CEACAM5 mRNA assay for patient selection in cancer therapy

Abstract

The present disclosure relates to methods and uses for selecting and treating a cancer patient, wherein the cancer expresses CEACAM5. These methods comprise determining a log2 converted, quantile normalized per million per thousand base transcript (TPM) value of CEACAM5 mRNA in a tumor isolation sample obtained from the subject, (ii) comparing the value to a reference value, and (iii) selecting the subject for cancer treatment if the determined value is higher than the reference value. The agent for use in the treatment of the selected subject may be tesutumab-raisine. Such agents may be used in combination with one or more additional agents to treat the cancer. In certain embodiments, the cancer is a non-squamous non-small cell lung cancer (NSQ NSCLC).

Description

[REFERENCE TO A SEQUENCE LISTING]

[0001] This application contains a Sequence Listing which has been submitted electronically in.xml format and which is hereby incorporated by reference in its entirety. The.xml copy, created on March 12, 2024, is named PI022916.WO SANOFI(S327)LISTING.xml. [TECHNICAL FIELD]

[0002] The present disclosure relates to the field of cancer therapy. [BACKGROUND]

[0003] Despite recent advances in cancer treatment, there remains an unmet need for effective new treatments when disease progresses after first-line therapy. Current follow-on systemic treatment options combining angiogenesis inhibitors with systemic cytotoxic agents such as docetaxel are associated with severe hematologic and other toxicities. Additional options offered by docetaxel, pemetrexed or gemcitabine as single cytotoxic agents are very limited. Therefore, targeted cytotoxic therapies can improve safety, tolerability, and efficacy.

[0004] Lung cancer is a leading cause of cancer-related mortality worldwide [1]. In the United States, non-squamous non-small cell lung cancer (NSQ-NSCLC) accounts for 45.4% of new lung cancer diagnoses, of which 47.7% present with distant metastases [2], with associated low 5-year relative survival rates of only 5-8% [3].

[0005] While some lung cancers respond to therapies targeting multiple oncogene drivers [4] or immune checkpoint inhibitors (ICIs) [5], there remains an unmet need for novel therapies for patients whose lung cancer has no actionable target or is unresponsive to ICIs.

[0006] Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are cell surface glycoproteins involved in cell adhesion, cell signaling, and promotion of cancer progression and metastasis [6]. As such, they are promising targets for novel anticancer agents.

[0007] While different CEACAMs show specific expression patterns in tissues, they exhibit similarities in structural homology and amino acid sequence [7]. This requires that molecules targeting CEACAMs must specifically bind to a single CEACAM to limit cross-reactivity and dose-limiting toxicities.

[0008] CEACAM5 was first described in 1965 as a tumor-associated antigen in human colon carcinoma tissue extracts (Gold P et al., J Exp Med. 1965; 122(3):467-481). Since then, high levels of CEACAM5 expression have been observed in several epithelial tumors, while in normal adult tissues, its expression is limited to few tissues (Hammarstrom S., Semin Cancer Biol. 1999; 9(2):67-81; Thompson JA., Tumor Biol. 1995; 16(1): 10-16).

[0009] Among the CEACAMs, therapies targeting CEACAM5 are of particular interest in lung cancer. CEACAM5 is overexpressed in many epithelial tumors [8], which promotes tumorigenesis and metastasis [9]. CEACAM5 is expressed at higher levels in NSCLC, but not in normal lung tissue [7, 10]. Moreover, higher CEACAM5 expression in human NSCLC tissues is associated with poorer histological grading

[10] , and higher CEACAM5 expression is associated with lower survival rates in NSCLC patients. [11, 12] Taken together, these findings suggest that CEACAM5 is a promising target for antibody drug conjugate (ADC) therapy.

[0010] Antibody drug conjugates (ADCs) have shown promise in improving the prognosis of lung cancer patients

[13] . Tusamitamab ravtansine (SAR408701) is a potential first-in-class ADC that selectively targets tumor cells expressing CEACAM5 without binding to CEACAM1, CEACAM6, or CEACAM8 glycoproteins [7]. It is composed of an anti-CEACAM5 humanized monoclonal antibody conjugated via a cleavable linker to a potent cytotoxic maytansinoid, N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4) payload [7]. The antibody component of tusamitamab ravtansine binds to the extracellular domain of CEACAM5, followed by internalization of the ADC and then release of DM4 into the tumor cell

[14] . DM4 inhibits microtubule assembly, leading to cell cycle arrest and apoptosis

[14] . Both DM4 and its active metabolite (S-methyl-DM4) cross the cell membrane, meaning that the cytotoxic effect of tusamitamab ravtansine acts both directly through specific binding to CEACAM5-expressing tumor cells and indirectly through local diffusion of DM4 and S-methyl-DM4 (bystander effect)

[14] .

[0011] An ongoing study (TED13751) demonstrated encouraging preliminary antitumor activity of tiragolumab-rysodic in participants with NSQ NSCLC who were heavily pretreated.

[0012] In the dose escalation phase of the first-in-human 1 / 2 phase study of tiragolumab-rysodic in patients with advanced solid tumors for which no standard alternative therapy was available (ClinicalTrials.gov NCT02187848), the primary dose-limiting toxicity was reversible keratopathy, and the maximum tolerated dose was 100 mg / m2every 2 weeks. 2

[15] .

[0013] In the expansion phase of the same study, in patients with advanced NSQ-NSCLC expressing high levels of CEACAM5 (defined as at least 50% of tumor cells with 2+ or 3+ staining intensity on immunohistochemistry (IHC)), tiragolumab-rysodic showed a promising objective response rate of 20.3% and good safety, with the most common treatment-emergent adverse events being asthenia, reversible keratopathy events, peripheral neuropathy, dyspnea, and diarrhea, with less hematologic toxicities compared to those reported in patients receiving docetaxel

[16] ; moreover, in patients achieving a partial response, 47% of treatment duration was over 1 year, suggesting that responses to tiragolumab-rysodic are durable and often sustained

[17] .

[0014] While these clinical trial data show promise, in clinical practice, a potential barrier to pre-screening patients with cancer (e.g., NSQ-NSCLC) who can respond to CEACAM5-targeted therapy includes the availability of archived tumor biopsies for measuring CEACAM5 expression by immunohistochemistry.

[0015] From the above 1 / 2 phase study, we explored the association of biomarkers with 1) tumor CEACAM5 expression measured by immunohistochemistry with tumor CEACAM5 mRNA levels; and 2) whether CEACAM5 mRNA predicted tumor objective response rate. [SUMMARY]

[0016] As shown in the examples, the clinical responses of patients with high CEACAM5 protein expression levels and treated with tiragolumab-rysodic (tiragolumab-rysodic responders) were rich.

[0017] In the present disclosure, the expression "patient clinical response enrichment" in relation to treatment with tisotumab vedotin, is intended to mean an increase in the overall response rate (ORR) following administration of a given treatment. ORR is defined as the proportion of patients who have a partial response or complete response to therapy; it does not include stable disease and is a direct measure of the tumoricidal activity of a drug.

[0018] Furthermore, a correlation was found between the level of CEACAM5 protein expression in the tumor (measured by immunohistochemistry (IHC) staining) and the CEACAM5 tumor mRNA level. CEACAM5 mRNA expression was significantly upregulated in patients with high versus intermediate CEACAM5 protein expression. Moreover, in tisotumab vedotin responders, patients with high CEACAM5 tumor mRNA level and high CEACAM5 immunohistochemistry (IHC) staining (i.e., >2+ intensity in >50% of tumor cells) had a clinical response enrichment.

[0019] Since a clinical response enrichment was shown for high CEACAM5 protein expression and a correlation between CEACAM5 protein expression and mRNA CEACAM5 expression level was shown, it is suggested that mRNA CEACAM5 expression level is a good biomarker to predict clinical response enrichment in patients to be treated with an anti-CEACAM5 antibody drug conjugate, such as tisotumab vedotin; alternatively, the results suggest that patients can be selected based on CEACAM5 mRNA level rather than IHC.

[0020] Therefore, the Examples section supports the use of CEACAM5 mRNA level as a biomarker for selecting and treating patients in need of cancer treatment with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.

[0021] Furthermore, the Examples section supports the use of CEACAM5 mRNA level as a biomarker for selecting patients in need of cancer treatment with an antibody drug conjugate (ADC comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent), for an additional selection step with CEACAM5 immunohistochemistry (IHC) staining and subsequent treatment of patients in need of cancer treatment with the ADC.

[0022] It is common knowledge to the skilled person that protein expression level is not necessarily correlated with the expression level of the corresponding mRNA.

[0023] The present disclosure is based, at least in part, on the observation that a particular value of the level of expression of the CEACAM5 gene (e.g. the level of transcription of the CEACAM5 gene, e.g. the level of CEACAM5 mRNA, e.g. the log2 transformed, quantile-normalized Transcripts Per Kilobase Million (TPM) value of CEACAM5 mRNA) can be used as a biomarker for selecting patients for cancer treatment, these cancers typically expressing the CEA cell adhesion molecule 5 (CEACAM5) on their tumor cells.

[0024] In some embodiments, the present disclosure relates to a method for selecting a subject in need of a treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0025] (i) determining a value of the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from said subject,

[0026] (ii) comparing said determined value to a reference value, and

[0027] (iii) selecting said subject for a treatment of a cancer if the determined value is higher than the reference value.

[0028] In the present disclosure, a subject in need is a subject suffering from a cancer. An isolated sample of a tumor of said cancer can be used.

[0029] Herein, the terms "subject", "patient", "subject in need" and "patient in need" can be used interchangeably.

[0030] In some embodiments, the present disclosure relates to a method for selecting a subject in need of a treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0031] (i) determining a value of the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from said subject,

[0032] (ii) comparing said value determined in step (i) to a reference value of the level of expression of the CEACAM5 gene,

[0033] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii),

[0034] (iv) determining the intensity of the level of CEACAM5 protein expression in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0035] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0036] (vi) selecting the subject for cancer treatment if the intensity determined in step (iv) is higher than the reference intensity.

[0037] The tumor sample of step (i) and step (iv) can be the same sample or different samples.

[0038] In some embodiments, the disclosure relates to a method for selecting and treating a subject in need of treatment of cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0039] (i) determining a value of the level of CEACAM5 gene expression in a tumor isolated sample obtained from the subject,

[0040] (ii) comparing the determined value to a reference value,

[0041] (iii) selecting the subject for cancer treatment if the determined value is higher than the reference value, and

[0042] (iv) administering to the selected subject an effective amount of the ADC.

[0043] In some embodiments, the disclosure relates to a method for selecting and treating a subject in need of treatment of cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0044] (i) determining a value of the level of CEACAM5 gene expression in a tumor isolated sample obtained from the subject,

[0045] (ii) comparing the value determined in step (i) to a reference value of the level of CEACAM5 gene expression,

[0046] (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii),

[0047] (iv) determining the intensity of the CEACAM5 protein expression level in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0048] (v) comparing the intensity determined in step (iv) with a reference intensity, and

[0049] (vi) selecting the subject for cancer treatment if the intensity determined in step (iv) is higher than the reference intensity, and

[0050] (vii) administering to the selected subject an effective amount of the ADC.

[0051] In some embodiments, the disclosure relates to an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,

[0052] The use comprises:

[0053] (i) determining a value of the CEACAM5 gene expression level in a tumor isolated sample obtained from the subject,

[0054] (ii) comparing the determined value with a reference value, and

[0055] (iii) administering to the subject an effective amount of the ADC if the determined value is higher than the reference value.

[0056] In some embodiments, the disclosure relates to an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,

[0057] The use comprises:

[0058] (i) determining a value of the CEACAM5 gene expression level in a tumor isolated sample obtained from the subject,

[0059] (ii) comparing the value determined in step (i) with a reference value of the CEACAM5 gene expression level,

[0060] (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii),

[0061] (iv) determining the intensity of the CEACAM5 protein expression level in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0062] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0063] (vi) if the determined intensity is higher than the reference intensity, administering to the subject an effective amount of the ADC.

[0064] In some embodiments, the disclosure relates to the use of a measure of the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from a subject in need thereof for characterizing the tumor as a CEACAM5 high expressing tumor.

[0065] In some embodiments, the disclosure relates to the use of a measure of the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from a subject in need thereof for selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test.

[0066] In some embodiments, the disclosure relates to the use of a measure of the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from a subject in need thereof for selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test.

[0067] In some embodiments, the disclosure relates to the use of a measure of the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from a subject in need thereof for selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test.

[0068] In some embodiments, the value of the level of expression of the CEACAM5 gene can be a measure of CEACAM5 gene transcripts.

[0069] In some embodiments, the CEACAM5 gene transcripts can be mRNA.

[0070] In some embodiments, the methods and uses of the disclosure comprise the step of determining a value of the level of CEACAM5 mRNA in a tumor isolated sample.

[0071] According to one of its objects, the disclosure relates to a method for selecting a subject in need of treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody coupled to a cytotoxic agent, the method comprising at least the steps of:

[0072] (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from said subject,

[0073] (ii) comparing said value to a reference value, and

[0074] (iii) selecting said subject for cancer treatment if the determined value is higher than the reference value.

[0075] In some embodiments, the value of the CEACAM5 gene expression level can be a measure of CEACAM5 gene transcript.

[0076] In some embodiments, the CEACAM5 gene transcript can be mRNA.

[0077] According to one of its objects, the disclosure relates to a method for selecting a subject in need of treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0078] (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from said subject,

[0079] (ii) comparing said value determined in step (i) to a reference value,

[0080] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii),

[0081] (iv) determining the intensity of the CEACAM5 protein expression level in a tumor isolated sample obtained from said subject with said CEACAM5 immunohistochemistry (IHC) staining test,

[0082] (v) comparing said intensity determined in step (iv) to a reference intensity, and

[0083] (vi) selecting said subject for cancer treatment if the determined intensity is higher than the reference intensity.

[0084] According to another of its objects, the disclosure relates to a method for selecting and treating a subject in need of treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0085] (i) determining a log2 transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject,

[0086] (ii) comparing the value to a reference value,

[0087] (iii) selecting the subject for cancer treatment if the determined value is higher than the reference value, and

[0088] (iv) administering to the selected subject an effective amount of the ADC.

[0089] Thereby, cancer can be treated.

[0090] According to another of its objects, the disclosure relates to a method for selecting and treating a subject in need of treatment of cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0091] (i) determining a log2 transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject,

[0092] (ii) comparing the value determined in step (i) to a reference value,

[0093] (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii),

[0094] (iv) determining the intensity of the level of CEACAM5 protein expression in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0095] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0096] (vi) selecting the subject for cancer treatment if the determined intensity is higher than the reference intensity, and

[0097] (vii) administering to the selected subject an effective amount of the ADC.

[0098] According to another of its objects, the disclosure relates to an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of cancer in a subject in need thereof,

[0099] The use comprises (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject, (ii) comparing the value to a reference value, and (iii) administering to the subject an effective amount of the ADC if the determined value is higher than the reference value.

[0100] According to another of its objects, the disclosure relates to an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,

[0101] The use comprises:

[0102] (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject,

[0103] (ii) comparing the value determined in step (i) to a reference value,

[0104] (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii),

[0105] (iv) determining the intensity of the level of CEACAM5 protein expression in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0106] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0107] (vi) administering to the subject an effective amount of the ADC if the determined intensity is higher than the reference intensity.

[0108] As shown in the Examples section, the expression of about 15,000 genes, including CEACAM5, was assessed using ribonucleic acid (RNA) sequencing. The RNA data was filtered and defined as log-transformed, quantile-normalized transcripts per million per kilobase (TPM). It was observed that differential gene expression analysis had determined that CEACAM5 mRNA was the most relevant gene (and also the only significant gene, adjusted P = 0.00265) with high versus moderate expression of CEACAM5 measured by IHC. There was an increase in CEACAM5 mRNA per million transcripts in high versus moderate CEACAM5 expression; and (C) CEACAM5 mRNA correlated with CEACAM5 IHC H-score. Thus, CEACAM5 gene expression level (RNA) can be used to identify and select patients who will respond to CEACAM5 -targeted therapy, such as an antibody drug conjugate comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.

[0109] In the present disclosure, “an antibody drug conjugate comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent”, “antibody drug conjugate” and “ADC” are used interchangeably.

[0110] In some embodiments, the ADC can be used in an effective amount.

[0111] In some embodiments, the use can be embodied in a subject in need thereof.

[0112] In the use as disclosed herein, the biomarker of CEACAM5 mRNA is measured in an isolated biological sample.

[0113] In some embodiments, the anti-CEACAM5 antibody can comprise a HCDR1 having the amino acid sequence of SEQ ID NO: 1, a HCDR2 having the amino acid sequence of SEQ ID NO: 2, a HCDR3 having the amino acid sequence of SEQ ID NO: 3, a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of NTR, and a LCDR3 having the amino acid sequence of SEQ ID NO: 5.

[0114] In some embodiments, the anti-CEACAM5 antibody can comprise a heavy chain variable domain (VH) consisting of SEQ ID NO: 6 and a light chain variable domain (VL) consisting of SEQ ID NO: 7.

[0115] In some embodiments, the anti-CEACAM5 antibody can be tisotumab.

[0116] In some embodiments, the cytotoxic agent can be selected from the group consisting of a radioisotope, a protein toxin, a small molecule toxin, and any combination thereof.

[0117] In some embodiments, the small molecule toxin can be selected from the group consisting of an antimetabolite, a DNA alkylating agent, a DNA cross-linking agent, a DNA intercalating agent, an anti-microtubule agent, a topoisomerase inhibitor, and any combination thereof.

[0118] In some embodiments, the anti-microtubule agent can be selected from the group consisting of taxanes, vinca alkaloids, maytansinoid alkaloids, colchicine, podophyllotoxin, griseofulvin, and any combination thereof.

[0119] In some embodiments, the cytotoxic agent can be a maytansinoid alkaloid or a maytansinoid alkaloid analog.

[0120] In some embodiments, the maytansinoid alkaloid can be selected from the group consisting of N2'-deacetyl-N2'-(3-mercapto-l-oxopropyl)-maytansine (DM1), N2'-deacetyl-N2'-(4-methyl-4- mercapto-l-oxopentyl)-maytansine (DM4), and combinations thereof.

[0121] In some embodiments, the anti-CEACAM5 antibody can be covalently attached to the at least one chemotherapeutic agent via a cleavable or non-cleavable linker.

[0122] In some embodiments, the linker can be selected from the group consisting of pyridyldithiobutyric acid N-succinimidyl ester (SPDB), 4-(pyridyl disulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and (N-maleimidomethyl) cyclohexane-l-carboxylate succinimidyl ester (SMCC).

[0123] In some embodiments, the anti-CEACAM5 antibody can be covalently attached to the at least one chemotherapeutic agent via a cleavable linker selected from the group consisting of pyridyldithiobutyric acid N-succinimidyl ester (SPDB), 4-(pyridyl disulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and (N-maleimidomethyl) cyclohexane-l-carboxylate succinimidyl ester (SMCC).

[0124] In some embodiments, the CEACAM5 antibody can comprise a heavy chain (VH) consisting of SEQ ID NO: 8 and a light chain (VL) consisting of SEQ ID NO: 9 (huMAb2-3), and it can be covalently linked to N2'-deacetyl-N-2'(4-methyl-4-mercapto-l-oxopentyl)-maytansine (DM4) via pyridyldithiobutyric acid N-succinimidyl ester (SPDB).

[0125] In some embodiments, the antibody drug conjugate can be characterized by a drug antibody ratio (DAR) ranging from 1 to 10.

[0126] In some embodiments, the antibody drug conjugate can be tisotumab vedotin.

[0127] In some embodiments, the reference value can be a log2 transformed, quantile normalized Transcripts Per Million per kilobase transcript (TPM) value of CEACAM5 mRNA.

[0128] In some embodiments, the reference value can be at least about 7 to about 13.

[0129] In some embodiments, the reference value can be at least about 7, or can be at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 13.

[0130] In some embodiments, the quantile normalization can be obtained by (i) ranking the transcripts of a sample by expression level, (ii) calculating the mean of genes occupying the same rank, and (iii) replacing the values of all genes occupying the same rank with this mean.

[0131] In some embodiments, the expression level of a transcript can be measured in Fragments Per Million per kilobase (FPKM) before being transformed into TPM.

[0132] In some embodiments, Fragments Per Million per kilobase (FPKM) can be obtained by counting the total transcripts in the sample, dividing the obtained transcript count by 1,000,000, and dividing the obtained value by the length of the gene in kilobases.

[0133] In some embodiments, the cancer can be selected from the group consisting of hepatocellular carcinoma, colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer, cervical cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer, prostate cancer, neuroendocrine cancer, and skin cancer.

[0134] In some embodiments, the cancer can be selected from the group consisting of colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer, cervical cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer, prostate cancer, neuroendocrine cancer, and skin cancer.

[0135] In some embodiments, the cancer can be selected from colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, pancreatic cancer, and lung cancer.

[0136] In some embodiments, the cancer can be gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, or esophageal cancer.

[0137] In some embodiments, the cancer can be gastric cancer.

[0138] In some embodiments, the cancer can be colorectal cancer.

[0139] In some embodiments, the cancer can be pancreatic cancer.

[0140] In some embodiments, the cancer can be gastroesophageal junction adenocarcinoma (GEJ).

[0141] In some embodiments, the cancer can be lung cancer.

[0142] In some embodiments, the lung cancer can be non-squamous non-small cell lung cancer (NSQ NSCLC).

[0143] In some embodiments, the non-squamous non-small cell lung cancer can be advanced or metastatic NSQ NSCLC.

[0144] In some embodiments, the non-squamous non-small cell lung cancer has no epidermal growth factor receptor (EGFR) sensitizing mutations or v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations or anaplastic lymphoma kinase / c-ros oncogene 1 (ALK / ROS) alterations.

[0145] In some embodiments, the anti-CEACAM5 antibody can be trastuzumab.

[0146] In some embodiments, the ADC can be trastuzumab-emtansine.

[0147] In some embodiments, the administration dose of the ADC can be > 80 mg / m2of body surface area of the subject, about once every two weeks, or the administration dose of the ADC can be > 80 mg / m2of body surface area of the subject, about once every three weeks. 2 2

[0148] In some embodiments, the ADC can be administered at a dose of 80 mg / m2to 210 mg / m2, 80 mg / m2to 170 mg / m2, 80 mg / m2to 150 mg / m2, 80 mg / m2to 120 mg / m2, or 80 mg / m2to 100 mg / m2. 2 2 2 2 2 2 2 2 2 2

[0149] ​​​​​​​​​​​​In some embodiments, the ADC can be administered at a dose of 80, 100, 120, 150, 170, 180, or 210 mg / m 2

[0150] In some embodiments, the method or use as described herein can further comprise administering to the subject an effective amount of at least one additional agent effective in treating cancer.

[0151] In some embodiments, the additional agent can be selected from the group consisting of an immune checkpoint inhibitor (ICI), platinum-based chemotherapy, pemetrexed, anti-VEGFR2, FOLFOX, FOLFIRI, TAS-102, anti-EGFR, and any combination thereof.

[0152] In some embodiments, the ICI can be an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0153] In some embodiments, the anti-PD-1 antibody can be selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sintilimab, dostarlimab, and tislelizumab.

[0154] In some embodiments, the anti-PD-L1 antibody can be selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0155] In some embodiments, the subject receives an effective amount of tisotumab vedotin and pembrolizumab.

[0156] In some embodiments, the method or use as described herein can further comprise administering to the subject an effective amount of platinum-based chemotherapy.

[0157] In some embodiments, the platinum-based chemotherapy can be selected from cisplatin and carboplatin.

[0158] In some embodiments, the method or use as described herein can further comprise administering to the subject an effective amount of pemetrexed. [SUMMARY]

[0159] Figure 1 : shows CEACAM5 mRNA per million transcript increase in high vs. intermediate CEACAM5 expression.

[0160] Figure 2 : shows correlation of CEACAM5 mRNA with CEACAM5 IHC H-score.

[0161] Figure 3 ​: Shows the correlation of CEACAM5 mRNA levels with CEACAM5 expression measured by immunohistochemistry as the sum of tumor cell percentage expressing target at at least 2+ intensity according to patient response to treatment with tisotumab vedotin-berdoxil.

[0162] Figure 4 : Shows the correlation of CEACAM5 mRNA levels with CEACAM5 expression measured by immunohistochemistry as the sum of tumor cell percentage expressing target at at least 2+ intensity in patients who responded to treatment with tisotumab vedotin-berdoxil.

[0163] Figure 5 : Shows the box plot of CEACAM family member expression according to clinical response of patients treated with tisotumab vedotin-berdoxil. [SEQUENCE SUMMARY]

[0164] SEQ ID NOs: 1-5 show the sequences CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 of the anti-CEACAM5 antibody (huMAb2-3).

[0165] SEQ ID NO: 6 shows the sequence of the heavy chain variable domain (VH) of the anti-CEACAM5 antibody (huMAb2-3).

[0166] SEQ ID NO: 7 shows the sequence of the light chain variable domain (VL) of the anti-CEACAM5 antibody (huMAb2-3).

[0167] SEQ ID NO: 8 shows the sequence of the heavy chain of the anti-CEACAM5 antibody (huMAb2-3).

[0168] SEQ ID NO: 9 shows the sequence of the light chain of the anti-CEACAM5 antibody (huMAb2-3). [DETAILED DESCRIPTION] DEFINITIONS

[0169] Unless otherwise defined, scientific and technical terms used in connection with the disclosure herein shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, can provide guidance to the meanings of many of the terms used in connection with the disclosure. Although definitions of terms can vary, these dictionaries can provide guidance to the meanings of the terms used in connection with the disclosure. It should be further understood that any methods disclosed can be used in connection with either or both of the genus and species of the disclosure, and comprises any sub or supplemental methods, or equivalents thereof, unless otherwise stated. If there is a conflict between the definitions in the specification and those in the patent, the definitions in the specification control. In general, the nomenclature used herein and the techniques utilized in cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic and medicinal chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well- known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0170] Units, prefixes, and symbols are denoted in their International System of Units (Système International des Unités, SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are to be given their broadest interpretation.

[0171] All publications and other references provided herein are incorporated by reference in their entirety. Although a number of documents are cited herein, such citation does not constitute an admission that any of these documents forms part of the prior art to the claim at issue. Prior art status of a document can be ascertained by a prior art search.

[0172] Throughout the specification and embodiments, the words "comprise" and "contain" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is understood that any foregoing description herein of aspects in terms of a single process, material or feature also applies similarly to aspects in terms of corresponding combinations of processes, materials or features.

[0173] It should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a" or "the" nucleotide sequence can include a single nucleotide sequence or a plurality of nucleotide sequences.

[0174] In addition, the use of "and / or", where ever used herein, is to be taken as a specific disclosure of each of the several enumerated terms. Thus "A and / or B" is a specific disclosure of A, B, or A and B. Likewise, "A, B, and / or C" is a specific disclosure of A, B, C, A and B, A and C, B and C, or A and B and C. The minimum intent of the term "and / or" is that "AND" and "OR" are both disclosed.

[0175] The term“about” or“approximately” is used herein to mean approximately, about, roughly, or in the vicinity of, when referring to a numerical value. When the term“about” is used in conjunction with a numerical value, it defines the range by extending the boundaries up and down from the recited numerical value. Generally, the term“about” can define a numerical value to be plus and minus variations of, for example, 10% up and down (higher or lower) from the stated value. In some embodiments, the term means a deviation of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01% from the indicated numerical value. In some embodiments,“about” means a deviation of ±10% from the indicated numerical value. In some embodiments,“about” means a deviation of ±5% from the indicated numerical value. In some embodiments,“about” means a deviation of ±4% from the indicated numerical value. In some embodiments,“about” means a deviation of ±3% from the indicated numerical value. In some embodiments,“about” means a deviation of ±2% from the indicated numerical value. In some embodiments,“about” means a deviation of ±1% from the indicated numerical value. In some embodiments,“about” means a deviation of ±0.9% from the indicated numerical value. In some embodiments,“about” means a deviation of ±0.8% from the indicated numerical value. In some embodiments,“about” means a deviation of ±0.7% from the indicated numerical value. In some embodiments,“about” means a deviation of ±0.6% from the indicated numerical value. In some embodiments,“about” means a deviation of ±0.5% from the indicated numerical value. In some embodiments,“about” means a deviation of ±0.4% from the indicated numerical value. In some embodiments,“about” means a deviation of ±0.3% from the indicated numerical value. In some embodiments,“about” means a deviation of ±0.1% from the indicated numerical value. In some embodiments,“about” means a deviation of ±0.05% from the indicated numerical value. In some embodiments,“about” means a deviation of ±0.01% from the indicated numerical value.

[0176] An "antibody" can be a natural or conventional antibody, in which two heavy chains are connected to each other by disulfide bonds and each heavy chain is connected to a light chain by a disulfide bond. There are two types of light chains, lambda (l) and kappa (k). There are five major classes (or isotypes) of heavy chains, which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chain includes two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine the binding recognition and specificity to the antigen. The light chain constant region domain (CL) and the heavy chain constant region domain (CH) confer important biological properties, such as antibody chain association, secretion, transplacental mobility, complement fixation, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody lies in the structural complementarity between the antibody combining site and the antigenic determinant. The antibody combining site is composed of residues from the hypervariable or complementarity determining regions (CDRs). Sometimes, residues from non-hypervariable or framework regions (FRs) influence the overall domain structure, which in turn affects the combining site. Thus, complementarity determining regions or CDRs refer to amino acid sequences within the naturally occurring Fv region of an immunoglobulin that collectively determine the binding affinity and specificity of the antibody. There are three CDRs for each of the light and heavy chains of an immunoglobulin, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen binding site includes six CDRs, comprising the CDR set from each of the heavy and light chain V regions.

[0177] As used herein, the term "antibody" is intended to refer to conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular the variable heavy chain of a single domain antibody, as well as chimeric antibodies, humanized antibodies, bispecific antibodies or multispecific antibodies. Antibody fragments contemplated herein are antigen binding fragments.

[0178] A "framework region" (FR) refers to the amino acid sequences that are relatively conserved among different immunoglobulins of a single species, i.e., those parts of the immunoglobulin light and heavy chain variable regions that are not involved in contacting the antigen. There are four FRs in each of the light and heavy chains of an immunoglobulin, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. A human framework region is a framework region that is substantially identical (about 85% or greater, in particular 90%, 95%, 97%, 99%, or 100%) to a framework region of a naturally occurring human antibody.

[0179] In the context of the present disclosure, the CDR / FR definition in the immunoglobulin light or heavy chain will be determined based on the IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1): 55-77; www.imgt.org).

[0180] As used herein, antibodies or immunoglobulins also include the recently described "single domain antibodies", which are antibodies where the complementarity determining regions are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, naturally light chain free antibodies, single domain antibodies derived from conventional four chain antibodies, engineered single domain antibodies. Single domain antibodies can be derived from any species, including but not limited to mouse, human, camel, llama, goat, rabbit, bovine. Single domain antibodies can be naturally occurring single domain antibodies, known as heavy chain antibodies devoid of light chains. In particular, camelid species (e.g. camels, dromedaries, llamas, alpacas and guanacos) produce heavy chain antibodies naturally devoid of light chains. Camelid heavy chain antibodies also lack a CH1 domain.

[0181] The variable heavy chains of these single domain antibodies devoid of light chains are referred to in the art as "VHH" or Similar to conventional VH domains, VHH contain four FRs and three CDRs. VHHs have the following advantages compared to conventional antibodies: they are about ten times smaller than IgG molecules and thus can be produced by in vitro expression of correctly folded functional VHHs while achieving high yields. Furthermore, VHHs are very stable and resistant to the action of proteases. The properties and production of VHHs have been reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov;77(1):13-22).

[0182] As used herein, the term "monoclonal antibody" or "mAb" refers to antibody molecules of single amino acid sequence, which are directed against a specific antigen, and should not be interpreted as requiring that the antibody is produced by any particular method. Monoclonal antibodies can be produced by a single clone of B cells or hybridomas, but can also be recombinant, i.e. produced by protein engineering.

[0183] The term "humanized antibody" refers to an antibody which is of fully or partially non-human origin and which is modified to replace certain amino acids, in particular in the framework regions of the VH and VL domains, to avoid or minimize the human immune response. The constant domains of a humanized antibody are mostly human CH and CL domains.

[0184] A "fragment" of a (conventional) antibody comprises a portion of an intact antibody, in particular an antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. Fragments of conventional antibodies can also be single domain antibodies, such as heavy chain antibodies or VHHs.

[0185] The term "Fab" denotes an antibody fragment having a molecular weight of about 50,000 and having antigen binding activity, in which about one-half of the N-terminal side of the heavy chain is bound together with the entire light chain through a disulfide bond. It is usually obtained in fragments by treating IgG with a protease such as papain.

[0186] The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and having antigen binding activity, which is slightly larger than 2 identical Fab fragments bound through a disulfide bond of the hinge region. It is usually obtained in fragments by treating IgG with a protease such as pepsin.

[0187] The term "Fab'" refers to an antibody fragment having a molecular weight of about 50,000 and having antigen binding activity, which is obtained by cleaving the disulfide bond of the F(ab')2 hinge region.

[0188] A single chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer, which is usually expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker. Human scFv fragments of the present disclosure include CDRs held in an appropriate conformation, in particular by use of genetic recombination techniques. Bivalent and multivalent antibody fragments can be formed either spontaneously by association of monomeric scFv, or can be generated by coupling of monomeric scFv via peptide linkers, such as bivalent sc(Fv)2. A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" denotes two dsFv coupled by a peptide linker.

[0189] The term "bispecific antibody" or "BsAb" denotes an antibody in which the antigen binding sites of two antibodies are combined in a single molecule. BsAbs are thus capable of binding two different antigens simultaneously. Genetic engineering has been used more and more frequently to design, modify and produce antibodies or antibody derivatives with a set of desired binding properties and effector functions, as described in, for example, EP 2 050 764 Al.

[0190] The term "multispecific antibody" denotes an antibody in which the antigen binding sites of two or more antibodies are combined in a single molecule.

[0191] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise 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 of the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0192] An amino acid sequence that is "at least 85% identical" to a reference sequence means a sequence that has 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the entire length of the reference amino acid sequence over its entire length.

[0193] The percent "sequence identity" between amino acid sequences is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions (i.e., gaps) to achieve the optimal alignment of the two sequences. The percent sequence identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. Optimal alignment of sequences for use in the comparison is conducted using, for example, global pairwise alignments using the algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970). The percent sequence identity can be readily determined using, for example, the program Needle with an BLOSUM62 matrix, and the following parameters: gap-open=10, gap-extend=0.5.

[0194] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with another amino acid residue having similar chemical properties (e.g., charge, size, or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. Examples of groups of amino acids that have 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 by amino acid size.

[0195] “Purified” and “isolated” when referring to a polypeptide (i.e., an antibody of the disclosure) or a nucleotide sequence means that the indicated molecule exists in the substantial absence of other biological molecules of the same type. As used herein, the term “purified” specifically means that at least 75%, 85%, 95%, or 98% by weight of the biological molecules of the same type are present. An “isolated” nucleic acid molecule encoding a particular polypeptide is a nucleic acid molecule that is substantially free of other nucleic acid molecules not encoding the polypeptide of interest; however, the molecule can include some additional bases or groups that do not materially affect the basic properties of the composition.

[0196] The term “subject” or “patient” as used herein means a mammal, such as a rodent, feline, canine, and primate. In particular, a subject according to the disclosure is a human.

[0197] “Administer” or “administering” as used herein means to deliver a composition described herein to a subject. The composition can be administered to a subject using methods known in the art. In particular, the composition can be administered intravenously, subcutaneously, intramuscularly, intradermally, or via any mucosal surface, e.g., orally, sublingually, buccally, nasally, rectally, vaginally, or via a pulmonary route. In some embodiments, administration is performed intravenously. In some embodiments, administration is performed subcutaneously.

[0198] The term “treat” or “treatment” or “therapy” means the administration of a compound or composition according to the disclosure with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a disorder, a symptom of a disorder, or to prevent or delay the onset of a symptom, a complication, or to stop or inhibit further development of a disorder in a statistically significant manner. More particularly, “treating” or “treatment” includes any method of obtaining a beneficial or desired result in a subject’s cancer disorder. Beneficial or desired clinical results can include, but are not limited to, alleviating or relieving one or more symptoms or conditions of cancer, reducing or mitigating the extent of a cancer disease or a cancer symptom, stabilizing (i.e., not worsening) the state of a cancer disease or a cancer symptom, preventing the spread of a cancer disease or a cancer symptom, delaying or slowing the progression of a cancer disease or a cancer symptom, remission or alleviation of a cancer disease state, reduction in recurrence of a cancer disease, and remission (whether partial or total, whether detectable or undetectable). In other words, “treatment” as used herein includes any curing, remission, or lessening of a cancer disease or symptom. “Lessening” of a symptom or disease means reducing the severity or frequency of a disease or symptom, or eliminating a disease or symptom.

[0199] As used herein, the term “effective amount” refers to an amount that provides a therapeutic benefit in the treatment, prevention, or management of the pathological process under consideration. The particular amount that is therapeutically effective can be readily determined by the ordinarily skilled medical practitioner and can vary depending on factors such as the type and stage of the pathological process under consideration, the patient’s medical history and age, and the administration of other therapeutic agents.

[0200] The unit “mg / m 2 ” indicates the amount of compound (in mg) administered per m 2 of body surface of the subject. The person skilled in the art knows how to determine the amount of compound needed for a subject to be treated depending on the body surface of the subject to be treated, which in turn can be calculated based on height and weight.

[0201] The unit “mg / kg” indicates the amount of compound (in mg) administered per kg of body of the subject per dose. The person skilled in the art knows how to determine the amount of compound needed for a subject to be treated depending on the body weight of the subject to be treated.

[0202] It is to be understood that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable

[0203] A “biomarker” is intended to refer to a biomolecule, such as a protein or metabolite, that is differentially present, increased, or decreased in a biological sample obtained from a subject or a group of subjects having a first phenotype (e.g., suffering from a disease, such as cancer) as compared to a biological sample from a subject or a group of subjects having a second phenotype (e.g., not suffering from the disease). In use, the biomarker is isolated from the subject.

[0204] A “sample” or “biological sample” is intended to refer to biological material isolated from a subject. The biological sample can contain any biological material suitable for detecting a biomarker (i.e., CEACAM5 mRNA) and can comprise cellular material and / or non-cellular material from the subject. The sample can be isolated from any suitable biological tissue or fluid, such as kidney tissue, blood, blood plasma / plasma, blood serum / serum, urine, or cerebrospinal fluid (CSF). In some embodiments, the biological sample is a plasma or serum sample.

[0205] A “reference value” or “threshold value” is intended to refer to a CEACAM5 mRNA level indicative of a particular disease state, phenotype (such as cancer), or lack thereof, and a combination of disease states, phenotypes, or lack thereof, in a subject of interest.

[0206] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless specifically defined otherwise. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, all publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0207] Lists of sources, ingredients and components as described below are listed such that combinations and mixtures thereof are also contemplated and within the scope herein.

[0208] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0209] All lists of items (such as, for example, lists of ingredients) are intended and should be interpreted to be Markush groups. Thus, all lists can be read and interpreted as “items selected from the group consisting of the list of items ‘and combinations and mixtures thereof’.

[0210] References herein to a component can be to a trade name of a component including various ingredients used in the present disclosure. The inventors herein do not intend to be limited to the material under any particular trade name. Equivalent materials (for example, materials obtained from a different source under a different name or reference number) to the materials mentioned by trade name can be substituted and used in the description herein. CEACAM mRNA measurements, methods and uses

[0211] According to one of its objects, the present disclosure relates to methods and uses for selecting a subject in need of a treatment of a cancer with a therapeutic agent targeting CEACAM5. The therapeutic agent targeting CEACAM5 can be an anti-CEACAM5 antibody coupled to a cytotoxic agent.

[0212] The methods and uses of the present disclosure can comprise the step of determining the amount of CEACAM5 gene expression (gene transcript or RNA) in a tumor sample.

[0213] The methods and uses of the present disclosure can comprise the step of determining the amount of CEACAM5 gene expression (such as CEACAM5 mRNA level) in a tumor sample.

[0214] The amount of CEACAM5 gene expression can be expressed as an amount relative to the total gene expression in the tumor sample and relative to the total length of expressed DNA or any other known method of expression.

[0215] In some embodiments, the disclosure relates to a method for selecting a subject in need of a treatment for a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0216] (i) determining a value for the level of CEACAM5 gene expression in a tumor isolated sample obtained from the subject,

[0217] (ii) comparing the determined value to a reference value, and

[0218] (iii) selecting the subject for a treatment for a cancer if the determined value is higher than the reference value.

[0219] In some embodiments, the disclosure relates to a method for selecting a subject in need of a treatment for a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0220] (i) determining a value for the level of CEACAM5 gene expression in a tumor isolated sample obtained from the subject,

[0221] (ii) comparing the determined value to a reference value, and

[0222] (iii) selecting the subject for a treatment for a cancer if the determined value is higher than the reference value.

[0223] (iv) determining an intensity of the level of CEACAM5 protein expression in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0224] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0225] (vi) selecting the subject for a treatment for a cancer if the intensity determined in step (iv) is higher than the reference intensity.

[0226] The tumor sample of step (i) and step (iv) can be the same sample or different samples.

[0227] In some embodiments, the disclosure relates to a method for diagnosing whether a subject in need thereof is eligible for treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0228] (i) determining a value for the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from the subject,

[0229] (ii) comparing the determined value to a reference value, and

[0230] (iii) selecting the subject for treatment of a cancer if the determined value is higher than the reference value.

[0231] In some embodiments, the disclosure relates to a method for diagnosing whether a subject in need thereof is eligible for treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0232] (i) determining a value for the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from the subject,

[0233] (ii) comparing the value determined in step (i) to a reference value for the level of expression of the CEACAM5 gene,

[0234] (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii),

[0235] (iv) determining an intensity of the level of expression of the CEACAM5 protein in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0236] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0237] (vi) selecting the subject for treatment of a cancer if the intensity determined in step (iv) is higher than the reference intensity.

[0238] In some embodiments, the disclosure relates to a method for selecting and treating a subject in need of treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0239] (i) determining a value for the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from the subject,

[0240] (ii) comparing the determined value to a reference value,

[0241] (iii) selecting the subject for cancer treatment if the determined value is higher than the reference value, and

[0242] (iv) administering to the selected subject an effective amount of the ADC.

[0243] In some embodiments, the disclosure relates to a method for selecting and treating a subject in need of treatment of cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0244] (i) determining a value for the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from the subject,

[0245] (ii) comparing the value determined in step (i) to a reference value for the level of expression of the CEACAM5 gene,

[0246] (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii),

[0247] (iv) determining the intensity of the level of expression of the CEACAM5 protein in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0248] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0249] (vi) selecting the subject for cancer treatment if the intensity determined in step (iv) is higher than the reference intensity, and

[0250] (vii) administering to the selected subject an effective amount of the ADC.

[0251] In some embodiments, the disclosure relates to an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of cancer in a subject in need thereof,

[0252] The use comprises:

[0253] (i) determining a value for the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from the subject,

[0254] (ii) comparing the value determined in step (i) to a reference value, and

[0255] (iii) if the determined value is higher than the reference value, administering to the subject an effective amount of the ADC.

[0256] In some embodiments, the disclosure relates to an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for use in the treatment of a cancer in a subject in need thereof,

[0257] The use comprises:

[0258] (i) determining a value of the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from the subject,

[0259] (ii) comparing the value determined in step (i) to a reference value of the level of expression of the CEACAM5 gene,

[0260] (iii) if the value determined in step (i) is higher than the reference value of step (ii), selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test,

[0261] (iv) determining the intensity of the level of expression of the CEACAM5 protein in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0262] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0263] (vi) if the determined intensity is higher than the reference intensity, administering to the subject an effective amount of the ADC.

[0264] In some embodiments, the disclosure relates to the use of a measured value of the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from a subject in need thereof for characterizing the tumor as a CEACAM5 high expressing tumor.

[0265] In some embodiments, the disclosure relates to the use of a measured value of the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from a subject in need thereof for selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test.

[0266] In some embodiments, the disclosure relates to the use of a measured value of the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from a subject in need thereof for selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test.

[0267] In some embodiments, the disclosure relates to the use of a measure of the level of CEACAM5 gene expression in a tumor isolated sample obtained from a subject in need thereof for selecting the subject for treatment of cancer with a CEACAM5 -targeted therapeutic (e.g., an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent).

[0268] In some embodiments, the value of the level of CEACAM5 gene expression can be a measure of CEACAM5 gene transcript.

[0269] In some embodiments, the CEACAM5 gene transcript can be mRNA.

[0270] In some embodiments, the methods and uses of the disclosure comprise the step of determining a value of the level of CEACAM5 mRNA in a tumor isolated sample.

[0271] In some embodiments, the amount of CEACAM5 gene expression can be expressed as a log2 transformed, quantile normalized transcripts per million per kilobase (TPM) value.

[0272] The determined value or level can be compared to a threshold or reference value.

[0273] A deviation from the reference value can indicate that a CEACAM5-expressing tumor is at a level sufficient for it to respond to a CEACAM5-targeted therapy.

[0274] In the case of a CEACAM5 immunohistochemistry (IHC) staining assay, the determined intensity or protein expression level can be compared to a threshold intensity or reference intensity.

[0275] A deviation from the reference intensity can indicate that a CEACAM5-expressing tumor is at a level sufficient for it to respond to a therapy targeting CEACAM5-expressing cancer.

[0276] The methods and uses of the disclosure can be used to characterize a subject in need thereof as responsive to a CEACAM5 -targeted therapy (e.g., an anti-CEACAM5 antibody conjugated to a cytotoxic agent).

[0277] The methods and uses of the disclosure can be used to select a subject in need thereof who is responsive to a CEACAM5 -targeted therapy (e.g., an anti-CEACAM5 antibody conjugated to a cytotoxic agent).

[0278] The methods and uses of the disclosure can be used to monitor a subject in need thereof for response to a CEACAM5 -targeted therapy (e.g., an anti-CEACAM5 antibody conjugated to a cytotoxic agent).

[0279] The methods and uses of the disclosure can be used to select a treatment targeting CEACAM5, e.g. an anti-CEACAM5 antibody coupled to a cytotoxic agent, depending on the level of CEACAM5 gene expression in a tumor of a subject in need thereof.

[0280] The methods and uses of the disclosure can be used to select a treatment targeting CEACAM5, e.g. an anti-CEACAM5 antibody coupled to a cytotoxic agent, depending on the intensity of CEACAM5 protein expression in a tumor of a subject in need thereof.

[0281] The methods and uses of the disclosure can be used to characterize a tumor of a subject in need thereof as responsive to a treatment targeting CEACAM5, e.g. an anti-CEACAM5 antibody coupled to a cytotoxic agent.

[0282] The methods and uses of the disclosure are performed on an isolated biological sample. The isolated sample can be a sample isolated from a tumor. The sample is isolated prior to performing the methods and uses as disclosed herein.

[0283] The methods and uses of the disclosure are performed in vitro.

[0284] According to one of its objects, the disclosure relates to a method for selecting a subject in need of a treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody coupled to a cytotoxic agent, the method comprising at least the steps of:

[0285] (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from said subject,

[0286] (ii) comparing said value to a reference value, and

[0287] (iii) selecting said subject for a treatment of a cancer if the determined value is higher than the reference value.

[0288] According to one of its objects, the disclosure relates to a method for selecting a subject in need of a treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody coupled to a cytotoxic agent, the method comprising at least the steps of:

[0289] (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from said subject,

[0290] (ii) comparing said value determined in step (i) to a reference value,

[0291] (iii) if the value determined in step (i) is higher than the reference value of step (ii), selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test,

[0292] (iv) determining the intensity of the CEACAM5 protein expression level in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0293] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0294] (vi) if the determined intensity is higher than the reference intensity, selecting the subject for a cancer treatment.

[0295] According to one of its objects, the disclosure relates to a method for diagnosing whether a subject in need is eligible for a treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0296] (i) determining a log2 transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject,

[0297] (ii) comparing the value to a reference value, and

[0298] (iii) if the determined value is higher than the reference value, selecting the subject as eligible for a cancer treatment.

[0299] According to one of its objects, the disclosure relates to a method for diagnosing whether a subject in need is eligible for a treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0300] (i) determining a log2 transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject,

[0301] (ii) comparing the value determined in step (i) to a reference value,

[0302] (iii) if the value determined in step (i) is higher than the reference value of step (ii), selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test,

[0303] (iv) determining the intensity of the level of CEACAM5 protein expression in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0304] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0305] (vi) selecting the subject for cancer treatment if the determined intensity is higher than the reference intensity.

[0306] According to another of its objects, the disclosure relates to a method for selecting and treating a subject in need of treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0307] (i) determining the log2 transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject,

[0308] (ii) comparing the value to a reference value,

[0309] (iii) selecting the subject for cancer treatment if the determined value is higher than the reference value, and

[0310] (iv) administering to the selected subject an effective amount of the ADC.

[0311] According to another of its objects, the disclosure relates to a method for selecting and treating a subject in need of treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:

[0312] (i) determining the log2 transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject,

[0313] (ii) comparing the value determined in step (i) to a reference value,

[0314] (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii),

[0315] (iv) determining the intensity of the level of CEACAM5 protein expression in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0316] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0317] (vi) selecting the subject for cancer treatment if the determined intensity is higher than the reference intensity, and

[0318] (vii) administering to the selected subject an effective amount of the ADC.

[0319] According to another of its objects, the disclosure relates to an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of cancer in a subject in need thereof,

[0320] The use comprises (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject, (ii) comparing the value to a reference value, and (iii) administering to the subject an effective amount of the ADC if the determined value is higher than the reference value.

[0321] According to another of its objects, the disclosure relates to an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of cancer in a subject in need thereof,

[0322] The use comprises:

[0323] (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject,

[0324] (ii) comparing the value determined in step (i) to a reference value,

[0325] (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii),

[0326] (iv) determining an intensity of the level of CEACAM5 protein expression in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test,

[0327] (v) comparing the intensity determined in step (iv) to a reference intensity, and

[0328] (vi) administering to the subject an effective amount of the ADC if the determined intensity is higher than the reference intensity.

[0329] In some embodiments, the reference value can be at least about 7 to about 13.

[0330] In some embodiments, the reference value can be at least about 7, or can be at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 13.

[0331] In some embodiments, quantile normalization can be obtained by (i) ranking the transcripts of a sample by expression level, (ii) calculating the mean of genes occupying the same rank, and (iii) replacing the values of all genes occupying said same rank with this mean.

[0332] In some embodiments, the expression level of a transcript can be measured in Fragments Per Kilobase of transcript per Million (FPKM) before conversion to TPM.

[0333] In some embodiments, Fragments Per Kilobase of transcript per Million (FPKM) can be obtained by counting the total transcripts in the sample, dividing the obtained transcript count by 1,000,000, and dividing the obtained value by the length of the gene in kilobases.

[0334] One step of the methods and uses disclosed herein is determining the relative expression of the CEACAM5 gene in the tumor sample relative to other genes. Many methods are available for such determination.

[0335] The relative amount of CEACAM5 gene transcripts is then compared to a reference value. A significant deviation from the reference value can indicate that the tumor expressing CEACAM5 can be responsive to a therapeutic agent specifically targeting CEACAM5.

[0336] The amount of gene transcripts can be measured by any method known in the art, such as microarray, large scale real-time reverse transcription PCR, RNA sequencing (RNA-Seq), next generation sequencing (NGS).

[0337] The gene expression of the tumor sample (RNA-seq) can be obtained by any method known in the art.

[0338] RNA-Seq is a sequencing method used to determine gene expression levels. The determined number of reads originating from each transcript (typically by alignment) is proportional to its expression level. RNA-Seq can be used to generate gene expression profiles for tumor samples of multiple cancer types and determine which gene expression levels contribute to tumor development. RNA-Seq data can be harmonized by aligning raw RNA reads to the GRCh38 reference genome build and calculating gene expression levels with a standardization scheme. RNA-Seq data can be provided as aligned reads (BAM) and expression levels: raw counts and normalized with TPM, FPKM, or FPKM-UQ.

[0339] In some embodiments, gene expression (RNA-seq) can be obtained as follows.

[0340] KAPA mRNA Platform can be used to sequence gene transcripts (RNA) from isolated tumor samples. RNA-seq data can be processed as follows: sequencing reads can be mapped to the reference genome GRCh 38 using, for example, the STAR aligner for Transcripts Alignment to a Reference

[25] . Gene expression can be first measured by CUFFLINK in FPKM (fragments per million per kilobase)

[26] , and FPKM at gene level can be converted to TPM (transcripts per million)

[27] . TPM values can be log2 transformed and quantile normalized for downstream analysis, including differential gene expression (DGE) analysis. In some embodiments, samples with a detected number of genes lower than 10,000 can be excluded from downstream analysis. RNA-seq can include microenvironment cell population [MCP] counter analysis according to published methods [28, 29].

[0341] In some embodiments, the measure of CEACAM5 gene expression level (measure of mRNA or gene transcript level) can be normalized.

[0342] In some embodiments, the measure of CEACAM5 gene expression level (measure of mRNA or gene transcript level) can be log2 transformed.

[0343] In some embodiments, the CEACAM5 gene expression level (mRNA) in a tumor sample is expressed as a log2 transformed, quantile normalized, transcripts per million (TPM) value.

[0344] Normalization of the measure of CEACAM5 gene expression level can be obtained by the following steps:

[0345] - log2 transformation of RNA-seq raw count values,

[0346] - quantile normalization of RNA-seq data by ranking and adjusting expression values in the sample to have the same distribution, and

[0347] - expression of data as Transcripts Per Million per kilobase transcript (TPM) by scaling normalized expression values by gene length and total reads sequenced, and then scaling to millions to make values more interpretable.

[0348] In some embodiments, the log2 transformed, quantile normalized Transcripts Per Million per kilobase transcript (TPM) value of the CEACAM5 gene expression level measured (or determined) in the tumor is compared to a reference value. If the determined value is higher than the reference value, the tumor can be qualified as responsive to CEACAM5 -targeted therapy. A subject in need of CEACAM5 -targeted therapy whose determined value is higher than the reference value can be selected.

[0349] In some embodiments, the reference value can be at least about 7 to about 13.

[0350] In some embodiments, the reference value can be at least about 7, or can be at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 13.

[0351] In some embodiments, the log2 transformed, quantile normalized Transcripts Per Million per kilobase transcript (TPM) reference value for CEACAM5 mRNA can be about = 7.

[0352] In some embodiments, the log2 transformed, quantile normalized Transcripts Per Million per kilobase transcript (TPM) reference value for CEACAM5 mRNA can be about = 7.5.

[0353] In some embodiments, the log2 transformed, quantile normalized Transcripts Per Million per kilobase transcript (TPM) reference value for CEACAM5 mRNA can be about = 8.

[0354] In some embodiments, the log2 transformed, quantile normalized Transcripts Per Million per kilobase transcript (TPM) reference value for CEACAM5 mRNA can be about = 8.5.

[0355] In some embodiments, the log2 transformed, quantile normalized Transcripts Per Million per kilobase transcript (TPM) reference value for CEACAM5 mRNA can be about = 9.

[0356] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 9.5.

[0357] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 10.

[0358] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 10.5.

[0359] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 11.

[0360] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 11.5.

[0361] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 12.

[0362] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 12.5.

[0363] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 13.

[0364] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 13.5.

[0365] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 14.

[0366] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 14.5.

[0367] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about = 15.

[0368] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 7.

[0369] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 7.5.

[0370] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 8.

[0371] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 8.5.

[0372] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 9.

[0373] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 9.5.

[0374] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 10.

[0375] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 10.5.

[0376] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 11.

[0377] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 11.5.

[0378] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 12.

[0379] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 12.5.

[0380] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 13.

[0381] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 13.5.

[0382] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 14.

[0383] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 14.5.

[0384] In some embodiments, the log2 transformed, quantile normalized transcripts per million per kilobase (TPM) reference value for CEACAM5 mRNA can be about > 15.

[0385] CEACAM5 protein expression levels (or intensity) can be measured by immunohistochemistry (IHC).

[0386] The level and pattern of CEACAM5 protein expression can be analyzed by immunohistochemistry (IHC) in a tumor sample isolated from a patient in need thereof, for example, as disclosed by LaPointe et al. (Journal of Clinical Oncology [Clinical Oncology Journal], Volume 39, Issue 15 Supplement, https: / / doi.org / 10.1200 / JCO.2021.39.15_suppl.e21030) or Blumenthal et al. (BMC Cancer. [BMC Cancer] 2007; 7:2. Published January 3, 2007. doi:10.1186 / 1471-2407-7-2).

[0387] CEACAM5 reactivity in tumor cells can be assessed using semi-quantitative percentage scores (calculated by summing the percentages with intensity ≥2+) of CEACAM5 plasma membrane staining (whole membrane or polarized membrane) or H scores.

[0388] Individuals with high CEACAM5 expression can be defined as those whose CEACAM5 protein expression intensity is ≥2+ in ≥50% of the tumor cell population of the tumor sample.

[0389] Patients with intermediate CEACAM5 expression can be defined as those whose CEACAM5 protein expression intensity is ≥2+ in ≥1% to <50% of the tumor cell population of the tumor sample.

[0390] In some embodiments, the reference strength of the CEACAM5 protein expression level measured by the CEACAM5 immunohistochemical (IHC) staining test can be ≥2+ in ≥50% of the tumor cell population.

[0391] The treatment may be selected for patients who require treatment with antibody-drug conjugates (ADCs, which contain an anti-CEACAM5 antibody conjugated to a cytotoxic agent) and whose CEACAM5 expression intensity is ≥2+ in ≥50% of their tumor cell population.

[0392] The treatment can be administered to patients who require cancer treatment with antibody-drug conjugates (ADCs, which contain an anti-CEACAM5 antibody conjugated to a cytotoxic agent) and whose CEACAM5 expression intensity is ≥2+ in ≥50% of their tumor cell population.

[0393] For patients requiring cancer treatment with antibody-drug conjugates (ADCs, which contain an anti-CEACAM5 antibody conjugated to a cytotoxic agent), the CEACAM5 gene expression level in tumor isolates obtained from the patient can be determined first. If the determined value of the CEACAM5 gene expression level in the tumor isolates obtained from the patient is higher than a reference value, the patient can be selected for further testing to determine the intensity of CEACAM5 protein expression using CEACAM5 immunohistochemical (IHC) staining.

[0394] If the determined intensity is higher than the reference intensity, the patient can be treated with an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent.

[0395] If the determined intensity is higher than the reference intensity, treatment with an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent can be administered to the patient. Antibody-drug conjugates containing anti-CEACAM5 antibodies.

[0396] The present disclosure relates to an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody or fragment thereof.

[0397] The antibody drug conjugate typically comprises an anti-CEACAM5 antibody and at least one chemotherapeutic agent. The antibody drug conjugate (ADC) comprises an anti-CEACAM5 antibody conjugated to at least one chemotherapeutic agent. In the antibody drug conjugate, the anti-CEACAM5 antibody can be covalently attached to the at least one chemotherapeutic agent via a cleavable or non-cleavable linker. Anti-CEACAM5 antibody

[0398] According to embodiments, the antibody drug conjugate comprises an anti-CEACAM5 antibody or fragment thereof.

[0399] According to embodiments, the antibody drug conjugate comprises a humanized anti-CEACAM5 antibody or fragment thereof.

[0400] According to embodiments, the anti-CEACAM5 antibody or fragment thereof comprises a CDR-H1 consisting of SEQ ID NO: 1, a CDR-H2 consisting of SEQ ID NO: 2, a CDR-H3 consisting of SEQ ID NO: 3, a CDR-L1 consisting of SEQ ID NO: 4, a CDR-L2 consisting of the amino acid sequence NTR, and a CDR-L3 consisting of SEQ ID NO: 5.

[0401] In further embodiments, the anti-CEACAM5 antibody or fragment thereof comprises a heavy chain variable domain (VH) having at least 90% identity to SEQ ID NO: 6 and a light chain variable domain (VL) having at least 90% identity to SEQ ID NO: 7, wherein CDR1-H consists of SEQ ID NO: 1, CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1-L consists of SEQ ID NO: 4, CDR2-L consists of the amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.

[0402] In a further embodiment, the anti-CEACAM5 antibody or fragment thereof comprises a heavy chain variable domain (VH) which is at least 92%, at least 95%, at least 98% identical to SEQ ID NO: 6 and a light chain variable domain (VL) which is at least 92%, at least 95%, at least 98% identical to SEQ ID NO: 7, wherein CDR1-H consists of SEQ ID NO: 1, CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1-L consists of SEQ ID NO: 4, CDR2-L consists of the amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.

[0403] In a further embodiment, the anti-CEACAM5 antibody or fragment thereof comprises a heavy chain variable domain (VH) which consists of SEQ ID NO: 6 and a light chain variable domain (VL) which consists of SEQ ID NO: 7.

[0404] In a further embodiment, the anti-CEACAM5 antibody or fragment thereof comprises:

[0405] - a heavy chain variable domain which consists of the sequence (SEQ ID NO: 6, CDRs shown in bold) wherein FR1-H spans amino acid positions 1 to 25, CDR1-H spans amino acid positions 26 to 33 (SEQ ID NO: 1), FR2-H spans amino acid positions 34 to 50, CDR2-H spans amino acid positions 51 to 58 (SEQ ID NO: 2), FR3-H spans amino acid positions 59 to 96, CDR3-H spans amino acid positions 97 to 109 (SEQ ID NO: 3), and FR4-H spans amino acid positions 110 to 120, and

[0406] - a light chain variable domain which consists of the sequence (SEQ ID NO: 7, CDRs shown in bold) wherein FR1-L spans amino acid positions 1 to 26, CDR1-L spans amino acid positions 27 to 32 (SEQ ID NO: 4), FR2-L spans amino acid positions 33 to 49, CDR2-L spans amino acid positions 50 to 52, FR3-L spans amino acid positions 53 to 88, CDR3-L spans amino acid positions 89 to 97 (SEQ ID NO: 5), and FR4-L spans amino acid positions 98 to 107.

[0407] In a further embodiment, the anti-CEACAM5 antibody or fragment thereof comprises a heavy chain (HC) having at least 90% sequence identity to SEQ ID NO: 8 and a light chain (LC) having at least 90% sequence identity to SEQ ID NO: 9, wherein CDR1-H consists of SEQ ID NO: 1, CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1-L consists of SEQ ID NO: 4, CDR2-L consists of the amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.

[0408] In a further embodiment, the anti-CEACAM5 antibody or fragment thereof comprises a heavy chain (HC) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO: 8 and a light chain (LC) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO: 9, wherein CDR1-H consists of SEQ ID NO: 1, CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1-L consists of SEQ ID NO: 4, CDR2-L consists of the amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.

[0409] In a further embodiment, the anti-CEACAM5 antibody or fragment thereof comprises a heavy chain (HC) consisting of SEQ ID NO: 8 and a light chain (LC) consisting of SEQ ID NO: 9.

[0410] The anti-CEACAM5 antibody can also be a single domain antibody or fragment thereof. In particular, the single domain antibody fragment can consist of a variable heavy chain (VHH) comprising CDR1-H, CDR2-H and CDR3-H of the antibody as described above. The antibody can also be a heavy chain antibody, i.e. an antibody without light chain, which can or can not contain a CH1 domain.

[0411] The single domain antibody or fragment thereof can further comprise the framework region of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody.

[0412] The anti-CEACAM5 antibody can also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2 and diabody.

[0413] The antibodies can also be bispecific or multispecific antibodies formed from antibody fragments, at least one of which is an antibody fragment according to the present disclosure. Multispecific antibodies are multivalent protein complexes, as described for example in EP 2 050 764 Al or US 2005 / 0003403 Al.

[0414] The anti-CEACAM5 antibodies and fragments thereof can be produced by any technique well known in the art. In particular, the antibodies are produced by techniques as described hereinafter.

[0415] The anti-CEACAM5 antibodies and fragments thereof can be isolated (e.g., purified) from or comprised in a vector, such as a membrane or a lipid vesicle (e.g., a liposome) for use.

[0416] The anti-CEACAM5 antibodies and fragments thereof can 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.

[0417] Knowing the amino acid sequence of the desired sequence, the anti-CEACAM5 antibodies and fragments thereof can be readily produced by the skilled person by standard techniques for polypeptide production. For example, they can be synthesized using well-known solid phase methods, in particular using commercially available peptide synthesis apparatus, such as that manufactured by Applied Biosystems, Foster City, California, following the manufacturer's instructions. Alternatively, the anti-CEACAM5 antibodies and fragments thereof can be synthesized by recombinant DNA techniques well known in the art. For example, after incorporating a DNA sequence encoding the desired (poly)peptide into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host, which will express the desired polypeptide, which can then be isolated therefrom using well-known techniques.

[0418] The anti-CEACAM5 antibodies and fragments thereof are suitably isolated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0419] Methods for generating 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, for example, the techniques disclosed in WO 2009 / 032661, CDR grafting (EP 239,400; PCT publication WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). General recombinant DNA techniques for making such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0420] Fabs of anti-CEACAM5 antibodies can be obtained by treating an antibody specifically reactive with CEACAM5 with a protease such as papain. In addition, Fabs of anti-CEACAM5 antibodies can be produced by inserting DNA sequences encoding both chains of the Fab of the anti-CEACAM5 antibody into a vector for prokaryotic expression or eukaryotic expression, and introducing the vector into a prokaryotic or eukaryotic cell (as the case can be) to express the Fab of the anti-CEACAM5 antibody.

[0421] F(ab')2 of anti-CEACAM5 antibodies can be obtained by treating an antibody specifically reactive with CEACAM5 with a protease such as pepsin. In addition, F(ab')2 of anti-CEACAM5 antibodies can be produced by binding the Fab' described below via a sulfide bond or a disulfide bond.

[0422] Fab' of anti-CEACAM5 antibodies can be obtained by treating F(ab')2 specifically reactive with CEACAM5 with a reducing agent such as dithiothreitol. In addition, Fab' of anti-CEACAM5 antibodies can be produced by inserting a DNA sequence encoding a Fab' chain of the antibody into a vector for prokaryotic expression or a vector for eukaryotic expression, and introducing the vector into a prokaryotic or eukaryotic cell (as the case can be) for its expression.

[0423] An scFv of an anti-CEACAM5 antibody can be produced by taking the sequences of the CDRs or VH and VL domains as previously described, 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 can be) to express the scFv. To produce a humanized scFv fragment, the well-known technique known as CDR grafting can be used, which involves selecting the complementarity determining regions (CDRs) according to the present disclosure and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, e.g., WO 98 / 45322; WO 87 / 02671; US 5,859,205; US 5,585,089; US 4,816,567; EP 0173494).

[0424] In embodiments, the anti-CEACAM5 antibody is tisotumab (CAS [2349294-95-5]). Chemotherapeutic agent

[0425] An antibody drug conjugate for use according to the present disclosure typically comprises at least one chemotherapeutic agent (also referred to herein as a cytotoxic agent). A chemotherapeutic agent as used herein refers to an agent that kills cells, including cancer cells. Such agents advantageously stop cancer cells from dividing and growing, and cause tumor size to shrink. The expression “chemotherapeutic agent” is used herein interchangeably with the expressions “cytotoxic agent”, “growth inhibitory agent”, or “cell inhibitory agent”.

[0426] The term “chemotherapeutic agent” as used herein refers to a substance that inhibits or stops cell function and / or causes cell destruction. The term “chemotherapeutic agent” is intended to include radioisotopes, enzymes, antibiotics, and toxins (such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof), as well as various antitumor or anticancer agents disclosed below. In some embodiments, the chemotherapeutic agent is an antimetabolite.

[0427] In further embodiments, the chemotherapeutic agent is selected from the group consisting of a radioisotope, a protein toxin, a small molecule toxin, and combinations thereof.

[0428] Radioisotopes include radioisotopes suitable for use in the treatment of cancer. Such radioisotopes typically emit beta-radiation primarily. In further embodiments, the radioisotope is selected from the group consisting of At 211 , Bi 212 , Er 169 , I 131 , I 125 , Y 90 , In 111 , P 32 , Re 186 , Re188 153 89 radioisotopes of Sm, and combinations thereof. In embodiments, the radioisotope is an alpha-emitter isotope, more particularly Th 227 emitting alpha-radiation.

[0429] In further embodiments, the small molecule toxin is selected from the group consisting of an antimetabolite, a DNA alkylating agent, a DNA crosslinking agent, a DNA intercalator, an anti-microtubule agent, a topoisomerase inhibitor, and combinations thereof.

[0430] In further embodiments, the anti-microtubule agent is selected from the group consisting of a taxane, a vinca alkaloid, a maytansinoid, colchicine, a podophyllotoxin, a griseofulvin, and combinations thereof.

[0431] In some embodiments, the chemotherapeutic agent can be a maytansinoid.

[0432] According to embodiments, the maytansinoid is selected from maytansinol, a maytansinol analog, and combinations thereof.

[0433] Examples of suitable maytansinol analogs include those with modified aromatic rings and those with modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

[0434] In further embodiments, the cytotoxic conjugate of the present disclosure utilizes a thiol- containing maytansinoid (DM1) (officially known as N2’-deacetyl-N2’-(3-mercapto-1-oxo- propyl)-maytansinol) as the cytotoxic agent. DM1 is represented by the following structural formula (I):

[0435] In further embodiments, the cytotoxic conjugate of the present disclosure utilizes a thiol- containing maytansinoid DM4 (officially known as N2’-deacetyl-N-2’(4-methyl-4-mercapto-1- oxopentyl)-maytansinol) as the cytotoxic agent. DM4 is represented by the following structural formula (II):

[0436] ​​In further embodiments of the disclosure, other maytansines can be used, including thiol and disulfide containing, mono- or di-alkyl substituted maytansine alkaloids having a sulfur atom bearing carbon. These maytansine alkaloids include maytansine alkaloids having acylated amino acid side chains at the C-3, C-14 hydroxymethyl, C-15 hydroxyl, or C-20 desmethyl positions, wherein the carbon atom of the acyl group bearing the thiol functional group has one or two substituents that are CH3, C2H5, straight chain or branched alkyl or alkenyl groups having from 1 to 10 agents and any aggregates that can be present in solution.

[0437] Examples of these cytotoxic agents and conjugation methods are further given in the application WO 2008 / 010101, which is incorporated by reference.

[0438] Immunoconjugates according to the disclosure can be prepared as described in the application WO 2004 / 091668, the entire contents of which are incorporated herein by reference.

[0439] Thus, in further embodiments, the maytansine alkaloid is selected from the group consisting of N2’-deacetyl-N2’-(3-mercapto-1-oxopropyl)-maytansine (DM1) or N2’-deacetyl-N-2’(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4) and combinations thereof.

[0440] In further embodiments, in the antibody drug conjugate, the anti-CEACAM5 antibody is covalently linked to the at least one chemotherapeutic agent via a cleavable or non-cleavable linker.

[0441] In further embodiments, the linker is selected from the group consisting of pyridyldithiobutyric acid N-succinimidyl ester (SPDB), 4-(pyridin-2- yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and (N-maleimidomethyl) cyclohexane-1-carboxylate succinimidyl ester (SMCC).

[0442] In further embodiments, the linker binds to a lysine or cysteine residue in the Fc region of the anti-CEACAM5 antibody. In further embodiments, the linker forms a disulfide bond or a thioether bond with the maytansine.

[0443] In particular, the anti-CEACAM5 antibody drug conjugate can be selected from the group consisting of:

[0444] The anti-CEACAM5-SPDB-DM4 antibody drug conjugate of formula (III):

[0445] anti-CEACAM5-sulfo-SPDB-DM4 antibody drug conjugate of formula (IV):

[0446] and

[0447] anti-CEACAM5-SMCC-DM1 antibody drug conjugate of formula (V):

[0448] In the above formulae (III), (IV) and (V), “n” corresponds to the number of chemotherapeutic agent molecules conjugated per antibody molecule. It corresponds to the “drug-to-antibody ratio” (or “DAR”) as defined below, and can range from 1 to 10.

[0449] In another embodiment, the antibody drug conjugate of the disclosure comprises an anti-CEACAM5 antibody comprising a heavy chain (VH) of SEQ ID NO: 8 and a light chain (VL) of SEQ ID NO: 9 (fam- trastuzumab), wherein fam-trastuzumab is covalently linked via pyridyldithiobutyric acid N- succinimidyl ester (SPDB) to N2’-deacetyl-N-2’(4-methyl-4-mercapto-l-oxopentyl)- maytansine (DM4). Thereby obtaining the antibody drug conjugate fam-trastuzumab-rsens (huMAb2-3-SPDB-DM4).

[0450] In an embodiment, the antibody drug conjugate of the disclosure is fam-trastuzumab-rsens (CAS [2254086-60-5]).

[0451] As used herein, “linker” means a chemical moiety comprising a covalent bond or chain of atoms that covalently attaches an antibody to a chemotherapeutic agent moiety (e.g., a cytostatic, cytotoxic, or growth inhibitory agent). Suitable linkers are well known in the art, including disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups.

[0452] Conjugates can be prepared by in vitro methods. To link a drug or prodrug to an antibody (e.g., a chemotherapeutic agent), a linker group is used. Suitable linker groups are well known in the art and include disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups, and esterase-labile groups. Conjugation of antibodies to the chemotherapeutic agents (e.g., cytotoxic agents) disclosed herein can be performed using a variety of bifunctional protein conjugates, including but not limited to pyridyl dithiobutyrate N-succinimide ester (SPDB), 4-[(5-nitro-2-pyridyl)dithio]-2,5-dioxo-1-pyrrolyl ester butyrate (nitro-SPDB), 4-(pyridyl-2-dithio)-2-sulfonyl-butyrate (sulfon-SPDB), (2-pyridyl dithio)propionate N-succinimide ester (SPDP), (N- Maleimide-methyl)cyclohexane-1-carboxylic acid succinimide (SMCC), iminothiones (IT), bifunctional derivatives of imine esters (such as dimethyl hexamethyleneimine ester HCl), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)-hexanediamine), diazide derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and difluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described by Vitetta et al. (1987). Carbon-labeled 1-isothiocyanate benzylmethyl diethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies (WO 94 / 11026).

[0453] Linkers can be “cleavable linkers” that facilitate the release of chemotherapeutic agents into cells. For example, acid-labile linkers, peptidase-sensitive linkers, esterase-labile linkers, light-labile linkers, or disulfide-containing linkers can be used (see, for example, U.S. Patent No. 5,208,020). Linkers can also be “non-cleavable linkers” (e.g., SMCC linkers), which may result in better tolerability in some cases.

[0454] Generally, couplings can be obtained through a process that includes the following steps:

[0455] (i) Contact an optional buffered aqueous solution of a cell binder (e.g., an antibody according to this disclosure) with a solution of a linker and a chemotherapeutic agent (such as a cytotoxic compound (or agent)).

[0456] (ii) The conjugate formed in (i) is then optionally separated from the unreacted cell binder (e.g., the antibody disclosed herein) and the unreacted chemotherapeutic agent (such as an unreacted cytotoxic compound (or agent)).

[0457] The aqueous solution of the cell-binding agent can be buffered with a buffer such as, for example, potassium phosphate, acetate, citrate or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer depends on the nature of the cell-binding agent (e.g. an antibody of the present disclosure). The chemotherapeutic agent, such as a cytotoxic compound (or agent), is in solution in an organic polar solvent (e.g. dimethylsulfoxide (DMSO) or dimethylacetamide (DMA)).

[0458] The reaction temperature is typically between 20°C and 40°C. The reaction time can vary from 1 hour to 24 hours. The reaction between the cell-binding agent and the chemotherapeutic agent (such as a cytotoxic agent) can be monitored by size exclusion chromatography (SEC) with a refractive and / or UV detector. If the yield of conjugate is too low, the reaction time can be prolonged.

[0459] The skilled person can use a variety of different chromatographic methods for the separation of step (ii): the conjugate can be purified, for example, from aggregates 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). Purification by dialysis or diafiltration can also be used.

[0460] As used herein, the term "aggregate" means an association that can form between two or more cell-binding agents, either modified by conjugation or not. Aggregates can form under the influence of many parameters, such as a high concentration of cell-binding agents (e.g. antibodies of the present disclosure) in solution, the pH of the solution, high shear forces, the number of bonded dimers and their hydrophobic properties, the temperature (see Wang and Gosh, 2008, J. Membrane Sci., 318: 311-316, and references cited therein); it should be noted that the relative influence of some of these parameters has not yet been determined definitively. In the case of proteins and antibodies, the skilled person will refer to Cromwell et al. (2006, AAPS Journal, 8(3): E572-E579). The content in aggregates can be determined with techniques well known to the skilled person, such as SEC (see Walter et al., 1993, Anal. Biochem., 212(2): 469-480).

[0461] After step (i) or (ii), the solution containing the conjugate can be submitted to a further step (iii) of chromatography, ultrafiltration and / or diafiltration.

[0462] At the end of these steps, the conjugate is recovered in aqueous solution.

[0463] In further embodiments, the antibody drug conjugates according to the disclosure are characterized by a "drug to antibody ratio" (or "DAR") ranging from 1 to 10, or from 2 to 5, or from 3 to 4. This is typically the case when the conjugate comprises a maytansinoid molecule.

[0464] This DAR value can vary depending on the nature of the antibody and drug (i.e. chemotherapeutic agent, such as a cytotoxic agent or a growth inhibitory agent) used, and on the experimental conditions used for the conjugation (like the ratio of chemotherapeutic agent (e.g. growth inhibitory agent) to antibody, the reaction time, the nature of the solvent and of the co-solvent if any). Thus, the contact between the antibody and the chemotherapeutic agent (e.g. cytotoxic agent or growth inhibitory agent) results in a mixture comprising several conjugates, which differ from each other by their drug to antibody ratio; optionally naked antibody; optionally aggregates. Thus, the determined DAR is an average value.

[0465] Methods that can be used to determine the DAR include spectrophotometric measurement of the absorbance of a substantially purified conjugate solution at λDand 280 nm. 280 nm is the wavelength usually used to measure the concentration of proteins (e.g. antibodies). The wavelength λDis chosen to allow the distinction between the drug and the antibody, i.e. λDis a wavelength at which the drug (i.e. chemotherapeutic agent) has a high absorbance, and λDis far enough from 280 nm to avoid a substantial overlap of the absorbance peaks of the drug and the antibody, as the skilled person will readily know. In the case of a maytansinoid molecule, λDmay be chosen to be 252 nm. The DAR calculation method can be derived from Antony S. Dimitrov (Editor), LLC, 2009, Therapeutic Antibodies and Protocols, Vol. 525, 445, Springer Science.

[0466] The absorbance of the conjugate is measured at λD(AλD) and 280 nm (A280) either on the monomeric peak of the size exclusion chromatography (SEC) analysis (allowing the calculation of the "DAR(SEC)" parameter) or using a classical spectrophotometer device (allowing the calculation of the "DAR(UV)" parameter). The absorbance can be expressed as follows:

[0467] AλD= (cD x εDλD) + (cA x εAλD)

[0468] A280= (cD x εD280) + (cA x εA280)

[0469] wherein:

[0470] cDand cAare the concentrations in the solution of the drug (i.e. chemotherapeutic agent) and of the antibody, respectively.

[0471] εDλDand εD280are the molar extinction coefficients of the drug at λDand 280 nm, respectively.

[0472] εAλDand εA280are the molar extinction coefficients of the antibody at λDand 280 nm, respectively.

[0473] Solving these two equations with two unknowns gives the following equation:

[0474] cD= [(εA280 x AλD) - (εAλD x A280)] / [(εDλD x εA280) - (εAλD x εD280)]

[0475] cA= [A280 - (cD x εD280)] / εA280

[0476] The average DAR is then calculated from the ratio of drug concentration to antibody concentration: DAR = cD / cA.

[0477] In some embodiments, the antibody drug conjugate can be administered at a dose of 80 mg / m 2 to 210 mg / m 2 , or at a dose of 80 mg / m 2 to 170 mg / m 2 , or at a dose of 80 mg / m 2 to 150 mg / m 2 , or at a dose of 80 mg / m 2 to 120 mg / m 2 , or at a dose of 80 mg / m 2 to 100 mg / m 2 .

[0478] In some embodiments, the antibody drug conjugate can be administered at a dose level of 80, 100, 120, 150, 170, 180, or 210 mg / m 2 .

[0479] In some embodiments, the dosage regimen can include administering the dose over a period of about 10 minutes to about 48 hours, or about 1 h to about 48 h, such as over a period of 1 h to 4 h. In some embodiments, the dosage regimen can include administering the dose over a period of about 1 h.

[0480] In some embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered over about 30 minutes to about 3 hours, about 1 hour to about 2 hours, or about 1.5 hours. ADC dosing

[0481] In some embodiments, an antibody-drug conjugate for the treatment of cancer in combination with an anti-CTLA4 antibody and an anti-PD-1 antibody or an anti-PD-L1 antibody (in the presence) is disclosed, comprising an anti-CEACAM5 antibody and a chemotherapeutic agent, wherein the antibody-drug conjugate can be administered at approximately 60 mg / m². 2 Approximately 210 mg / m 2 or approximately 80 to approximately 170 mg / m³ 2 or approximately 100 to approximately 170 mg / m³ 2 or approximately 120 to approximately 170 mg / m³ 2 or approximately 135 to approximately 170 mg / m³ 2 or approximately 150 to approximately 170 mg / m³ 2 Dosage administration based on the required body surface area of ​​the subject.

[0482] In some embodiments, the antibody-drug conjugate may be administered at a concentration of about 60 to about 210 mg / m³. 2 Or approximately 80 to approximately 170 mg / m³ 2 or approximately 100 to approximately 150 mg / m³ 2 Dosage administration.

[0483] In several embodiments, the antibody-drug conjugate containing the anti-CEACAM5 antibody may be in the form of about 60, 70, 80, 90, 100, 110, 120, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, or about 210 mg / m³. 2 Dosage administration.

[0484] In several embodiments, the antibody-drug conjugate containing the anti-CEACAM5 antibody may be in the form of about 60, 80, 100, 120, 135, 150, 170, 180, 190, or about 210 mg / m³. 2 Dosage administration.

[0485] In some embodiments, the ADC can be at approximately 80 mg / m³ 2 Approximately 170 mg / m 2 Administer at a dose of approximately 80 mg / m³. 2 Approximately 150 mg / m 2 Administer at a dose of approximately 100 mg / m³. 2 Approximately 120 mg / m 2 Dosage administration.

[0486] In some embodiments, the ADC can be at approximately 80 mg / m³ 2 or approximately 100 mg / m 2 or approximately 120 mg / m2 or about 150 mg / m 2 or about 170 mg / m 2 .

[0487] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 80 mg / m 2 .

[0488] In some embodiments, the ADC can be administered at a dose of about 100 mg / m 2 .

[0489] In some embodiments, the ADC can be administered at a dose of about 120 mg / m 2 .

[0490] In some embodiments, the ADC can be administered at a dose of about 150 mg / m 2 .

[0491] In some embodiments, the ADC can be administered at a dose of about 170 mg / m 2 .

[0492] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 80, 100, 120, 150, or about 170 mg / m 2 as a loading dose (or first dose).

[0493] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 80 mg / m 2 as a loading dose.

[0494] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 100 mg / m 2 as a loading dose.

[0495] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 120 mg / m 2 as a loading dose.

[0496] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 150 mg / m 2 as a loading dose.

[0497] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 170 mg / m 2 as a loading dose.

[0498] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 80, 100, 120, 150, or about 170 mg / m 2 as a subsequent dose (or second dose).

[0499] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 80 mg / m 2 as a subsequent dose.

[0500] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 100 mg / m 2 as a subsequent dose.

[0501] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 120 mg / m 2 as a subsequent dose.

[0502] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 150 mg / m 2 as a subsequent dose.

[0503] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 170 mg / m 2 as a subsequent dose.

[0504] The subsequent dose can be administered on day 1 of a cycle (a subsequent or additional cycle) after the first cycle.

[0505] According to embodiments, the antibody drug conjugate comprising an anti-CEACAM5 antibody can be administered at a dose of about 80, 100, 120, 150, or about 170 mg / m 2 as a loading dose in a first treatment cycle (e.g., on day 1), and then at a dose of about 80, 100, 120, 150, or about 170 mg / m 2 as a subsequent dose in additional cycles (e.g., on day 1).

[0506] In certain embodiments, for subjects with a body surface area (BSA) > 2.2 m 2 , the dose of the antibody drug conjugate comprising an anti-CEACAM5 antibody can be calculated based on a BSA of 2.2 m 2 .

[0507] The antibody drug conjugate can be tisotumab vedotin (huMAb2-3-SPDB-DM4).

[0508] In certain embodiments, the ADC is administered once about every two weeks. In certain embodiments, the ADC is administered once about every three weeks. In certain embodiments, the ADC is administered once about every four weeks. In certain embodiments, the ADC is administered once about every five weeks. In certain embodiments, the ADC is administered once about every six weeks. Additional Agents

[0509] In certain embodiments, the methods and uses further comprise administering to the subject an effective amount of at least one additional agent effective to treat cancer.

[0510] In certain embodiments, the additional agent is selected from the group consisting of an immune checkpoint inhibitor (ICI), a platinum-based chemotherapy (e.g., cisplatin or carboplatin), pemetrexed, anti-VEGFR2, FOLFOX, FOLFIRI, TAS-102, anti-EGFR, and any combination thereof.

[0511] In certain embodiments, the ICI is an anti-PD-1 antibody.

[0512] In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sintilimab, dostarlimab, and tislelizumab.

[0513] In certain embodiments, the anti-PD-1 antibody is pembrolizumab.

[0514] In certain embodiments, the ICI is an anti-PD-L1 antibody.

[0515] In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, durvalumab, envafolimab, BMS-936559, CK-301, CS-1001, SHR-1316 (HTI-1088), CBT-502 (TQB-2450), and any combination thereof.

[0516] In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0517] In certain embodiments, the method comprises administering to the subject an effective amount of tisotumab vedotin and pembrolizumab.

[0518] In certain embodiments, the method further comprises administering to the subject an effective amount of a platinum-based chemotherapy.

[0519] In certain embodiments, the platinum-based chemotherapy is selected from cisplatin and carboplatin.

[0520] In certain embodiments, the method further comprises administering to the subject an effective amount of pemetrexed.

[0521] In certain embodiments, the method comprises administering to the subject an effective amount of tiragolumab, atezolizumab, and cisplatin.

[0522] In certain embodiments, the method comprises administering to the subject an effective amount of tiragolumab, atezolizumab, cisplatin, and pemetrexed.

[0523] In certain embodiments, the method comprises administering to the subject an effective amount of tiragolumab, atezolizumab, and carboplatin.

[0524] In certain embodiments, the method comprises administering to the subject an effective amount of tiragolumab, atezolizumab, carboplatin, and pemetrexed.

[0525] In one embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody or fragment thereof is a monoclonal antibody having activity that interferes with the interaction between PD-1 and PD-L1. In one embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody is an IgG.

[0526] Anti-PD-1 antibodies and anti-PD-L1 antibodies that are capable of interfering with the interaction between PD-1 (expressed on the surface of immune cells) and PD-L1 (expressed on the surface of cancer cells) can be used as immune checkpoint inhibitors, thereby blocking the pathway that protects tumor cells from components of the immune system that are capable and prepared to fight cancer. When PD-1 and PD-L1 interact, they form a biochemical “shield” that protects tumor cells from destruction by the immune system. Thus, blocking PD-1 or PD-L1 results in blocking the interaction between PD-1 and PD-L1, thereby preventing or lifting the biochemical “shield” that protects tumor cells from destruction by the immune system.

[0527] Many anti-PD-1 antibodies have been approved for clinical use in treating cancer. These anti-PD-1 antibodies include pembrolizumab nivolumab cemiplimab sintykirumab dostarlimab and tislelizumab.

[0528] Likewise, many anti-PD-L1 antibodies have been approved for clinical use in treating cancer. These anti-PD-L1 antibodies include atezolizumab avelumab and durvalumab

[0529] In one embodiment, the anti-PD-1 antibody is pembrolizumab or sintykirumab.

[0530] In one embodiment, the anti-PD-1 antibody is pembrolizumab. It is a fully human monoclonal IgGl antibody against human PD-1.

[0531] In one embodiment, the anti-PD-1 antibody or fragment thereof comprises the light chain and heavy chain CDRs of pembrolizumab.

[0532] In one embodiment, the anti-PD-1 antibody or fragment thereof comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of pembrolizumab.

[0533] In one embodiment, the anti-PD-1 antibody is sintilimab. In one embodiment, the anti-PD-1 antibody or fragment thereof comprises the light chain and heavy chain CDRs of sintilimab. In one embodiment, the anti-PD-1 antibody or fragment thereof comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of sintilimab.

[0534] The anti-PD-1 antibody or anti-PD-L1 antibody or fragment thereof can also be a single domain antibody or fragment thereof. In particular, the single domain antibody fragment can consist of a variable heavy chain (VHH) comprising the CDR1-H, CDR2-H and CDR3-H of the antibody as described above. The antibody can also be a heavy chain antibody, i.e. an antibody without light chain, which can or can not contain a CH1 domain.

[0535] The single domain antibody or fragment thereof can further comprise the framework regions of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody.

[0536] The anti-PD-1 antibody or anti-PD-L1 antibody can also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2 and diabodies.

[0537] The antibody can also be a bispecific or multispecific antibody formed from antibody fragments, wherein at least one antibody fragment is an antibody fragment according to the present disclosure. The anti-PD-1 antibody or anti-PD-L1 antibody and fragments thereof can be produced by any technique well known in the art. In particular, the antibodies are produced by techniques already described.

[0538] The anti-PD-1 antibody or anti-PD-L1 antibody and fragments thereof can be isolated (e.g. purified) from a vector or comprised in a vector for use, such as a membrane or a lipid vesicle (e.g. a liposome).

[0539] The anti-PD-1 antibody or anti-PD-L1 antibody and fragments thereof can 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).

[0540] In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody can be administered at a dose of 150 mg to 400 mg, or at a dose of 150 mg to 300 mg.

[0541] In one embodiment, the anti-VEGFR-2 antibody is a monoclonal antibody or fragment thereof having antagonistic activity against VEGFR-2. In one embodiment, the anti-VEGFR-2 antibody is an IgG.

[0542] The anti-VEGFR-2 antibody is preferably suitable for use in a patient. For example, an anti-mouse VEGFR-2 antibody, such as DC-101, is preferably used in mice, and an anti-human VEGFR-2 antibody is preferably used in humans.

[0543] In one embodiment, the anti-VEGFR-2 antibody is Ramucirumab (CAS No. 947687-13-0). It is a fully human monoclonal IgGl antibody against human VEGFR-2.

[0544] In one embodiment, the anti-VEGFR-2 antibody or fragment thereof comprises the light chain and heavy chain CDRs of Ramucirumab.

[0545] In one embodiment, the anti-VEGFR-2 antibody or fragment thereof comprises the heavy chain variable domain (VH) and the light chain variable domain (VL) of Ramucirumab.

[0546] The anti-VEGFR-2 antibody or fragment thereof can also be a single domain antibody or fragment thereof. In particular, the single domain antibody fragment can consist of a variable heavy chain (VHH) comprising the CDR1-H, CDR2-H and CDR3-H of the antibody as described above. The antibody can also be a heavy chain antibody, i.e. an antibody without light chain, which can or can not contain a CH1 domain.

[0547] The single domain antibody or fragment thereof can further comprise the framework regions of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody.

[0548] The anti-VEGFR-2 antibody can also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2 and diabodies.

[0549] The antibody can also be a bispecific or multispecific antibody formed from antibody fragments, wherein at least one antibody fragment is an antibody fragment according to the present disclosure. The anti-VEGFR-2 antibody and fragments thereof can be produced by any technique well known in the art. In particular, the antibody is produced by techniques already described.

[0550] The anti-VEGFR-2 antibodies and fragments thereof can be isolated (e.g., purified) from or contained in a vector for use, such as a membrane or a lipid vesicle (e.g., a liposome).

[0551] The anti-VEGFR-2 antibodies and fragments thereof can 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.

[0552] The immunoconjugates comprising the anti-CEACAM5 antibodies will be used in combination with cetuximab to treat cancer.

[0553] Cetuximab (CAS No. 205923-56-4) is a chimeric monoclonal IgGl antibody directed against the epidermal growth factor receptor (EGFR). Cetuximab itself has been used to treat metastatic colorectal cancer, metastatic non-small cell lung cancer, and head and neck cancer.

[0554] The immunoconjugates comprising the anti-CEACAM5 antibodies will be used in combination with TAS-102 to treat cancer.

[0555] TAS-102 itself is a known approved chemotherapy regimen for human use comprising the combined administration of trifluoridine and tipiracil, and is typically administered in 4-week cycles. TAS-102 combines trifluoridine and tipiracil, and has been used to treat colorectal cancer.

[0556] Trifluoridine (CAS Registry No. 70-00-8) is a nucleoside analog that is incorporated into DNA. The modified DNA binds to thymidiylate synthase, inhibiting the activity of the enzyme. Tipiracil (CAS Registry No. 183204-74-2) is a thymine analog that prevents thymidine phosphorylase from degrading trifluoridine.

[0557] The immunoconjugates comprising the anti-CEACAM5 antibodies will be used in combination with FOLFIRI to treat cancer.

[0558] FOLFIRI itself is a known approved chemotherapy regimen for human use comprising the combined administration of folinic acid, 5-fluoro-uracil, and irinotecan, and is typically administered in up to 12 two-week cycles. FOLFIRI combines multiple drugs, each with a different mechanism of action, and advantageously has a synergistic effect, leading to cancer cell death.

[0559] 5-fluorouracil (CAS Registry Number 51-21-8) is an antimetabolite that primarily inhibits thymidylate synthase and thereby blocks the synthesis of thymidine. 5-fluorouracil has been used to treat colon cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and cervical cancer.

[0560] Folinic acid, also known as leucovorin (CAS Registry Number 58-05-9), stabilizes the complex between 5-fluorouracil and thymidylate synthase, thereby increasing the cytotoxicity of 5-fluorouracil. In one embodiment, the folinic acid is L-folinic acid (N-[4-[[[(6S)-2-amino-5-formyl-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl]methyl]amino]benzoyl]-L-glutamic acid). In another embodiment, the folinic acid is a calcium salt of L-folinic acid. The folinic acid can also comprise a mixture of two or more stereoisomers.

[0561] Irinotecan (CAS Number 97682-44-5) is a cytotoxin that is a semi-synthetic derivative of the alkaloid camptothecin and inhibits topoisomerase I, thereby inhibiting DNA replication and transcription, and which has been used to treat colon cancer and small cell lung cancer.

[0562] The immunoconjugate comprising an anti-CEACAM5 antibody will be used in combination with FOLFOX to treat cancer.

[0563] FOLFOX is itself a known approved chemotherapy regimen for human use that comprises the combined administration of folinic acid, 5-fluorouracil, and oxaliplatin, and is typically administered in cycles of up to 12 weeks. FOLFOX combines multiple drugs, each with a different mechanism of action, and advantageously has a synergistic effect, resulting in cancer cell death.

[0564] 5-fluorouracil (CAS Registry Number 51-21-8) is an antimetabolite that primarily inhibits thymidylate synthase and thereby blocks the synthesis of thymidine. 5-fluorouracil has been used to treat colon cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and cervical cancer.

[0565] Folinic acid, also known as leucovorin (CAS Registry Number 58-05-9), stabilizes the complex between 5-fluorouracil and thymidylate synthase, thereby increasing the cytotoxicity of 5-fluorouracil. In one embodiment, the folinic acid is L-folinic acid (N-[4-[[[(6S)-2-amino-5-formyl-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl]methyl]amino]benzoyl]-L-glutamic acid). In another embodiment, the folinic acid is a calcium salt of L-folinic acid. The folinic acid can also comprise a mixture of two or more stereoisomers.

[0566] Oxaliplatin (CAS No. 61825-94-3) is known to form crosslinks in DNA strands, thereby preventing DNA replication and transcription, and has been used to treat colorectal cancer. cancer

[0567] In embodiments, the cancer is a carcinoma, sarcoma, or blastoma. In further embodiments, the cancer is a carcinoma.

[0568] According to embodiments, the cancer is a CEACAM5-expressing cancer. A CEACAM5-expressing cancer can also be referred to as a CEACAM5-positive cancer.

[0569] In some embodiments, the cancer is a CEACAM5-positive cancer.

[0570] A CEACAM5-positive cancer is defined as a cancer with CEACAM5 immunohistochemistry [IHC] intensity of >2+ in >50% of cancer cells or this intensity of >2+ in >1% and <50% of tumor cells (or cancer cells).

[0571] In certain embodiments, the cancer has negative or low CEACAM5 expression on tumor cells. Negative or low CEACAM5 expression on tumor cells is defined as CEACAM5 immunohistochemistry [IHC] intensity of >2+ in <1% of cells as measured by immunohistochemistry (IHC).

[0572] In certain embodiments, the cancer has moderate CEACAM5 expression on tumor cells. Moderate CEACAM5 expression on tumor cells can be defined as CEACAM5 immunohistochemistry [IHC] intensity of >2+ in >1% and <50% of cancer cells as measured by immunohistochemistry.

[0573] In certain embodiments, the cancer has high CEACAM5 expression on tumor cells. High CEACAM5 expression on tumor cells can be defined as CEACAM5 immunohistochemistry [IHC] intensity of >2+ intensity in >50% of cancer cells as measured by immunohistochemistry.

[0574] Immunohistochemical techniques for the detection of antigens on cells or tissue sections by immunological and chemical reactions are well known in the art. These techniques have a high sensitivity and specificity and can detect a variety of antigens. The immunohistochemical method comprises the following steps: binding of the antibody to the specific antigen; formation of the antibody-antigen complex by incubation with a secondary antibody coupled to an enzyme, and production of a colored deposit at the antibody-antigen binding site in the presence of a substrate and chromogen catalyzed by the enzyme.

[0575] CEACAM5 tumor expression can be determined by using an immunohistochemistry (IHC) assay. The assay can be performed using an anti-CEACAM5 antibody, such as the antibody clone 769 from SANOFI. Anti-CEACAM5 clone 769 is a murine monoclonal antibody with the same specificity for the CEACAM5 target as tisotumab-raltritux. The assay can be performed on the Techmate platform or the Dako / Agilent Autostainer Link 48 IHC or any other immunohistochemistry platform. Interpretation of CEACAM5 reactivity will be performed using a semi-quantitative percentage score of CEACAM5 plasma membrane staining (whole membrane or polarized membrane) in tumor cells (calculated by summing the percentage of intensity > 2+) or H-score.

[0576] According to embodiments, the cancer is selected from hepatocellular carcinoma, colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer (e.g. non-squamous non-small cell lung cancer), cervical cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer (e.g. cholangiocarcinoma), prostate cancer, neuroendocrine cancer and skin cancer.

[0577] The cancer can be selected from colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, pancreatic cancer and lung cancer.

[0578] In some embodiments, the cancer can be colorectal cancer.

[0579] In some embodiments, the cancer can be pancreatic cancer.

[0580] In some embodiments, the cancer can be selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer and lung cancer.

[0581] In some embodiments, the cancer can be gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma or esophageal cancer.

[0582] According to embodiments, the cancer is gastric cancer or gastroesophageal junction adenocarcinoma (GEJ).

[0583] According to embodiments, the cancer is gastric cancer.

[0584] According to embodiments, the cancer is lung cancer.

[0585] The lung cancer can be non-squamous non-small cell lung cancer (NSQ NSCLC).

[0586] Non-small cell lung cancer is a disease in which malignant (cancer) cells form in the tissues of the lung. Smoking is the major cause of this disease. It is a kind of lung cancer that is not small cell lung cancer. There are several types of non-small cell lung cancer. Each type of non-small cell lung cancer has different kinds of cancer cells. Each type of cancer cell grows and spreads in different ways. The types of non-small cell lung cancer are named for the kind of cells found in the cancer and how the cells look under a microscope: (1) Squamous cell carcinoma: cancer that forms in the thin, flat cells that line the inside of the lungs. This also is called epidermoid carcinoma. (2) Large cell carcinoma: cancer that can form from several types of large cells. (3) Adenocarcinoma: cancer that forms in the cells that line the inside of the lung's air sacs (alveoli) and produce substances such as mucus.

[0587] In some embodiments, the non-squamous non-small cell lung cancer can be advanced or metastatic NSQ NSCLC.

[0588] According to embodiments, the subject is a patient having a malignant tumor, in particular a malignant solid tumor, and more particularly a locally advanced or metastatic solid malignant tumor. The metastatic solid malignant tumor can be a metastatic cancer, e.g. a metastatic carcinoma. The cancer or carcinoma can be as indicated above.

[0589] In some embodiments, the non-squamous non-small cell lung cancer has no epidermal growth factor receptor (EGFR) sensitizing mutations or v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations or anaplastic lymphoma kinase / c-ros oncogene 1 (ALK / ROS) alterations. Pharmaceutical compositions or combinations

[0590] In some embodiments, in the uses and methods as disclosed herein, the administration of the ADC can be by a parenteral route. A suitable parenteral route can be intravenous infusion.

[0591] The disclosure also relates to an ADC for use in the manufacture of a medicament for the treatment of cancer, combination comprising.

[0592] In some embodiments, the disclosure relates to a pharmaceutical composition comprising (i) an ADC as disclosed herein and a pharmaceutically acceptable excipient.

[0593] The ADC of the disclosure can be combined with a pharmaceutically acceptable excipient and, optionally, a sustained release matrix, such as a biodegradable polymer, to form a therapeutic composition.

[0594] Accordingly, another object of the disclosure relates to a pharmaceutical composition comprising an ADC of the disclosure and a pharmaceutically acceptable carrier or excipient. The ADC or immunoconjugate according to the disclosure for use as a medicament.

[0595] The present disclosure also relates to an ADC according to the present disclosure for use in the treatment of cancer.

[0596] A "pharmaceutical excipient" or "pharmaceutically acceptable excipient" refers to a molecular entity and composition that does not produce an adverse, allergic, or other untoward reaction when administered to a mammal, particularly a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0597] As used herein, "pharmaceutically acceptable carrier or excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents and / or excipients include 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, NaCl, calcium chloride; sugars or polyols, such as glucose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants, such as polysorbate 80, polysorbate 20, poloxamer 188; and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition, and the formulation can also contain adjuvants, such as amines, and stabilizers, such as tryptane and Tween 20.

[0598] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen naturally depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the subject, etc.

[0599] The pharmaceutical compositions of the present disclosure can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, etc. In embodiments, the pharmaceutical compositions and combinations of the present disclosure are formulated for intravenous administration.

[0600] In particular, the pharmaceutical compositions contain a pharmaceutically acceptable vehicle or excipient for a formulation capable of injection. These can be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or mixtures of such salts) or dry, especially freeze-dried compositions which, on addition of sterile water or physiological saline, allow to form isotonic solutions. The compositions will be presented in unit dosage form, e.g. the form of tablets, pills, capsules, powders, granules, sterile parenteral solutions, or suspensions, or suppositories, for oral, parenteral, intranasal, intramuscular, subcutaneous, or rectal administration, or in any other form suitable for use.

[0601] The pharmaceutical composition can be administered by a pharmaceutical combination device.

[0602] The dose for administration can be adjusted according to various parameters, in particular according to the mode used, the pathology concerned or alternatively the duration of treatment desired.

[0603] For preparing pharmaceutical compositions, an effective amount of an antibody drug conjugate comprising an anti-CEACAM5 antibody and / or an anti-CTLA4 antibody and / or an anti-PD-1 antibody or an anti-PD-L1 antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0604] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including peanut oil, safflower oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists, 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. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0605] Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water or physiologically compatible solvents such as Ringer's solution, or isotonic sodium chloride solution. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0606] The antibody drug conjugate comprising an anti-CEACAM5 antibody can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and which are derived from inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid) or organic acids (for example, acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like). Salts formed with the free carboxyl groups can also be derived from inorganic bases (for example, sodium, potassium, ammonium, calcium, or ferric hydroxides) and such organic bases as isopropylamine, trimethylamine, glycerol, histidine, procaine and the like.

[0607] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0608] A sterile injectable solution can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0609] Further preparation of more or highly concentrated solutions for direct injection is also contemplated, wherein the use of DMSO as a solvent is envisioned to result in very fast penetration to deliver high concentrations of the activating agent to small tumor areas.

[0610] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules or the like can also be employed.

[0611] For instance, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0612] Antibody drug conjugates comprising an anti-CEACAM5 antibody are 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 in use.

[0613] In certain embodiments, the use of liposomes and / or nanoparticles to introduce the polypeptide into the host cell is contemplated. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0614] Nanocapsules can generally capture compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such submicron particles (size of about 0.1 pm) are usually designed using polymers that are able to degrade in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles, or biodegradable polylactide or polylactide-co-glycolide nanoparticles that meet these requirements are contemplated for use in the present disclosure, and such particles can be readily prepared.

[0615] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also known as multilamellar vesicles (MLV)). The diameter of MLV is typically 25 nm to 4 pm. Sonication of MLV results in the formation of small unilamellar vesicles (SUV) with a core containing an aqueous solution, having a diameter in the range of 200 to 400 nm. Methods of administration and formulations

[0616] The methods described herein include administering to a subject a therapeutically effective amount of an anti-CEACAM5 ADC. As used herein, an “effective amount” or “therapeutically effective amount” is a therapeutic dose that results in a detectable improvement in one or more symptoms associated with a CEACAM5-expressing cancer (e.g., lung cancer, gastric cancer, gastroesophageal junction cancer, or esophageal cancer), or that results in a biological effect associated with the underlying pathogenic mechanism that causes the condition or symptom (e.g., a decrease in the level of a particular biomarker). For example, a dose of an anti-CEACAM5 ADC that results in an improvement in any of the following symptoms or conditions associated with a CEACAM5-expressing cancer is considered a “therapeutically effective amount”:

[0617] In another example, a treatment is ineffective when a dose of an anti-CEACAM5 ADC does not result in a detectable improvement in one or more parameters or symptoms associated with a CEACAM5-expressing cancer (e.g., lung cancer, gastric cancer, gastroesophageal junction cancer, or esophageal cancer), or that does not result in a biological effect associated with the underlying pathogenic mechanism that causes the condition or symptom of the cancer.

[0618] According to some of these embodiments, the anti-CEACAM5 ADC is administered intravenously.

[0619] According to the methods of the present disclosure, the therapeutically effective amount of an anti-CEACAM5 ADC administered to a subject will vary depending on the age and size (e.g., weight or body surface area) of the subject, as well as the route of administration and other factors well known to those of ordinary skill in the art.

[0620] ​In certain embodiments, the dose of the ADC varies according to the body surface area of the subject. In certain embodiments, the dose of the anti-CEACAM5 ADC administered to the subject is about 1 mg / m 2 to about 500 mg / m 2 . In some embodiments, the dose of the ADC administered to the subject is about 5 mg / m 2 to about 300 mg / m 2 . In various embodiments, the dose of the ADC administered to the subject is about 5 mg / m 2 to about 250 mg / m 2 . In various embodiments, the dose of the ADC administered to the subject is about 60 mg / m 2 to about 190 mg / m 2 . In various embodiments, the dose is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 210 mg / m 2 . In certain embodiments, the dose of the ADC is about 100 mg / m 2 . In certain embodiments, the dose of the ADC is about 150 mg / m 2 . In certain embodiments, the dose of the ADC is about 170 mg / m 2 . In certain embodiments, the dose of the ADC is about 190 mg / m 2 .

[0621] In various embodiments, the dose of the ADC is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 210 mg / m 2 . In certain embodiments, the dose of the ADC is 100 mg / m 2 . In certain embodiments, the dose of the ADC is 150 mg / m 2 . In certain embodiments, the dose of the ADC is 170 mg / m 2 . In certain embodiments, the dose of the ADC is 190 mg / m 2 . [Examples]

[0622] The following examples illustrate the embodiments of the disclosure presently best known. However, it is to be understood that the following are merely examples or illustrations of applications of the principles of the present disclosure. Numerous modifications can be made by those skilled in the art without departing from the spirit and scope of the disclosure. Therefore, although the disclosure has been described in particular detail with specific reference to certain versions thereof, the following examples provide further detail in connection with what are presently believed to be the most practical and preferred embodiments of the present disclosure. Example 1: Materials and Methods Study design and patients

[0623] Details of the study design of this phase 1 / 2 first-in-human, open-label, dose-escalation and expansion trial of tisotumab vedotin-ldodomed have been published elsewhere.

[15] Ethical statement

[0624] Briefly, patients were aged > 18 years, had locally advanced or metastatic solid tumors for which there were no available standard alternative therapies, and Eastern Cooperative Oncology Group (ECOG PS) of 0 or 1, who met the conditions for the dose-escalation and expansion phases.

[0625] The population of the dose-escalation phase was enriched (but not limited to) patients with tumor types known to express CEACAM5 and expression was retrospectively confirmed using IHC on the most recent archival tissue sample at a central laboratory.

[0626] The population of the expansion phase was limited to separate cohorts of patients with advanced colorectal cancer, NSQ-NSCLC, small cell lung cancer, and gastric adenocarcinoma.

[0627] In this report, we present results for NSQ-NSCLC patients only. Based on IHC analysis of tumor tissue, there were two separate NSQ-NSCLC expansion phase populations:

[0628] CEACAM5 high expressers, defined as patients with > 50% of the tumor cell population with CEACAM5 expression intensity > 2+ and

[0629] CEACAM5 intermediate expressers, defined as patients with > 1% to < 50% of the tumor cell population with CEACAM5 expression intensity > 2+.

[0630] All patients were required to have at least one measurable lesion according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1.

[0631] Key exclusion criteria included: life expectancy < 12 weeks; known or symptomatic brain metastases; receiving other cancer treatment; prior therapy targeting CEACAM5; prior treatment with maytansinoid; and poor bone marrow reserve or organ dysfunction. Treatment

[0632] During the expansion phase, all patients received intravenous (IV) tesidolumab-rsxd 100 mg / m2 Q2W, as determined in the dose escalation phase of the study

[15] . Tesidolumab-rsxd was infused over 30 minutes at a rate of 2.5 mg / min, followed by 5 mg / min, provided there were no signs or symptoms of hypersensitivity.

[0633] To prevent hypersensitivity reactions, patients were premedicated with oral antihistamines 1 hour prior to receiving tesidolumab-rsxd.

[0634] Treatment was continued until disease progression, unacceptable toxicity, or the patient elected to stop treatment. Results

[0635] The primary outcome during the expansion phase was the objective response rate assessed according to RECIST vl.l criteria.

[0636] Key secondary outcomes assessed safety, which will be reported in a separate publication.

[0637] Exploratory biomarker objectives (reported herein) included exploring potential links between CEACAM5 expression signatures and response (expansion phase), investigating potential biomarkers (other than CEACAM5) that can predict tesidolumab-rsxd activity (expansion phase), and evaluating the potential of circulating CEA levels as a convenient companion diagnostic for tesidolumab-rsxd treatment (by assessing the correlation between circulating CEA levels and tumor CEACAM5 expression, as well as the correlation between this biomarker and treatment response). Tumor samples

[0638] Tumor samples were obtained using the most recent archival tumor sample (i.e., tumor tissue archived at the time of diagnosis, surgery, or collected prior to the patient’s inclusion in the study but prior to receiving anticancer treatment); for patients with lesions amenable to biopsy, a fresh biopsy at baseline was optional. Measurement of CEACAM5 expression by immunohistochemistry

[0639] Archival tumor samples were analyzed by IHC for the level and pattern of CEACAM5 expression using mouse anti-CEACAM5 clone 769 (which has the same specificity for the CEACAM5 target as tesidolumab-rsxd) locally at the clinical site and / or centrally in the laboratory.

[0640] At least six 5 pm slides and three additional 10 pm slides (or six 5 pm slides) of formalin-fixed paraffin-embedded (FFPE) tissue were provided for each patient. CEACAM5 expression was centrally determined by using a validated IHC assay with 0.5 pg / mL of the anti-CEACAM5 clone 769 antibody.

[0641] CEACAM5 reactivity in tumor cells was assessed using semi-quantitative percentage score of CEACAM5 plasma membrane staining (whole membrane or polarized membrane) (calculated by summing the percentage of intensity > 2+) or H-score; CEACAM5 cytoplasmic staining (only concentrated) was also assessed. Tissue RNA / DNA extraction

[0642] If provided as a block, FFPE tumor tissue was cut into 10 pm thick using a microtome and 3 sections were mounted on adhesive microscope slides. After trimming off the excess paraffin from the sample slides using a sterile scalpel, the tumor tissue was macrodissected and collected in separate Eppendorf DNA LoBind tubes. Genomic DNA and total RNA were extracted from each lung cancer FFPE tissue sample using the AllPrep® DNA / RNA FFPE Kit (reference 80234, QIAGEN) according to the manufacturer’s instructions, with a starting manual processing step followed by a final automated extraction step using the QIACUBE automated nucleic acid purification instrument. Genomic DNA was eluted in 30 pL ATE buffer and total RNA was eluted in 20 pL RNase-free water. Genomic DNA and total RNA were extracted from each lung cancer FFPE tissue sample using the AllPrep® DNA / RNA FFPE Kit (reference 80234, QIAGEN) according to the manufacturer’s instructions, with a starting manual processing step followed by a final automated extraction step using the QIACUBE automated nucleic acid purification instrument. Genomic DNA was eluted in 30 pL ATE buffer and total RNA was eluted in 20 pL RNase-free water.

[0643] Seventy-one RNA samples were generated for RNA sequencing analysis. Gene expression (RNA-seq)

[0644] Tissue samples with known CEACAM5 expression levels (measured by IHC) were analyzed using RNA-seq. Total RNA was extracted from each sample using the KAPA mRNA HyperPrep® Kit (reference KK8500, KAPA Biosystems) according to the manufacturer’s instructions. RNA-seq data was processed as follows: Sequencing reads were mapped to the reference genome GRCh 38 using the STAR aligner to the spliced transcripts of the reference (STAR)

[25] . Gene expression was initially measured by CUFFLINK in FPKM (fragments per million per kilobase)

[26] and gene-level FPKM was converted to TPM (transcripts per million)

[27] . TPM values were log2 transformed and quantile normalized for downstream analysis, including differential gene expression (DGE) analysis. Samples with a detected number of genes below 10,000 were excluded from downstream analysis. RNA-seq included microenvironment cell population [MCP] counter analysis according to published methods [28, 29]. Statistical analysis

[0645] All analyses were performed in the biomarker population, defined as patients who received treatment and had at least one evaluable CEACAM5 expression measurement or at least one valid RNA assessment. CEACAM5 expression in pre-treatment tumor samples

[0646] The percentage of positive tumor cells with intensity > 2+ across the membrane defined CEACAM5 expression in tumor tissue, which was analyzed using descriptive statistics.

[0647] The association between CEACAM5 expression signatures was assessed by computing the P-value of the Mann-Whitney U test (only when there are 2 groups) or Kendall tau.

[0648] Adjusted P-values were also computed using the Benjamini-Hochberg (BH) multiple correction procedure to control the false discovery rate. Association between baseline biomarkers and tumor response

[0649] The statistical significance of the association between CEACAM5 expression (moderate vs. high) and overall response rate (ORR) was assessed by a two-sided Fisher’s exact test. Example 2: Results Biomarker evaluable patients

[0650] In this exploratory biomarker analysis of the cohort of patients with advanced non-squamous NSCLC in the dose expansion phase, the first patient was enrolled on January 2, 2017, and the data cutoff date for these analyses was December 2020.

[0651] Of the 888 pre-screened NSQ-NSCLC patients, 172 (19%) had high CEACAM5 expression and 210 (24%) had moderate CEACAM5 expression.

[0652] 92 patients were treated: 64 with high CEACAM5 expression (high expressers), 28 with intermediate CEACAM5 expression (intermediate expressers)

[0653] Overall, the median age was 62.5 years (range 31-91 years; 42% > 65 years); 51% were male; 72% had an ECOG PS > 1; patients had received a median of 3 lines of therapy (range 1-10) for advanced disease, including anti-tubulin agents (61%) and anti-PD1 / PD-L1 agents (75%).

[0654] Clinical outcomes showed that patients with high CEACAM5 protein expression levels measured by immunohistochemistry (IHC) (i.e., > 50% of tumor cells with intensity > 2+ by CEACAM5 IHC) (responders) had a rich clinical response. Non-responders were CEACAM5 protein intermediate expressers, defined as > 1% to < 50% of tumor cell population with CEACAM5 protein expression intensity > 2+ measured by IHC. CEACAM5 expression: staining and distribution patterns in tumors

[0655] High CEACAM5 expressers showed a predominance of full-membrane expression over polarized membrane expression. In contrast, intermediate CEACAM5 expressers had similar full-membrane and polarized membrane expression (Table 1).

[0656] At initial diagnosis, the predominant histology type was adenocarcinoma, and most (91.3%) of the tumors expressing CEACAM5 were in stage III or higher, regardless of CEACAM5 expression level (Table 1). Table 1. CEACAM5 expression: staining and distribution patterns in tumors

[0657] a For polarized membrane, N = 63.

[0658] CEACAM5, Carcinoembryonic Antigen Related Cell Adhesion Molecule 5; NSCLC, non-small cell lung cancer; SD, standard deviation. Correlation between CEACAM5 expression (measured by IHC) and gene expression (RNA-seq)

[0659] Differential gene expression analysis has identified CEACAM5 mRNA as the most relevant gene (and also the only significant gene, adjusted P = 0.00265) with high vs. moderate expression of CEACAM5 measured by IHC. After multiple testing correction, there was no significant association of expression of other CEACAM family members or other genes with CEACAM5 IHC (Table 2). Table 2. RNA-seq gene expression fold change for high vs. moderate CEACAM5 expression (measured by IHC): top 10 genes, including other CEACAM family members ranked closest.

[0660] CEACAM, carcinoembryonic antigen-related cell adhesion molecule; CL, confidence limit; logFC, log fold change. Association between CEACAM5 expression, objective response rate, and CEACAM5 mRNA level

[0661] The correlation of CEACAM5 mRNA level with CEACAM5 expression measured by immunohistochemistry, recorded as the sum of percentage of tumor cells expressing the target at at least 2+ intensity, was observed in patients who responded to treatment with tisotumab vedotin-beridotin. Example 3: Discussion

[0662] Nearly 20% of prescreened NSCLC patients had high CEACAM5 expression, i.e., > 50% of tumor cells staining at intensity > 2+, measured by IHC. The clinical response was observed to be enriched in patients with high CEACAM5 protein expression level and treated with tisotumab vedotin-beridotin.

[0663] A correlation was found between CEACAM5 expression, cCEA, cCEACAM5, and CEACAM5 tumor mRNA level.

[0664] In addition, CEACAM5 mRNA expression was significantly upregulated in patients with high vs. moderate CEACAM5 protein expression, but not other genes, including other CEACAM family genes (Table 1). Figure 1 and Table 1).

[0665] Higher levels of CEACAM5 mRNA were observed in CEACAM5 high expressers compared to moderate expressers (P = 0.0027) Figure 1 ).

[0666] A correlation was found between CEACAM5 mRNA levels in tumor cells and CEACAM5 IHC staining in tumor cells. Figure 2 ).

[0667] Furthermore, as measured by IHC, patients treated with ADCs who had high CEACAM5 mRNA levels and high CEACAM5 protein expression showed rich clinical responses. Figure 3 and Figure 4 ).

[0668] like Figure 5 As shown, based on clinical response to teretumab-lexacin, the median CEACAM5 mRNA level was higher in the responder group compared to the non-responder group, indicating a richer clinical response in patients treated with teretumab-lexacin based on CEACAM5 mRNA levels. This result suggests that, based on CEACAM5 mRNA levels, patients treated with teretumab-lexacin had a richer clinical response. This was not observed in other CEACAM family members except for a minor trend observed in CEACAM3.

[0669] Clinical results from the dose escalation and expansion phases of the clinical study showed that patients with high CEACAM5 protein expression (responders) and treated with teretetumab-Rasin had richer clinical responses compared to patients treated with ADCs and with moderate CEACAM5 protein expression (non-responders).

[0670] Further results from the biomarker analysis support the association between mRNA CEACAM5 expression levels and CEACAM5 protein expression levels.

[0671] Because a correlation was shown between CEACAM5 protein expression levels and rich clinical response in patients treated with teretumab-Lexacin, and because a correlation was shown between CEACAM5 protein expression levels and mRNA CEACAM5 expression levels, mRNA CEACAM5 expression levels are considered a good biomarker for rich clinical response and can be used to select patients for treatment with anti-CEACAM5 antibody-drug conjugates (such as teretumab-Lexacin).

[0672] In addition, CEACAM5 mRNA level is a good biomarker for pre-selecting patients who need cancer treatment with antibody-drug conjugates (ADCs) containing anti-CEACAM5 antibodies conjugated with cytotoxic agents, for additional selection steps using CEACAM5 immunohistochemical (IHC) staining, and for treating patients who need cancer treatment with ADCs. [References] 1. International Agency for Research on Cancer and WHO. Globocan: Cancer today; mortality. 2020 October 12, 2021] ; Available from: https: / / gco.iarc.fr / today / online-analysis-pie? v=2020&mode=cancer&mode_population=continents&population=900&populations=900&key=total&sex=0&cancer=39&type=1&statistic=5&prevalence=0&population_group=0&ages_group%5B%5D=0&ages_group%5B%5D=17&nb_items=15&group_cancer=1&include_nmsc=1&include_nmsc_other=1&half_pie=0&donut=0. 2. National Cancer Institute. Surveillance, Epidemiology, and End Results Program. Lung and bronchus: SEER 5-year age-adjusted incidence rates, 2015-2019. 2021 October 12, 2021] ; Available from: https: / / seer.cancer.gov / explorer / application.html?site=47&data_type=1&graph_type=10&compareBy=subtype&chk_subtype_47=47&chk_subtype_612=612&chk_subtype_613=613&chk_subtype_611=611&chk_subtype_610=610&series=stage&chk_stage_101=101&chk_stage_104=104&chk_stage_105=105&chk_stage_106=106&chk_stage_107=107&sex=1&race=1&age_range=1&advopt_precision=1. 3. National Cancer Institute. Surveillance, Epidemiology, and End Results Program. Lung and bronchus: SEER 5-year relative survival rates, 2012-2018. 2021 October 12, 2021] Available from: https: / / seer.cancer.gov / explorer / application.html?site=47&data_type=4&graph_type=5&compareBy=subtype&chk_subtype_47=47&chk_subtype_612=612&chk_subtype_613=613&chk_subtype_611=611&chk_subtype_610=610&series=stage&chk_stage_101=101&chk_stage_104=104&chk_stage_105=105&chk_stage_106=106&chk_stage_107=107&sex=1&race=1&age_range=1&advopt_precision=1. 4. Chu, QS. Targeting non-small cell lung cancer: driver mutation beyond epidermal growth factor mutation and anaplastic lymphoma kinase fusion. Ther Adv Med Oncol, 2020, 12: 1758835919895756. [PubMed ID: 32047535] 5. Rodak, O, Peris-Diaz, MD, Olbromski, M, et al. Current Landscape of Non-Small Cell Lung Cancer: Epidemiology, Histological Classification, Targeted Therapies, and Immunotherapy. Cancers (Basel), 2021, 13: 4705. [PubMed ID: 34572931] 6. Beauchemin, N and Arabzadeh, A. Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis. Cancer Metastasis Rev, 2013, 32:643-71. [PubMed ID: 23903773] 7. Decary, S, Berne, PF, Nicolazzi, C, et al. Preclinical Activity of SAR408701 : A Novel Anti-CEACAM5-maytansinoid Antibody-drug Conjugate for the Treatment of CEACAM5-positive Epithelial Tumors. Clin Cancer Res, 2020, 26:6589-6599. [PubMed ID: 33046521] 8. Decary, S, Berne, PF, Nicolazzi, C, et al. A novel anti-CEACAM5 maytansinoid-antibody-drug conjugate for the treatment of colorectal, lung and gastric tumors. Cancer Research, 2015, 75: Abs 1688. [PubMed ID: WOS:000371578503178] 9. Blumenthal, RD, Hansen, HJ, and Goldenberg, DM. Inhibition of adhesion, invasion, and metastasis by antibodies targeting CEACAM6 (NCA-90) and CEACAM5 (Carcinoembryonic Antigen). Cancer Res, 2005, 65:8809-17. [PubMed ID: 16204051] 10. Zhang, X, Han, X, Zuo, P, et al. CEACAM5 stimulates the progression of non-small-cell lung cancer by promoting cell proliferation and migration. J Int Med Res, 2020, 48: 300060520959478. [PubMed ID: 32993395] 11. Hu, R, Huffman, KE, Chu, M, et al. Quantitative Secretomic Analysis Identifies Extracellular Protein Factors That Modulate the Metastatic Phenotype of Non-Small Cell Lung Cancer. J Proteome Res, 2016, 15: 477-86. [PubMed ID: 26736068] 12. Papadaki, MA, Messaritakis, I, Fiste, O, et al. Assessment of the Efficacy and Clinical Utility of Different Circulating Tumor Cell (CTC) Detection Assays in Patients with Chemotherapy-Naive Advanced or Metastatic Non-Small Cell Lung Cancer (NSCLC). Int J Mol Sci, 2021, 22. [PubMed ID: 33477700] 13. Desai, A, Abdayem, P, Adjei, AA, et al. Antibody-drug conjugates: A promising novel therapeutic approach in lung cancer. Lung Cancer, 2022, 163: 96-106. [PubMed ID: 34942494] 14. Pouzin, C, Gibiansky, L, Fagniez, N, et al. Integrated multiple analytes and semi-mechanistic population pharmacokinetic model of tusamitamab ravtansine, a DM4 anti-CEACAM5 antibody-drug conjugate. J Pharmacokinet Pharmacodyn, 2022, 49: 381-394. [PubMed ID: 35166967] 15. Gazzah, A, Bedard, PL, Hierro, C, et al. Safety, pharmacokinetics, and antitumor activity of the anti-CEACAM5-DM4 antibody-drug conjugate tusamitamab ravtansine (SAR408701) in patients with advanced solid tumors: first-in-human dose-escalation study. Ann Oncol, 2022, 33: 416-425. [PubMed ID: 35026412] 16. Gazzah, A, Ricordel, C, Cousin, S, et al. Efficacy and safety of the antibody-drug conjugate (ADC) SAR408701 in patients (pts) with non-squamous non-small cell lung cancer (NSQ NSCLC) expressing carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5). Journal of Clinical Oncology, 2020, 38: Abs9505. [PubMed ID: WOS:000560368303410] 17. Ricordel, C, Barlesi, F, Cousin, S, et al. Safety and efficacy of tusamitamab ravtansine (SAR408701) in long-term treated patients with nonsquamous non- small cell lung cancer (NSQ NSCLC) expressing carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5). Journal of Clinical Oncology, 2022, 40: 9039-9039. [PubMed ID: 36588657] 18. Sefrioui, D, Beaussire, L, Gillibert, A, et al. CEA, CA19-9, circulating DNA and circulating tumour cell kinetics in patients treated for metastatic colorectal cancer (mCRC). Br J Cancer, 2021, 125: 725-733. [PubMed ID: 34112948] 19. Gherman, A, Balacescu, L, Gheorghe-Cetean, S, et al. Current and New Predictors for Treatment Response in Metastatic Colorectal Cancer. The Role of Circulating miRNAs as Biomarkers. Int J Mol Sci, 2020, 21. [PubMed ID: 32197436] 20. Locker, GY, Hamilton, S, Harris, J, et al. ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. J Clin Oncol, 2006, 24: 5313-27. [PubMed ID: 17060676] 25. 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Claims

1. A method for selecting a subject in need of treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of: (i) determining a value for the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from the subject, (ii) comparing the determined value to a reference value, and (iii) selecting the subject for treatment of the cancer if the determined value is higher than the reference value.

2. An antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for use in the treatment of a cancer in a subject in need thereof, the use comprising: (i) determining a value for the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from the subject, (ii) comparing the determined value to a reference value, and (iii) administering to the subject an effective amount of the ADC if the determined value is higher than the reference value.

3. A method for selecting a subject in need of treatment of a cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of: (i) determining a value for the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from the subject, (ii) comparing the value determined in step (i) to a reference value for the level of expression of the CEACAM5 gene, (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii), (iv) determining the intensity of the expression level of the CEACAM5 protein in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test, (v) comparing the intensity determined in step (iv) to a reference intensity, and (vi) selecting the subject for treatment of the cancer if the intensity determined in step (iv) is higher than the reference intensity.

4. An antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for use in the treatment of a cancer in a subject in need thereof, the use comprising: (i) determining a value for the level of expression of the CEACAM5 gene in a tumor isolated sample obtained from the subject, (ii) comparing the value determined in step (i) to a reference value for the level of expression of the CEACAM5 gene, (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii), (iv) determining the intensity of the expression level of the CEACAM5 protein in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test, (v) comparing the intensity determined in step (iv) to a reference intensity, and (vi) administering to the subject an effective amount of the ADC if the intensity determined in step (iv) is higher than the reference intensity. (vi) if the determined intensity is higher than the reference intensity, administering to the subject an effective amount of the ADC.

5. The method according to claim 1 or 3 or the antibody drug conjugate (ADC) for use according to claim 2 or 4, wherein the value of the CEACAM5 gene expression level is a measure of CEACAM5 gene transcript.

6. The method or antibody drug conjugate (ADC) for use according to claim 3, wherein the CEACAM5 gene transcript is mRNA.

7. A method for selecting a subject in need of treatment of cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody coupled to a cytotoxic agent, the method comprising at least the steps of: (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject, (ii) comparing the value to a reference value, and (iii) selecting the subject for cancer treatment if the determined value is higher than the reference value.

8. An antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody coupled to a cytotoxic agent for use in the treatment of cancer in a subject in need thereof, the use comprising: (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject, (ii) comparing the value to a reference value, and (iii) administering to the subject an effective amount of the ADC if the determined value is higher than the reference value.

9. A method for selecting a subject in need of treatment of cancer with an antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody coupled to a cytotoxic agent, the method comprising at least the steps of: (i) determining a log2-transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject, (ii) comparing the value determined in step (i) to a reference value, (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii), (iv) determining an intensity of CEACAM5 protein expression level in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test, (v) comparing the intensity determined in step (iv) to a reference intensity, and (vi) selecting the subject for cancer treatment if the determined intensity is higher than the reference intensity.

10. An antibody drug conjugate (ADC) comprising an anti-CEACAM5 antibody coupled to a cytotoxic agent for use in the treatment of cancer in a subject in need thereof, the use comprising: (i) determining a log2 transformed, quantile-normalized transcripts per million per kilobase (TPM) value of CEACAM5 mRNA in a tumor isolated sample obtained from the subject, (ii) comparing the value determined in step (i) to a reference value, (iii) selecting the subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined in step (i) is higher than the reference value of step (ii), (iv) determining the intensity of the CEACAM5 protein expression level in a tumor isolated sample obtained from the subject with the CEACAM5 immunohistochemistry (IHC) staining test, (v) comparing the intensity determined in step (iv) to a reference intensity, and (vi) administering to the subject an effective amount of the ADC if the determined intensity is higher than the reference intensity.

11. The method of any one of claims 1, 3, 5-7, and 9 or the antibody drug conjugate (ADC) for use of any one of claims 2, 4-6, 8, and 10, wherein the anti-CEACAM5 antibody comprises a HCDR1 having the amino acid sequence of SEQ ID NO: 1, a HCDR2 having the amino acid sequence of SEQ ID NO: 2, a HCDR3 having the amino acid sequence of SEQ ID NO: 3, a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of NTR, and a LCDR3 having the amino acid sequence of SEQ ID NO:

5.

12. The method of any one of claims 1, 3, 5-7, 9, and 11 or the antibody drug conjugate (ADC) for use of any one of claims 2, 4-6, 8, and 10-11, wherein the cytotoxic agent is a maytansinoid or a maytansinoid analog.

13. The method of any one of claims 7, 9, and 11-12 or the antibody drug conjugate (ADC) for use of any one of claims 8 and 10-12, wherein the reference value is at least about 7 to about 13.

14. The method of any one of claims 7, 9, and 11-13 or the antibody drug conjugate (ADC) for use of any one of claims 8 and 10-13, wherein the reference value is at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 13.

15. The method of any one of claims 7, 9, and 11-14 or the antibody drug conjugate (ADC) for use of any one of claims 8 and 10-13, wherein the quantile normalization is obtained by (i) ranking the transcripts of the sample by expression level, (ii) calculating the mean of genes occupying the same rank, and (iii) replacing the values of all genes occupying the same rank with the mean.

16. The method of any one of claims 7, 9, and 11-15 or the antibody drug conjugate (ADC) for use of any one of claims 8 and 10-15, wherein the expression level of the transcripts is measured in fragments per kilobase per million (FPKM) prior to conversion to TPM.

17. The method or antibody drug conjugate (ADC) for use of claim 16, wherein the fragments per kilobase per million (FPKM) is obtained by counting the total transcripts in the sample, dividing the obtained transcript count by 1,000,000, and dividing the obtained value by the length of the gene in kilobases.

18. The method or antibody drug conjugate (ADC) for use of any one of claims 1-17, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer, cervical cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer, prostate cancer, neuroendocrine cancer, and skin cancer.

19. The method or antibody drug conjugate (ADC) for use of any one of claims 1-18, wherein the ADC is tisotumab vedotin.

20. The method or antibody drug conjugate (ADC) for use of any one of claims 1-19, wherein the administered dose of the ADC is > 80 mg / m2relative to the body surface area of the subject 2 , in particular a dose of 80 mg / m2 2 to 210 mg / m2 2 , 80 mg / m2 2 to 170 mg / m2 2 , or a dose of 80 mg / m2 2 to 150 mg / m2 2 , or a dose of 80 mg / m2 2 to 120 mg / m2 2 , or a dose of 80 mg / m2 2 to 100 mg / m2 2 , and in particular a dose of 80, 100, 120, 150, 170, 180, or 210 mg / m2 2 , about once every two weeks, or the administered dose of the ADC is > 80 mg / m2relative to the body surface area of the subject 2 , about once every three weeks.

21. The method or antibody drug conjugate (ADC) for use of any one of claims 2, 4-6, and 8, 10-16, further comprising administering to the subject an effective amount of at least one additional agent effective to treat the cancer.

22. The method or antibody drug conjugate (ADC) for use of claim 21, wherein the additional agent is selected from the group consisting of an immune checkpoint inhibitor (ICI), particularly an anti-PD-1 antibody or an anti-PD-L1 antibody, a platinum-based chemotherapy, pemetrexed, an anti-VEGFR2, FOLFOX, FOLFIRI, TAS-102, an anti-EGFR, and any combination thereof.

23. The method or antibody drug conjugate (ADC) for use of claim 22, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sintilimab, dostarlimab, and tiragolumab, or wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

24. The method or antibody drug conjugate (ADC) for use of claim 23, comprising administering to the subject an effective amount of tisotumab vedotin and pembrolizumab.

25. The method or antibody drug conjugate (ADC) for use of claim 22, further comprising administering to the subject an effective amount of a platinum-based chemotherapy.

26. The method or antibody drug conjugate (ADC) for use of claim 25, wherein the platinum-based chemotherapy is selected from cisplatin and carboplatin.

27. The method or antibody drug conjugate (ADC) for use of claim 25 or 26, further comprising administering to the subject an effective amount of pemetrexed.

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