence reads as follows: Five non-synonymous SNPs have been identified with a frequency higher than 2% in the Caucasian population, four of which are located in the N domain of human CEACAM5 (at positions 80, 83, 112, 113) and the last one in the A2 domain of human CEACAM5 (at position 398). GenBank AAA51967.1 contains the major haplotypes (I80, V83, I112, I113 and E398).

[0071] A "domain" or "region" can be any region of a protein, which is generally defined based on sequence homology, and is generally associated with a specific structural or functional entity. It is known that CEACAM family members are composed of Ig-like domains. The term domain is used herein to refer to a single Ig-like domain, such as an "N domain", or to a group of consecutive domains, such as an "A2-B2 domain".

[0072] The domain sequence of human CEACAM5 is as follows (based on GenBank AAA51967.1 sequence; SEQ ID NO: 1): Human CEACAM5 domain Position in SEQ ID NO:1 N 35-142 A1 143-237 B1 238-320 A2 321-415 B2 416-498 A3 499-593 B3 594-685 Thus, the A2-B2 domain of human CEACAM5 consists of amino acids 321-498 of SEQ ID NO:1.

[0073] The reference sequence of the cynomolgus macaque CEACAM5 protein is available (NCBI reference sequence XP_005589491.1), and the amino acid sequence reads as follows: mgspsaplhrwcipwqtllltaslltfwnppttaqltiesrpfnvaegkevlllahnvsqnlfgyiwykgervdasrrigscvirtqqitpgpahsgretidfnasllihnvtqsdtgsytiqvike dlvneeatgqfrvypelpkpyissnnsnpvedkdavaltcepetqdttylwwvnnqslpvsprlelssdnrtltvfniprndttsykcetqnp vsvrrsdpvtlnvlygpdaptisplntpyragenlnlschaasnptaqyfwfvngtfqqstqelfipnitvnnsgsymcqahnsatglnrttvt aitvyaelpkpyitsnnsnpiedkdavtltcepetqdttylwwvnnqslsvssrlelsndnrtltvfniprndttfyecetqnpvsvrrsdpv tlnvlygpdaptisplntpyragenlnlschaasnpaaqyswfvngtfqqstqelfipnitvnnsgsymcqahnsatglnrttvtaitvyvelp kpyissnnsnpiedkdavtltcepvaenttylwwvnnqslsvsprlqlsngnriltllsvtrndtgpyecgiqnsesakrsdpvtlnvtygpd tpiisppdlsyrsganlnlschsdsnpspqyswlingtlrqhtqvlfiskitsnnngayacfvsnlatgrnnsivknisvssgdsapgssglsa ratvgiiigmlvgvalm (SEQ ID NO: 2) (Signal peptide is italic; A2-B2 domain is bold; GPI anchor is Underline ; N-A1-B1 domain is in regular font between the signal peptide and the A2-B2 domain; A3-B3 domain is in regular font between the A2-B2 domain and the GPI anchor).

[0074] A "coding sequence" or a sequence "encoding" an expression product, such as a polypeptide, protein or enzyme, is a nucleotide sequence that, when expressed, results in the production of the polypeptide, protein or enzyme, i.e., the nucleotide sequence encodes the amino acid sequence of the polypeptide, protein or enzyme. The coding sequence of a protein may include a start codon (usually ATG) and a stop codon.

[0075] As used herein, reference to a particular protein (e.g., an antibody) may include polypeptides having a native amino acid sequence, as well as variants and modified forms that are unrelated to their source or mode of preparation. A protein having a native amino acid sequence is a protein having the same amino acid sequence as obtained from nature. Such native sequence proteins can be isolated from nature, or can be prepared using standard recombinant and / or synthetic methods. Native sequence proteins particularly include naturally occurring truncated or soluble forms, naturally occurring variant forms (e.g., alternative splicing forms), naturally occurring allelic variants, and forms including post-translational modifications. Native sequence proteins include proteins that carry post-translational modifications, such as glycosylation or phosphorylation, or other modifications of certain amino acid residues.

[0076] The term "gene" refers to a DNA sequence encoding or corresponding to a specific amino acid sequence comprising all or part of one or more proteins or enzymes, and may or may not include a regulatory DNA sequence, such as a promoter sequence, which determines the condition of, for example, expressing a gene. Some genes that are not structural genes can be transcribed into RNA from DNA, but are not translated into amino acid sequences. Other genes can serve as regulators of structural genes or regulators of DNA transcription. In particular, the term gene can be used for the genomic sequence of coded proteins, i.e., sequences comprising regulators, promoters, introns and exon sequences.

[0077] In this article, a sequence "having at least 85% identity" to a reference sequence is a sequence having 85% or higher, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity over its full length to the reference sequence. The percentage of "sequence identity" can thus be determined by comparing two such sequences over their full length using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443 (1970)), e.g., using the program NeedleE (EMBOSS) with the BLOSUM62 matrix and a global pairwise alignment of the following parameters: Gap Opening=10, Gap Extension=0.5, Terminal Gap Penalty=False, Terminal Gap Opening=10, Terminal Gap Extension=0.5 (which is the standard setting).

[0078] A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a side chain with similar chemical properties (e.g., charge, size, or hydrophobicity). Typically, conservative amino acid substitutions do not substantially change the functional properties of a protein. Examples of amino acid groups 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 substitution groups can also be defined based on amino acid size.

[0079] An "antibody" (also referred to as an "immunoglobulin") may be, 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, λ (I) and κ (κ). There are five major heavy chain classes (or isotypes) that determine aspects of the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each antibody chain contains different sequence domains (or regions). The light chain of a typical IgG antibody includes two regions, a variable region (VL) and a constant region (CL). The heavy chain of a typical IgG antibody includes four regions, a variable region (VH) and a constant region (CH), the latter consisting of three constant domains (CH1, CH2, and CH3). The variable regions of the light and heavy chains determine the binding and specificity to the antigen. The constant regions of the light and heavy chains may provide 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.

[0080] The specificity of antibody lies in the structural complementarity between antibody binding site and antigenic determinant.Antibody binding site is made up of residues mainly from so-called hypervariable region or complementary determining region (CDR).Complementary determining region (CDR) therefore refers to the amino acid sequence that defines the binding affinity and specificity of the Fv region of antibody together.The light (L) chain and heavy (H) chain of antibody each have three CDRs, respectively named as CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H.Therefore, the antigen binding site of conventional antibody includes six CDRs, which include CDR groups from each of heavy chain and light chain variable region.

[0081] "Framework region" (FR) refers to the amino acid sequence inserted between CDRs, i.e., those parts of the immunoglobulin light chain and heavy chain variable regions that are relatively conservative in different immunoglobulins in a single species. The light chain and heavy chain of an immunoglobulin each have four FRs, respectively named FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H. As used herein, "human framework region" is a framework region that is substantially identical (about 85% or higher, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to the framework region of a naturally occurring human antibody.

[0082] In the context of the present invention, the CDR / FR definition in an immunoglobulin light or heavy chain is determined based on the IMGT definition (Lefrnc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org).

[0083] The term "antibody" as used herein includes conventional antibodies and fragments thereof, and single domain antibodies and fragments thereof, such as the variable heavy chain of a single domain antibody; the term "antibody" as used herein also includes chimeric, humanized, bispecific or multispecific antibodies, and other types of engineered antibodies. The term "antibody" includes monoclonal antibodies.

[0084] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule with a single amino acid sequence directed against a specific antigen, and should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies can be produced, for example, by a single clone of a B cell or hybridoma, but can also be recombinant, for example, produced by methods involving genetics or protein engineering.

[0085] The term "chimeric antibody" 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 the broadest sense. In one embodiment, a chimeric antibody comprises the VH and VL of an antibody derived from a non-human animal associated with the CH and CL of another antibody (which is a human antibody in some embodiments). As non-human animals, any animal such as mouse, rat, hamster, rabbit, etc. can be used. Chimeric antibodies can also represent multispecific antibodies that are specific for at least two different antigens.

[0086] The term "humanized antibody" refers to an antibody that is wholly or partially non-human in origin and has been modified to replace certain amino acids (e.g., amino acids in the framework regions of VH and VL) to avoid or minimize immune responses in humans. The constant regions of humanized antibodies are typically human CH and CL regions.

[0087] A "fragment" of an antibody (e.g., a conventional antibody) comprises a portion of an intact antibody such as IgG, in particular the antigen binding region 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 by antibody fragments. A conventional antibody fragment may also be a single domain antibody, such as a heavy chain antibody or VHH.

[0088] The term "Fab" means an antibody fragment having a molecular weight of about 50,000 Da and antigen binding activity, in which about half of the N-terminal side of the heavy chain and the entire light chain are bound together by a disulfide bond. It is usually obtained in the fragment by treating IgG with the protease papain.

[0089] The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 Da and antigen binding activity, which is slightly larger than two identical Fab fragments bound via a disulfide bond in the hinge region. It is usually obtained in the fragment by treating IgG with the protease pepsin.

[0090] 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 of the hinge region of F(ab')2.

[0091] Single-chain Fv ("scFv") is a covalently linked VH::VL heterodimer, which is usually expressed by a gene fusion comprising genes encoding VH and VL connected by a linker encoding a peptide. The human scFv fragments of the present invention include CDRs maintained in an appropriate conformation, for example, by using genetic recombination techniques. Divalent and multivalent antibody fragments can be formed spontaneously by the association of monovalent scFvs, or can be generated by coupling monovalent scFvs (such as divalent sc(Fv)2) via a peptide linker. "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" represents two dsFvs coupled by a peptide linker.

[0092] The term "bispecific antibody" or "BsAb" refers to an antibody comprising two different antigen binding sites. Thus, BsAb can, for example, simultaneously bind two different antigens. Genetic engineering has been increasingly used to design, modify and produce antibodies or antibody derivatives with desired binding properties and effector function groups, as described, for example, in EP 2 050 764 A1.

[0093] The term "multispecific antibody" refers to an antibody that contains two or more different antigen binding sites.

[0094] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in 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 the complementary domains of another chain and generate two antigen-binding sites.

[0095] The term "hybridoma" refers to a cell obtained by fusion of B cells prepared by immunizing a non-human mammal with an antigen and a myeloma cell derived from a mouse or the like that produces a desired monoclonal antibody having antigen specificity.

[0096] "Purified" or "isolated" when referring to polypeptides (e.g., antibodies) or nucleotide sequences refers to the presence of the molecule in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" refers to the presence of at least 75%, 85%, 95%, 96%, 97%, or 98% by weight of biological macromolecules of the same type. 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 subject polypeptide; however, the molecule may include some additional bases or moieties that do not adversely affect the basic properties of the composition.

[0097] The term "subject" as used herein refers to a mammal, such as a rodent, a cat, a dog, a primate or a human. In an embodiment of the present invention, the subject (or patient) is a human. Antibodies of the present invention

[0098] The present inventors have successfully generated, screened and selected specific anti-CEACAM5 antibodies that unexpectedly exhibit a combination of several properties that make them ideally suited for cancer therapy, particularly as part of an immunoconjugate (antibody-drug conjugate). For example, the antibodies of the present invention exhibit high affinity for both human and cynomolgus macaque CEACAM5 proteins, and they do not significantly cross-react with human CEACAM1, CEACAM6, CEACAM7 and CEACAM8 proteins or cynomolgus macaque CEACAM6 protein. The present inventors have determined the amino acid sequences of such monoclonal antibodies according to the present invention.

[0099] The present invention provides an isolated antibody that binds to human CEACAM5 protein; and 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), and preferably comprising the 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 mutation); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y, S254T, and T256E (YTE mutation); (e) K222R; and wherein Eu numbering is used for said amino acid substitutions; And wherein 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.

[0100] The present invention further provides an isolated antibody that binds to human CEACAM5 protein; and 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), and preferably comprising the 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 mutation); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y, S254T, and T256E (YTE mutation); (e) K222R; and wherein Eu numbering is used for said amino acid substitutions; And wherein 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.

[0101] Preferably, the isolated antibody of the present 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; wherein 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.

[0102] The present invention further provides an isolated antibody that binds to human CEACAM5 protein. wherein the isolated antibody preferably 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; and wherein 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; wherein FR1 consists of SEQ ID NO:54 (EVQLQESGPGLVKPSQTLSLTCTVS), FR2 consists of SEQ ID NO:55 (LTWIRQHPGKGLEWIGY), 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 (EIVLTQSPATLSVSPGERATLSCRTS), and FR7 consists of SEQ ID NO:60 (EIVLTQSPATLSVSPGERATLSCRTS). NO:59 (LAWYQQKPGQAPRLLIY), FR7 consists of SEQ ID NO:60 (TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC) and FR8 consists of SEQ ID NO:61 (FGPGTKVDIK).

[0103] The present invention also provides an isolated antibody that binds to 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, and most preferably the sequence GGTLQSPP, and preferably comprising this sequence at the C-terminus of said 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 mutation); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y, S254T, and T256E (YTE mutation); (e) K222R; and wherein Eu numbering is used for said amino acid substitutions; And wherein 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; And wherein preferably the isolated antibody comprises framework regions FR1, FR2, FR3, FR4, FR5, FR6, FR7 and FR8 having the structure 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, 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.

[0104] The present invention also provides an isolated antibody that binds to human CEACAM5 protein and 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); and 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 mutation); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y, S254T, and T256E (YTE mutation); (e) K222R; and / or (ii) at least one light chain constant region (CL) comprising the sequence GGTLQSPP, and preferably comprising this sequence at the C-terminus of said light chain constant region; And wherein 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 edition; DIANE Publishing: Collingdale, PA, USA, 1991), and the positions of the amino acid substitutions shown follow this numbering system. Amino acid substitutions are specified using single-letter amino acid codes. GGTLQSPP may also be included multiple times in the light chain constant region (CL), and may also be included alternatively or additionally in the heavy chain constant region (CH). Preferably, in the two light chain constant regions (CL) of the antibodies of the present invention, each light chain constant region (CL) comprises GGTLQSPP once.

[0105] The antibodies of the present invention can also preferably bind to the cynomolgus monkey CEACAM5 protein.

[0106] In one embodiment of the isolated antibody of the present invention, both heavy chain constant regions (CH) comprise one or more of the amino acid substitutions (a) to (e) and / or both light chain constant regions comprise the sequence GGTLQSPP. Preferred combinations of modifications of the CL and CH chains are summarized in Table 4 below, which shows combinations of modifications of antibodies mAb1-M, mAb2-M, mAb3-M, mAb6-M and mAb7-M. Preferably, the antibodies of the present invention comprise any of the following heavy chain constant region (CH) and light chain constant region (CL) modifications: (a) CH comprises amino acid substitutions L234A, L235A (LALA mutation) and M252Y, S254T and T256E (YTE mutation); or (b) CH comprises the amino acid substitutions L234A, L235A (LALA mutation) and M252Y, S254T and T256E (YTE mutation); and the light chain constant region (CL) comprises the sequence GGTLQSPP (preferably at the C-terminus); or (c) CH comprises the amino acid substitutions L234A, L235A (LALA mutation) and M252Y, S254T and T256E (YTE mutation) and K222R; and the light chain constant region (CL) comprises the sequence GGTLQSPP (preferably at the C-terminus); or (d) CH comprises amino acid substitutions L234A, L235A (LALA mutation); or (e) CH comprises the amino acid substitutions L234A, L235A (LALA mutation); and the light chain constant region (CL) comprises the sequence GGTLQSPP (preferably at the C-terminus).

[0107] Preferably, both CL regions and both CH regions of the antibody of the present invention comprise modifications as outlined in (a) to (e) above.

[0108] In another embodiment of the isolated antibody of the present invention, at least one heavy chain constant region (CH) comprises the amino acid sequence or and / or at least one of the light chain constant regions (CL) comprises the amino acid sequence

[0109] In another embodiment of the isolated antibody of the present invention, the heavy chain constant region (CH) and the light chain constant region (CL) have any one of the following sequence combinations: (1) both CHs contain the sequence of SEQ ID NO: 31 and both CLs contain the sequence of SEQ ID NO: 12; or (2) both CHs contain the sequence of SEQ ID NO: 31 and both CLs contain the sequence of SEQ ID NO: 33; or (3) both CHs contain the sequence of SEQ ID NO: 32 and both CLs contain the sequence of SEQ ID NO: 33; or (4) Both CHs contain and both CLs comprise the sequence of SEQ ID NO: 12; or (5) both CHs contain the sequence of SEQ ID NO: 50 and both CLs contain the 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.

[0110] In an embodiment of the present invention, an antibody having the above-mentioned six CDR sequences comprises a polypeptide having the amino acid sequence (CDRs are shown in bold) and a heavy chain variable region (VH) having an amino acid sequence at least 85% identical to the amino acid sequence (CDRs are shown in bold) Light chain variable region (VL) having an amino acid sequence that is at least 85% identical.

[0111] In an embodiment of the present invention, the antibody having the above-mentioned six CDR sequences 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.

[0112] In an embodiment of the present invention, the antibody further comprises A heavy chain constant region (CH) having an amino acid sequence that is at least 85% identical to the amino acid sequence A light chain constant region (CL) having an amino acid sequence that is at least 85% identical.

[0113] In an embodiment of the present 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.

[0114] It is known that the heavy chain constant region (CH) of an antibody may contain a C-terminal lysine (K) without losing any binding function. Therefore, in the sequences described herein of the antibodies of the present invention, the heavy chain constant region (CH) may 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.

[0115] In a more specific embodiment, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and comprises a polypeptide having an amino acid sequence A heavy chain (HC) having an amino acid sequence that is at least 85% identical to the amino acid sequence In an even more particular 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) 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; and a light chain (LC) comprising the amino acid sequence (SEQ ID NO: 36); or (iii) comprising the amino acid sequence The heavy chain (HC) and the amino acid sequence Light chain (LC); or (iv) Include (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.

[0116] In a still more particular embodiment 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 single amino acids of the antibodies of the invention may be altered in one or more of the above-mentioned sequences (including CDR sequences) by substitution, particularly by conservative substitution. Such changes may be intended, for example, to remove glycosylation sites or deamidation sites, for example, in connection with the humanization of the antibody.

[0117] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and 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 specific antibody is also referred to herein as "mAb1".

[0118] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and 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 specific antibody is also referred to herein as "mAb1-M".

[0119] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and 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 specific antibody is also referred to herein as "mAb2-M".

[0120] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and 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 specific antibody is also referred to herein as "mAb3-M".

[0121] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and 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 specific antibody is also referred to herein as "mAb6-M".

[0122] In some embodiments, the antibody of the present invention is an isolated antibody that binds to human CEACAM5 protein and 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 specific antibody is also referred to herein as "mAb7-M".

[0123] In some embodiments, the antibodies of the present invention bind to the A2-B2 domain of human and cynomolgus macaque CEACAM5. The present invention also provides an antibody that competes for binding to the A2-B2 domain of human and cynomolgus macaque CEACAM5 protein with an antibody comprising the heavy chain 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 one of mAb1-M, mAb2-M, mAb3-M, mAb6-M or mAb7-M.

[0124] The ability of a candidate antibody to compete with an antibody comprising the VH and VL of mAb1 (hereinafter, referred to as a "reference" antibody in the case of competition with the candidate antibody) for binding to the A2-B2 domain of human and / or cynomolgus monkey CEACAM5 protein can be easily determined, for example, by competitive ELISA, in which an antigen (i.e., the A2-B2 domain of human or cynomolgus monkey CEACAM5, or a fragment of human or cynomolgus monkey CEACAM5 comprising the A2-B2 domain, particularly the extracellular domain of human or cynomolgus monkey CEACAM5, or a polypeptide consisting of said fragment) is bound to a solid support, and two solutions containing the candidate antibody and the reference antibody are added separately, 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 with the amount of the reference antibody bound to the antigen when measured for a negative control (e.g., a solution without an antibody). A reduction in the amount of the reference antibody bound in the presence of the candidate antibody compared to the amount of the reference antibody bound in the presence of the negative control indicates that the candidate antibody competes with the reference antibody. Conveniently, the reference antibody may be labelled (eg fluorescently labelled) to facilitate detection of bound reference antibody.Replicate measurements may be performed with serial dilutions of the candidate antibody and / or the reference antibody.

[0125] In some embodiments, the antibodies of the present invention do not bind to or significantly cross-react with human CEACAM1, human CEACAM6, human CEACAM7, human CEACAM8, and cynomolgus monkey CEACAM6 proteins. In some embodiments, the antibodies do not bind to or significantly cross-react with the extracellular domains of the aforementioned human and cynomolgus monkey CEACAM proteins other than CEACAM5.

[0126] "Affinity" is theoretically defined as the equilibrium association between whole antibody and antigen. It can be assessed experimentally by a variety of known methods, such as measuring association and dissociation rates with surface plasmon resonance, or measuring EC in immunochemical assays (ELISA, FACS). 50 (or apparent K D ). In these assays, EC 50 It is the antibody concentration that induces a response halfway between baseline and maximum after some specified exposure time by ELISA (enzyme-linked immunosorbent assay) against a defined concentration of antigen or by FACS (fluorescence-activated cell sorting) against cells expressing the antigen.

[0127] When EC of two antigens 50A monoclonal antibody that binds to antigen 1 (Ag1) is "cross-reactive" with antigen 2 (Ag2) when the affinity of the monoclonal antibody that binds to Ag1 to Ag2 is within 10 times (e.g., 5 times) of its affinity for Ag1, and the affinity for the two antigens is measured by the same method.

[0128] A monoclonal antibody that binds to Ag1 is "not significantly cross-reactive" with 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 with 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 may be EC 50 Or the concentration required to reach a saturation plateau obtained with Ag 1. A monoclonal antibody "specifically binds" (or is "specific for") Ag 1 when it does not significantly cross-react with Ag2.

[0129] In some embodiments, antibodies according to the invention have an affinity for cynomolgus macaque CEACAM5 that is within 10-fold (e.g., 5-fold) of its affinity for human CEACAM5. Thus, antibodies according to the invention can be used for toxicology studies in monkeys, as the toxicity profile observed in monkeys will be relevant to potential adverse reactions expected in humans.

[0130] In some embodiments, the antibodies of the invention have an affinity for human CEACAM5 and / or cynomolgus monkey CEACAM5 that is ≤10 nM; for example, the antibodies of the invention may have an affinity for human CEACAM5 that is 1 to 10 nM, such as an affinity for human CEACAM5 of about 6 nM.

[0131] The affinity for human CEACAM5 or for cynomolgus macaque CEACAM5 can be measured, for example, as an EC in an ELISA using soluble recombinant CEACAM5 as capture antigen. 50 value to measure.

[0132] Alternatively, for the antibodies of the invention, the apparent dissociation constant (apparent K) can be determined by FACS analysis, for example on the tumor cell line MKN45 (DSMZ, ACC 409). D ).

[0133] Furthermore, the antibodies according to the invention have been shown to be able to detect CEACAM5 expression by immunohistochemistry, for example in frozen and formalin-fixed and paraffin-embedded (FFPE) tissue sections.

[0134] Any combination of the embodiments described above and below forms part of the present invention.

[0135] In some embodiments, the antibodies according to the invention are conventional antibodies, such as conventional monoclonal antibodies, or antibody fragments, bispecific or multispecific antibodies.

[0136] In some embodiments, the antibodies according to the invention comprise or consist of IgG or fragments thereof.

[0137] In some embodiments, the antibody of the present invention can be, for example, a mouse antibody, a chimeric antibody, a humanized antibody or a human antibody. Many methods for humanization of antibody sequences are known in the art; see, for example, Almagro & Fransson (2008) Front Biosci.13:1619-1633. A commonly used method is CDR transplantation or antibody remodeling, which includes transplanting the CDR sequence of a donor antibody (usually a mouse antibody) into a framework scaffold of a human antibody of different specificity. Since CDR transplantation can reduce the binding specificity and affinity of a non-human antibody transplanted with CDR, and thereby reduce its biological activity, a back mutation can be introduced at the selected position of the CDR transplanted antibody to maintain the binding specificity and affinity of the parent antibody. The position of the possible back mutation can be identified using the information available in the literature and antibody database. The amino acid residues that are candidates for back mutations are usually those located on the surface of the antibody molecule, while residues that are buried or have a low degree of surface exposure are usually not changed. The alternative humanization technique of CDR transplantation and back mutation is surface reconstruction, wherein retains the non-surface exposed residues of non-human origin, and surface residues are changed to human residues.Another alternative technique is called "directed selection (guided selection)" (Jespers et al. (1994) Biotechnology12,899), and can be used to obtain a complete human antibody retaining the epitope and binding properties of the parent antibody from a mouse antibody.

[0138] For chimeric antibodies, humanization generally involves modification of the framework regions of the variable region sequences.

[0139] The amino acid residues that are part of the CDR are usually not changed due to humanization, although in some cases it may be desirable to change a single CDR amino acid residue, for example to remove a glycosylation site, a deamidation site or an undesirable cysteine ​​residue. N-linked glycosylation occurs by attaching an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. Removal of N-glycosylation sites can be achieved by mutating the Asn or Ser / Thr residues to different residues, for example by conservative substitutions. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, mainly when present in the sequence Asn-Gly, while the degree of deamidation is lower when present in other dipeptide sequences such as Asn-Ala. When such deamidation sites, such as Asn-Gly, are present in the CDR sequence, it may therefore be desirable to remove the site, usually by conservative substitutions to remove one of the related residues. The present invention is also intended to include substitutions in the CDR sequences that remove one of the relevant residues.

[0140] In a humanized antibody or fragment thereof, the variable domains of the heavy and light chains may comprise human acceptor framework regions. Where present, the humanized antibody may further comprise human constant heavy and light chain domains.

[0141] In some embodiments, the antibody according to the present invention may be an antibody fragment (eg, a humanized antibody fragment) selected from Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies.

[0142] In some embodiments, the antibody according to the present invention can be a bispecific or multispecific antibody formed by antibody fragments, at least one antibody fragment is a fragment of an antibody according to the present invention. Multispecific antibodies are multivalent protein complexes, such as described in EP 2 050 764 A1 or US2005 / 0003403 A1.

[0143] The bispecific or multispecific antibodies according to the invention may be specific for (a) human and cynomolgus macaque CEACAM5 proteins and (b) at least one other antigen. In some embodiments, the at least one other antigen is not a member of the human or cynomolgus macaque CEACAM family. In other embodiments, the at least one other antigen may be an epitope on human or cynomolgus macaque CEACAM5 other than the epitope targeted by mAb1.

[0144] 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 an isolated (eg purified) form or contained in a carrier such as a membrane or lipid vesicle (eg liposome). Nucleic acids and host cells of the invention

[0145] Another aspect of the invention relates to an isolated nucleic acid comprising or consisting of a nucleic acid sequence encoding an antibody of the invention as defined above.

[0146] Typically, the nucleic acid is a DNA or RNA molecule, which may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, bacteriophage or viral vector.

[0147] The terms "vector", "cloning vector" and "expression vector" refer to a vector through which DNA or RNA sequences (eg, foreign genes) can be introduced into host cells in order to transform the host and promote the expression (eg, transcription and translation) of the introduced sequences.

[0148] Therefore, another aspect of the present invention relates to a vector comprising the nucleic acid of the present invention as defined above.

[0149] Such vectors may include regulatory elements, such as promoters, enhancers, terminators, etc., to cause or guide the expression of the polypeptide after administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include early promoters and enhancers of SV40 (Mizukami T et al., 1987), LTR promoters and enhancers of Moloney murine leukemia virus (Mizukami T et al., 1987), promoters of immunoglobulin H chains (Mason JO et al., 1985) and enhancers (Gillies SD et al., 1983), etc.

[0150] 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), pSG1βd2-4- (Miyaji H et al., 1990), and the like.

[0151] Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrating plasmids such as pUC, pcDNA, pBR, and the like.

[0152] Other examples of viral vectors include adenovirus, retrovirus, herpes virus and AAV vector. Such recombinant viruses can be produced by techniques known in the art, such as by transfection of packaging cells or by transient transfection with helper plasmids or viruses. 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 are for example found in WO 95 / 14785, WO 96 / 22378, US5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0153] Another object of the present invention relates to a host cell which has been transfected, infected or transformed with a nucleic acid and / or a vector according to the invention.

[0154] The term "transformation" refers to the introduction of a "foreign" (i.e., foreign) gene, DNA or RNA sequence into a host cell so that the host cell will express the introduced gene or sequence to produce a desired substance, usually a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is "transformed."

[0155] 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" refers to host cells and compatible vectors under suitable conditions (eg, suitable for the expression of proteins encoded by foreign DNA carried by the vector and introduced into the host cell).

[0156] 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, but are not limited to, 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 cerevisiae, mammalian cell lines (such as Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced by lymphocytes, fibroblasts, embryonic cells, epithelial cells, neurons, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells in which the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is deficient (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 these cells.

[0157] In order to express humanized antibodies, the expression vector can be a type in which the gene encoding the heavy chain of the antibody and the gene encoding the light chain of the antibody are present on separate vectors, or a type in which the two genes are present on the same vector (tandem type). With respect to the ease of constructing a humanized antibody expression vector, the ease of introducing animal cells, and the balance between 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 humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354), pEE18, etc.

[0158] The present invention also relates to a method for producing a recombinant host cell expressing an antibody according to the present invention, the method comprising the steps consisting of: (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell in vitro or ex vivo, (ii) culturing the obtained recombinant host cell in vitro or ex vivo, and (iii) optionally, selecting cells that express and / or secrete the antibody.

[0159] Such recombinant host cells can be used to produce the antibodies of the invention. Methods for producing antibodies of the invention

[0160] The antibodies of the present invention may be produced by any technique known in the art, such as but not limited to any chemical, biological, genetic or enzymatic technique, alone or in combination.

[0161] Knowing the amino acid sequence of the desired antibody, those skilled in the art can easily produce the antibody or immunoglobulin chain using standard techniques for producing polypeptides. For example, they can be synthesized using a commercially available peptide synthesis device (such as manufactured by A Applied Biosystems, Foster City, California) and according to the manufacturer's instructions using a known solid phase method. Alternatively, as is well known in the art, the antibodies and immunoglobulin chains of the present invention can be produced by recombinant DNA technology. For example, after the DNA sequence encoding the desired polypeptide is incorporated into an expression vector and such vectors are introduced into a suitable eukaryotic or prokaryotic host that will express the desired polypeptide, these polypeptides (e.g., antibodies) as DNA expression products can be obtained, and then known techniques can be used to separate them therefrom.

[0162] For example, the present invention provides the following DNA sequence encoding antibody mAb1: mAb1 heavy chain nucleotide sequence The mVk signal peptide is Underline , Start and stop codons are in italics. The VH region sequences are in bold, and For CDR express: GAAGAGCCTGAGCCTGTCCCCCGGCTGA (SEQ ID NO:15) mAb1 light chain nucleotide sequence The uPA signal peptide is Underline , Start and stop codons are in italics. The VL region sequence is in bold, and For CDR express: The present invention also provides the following DNA sequences encoding antibodies mAb1-M, mAb2-M, mAb3-M, mAb6-M, and mAb7-M: ------- SEQ ID NO 37: HC mAb1-M & mAb2-M (HC-LALA-YTE) (nucleotides) The signal peptide is Underline , Start and stop codons are in italics. The VH region sequences are in bold, and For CDR express: SEQ ID NO 38: HC mAb3-M (HC-K222R-LALA-YTE) (nucleotide) The signal peptide is Underline , Start and stop codons are in italics. The VH region sequences are in bold, and For CDR express: SEQ ID NO 39: LC mAb2-M, mAb3-M and mAb7-M (LC-Tag) (nucleotides) The signal peptide is Underline , Start and stop codons are in italics. The VL region sequence is in bold, and For CDR express: SEQ ID NO 52: HC mAb6-M & HC mAb7-M (nucleotides) The signal peptide is Underline , Start and stop codons are in italics. The VL region sequence is in bold, and For CDR express: SEQ ID NO 53: LC mAb1-M & LC mAb6-M (nucleotides) The signal peptide is underlined. Start and stop codons are in italics. The VL region sequence is in bold, and For CDR express: CD20 isotype control SEQ ID NO 40: HC mAb5-M (anti-CD20-HC) SEQ ID NO 41: LC mAb5-M (anti-CD20-LC) SEQ ID NO 42: HC mAb5-M (anti-CD20-HC) (nucleotide) Signal peptide is underlined Start and stop codons are in italics SEQ ID NO 43: LC mAb5-M (anti-CD20-LC) (nucleotide) Signal peptide is underlined Start and stop codons are in italics SEQ ID NO 64: Rituximab HC SEQ ID NO 65: Rituximab LC The present invention further relates to a method of producing an antibody of the present invention, the method comprising the steps consisting of: (i) culturing a transformed host cell according to the present invention; (ii) expressing the antibody; and (iii) recovering the expressed antibody.

[0163] The antibodies of the present invention can be suitably separated from the culture medium by conventional immunoglobulin purification procedures (such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0164] In some embodiments, the humanized chimeric antibodies of the present invention can be produced by obtaining nucleic acid sequences encoding 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 having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into animal cells.

[0165] As the CH domain of the human chimeric antibody, any region belonging to the heavy chain of human immunoglobulin can be used, for example, those of the IgG class are suitable, and any subclass of the IgG class, such as IgG1, IgG2, IgG3, and IgG4, can be used. Likewise, as the CL of the human chimeric antibody, any region belonging to the light chain of human immunoglobulin can be used, and those of the κ class or λ class can be used.

[0166] Methods for producing humanized or chimeric antibodies may include conventional recombinant DNA and gene transfection techniques well known in the art (see, for example, Morrison SL et al., (1984) and patent documents US Pat. No. 5,202,238; and US Pat. No. 5,204,244).

[0167] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, e.g., Riechmann L et al., 1988; Neuberger MS et al., 1985). Antibodies can be humanized using a variety of techniques known in the art, including, e.g., techniques disclosed in WO2009 / 032661, CDR transplantation (EP239,400; PCT Publication WO91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or surface reconstruction (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA et al. (1994)) and chain shuffling (U.S. Patent No. 5,565,332). General recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0168] Fab of the present invention can be obtained by treating the antibody of the present invention (e.g., IgG) with a protease such as papain. In addition, Fab can be produced by inserting the DNA sequence of the two chains of the Fab encoding the antibody into a vector for prokaryotic expression or for eukaryotic expression, and introducing the vector into a prokaryotic or eukaryotic cell (as the case may be) to express Fab.

[0169] The F(ab')2 of the present invention can be obtained by treating the antibody of the present invention (eg, IgG) with the protease pepsin. In addition, F(ab')2 can be produced by binding the following Fab' via a thioether bond or a disulfide bond.

[0170] 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. In addition, Fab' can be produced by inserting a DNA sequence encoding the Fab' chain of an antibody into a vector for prokaryotic expression or a vector for eukaryotic expression, and introducing the vector into a prokaryotic or eukaryotic cell (as the case may be) for expression thereof.

[0171] The scFv of the present invention can be produced by taking the sequence of the CDR or VH and VL domains of the antibody of the present invention as described above, then constructing a 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 the case may be) to express the scFv. In order to produce a humanized scFv fragment, a well-known technique called CDR transplantation can be used, which includes selecting the complementary determining regions (CDRs) according to the present invention and transplanting them onto a human scFv fragment framework having a known three-dimensional structure (see, for example, WO98 / 45322; WO87 / 02671; US5,859,205; US5,585,089; US4,816,567; EP0173494). Modification of the Antibodies of the Present Invention

[0172] Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody.

[0173] The structure of the antibodies of the invention and the DNA sequences encoding them may be modified and altered and still produce a functional antibody or polypeptide having the desired properties.

[0174] When making changes in the amino sequence of a polypeptide, the hydropathic index of the amino acids may be considered. The importance of the hydropathic index of amino acids to the interactive biological functions of proteins is generally understood in the art. It is generally recognized that the relative hydrophilicity characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn defines 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 properties, which 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); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0175] Another aspect of the present invention also includes function conservative variants of the polypeptides of the present invention.

[0176] For example, certain amino acids in the protein structure may be replaced by other amino acids without significant loss of activity. Since the interaction ability and properties of the protein define its biological function activity, certain amino acid substitutions may be made in the protein sequence, and certainly also in its encoding DNA sequence, while still obtaining a protein with similar properties. It is thus conceivable that various changes may be made to the antibody sequence of the present invention or the corresponding DNA sequence encoding the polypeptide without significant loss of their biological activity.

[0177] It is known in the art that certain amino acids can be replaced by other amino acids with similar hydropathic indexes or scores, and still produce proteins with similar biological activity, i.e., still obtain proteins with equivalent biological functions. It is also possible to use established techniques, such as alanine scanning methods, to identify all amino acids that can be replaced without significantly losing the combination with the antigen in the antibody or polypeptide of the present invention. Such residues can be considered to be neutral because they do not participate in antigen binding or maintain the structure of the antibody. One or more of these neutral positions can be replaced by alanine or another amino acid without changing the main characteristics of the antibody or polypeptide of the present invention.

[0178] Neutral positions can be viewed as positions where any amino acid substitution can be incorporated. In fact, in the principle of alanine scanning, alanine is selected because this residue does not carry specific structural or chemical features. It is generally believed that if alanine can replace a specific amino acid without changing the properties of the protein, many other amino acid substitutions (even if not all) may also be neutral. In the opposite case where alanine is the wild-type amino acid, if a specific substitution can be shown to be neutral, other substitutions may also be neutral.

[0179] As described above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account any of the foregoing properties are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0180] It may also be desirable to modify the antibodies of the invention in terms of effector function, for example to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody, or, for example, to change the binding to Fc receptors. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or in addition, cysteine ​​residues may be introduced in the Fc region, thereby enabling the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus produced may have improved internalization capacity and / or enhanced complement-mediated cell killing and / or antibody-dependent cellular cytotoxicity (ADCC) (Caron PC et al., 1992; and Shopes B. 1992). In some embodiments, the antibodies of the invention may be antibodies with modified amino acid sequences that result in reduced or eliminated binding to most Fcγ receptors, which can reduce uptake and toxicity in normal cells and tissues expressing such receptors (e.g., macrophages, liver sinusoidal cells, etc.). An example of such an antibody is an antibody comprising substitution of two leucine (L) residues to alanine (A) (i.e., LALA) at positions 234 and 235; this double substitution has been shown to reduce the binding of Fc to FcγR, and thus reduce ADCC and reduce complement binding / activation. Another example of such an antibody is an antibody comprising substitution of P329G (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, Vol. 29, No. 10, October 2016, pp. 457–466) in addition to the LALA double substitution. In some embodiments, the antibody of the present invention can thus be an antibody having an amino acid sequence, the amino acid sequence: (i) containing, e.g., a LALA or PG-LALA substitution group, and (ii) otherwise identical to the amino acid sequence of one of the antibodies of the present invention described above with reference to the corresponding SEQ ID NO.

[0181] Another type of amino acid modification of the antibodies of the present invention can be used to change the original glycosylation pattern of the antibody, i.e., by deleting one or more carbohydrate moieties present in the antibody, and / or adding one or more glycosylation sites not present in the antibody. The presence of any of the tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) creates a potential glycosylation site. Adding or deleting glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites).

[0182] Another type of modification includes removing sequences identified as heterogeneous that may lead to degradation products or antibody preparations on a computer or experimentally. As an example, the deamidation of asparagine and glutamine residues may occur depending on factors such as pH and surface exposure. Asparagine residues are particularly prone to deamidation, mainly when present with the sequence Asn-Gly, while the degree of deamidation when present with other dipeptide sequences such as Asn-Ala is lower. When such deamidation sites, particularly Asn-Gly, are present in antibodies or polypeptides, it may therefore be considered to remove the site, typically by conservative substitution to remove one of the associated residues. The present invention is also intended to include such substitutions in the sequence that remove one or more of the associated residues.

[0183] Another type of covalent modification includes chemical or enzymatic coupling of glycosides to antibodies. The advantage of these methods is that they do not require the production of antibodies in host cells with glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyls such as those of serine, threonine or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan, or (f) amide groups of glutamine. For example, such methods are described in WO87 / 05330.

[0184] Removal of carbohydrate moieties present on antibodies can be achieved 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 linked sugar (N-acetylglucosamine or N-acetylgalactosamine), while retaining the antibody intact. Sojahr H et al. (1987) and Edge, AS et al. (1981) describe chemical deglycosylation. Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by using a variety of endo- and exo-glycosidases as described by Thotakura, NR. et al. (1987).

[0185] Another type of covalent modification of the antibody involves linking the antibody to one of a variety of nonprotein polymers (eg, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes), for example 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.

[0186] Other amino acid sequence modifications known in the art may also be applied to the antibodies of the invention. Immunoconjugates of the present invention

[0187] The present invention provides immunoconjugates, which are also referred to herein as antibody-drug conjugates, or more simply as conjugates. As used herein, all of these terms have the same meaning and are interchangeable. Methods suitable for preparing immunoconjugates are known in the art. The immunoconjugates of the present invention can be prepared by in vitro methods (e.g., as described herein).

[0188] The present invention provides an immunoconjugate comprising an antibody of the present invention (such as mAb1, or an antibody having the same six CDRs as mAb1) covalently linked to at least one growth inhibitory agent via a linker.

[0189] The term "growth inhibitory agent" (also called "antiproliferative 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.

[0190] In some embodiments, the growth inhibitory agent is a cytotoxic drug (also known as a cytotoxic agent). In some embodiments, the growth inhibitory agent is a radioactive moiety.

[0191] As used herein, the term "cytotoxic drug" refers to a substance that directly or indirectly inhibits or hinders the function of a cell and / or causes cell destruction. The term "cytotoxic drug" includes, for example, chemotherapeutic agents, enzymes, antibiotics, toxins such as small molecule toxins or enzyme-active toxins, toxoids, vincas, taxanes, maytansinoids or maytansinoid analogs, tomamycin or pyrrolobenzodiazepines. derivatives, nostoc derivatives, leptomycin derivatives, auristatins or dolastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, anti-tubulin agents, CC-1065 and CC-1065 analogs.

[0192] Topoisomerase I inhibitors are molecules or compounds that inhibit the human enzyme topoisomerase I, which is involved in changing the topological structure of DNA by catalyzing the transient breakage and reconnection of single strands of DNA. Topoisomerase I inhibitors are highly toxic to, for example, dividing cells of mammals. Examples of suitable topoisomerase I inhibitors include camptothecin (CPT) and its analogs, such as topotecan, irinotecan, silatecan, cositecan, exatecan, lortotecan, gimatecan, belotecan and rubitecan.

[0193] In some embodiments, the immunoconjugates of the present invention include the cytotoxic drug exatecan as a growth inhibitor. 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 is represented by the following structural formula (I): In other embodiments of the invention, other CPT analogs and other cytotoxic drugs may be used, for example as listed above. Some examples of cytotoxic drugs and conjugation methods are further given in the application WO2008 / 010101, incorporated by reference.

[0194] The term "radioactive moiety" refers to a chemical entity (such as a molecule, compound or composition) that comprises or consists of a radioactive isotope suitable for treating cancer, such as At 211 、Bi 212 , Er 169 ,I 131 ,I 125 , Y 90 、In 111 , P 32 、Re 186 、Re 188 、Sm 153 , Sr 89 , or a radioisotope of Lu. Such radioisotopes generally emit mainly β-radiation. In some embodiments, the radioisotope is an α radioisotope, such as thorium 227, which emits α radiation. The immunoconjugates can be prepared, for example, as described in application WO2004 / 091668.

[0195] In the immunoconjugates of the present invention, the antibodies of the present invention are covalently linked to at least one growth inhibitor via a linker. As used herein, "linker" refers to a chemical part comprising a covalent bond and / or any atomic chain that covalently attaches a growth inhibitor to an antibody. Linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. The conjugation of the antibodies of the present invention to cytotoxic drugs or other growth inhibitors can be carried out, for example, using a variety of bifunctional protein coupling agents, including but not limited to N-succinimidyl pyridyl dithiobutyrate (SPDB), butyric acid 4-[(5-nitro-2-pyridyl) dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2-yl disulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl (2-pyridyl dithio) propionate (SPDP), succinimidyl (N 1-(p-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 (such as 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 chelator for conjugating radionucleotides to antibodies (WO 94 / 11026).

[0196] In embodiments of the present invention, the linker can be a "cleavable linker" that can promote the release of cytotoxic drugs or other growth inhibitors inside or near cells (e.g., tumor cells). In some embodiments, the linker is a linker that is cleavable in the endosome of a mammalian cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase-labile linker, a photolabile linker, or a disulfide-containing linker (see, e.g., U.S. Pat. No. 5,208,020) can be used.

[0197] When referring to structural formulas representing immunoconjugates, the following nomenclature is also used herein: the growth inhibitory agent and the linker together are also referred to as the [(linker)-(growth inhibitory agent)] moiety; for example, the exatecan molecule and the linker together are also referred to as the [(linker)-(exatecan)] moiety.

[0198] In some particular embodiments of the invention, the linker is a linker that can be cleaved by the human enzyme glucuronidase. For example, the immunoconjugate of the invention may thus have the following formula (II) or formula (IIA), which includes a linker that can be cleaved by glucuronidase: Wherein the antibody is an antibody of the invention, wherein S is a sulfur atom of the antibody, and wherein n is the number of [(linker)-(growth inhibitor)] moieties covalently attached to the antibody. The number n can be, for example, 1 to 10; in more particular embodiments comprising the above formula (II), n is 7 to 8; in even more particular embodiments using the above formula (II), n is 7.5 to 8.0 (i.e., about 8). In embodiments using the above formula IIA, n is preferably 3 to 4 and most preferably 3.5 to 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.

[0199] The number n is also referred to as the "drug / antibody ratio" (or "DAR"); this number n should always be understood as an average for any given (preparation of) immunoconjugate.

[0200] In the above formula (II), the chemical structure between the sulfur atom of the antibody and the growth inhibitory agent is a linker. One of these linkers is also included in each of formulas (IV) to (VIIIA) further depicted below.

[0201] In any of the embodiments having a linker cleavable by glucuronidase, as described above, the growth inhibitory agent can be, for example, exatecan.

[0202] Thus, in some embodiments, the present invention provides an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, wherein the conjugate has the following formula (IV) or formula (IVA): Wherein S is a sulfur atom of the antibody, and wherein n is the number of [(linker)-(exatecan)] moieties covalently attached to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10; in more particular embodiments comprising Formula IV, n is 7 to 8; in even more particular embodiments based on Formula IV, n is 7.5 to 8.0 (i.e., about 8). In embodiments using the above Formula IVA, n is preferably 3 to 4 and most preferably 3.5 to 4.0 (i.e., about 4). In some embodiments, the antibody is mAb1-M, mAb2-M, mAb3-M, mAb6-M, or mAb7-M.

[0203] In some embodiments, in the immunoconjugates of the invention (such as the exatecan conjugates with a glucuronidase cleavable linker as described above), the linker is covalently attached to the antibody at the sulfur atom of a cysteine ​​residue of the antibody. For example, the cysteine ​​residue of the antibody may be one of the cysteine ​​residues capable of forming an interchain disulfide bond (also referred to herein as an interchain disulfide bridge). Since there are four interchain disulfide bonds in IgG1 antibodies, involving a total of eight cysteine ​​residues, attaching the linker to the antibody at the sulfur atom of such cysteine ​​residues provides a DAR of up to 8, and in such cases, the DAR is typically 7 to 8, such as 7.5 to 8.0 (i.e., about 8), provided that the antibody is IgG1 or has the same number of interchain disulfide bonds as IgG1.

[0204] Thus, in some embodiments, the present invention provides an immunoconjugate comprising an antibody according to the present invention covalently linked to exatecan via a linker, wherein the conjugate has the following formula (VI) or formula (VIA): wherein S is the sulfur atom of a cysteine ​​of the antibody, and wherein n is the number of [(linker)-(exatecan)] moieties covalently attached to the antibody. The number n (also referred to as DAR) can be, for example, 1 to 10; in more particular embodiments based on Formula VI above, n is 7 to 8; in even more particular embodiments using Formula VI, n is 7.5 to 8.5 (preferably 8). In embodiments using Formula VIA above, n is preferably 3 to 5, and more preferably 3.5 to 4.5, and most preferably 4.

[0205] In any of the above immunoconjugates, any antibody of the invention (as described above and below) may be used. In some embodiments, the immunoconjugate of the invention comprises mAb1-M, mAb2-M, mAb3-M, mAb6-M or mAb7-M as the antibody.

[0206] Therefore, in some embodiments, the present invention provides an immunoconjugate comprising an antibody according to the present invention (preferably selected from mAb1-M, mAb2-M, mAb3-M, mAb6-M and mAb7-M) covalently linked to exatecan via a linker, wherein the conjugate has the following formula (VIII) or formula (VIIIA): Wherein S is the sulfur atom of cysteine ​​of the antibody (preferably mAb1-M, mAb2-M, mAb3-M, mAb6-M or mAb7-M), and wherein n is the number of [(linker)-(exatecan)] moieties covalently attached 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, from 1 to 10; in more particular embodiments, n is from 7 to 8; in even more particular embodiments, n is from 7.5 to 8.0 (i.e., about 8). In some embodiments, S is the sulfur atom of cysteine ​​of the 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 present invention are listed below: In another 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 and wherein exatecan is linked to the antibody via a linker type and conjugation 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 and wherein exatecan is linked to the antibody via a linker type and conjugation 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 and wherein exatecan is linked to the antibody via a linker type and a conjugation 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 and wherein exatecan is linked to the antibody via a linker type and a conjugation 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 and wherein exatecan is linked to the antibody via a linker type and a conjugation type shown for ADC7-M in Table 4.

[0207] In another preferred embodiment, the present invention provides an antibody-drug conjugate selected from ADC1-M, ADC2-M, ADC3-M, ADC6-M and ADC7-M, which has all the characteristics of the corresponding ADC as summarized in Table 4, including the corresponding DAR as shown in Table 4.

[0208] In other embodiments of the invention, the linker may be a "non-cleavable linker" (eg, an SMCC linker). Release of the growth inhibitory agent from the antibody may occur upon lysosomal degradation of the antibody.

[0209] In other embodiments of the invention, the immunoconjugate may be a fusion protein comprising an antibody of the invention and a cytotoxic or growth inhibitory polypeptide (as a growth inhibitor); such fusion proteins may be prepared by recombinant techniques or by peptide synthesis, i.e., methods well known in the art. The molecule encoding the DNA may comprise the corresponding regions encoding the two parts of the conjugate (the antibody and the cytotoxic or growth inhibitory polypeptide, respectively), the regions being adjacent to each other or separated by a region encoding a linker peptide.

[0210] The antibodies of the invention can also be used in directed enzyme prodrug therapy, such as antibody-directed enzyme prodrug therapy, by conjugating the antibodies to prodrug-activating enzymes that convert prodrugs (e.g., peptidyl chemotherapeutic agents, see WO81 / 01145) into active cytotoxic drugs (see, e.g., WO 88 / 07378 and U.S. Pat. No. 4,975,278). The enzyme component of the immunoconjugate useful in ADEPT may include any enzyme that can act on a prodrug in a manner that converts the prodrug into its more active cytotoxic form. Enzymes useful in this context include, but are not limited to, alkaline phosphatases, which can be used to convert phosphate-containing prodrugs to free drugs; arylsulfatases, which can be used to convert sulfate-containing prodrugs to free drugs; cytosine deaminases, which can be used to convert nontoxic fluorocytosine to the anticancer drug 5-fluorouracil; proteases, such as serratia proteases, thermolysin, subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B and L), which can be used to convert peptide-containing prodrugs to free drugs; D-alanyl carboxypeptidases, which can be used to convert prodrugs containing D-amino acid substituents; carbohydrate cleavage enzymes, such as O-galactosidases and neuraminidase, which can be used to convert glycosylated prodrugs to free drugs; β-lactamases, which can be used to convert β-lactam-derivatized drugs to free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, which can be used to convert drugs derivatized with phenoxyacetyl or phenylacetyl groups, respectively, at the amine nitrogen of the drug to free drugs. The enzyme can be covalently bound to the antibody of the invention by techniques well known in the art, such as using the linkers described above.

[0211] Methods suitable for preparing the immunoconjugates of the present invention are well known in the art (see, e.g., Hermanson GT, Bioconjugate Techniques, 3rd edition, 2013, Academic Press). For example, methods for conjugating cytotoxic drugs to antibodies via linkers covalently attached to cysteine ​​residues of the interchain disulfide bridges of the antibodies are well known.

[0212] Generally, the immunoconjugate of the present invention can be obtained, for example, by a method comprising the following steps: (i) preparing a compound comprising a linker and a growth inhibitory agent (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 the drug-linker compound; (iii) optionally, the conjugate formed in (ii) is then separated from unreacted antibody and / or drug-linker compound.

[0213] The aqueous solution of the antibody can be buffered with a buffer such as histidine, potassium phosphate, acetate, citrate or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer can be selected according to the properties of the antibody. The drug-linker compound can be dissolved in, for example, an organic polar solvent such as dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).

[0214] For conjugation to cysteine ​​residues of the antibody, the antibody is reduced (eg using TCEP) prior to step (ii). Reducing conditions suitable for reducing only interchain disulfide bonds are known in the art.

[0215] The reaction temperature for conjugation is generally 20 to 40° C. The reaction time may vary and is generally 1 to 24 hours. The reaction between the antibody and the drug-linker compound may be monitored by size exclusion chromatography (SEC) with a refractometer and / or UV detector. If the conjugate yield is too low, the reaction time may be extended.

[0216] A number of different chromatography methods are available to those skilled in the art for separation in step (iii): the conjugate may be purified, for example, by SEC, adsorption chromatography (such as 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 may also be used.

[0217] After step (ii) and / or (iii), the solution containing the conjugate may be subjected to an additional purification step (iv), for example by chromatography, ultrafiltration and / or diafiltration. In the case where reduction is performed prior to conjugation, such additional purification steps, for example by chromatography, ultrafiltration and / or diafiltration, may also be performed with the solution containing the antibody after the reduction reaction.

[0218] At the end of such a process, the conjugate is recovered in aqueous solution. The drug / antibody ratio (DAR) is a numerical value that can vary with the nature of the antibody and drug-linker compound used, as well as the experimental conditions used for conjugation, such as the ratio of (drug-linker compound) / (antibody), reaction time, the nature of the solvent and cosolvent (if any). Thus, the contact between the antibody and the drug-linker compound can result in a mixture containing several conjugates that differ from each other by different drug / antibody ratios. Thus, the determined DAR is an average value.

[0219] Using an antibody with four interchain disulfide bridges (e.g. mAb1 or any IgG1 antibody) for conjugation at the cysteine ​​residues of the interchain disulfide bridges (a method well known in the art) offers the advantage that a relatively uniform DAR of about 8 can be achieved by choosing reaction conditions that allow the conjugation to proceed to completion (or at least close to completion).

[0220] An exemplary method that can be used to determine DAR consists of spectrophotometrically measuring the concentration of a solution of the purified conjugate at D The ratio of the absorbance at λ and 280 nm. 280 nm is a wavelength commonly used to measure protein concentrations, such as antibody concentrations. D To enable the drug to be distinguished from the antibody, that is, as is readily known to those skilled in the art, D is the wavelength at which the drug has high absorbance, and λ D The distance from 280 nm is far enough to avoid significant overlap of the absorption peaks of the drug and the antibody. For example, for exatecan (or for camptothecin or other camptothecin analogs), λ D You can choose 370nm, or for maytansinoid molecules, λ D It can be selected as 252nm.

[0221] The method of DAR calculation can be derived, for example, from Antony S. Dimitrov (ed.), LLC, 2009, Therapeutic Antibodies and Protocols, Vol. 525, 445, Springer Science: The DAR of the conjugate at λ is measured on the monomer peak analyzed by size exclusion chromatography (SEC) (allowing calculation of the “DAR(SEC)” parameter) or using a classical spectrophotometer setup (allowing calculation of the “DAR(UV)” parameter). D (A λD ) and at 280nm(A 280 ) at . The absorbance can be expressed as follows: A λD =(C D Xε DλD )+(C A Xε AλD ) A 280 =(C D Xε D280 )+(C A Xε A280 ) in: ·C D and C A are the concentrations of drug and antibody in solution, respectively. ·ε DλD and εD280 The drugs are D and the molar extinction coefficient at 280 nm ·ε AλD and ε A280 Antibody λ D and the molar extinction coefficient at 280 nm.

[0222] Solving these two equations with two unknowns yields the following equations: C D =[(ε A280 XA λD )-(ε AλD XA 280 )] / [(ε DλD Xε A280 )-(ε AλD Xε D280 )] C A =[A 280 -(C D Xε D280 )] / ε A280 The average DAR was then calculated from the ratio of drug concentration to antibody concentration: DAR = C D / C A .

[0223] An alternative method for preparing the immunoconjugates of the invention is described below. The method can be particularly useful for antibodies 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 in at least one of its heavy chain constant regions (CH). Therefore, another aspect of the invention relates to a method for producing an antibody-linker-conjugate comprising the following steps: (1) providing an antibody comprising an amino acid sequence selected from the group consisting of GGTLQSPP, LLQGA, GGLLQGPP, TLQSG, TLQSPP and TLQSA, and more preferably the amino acid sequence TLQSPP or GGTLQSPP (most preferably the sequence GGTLQSPP) in at least one and preferably both of its light chain constant regions (CL) and / or in at least one and preferably both of its heavy chain constant regions (CH); (2) Mix 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: or as well as (c) a linker comprising an H2N-moiety capable of reacting with the antibody from step (1) in the presence of said transglutaminase, and wherein the linker is preferably a drug-linker, wherein said linker is covalently attached to a drug; (3) separating the antibody-linker-conjugate produced in step (2) from unreacted linker and the transglutaminase, preferably by subjecting the mixture from step (2) to size exclusion chromatography.

[0224] In one embodiment, the transglutaminase is encoded by the following polynucleotide:

[0225] In a preferred embodiment of the method, the reaction buffer is 7% DMSO, 24 mM HEPES, pH 7.0.

[0226] Preferably, the antibody used in the method of the present invention further comprises LLQGA and / or GGLLQGPP sequences in at least one of its light chain constant regions (CL) and / or at least one of its heavy chain constant regions (CH).

[0227] In the method of the present invention, the linker is a linker having the formula: wherein R is the remainder of the linker and may optionally further comprise a drug, wherein the drug is preferably exatecan. In a more preferred embodiment of the method of the present invention, the linker is NH2-GGG-β-glucuronide.

[0228] In a preferred embodiment of the method of the present invention, in step (2), the mixture comprises the following drug-linker: In another embodiment of the method of the present invention, the mixture in step (2) comprises 5 molar equivalents of a 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 the heavy chain constant region (CH) of the antibody.

[0229] In another embodiment of the method of the invention, the antibody is an anti-CEACAM5 antibody of the invention as described herein and / or the drug is a growth inhibitory agent as defined herein.

[0230] Another aspect of the invention relates to an antibody-linker conjugate which can be produced according to the method of the invention, wherein the linker is preferably a linker or a drug-linker as described herein in the context of the ADC of the invention.

[0231] Exemplary methods for preparing the immunoconjugates of the invention are described in the Examples. Drug-Linker Compounds

[0232] The present invention also provides compounds comprising a linker and a growth inhibitor (e.g., a cytotoxic drug), which are also referred to herein as "drug-linker compounds." For example, the present invention provides compounds of the following formula (X) or formula (XA): or a physiologically acceptable salt thereof; the compound according to Formula X is also referred to herein as “Drug-Linker Compound 1,” “Compound DL1,” or “DL1,” and the compound according to Formula XA is also referred to herein as “Drug-Linker Compound 1-M,” “Compound DL1-M,” or “DL1-M.”

[0233] These drug-linker compounds can be used to prepare the immunoconjugates of the invention as described above and below.

[0234] The 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. Pharmaceutical composition

[0235] The antibodies or immunoconjugates of the present invention can be combined with pharmaceutically acceptable carriers, diluents and / or excipients, and optionally with sustained-release matrices to form pharmaceutical compositions, wherein the sustained-release matrices include, but are not limited to, biodegradable polymers, non-biodegradable polymers, lipids or sugars.

[0236] Thus, another aspect of the present invention relates to a pharmaceutical composition comprising an antibody or immunoconjugate of the present invention and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0237] "Pharmaceutical" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other undesirable reactions when administered to mammals, especially humans, under appropriate circumstances. Pharmaceutically acceptable carriers, diluents or excipients refer to non-toxic solid, semisolid or liquid fillers, diluents, encapsulating materials or formulation auxiliary agents of any type.

[0238] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, etc. that are physiologically compatible. Examples of suitable carriers, diluents, and / or excipients include, but are not limited to, one or more of the following: water, amino acids, saline, phosphate buffered saline, buffered phosphate, acetate, citrate, succinate; amino acids and derivatives such as histidine, arginine, glycine, proline, glycylglycine; inorganic salts such as NaCl or calcium chloride; sugars or polyols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as polysorbate 80, polysorbate 20, poloxamer 188; etc., and combinations thereof. In many cases, it will be useful to include an isotonic agent such as a sugar, a polyol, or sodium chloride in the pharmaceutical composition, and the formulation may also contain an antioxidant such as a tryptamine and / or a stabilizer such as Tween 20.

[0239] The form of the pharmaceutical composition, route of administration, dosage and regimen will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, and the like.

[0240] The pharmaceutical compositions of the present invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, and the like.

[0241] In one embodiment, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for injection preparations. These can be isotonic, sterile, saline solutions (sodium dihydrogen phosphate or disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of such salts), or dried, especially freeze-dried compositions, which enable the constitution of injectable solutions upon the addition of sterile water or physiological saline, as the case may be.

[0242] The pharmaceutical composition may be administered via a pharmaceutical combination device.

[0243] The dosage for administration can be adjusted according to various parameters, and for example according to the mode of administration adopted, the relevant pathology, or, alternatively, the desired duration of treatment.

[0244] To prepare a pharmaceutical composition, an effective amount of the antibody or immunoconjugate of the present invention can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0245] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol solutions; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions; in all such cases, the form must be sterile and must not degrade by injection using a device or system suitable for the delivery of the drug, and it must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0246] Solutions of the active compound as a free base or a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under normal conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0247] The antibody or immunoconjugate of the present invention can be formulated as a pharmaceutical composition 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 free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, glycine, histidine, procaine, etc.

[0248] The carrier can also be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, by using a coating (such as lecithin), by maintaining the required particle size in the case of a dispersion, and by using a surfactant, suitable fluidity can be maintained. The effect of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In some cases, it is desirable to include isotonic agents, such as sugar or sodium chloride. By using an agent that delays absorption in the composition, such as aluminum monostearate and gelatin, the extended absorption of the injectable composition can be achieved.

[0249] Sterile injectable solutions are prepared by mixing the active compound in the desired amount into an appropriate solvent and any other ingredients listed above (if necessary), followed by filtration sterilization. Typically, dispersions are prepared by mixing various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze drying techniques that produce powders of the active ingredient and any additional required ingredients from previously sterile filtered solutions.

[0250] It is also contemplated that more concentrated or highly concentrated solutions are prepared for direct injection, wherein it is contemplated that DMSO is used as a solvent to produce extremely rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0251] When formulated, the solution may be administered in a manner compatible with the dosage formulation and in an amount such as to be therapeutically effective. The formulation is readily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like may also be used.

[0252] For example, for parenteral administration with an aqueous solution, if necessary, the solution can be appropriately buffered, and first the liquid diluent is isotonic with enough saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, in view of the disclosure, the sterile aqueous matrix that can be used is known to those skilled in the art. For example, a dose can be dissolved in 1ml isotonic NaCl solution, and added to 1000ml subcutaneous infusion or injected at the infusion site of suggestion (see, for example, " Remington's Pharmaceutical Sciences ", the 15th edition, pages 1035-1038 and 1570-1580). Depending on the patient's condition of the subject for treatment, dosage is bound to change. In any case, the personnel responsible for administration will determine the appropriate dose for individual subjects.

[0253] The antibodies or immunoconjugates of the invention can be formulated in therapeutic mixtures to contain, for example, about 0.01 to 100 mg per dose.

[0254] In addition to antibodies or immunoconjugates formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration, time-release capsules, and any other form currently used.

[0255] In some embodiments, the use of liposomes and / or nanoparticles is contemplated for introducing polypeptides into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

[0256] Nanocapsules can generally capture compounds in a stable and reproducible manner. In order to avoid side effects caused by intracellular polymer overload, such ultrafine particles (about 0.1 μm in size) are generally designed using polymers that can degrade in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles or biodegradable polylactic acid or polylactic acid polyglycolic acid copolymer nanoparticles that meet these requirements for use in the present invention are contemplated, and such particles can be easily prepared by those skilled in the art.

[0257] Liposomes can be formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles, also known as multilamellar vesicles (MLVs). MLVs typically have diameters ranging from 25 nm to 4 μm. Ultrasonication of MLVs results in the formation of vesicles with diameters ranging from 200 to 4 μm containing an aqueous solution in the core. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0258] In addition to the examples mentioned above, other drug forms such as nanoparticles, microparticles and microcapsules, implants (eg lipid implants), or self-solidifying or self-emulsifying systems are also envisaged. Treatments and uses

[0259] The inventors have found that the antibodies of the invention (e.g., mAb1) can be internalized as part of a CEACAM5-antibody complex after binding. In addition, they have shown that such antibodies conjugated to a cytotoxic drug (exatecan) mediate cytotoxic effects on tumor cells in vitro. The inventors have also shown that, in the case of a single injection, these immunoconjugates of the invention induce significant antitumor activity in vivo, for example in a mouse xenograft model of human colorectal cancer derived from a patient, when used at a dose of 10 mg / kg. In fact, the immunoconjugates of the invention show a wide range of activity in a large group of in vitro and in vivo models. The cytotoxic potency is closely related to 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. In a dose range discovery study in non-human primates, the immunoconjugates were well tolerated with a side effect profile typical of topoisomerase-I inhibitor chemotherapy. Preclinical data indicate a good therapeutic window for subsequent clinical testing. The antibodies, immunoconjugates and pharmaceutical compositions of the invention are thus useful for treating cancer.

[0260] Therefore, the present invention provides antibodies, immunoconjugates or pharmaceutical compositions of the present invention for use as medicines. For example, the present invention provides antibodies, immunoconjugates or pharmaceutical compositions of the present invention for use in treating cancer. The present invention further provides a method for treating cancer, comprising administering an antibody, immunoconjugate or pharmaceutical composition of the present invention to a subject in need thereof.

[0261] The cancer to be treated with the antibody, immunoconjugate or pharmaceutical composition of the 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. The CEACAM5 expression of a cell can be easily determined, for example by using an antibody according to the invention (or a commercially available anti-CEACAM5 antibody), for example as described in the "Diagnostic Use" section below, and for example by immunohistochemical methods.

[0262] In some embodiments, the cancer to be treated with the antibodies, immunoconjugates or pharmaceutical compositions of the invention is colorectal 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 cancer of the stomach, uterus, endometrium, thyroid or skin. In some specific embodiments, the cancer to be treated with the antibodies, immunoconjugates or pharmaceutical compositions of the invention is colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer or prostate cancer.

[0263] The antibodies or immunoconjugates of the invention may be used in cancer therapy alone or in combination with any suitable growth inhibitory agent.

[0264] As described above, the antibodies of the invention may be conjugated (linked) to a growth inhibitory agent. The antibodies of the invention may thus be used to target said growth inhibitory agent to cancer cells that express or overexpress CEACAM5 on their surface.

[0265] It is also well known that therapeutic monoclonal antibodies can lead to the depletion of cells bearing the antigen specifically recognized by the antibody. Such depletion can be mediated by at least three mechanisms: antibody-mediated cellular cytotoxicity (ADCC), complement-dependent lysis, and direct inhibition of tumor growth through signals mediated by the antigen targeted by the antibody.

[0266] "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, an antigen-bearing target cell. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337, may be performed.

[0267] "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 an antibody that binds to its cognate antigen. To assess complement activation, a CDC assay can be performed, for example as described in Gazzano-Santoro et al. (Journal of Immunological Methods. 1997 March; 202(2): 163-171).

[0268] In some embodiments, the antibodies of the present invention may be antibodies with modified amino acid sequences that result in reduced or eliminated binding to most Fcγ receptors, which can reduce uptake and toxicity by normal cells and tissues expressing such receptors (e.g., macrophages, liver sinusoidal cells, etc.).

[0269] One aspect of the invention relates to a method 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.

[0270] In the context of the present invention, the terms "treating" or "treatment" as used herein refer to reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such terms apply, or reversing, alleviating, inhibiting one or more symptoms of, or preventing one or more symptoms of, the disorder or condition. The term "treating cancer" as used herein refers to inhibiting the growth of malignant cells of a tumor and / or the metastatic progression of the tumor. Such treatments may also result in regression of tumor growth, i.e., a reduction in the size of a measurable tumor. For example, such treatments may result in complete regression of a tumor or metastasis.

[0271] In the context of the therapeutic applications of the present invention, the term "subject" or "patient" or "subject in need" or "patient in need" refers to a subject (e.g., a human or non-human mammal) who is or may be affected by a tumor. For example, the patient may be a patient who has been determined to be susceptible to a therapeutic agent targeting CEACAM5, in particular an antibody or immunoconjugate according to the present invention, for example according to the methods described below.

[0272] "Therapeutically effective amount" refers to an amount sufficient to treat the cancer disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it is to be understood that the total daily dosage 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 reasonable medical judgment. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific therapeutic agent used; the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the specific therapeutic agent used; the duration of treatment; drugs used in combination or concomitantly with the specific therapeutic agent used; and similar factors well known in the medical field. For example, it is well known to those skilled in the art to start the dosage of the 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.

[0273] The antibodies, immunoconjugates or pharmaceutical compositions of the present invention can also be used to inhibit the progression of cancer metastasis.

[0274] The antibodies, immunoconjugates or pharmaceutical compositions of the present invention may also be used in combination with any other therapeutic interventions for treating cancer (e.g., adjuvant therapy) and / or for reducing the growth of metastatic cancer. For example, other therapeutic interventions for such combinations may be standard of care (SOC) therapeutic agents for the cancer to be treated.

[0275] The efficacy of treatment with the antibodies or immunoconjugates or pharmaceutical compositions according to the invention can be readily determined in vivo (e.g., in a mouse model of cancer) by measuring, for example, the change in tumor volume, % tumor regression, partial regression or complete regression between treatment and control groups. Diagnostic Uses

[0276] CEACAM5 has been reported to be highly expressed on the surface of cancer cells such as colorectal, gastric, lung and pancreatic tumor cells, and expression in normal tissues is restricted to a few normal epithelial cells such as colon and esophageal epithelial cells.

[0277] Therefore, CEACAM5 constitutes a cancer marker and has the potential to be used, for example, to indicate the effectiveness of anticancer therapy or to detect disease recurrence.

[0278] In one embodiment, the antibodies of the invention can be used as a component of an assay in the context of a therapy targeting a tumor expressing CEACAM5 to determine a patient's susceptibility to a therapeutic agent, to monitor the effectiveness of an anti-cancer therapy, or to detect recurrence of disease following treatment. In some embodiments, the same antibody of the invention can be used as a component of a therapeutic agent and a component of a diagnostic assay.

[0279] Thus, another aspect of the invention relates to the use of an antibody according to the invention for detecting CEACAM5 expression in vitro in a biological sample from a subject. Another aspect of the invention relates to the use of an antibody according to the invention for detecting CEACAM5 expression in vivo in a subject. When used to detect CEACAM5, the antibody may be labeled with a detectable molecule such as a fluorophore or an enzyme.

[0280] Detection of CEACAM5 can be intended to be used, for example, for detecting the expression of surface protein CEACAM5 on tumor cells: a) diagnosing the presence of cancer in a subject, or b) determining the susceptibility of a patient suffering from cancer to a therapeutic agent targeting CEACAM5, in particular an antibody or immunoconjugate according to the invention, or c) monitoring the effectiveness of an anti-CEACAM5 cancer therapy or detecting cancer recurrence following an anti-CEACAM5 cancer therapy, in particular wherein the therapy is a therapy with an antibody or immunoconjugate according to the invention.

[0281] In an embodiment, the antibody is intended for in vitro or ex vivo diagnostic use. For example, CEACAM5 can be detected in vitro or ex vivo in a biological sample obtained from a subject using the antibody of the invention. The use according to the invention can also be an in vivo use. For example, an antibody according to the invention can be administered to a subject, and antibody-cell complexes can be detected and / or quantified, wherein the detection of the complex indicates cancer.

[0282] The present invention further relates to an in vitro or ex vivo method for 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 conditions suitable for the antibody to form a complex with the biological sample; (b) measuring the level of antibodies bound to the biological sample; and (c) detecting the presence of cancer by comparing the measured level of bound antibody to a control, an increase in the level of bound antibody compared to the control being indicative of cancer.

[0283] The present invention also relates to an in vitro or ex vivo method for determining the susceptibility of a patient suffering from cancer to a therapeutic agent targeting CEACAM5, in particular an antibody or immunoconjugate according to the present invention, the method comprising the following steps: (a) contacting a biological sample from a patient suffering from cancer with an antibody according to the invention, in particular under conditions suitable for the antibody to form a complex with said biological sample; (b) measuring the level of antibodies bound to the biological sample; and (c) comparing the measured level of antibody binding to the biological sample with the level of antibody binding to a control; Wherein an increased level of antibody binding to the biological sample compared to a control indicates that the patient is susceptible to a therapeutic agent targeting CEACAM5.

[0284] In the above methods, the control may be a normal non-cancerous biological sample of the same type, or a reference value determined to represent the antibody binding level in a normal biological sample of the same type.

[0285] In one embodiment, the antibodies of the invention can be used to diagnose cancers expressing CEACAM5, such as colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer or other solid tumors expressing CEACAM5.

[0286] The present invention further relates to an in vitro or ex vivo method for monitoring the effectiveness of an anti-CEACAM5 cancer therapy comprising the following steps: (a) contacting a biological sample from a subject undergoing anti-CEACAM5 cancer therapy with an antibody according to the invention, in particular under conditions suitable for the antibody to form a complex with said biological sample; (b) measuring the level of antibodies bound to the biological sample; and (c) comparing the measured level of bound antibody to the level of antibody bound to a control; wherein a decrease in the level of antibody binding to the biological sample compared to a control indicates that the anti-CEACAM5 cancer therapy is effective. In the method, an increase in the level of antibody binding to the biological sample compared to a control will indicate that the anti-CEACAM5 cancer therapy is not effective. In one embodiment of this method of monitoring effectiveness, the control is a biological sample of the same type as the biological sample being analyzed, but obtained from the subject at an earlier time point during the course of the anti-CEACAM5 cancer therapy.

[0287] The present invention further relates to an in vitro or ex vivo method for detecting cancer recurrence after anti-CEACAM5 cancer therapy, comprising the following steps: (a) contacting a biological sample from a subject (the subject having completed anti-CEACAM5 cancer therapy) with an antibody according to the invention, in particular under conditions suitable for the antibody to form a complex with the biological sample; (b) measuring the level of antibodies bound to the biological sample; and (c) comparing the measured level of bound antibody to the level of antibody bound to a control; wherein an increased level of antibody binding to the biological sample compared to a control indicates cancer recurrence after anti-CEACAM5 cancer therapy. The control may in particular be a biological sample of the same type as the biological sample analyzed, but obtained from the subject previously, i.e. upon or after completion of anti-CEACAM5 cancer therapy.

[0288] The anti-CEACAM5 cancer therapy is, for example, a therapy using an antibody or immunoconjugate according to the invention. The anti-CEACAM5 cancer therapy targets cancers expressing CEACAM5, such as colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer or other solid tumors expressing CEACAM5.

[0289] In some embodiments, the antibodies of the 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 a signal (directly or indirectly).

[0290] With respect to the antibodies according to the present invention, the term "labeled" as used herein is intended to include 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 reaction with a detectable substance.

[0291] The antibodies of the present invention can be labeled with radioactive molecules by any method known in the art. For example, radioactive molecules include, but are not limited to, radioactive atoms used in scintigraphic studies, such as I 123 ,I 124 、In 111 、Re 186 、Re 188 , Tc 99 The antibodies of the invention may also be labeled with spin labels for use in nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0292] "Biological samples" include a variety of 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 include clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples, such as tumor samples.

[0293] In some embodiments, the biological sample can be a formalin-fixed and paraffin-embedded (FFPE) tissue sample.

[0294] The present invention also relates to an in vivo method for 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 the antibody in the patient by imaging, e.g. by detecting a detectable molecule.

[0295] In the method, the cancer may be a cancer expressing CEACAM5, such as colorectal cancer, gastric cancer, non-small cell lung cancer, pancreatic cancer, esophageal cancer, prostate cancer, or other solid tumors expressing CEACAM5.

[0296] The antibodies of the invention may also be used for staging of cancer (eg in radiological imaging). They may be used alone or in combination with other cancer markers.

[0297] The term "detecting" or "detecting" as used herein includes qualitative and / or quantitative detection (ie, measuring levels) with or without reference to a control.

[0298] In the context of the present invention, the term "diagnosis" as used herein refers to the determination of the nature of a medical condition based on a number of collected data, which aims to identify the pathology affecting a subject. Reagent test kit

[0299] 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 be used to detect the surface protein CEACAM5, or for treatment or diagnostic determination. The kit of the present invention can contain antibodies coupled to a solid support such as a tissue culture plate or beads (e.g., agarose beads). A kit can be provided, which contains antibodies for detecting and quantifying the surface protein CEACAM5 in vitro, such as in ELISA or Western blot. Such antibodies that can be used for detection can have a label, such as a fluorescent or radioactive label. Sequence Description

[0300] 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 Nucleic acid sequence: SEQ ID NO: 15 DNA sequence encoding HC of mAb1 SEQ ID NO: 16 DNA sequence encoding LC of mAb1 Amino Acid Sequence: SEQ ID NO: 17 HC of antibody hu8G4 SEQ ID NO: 18 LC of antibody hu8G4 SEQ ID NO: 19 HC of optimized antibody variant 1 SEQ ID NO: 20 Optimized LC of antibody variant 1 SEQ ID NO:21 Optimized LC of antibody variant 2 SEQ ID NO:22 Optimized LC of antibody variant 4 SEQ ID NO: 23 Optimized LC of antibody variant 5 SEQ ID NO: 24 HC of optimized antibody variant 6 SEQ ID NO:25 HC of huMab2-3 (allotype) SEQ ID NO:26 LC of huMab2-3 SEQ ID NO: 27 HC of hmn-14 SEQ ID NO:28 LC of hmn-14 SEQ ID NO: 29rb8G4 HC SEQ ID NO:30 LC of rb8G4 SEQ ID NO: 31 CH(CH-LALA-YTE) of mAb1-M&mAb2-M SEQ ID NO: CH of 32mAb3-M (CH-K222R-LALA-YTE) SEQ ID NO:33 CL of mAb2-M, mAb3-M and mAb7-M (CL-tag) SEQ ID NO:34 HC of mAb1-M and mAb2-M (HC-LALA-YTE) SEQ ID NO: HC of 35mAb3-M (HC-K222R-LALA-YTE) SEQ ID NO:36 LC of mAb2-M, mAb3-M and mAb7-M (LC-tag) 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 Nucleic acid sequence: SEQ ID NO:37 DNA sequence encoding HC (HC-LALA-YTE) of mAb1-M and mAb2-M SEQ ID NO:38 DNA sequence encoding HC of mAb3-M (LC-K222R-LALA-YTE) SEQ ID NO: 39 DNA sequence encoding LC (LC-tag) of mAb2-M, mAb3-M and mAb7-M SEQ ID NO:42 DNA sequence encoding HC of mAb5-M (anti-CD20-HC) SEQ ID NO:43 DNA sequence encoding LC of mAb5-M (anti-CD20-LC) SEQ ID NO:46 DNA sequence encoding transglutaminase SEQ ID NO:52 DNA sequence encoding the HC of mAb6-M and mAb7-M (HC-LALA) SEQ ID NO:53 DNA sequence encoding LC of mAb1-M and mAb6-M 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 Example Example 1: Anti-CEACAM5 Antibodies 1.1 Immunization of transgenic rats and isolation of hybridomas

[0301] To generate monoclonal antibodies against human CEACAM5 protein (carcinoembryonic antigen-related cell adhesion molecule 5; CD66e), human immunoglobulin gene transgenic rats (OmniRat TM ) was obtained from CHARLES RIVER LABORATORIES INTERNATIONAL INC (WILMINGTON, MA). Five animals were immunized four times with CEACAM5 cDNA (UniProt ID no. P06731 encoding amino acids 35-675 of the human CEACAM5 protein sequence; the sequence of P06731 is identical to SEQ ID NO: 1, except that E398 of SEQ ID NO: 1 was replaced by K398) cloned into an Aldevron proprietary immunization vector (pB8-CEA-hum-MC) and transiently transfected into OMT rat cells using a gene gun.

[0302] Anti-CEACAM5 titers were assessed by cell-based ELISA (CELISA) assay using cells expressing CEACAM5 on their cell membranes (titer results are shown below). Sera from immunized animals were collected on day 31 of the immunization protocol after 4 rounds of genetic material immunization (IS31d-4). Sera diluted in PBS + 3% FBS were tested by flow cytometry on mammalian cells transiently transfected with CEACAM5 cDNA cloned into an Aldevron proprietary expression vector (pB1-CEA-hum-MC). 10 μg / ml of goat anti-rat IgG R-phycoerythrin conjugate (Southern Biotech, #3030-09) was used as a secondary antibody.

[0303] 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. The hybridoma cells from this fusion were then transferred to ten 96-well plates. 1.2 CEACAM5 specificity

[0304] Hybridoma supernatants were screened using a cell-based ELISA (CELISA) assay to detect anti-CEACAM5 antibodies that did not bind to CEACAM1 (BGP), CEACAM3 (CGM1a), CEACAM4 (CGM7), CEACAM6 (NCA), and CEACAM8 (NCA-95). 10 μg / ml of goat anti-rat IgG R-phycoerythrin conjugate (Southern Biotech, #3030-09) was used as a secondary antibody.

[0305] Clones showing specificity for human CEACAM5 but not for its related proteins were transferred to a 96-well plate and the hybridoma supernatants were evaluated for specificity and cross-reactivity in an ELISA assay. In this assay, the 8G4 hybridoma clone and its subclones showed specificity for human CEACAM5 and cross-reactivity for cynomolgus monkey CEACAM5. 1.3 Detection of antibody sequences and clones

[0306] Total RNA was prepared from each hybridoma clone by Qiagen according to the RNeasy 96 protocol. Random hexamers and III transcribes total RNA into cDNA.

[0307] The quality of the resulting cDNA was controlled by qPCR, and VH and Vk were amplified by PCR. PCR products were purified using the AMpure XP PCR purification kit in combination with KingFisher instruments.

[0308] 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"). Mach1 TM The reaction mixture was transformed into -T1R chemically competent E. coli. Correctly recombined clones were confirmed by Sanger sequencing. 1.4 Humanization and biochemical characterization of hits, and candidate selection

[0309] 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, a humanized candidate antibody (named hu8G4) was selected for amino acid sequence optimization to improve manufacturability and affinity.

[0310] The amino acid sequence of the humanized candidate antibody hu8G4 is as follows: Heavy Chain: Light chain: 1.5 Biophysical improvement strategies for hu8G4, particularly leading to mAb1

[0311] Evaluation of the variable region sequence of hu8G4 identified six non-germline amino acid residues in the light chain framework and two non-germline amino acid residues in the heavy chain framework. The evaluation of amino acids and sequence motifs that may be prone to post-translational modification, such as deamidation motifs, surface accessible methionine and free cysteine, did not identify any amino acid residues with increased susceptibility (liability). Several designed antibody sequences were generated, in which certain amino acids were replaced by germline-related amino acids at this position. Subsequently, different VH and VL optimized designs were co-expressed as Fab and complete IgG1 molecules in HEK 293 6E cells, purified and tested (see, for example, optimized variants 1-10 below).

[0312] The amino acid sequences of the 10 optimized antibody variants in full IgG1 format are as follows: Variant 1 (VH1.00 / VL1.00) HC: LC: Variant 2 (VH1.00 / VL1.01) HC: SEQ ID NO: 19 LC: 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 LC: Variant 5 (VH1.00 / VL1.04) HC: SEQ ID NO: 19 LC: Variant 6 (VH1.02 / VL1.00) HC: 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

[0313] All optimized variants 1 to 10 performed similarly well in terms of maintaining quality as determined by percent aggregation by size exclusion chromatography, maintaining stability based on fluorescence-monitored thermal unfolding (FMTU), maintaining binding to the MKN-45 cancer cell line, and maintaining selectivity for the target. As an optimized variant with a sequence particularly similar to the germline, variant 8 (i.e., a variant comprising VH1.02 and VL1.02) was selected for further development.

[0314] The selected variant 8 was then subjected to further sequence optimization, in particular to reduce IgG Fc effector function. The resulting final sequence optimized (so) clone, which was designated so8G4 (also referred to herein as mAb1), showed improved affinity and improved manufacturability compared to the parental clone, and also showed reduced or no binding to FcγRI, FcγRIIa, FcγRIIIa, FcγRIIIa / complex, C1q, FcγRIIb and FcγRIIIb, while maintaining affinity for 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 above) Light chain (LC): SEQ ID NO: 14 (as defined above) 1.6 In vitro characterization of mAb1

[0315] Several properties of antibody mAb1 were characterized using in vitro assays including: binding affinity, selectivity, and internalization. 1.6.1 Binding affinity

[0316] To determine the binding affinity of soluble antibody analytes to captured target protein CEACAM5 (human or cynomolgus macaque), the following experimental conditions were used on the Octet Red instrument: • Biosensors coated with streptavidin. Biotinylated target protein concentration (ECD stands for extracellular domain): o Capture human_CEACAM5_ECD-his-biotin R&D system (biotinylated using conventional methods) at 2.5 μg / ml for 900 seconds at 1000 rpm. o Recombinant cynomolgus monkey CEACAM5_ECD-His-biotin (biotinylated using conventional methods) obtained from Syngene was captured at 5 μg / ml for 900 seconds at 1000 rpm. Analyte antibody concentration: 200, 100, 50, 25, 12.5, 6.25, 0 nM. The binding affinity KD (equilibrium dissociation constant) value was determined from the measured binding kinetic association (ka) and dissociation (kd) rate constants using Octet evaluation software, where KD = kd / ka Use of Fab-form antibodies

[0317] 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. 1.6.2. Selectivity a) Species and domains

[0318] The selectivity of mAb1 was determined by titrating the antibody from 4 nM to 0.25 pM in an ELISA assay and applying it to 1 μg / ml of conjugated recombinant human (rh) CEACAM5 ECD or its domains N-A1-B1, A2-B2, A3-B3 or conjugated recombinant cynomolgus macaque (mf) CEACAM5 ECD (all obtained from Syngene). The results are shown in Figure 1 The following is a summary: The binding EC50 for rhCEACAM5 was 153.4 pM. The binding EC50 to the rhA2-B2 domain was 166.9 pM. The binding EC50 for mfCEACAM5 was 324.3 pM. No binding was detected to rhN-A1-B1 or rhA3-B3 or BSA (bovine serum albumin, used as a negative control). b) Different CEACAM proteins

[0319] The selectivity of mAb1 Fab for human CEACAM5 and other human CEACAM family members was determined in an ELISA assay. The proteins were coated on a 96-well assay plate: 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 at a concentration of 12 nM on the plate. result:

[0320] In an ELISA assay, mAbl Fab bound to human CEACAM5 (EC50 of 3.04 nM), but not to other human CEACAM family members, even when 1000 nM of mAbl Fab was used, which is a concentration more than 300-fold the EC50 for binding to human CEACAM5.

[0321] In the ELISA assay, the Fab of mAbl also did not bind to an irrelevant protein (BSA) at all concentrations tested. 1.6.3 Cell Binding of mAb1

[0322] The ability of an antibody to bind to its target protein on cells is determined by titrating the antibody on cells expressing the target (e.g., human CEACAM5) and measuring the fluorescent MFI of the cells. The model cells used for antibody binding comparison are the MKN45 cell line expressing human CEACAM5 and the CHO cell line expressing mfCEACAM5. The titration is performed at 10 points × 4 dilutions, with a curve starting concentration of 2000 nM in assay buffer (PBS × 1 containing 1% BSA).

[0323] Example data: mAb1 bound to the MKN-45 cell line expressing human CEACAM5 with EC50 = 10.62 ± 1.6 nM. mAb1 bound to the CHO cell line expressing mfCEACAM5 with EC50 = 4.8 ± 0.6 nM. 1.6.4 Comparison of cell binding with known antibodies

[0324] The cellular binding of our lead antibody mAb1 was compared to the known ADC-related antibodies huMab2-3 (as in the known ADC SAR408701) and hMN14 (also referred to herein as hman-14) (as in the known ADC govenotecan-labetuzumab or IMMU-130) on the CEACAM5-expressing MKN45 cell line.

[0325] In the experiments described below, the following amino acid sequence was used for the above-mentioned known antibody: huMab2-3: HC (allogeneic): LC: HMN-14: HC: LC:

[0326] Rituximab was included in the comparison as a control. The results of antibody binding to cells are shown in Figure 2 The following is a summary: mAb1(so8G4)=8.3nM huMab2-3=6nM hmn-14=11.8nM

[0327] Average of several experiments (EC50): mAb1(so8G4)=10.4nM±1.6nM(n=12) huMab2-3=4.9nM±1.6nM(n=3) hmn-14=16nM(n=2)

[0328] Cellular binding of anti-CEACAM5 antibodies to the MKN45 cell line showed that the binding of mAbl was similar to that of known antibodies. 1.6.5 Internalization assay using Cell Discoverer

[0331] A relevant property of an ADC is its internalization into cells and lysosomes expressing the target, and thus internalization is a relevant property of antibodies 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 emits strong fluorescence at a pH below 6.0 after excitation. This fluorescence can be imaged in a Cell-Discoverer7 (Zeiss), and the internalization rate can be calculated.

[0329] To analyze the internalization rate of several antibodies, MKN45 cells were seeded in 96-well, dark, flat transparent bottom plates (Cellvis) at 25,000 cells / well. Cells were cultured overnight with 100 μl / well of RMPI-1640+10% FBS (Thermo). Cell culture medium was removed and cells were stained with 100 μl of 10 μg / ml Hoechst dye diluted in PBS×1 at room temperature (RT) for 15 min in the dark. Cells were subsequently washed twice with PBS×1.

[0330] 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 warm RPMI1640 + 10% FBS without phenol red and added to their corresponding wells. The plates were incubated in Cell Discoverer at 37° C., 5% CO2 for 20 hours, and images were acquired every 20 minutes, as described further below.

[0332] pHrodo-labeled antibodies were internalized into late endosomes and lysosomes of cells and imaged by Cell-Discoverer7 (Zeiss) using fluorescence at 567 nm excitation and 592 / 25 nm emission detectors. Cell nuclei were labeled with Hoechst and imaged at 385 nm excitation and 425 / 30 nm emission detectors.

[0333] The fluorescence of each well was recorded every 20 min for 20 h. The summed fluorescence intensity (SFI) of each cell was calculated by Excel analysis using Zen Software (ZEN 3.1) and linear regression.

[0334] The results are shown in Figure 3 and Figure 4 and the slope of the linear part of the curve (see Figure 4 ) are also summarized in the following table: in conclusion:

[0335] 1.so8G4 (mAb1) has a higher average binding rate (28958±766) than humab2-3 (18917±1416) and hmn-14 (22268±3060).

[0336] 2. Compared with humab2-3 and hmn-14, so8G4 (mAb1) also has a higher internalization strength.

[0337] 3. mAb1-M, mAb2-M, mAb3-M, mAb6-M and mAb7-M are expected to show internalization properties corresponding to mAb1. 1.7 Exemplary Methods for Producing mAb1 (eg, mAb1 for Conjugation to a Drug-Linker Compound)

[0338] Anti-CEACAM5 antibody mAb1 was produced in a recombinant CHO-K1Sv cell line. Cell culture was performed in a 200 l disposable bioreactor in batch mode. Cells were grown at 37°C in a proprietary CHO batch fed growth medium supplemented with glucose. The culture was fed with a mixture of proprietary feed components on days 3, 5, 7 and 10 after inoculation.

[0339] Use 3×1.1m 2 Millistak+Pod DOHC (Millipore MD0HC10FS1) and 1.1m 2 Crude conditioned media from the bioreactor run was clarified with a Millistak+Pod XOHC (Millipore #MX0HC01 FS1) filter, followed by a final filtration with a Millipore Opticap XL3 0.5 / 0.2 μm filter (Millipore #KHGES03HH3).

[0340] After clean-up, antibody mAb1 was purified using a standard antibody purification method consisting of a protein A capture step and an ion exchange chromatography step. Anti-CEACAM5 antibody mAb1 was used as an intermediate for the production of ADC molecules. 1.8 Expression and purification of human / rabbit chimeric variants of mAb1 and their use in immunohistochemistry (IHC) on formaldehyde-fixed and paraffin-embedded cell lines and human tumor tissues

[0341] The 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") has the following amino acid sequence: Heavy chain Light chain

[0342] The antibody rb8G4 was expressed in HEK cells (Expi 293 suspension cells) by transient transfection and purified using MabSelect SuRe and citrate buffer. rb8G4 was subsequently used for IHC on formaldehyde-fixed and paraffin-embedded cell lines and human tumor tissues: Materials and methods Cell lines and tissues Human cancer cell lines were cultured from Merck Cell Bank, fixed in 4% buffered formaldehyde, and embedded in paraffin (FFPE). Paraffin-embedded cell lines were arrayed into cell line microarrays (CMA) (Zytomed). FFPE tissue sections with tissue microarrays (TMA) of human organs were from amsbio (FDA Standard Tissue Array, T8234701). FFPE human tumor samples were provided by BioIVT and Indivumed GmbH. method

[0343] 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 fixed 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 dewaxing, the sections were heated in Tris-EDTA buffer pH 8 (CC1, Roche Diagnostics) for epitope retrieval. The sections were incubated with monoclonal primary 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) was used as an isotype control antibody. The primary antibody was followed by the HQ anti-rabbit IgG detection kit (Roche Diagnostics). Slides were counterstained with hematoxylin, washed in tap water, dehydrated, and mounted on glass coverslips in Entellan Neu (VWR) permanent mounting medium.

[0344] CMA and TMA with human organ tissue were stained and scanned with NanoZoomeR (Hamamatsu) at a resolution of 0.46 μm / pixel. Human tumor sections were stained and scanned with an AxioScan.Z1 (Zeiss) instrument at a resolution of 0.44 μm / pixel. The scanning of CMA was analyzed with image analysis software HALO (Indica Labs, USA). In order to determine the amount of antigen present, the positive brown staining area was calculated as the area percentage of the living tissue area. Staining (arbitrary units) was calculated as antibody staining (AU) =% positive tissue area * average optical density of brown (OD range of 0 to 1). The maximum value of antibody staining is 100 = 100% of the tissue area is black (grayscale OD value is 1).

[0345] CEACAM-5 mRNA data of cancer cell lines were obtained from Cancer Cell Line Encyclopedia (CCLE; Broad Institute of MIT & Harvard). result Validation in cancer cell lines and normal human tissues

[0346] Antibody rb8G4 showed signals in the cytoplasm and plasma membrane on FFPE cancer cell lines ( Figure 5 ).

[0347] The specificity of antibody rb8G4 for FFPE tissues / cells was shown by comparing the staining signals of 104 cancer cell lines with the mRNA expression of these cell lines (CCLE dataset). The resulting Pearson correlation coefficient r = 0.88 supports the conclusion that antibody rb8G4 (also known as SO8G4AB323) detects the CEACAM-5 epitope in FFPE tissues / cells ( Figure 6 ). The cancer cell line microarray and individually selected positive and negative cell lines were used as control matrices in staining procedures for human normal and tumor tissues.

[0348] In normal human tissues, staining with antibody rb8G4 ( Figure 7 ) and CEACAM-5 mRNA expression ( Figure 8 )(Source: http: / / www.proteinatlas.org / ENSG00000105388-CEACAM5 / tissue), further supporting the specificity of the antibody for CEACAM-5. Human tumor tissue

[0349] Such as colorectal cancer ( Fig. 9 )、Gastric cancer( Fig.10 ), esophageal cancer( Fig.11 ) and non-small cell lung cancer ( Fig.12 ), antibody rb8G4 stained positive in several human tumor indications. The signal was localized in the cytoplasm and at the plasma membrane. 1.9 Flow cytometry and western blotting using mAb1 and rb8G4

[0350] The binding of mAbl, rb8G4 and commercially available anti-CEACAM5 antibodies to CEACAM5 positive and negative cell lines was compared.

[0351] Methods used: 5E5 to 1E6 cells were used for flow cytometry analysis using BD FACSCanto II (BD Biosciences) in 5 ml polystyrene tubes. Staining with 10 μg / mL primary antibodies (mAb1, rb8G4, mouse monoclonal Agilent Dako #M7072 clone #IL7) and corresponding 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 at 4°C in 50 μL 1% PBS / BSA for 20-30 min. Between and after the staining steps, cells were washed three times with 1% PBS / BSA and resuspended in 500 μL 1% PBS / BSA (containing 0.2 μg / mL DAPI for live cell gating) for flow cytometry analysis. For data evaluation, FlowJo software (BD Biosciences) was used.

[0352] Results: mAbl and rb8G4 showed binding corresponding to mRNA expression level data only on CEACAM5-positive cell lines (Table 1 below; MKN-45, NCI-H441). In contrast, for the commercial antibodies, binding was weak and limited to high CEACAM5 cell lines (Table 1 below; MKN-45). In conclusion, mAbl and rb8G4 specifically detect CEACAM5-positive cancer cells and can be used as detection agents. Table 1. Binding of different antibodies to CEACAM5-positive and CEACAM5-negative cell lines. For each cell line the median fluorescence intensity (MFI) of the corresponding antibody is divided by the MFI of the secondary antibody only control.

[0353] Binding of human mAbl and rb8G4 to lysates of CEACAM5 positive and negative cell lines was also investigated by Western blotting.

[0354] Methods used: Western blotting was performed according to standard protocols (Sambrook, J. & Russell, DW, 2001. Molecular Cloning: A Laboratory Manual, Vol. 1, CSHL Press). For SDS-PAGE and subsequent membrane wet blotting, 15 μg of total protein from RIPA cell lysates quantified by BCA kit (Thermo Scientific, #23227) was loaded per lane. Standard XT 4-12% gels (Bio-Rad, #3450125) using MOPS running buffer (Bio-Rad, #1610788) were used in standard electrophoresis pools (Bio-Rad, #1656001). Transfer of proteins was confirmed by Ponceau staining. Membranes were washed before and between staining with 0.5 μg / mL to 1 μg / mL of primary antibody (mAb1 or rb8G4) and secondary antibody (anti-human IgG, Jackson ImmunoResearch #109-035-098 or anti-rabbit IgG, Cell Signaling #7074). Stained membranes were visualized by ECL detection reagent using the Fusion FX imaging system (Vilber).

[0355] The results are shown in Fig.13A and Fig. 13B Middle: Both antibodies bind with similar patterns corresponding to the expected migration speed of heavily glycosylated CEACAM5. mAb1( Fig.13A ) and rb8G4( Fig. 13B ) is specific for CEACAM5-positive cell lines, and the intensity correlates with mRNA expression levels. A second band observed at a lower intensity corresponds to a potential second subtype 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). Example 2: Synthesis of Drug-Linker Compounds with Glucuronide-Based Linkers: Drug-Linker Compound 1 (DL1) and drug-linker compound 1-M (DL1-M)

[0356] Synthetic route to compound 9 (also referred to herein as drug-linker compound 1 (DL1))

[0357] Synthetic Pathway to Compound 11 (also referred to herein as Drug-Linker Compound 1-M (DL1-M)) Scheme for the chemical preparation of compound 9 Step 1: Compound 1

[0358] 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 room temperature for 16 hours. The reaction mixture was filtered through celite. The filtrate was concentrated under vacuum to obtain a solid. The solid was dissolved in EtOAc and washed with a 10% aqueous solution of NaHCO3 to remove excess 4-hydroxy-3-nitro-benzaldehyde. The organic layer was concentrated under vacuum to obtain compound 1 as a sandy solid. Yield: 9.0g Percent yield: 89.1% Analyze the data: NMR: 1 H-NMR (400MHz, 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=10.00Hz,1H),5.51-5.44(m,1H),5.20-5.09(m,2H),4.80(d,J=13.20Hz,1H),3.64(s,3H),2.09(s,9H). Step 2: Compound 2

[0359] To a stirred solution of compound 1 (9.00 g; 18.62 mmol; 1.00 eq) in propane-2-ol (33.00 ml; 3.67 V) and CHCl (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 room temperature for 1 hour. After completion, the reaction mixture was quenched with cooled H2O and filtered through celite. The filtrate was extracted with dichloromethane and dried over Na2SO4. The solvent was concentrated to obtain compound 2 as an off-white powder. Yield: 8.70g Percent yield: 92.4% Analyze the data LCMS: Column: ATLANTIS dC18 (50×4.6 mm) 5 μm; Mobile phase A: 0.1% HCOOH:ACN (95:5) in H2O; B: ACN RT(min):2.05; M+H:503.2, purity:96.6% Step 3: Compound 3

[0360] 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 at room temperature for 3 hours under hydrogen atmosphere. After completion, the reaction mixture was filtered through celite. The solvent was concentrated under vacuum to obtain compound 3 as an off-white solid. Yield: 8.5g Percent yield: 100% Analyze the data: LCMS: Column: ATLANTIS dC18 (50×4.6 mm) 5 μm; Mobile phase A: 0.1% HCOOH:ACN (95:5) in H2O; B: ACN RT (min): 1.73; M+H: 456.10, purity: 95.1% Step 4: Compound 4

[0361] To a stirred solution of compound 3 (10.00 g; 20.89 mmol; 1.00 equiv) and (9H-fluorene-9-ylmethoxycarbonylamino)-acetic acid (7.60 g; 25.06 mmol; 1.20 equiv) in DCM (250.00 ml; 25.00 V) at 0 ° C was added 2-ethoxy-2H-quinoline-1-carboxylic acid ethyl ester (15.65 g; 62.66 mmol; 3.00 equiv). The reaction mixture was stirred at room temperature for 16 hours. After completion, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (56% EtOAc: petroleum ether) to obtain a compound with a purity of 80%. The compound was further purified by washing with 30% EtOAc and petroleum ether to obtain compound 4 as a white solid. Yield: 8.5g Percent yield: 50.7% Analyze the data: LCMS: Column: ATLANTIS dC18 (50×4.6 mm) 5 μm; Mobile phase A: 0.1% HCOOH:ACN (95:5) in H2O; B: ACN RT (min): 3.03; M+H: 735.2, purity: 81.9% Step 5: Compound 5

[0362] To a stirred solution of compound 4 (2.00 g; 2.49 mmol; 1.00 equiv) in THF (40.00 ml; 20.00 V) was added carbonate bis-(4-nitro-phenyl) ester (3.06 g; 9.97 mmol; 4.00 equiv) and DIPEA (4.40 ml; 24.92 mmol; 10.00 equiv) at 0 ° C. The reaction mixture was stirred at room temperature for 12 hours. After the reaction was completed, the reaction mixture was concentrated under vacuum. The crude product was purified by column chromatography using silica gel (230-400) and petroleum ether / ethyl acetate as eluent to obtain compound 5 as a light yellow solid. Yield: 2.0g Percent yield: 84.6% Analyze the data: LCMS: Column: X-Bridge C8 (50×4.6) mm, 3.5 μm; Mobile phase A: 0.1% TFA in MilliQ water; B: ACN RT(min):3.24; M+H:900.20, purity:94.9% Step 6: Compound 6

[0363] Compound 5 (1,369 g; 1,00 equivalent) was dissolved in N,N-dimethylformamide (15,00 ml), exatecan mesylate (679,7 mg; 1,00 equivalent), 4-methylmorpholine (0,422 ml; 3,00 equivalent) and 1-hydroxybenzotriazole (172,8 mg; 1,00 equivalent) for synthesis were added. The reaction mixture was stirred at room temperature overnight. After the stirring time, the reaction suspension became a brown solution. The reaction was monitored by LC-MS (which showed complete conversion of the starting material). The reaction mixture was purified by RP flash chromatography. The fractions containing the product were combined, concentrated in vacuo and lyophilized overnight to obtain compound 6 as a yellow solid. Yield: 1.59 g Percent yield: 87.5% Analyze the data: LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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): 1.95; M+H: 1196.40, purity: 84.4% Step 7: Compound 7

[0364] Compound 6 (1,586 g; 1,00 equiv) was dissolved in tetrahydrofuran (50,00 ml) and LiOH solution (0.1 M) (containing lithium hydroxide hydrate (281,77 mg; 6,00 equiv) 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 LiOH solution was completed after 1.5 hours. 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 product was purified by preparative HPLC. The fractions containing the product were combined and lyophilized to obtain compound 7 as a dark yellow solid. Yield: 728 mg Percent yield: 54.8% Analyze the data: LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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): 1.68; M+H: 1056.30, Purity: 98.5%. Step 8: Compound 8

[0365] Compound 7 (728,000 mg; 1,00 equiv) was dissolved in N,N-dimethylformamide (20,00 ml). Piperidine (136,513 μl; 2,00 equiv) was added and the solution was stirred at room temperature for a total of 4 hours. 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 fractions containing the product were combined, the solvent was partially removed, and it was lyophilized overnight to obtain compound 8 as a yellow solid. Yield: 706 mg Percent yield: 100% Analyze the data: LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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: 834.30, purity: 97.6%. Step 9: Compound 9

[0366] To a solution of compound 8 (854 mg; 1,00 equiv.) in dimethylformamide (30,00 ml) was added N-ethyldiisopropylamine (149,234 μl; 1,00 equiv.) 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 equiv.). The reaction mixture was stirred at room temperature for 3 hours. 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 fractions containing the product were combined, concentrated and lyophilized to obtain the desired product with a purity of 91%. The material was purified again by RP chromatography to obtain compound 9 as a yellow solid. Yield: 580 mg Percent yield: 60.1% Analyze the data: LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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% (another 10% of isomers can be removed by HPLC) 1 H NMR (500MHz, DMSO-d6) δ13.10–12.44(m,1H),9.08(s,1H),8.32(t,J=5.8Hz,1H),8.16(s,1H),8.02(d,J=8.8Hz,1H),7.76(d,J= 10.9Hz,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 .6Hz,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=7.3Hz,3H). Scheme for the chemical preparation of compound 11 Step 1: Compound 10

[0367] To a solution of compound 8 (289.00 mg; 1.00 equiv) in dimethylformamide (10.00 ml) was added N-ethyldiisopropylamine (0.104 ml, 2.00 equiv) and (tert-butoxycarbonyl)glycylglycine 2,5-dioxopyrrolidin-1-yl ester (105.70 mg; 1.00 equiv). The reaction mixture was stirred at room temperature for 1 hour, and the reaction was monitored by LC-MS. After completion, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC on a Sunfire column. The fractions containing the product were combined and lyophilized to obtain the trifluoroacetate salt of compound 10 as a yellow solid. Yield: 162.00 mg, 0.14 mmol Percent yield: 44.8% Analyze the data: LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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% Step 2: Compound 11

[0368] To a suspension of compound 10 (162.00 mg; 1.00 equiv) in dichloromethane (5.00 ml) was added a 4.0 M solution of hydrogen chloride in 1,4-dioxane (682.43 μl; 20.00 equiv) and stirred at room temperature for 3.5 hours and the reaction was monitored by LC-MS. After completion, the reaction mixture was concentrated in vacuo and the crude product was purified by preparative HPLC. The fractions containing the product were combined and lyophilized to obtain compound 11 trifluoroacetate as a yellow solid. Yield: 113 mg; 0.11 mmol Percent yield: 77.0% Analyze the data: LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O 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% 1 H NMR(700MHz,DMSO-d6)δ12.93–12.79(m,1H),9.14(s,1H),8.61(t,J=5.8H z,1H),8.41(t,J=5.9Hz,1H),8.14(s,1H),8.05(d,J=8.8Hz,1H),8.03–7.9 3(m,3H),7.79(d,J=10.8Hz,1H),7.31(s,1H),7.13(s,2H),6.58–6.47(m,1 H),5.80–5.61(m,1H),5.48–5.40(m,2H),5.31–5.24(m,3H),5.08(d,J=12. 2Hz,1H),5.03(d,J=12.3Hz,1H),4.91(d,J=7.7Hz,1H),3.96(d,J=5.8Hz,2 H),3.95–3.86(m,3H),3.62(q,J=5.8Hz,2H),3.43(t,J=9.3Hz,1H),3.41(t ,J=8.5Hz,1H),3.35(t,J=9.0Hz,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=7.3Hz,3H). Example 3: Synthesis of Drug-Linker Compounds with Asparagine Endopeptidase-Cleavable Linkers: Drug-Linker Compound 2 (DL2) Step 1 4-Nitrophenylcarbonic acid {4-[(2S)-3-carbamoyl-2-[(2S)-2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propionamido]propionamido]propionamido]phenyl}methyl ester (400 mg; 0.52 mmol; 1.00 equiv) [commercially available from Levena Biopharma US] was dissolved in N,N-dimethylformamide (5.00 ml). Exatecan mesylate (277.30 mg; 0.52 mmol; 1.00 equiv), N-ethyldiisopropylamine (0.27 ml; 1.57 mmol; 3.00 equiv) and 1-hydroxybenzotriazole (HOBT) (3.52 mg; 0.03 mmol; 0.05 equiv) were added. The reaction mixture was stirred at room temperature overnight. LC / MS indicated complete conversion.

[0369] The crude reaction mixture was purified via preparative HPLC and lyophilized to afford 365 mg (0.343 mmol) of ((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)carbamic acid (9H-fluoren-9-yl)methyl ester LC / MS: [M+H]=1064.2 Preparative HPLC: Column: sunfire prep c18 obd-75.0g (250bar) Solvent A: Wasser 0.1% TFA Solvent C: Solvent B: Acetonitrile 0.1% TFA Step 2 ((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)carbamic acid (9H-fluoren-9-yl)methyl ester (365 mg; 0.34 mmol; 1.00 equiv) was dissolved in N,N-(4.00 ml). Piperidine (0.07 ml; 0.69 mmol; 2.00 eq.) used for synthesis was added, and the reaction solution was stirred at room temperature for 1 hour.

[0370] The reaction mixture was purified via preparative HPLC to yield 300 mg (0.314 mmol) of 4-((S)-4-amino-2-((S)-2-((S)-2-aminopropionamido)propionamido)-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.

[0371] LC / MS: [M+H]: 841.3 Preparative HPLC for purification: RediSep C18 130g SN: E0410A0D24BE1 Batch number: 262118923W Flow rate: 75ml / min Conditions - Volume: 390,0ml Eluent: A1 water 0.1% TFA Eluent: B1 acetonitrile 0.1% TFA Step 3 To ((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)carbamic acid 4-((S)-4-amino-2-((S)-2-((S)-2-aminopropanamide To a solution of (4-oxobutyramido)benzyl propionamido)-4-oxobutyramido)benzyl ester (571 mg; 0.60 mmol; 1.00 equiv) in N,N-dimethylformamide (20 ml) were added N-ethyldiisopropylamine (203 μl; 1.20 mmol; 2.00 equiv) and N-succinimidyl 3-maleimidopropionate (162 mg; 0.60 mmol; 1.00 equiv). The reaction mixture was then stirred for 10 min and monitored by LC / MS.

[0372] The reaction mixture was purified by preparative HPLC to obtain 378 mg (0.35 mmol) of DL2.

[0373] LC / MS: [M+H]: 992.4 Preparative HPLC for purification: RediSep column: C18 86g SN: E0410A8B46130 Batch number: 281729189W Flow rate: 60ml / min Conditions - Volume: 264,0ml Eluent 1: A1 water 0.1% TFA Eluent 2: B1 acetonitrile 0.1% TFA Sequence analysis: Method information: A: H2O + 0,05% HCOOH | B: MeCN + 0,04% HCOOH + 1% H2O T: 40°C | Flow rate: 3,3ml / min | MS: 100-2000amu positive Column: Chromolith HR RP-18e 50-4,6mm0%->100% B: 0->2,0min|100% B: 2,0->2,5min Example 4: Preparation of immunoconjugates:Glucuronide-based conjugates of mAb1 (referred to as ADC1) and glucuronide-based conjugates of 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) Although the following methods are described in the context of mAb1 (the resulting immunoconjugate is referred to as ADC1), the same methods can 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). 4.1 Conjugation method Antibody preparation

[0374] Antibody mAb1 (as defined above) was thawed at 2-8°C for up to 3 days before conjugation and stored at 2-8°C until use. Before use, on the day of conjugation, mAb (>10g) was balanced at room temperature. mAb (9.6mg / ml) was divided into (10.0g, 1041.7ml) 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 while stirring at 50rpm. Antibody reduction

[0375] 7.0 molar equivalents (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. Conjugation

[0376] The drug-linker compound 1 (DL1) of formula (X) was weighed and dissolved in DMSO to prepare a 20mM solution. 90% (148.6ml) of the required DMSO was added to the reactor. Immediately after the addition of DMSO, 10.0 molar equivalents (38.2ml) of the 20mM drug-linker solution was added to the reactor. Subsequently, 10% (18.2ml) 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 at 25±2°C for 1 hour. The total volume during conjugation was 1997.0ml. Note: The total DMSO concentration of the reaction was 10% (v / v) (DMSO + drug linker solution). Quenching

[0377] 35 molar equivalents (48.5 ml) of 50 mM NAC were added to the reactor and the reaction was allowed to proceed at 25 ± 2 °C for 30 minutes. filter

[0378] The filtered crude conjugate solution was transferred from the reactor and then filtered using Millipak Gamma Gold 60 (MPGL06GH2) to obtain 1993.6 ml (filter load: 324.7 g / m 2 [Protein], 66.5 L / m 2 [solution]) of the filtered crude conjugate. percolation

[0379] Pellicon 3 (30 kDa) Biomax membrane (1×0.11 m 2 ,300LMH (550mL / min),16psi TMP,actual load 88.5g / m 2 ) The filtered crude conjugate solution was buffer exchanged (DV = 1993.6 mL). Diafiltration buffer (10 mM histidine, pH 5.5) was used to buffer exchange the crude conjugate for 16 diafiltration volumes. After buffer exchange, the solution was concentrated to ≥ 25 mg / ml, transferred to a bottle, and the membrane was rinsed with diafiltration buffer. The total volume recovered from UF / DF was 361.5 ml. Preparation

[0380] 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), so that the final target drug substance (BDS) concentration was 15.0 mg / ml. filter

[0381] The final formulated ADC was filtered using a 0.2 μm Millipak Gamma Gold 40 (MPGL04GH2) filter to obtain 619.6 ml (filter load: 464.6 g / m 2 [Protein], 31.0 L / m 2 [Solution]) of ADC1 BDS. The material was packaged into HDPE bottles and stored at ≤-65°C. 4.2 Methods: Drug Substance Characterization: ADC1

[0382] Size Exclusion Chromatography (SEC) Method SEC method parameters Wavelength 280nm Column Tosoh TSKgel 7.8mm×300mm, 5μm (P / N 0008541) Mobile phase: 0.14 M potassium dihydrogen phosphate 50mM Sodium dihydrogen phosphate 0.06M Potassium Hydrogen Phosphate 0.25M potassium chloride 5% IPA Injection volume 20 μL Temperature 25℃ Flow rate 0.5mL / min Running time 30min Typical SEC chromatograms showing purity of stock mAb, post-UF conjugate and final BDS: Fig.14 .

[0383] For the BDS material shown above, 1.7% HMWS and 96.9% monomer purity have been reported.

[0384] Reverse Phase HPLC (RP HPLC) Method RP HPLC method parameters Wavelength 280nm Column PLRP-S (50×2.1mm, 8μm) column, Agilent (P / N PL1912-1802) Mobile phase A: 0.1% formic acid in water containing 0.01% TFA Mobile phase B contained 0.1% formic acid in ACN with 0.01% TFA gradient Injection volume 10 μL Column temperature 80℃ Flow rate 1.0mL / min Running time 30min Sample Preparation Dilute sample to 2 mg / ml and add 40 μL to a microcentrifuge tube. Add 60 μL of ~8 M guanidine hydrochloride, ~130 mM Tris, ~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 minutes.

[0385] Typical RP-HPLC chromatogram showing separation of light and heavy chains: Fig.15 This chromatogram shows the overlay of the stock mAb, crude ADC, and final BDS.

[0386] For the ADC1 BDS material described above, a DAR of 7.9 was reported. Free Drug Method Free Drug Method Parameters Wavelength 254nm Column Phenomenex Gemini, C18, 2×150 mm, 3 μm (P / N 00F-4439-B0) Mobile phase A 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile gradient Injection volume 10.00 μL Column temperature 50℃ Flow rate 0.75mL / min Sample preparation Protein drop: 100 μL of drug substance + 250 μL of cold MeOH + 50 μL of 3M MgCl2. Spin at 20,000 rpm for 10 min Standard Preparation Mix 20 μL of 20 mM DL1 (Drug-Linker Compound 1 in DMSO) + 20 μL DMSO + 40 μL MeOH + 20 μL of 200 mM NAC in diafiltration buffer. Incubate overnight to obtain 4 mM DL-NAC. Dilute 4 mM DL-NAC in MeOH to obtain 4 μM DL-NAC standard.

[0387] Typical chromatograms showing free drug levels of NAC standard and final BDS: Fig.16 .

[0388] For the ADC1 BDS material shown above, residual free drug levels of less than 2.4% (on a molar basis) have been reported. Example 5: Preparation of immunoconjugates: peptide-based conjugates of mAbl (referred to as ADC2) 5.1 Conjugation method Antibody preparation

[0389] Antibody mAb1 (as defined above) was thawed at 2-8°C for up to 3 days before conjugation and stored at 2-8°C until use. Before use, on the day of conjugation, mAb (>9.5g) was balanced at room temperature. mAb (9.6mg / ml) was divided into 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 while stirring at 50rpm.

[0390] Antibody reduction 8.0 molar equivalents (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. percolation

[0391] Pellicon 3 (30 kDa) Biomax membrane (1×0.11 m 2 ,300LMH (550mL / min),16psi TMP,actual load 86.3g / m 2 ) The reduced mAb solution was buffer exchanged against 6 DV (DV = 1706.9 mL). Conjugation buffer (200 mM histidine, pH 6.5) was used to buffer exchange the reduced antibody. After the buffer exchange, the reduced mAb solution was recovered back into the reactor and the membrane was rinsed with conjugation buffer. Conjugation

[0392] Drug-linker compound 2 (DL2) was weighed and dissolved in DMSO to prepare a 20mM drug-linker solution. 90% (142.6 ml) of the required DMSO was added to the reactor. 9.5 molar equivalents (31.2 ml) of the 20mM drug-linker solution were added to the reactor immediately after the addition of DMSO. Subsequently, the drug-linker vial was rinsed with 10% (15.8 ml) of the remaining required DMSO to ensure complete transfer. After the final addition, the reaction was allowed to proceed at 25 ± 2 ° C for 2 hours. The total volume during the conjugation reaction was 1894.5 ml. Quenching

[0393] 35 molar equivalents (46 ml) of 50 mM NAC were added to the reactor and the reaction was allowed to proceed at 25 ± 2 °C for 45 minutes. filter

[0394] The crude conjugate solution was transferred from the reactor and filtered using Millipak Gamma Gold 60 (MPGL06GH2) to obtain 1897.3 ml (filter load: 308.9 g / m 2 [Protein], 63.2 L / m 2 [solution]) of the filtered crude conjugate. percolation

[0395] Pellicon 3 (30 kDa) Biomax membrane (1×0.11 m 2 ,300LMH(550mL / min),16psi TMP,actual load 84.2g / m 2) The filtered crude conjugate solution was buffer exchanged (DV=1897.3 mL). An initial 12 DV was performed using conjugation buffer (200 mM histidine, pH 6.5), and then switched to standard diafiltration buffer (10 mM histidine, pH 5.5) for 8 additional DVs. After the buffer was complete for buffer exchange, the solution was then concentrated to ≥25 mg / ml, transferred to a bottle, and the membrane was rinsed with diafiltration buffer. The total pooled volume recovered from UF / DF was 335.7 ml. Preparation

[0396] 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. filter

[0397] The final formulated ADC was sterile filtered using Millipak Gamma Gold 60 (MPGL06GH2) to obtain 549.3 ml (filter load: 411.4 g / m 2 [Protein], 27.5 L / m 2 [Solution]) of ADC2 BDS. The material was packaged into HDPE bottles and stored at ≤-65°C. 5.2 Methods: Drug Substance Characterization: ADC2 Size Exclusion Chromatography (SEC) Method

[0398] SEC method parameters Wavelength 280nm Column Tosoh TSKgel 7.8mm×300mm, 5μm (P / N 0008541) Mobile phase: 50 mM potassium dihydrogen phosphate 0.4M Sodium Perchlorate pH 6.3 Injection volume 1 μL Column temperature 25℃ Flow rate 0.5mL / min Running time 30min Typical SEC chromatograms showing purity of stock mAb and final BDS: Fig.17 .

[0399] For the ADC2 BDS material shown above, 4.2% HMWS and 95.8% monomer purity have been reported.

[0400] Reverse Phase HPLC (RP HPLC) Method RP HPLC method parameters Wavelength 280nm Column PLRP-S (50×2.1mm, 8μm) column, Agilent (P / N PL1912-1802) Mobile phase A: 0.1% formic acid in water containing 0.01% TFA Mobile phase B contained 0.1% formic acid in ACN with 0.01% TFA gradient Injection volume 10 μL Column temperature 80℃ Flow rate 1.0mL / min Running time 30min Sample Preparation Dilute sample to 2 mg / ml and add 40 μL to a microcentrifuge tube. Add 60 μL of ~8 M guanidine hydrochloride, ~130 mM Tris, ~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 minutes.

[0401] Typical RP-HPLC chromatogram showing separation of light and heavy chains: Fig.18 This chromatogram shows the overlay of the stock mAb and the final BDS.

[0402] For the above ADC2 BDS material, a DAR of 7.6 was reported. Free Drug Method

[0403] Free Drug Method Parameters Wavelength 254nm Column Phenomenex Gemini, C18, 2×150 mm, 3 μm (P / N 00F-4439-B0) Mobile phase A 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile gradient Injection volume 10.00 μL Column temperature 50℃ Flow rate 0.75mL / min Sample preparation Protein drop: 100 μL of drug substance + 250 μL of cold MeOH + 50 μL of 3M MgCl2. Spin at 20,000 rpm for 10 min Standard Preparation Mix 20 μL of 20 mM DL2 (Drug-Linker Compound 2 in DMSO) + 20 μL DMSO + 40 μL MeOH + 20 μL of 200 mM NAC in diafiltration buffer. Incubate overnight to obtain 4 mM DL-NAC. Dilute 4 mM DL-NAC in MeOH to obtain 4 μM DL-NAC standard.

[0404] Typical chromatograms showing free drug levels of NAC standard and final BDS: Fig.19 .

[0405] For the ADC2 BDS material shown above, residual free drug levels of less than 1.9% (on a molar basis) have been reported. Example 6: Analogs of ADC SAR408701 6.1 Antibodies

[0406] For further comparative experiments, Sanofi's antibody was prepared based on a monoclonal antibody with the following sequence: Analogs of CEACAM5 ADC SAR408701: Heavy chain: SEQ ID NO: 25 Light chain: SEQ ID NO: 26 6.2 Drug-Linker Compounds As the drug-linker molecule to be conjugated to the above-mentioned antibody, SPDB-DM4 (obtained from Levena Biopharma) was used: Product Name: SPDB-DM4 structure: Expected quality: 994.35 Observed average mass: 995.5 (Ms+H + ) Mass spectrometry: consistent, showing correct MW HPLC analysis: purity>95% Appearance: white powder 6.3 Conjugation The antibodies were thawed at 2-8°C for up to 3 days before conjugation and stored at 2-8°C until use. Before use, on the day of conjugation, the antibodies (175 mg) were equilibrated at room temperature. The antibodies (7.9 mg / ml) were diluted to 5 mg / ml using a 5 mM DMSO solution of conjugation buffer (PBS pH 7.4) and SPDB-DM4 (Levena Biopharma) (8 molar equivalents relative to the antibody). The reaction solutions were mixed and incubated at 25°C for 4 hours. 6.4 Preparative size exclusion chromatography, desalting and filtration

[0407] The reaction mixture was purified using preparative size exclusion chromatography. A Superdex 200 pg (50 / 60) column was attached to Avant 25 system (GE Healthcare) and balanced with PBS pH 7.4 according to the manufacturer's instructions. Subsequently, the reaction mixture was injected and run through the column at a flow rate of 10 ml / min and PBS pH 7.4 as running buffer. The fractions containing ADC were determined by UV light absorption at 280 nm, pooled and concentrated. The ADC material was concentrated using 15 ml Amicon Ultra 50 kDa cutoff centrifugal device (Merck Millipore) according to the manufacturer's instructions. 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) on an Avant 25 system (GE Healthcare) at a flow rate of 10 ml / min. The resulting ADC material was filtered using a 0.2 μm filter (Merck Millipore), aliquoted and then snap 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 obtained from this work is also referred to herein as "ADC SAR DM4", or simply "ADC SAR"; the ADC is an analog of SAR408701. Example 7: ADC based on mAb1 and SPDB-DM4

[0408] Another ADC was prepared based on antibody mAb1 (described above) and drug-linker compound SPDB-DM4 (i.e., the same drug-linker compound as in ADC SAR DM4 described above). The ADC obtained from this work is referred to herein as "ADC mAb1DM4" and was prepared as follows: 7.1 Materials used: Antibody: mAb1, 1 mg / ml in 10 mM HEPES, pH 5.8 Conjugation buffer: 10 mM HEPES, pH 5.8 Drug-linker compound: SPDB-DM4, 2 mg / ml in DMF 7.2 Methods: Conjugation: About 30-fold molar excess of drug-linker molecules was used for conjugation (75 mL antibody + 7.1 mL SPDB-DM4 drug-linker), incubated at room temperature for 5 hours with slow shaking Purification: ADC buffer exchange into 20 mM histidine, 150 mM NaCl, pH 6.0 to remove free drug Preparation buffer: 20 mM histidine, 150 mM NaCl, pH 6.0 7.3 Analysis details of purified ADC: Final yield: 40 mg Concentration: 2.2mg / ml DAR: 4.4 Example 8: Characterization of drug release from ADC1 and ADC2 8.1 Materials and methods 8.1.1 Test products 8.1.2 Materials All reagents and buffers were stored according to the manufacturer's instructions and used before the batch expiration date. 8.1.3 Instruments 8.1.4 Procedure 8.1.4.1 Serum sample preparation

[0409] 2M HEPES solution: 52.1 g HEPES was dissolved in 75 mL MiliQ water and 15 mL 25% HCl, adjusted to pH 7.55, and added to 100 ml. The solution was mixed with serum at 15% v / v to obtain a stabilized serum with a pH of 7.3-7.4. Human serum from Biowest (lot S15594S4200) was thawed. 100 mL serum was mixed with 15 mL 2M HEPES buffer. Mouse serum from Biowest (lot S18169S2160) was thawed. 100 mL serum was mixed with 15 mL 2M HEPES buffer. Cynomolgus monkey serum was thawed and 8.5 mL serum was mixed with 1.5 mL 2M HEPES buffer. The pH was measured (7.37) and the serum was sterile filtered. 2 mL aliquots were frozen at -20°C.

[0410] The prepared serum was thawed at room temperature. The required ADC protein concentration was prepared in triplicate at 180 μg / mL for subsequent free payload analysis via LC-MS. After ADC was added to the serum, the single batch was mixed and divided into 20 μL aliquots. In addition, a 96-hour sample of 20 μL was drawn for each ADC and used for total post-processing analysis to measure the recovery. The 0-hour sample was directly frozen at -80°C, and the remaining samples were incubated at 37°C and 5% CO2, and the reaction was stopped when stored at -80°C for 2 / 4 / 6 / 24 / 48 / 72 / 96 hours of incubation. 8.1.4.2 Preparation of human liver lysosome samples

[0411] The pH was adjusted to pH 5.0 or pH 4.0 using assay buffer. Lysosomal stability preparation: As exemplified for ADC1 (n=3), 80 μL human liver lysosomes were prepared for triplicate measurements: 2.76 μL ADC1 + 6 μL human liver lysosomes + 71.2 μL asparagine endopeptidase assay buffer. Preparation of MeOH + PIC (1:200): 10 μL PIC III + 1990 μL MeOH. The reaction was started when the Eppendorf tube was transferred 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). 8.2 Results

[0412] ADC stability in human, mouse and cynomolgus monkey serum ( Fig. 20). Conjugated exatecan concentrations (initial dose -10 μM) were calculated using free exatecan (normalized data). Similar profiles were obtained for human, cynomolgus monkey, and mouse sera. Only minor warhead release was observed, with ADC2 (5.9% free of initial conjugate payload at 96 hours) and ADC1 (1.4%) being the most prominent in mouse serum.

[0413] ADC3 stability control in mouse serum and buffer ( Fig.21 ). The conjugated SN38 concentration (initial dose 50 μg / mL ADC protein concentration) was calculated using free SN38 (unnormalized). Significant SN-38 release was observed for both matrices.

[0414] Payload release profiles of ADC1 and ADC2 in human liver lysosomes (pH 5.0) Fig. 22 ). Conjugated drug concentrations were calculated using normalized data, for example, of free exatecan (initial concentration -10 μM exatecan). Moderate levels of payload release were observed for both ADC1 and ADC2 cleavage-mediated payload release (both -40% of initial total conjugate payload).

[0415] Analysis of ADC metabolites confirmed that free exatecan was a lysosomal release product ( Fig.23 ). To confirm that exatecan was the major released product, ADC1 catabolite profiling studies were performed in human lysosomal extracts. Here, comparison of TIC-MS and extracted ion chromatograms at different time points showed that the expected exatecan catabolites were subsequently released from ADC1 during the incubation period (0 h, 4 h, 24 h). Retention time 9.33 min, detection mass m / z 436.1671 ([M+H] + ,C 24 H 23 O4N4 F) and MS / MS spectra were consistent with those of exatecan. Example 9: ADC1 and ADC2 specifically kill cancer cells in vitro with high efficacy

[0416] Human cancer cell lines were used to evaluate the potential of ADC1 and ADC2 to kill cancer cells. ADC1 and ADC2 showed subnanomolar in vitro potency against different CEACAM5-positive cell lines, while showing less effect on CEACAM5-negative cell lines (Table 2 below). As shown in the exemplary dose-response curve ( Fig.24A / B), ADC1 and ADC2 were very effective against the CEACAM5-positive cell lines SK-CO-1 and SNU-16. In contrast, the effects of ADC1 and ADC2 on antigen-negative MDA-MB-231 were limited to the highest concentrations tested ( Fig.24C ).

[0417] Rituximab isotype control ADCs using linkers and payloads corresponding to ADC1 and ADC2 showed much lower effects on the SK-CO-1 cell line ( Fig.25 ).

[0418] In conclusion, ADC1 and ADC2 specifically kill CEACAM5-expressing human cancer cell lines in vitro with high potency. Table 2. Efficacy of ADC1, ADC2 and free payload on various human cell lines. The maximum effect compared to untreated control at the highest test compound concentration is indicated in parentheses. For each cell line, CEACAM5 expression is indicated. Methods - Viability assay:

[0419] The cytotoxic effect of ADC on cancer cell lines was measured by cell viability assay. One day before treatment, cells were seeded in a 96-well plate in a volume of 90 μL. Test compounds (ADC or free payload) were prepared in cell culture medium at 10 times 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 incubated at 37°C in a CO2 incubator for six days. For cell viability measurements, Cell Titer- Reagents (Promega TM Corp, Madison, WI) was added to each well and the plate was processed according to the manufacturer's instructions. The luminescent signal was measured using a Varioskan plate reader (Thermo Fisher). The luminescent readings were converted to viability % relative to untreated cells. Log (inhibitor) was used relative to reaction, variable slope, using the 4-parameter fitting equation of GraphPad Prism, and the data were fitted using nonlinear regression analysis. Data are displayed as relative cell viability % relative to molar compound concentration, and error bars indicate the standard deviation (SD) of triplicate. The geometric mean of the IC50 derived from multiple experiments was calculated.

[0420] Using the same method as above, ADC1 and ADC2 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 and ADC2 showed 2.9 and 2.7 times the potency of ADC SAR DM4 against SK-CO-1 cancer cells, respectively ( Fig.26A ). Compared with ADC1 and ADC2, ADCSAR DM4 had slightly higher nonspecific effect against antigen-negative MDA-MB-231 ( Fig.26B ADC SAR DM4 and ADC mAb1DM4 showed comparable potency against SK-CO-1, with ADC mAb1 DM4 tending to be slightly more potent ( Fig.26A ). Example 10: ADC1 and ADC2 mediate a potent bystander effect against antigen-negative cells in co-culture with antigen-positive cells

[0421] The potential of ADC1 and ADC2 to mediate a bystander effect against antigen-negative cells in close proximity to antigen-positive cells was evaluated in a bystander assay. In the presence of CEACAM5-positive SK-CO-1, ADC1 and ADC2 demonstrated a potent bystander effect against CEACAM5-negative MDA-MB-231 cells ( Fig.27A ). The co-culture experiment was carried out at a concentration of 1 nM ADC, and for ADC1, the concentration caused the maximum inhibition of CEACAM5-positive SK-CO-1 cell viability ( Fig.26A ), but had no effect on CEACAM5-negative MDA-MB-231 cells ( Fig.26B Consistent with these findings, no nonspecific effects of ADC1 or ADC2 were observed in the bystander assay setting with MDA-MB-231 controls only ( Fig.27B ).

[0422] In conclusion, ADC1 and ADC2 mediate a potent bystander effect against antigen-negative cancer cells in co-culture with antigen-positive cells. These findings indicate the potential for effective targeting of tumors with heterogeneous target expression.

[0423] Given that the same drug-linker and Fv region are utilized, ADC1-M, ADC2-M, ADC3-M, ADC6-M, and ADC7-M are expected to mediate a potent bystander effect similar to ADC1 and ADC2.

[0424] Compared with ADC SAR, ADC1 and ADC2 mediated a much stronger bystander effect on antigen-negative cells in co-culture with antigen-positive cells ( Fig.28A , Fig.28B ADC mAb1 DM4, which uses the same antibody as in ADC1 and ADC2 (i.e., mAb1) and the drug-linker molecule used in ADC SAR DM4 (i.e., SPDB-DM4), also showed a more significant bystander effect than ADC SAR DM4 ( Fig.28A , Fig.28B). This indicates that mAb1 contributes to the higher bystander effect observed for ADC1 and ADC2 compared to ADC SAR using different antibodies. It is therefore expected that ADC1-M, ADC2-M, ADC3-M, ADC6-M and ADC7-M also mediate a more potent bystander effect than ADC SAR.

[0425] For all ADCs tested, the extent of the bystander effect increased with increasing numbers of antigen-positive cells added to a constant number of antigen-negative cells (compare Fig.28A and Fig.28B Without wishing to be bound by theory, this could be a result of more ADC being processed by a higher number of antigen-positive cells to release more free payload, which is responsible for the bystander effect on antigen-negative cells.

[0426] No nonspecific effects of the tested ADCs on MDA-MB-231 cells alone were observed ( Fig.28C ). Methods – Bystander measurement

[0427] The cytotoxic effects of ADC on antigen-negative cancer cell lines co-cultured with antigen-positive cancer cell lines were measured by bystander assay. One thousand CEACAM5-negative MDA-MB-231 cells were seeded in co-culture experiments with 750 or 3000 CEACAM5-positive SK-CO-1 cells per well. As a control, only 1000 MDA-MB-231 cells were seeded in parallel. One day before treatment, cells were seeded in a total volume of 90 μL in 96-well plates. The test compound was formulated in cell culture medium at 10 times the final concentration of 1E-9M, and 10 μL was added to the cells in duplicate. The plate was cultured at 37°C in a CO2 incubator for six days.

[0428] Prior to immunofluorescence staining, the culture medium was removed and the cells were treated with 100% methanol (-20°C) for 30 minutes. After removing the methanol and one PBS washing step, the cells were treated with 2.5% paraformaldehyde (PFA) and 0.2% Triton X-100 in PBS for 15 minutes at room temperature. After removing the solution and one PBS washing step, the cells were treated with 1% BSA / 0.1% Tween / 0.1% sodium azide in PBS for at least one hour at room temperature. Antigen-positive and antigen-negative cells were distinguished by immunofluorescence staining with 10 μg / mL human anti-CEACAM5 (mAb1) primary antibody and 1:2000 diluted 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 PBS solution for 30 minutes 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] The confocal quantitative imaging cytometer CQ1 ( Electric Corporation, Tokyo, Japan) to image the plate. The analysis was adjusted by CQ1 software (Yokogawa) template "nucleus and pseudocell body", and the FCS output file was analyzed using FlowJo (BD). Based on the staining or non-staining of fluorescent labeled antibodies around the nucleus, antigen-positive and antigen-negative cells were distinguished and quantified. The bar graph shows the number of antigen-positive and antigen-negative cells identified for each treatment condition. Example 11: Efficacy of ADC1 and ADC2 in a colorectal cancer (CRC) patient-derived xenograft (PDX) mouse model

[0430] The in vivo antitumor efficacy has been evaluated in the human patient-derived CRC xenograft model COPF217 (Shanghai LideBiotech CO., LTD). COPF217 tumor fragments were subcutaneously implanted into the right flank of six to eight-week-old immunodeficient female mice (NU-Foxn1nu, Charles River). When the tumor reached 165 mm 3 When the mean volume of 6 mice / group was 10 mg / kg, 6 mice / group were treated intravenously once with vehicle (saline solution) or with 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×(W^2) / 2.

[0431] A single treatment with ADC1 or ADC2 at a dose of 10 mg / kg produced a significant antitumor effect. Both substances showed comparable effects in causing tumor stasis ( Fig.29 Treatment with ADC1 or ADC2 had no significant effect 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 tumor regression in 12 other CRC PDX models with high CEACAM5 expression. Example 12: Efficacy of ADC1 in a non-small cell lung cancer (NSCLC) PDX mouse model

[0433] The in vivo antitumor efficacy has been evaluated in a human patient-derived NSCLC xenograft model, LUPF160151 (Shanghai Lide Biotech CO., LTD). LUPF160151 tumor fragments were subcutaneously implanted into the right flank of six to eight-week-old immunodeficient female mice (NU-Foxn1nu, Charles River). When the tumor reached 180 mm 3 When the mean volume of 5 mice / group was 1:1, 5 mice / group were treated once with vehicle (saline solution) or with ADC1 (6 mg / kg; day 0) intravenously. Tumor length (L) and width (W) were measured with calipers, and tumor volume was calculated using the formula L×(W^2) / 2.

[0434] A single treatment with ADC1 at a dose of 6 mg / kg produced a significant antitumor effect ( Fig.30 ), with no effect on body weight (data not shown). Model LUPF160151 showed heterogeneous CEACAM5 expression, with CEACAM5-negative tumor cells adjacent to CEACAM5-positive tumor cells. The good efficacy of ADC1 in this model thus indicates a potent bystander effect of ADC. Example 13: Efficacy of ADC1 in a gastric cancer PDX mouse model

[0435] The 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). When the tumor reached 220 mm 3When the mean volume of 6 mice / group was reached, 6 mice / group were treated once with vehicle (saline solution) or with ADC1 (3 or 10 mg / kg; day 0) intravenously. Tumor length (L) and width (W) were measured with calipers, and tumor volume was calculated using the formula L×(W^2) / 2.

[0436] A single treatment with 3 or 10 mg / kg ADC1 induced a significant antitumor effect ( Fig.31 ), with no effect on body weight (data not shown). In five of six tumors, treatment with 10 mg / kg resulted in complete tumor regression. Example 14: Efficacy of ADC1 compared to ADC3 in a pancreatic cell line-derived tumor model

[0437] The efficacy of ADC1 compared with ADC3 has been 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 six to eight-week-old immunodeficient female mice (Hsd: Athymic Nude-Foxn1nu, Envigo). 3 When the mean volume of 10 mice / group was reached, 10 mice / group were treated intravenously once with vehicle (saline solution) or with ADC1 (1 mg / kg or 6 mg / kg; day 0) or with 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 the formula L×(W^2) / 2.

[0438] A single treatment with ADC1 at a dose of 6 mg / kg resulted in a significant anti-tumor effect. The effect was dose-dependent, as a single treatment with 1 mg / kg resulted in only a modest but significant, transient anti-tumor effect. In contrast, a single treatment with the same dose of ADC3 showed no significant anti-tumor effect at either dose ( Fig.32 ). All treatments had no significant effect on body weight (data not shown). ADC1-M, ADC2-M, ADC3-M, ADC6-M and ADC7-M are expected to be similarly more favorable than ADC3 in this regard as ADC1. Example 15: Efficacy of ADC1 compared to ADC SAR DM4 in two CRC PDX mouse models

[0439] In vivo antitumor efficacy was evaluated in human patient-derived CRC xenograft models COPF230 and REPF210 (Shanghai Lide Biotech CO., LTD). Tumor fragments were subcutaneously implanted into the right flank of six to eight-week-old immunodeficient female mice (NU-Foxn1nu, Charles River). When the tumor reached approximately 170 mm 3 When the average volume of 6 mice / group was 2.5 μg / kg, 6 mice / group were treated intravenously with vehicle (saline solution), ADC1 (6 mg / kg) or ADC SAR DM4 (6 mg / kg) once (day 0). Tumor length (L) and width (W) were measured with calipers, and tumor volume was calculated using the formula L×(W^2) / 2.

[0440] A single treatment with ADC1 in the PDX model COPF230 ( Fig.33 ) and REPF210( Fig.34 ) resulted in significant antitumor effects in both. In contrast, a single treatment with the same dose of ADC SAR DM4 showed no antitumor effects in either CRC PDX model ( Fig.33 , Fig.34 ). No significant effect on body weight was observed in any treatment group (data not shown). ADC1-M, ADC2-M, ADC3-M, ADC6-M and ADC7-M are expected to be similarly more favorable than ADCSAR DM4 in this regard as ADC1. Example 16: Efficacy of ADC1 compared to ADC SAR DM4 in a gastric PDX mouse model (GAPF313)

[0441] In vivo antitumor efficacy was evaluated in a human patient-derived gastric xenograft model, GAPF313 (Shanghai LideBiotech CO., LTD). Tumor fragments were subcutaneously implanted into the right flank of six to eight-week-old immunodeficient female mice (NU-Foxn1nu, Charles River). When tumors reached approximately 180 mm 3 When the mean volume of 6 mice / group was 2.5 μg / kg, ADC1 (4 mg / kg or 7 mg / kg Q2W×3) or ADC SAR DM4 (4.7 mg / kg Q2W×3) was intravenously treated 3 times every two weeks starting from day 0. Tumor length (L) and width (W) were measured with a caliper, and tumor volume was calculated using the formula L×(W^2) / 2.

[0442] Interim data analysis from ongoing trials demonstrates clear antitumor efficacy of ADC1 in this model, and no effect of ADC SAR DM4 ( Fig.35). All treatments were well tolerated (data not shown). ADC1-M, ADC2-M, ADC3-M, ADC6-M and ADC7-M are expected to be similarly advantageous over ADC SAR DM4 in this regard as ADC1. Example 17: Safety profile of ADC1 - preliminary toxicity study in cynomolgus monkeys

[0443] To study the safety profile, ADC1 was administered to cynomolgus monkeys at doses of 0, 3, 10, and 30 mg / kg by 30-min intravenous infusion three times at 3-week intervals (on days 1, 22, and 43), and the animals were sacrificed for macroscopic and histopathological examinations on day 50. As a result, it was found that ADC1 had a relatively favorable safety profile because ADC1 had no toxicity in certain organs affected by the toxic side effects of known ADCs. Example 18: Expression and purification of modified antibodies and transglutaminase

[0444] In the following, the expression and purification of modified antibodies will be described. These modified antibodies can be used to produce corresponding antibody-drug-conjugates. The characteristics of modified antibodies mAb1-M, mAb2-M, mAb3-M, mAb4-M, mAb5-M, mAb6-M and mAb7-M are summarized in the following Table 4: Table 4

[0445] The DNA sequence of synthetic coding antibody mAb1-M, mAb2-M, mAb3-M, mAb5-M, mAb6-M and mAb7-M is also cloned into pTT5 plasmid for recombinant expression at GeneArt (Life Technologies). The plasmid produced is used for transient transfection and recombinant protein expression in shaking flask using ExpiCHO expression system (GibcoTM, Thermo Fisher Scientific Inc.). After transfection 7 days, harvest supernatant and use standard step-by-step method to purify the antibody expressed, and the standard step-by-step method includes protein A affinity chromatography (HiTrap MabSelect SuRe post, Cytiva) and size exclusion chromatography (HiLoadSuperdex 200pg post, Cytiva).

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

[0447] A DNA sequence encoding transglutaminase mTG (Seq ID 46) was synthesized and cloned into the pET30a plasmid for recombinant expression at GeneArt (Life Technologies). E. coli BL21 (DE3) strain transformed with the resulting plasmid was cultured overnight in a shake flask at 28°C and 130 rpm in a lysogeny broth medium supplemented with 5 g / l glucose, 10 mL / 100 mL 10× phosphate buffered saline and 30 mg / l kanamycin (50 mm throw). The culture was used to inoculate a fermenter containing 9.5 liters of growth medium (50 g / l yeast extract, 10 g / l peptone, 0.5 g / l MgSO4×7H2O and 2 ml 50% Desmophen (defoamer from Rhein Chemie Rheinau)) to an optical density of 0.00002. The fermenter was run overnight (16 h) at 28°C at 800 U / min, 5 Nl / min aeration and pH 7.0-7.4. At OD 5, the culture was induced with 0.1 mM IPTG until an OD of ~30 was reached (5-6 h). In the event of foam formation or oxygen concentration falling below 2 mg / ml, more Desmophen was added or the speed was increased to 1000 rpm, respectively. The cell pellet was harvested by continuous flow-through centrifugation.

[0448] 50 g of cell pellet was resuspended in 250 ml 50 mM sodium 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), pH adjusted to 5.5 and loaded onto SO3-(M) 85ml column (Millipore), followed by 20CV 0-1M NaCl linear gradient elution. The fractions of purified protein with the expected size were identified by SDS-PAGE, combined and dialyzed against 50mM Tris / HCl, 300mM NaCl, pH 8.0 at 4°C overnight. Subsequently, 0.5U / ml I (Sigma Aldrich) and 2 mM CaCl2 were used to proteolytically process transglutaminase at a final concentration of 2 mg / ml at 37°C for 60 min, followed by the addition of 5 mM EDTA. The reaction mixture was dialyzed against 50 mM sodium phosphate buffer pH 6.0 at 4°C overnight and loaded onto SO3-column (Millipore), and eluted with a linear gradient of 20CV 0-1M NaCl. The fractions with effective cleavage and purification of the protein were identified by SDS-PAGE, merged, concentrated and purified using HiLoadSuperdex 75pg size exclusion column (Cytiva), using 24HEPES pH 7, 100mM NaCl as running buffer. The fractions containing transglutaminase were merged, concentrated to>20mg / ml, quickly frozen in liquid nitrogen and stored at -80°C. Enzyme activity was determined using ZediXclusive microbial transglutaminase assay (Zedira). Example 19: ADC preparation, conjugation and characterization of ADC1-M, ADC4-M and ADC6-M 19.1. Antibody Preparation and Conjugation

[0449] The monoclonal antibody (mAb) prepared in 50mM histidine, 100mM NaCl, pH 6.5 was stored at -80°C. Before conjugation, mAb was thawed at room temperature and the protein concentration was adjusted to 5mg / ml via dilution with formulation buffer. Subsequently, 10-12 molar equivalents of excess (relative to mAb) of TCEP were added to reduce mAb and incubated at 37°C for 2-3 hours. 16-24 molar equivalents (relative to mAb) of DL1 (in 10mM stock solution in DMSO) were added and incubated at 25°C for 60min. 25 molar equivalents (relative to mAb) of N-acetyl-cysteine ​​(from 25mM DMSO stock solution) were added to quench the reaction mixture and incubated at 25°C for 30 minutes. ADC was separated from DL1 and possible high molecular weight species (HMWS) via size exclusion chromatography (SEC). Prior to SEC purification, samples were centrifuged at 4000 × g for 2 min to remove possible precipitates. LC system (Cytiva) combined with HiLoad Superdex 200 26 / 60 Increase column, at a flow rate of 2.5 mL / min and 50 mM histidine, 100 mM NaCl, pH 6.5 as running buffer. Fractions containing ADC material were pooled and concentrated to 6 mg / ml using an Amicon Ultra 15 50K centrifuge (Millipore) and then centrifuged using the same The final buffer exchange was into 10 mM histidine, 40 mM NaCl, 6% trehalose, 0.05% Tween, pH 5.5 in combination with a HiTrap desalting column of an LC system (Cytiva). The final drug substance (BDS) was filtered through a 0.22 μm sterile filtration unit (Millipore) and snap frozen in liquid nitrogen until further use.

[0450] 19.2. Quality attributes ADC1-M Concentration [mg / ml] 6.0 Drug / antibody ratio 8.16 PuritySEC[%] 98.9 Free drug content [%] 0.08 Endotoxin content [EU / mg] 1.84 ADC6-M Concentration [mg / ml] 7.09 Drug / antibody ratio 8.14 PuritySEC[%] 100 Free drug content [%] 0.01 Endotoxin content [EU / mg] 0.39 ADC4-M Concentration [mg / ml] 5.82 Drug / antibody ratio 7.87 PuritySEC[%] 97.8 Free drug content [%] 0.03 Endotoxin content [EU / mg] 0.13 9.3 Drug Substance Characterization Size Exclusion Chromatography (SEC) Method Monomer content and purity were assessed by size exclusion chromatography on a Waters Bio Resolve SEC mAb column.

[0451] Wavelength: 214nm ColumnWaters Bio Resolve SEC mAb Column, 2.5μm,4.6×150mm Mobile phase: 50 mM sodium dihydrogen phosphate 0.4M sodium perchlorate, pH 6.3 Injection volume 5 μL Column temperature 25℃ Flow rate 0.35mL / min Typical SEC chromatograms showing the purity of the input mAb and final BDS are shown in Fig.36 middle.

[0452] Reverse Phase HPLC (RP HPLC) Method RP HPLC method parameters Wavelength: 214nm Column PLRP-S (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 in 7.5 min Injection volume 10 μL Column temperature 65°C Flow rate: 1.0 mL / min Sample preparation 40 μL of sample (2.2 mg / ml) was mixed with 4 μL of 0.5 M TCEP and incubated at room temperature for 5 minutes. Then, 40 μL of 0.5 M iodoacetamide was added and incubated at 37° C. for 15 minutes. 10 μL of the reaction was injected.

[0453] A typical RP-HPLC chromatogram showing the DAR determination of the final BDS is shown in Fig.37 middle.

[0454] Free drug method Wavelength 254nm Column Phenomenex Gemini, C18, 2×150 mm, 3 μm (P / N 00F-4439-B0) Mobile phase A 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile gradient Injection volume 10.00 μL Column temperature 50℃ Flow rate 0.75mL / min Sample preparation Protein drop: 100 μL of API + 250 μL of cold MeOH + 50 μL of 3M MgCl2. Spin at 20,000 rpm for 10 min Standard preparation Mix 20 μL of 20 mM DL(MSC2702209A in DMSO) + 20 μL DMSO + 40 μL MeOH + 20 μL of 200 mM NAC in diafiltration buffer. Incubate overnight to obtain 4 mM DL-NAC. Dilute 4 mM DL-NAC in MeOH to obtain 4 μM DL-NAC standard. Endotoxin characterization

[0455] Endotoxin was determined by kinetic chromogenic LAL assay using the Endosafe PTS Endotoxin System (Charles River). Buffer and antibody were diluted 10-fold in LAL reagent water. ADC was diluted 10-fold in LAL reagent water. All samples were analyzed on 0.01-1 EU / mL cartridges. EU / mL values ​​were converted to EU / mg by dividing by ADC[P]mg / mL. Example 20: ADC Preparation, Conjugation and Characterization of ADC7-M, ADC2-M, ADC5-M and ADC3-M 20.1. Antibody Preparation and Conjugation

[0456] Monoclonal antibodies (mAbs) were stored at -80°C. Prior to conjugation, mAbs were thawed at room temperature and used with The buffer was exchanged to 24 mM HEPES, pH 7.0 using a HiTrap desalting column in combination with a liquid chromatography (LC) system (Cytiva). To conjugate the drug linker (DL) to the antibody, microbial transglutaminase (mTG) was used. The reaction was set up 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 performed at 37°C for 18 hours. The ADC was separated from DL and mTG via size exclusion chromatography (SEC). Prior to SEC purification, the NaCl concentration of the sample was adjusted to 100 mM using a 5 M NaCl stock solution. SEC was performed using a HiLoad Superdex 200 26 / 60 Increase column in combination with a Cytiva 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. Fractions containing ADC material were pooled and concentrated to 8 mg / ml using an Amicon Ultra 1550K centrifuge (Millipore) and then centrifuged using the same centrifuge as described above. The final buffer exchange was into 10 mM histidine, 40 mM NaCl, 6% trehalose, 0.05% Tween, pH 5.5 in combination with a HiTrap desalting column of an LC system (Cytiva). The final drug substance (BDS) was filtered through a 0.22 μm sterile filtration unit (Millipore) and snap frozen in liquid nitrogen until further use.

[0457] 20.2 Quality attributes ADC7-M Concentration [mg / ml] 7.11 Drug / antibody ratio 3.87 PuritySEC[%] 99.9 Free drug content [%] 0.12 Endotoxin content [EU / mg] 0.34 ADC2-M Concentration [mg / ml] 6.9 Drug / antibody ratio 3.86 PuritySEC[%] 99.7 Free drug content [%] 0.03 Endotoxin content [EU / mg] 0.19 ADC3-M Concentration [mg / ml] 7.98 Drug / antibody ratio 4.2 PuritySEC[%] 100 Free drug content [%] 0.13 Endotoxin content [EU / mg] 0.14 ADC5-M 20.3. Methods: Drug Substance Characterization

[0458] Size Exclusion Chromatography (SEC) Method 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×300mm, 5μm Mobile phase: 50 mM sodium dihydrogen phosphate, 0.4 M sodium perchlorate, pH 6.3 Injection volume 1-5 μL Column temperature 25℃ Flow rate 0.5mL / min Typical SEC chromatograms showing the purity of the input mAb and final BDS are shown in Fig.38 middle.

[0459] Reverse Phase HPLC (RP HPLC) Method RP HPLC method parameters Wavelength: 214nm Column PLRP-S (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 in 7.5 min Injection volume 10 μL Column temperature 65°C Flow rate: 1.0 mL / min Sample preparation 40 μl of sample (2.2 mg / ml) was mixed with 4 μl of 0.5 M TCEP and incubated at room temperature for 5 minutes. Then, 40 μL of 0.5 M iodoacetamide was added and incubated at 37° C. for 15 minutes. 10 μl of the reaction was injected.

[0460] A typical RP-HPLC chromatogram showing the DAR determination of the final BDS is shown in Fig.39 middle.

[0461] Free drug method Wavelength 254nm Column Phenomenex Gemini, C18, 2×150 mm, 3 μm (P / N 00F-4439-B0) Mobile phase A 0.1% formic acid in water Mobile phase B: 0.1% formic acid in acetonitrile gradient Injection volume 10.00 μL Column temperature 50℃ Flow rate 0.75mL / min Sample preparation Protein drop: 100 μL of API + 250 μL of cold MeOH + 50 μL of 3M MgCl2. Spin at 20,000 rpm for 10 min Standard preparation Mix 20 μL of 20 mM DL(MSC2702209A in DMSO) + 20 μL DMSO + 40 μL MeOH + 20 μL of 200 mM NAC in diafiltration buffer. Incubate overnight to obtain 4 mM DL-NAC. Dilute 4 mM DL-NAC in MeOH to obtain 4 μM DL-NAC standard. Endotoxin characterization

[0462] Endotoxin was determined by kinetic chromogenic LAL assay using the Endosafe PTS Endotoxin System (Charles River). Buffer and antibody were diluted 10-fold in LAL reagent water. ADC was diluted 10-fold in LAL reagent water. All samples were analyzed on 0.01-1 EU / mL cartridges. EU / mL values ​​were converted to EU / mg by dividing by ADC[P]mg / mL. Example 21: ADC1-M and ADC2-M specifically kill cancer cells in vitro with high potency

[0463] 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 showed subnanomolar and subnanomolar to single-digit nanomolar in vitro potency against different CEACAM5-positive cell lines, respectively (Table 3). In contrast, ADC1-M and ADC2-M had less effect on the CEACAM5-negative cell line MDA-MB-231 (Table 3). As shown in the exemplary dose-response curves, ADC1-M and ADC2-M were very potent against the CEACAM5-positive cell lines SK-CO-1, SNU-16, MKN-45, and LS174T ( Fig.40 ad and Fig.41 ad). In contrast, ADC1-M and ADC2-M had only minor effects on the viability of antigen-negative MDA-MB-231 cells ( Fig.40 e and Fig.41 e).

[0464] Isotype control ADCs using the same linker payload as ADC1-M and ADC2-M showed much lower efficacy against the CEACAM5-positive cell lines tested ( Fig.40 and Fig.41 ).

[0465] In conclusion, ADC1-M and ADC2-M specifically kill CEACAM5-expressing human cancer cell lines in vitro with high potency. Table 3. Efficacy of ADC1-M, ADC2-M and free payload on various human cell lines. The maximum effect compared to untreated control at the highest test compound concentration is indicated in parentheses. The cutoff for efficacy reporting is -50% effect, otherwise described as "n / a". For each cell line, CEACAM5 expression is indicated. Methods - Viability assay:

[0466] The cytotoxic effect of ADC on cancer cell lines was measured by cell viability assay. One day before treatment, cells were seeded in a 96-well plate in a volume of 90 μL. Test compounds (ADC or free payload) were prepared in cell culture medium at 10 times 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 incubated at 37°C in a CO2 incubator for six days. For cell viability measurements, Cell Titer- Reagents (Promega TM Corp, Madison, WI) was added to each well and the plate was processed according to the manufacturer's instructions. The luminescent signal was measured using a Varioskan plate reader (Thermo Fisher). The luminescent readings were converted to viability % relative to untreated cells using loG (inhibitor) relative to reaction, variable slope, using the 4-parameter fitting equation of Genedata Screener or GraphPadPrism, and the data were fitted using nonlinear regression analysis. Data are displayed as relative cell viability % relative to molar compound concentration, and error bars indicate the standard deviation (SD) of duplicate or triplicate. The geometric mean of the IC50 derived from multiple experiments was calculated.

[0467] 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. Against SK-CO-1 cancer cells, ADC1-M and ADC2-M showed similar potency to ADC SAR DM4 ( Fig.40 a and Fig.41 a, compared with Figure 26a). Compared with ADC1-M and ADC2-M, ADCSAR DM4 has a higher non-specific effect on antigen-negative MDA-MB-231 ( Fig.40 e and Fig.41 e, compared with Figure 26b), and the difference of ADC2-M is more significant than that of ADC1-M.

[0468] ADC1-M and ADC2-M were also generated using an antibody backbone lacking the YTE mutation. Both of these ADCs (ADC6-M and ADC7-M) showed comparable results to the corresponding ADCs (ADC1-M and ADC2-M) with the YTE mutation. Example 23: Pharmacokinetic study of ADC

[0469] Pharmacokinetic studies were performed in human FcRn transgenic (276 hemizygous model) mice after a single intravenous administration of 3 mg / kg of ADC1, ADC1-M, ADC6-M, ADC7-M, ADC2-M. For ADC1-M, ADC6-M, ADC7-M, and ADC2-M, samples were collected from 6 animals in each treatment group at 1, 24, 48, 72, 144, 168, 240, 336, and 504 hours after dosing, and for ADC1, samples were collected from 9 animals at 0.08 (approximately 5 minutes), 4, 24, 48, 72, 168, 240, 336, and 504 hours after dosing. Pooled data were used for PK analysis, and plasma concentrations for each treatment are shown in Table 1. Fig.42 Back extrapolation of plasma concentrations at time 0 (C0) resulted in slightly below theoretical maximum concentrations for ADC1-M, ADC7-M, and ADC2-M, and close to theoretical maximums for ADC6-M and ADC1 (assuming dilution in 40 mL / kg plasma volume). Plasma concentrations decreased in a biphasic curve: a sharp decrease in plasma concentrations was observed up to 24 hours in all molecule groups, followed by a slower decrease until the last sampling time (504 hours), except for animals treated with ADC6-M, which were quantifiable until 336 hours.

[0470] The longest terminal half-life (t1 / 2) value was calculated for ADC2-M (190 hours) (Lala-YTE mutant with DAR=4), followed by ADC7-M (122 hours) (Lala-mutant with DAR=4), ADC1-M (64.5 hours) (Lala-mutant with DAR=8), and ADC6-M (33.8 hours) (Lala-mutant with DAR=8), and finally ADC1 (29.6 hours) (IgG1.4 with DAR=8). The % of extrapolated AUCinf was less than 20%, allowing reliable calculation of AUC0-inf and derived parameters (Cl, Vz, and Vss). The highest AUC0-inf and lowest Cl values ​​were calculated for ADC2-M (Lala-YTE-mutant, DAR=4) in transgenic mice, followed by the highest AUC0-inf and lowest Cl values ​​for ADC7-M, ADC6-M, ADC1-M, and ADC1. AUC0-inf and Cl values ​​ranged from 1360000 to 10200000 h*ng / mL and 0.293 to 1.16 mL / h / kg, respectively. No relevant differences in volume of distribution (Vss) were observed, with Vss ranging from 49.8 to 113 mL / kg.

[0471] In summary, ADC2-M, which had the highest plasma exposure, lowest Cl, and longest t1 / 2, showed the best PK profile after a single iv administration of 3 mg / kg in transgenic mice. The PK profile was improved by moving from IgG1.4 to the Lala mutant and further by including the YTE mutant, with the strongest effect observed by reducing the DAR from 8 to 4.

[0472] Table 5: PK parameters of Ceacam5 backup molecules after 3 mg / kg iv administration. Antibody-drug conjugate (ADC) and drug / antibody ratio (DAR) are also indicated. Example 24: Efficacy of ADC1-M and ADC2-M in a pancreatic cell line-derived tumor model

[0473] The efficacy of ADC1-M and ADC2-M has been evaluated in the human pancreatic cell line-derived xenograft model BxPC3 (ATCC, CRL-1687). 6 BxPC3 cells were injected subcutaneously into the right flank of 6- to 8-week-old immunodeficient female mice (Hsd: Athymic Nude-Foxn1nu, Envigo). 3When the mean volume of the tumor was 2.34 × 10 cm, 10 mice / group were treated once intravenously with vehicle (saline solution) or ADC1-M (5 mg / kg; day 0) or 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 the formula L×(W^2) / 2.

[0474] Single treatments with ADC1-M at a dose of 5 mg / kg and ADC2-M at a dose of 5 mg / kg or 10 mg / kg produced significant antitumor effects. Both ADCs had comparable efficacy in this model ( Fig.43 Treatment had no significant effect on body weight (data not shown). Example 25: Preparation of ADC8, an analog of ADC govetican-labetuzumab 25.1 Antibodies

[0475] For the purpose of further comparative experiments, another analog of the ADC govetican-labetuzumab was prepared based on a monoclonal antibody with the following sequence: Heavy Chain: Light chain: 25.2 Drug-Linker Compounds

[0476] As the drug-linker molecule to be conjugated to the above-mentioned antibody, a molecule of the following structure was used: The drug-linker molecule was purchased from SyntaBio LLC, 10239 Flanders Ct, San Diego, CA 92121. Lot No. S041070422. 25.3 Conjugation

[0477] The monoclonal antibody (mAb) was thawed at 2-8°C for up to 3 days before conjugation and stored in PBS pH 6.8 at 2-8°C 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 adding 0.5 M Tris, 0.025 M EDTA, pH 8.5. After pH adjustment, 10 molar equivalents of TCEP were used to reduce mAb and incubated 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 16 molar equivalents of the above-mentioned drug-linker were added to start the reaction. The reaction was incubated at 20°C for 60 min and finally quenched by adding 100 mM NAC (N-acetyl-cysteine). Then, the conjugated mAb (i.e., ADC) was processed via preparative size exclusion chromatography. 25.4 Preparative Size Exclusion Chromatography, Desalting, and Filtration

[0478] The reaction mixture was purified using preparative size exclusion chromatography. A GE HiLoad 26 / 60 Superdex S200 column was attached to Avant 25 system (GE Healthcare) and equilibrated with 20 mM histidine, 80 mM NaCl, pH 5.5 according to the manufacturer's instructions. Subsequently, the reaction mixture was 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 running buffer. The fractions containing ADC 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. 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) using a HiPrep 26 / 10 desalting column (GE Healthcare) on an Avant 25 system (GE Healthcare) at a flow rate of 10 ml / min. The final ADC material was filtered using a 0.2 μm filter (0.2 μm PES filter, Merck Millipore), aliquoted and then snap 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 obtained from this work is also referred to herein as "ADC8"; this ADC is an analog of govetican-labetuzumab. 25.5 Further Characterization of ADC8 API

[0479] The ADC8 drug substance obtained above was further analyzed by (a) size exclusion chromatography (SEC) (showing 99.3% monomer purity), (b) reverse phase HPLC (RP HPLC) (showing a DAR of 7.7), and (c) RP HPLC-based free drug method (showing a residual free drug level of less than 0.02% (on a molar basis)). Example 26: ADC1-M, ADC2-M, ADC6-M and ADC7-M kill cancer cells with higher specificity than ADC SAR DM4 and ADC8

[0480] Using the same method as 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 ADC SAR DM4 and ADC8, on cancer cells with CEACAM5 expression relative to the effects on cancer cells lacking CEACAM5 expression. The fold reduction in IC50, defined as the specificity factor, was calculated by dividing the IC50 for CEACAM5-negative MDA-MB-231 cells by the IC50 for each CEACAM5-positive cell line (see Table 6). The larger the value of the specificity factor, the higher the specificity of the ADC tested. ADC1-M, ADC2-M, ADC6-M and ADC7-M showed much lower IC50s in CEACAM5-positive SK-CO-1, SNU-16 and MKN-45 cells than in CEACAM5-negative MDA-MB-231 cells, resulting in specificity factors ranging from 116 to 874. The specificity factors of ADC2-M and ADC7-M may be underestimated due to the lack of effect on MDA-MB-231 cells in the concentration range tested (Table 3 and Fig.41 e for ADC2-M). For this reason, only for ADC2-M and ADC7-M, the IC50 was set to the highest tested concentration of 100 nM to calculate the specificity factor. In contrast, ADC SAR DM4 and ADC8 showed lower specificity factors ranging from 26 to 73 and 1.8 to 4.8, respectively. In summary, ADC1-M, ADC2-M, ADC6-M and ADC7-M showed more selective killing of CEACAM5-positive cancer cells in vitro compared to ADC SAR DM4 and ADC8. Table 6. Relative potency improvements (specificity factors) of ADCs in CEACAM5-positive cell lines compared to CEACAM5-negative MDA-MB-231 cell lines. The corresponding specificity factor for each ADC was calculated by dividing the IC50 in MDA-MB-231 by the IC50 in each of the three CEACAM5-positive cell lines (for illustration only, the theoretical factor for MDA-MB-231 is reported as 1). Calculations are based on the geometric mean of the IC50s from 2 to 4 separate experiments. Example 27: ADC1-M, ADC2-M, ADC6-M, and ADC7-M mediate a more potent bystander effect than ADC SAR DM4 for antigen-negative cells co-cultured with antigen-positive cells

[0481] The potential of ADC1-M, ADC2-M, ADC6-M, ADC7-M and ADC SAR DM4 to mediate a bystander effect against antigen-negative cells adjacent to antigen-positive cells was evaluated in a bystander assay using the same method as in Example 10. In the presence of CEACAM5-positive SK-CO-1, ADC1-M, ADC2-M, ADC6-M and ADC7-M showed a potent bystander effect against CEACAM5-negative MDA-MB-231 cells ( Fig.44 ). The co-culture experiment was carried out at a concentration of 1 nM ADC, and for ADC1, the concentration caused the maximum inhibition of CEACAM5-positive SK-CO-1 cell viability ( Fig.26A ), but had no effect on CEACAM5-negative MDA-MB-231 cells alone ( Fig.26B ). Consistent with these findings, no non-specific effects of ADC1-M, ADC2-M, ADC6-M, ADC7-M, and ADC SAR DM4 were observed in the bystander assay setting with MDA-MB-231 only control.

[0482] In conclusion, ADC1-M, ADC2-M, ADC6-M, ADC7-M mediate a potent bystander effect against antigen-positive cancer cells co-cultured with antigen-positive cells. These findings indicate the potential for effective targeting of tumors with heterogeneous target expression.

[0483] Compared with ADC SAR DM4, ADC1-M, ADC2-M, ADC6-M, and ADC7-M mediated much stronger bystander effects on antigen-negative cells in co-culture with antigen-positive cells ( Fig.44 ).

[0484] For all ADCs tested, the extent of the bystander effect increased with increasing numbers of antigen-positive cells added to a constant number of antigen-negative cells. Without wishing to be bound by theory, this may be a result of more ADC being processed by a higher number of antigen-positive cells to release more free payload, which is responsible for the bystander effect on antigen-negative cells.

[0485] In the bystander assay, no non-specific effects of the tested ADCs on MDA-MB-231 cells alone were observed for all tested ADCs at 1E-9 M tested concentration. Example 28: Efficacy of ADC1-M and ADC3-M compared to ADC8

[0486] The efficacy of ADC1-M and ADC2-M compared with ADC8 was evaluated in the HPAF-II xenograft model derived from the human pancreatic adenocarcinoma cell line (ATCC, CRL-1997). 6 HPAF-II cells were injected subcutaneously in the right flank of 6- to 8-week-old immunodeficient female mice (Hsd: Athymic Nude-Foxn1nu, Envigo). 3 When the average volume of 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) was reached, 10 mice / group were treated intravenously once. Tumor length (L) and width (W) were measured with a caliper, and tumor volume was calculated using the formula L×W^2 / 2.

[0487] Compared with vehicle control, single treatment with ADC1-M or ADC3-M at a dose of 1 or 6 mg / kg resulted in significant anti-tumor effects. The effect was dose-dependent, as a single treatment with 1 mg / kg resulted in only a slight and temporary anti-tumor effect, while 6 mg / kg showed a much stronger anti-tumor effect. In contrast, a single treatment with ADC8 did not show a significant anti-tumor effect at any dose ( Fig.45 All treatments had no significant effect on body weight (data not shown). Example 29: Efficacy of ADC1-M and ADC3-M compared to ADC SAR DM4 in the CRC PDX mouse model

[0488] The antitumor efficacy of ADC1-M and ADC3-M compared to ADC SAR DM4 was evaluated in the human CRC xenograft model COPF230. 6- to 8-week-old immunodeficient female mice (Crl:NU-Foxn1nu) were subcutaneously implanted with COPF230 tumor fragments in the right flank. When tumors reached 165 mm3 When the average volume of 6 mice / group was 6 mg / kg, 6 mice / group were treated intravenously once with vehicle (saline solution) or ADC1-M, ADC3-M or ADC SAR DM4 (each at 6 mg / kg). Tumor length (L) and width (W) were measured with calipers, and tumor volume was calculated using the formula L×W^2 / 2.

[0489] Compared with the vehicle control group, a single treatment with 6 mg / kg ADC1-M or ADC3-M resulted in comparable and significant antitumor effects. In contrast, a single treatment with 6 mg / kg ADC SAR DM4 showed only a minor antitumor effect ( Fig.46 All treatments had no significant effect on body weight (data not shown). Example 30: Efficacy of ADC1-M and ADC3-M compared to ADC SAR DM4 in the GC PDX mouse model

[0490] 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). 6- to 8-week-old immunodeficient female mice (Crl:NU-Foxn1nu) were subcutaneously transplanted with GAPF313 tumor fragments in the right flank. When the tumor reached 176 mm 3 When the average volume of 6 mice / group was 1.5 mg / kg, ADC1-M or ADC3-M or ADC SAR DM4 was 4.7 mg / kg, ADC SAR DM4 was used to treat 6 mice / group (3 cycles) every other week intravenously. Tumor length (L) and width (W) were measured with calipers, and tumor volume was calculated using the formula L×W^2 / 2.

[0491] Multiple treatments with ADC1-M or ADC3-M resulted in comparable and significant antitumor effects compared to vehicle control. ADC SAR DM4 was significantly less effective than ADC1-M and ADC3-M ( Fig.47 All treatments had no significant effect on body weight (data not shown).

Claims

1. An isolated antibody that binds to human CEACAM5 protein; and 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, and preferably comprising said sequence at the C-terminus of said 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 mutation); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y, S254T, and T256E (YTE mutation); (e) K222R; and wherein Eu numbering is used for said amino acid substitutions; And wherein 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.

2. The isolated antibody of claim 1, wherein 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; 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.

3. An isolated antibody that binds to human CEACAM5 protein; and wherein the isolated antibody comprises: (i) at least one light chain constant region (CL) comprising a sequence selected from the group consisting of GGTLQSPP, TLQSPP, TLQSA and TLQSG, and preferably comprising said sequence at the C-terminus of said 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 mutation); (b) L234A and L235A and P329G (LALA-PG mutation); (c) L235A and G237A (LAGA mutations); (d) M252Y, S254T, and T256E (YTE mutation); (e) K222R; and wherein Eu numbering is used for said amino acid substitutions; And wherein 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; And wherein 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; wherein 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.

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

5. The isolated antibody of any one of claims 1-3, wherein at least one heavy chain constant region (CH) comprises the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:32 and / or wherein at least one of the light chain constant regions (CL) comprises the amino acid sequence of SEQ ID NO:

33.

6. The isolated antibody of any one of claims 1 to 3, wherein the heavy chain constant region (CH) and the light chain constant region (CL) have any one of the following sequence combinations: (1) both CHs contain the sequence of SEQ ID NO: 31 and both CLs contain the sequence of SEQ ID NO: 12; or (2) both CHs contain the sequence of SEQ ID NO: 31 and both CLs contain the sequence of SEQ ID NO: 33; or (3) both CHs contain the sequence of SEQ ID NO: 32 and both CLs contain the sequence of SEQ ID NO: 33; or (4) both CHs contain the sequence of SEQ ID NO: 50 and both CLs contain the sequence of SEQ ID NO: 12; or (5) both CHs contain the sequence of SEQ ID NO: 50 and both CLs contain the sequence of SEQ ID NO: 33; or (6) one CH comprises the sequence of SEQ ID NO:31 and one CL comprises the sequence of SEQ ID NO:12; or (7) one CH comprises the sequence of SEQ ID NO:31 and one CL comprises the sequence of SEQ ID NO:33; or (8) one CH comprises the sequence of SEQ ID NO: 32 and one CL comprises the sequence of SEQ ID NO: 33; or (9) one CH comprises the sequence of SEQ ID NO:50 and one CL comprises the sequence of SEQ ID NO:12; or 10) One CH comprises the sequence of SEQ ID NO:50 and one CL comprises the sequence of SEQ ID NO:

33.

7. The antibody according to any one of claims 1 to 6, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 9 and a light chain variable region (VL) comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

10.

8. The antibody according to claim 7, comprising 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.

9. The antibody according to any one of claims 1 to 8, comprising: (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) 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: 36; or (iii) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 35 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 36; or (iv) 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: 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.

10. An isolated antibody that competes for binding to the A2-B2 domain of human CEACAM5 protein with 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, and wherein the heavy chain constant region (CH) and the light chain constant region (CL) are as defined in any one of claims 1 to 9.

11. The antibody according to claim 10, wherein the antibody also competes for binding to the A2-B2 domain of cynomolgus monkey CEACAM5 protein with 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, and wherein the heavy chain constant region (CH) and the light chain constant region (CL) are as defined in any one of claims 1 to 9.

12. The antibody according to any one of claims 1 to 11, wherein the antibody does not significantly cross-react with human CEACAM1, human CEACAM6, human CEACAM7, human CEACAM8 and cynomolgus monkey CEACAM6.

13. The antibody according to any one of claims 1 to 12, wherein the antibody is an antibody fragment.

14. The antibody according to claim 13, wherein the antibody is an antibody fragment selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2 and diabodies.

15. The antibody according to any one of claims 1 to 14, which is a bispecific or multispecific antibody.

16. An isolated antibody that binds to human CEACAM5 protein and: It consists of 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 It consists of 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 It consists of 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 It consists of 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 It consists of 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.

17. An isolated nucleic acid comprising a nucleic acid sequence encoding the antibody according to any one of claims 1 to 16.

18. A host cell which has been transformed with the nucleic acid according to claim 17.

19. An immunoconjugate comprising the antibody according to any one of claims 1 to 16 covalently linked to at least one growth inhibitory agent via a linker.

20. The immunoconjugate of claim 19, wherein the growth inhibitory agent is a cytotoxic drug or a radioactive moiety.

21. The immunoconjugate of claim 19 or 20, wherein the growth inhibitory agent is selected from a chemotherapeutic agent, an enzyme, an antibiotic, a toxin, such as a small molecule toxin or an enzymatically active toxin, a toxoid, a vinca, a taxane, a maytansinoid or a maytansinoid analog, tomamycin or a pyrrolobenzodiazepine derivatives, nostoc derivatives, leptomycin derivatives, auristatins or dolastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, anti-tubulin agents, CC-1065 and CC-1065 analogs.

22. The immunoconjugate of any one of claims 19 to 21, wherein the growth inhibitory agent is exatecan.

23. The immunoconjugate of any one of claims 19 to 22, wherein the linker is a cleavable linker.

24. The immunoconjugate of any one of claims 19 to 23, wherein the linker is a linker cleavable in the endosome of a mammalian cell.

25. The immunoconjugate of any one of claims 19 to 24, wherein the linker is a linker cleavable by the human enzyme glucuronidase.

26. The immunoconjugate according to any one of claims 19 to 25, wherein the immunoconjugate has the following formula (II) or formula (IIA): wherein S is a sulfur atom of the antibody and wherein n is the number of [(linker)-(growth inhibitory agent)] moieties covalently attached to the antibody.

27. The immunoconjugate according to claim 26, wherein n is 1 to 10, and preferably 4.

28. The immunoconjugate according to any one of claims 19 to 26, wherein the immunoconjugate has the following formula (IV) or formula (IVA): wherein S is a sulfur atom of the antibody, and wherein n is the number of [(linker)-(exetatecan)] moieties covalently attached to the antibody.

29. The immunoconjugate of claim 28, wherein the immunoconjugate has formula (IVA) and n is 1 to 10, and preferably 4.

30. The immunoconjugate of any one of claims 19 to 29, wherein S is the sulfur atom of a cysteine ​​of the antibody.

31. The immunoconjugate of claim 30, wherein the cysteine ​​of the antibody is one of the cysteines capable of forming an interchain disulfide bond.

32. The immunoconjugate of any one of claims 26 to 31, wherein for formula (II) and (IV), n is 7 to 8; and wherein for formula (IIA) and (IVA), n is 3 to 5.

33. The immunoconjugate of any one of claims 26 to 32, wherein for formula (II) and (IV), n is 7.5 to 8.0; and wherein for formula (IIA) and (IVA), n is 3.5 to 4.5, and preferably for formula (IIA) and (IVA), n is 4.

34. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 16 or an immunoconjugate according to any one of claims 19 to 33, further comprising a pharmaceutically acceptable carrier, diluent and / or excipient.

35. An antibody according to any one of claims 1 to 16 or an immunoconjugate according to any one of claims 19 to 33 or a pharmaceutical composition according to claim 34 for use as a medicament.

36. The antibody according to any one of claims 1 to 16 or the immunoconjugate according to any one of claims 19 to 33 or the pharmaceutical composition according to claim 34 for use in treating cancer.

37. The antibody, immunoconjugate or pharmaceutical composition for use according to claim 36, wherein the cancer is a CEACAM5 expressing cancer.

38. The antibody, immunoconjugate or pharmaceutical composition for use according to claim 36 or 37, wherein the cancer is colorectal cancer, gastric cancer, lung cancer, pancreatic cancer, esophageal cancer or prostate cancer.

39. A method of treating cancer comprising administering to a subject an antibody according to any one of claims 1 to 16, an immunoconjugate according to any one of claims 19 to 33, or a pharmaceutical composition according to claim 34.

40. The method of claim 39, wherein the cancer is a CEACAM5 expressing cancer.

41. The method of claim 39 or 40, wherein the cancer is colorectal cancer, gastric cancer, lung cancer, pancreatic cancer, esophageal cancer, or prostate cancer.

42. A method for detecting CEACAM5 expression ex vivo in a biological sample from a subject using the antibody according to any one of claims 1 to 16.

43. The method of claim 42, wherein the antibody is labeled with a detectable molecule.

44. Use of the antibody according to any one of claims 1 to 16 for detecting CEACAM5 expression ex vivo in a biological sample from a subject.

45. The use according to claim 44, wherein the antibody is labeled with a detectable molecule.

Images