CEACAM5 mRNA assay for patient selection in cancer treatment

Abstract

This disclosure relates to a method and use for selecting and treating patients having cancer expressing CEACAM5. The method includes (ii) determining a log2-converted and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA in an isolated tumor sample obtained from a subject, (ii) comparing the value to a reference value, and (iii) selecting the subject for treatment of the cancer if the determined value is greater than the reference value. The agent for treating the selected subject may be tusamitamaburabutansine. Such an agent may be used in combination with one or more additional agents for treating the cancer. In certain embodiments, the cancer is non-squamous non-small cell lung cancer (NSQ NSCLC).

Description

[Technical Field]

[0001] References to sequence listings This application includes a sequence listing submitted electronically in XML format, the entirety of which is incorporated herein by reference. The .xml copy prepared on 12 March 2024 is named PI022916.WO SANOFI(S327)LISTING.xml.

[0002] This disclosure relates to the field of cancer treatment. [Background technology]

[0003] Despite recent advances in cancer treatment, there is still a need for new treatments that are effective when disease progresses after first-line therapy. Current therapeutic approaches for subsequent systemic options, combining angiogenesis inhibitors with systemic cytotoxic agents such as docetaxel, are associated with serious hematological and other toxicities. Docetaxel, pemetrexed, or gemcitabine, used as single cytotoxic agents, offer very limited alternatives. Therefore, targeted cytotoxic therapy may offer improved safety, tolerability, and efficacy.

[0004] Lung cancer is the 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% have distant metastases.[2] The associated 5-year relative survival rate is poor, between 5 and 8%.[3]

[0005] While some lung cancers respond to therapies targeting various oncogene drivers[4] or immune checkpoint inhibitors (ICIs)[5], there is a continuing unmet need for novel therapies for patients whose lung cancer has no viable targets or who do not respond to ICIs.

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

[0007] Different CEACAMs exhibit specific expression patterns across tissues, yet they show similarities in terms of structural homology and amino acid sequence [7]. This necessitates that molecules targeting CEACAMs must specifically bind to individual CEACAMs to limit cross-reactivity and dose-limiting toxicity.

[0008] CEACAM5 was first described in 1965 as a tumor-associated antigen in human colon cancer tissue extracts (Gold P et al., J Exp Med. 1965;122(3):467-481). Subsequently, high levels of CEACAM5 expression have been observed in some epithelial tumors, but in normal adult tissues, its expression is limited to a 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] and promotes tumorigenesis and metastasis[9]. CEACAM5 is expressed at higher levels in NSCLC but not in normal lung tissue[7,10]. Furthermore, higher expression of CEACAM5 in human NSCLC tissue correlates with a worse histological grade

[10] , and higher expression of CEACAM5 is associated with poor survival 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 outcomes in lung cancer patients

[13] . Tusamitamablubutansin (SAR408701) is a potential first-in-class ADC that selectively targets CEACAM5-expressing tumor cells without binding to CEACAM1, 6, or 8 glycoproteins [7]. It consists of an anti-CEACAM5 humanized monoclonal antibody conjugated to a potent cytotoxic maytansinoid N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansin (DM4) payload via a cleavable linker [7]. The antibody component of tusamitamablubutansin binds to the extracellular domain of CEACAM5, followed by internalization of the ADC and subsequent release of DM4 into tumor cells

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

[14] . Both DM4 and its active metabolite, S-methyl-DM4, can cross the cell membrane, meaning that the cytotoxic effect of tusamitamabrabutansine is both direct, via specific binding to CEACAM5-expressing tumor cells, and indirect, via local diffusion of DM4 and S-methyl-DM4 (bystander effect)

[14] .

[0011] The promising preliminary antitumor activity of tusamitamablubutansine in heavily pre-treated participants with NSQ NSCLC has been demonstrated in an ongoing study (TED13751).

[0012] In the first-in-human phase 1 / 2 trial of tusamitamablubutansine among patients with advanced solid tumors for whom standard alternative therapies were unavailable, the primary dose-limiting toxicity at the dose-escalation stage (ClinicalTrials.gov NCT02187848) was reversible keratopathy, and the maximum tolerated dose was 100 mg / m2 every two weeks

[15] .

[0013] In the expanded phase of the same trial among patients with advanced NSQ-NSCLC expressing high levels of CEACAM5, defined as at least 50% of tumor cells having a staining intensity of 2+ or 3+ by immunohistochemistry (IHC), tusamitamablutansine demonstrated a promising objective response rate of 20.3% and a favorable safety profile. The most common adverse events reported under treatment were asthenia, reversible corneal events, peripheral neuropathy, dyspnea, and diarrhea, with hematological toxicity being rarer compared to that reported with docetaxel

[16] ; in addition, 47% of patients who achieved a partial response were treated for more than one year, suggesting that the response to tusamitamablutansine was permanent and often sustained

[17] . [Overview of the project] [Problems that the invention aims to solve]

[0014] While these clinical trial data show promise in clinical practice, potential barriers to pre-screening patients with cancers likely to respond to CEACAM5-targeted therapy, such as NSQ-NSCLC, include the availability of stored tumor biopsies for measuring CEACAM5 expression by immunohistochemistry.

[0015] Based on the aforementioned Phase 1 / 2 trial, the inventors explored the correlation between biomarkers: 1) tumor CEACAM5 mRNA levels and tumor CEACAM5 expression as determined by immunohistochemistry; and 2) whether CEACAM5 mRNA predicted the objective response rate of tumors. [Means for solving the problem]

[0016] As shown in the examples, clinical response enrichment was observed in patients with high CEACAM5 protein expression levels who were treated with tusamitamabrabutansine (tusamitamabrabutansine responders).

[0017] Within this disclosure, the expression “enrichment of clinical response in patients” with respect to treatment with tusamitamabrabutansine is intended to refer to an increase in the overall response rate (ORR) after administration of the given treatment. ORR is defined as the proportion of patients having a partial or complete response to treatment; it does not include stable disease and is a direct measure of drug-induced tumor activity.

[0018] Furthermore, a correlation was found between CEACAM5 protein expression levels (measured by immunohistochemical (IHC) staining) and CEACAM5 tumor mRNA levels in tumors. CEACAM5 mRNA expression was significantly upregulated in patients with high CEACAM5 protein expression versus moderate CEACAM5 protein expression. In addition, enrichment of clinical responses was observed in tussami tamaburabutansin responders in patients with high CEACAM5 tumor mRNA levels and high CEACAM5 immunohistochemical (IHC) staining (i.e., intensity of 2+ or higher in more than 50% of tumor cells).

[0019] High CEACAM5 protein expression was associated with enriched clinical response, and a correlation was demonstrated between CEACAM5 protein expression and mRNA CEACAM5 expression levels. Therefore, mRNA CEACAM5 expression levels are a good biomarker for predicting enriched clinical response in patients treated with anti-CEACAM5 antibody-drug conjugates, such as tusamitamablubutansine; or the results suggest that patients may be selected based on CEACAM5 mRNA levels instead of IHC.

[0020] Therefore, the Examples section supports the use of CEACAM5 mRNA levels as a biomarker for selecting and treating patients requiring cancer treatment with antibody-drug conjugates (ADCs) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent.

[0021] Furthermore, the Examples section supports the use of CEACAM5 mRNA levels as a biomarker for selecting patients requiring cancer treatment with antibody-drug conjugates (ADCs) containing anti-CEACAM5 antibodies conjugated to cytotoxic agents, for further selection by CEACAM5 immunohistochemistry (IHC) staining and subsequent cancer treatment with ADCs.

[0022] It is common knowledge to those skilled in the art that protein expression levels do not necessarily correlate with the expression levels of the corresponding mRNAs.

[0023] This disclosure is at least in part based on the observation that specific values ​​of log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) values ​​for CEACAM5 gene expression levels, e.g., CEACAM5 gene transcript levels, e.g., CEACAM5 mRNA levels, e.g., CEACAM5 mRNA, may be useful as biomarkers for selecting patients for cancer treatment who typically express CEA cell adhesion molecule 5 (CEACAM5) on their tumor cells.

[0024] In some embodiments, the present disclosure relates to a method for selecting a target for cancer treatment using an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the determined value with a reference value, (iii) If the determined value exceeds the reference value, the step of selecting the subject for cancer treatment. Methods that include at least

[0025] In this disclosure, the subject requiring this is a subject having cancer. Isolated samples of the tumor of the cancer may be used.

[0026] In this specification, the terms “subject,” “patient,” “subject in need,” and “patient in need” are used interchangeably.

[0027] In some embodiments, the present disclosure relates to a method for selecting a target for cancer treatment using an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value for the CEACAM5 gene expression level, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the intensity determined in step (iv) exceeds the reference intensity, the step of selecting the target for cancer treatment. Methods that include at least

[0028] The tumor samples used in steps (i) and (iv) may be the same sample or different samples.

[0029] In some embodiments, the present disclosure relates to a method for selecting and treating subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the determined value with a reference value, (iii) If the determined value exceeds the reference value, the step of selecting the subject for cancer treatment, and (iv) The step of administering an effective amount of the ADC to the selected subject. Methods that include at least

[0030] In some embodiments, the present disclosure relates to a method for selecting and treating subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value for the CEACAM5 gene expression level, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, (vi) If the intensity determined in step (iv) exceeds the reference intensity, the step of selecting the subject for cancer treatment, and (vii) The step of administering an effective amount of the ADC to the selected subject. Methods that include at least

[0031] In some embodiments, the present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for use in the treatment of cancer in subjects requiring such treatment. Use (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the determined value with a reference value, (iii) If the determined value exceeds the reference value, the step of administering an effective amount of the ADC to the subject. Includes.

[0032] In some embodiments, the present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for use in the treatment of cancer in subjects requiring such treatment. Use (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value for the CEACAM5 gene expression level, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the determined strength exceeds the reference strength, administer an effective amount of the ADC to the subject. Includes.

[0033] In some embodiments, the present disclosure relates to the use of measured values ​​of CEACAM5 gene expression levels in isolated tumor samples obtained from subjects requiring such expression, in order to characterize the tumor as a CEACAM5-highly expressing tumor.

[0034] In some embodiments, the present disclosure relates to the use of a measure of CEACAM5 gene expression levels in isolated tumor samples obtained from said subjects requiring CEACAM5 immunohistochemical (IHC) staining for the selection of subjects.

[0035] In some embodiments, the present disclosure relates to the use of measured values ​​of CEACAM5 gene expression levels in isolated tumor samples obtained from subjects requiring treatment with CEACAM5-targeted therapeutic agents, such as antibody-drug conjugates (ADCs) containing anti-CEACAM5 antibodies conjugated to cytotoxic agents.

[0036] In some embodiments, the present disclosure relates to the use of measured values ​​of CEACAM5 gene expression levels in tumor isolation samples obtained from subjects requiring CEACAM5 immunohistochemical (IHC) staining for the selection of subjects for a CEACAM5 immunohistochemical (IHC) staining test, wherein the staining test is for the selection of subjects for treatment of cancer with a CEACAM5-targeted therapeutic agent, such as an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent.

[0037] In some embodiments, the value of the CEACAM5 gene expression level may be a measure of the CEACAM5 gene transcript.

[0038] In some embodiments, the CEACAM5 gene transcript may be mRNA.

[0039] In some embodiments, the methods and uses of the present disclosure include the step of determining the value of CEACAM5 mRNA levels in an isolated tumor sample.

[0040] According to one of its objectives, this disclosure relates to a method for selecting subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent. (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the above value with a reference value, and (iii) If the determined value exceeds the reference value, the step of selecting the subject for cancer treatment. Methods that include at least

[0041] In some embodiments, the value of the CEACAM5 gene expression level may be a measure of the CEACAM5 gene transcript.

[0042] In some embodiments, the CEACAM5 gene transcript may be mRNA.

[0043] According to one of its objectives, this disclosure relates to a method for selecting subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent. (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the determined intensity exceeds the reference intensity, the step of selecting the target for cancer treatment. Methods that include at least

[0044] In another purpose, the present disclosure relates to a method for selecting and treating subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent. (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the above value with a reference value, (iii) If the determined value exceeds the reference value, the step of selecting the subject for cancer treatment, and (iv) The step of administering an effective amount of the ADC to the selected subject. Methods that include at least

[0045] This allows us to treat cancer.

[0046] In another purpose, the present disclosure relates to a method for selecting and treating subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent. (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, (vi) If the determined intensity exceeds the reference intensity, the step of selecting the subject for cancer treatment, and (vii) The step of administering an effective amount of the ADC to the selected subject. Methods that include at least

[0047] In another object, 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, the use comprising (i) determining a log2-converted and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA in an isolated tumor sample obtained from the subject, (ii) comparing the value thereof to a reference value, and (iii) if the determined value exceeds the reference value, administering an effective amount of the ADC to the subject.

[0048] For another purpose, this 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 subjects where it is needed. Use (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the determined intensity exceeds the reference intensity, administer an effective amount of the ADC to the subject. Includes.

[0049] As shown in the Examples section, ribonucleic acid (RNA) sequencing was used to evaluate the expression of approximately 15,000 genes, including CEACAM5. RNA data were filtered and defined as log-transformed quantile-normalized TPM (transcripts per kilobase million). Differential gene expression analysis confirmed that CEACAM5 mRNA was the gene most associated with high versus moderate CEACAM5 expression by IHC (and the only significant adjusted P=0.00265). Parts per million of CEACAM5 mRNA transcripts were elevated in high versus moderate CEACAM5 expression; and (C)CEACAM5 mRNA correlated with the CEACAM5 IHC H score. Therefore, the gene expression levels of CEACAM5 (RNA) can be used to identify and select patients responsive to CEACAM5-targeted treatments, such as antibody-drug conjugates, including anti-CEACAM5 antibodies conjugated to cytotoxic agents.

[0050] In this disclosure, the terms "antibody-drug conjugate containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent," "antibody-drug conjugate," and "ADC" are used interchangeably.

[0051] In some embodiments, the ADC may be used in an effective amount.

[0052] In some embodiments, use may be in an object that requires it.

[0053] In the uses disclosed herein, the biomarker CEACAM5 mRNA is measured in isolated biological samples.

[0054] In some embodiments, the anti-CEACAM5 antibody may include HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, HCDR3 having the amino acid sequence of SEQ ID NO: 3, LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence NTR, and LCDR3 having the amino acid sequence of SEQ ID NO: 5.

[0055] In some embodiments, the anti-CEACAM5 antibody may include a variable heavy chain domain (VH) consisting of SEQ ID NO: 6 and a variable light chain domain (VL) consisting of SEQ ID NO: 7.

[0056] In some embodiments, the anti-CEACAM5 antibody may be tusamitamab.

[0057] In some embodiments, the cytotoxic agent may be selected from the group consisting of radioisotopes, protein toxins, low molecular weight toxins, and combinations thereof.

[0058] In some embodiments, the low molecular weight toxin may be selected from antimetabolites, DNA alkylating agents, DNA crosslinking agents, DNA intercalating agents, antimicrotubule agents, topoisomerase inhibitors, and combinations thereof.

[0059] In certain embodiments, the antimicrotubule agent is selected from taxanes, vinca alkaloids, maytansinoids, colchicine, podophyllotoxin, griseofulvin, and any combination thereof.

[0060] In some embodiments, the cytotoxic agent may be a meitansinoid or a meitansinoid analog.

[0061] In some embodiments, the maytansinoid may be selected from N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) or N2'-deacetyl-N2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4) and combinations thereof.

[0062] In some embodiments, an anti-CEACAM5 antibody may be covalently bound to at least one chemotherapeutic agent via a cleavable or non-cleavable linker.

[0063] In some embodiments, the linker may be selected from N-succinimidylpyridyl dithiobutyrate (SPDB), 4-(-iridin-2-yldisulfanyl)-2-sulfobutyrate (sulfo-SPDB), and succinimidyl (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0064] In some embodiments, an anti-CEACAM5 antibody may be covalently bound to at least one chemotherapeutic agent via a cleavable linker, the linker being selected from N-succinimidylpyridyl dithiobutyrate (SPDB), 4-(pyridine-2-yldisulfanyl)-2-sulfobutyrate (sulfo-SPDB), and succinimidyl (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0065] In some embodiments, the CEACAM5 antibody may comprise a heavy chain (VH) consisting of SEQ ID NO: 8 and a light chain (VL) consisting of SEQ ID NO: 9 (huMAb2-3), which are covalently bound to N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4) via N-succinimidylpyridyl dithiobutyrate (SPDB).

[0066] In certain embodiments, the antibody-drug conjugate is characterized by a drug-antibody ratio (DAR) in the range of 1 to 10.

[0067] In some embodiments, the antibody-drug conjugate may be tusamitamabrabutansine.

[0068] In some embodiments, the reference value may be a log2-transformed, quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA.

[0069] In some embodiments, the reference value may be at least about 7 to about 13.

[0070] In some embodiments, the reference value may be 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.

[0071] In some embodiments, quantile normalization can be achieved by (i) ranking the transcripts of a sample by their expression levels, (ii) calculating the mean value for genes of the same rank, and (iii) replacing the values ​​of all genes of the same rank with this mean value.

[0072] In some embodiments, the expression level of the transcript may be measured in FPKM (Fragments Per Kilobase Million) and then converted to TPM.

[0073] In some embodiments, FPKM (Fragments Per Kilobase Million) can be obtained by counting all transcripts in the sample, dividing the resulting transcript count by 1,000,000, and then dividing the resulting value by the gene length in kilobases.

[0074] In some embodiments, cancer may be selected from the group consisting of hepatocellular carcinoma, colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ) cancer, esophageal cancer, lung cancer, cervical cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer (e.g., bile duct cancer), prostate cancer, neuroendocrine cancer, and skin cancer.

[0075] In some embodiments, cancer may be selected from the group consisting of colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ) cancer, esophageal cancer, lung cancer, cervical cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer (e.g., bile duct cancer), prostate cancer, neuroendocrine cancer, and skin cancer.

[0076] In some embodiments, the cancer may be selected from colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, pancreatic cancer, and lung cancer.

[0077] In some embodiments, the cancer is gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, or esophageal cancer.

[0078] In some embodiments, the cancer may be gastric cancer.

[0079] In some embodiments, the cancer may be colorectal cancer.

[0080] In some embodiments, the cancer may be pancreatic cancer.

[0081] The cancer could be gastroesophageal junction adenocarcinoma (GEJ).

[0082] In some embodiments, the cancer may be lung cancer.

[0083] In some embodiments, the lung cancer may be non-squamous non-small cell lung cancer (NSQ NSCLC).

[0084] In some embodiments, non-squamous non-small cell lung cancer may be advanced or metastatic NSQ NSCLC.

[0085] In certain embodiments, non-squamous non-small cell lung cancer does not have epidermal growth factor receptor (EGFR) sensitization mutations, v-raf mouse sarcoma virus oncogene homolog B1 (BRAF) mutations, or anaplastic lymphoma kinase / c-ros oncogene 1 (ALK / ROS) modifications.

[0086] In some embodiments, the anti-CEACAM5 antibody may be tusamitamab.

[0087] In some embodiments, the ADC may be tusamitamaburabutancin.

[0088] In some embodiments, the ADC is 80 mg / m² relative to the surface area of ​​the target. 2 Even when administered at the above dose approximately once every two weeks, or when ADC is 80 mg / m² relative to the surface area of ​​the subject,2 It may be administered once every about three weeks at the above dosage.

[0089] In some embodiments, the ADC is 80 mg / m 2 ~210 mg / m 2 、80 mg / m 2 ~170 mg / m 2 of dosage, or 80 mg / m 2 ~150 mg / m 2 of dosage, or 80 mg / m 2 ~120 mg / m 2 of dosage, or 80 mg / m 2 ~100 mg / m 2 and may be administered at a dosage of.

[0090] In some embodiments, the ADC can be administered at a dosage of 80, 100, 120, 150, 170, 180 or 210 mg / m 2 and may be administered at a dosage of.

[0091] In some embodiments, the methods and uses described herein may further comprise administering to the subject an effective amount of at least one additional agent effective to treat cancer.

[0092] In some embodiments, the additional agent may be selected from the group consisting of immune checkpoint inhibitors (ICIs), platinum-based chemotherapy, pemetrexed, anti-VEGFR2, FOLFOX, FOLFIRI, TAS-102, anti-EGFR and any combination thereof.

[0093] In some embodiments, the ICI can be an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0094] In some embodiments, the anti-PD-1 antibody may be selected from the group consisting of pembrolizumab, nivolumab, semipramab, sintilimab, dostarlimab and tislelizumab.

[0095] In some embodiments, the anti-PD-L1 antibody may be selected from the group consisting of atezolizumab, avelumab and durvalumab.

[0096] In some embodiments, the subjects are effective amounts of tusamitamabrabutansine and pembrolizumab.

[0097] In some embodiments, the methods or uses described herein may further include administering an effective amount of platinum-based chemotherapy to a target.

[0098] In some embodiments, platinum-based chemotherapy may be selected from cisplatin and carboplatin.

[0099] In some embodiments, the methods or uses described herein may further include administering an effective amount of pemetrexed to a target. [Brief explanation of the drawing]

[0100] [Figure 1] This shows that the parts per million of CEACAM5 mRNA transcript is elevated in high CEACAM5 expression versus moderate CEACAM5 expression. [Figure 2] This shows the correlation between CEACAM5 mRNA and the CEACAM5 IHC H score. [Figure 3] The CEACAM5 mRNA levels correlate with CEACAM5 expression as recorded by immunohistochemistry, specifically as the sum of the percentages of tumor cells expressing the target at an intensity of at least 2+ in response to tusamitamabrabutansine treatment. [Figure 4] The CEACAM5 mRNA levels correlate with immunohistochemical CEACAM5 expression, recorded as the sum of the percentages of tumor cells expressing the target at least 2+ intensity in patients who responded to tusamitamabrabutansine treatment. [Figure 5] This shows a box plot of CEACAM family member expression based on clinical response in patients treated with tusamitamabrabutansine. [Modes for carrying out the invention]

[0101] A brief explanation of arrays Sequence IDs 1-5 show the sequences CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 of the anti-CEACAM5 antibody (huMAb2-3).

[0102] Sequence ID 6 shows the sequence of the variable domain (VH) of the heavy chain of the anti-CEACAM5 antibody (huMAb2-3).

[0103] Sequence ID 7 shows the sequence of the variable domain (VL) of the light chain of the anti-CEACAM5 antibody (huMAb2-3).

[0104] Sequence ID 8 shows the heavy chain sequence of the anti-CEACAM5 antibody (huMAb2-3).

[0105] Sequence ID 9 shows the light chain sequence of the anti-CEACAM5 antibody (huMAb2-3).

[0106] definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled 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 may provide those skilled in the art with a general dictionary of many of the terms used in this disclosure. Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used in the practice or testing of this disclosure. In the event of any conflict, this specification, including definitions, shall prevail. In general, the terminology used in relation to and for the cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical chemistry and medicinal chemistry, and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art. Enzyme reactions and purification techniques are performed as commonly practiced in the art or as described herein, in accordance with the manufacturer's specifications. Furthermore, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include singular terms.

[0107] Units, prefixes, and symbols are given in their International System of Units (SI) approved forms. Numerical ranges include the number defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino-carboxyl direction. The headings provided herein are not limitations on the various aspects of this disclosure. Thus, terms defined immediately thereafter are defined in more detail by referring to this specification as a whole.

[0108] All publications and other references mentioned herein are invoked by reference as a whole. While many sources are cited herein, such citations do not constitute an acknowledgment that any of those sources constitute common technical knowledge in the art.

[0109] Throughout this specification and its embodiments, variations of the words “have” and “comprise,” or “has,” “having,” “comprises,” or “comprises,” will be understood to mean that they encompass the specified integer or group of integers, but do not exclude any other integer or group of integers. Whenever an aspect is described herein in the language of “comprise,” it will be understood that other similar aspects are also provided, described in the terms “consisting of” and / or “essentially consisting of.”

[0110] It should be noted that the terms “one (a)” or “one (an)” entity refer to one or more of those entities; for example, “nucleotide sequence” is understood to represent one or more nucleotide sequences. Thus, the terms “one (a)” (or “one (an)”), “one or more” and “at least one” can be used interchangeably herein.

[0111] Furthermore, when used herein, “and / or” should be interpreted as the specific disclosure of each of two particular features or components that have or do not have the other. Thus, the term “and / or” as used in phrases such as “A and / or B” is intended herein to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include each of the following situations: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0112] The terms “approximately” or “about” are used herein to mean roughly, roughly, around, within or within a range. When the term “about” is used with a numerical range, it modifies that range by extending the boundary above and below the indicated number. Generally, the term “about” can modify a number above and below a stated value by, for example, a difference of 10 percent above or below (higher or lower). In some embodiments, the term indicates 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 number. In some embodiments, “about” indicates a deviation of ±10% from the indicated number. In some embodiments, “about” indicates a deviation of ±5% from the indicated number. In some embodiments, “about” indicates a deviation of ±4% from the indicated number. In some embodiments, "approximately" indicates a deviation of ±3% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±2% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±1% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.9% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.8% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.7% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.6% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.5% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.4% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.3% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.1% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.05% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.01% from the indicated value.

[0113] An antibody can be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by disulfide bonds. There are two types of light chains: lambda(l) and kappa(k). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chain contains two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively called CH). The variable regions of both the light (VL) and heavy (VH) chains determine the binding recognition and specificity to the antigen. The constant domains of the light chain (CL) and heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental migration, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of immunoglobulins and consists of a variable region of one light chain and one heavy chain. Antibody specificity lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is mainly composed of residues derived from the hypervariable or complementarity-determining region (CDR). Occasionally, residues derived from the non-hypervariable region or framework region (FR) affect the entire domain structure and therefore the binding site. Thus, the complementarity-determining region or CDR refers to the amino acid sequence that together defines the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of immunoglobulins each contain three CDRs, called CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, and CDR3-H, respectively. Therefore, a conventional antibody-antigen binding site contains six CDRs, including sets of CDRs from the heavy chain V region and the light chain V region, respectively.

[0114] As used herein, the term “antibody” is intended to refer to conventional antibodies and their fragments, as well as single-domain antibodies and their fragments, in particular the variable heavy chains of single-domain antibodies, and chimeric, humanized, bispecific, or multispecific antibodies. The antibody fragments considered herein are antigen-binding fragments.

[0115] The "framework region" (FR) refers to the amino acid sequence interposed between CDRs, i.e., the relatively conserved portions of the immunoglobulin light chain variable region and immunoglobulin heavy chain variable region across different immunoglobulins of the same species. The light and heavy chains of immunoglobulins each have four FRs, called FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively. Human framework regions are substantially identical (approximately 85% or more, particularly 90%, 95%, 97%, 99%, or 100%) to the framework regions of naturally occurring human antibodies.

[0116] In the context of this disclosure, the definition of CDR / FR in immunoglobulin light chains or heavy chains should be determined based on the definition by IMGT (Lefranc et al. Dev.Comp.Immunol., 2003, 27(1):55-77; www.imgt.org).

[0117] As used herein, antibodies or immunoglobulins also include "single-domain antibodies," which are antibodies whose complementarity-determining region is part of a single-domain polypeptide, as described more recently. Examples of single-domain antibodies include heavy-chain antibodies, antibodies that naturally lack a light chain, single-domain antibodies derived from conventional four-chain antibodies, and engineered single-domain antibodies. Single-domain antibodies may be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. Single-domain antibodies may be naturally occurring single-domain antibodies known as light-chain-lacking heavy-chain antibodies. In particular, camelidae species, such as camels, dromedaries, llamas, alpacas, and guanacos, produce light-chain-lacking heavy-chain antibodies. Camel heavy-chain antibodies also lack the CH1 domain.

[0118] The variable heavy chains of these single-domain antibodies lacking light chains are known in the art as "VHH" or "Nanobody®". Similar to conventional VH domains, VHH contains four FRs and three CDRs. VHH offers advantages over conventional antibodies. They are approximately 10 times smaller than IgG molecules, and as a result, well-folded functional VHH can be produced by in vitro expression with high yields. Furthermore, VHH is highly stable and resistant to protease action. The characterization and production of VHH are outlined in Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov;77(1):13-22).

[0119] As used herein, the terms “monoclonal antibody” or “mAb” refer to an antibody molecule with a single amino acid sequence against a specific antigen and should not be interpreted as requiring antibody production by any particular method. Monoclonal antibodies can be produced by a single clone of a B cell or hybridoma, but they can also be produced by recombinants, i.e., protein engineering.

[0120] The term "humanized antibody" refers to an antibody that is entirely or partially of non-human origin and has been modified to evade or minimize the immune response in humans, particularly by substituting specific amino acids in the framework regions of the VH and VL domains. The constant domains of humanized antibodies are, in most cases, the human CH and CL domains.

[0121] A (conventional) antibody "fragment" is a part of an intact antibody, particularly the antigen-binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. Conventional antibody fragments can also be heavy chain antibodies or single-domain antibodies such as VHH.

[0122] The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, in which approximately half of the N-terminal side of the heavy chain and the entire light chain are linked by disulfide bonds. It is typically obtained by treating IgG with a protease such as papain.

[0123] The term "F(ab')2" refers to an antibody fragment with a molecular weight of approximately 100,000 and slightly greater antigen-binding activity than two identical Fab fragments linked via a disulfide bond in the hinge region. This is typically obtained by treating IgG with a protease such as pepsin.

[0124] The term "Fab'" refers to an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity, obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0125] A single-stranded Fv ("scFv") polypeptide is a VH::VL heterodimer typically expressed from a gene fusion containing VH and VL encoding genes linked by a covalently bonded peptide-encoding linker. The human scFv fragments of this disclosure contain a CDR maintained in an appropriate conformation, particularly by using recombination techniques. Divalent and multivalent antibody fragments can be spontaneously formed by the association of monovalent scFv or produced by coupling monovalent scFv with a peptide linker such as divalent sc(Fv)2. "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" indicates two dsFv coupled by a peptide linker.

[0126] The term "bispecific antibody" or "BsAb" refers to an antibody that combines the antigen-binding sites of two antibodies within a single molecule. Therefore, a BsAb can bind to two different antigens simultaneously. Genetic engineering is increasingly used to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions, as described, for example, in European Patent Application Publication No. 2050 764A1.

[0127] The term "multispecific antibody" refers to an antibody that combines two or more antibody antigen-binding sites within a single molecule.

[0128] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, which contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with a complementary domain on another chain, creating two antigen-binding sites.

[0129] An amino acid sequence that is "at least 85% identical to the reference sequence" is a sequence that, in its entire length, has sequence identity of 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the full-length reference amino acid sequence.

[0130] The percentage of "sequence identity" between amino acid sequences can be determined by comparing two sequences that are optimally aligned across a comparison window. The portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences, obtaining the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for comparison is performed by global pairwise alignment, for example, using the algorithm in Needleman and Wunsch J.Mol.Biol.48:443 (1970). The percentage of sequence identity can be easily determined, for example, using the program Needle with the BLOSUM62 matrix and the following parameters: gap-open=10, gap-extend=0.5.

[0131] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge, size, or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. Examples of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitutions can also be defined based on amino acid size.

[0132] "Purified" and "isolated," when referring to polypeptides (i.e., the antibodies of this disclosure) or nucleotide sequences, mean that the indicated molecule exists in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" specifically means that at least 75%, 85%, 95%, or 98% (by weight) of the same type of biological macromolecule is present. An "isolated" nucleic acid molecule encoding a particular polypeptide means a nucleic acid molecule that substantially does not contain other nucleic acid molecules that do not encode the polypeptide of interest; however, the molecule may contain several additional bases or parts that do not adversely affect the fundamental characteristics of the composition.

[0133] As used herein, the terms “subject” or “patient” mean mammals such as rodents, cats, dogs, and primates. In particular, the subject as disclosed herein is human.

[0134] As used herein, “administer” or “to administer” means to deliver the compositions described herein, for example, lipid nanoparticles, to a target. The compositions may be administered to a target using methods known in the art. In particular, the compositions may be administered intravenously, subcutaneously, intramuscularly, intradermally, or via any mucosal surface, for example, orally, sublingually, phalanxally, transnasally, rectally, transvaginally, or via the pulmonary route. In some embodiments, the administration is intravenous. In some embodiments, the administration is subcutaneous.

[0135] In this specification, the terms “to treat” or “treatment” or “therapy” mean the administration or consumption of any composition disclosed herein for the purpose of curing, healing, alleviating, reducing, altering, correcting, improving, enhancing, or influencing the symptoms of a disorder or condition, or for the purpose of preventing or delaying the onset of symptoms or complications, or otherwise stopping or inhibiting the further onset of the disorder in a statistically significant manner. More specifically, “to treat” or “treatment” includes any approach to obtain a beneficial or desired outcome in the cancerous condition of interest. Beneficial or desired clinical outcomes may include, but are not limited to, the reduction or improvement of one or more cancerous symptoms or conditions, the reduction or decrease in the severity of the cancerous disease or cancerous symptoms, the stabilization of the cancerous disease or cancerous symptoms, i.e., no worsening, the prevention of the spread of the cancerous disease or cancerous symptoms, the delay or delay of the progression of the cancerous disease or cancerous symptoms, whether partial or whole, and whether detectable or undetectable, the improvement or alleviation of the cancerous condition, the reduction of cancerous disease recurrence, and remission. In other words, as used herein, “treatment” includes any cure, improvement, or alleviation of the cancerous disease or symptoms. "Reduction" of symptoms or disease means a decrease in the severity or frequency of the disease or symptoms, or the elimination of the disease or symptoms.

[0136] As used herein, the term “effective dose” refers to the amount that provides a therapeutic benefit in the treatment, prevention, or management of the pathological process under consideration. The specific therapeutically effective dose can be readily determined by a typical healthcare professional and may 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.

[0137] Unit: mg / m 2 "This refers to the amount of the patient's body surface covered per dose (1 m²). 2 This indicates the amount of compound in milligrams per unit. Those skilled in the art are aware of methods for determining the required amount of compound to be administered based on the patient's body surface, which can be calculated based on height and weight.

[0138] The unit "mg / kg" indicates the amount of compound in milligrams per kilogram of body weight administered per dose to a patient. Those skilled in the art are familiar with methods for determining the required amount of compound to be treated based on the patient's body weight.

[0139] For clarity, it should be understood that certain features of the Disclosure, described in relation to separate embodiments, may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the Disclosure, described in relation to a single embodiment, may also be provided separately or in any preferred secondary combination.

[0140] "Biomarker" is intended to refer to a biological molecule, such as a protein or metabolite, that is differentially present, increased, or decreased in a biological sample obtained from a subject or group of subjects having a first phenotype, such as a disease like cancer, compared to a biological sample from a subject or group of subjects having a second phenotype, such as a disease-free subject or group of subjects. In use, the biomarker is isolated from the subject.

[0141] "Sample" or "biological sample" is intended to refer to biological material isolated from a subject. A biological sample may contain any biological material suitable for detecting a biomarker, i.e., CEACAM5 mRNA, and may include cellular and / or non-cellular material from a subject. A sample may be isolated from any suitable biological tissue or body fluid, such as kidney tissue, blood, plasma of blood, serum of blood, urine, or cerebrospinal fluid (CSF). In some embodiments, the biological sample is a plasma or serum sample.

[0142] The term "reference value" or "threshold" is intended to refer to the level of CEACAM5 mRNA indicating a specific disease state, phenotype, e.g., cancer or its absence, or a combination of disease states, phenotypes, or their absence, in the subject concerned.

[0143] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. However, any methods and materials similar to or equivalent to those described herein may also be used in the implementation or testing of this disclosure. All publications referenced herein are incorporated herein by reference to disclose and explain such methods and / or materials in the context in which those publications are cited.

[0144] The following list of suppliers, raw materials, and ingredients, including their combinations and mixtures, is enumerated as is within the scope of this specification.

[0145] It should be understood that any upper limit of any numerical limitation provided throughout this specification encompasses all lower numerical limitations, as those expressly stated herein. Any lower limit of any numerical limitation provided throughout this specification encompasses all higher numerical limitations, as those expressly stated herein. Any numerical range provided throughout this specification encompasses all narrower numerical ranges within such wider ranges, as those expressly stated herein.

[0146] For example, all lists of items, such as a list of raw materials, are intended to be and must be interpreted as Markush groups. Therefore, all lists can be read and interpreted as items "selected from a group consisting of lists of items and combinations and mixtures thereof."

[0147] In this specification, trademark names may be used to refer to components, including various raw materials, used in this disclosure. The inventors do not intend to limit themselves in this specification to any particular trademarked material. In the description herein, materials equivalent to those referenced by trademark names (e.g., those available from different suppliers under different names or reference numbers) may be substituted and used.

[0148] Measurement, methods, and use of CEACAM mRNA According to one of its objectives, this disclosure relates to a method and use for selecting targets that require a CEACAM5-targeted therapeutic agent for treating cancer. The CEACAM5-targeted therapeutic agent may be an anti-CEACAM5 antibody conjugated to a cytotoxic agent.

[0149] The methods and uses of the present disclosure may include a step of determining the amount of CEACAM5 gene expression (gene transcript or RNA) in a tumor sample.

[0150] The methods and uses of the present disclosure may include a step of determining the amount of CEACAM5 gene expression, such as the CEACAM5 mRNA level, in a tumor sample.

[0151] The amount of CEACAM5 gene expression can be expressed as the amount relative to the total gene expression in the tumor sample, the amount relative to the total length of the expressed DNA, or any other known method.

[0152] In some embodiments, the present disclosure relates to a method for selecting a target for cancer treatment using an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the determined value with a reference value, (iii) If the determined value exceeds the reference value, the step of selecting the subject for cancer treatment. A method that includes at least the following.

[0153] In some embodiments, the present disclosure relates to a method for selecting a target for cancer treatment using an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value for the CEACAM5 gene expression level, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the intensity determined in step (iv) exceeds the reference intensity, the step of selecting the target for cancer treatment. Methods that include at least

[0154] The tumor samples used in steps (i) and (iv) may be the same sample or different samples.

[0155] In some embodiments, the present disclosure relates to a method for diagnosing a subject who is eligible for and in need of treatment for cancer using an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the determined value with a reference value, (iii) If the determined value exceeds the reference value, the step of selecting the subject as eligible for cancer treatment. Methods that include at least

[0156] In some embodiments, the present disclosure relates to a method for diagnosing a subject who is eligible for and in need of treatment for cancer using an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value for the CEACAM5 gene expression level, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the intensity determined in step (iv) exceeds the reference intensity, the step of selecting the target for cancer treatment. Methods that include at least

[0157] In some embodiments, the present disclosure relates to a method for selecting and treating subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the determined value with a reference value, (iii) If the determined value exceeds the reference value, the step of selecting the subject for cancer treatment, and (iv) The step of administering an effective amount of the ADC to the selected subject. Methods that include at least

[0158] In some embodiments, the present disclosure relates to a method for selecting and treating subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value for the CEACAM5 gene expression level, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, (vi) If the intensity determined in step (iv) exceeds the reference intensity, the step of selecting the subject for cancer treatment, and (vii) The step of administering an effective amount of the ADC to the selected subject. Methods that include at least

[0159] In some embodiments, the present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for use in the treatment of cancer in subjects requiring such treatment. Use (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the determined value with a reference value, (iii) If the determined value exceeds the reference value, the step of administering an effective amount of the ADC to the subject. Includes.

[0160] In some embodiments, the present disclosure relates to antibody-drug conjugates (ADCs) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for use in the treatment of cancer in subjects requiring such treatment. Use (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value for the CEACAM5 gene expression level, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the determined strength exceeds the reference strength, administer an effective amount of the ADC to the subject. Includes.

[0161] In some embodiments, the present disclosure relates to the use of measured values ​​of CEACAM5 gene expression levels in isolated tumor samples obtained from subjects requiring such expression, in order to characterize the tumor as a CEACAM5-highly expressing tumor.

[0162] In some embodiments, the present disclosure relates to the use of a measure of CEACAM5 gene expression levels in isolated tumor samples obtained from said subjects requiring CEACAM5 immunohistochemical (IHC) staining for the selection of subjects.

[0163] In some embodiments, the present disclosure relates to the use of measured values ​​of CEACAM5 gene expression levels in isolated tumor samples obtained from subjects requiring treatment with CEACAM5-targeted therapeutic agents, such as antibody-drug conjugates (ADCs) containing anti-CEACAM5 antibodies conjugated to cytotoxic agents.

[0164] In some embodiments, the present disclosure relates to the use of measured values ​​of CEACAM5 gene expression levels in isolated tumor samples obtained from subjects requiring treatment with CEACAM5-targeted therapeutic agents, such as antibody-drug conjugates (ADCs) containing anti-CEACAM5 antibodies conjugated to cytotoxic agents.

[0165] In some embodiments, the value of the CEACAM5 gene expression level may be a measure of the CEACAM5 gene transcript.

[0166] In some embodiments, the CEACAM5 gene transcript may be mRNA.

[0167] In some embodiments, the methods and uses of the present disclosure include the step of determining the value of CEACAM5 mRNA levels in an isolated tumor sample.

[0168] In some embodiments, the level of CEACAM5 gene expression may be expressed as a log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value.

[0169] The determined value or level may be compared to a threshold or reference value.

[0170] Deviations from normal levels may indicate a tumor expressing CEACAM5 at levels sufficient to respond to CEACAM5-targeted treatments.

[0171] In the case of CEACAM5 immunohistochemistry (IHC) staining, the determined intensity or protein expression level can be compared to the threshold intensity or reference intensity.

[0172] Deviations from baseline intensity may indicate tumors that express CEACAM5 at levels sufficient to respond to treatments targeting CEACAM5-expressing tumors.

[0173] The methods and uses described herein may be used to characterize subjects requiring treatment that is responsive to CEACAM5, for example, an anti-CEACAM5 antibody conjugated to a cytotoxic agent.

[0174] The methods and uses described herein may be used to select subjects that are responsive to treatment targeting CEACAM5, for example, an anti-CEACAM5 antibody conjugated to a cytotoxic agent.

[0175] The methods and uses described herein may be used to monitor the response of subjects requiring treatment targeting CEACAM5, for example, an anti-CEACAM5 antibody conjugated to a cytotoxic agent.

[0176] The methods and uses described herein may be for selecting a treatment that targets CEACAM5, for example, an anti-CEACAM5 antibody conjugated to a cytotoxic agent, according to the level of CEACAM5 gene expression in the target tumor requiring it.

[0177] The methods and uses described herein may be for selecting a treatment that targets CEACAM5, for example, an anti-CEACAM5 antibody conjugated to a cytotoxic agent, according to the CEACAM5 protein expression level in the target tumor requiring it.

[0178] The methods and uses described herein may be used to characterize tumors in subjects requiring responsiveness to treatment targeting CEACAM5, for example, an anti-CEACAM5 antibody conjugated to a cytotoxic agent.

[0179] The methods and uses disclosed herein are performed on isolated biological specimens. The isolated specimens may be specimens isolated from tumors. The specimens are isolated before performing the methods and uses disclosed herein.

[0180] The methods and uses described herein shall be performed in vitro.

[0181] According to one of its objectives, this disclosure relates to a method for selecting subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent. (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the above value with a reference value, and (iii) If the determined value exceeds the reference value, the step of selecting the subject for cancer treatment. Methods that include at least

[0182] According to one of its objectives, this disclosure relates to a method for selecting subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent. (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the determined intensity exceeds the reference intensity, the step of selecting the target for cancer treatment. Methods that include at least

[0183] According to one of its purposes, this disclosure relates to a method for diagnosing a subject who is eligible for and in need of treatment for cancer using an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the above value with a reference value, and (iii) If the determined value exceeds the reference value, the step of selecting the subject as eligible for cancer treatment. Methods that include at least

[0184] According to one of its purposes, this disclosure relates to a method for diagnosing a subject who is eligible for and in need of treatment for cancer using an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the determined intensity exceeds the reference intensity, the step of selecting the target for cancer treatment. Methods that include at least

[0185] In another purpose, the present disclosure relates to a method for selecting and treating subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent. (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the above value with a reference value, (iii) If the determined value exceeds the reference value, the step of selecting the subject for cancer treatment, and (iv) The step of administering an effective amount of the ADC to the selected subject. Methods that include at least

[0186] In another purpose, the present disclosure relates to a method for selecting and treating subjects requiring an antibody-drug conjugate (ADC) for the treatment of cancer, comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent. (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, (vi) If the determined intensity exceeds the reference intensity, the step of selecting the subject for cancer treatment, and (vii) The step of administering an effective amount of the ADC to the selected subject. Methods that include at least

[0187] For another purpose, this 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 subjects requiring such treatment. Use includes (i) determining a log2-converted and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA in an isolated tumor sample obtained from the subject, (ii) comparing the value with a reference value, and (iii) administering an effective amount of the ADC to the subject if the determined value exceeds the reference value.

[0188] For another purpose, this 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 subjects requiring such treatment. Use (i) A step of determining the log2-converted, quantile-normalized TPM (Transcripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemistry (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the determined intensity exceeds the reference intensity, administer an effective amount of the ADC to the subject. Includes.

[0189] In some embodiments, the reference value may be at least about 7 to about 13.

[0190] In some embodiments, the reference value may be 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.

[0191] In some embodiments, quantile normalization can be achieved by (i) ranking the transcripts of a sample by their expression levels, (ii) calculating the mean value for genes of the same rank, and (iii) replacing the values ​​of all genes of the same rank with this mean value.

[0192] In some embodiments, the expression level of the transcript may be measured in FPKM (Fragments Per Kilobase Million) and then converted to TPM.

[0193] In some embodiments, FPKM (Fragments Per Kilobase Million) can be obtained by counting all transcripts in the sample, dividing the resulting transcript count by 1 million, and then dividing the resulting value by the gene length in kilobases.

[0194] The methods and steps of use disclosed herein involve determining the relative expression of the CEACAM5 gene to other genes in a tumor sample. Many methods can be used for such determination.

[0195] Next, the relative amount of the CEACAM5 gene transcript is compared to the reference value. A significant deviation from the reference value may indicate a tumor expressing CEACAM5 that may be responsive to therapeutic agents that specifically target CEACAM5.

[0196] The amount of gene transcripts can be measured by any method known in the art, such as microarrays, large-scale real-time reverse transcription PCR, RNA sequencing (RNA-Seq), and next-generation sequencing (NGS).

[0197] Gene expression (RNA-seq) of tumor samples can be obtained by any method known in the art.

[0198] RNA-Seq is a sequencing method used to determine gene expression levels. The number of reads determined to originate from each transcript (usually by alignment) is proportional to their expression levels. RNA-Seq can generate gene expression profiles of tumor samples across many cancer types and be used to determine which gene expression levels are responsible for tumorigenesis. RNA-Seq data can be standardized by aligning raw RNA reads to a GRCh38 reference genome build and calculating gene expression levels using standardized protocols. RNA-Seq data may be available as aligned reads (BAM), and expression levels may be available as raw counts, which are normalized using TPM, FPKM, or FPKM-UQ.

[0199] In some embodiments, gene expression (RNA-seq) can be obtained as follows:

[0200] Gene transcripts (RNA) from isolated tumor samples can be sequenced using the KAPA mRNA HYPERPREPKITILLUMINA® Platform. RNA-seq data can be processed as follows: Sequencing reads can be mapped to the reference genome GRCh 38 using, for example, a Spliced ​​Transcripts Alignment to a Reference (STAR) aligner

[25] . Gene expression can be initially measured in FPKM (Fragments Per Kilobase Million) by CUFFLINK

[26] , and gene-level FPKM can be converted to TPM (Transcripts Per Kilobase Million)

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

[0201] In some embodiments, the measured value of the CEACAM5 gene expression level (mRNA or the measured value of the gene transcription level) can be normalized.

[0202] In some embodiments, the measured value of the CEACAM5 gene expression level (mRNA or the measured value of the gene transcription level) can be log2-transformed.

[0203] In some embodiments, the CEACAM5 gene expression level in a tumor sample can be represented as a log2-transformed, quantile-normalized Transcripts Per Kilobase Million (TPM) value.

[0204] Normalization of the CEACAM5 gene expression level scale involves the following steps: - A Log2 transformation step of RNA-seq raw count values, - A quantile normalization step of RNA-seq data by ranking the values and adjusting them to have the same distribution to match the distribution of expression values across samples, and - An expression step of the data as Transcripts Per Kilobase Million (TPM) by scaling the normalized expression values by the gene length and the total number of sequenced reads and then scaling the values to 1 million to make them more interpretable can be obtained by.

[0205] In some embodiments, the log2-transformed, quantile-normalized Transcripts Per Kilobase Million (TPM) value for the CEACAM5 gene expression level measured (or determined) in a tumor is compared to a reference value. If the determined value exceeds the reference value, the tumor can be determined to be responsive to a CEACAM5-targeted treatment. A subject who needs it and whose determined value exceeds the reference value can be selected for a CEACAM5-targeted treatment.

[0206] In some embodiments, the reference value may be at least about 7 to about 13.

[0207] In some embodiments, the reference value may be 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.

[0208] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately = 7.

[0209] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 7.5.

[0210] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately = 8.

[0211] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 8.5.

[0212] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA can be approximately = 9.

[0213] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 9.5.

[0214] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately = 10.

[0215] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately = 10.5.

[0216] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately = 11.

[0217] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 11.5.

[0218] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately = 12.

[0219] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 12.5.

[0220] In some embodiments, the log2-transformed, quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA can be approximately = 13.

[0221] In some embodiments, the log2-transformed, quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA can be approximately = 13.5.

[0222] In some embodiments, the log2-transformed, quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA can be approximately = 14.

[0223] In some embodiments, the log2-transformed, quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA can be approximately = 14.5.

[0224] In some embodiments, the log2-transformed, quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA can be approximately = 15.

[0225] In some embodiments, the log2-transformed, quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA can be about 7 or more.

[0226] In some embodiments, the log2-transformed, quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA can be about 7.5 or more.

[0227] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 8 or greater.

[0228] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 8.5 or greater.

[0229] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 9 or greater.

[0230] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 9.5 or higher.

[0231] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 10 or greater.

[0232] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 10.5 or greater.

[0233] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 11 or greater.

[0234] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 11.5 or greater.

[0235] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 12 or greater.

[0236] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 12.5 or greater.

[0237] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 13 or greater.

[0238] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 13.5 or greater.

[0239] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 14 or greater.

[0240] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 14.5 or greater.

[0241] In some embodiments, the log2-transformed and quantile-normalized TPM (Transcripts Per Kilobase Million) value for CEACAM5 mRNA may be approximately 15 or greater.

[0242] The expression level (or intensity) of the CEACAM5 protein can be measured by immunohistochemistry (IHC).

[0243] The levels and patterns of CEACAM5 protein expression in tumor samples isolated from patients requiring CEACAM5 protein can be analyzed by immunohistochemistry (IHC), as disclosed, for example, by LaPointe et al. (Journal of Clinical Oncology, Volume 39, Number 15_suppl., https: / / doi.org / 10.1200 / JCO.2021.39.15_suppl.e21030) or Blumenthal et al. (BMC Cancer. 2007;7:2. Published January 3, 2007. doi:10.1186 / 1471-2407-7-2)).

[0244] CEACAM5 responsiveness can be assessed in tumor cells using a semi-quantitative percentage score (calculated by summing the percentages of intensities of 2+ or higher) or an H-score for CEACAM5 cell membrane staining (whole or polarized).

[0245] High CEACAM5 expression can be defined as a patient in whom more than 50% of the tumor cell population in a tumor sample expresses the CEACAM5 protein at an intensity of 2+ or higher.

[0246] A moderate CEACAM5 expression can be defined as a patient who expresses the CEACAM5 protein at an intensity of 2+ or higher in 1% to less than 50% of the tumor cell population in a tumor sample.

[0247] In some embodiments, the baseline intensity of CEACAM5 protein expression levels, as measured by CEACAM5 immunohistochemistry (IHC) staining, can be 2+ or higher in more than 50% of the tumor cell population.

[0248] Patients requiring cancer treatment with an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent, which exhibits CEACAM5 expression at a strength of 2+ or higher in more than 50% of the tumor cell population, can be selected for the aforementioned treatment.

[0249] The aforementioned treatment can be administered to patients requiring cancer treatment with an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent, wherein CEACAM5 expression is 2+ or higher in 50% or more of the tumor cell population.

[0250] Patients requiring cancer treatment with an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent can first be subjected to measurement of CEACAM5 gene expression levels in isolated tumor samples obtained from said patients. If the measured CEACAM5 gene expression levels in isolated tumor samples obtained from said patients exceed a reference value, the patients can be selected for subsequent testing to determine the intensity of CEACAM5 protein expression levels by CEACAM5 immunohistochemistry (IHC) staining.

[0251] If the determined intensity exceeds the baseline intensity, the patient may be selected for treatment with an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated with a cytotoxic agent.

[0252] If the determined intensity exceeds the baseline intensity, the patient may be treated with an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated with a cytotoxic agent.

[0253] Antibody-drug conjugate containing anti-CEACAM5 antibody This disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody or a fragment thereof.

[0254] Antibody-drug conjugates typically comprise an anti-CEACAM5 antibody and at least one chemotherapeutic agent. An antibody-drug conjugate (ADC) comprises an anti-CEACAM5 antibody conjugated to at least one chemotherapeutic agent. Specifically, in an antibody-drug conjugate, the anti-CEACAM5 antibody is covalently bound to at least one chemotherapeutic agent via a cleavable or non-cleavable linker.

[0255] Anti-CEACAM5 antibody According to one embodiment, the antibody-drug conjugate comprises a humanized anti-CEACAM5 antibody or a fragment thereof.

[0256] According to one embodiment, the antibody-drug conjugate comprises a humanized anti-CEACAM5 antibody or a fragment thereof.

[0257] In some embodiments, the anti-CEACAM5 antibody may include CDR-H1, corresponding to SEQ ID NO: 1; CDR-H2, corresponding to SEQ ID NO: 2; CDR-H3, corresponding to SEQ ID NO: 3; CDR-L1, corresponding to SEQ ID NO: 4; CDR-L2, corresponding to the amino acid sequence NTR; and CDR-L3, corresponding to SEQ ID NO: 5.

[0258] In a further embodiment, the anti-CEACAM5 antibody or its fragment comprises a heavy chain variable domain (VH) having at least 90% identity with SEQ ID NO: 6, and a light chain variable domain (VL) having at least 90% identity with 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.

[0259] In further embodiments, the anti-CEACAM5 antibody or its fragment comprises a heavy chain variable domain (VH) having at least 92%, at least 95%, and at least 98% identity with SEQ ID NO: 6, and a light chain variable domain (VL) having at least 92%, at least 95%, and at least 98% identity with 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.

[0260] In some embodiments, the anti-CEACAM5 antibody or its fragment comprises a variable heavy chain domain (VH) consisting of SEQ ID NO: 6 and a variable light chain domain (VL) consisting of SEQ ID NO: 7.

[0261] In further embodiments, the anti-CEACAM5 antibody or its fragment is, -FR1-H extends to amino acid positions 1-25, CDR1-H extends to amino acid positions 26-33 (SEQ ID NO: 1), FR2-H extends to amino acid positions 34-50, CDR2-H extends to amino acid positions 51-58 (SEQ ID NO: 2), FR3-H extends to amino acid positions 59-96, CDR3-H extends to amino acid positions 97-109 (SEQ ID NO: 3), and FR4-H extends to amino acid positions 110-120, sequence [ka] A variable domain of the heavy chain consisting of, -FR1-L extends to amino acid positions 1-26, CDR1-L extends to amino acid positions 27-32 (SEQ ID NO: 4), FR2-L extends to amino acid positions 33-49, CDR2-L extends to amino acid positions 50-52, FR3-L extends to amino acid positions 53-88, CDR3-L extends to amino acid positions 89-97 (SEQ ID NO: 5), and FR4-L extends to amino acid positions 98-107. [ka] A variable domain of a light chain consisting of Includes.

[0262] In a further embodiment, the anti-CEACAM5 antibody or its fragment comprises a heavy chain (HC) having at least 90% sequence identity with SEQ ID NO: 8 and a light chain (LC) having at least 90% sequence identity with SEQ ID NO: 9, where CDR1-H is derived from SEQ ID NO: 1, CDR2-H is derived from SEQ ID NO: 2, CDR3-H is derived from SEQ ID NO: 3, CDR1-L is derived from SEQ ID NO: 4, CDR2-L is derived from the amino acid sequence NTR, and CDR3-L is derived from SEQ ID NO: 5.

[0263] In further embodiments, the anti-CEACAM5 antibody or its fragment comprises a heavy chain (HC) having at least 92%, at least 95%, and at least 98% identity with SEQ ID NO: 8 and a light chain (LC) having at least 92%, at least 95%, and at least 98% identity with 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.

[0264] In certain embodiments, the anti-CEACAM5 antibody comprises a heavy chain (HC) consisting of SEQ ID NO: 8 and a light chain (LC) consisting of SEQ ID NO: 9.

[0265] Anti-CEACAM5 antibodies may also be single-domain antibodies or fragments thereof. In particular, single-domain antibody fragments may consist of a variable heavy chain (VHH) containing CDR1-H, CDR2-H, and CDR3-H of the antibody described above. The antibody may also be a heavy-chain antibody, i.e., an antibody lacking a light chain, which may or may not contain the CH1 domain.

[0266] A single-domain antibody or its fragment may include the framework region of a single-domain antibody of a camelid, and optionally the constant domain of a single-domain antibody of a camelid.

[0267] Anti-CEACAM5 antibodies may also be antibody fragments selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies, particularly humanized antibody fragments.

[0268] The antibody may also be a bispecific or multispecific antibody formed from antibody fragments, where at least one antibody fragment is an antibody fragment according to the present disclosure. A multispecific antibody is a multivalent protein complex, such as those described, for example, in European Patent Application Publication No. 2050764A1 or U.S. Patent Application Publication No. 2005 / 0003403A1.

[0269] Anti-CEACAM5 antibodies and their fragments can be produced by any technique known in the art. In particular, the antibodies can be produced by techniques such as those described below.

[0270] Anti-CEACAM5 antibodies and their fragments can be isolated (e.g., purified) from a vector such as a membrane or lipid vesicle (e.g., liposome), or used contained within a vector.

[0271] Anti-CEACAM5 antibodies and their fragments can be produced alone or in combination by any technique known in the art, for example, any chemical, biological, genetic, or enzymatic technique, though not limited to these.

[0272] By knowing the amino acid sequence of a desired sequence, those skilled in the art can readily produce anti-CEACAM5 antibodies and their fragments using standard techniques for polypeptide production. For example, they can be synthesized using well-known solid-phase methods, particularly using commercially available peptide synthesizers (e.g., those manufactured by Applied Biosystems, Foster City, California), according to the manufacturer's instructions. Alternatively, anti-CEACAM5 antibodies and their fragments can be synthesized by recombinant DNA techniques, as is well known in the art. For example, these fragments can be obtained as DNA expression products after incorporating the DNA sequence encoding the desired (poly)peptide into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host expressing the desired polypeptide, from which they can later be isolated using well-known techniques.

[0273] Anti-CEACAM5 antibodies and their fragments can be appropriately isolated from the culture medium by conventional immunoglobulin purification procedures such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0274] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using various techniques known in the art, such as those disclosed in International Publication No. 2009 / 032661, CDR grafting (European Patent No. 239,400; International Publication No. 91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (European Patent Nos. 592,106; European Patent Nos. 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Patent No. 5,565,332). Common recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application No. 125023 and International Publication No. 96 / 02576).

[0275] The anti-CEACAM5 antibody Fab can be obtained by treating an antibody that specifically reacts with CEACAM5 with a protease such as papain. Alternatively, the anti-CEACAM5 antibody Fab can be produced by inserting the DNA sequences encoding both strands of the anti-CEACAM5 antibody Fab into a prokaryotic or eukaryotic expression vector, and then introducing the vector into prokaryotic or eukaryotic cells (if necessary) to express the anti-CEACAM5 antibody Fab.

[0276] Anti-CEACAM5 antibody F(ab')2 can be obtained by treating an antibody that specifically reacts with CEACAM5 with a protease and pepsin. Alternatively, anti-CEACAM5 antibody F(ab')2 can be produced by conjugating it to the following Fab' via a thioether bond or disulfide bond.

[0277] The Fab' of an anti-CEACAM5 antibody can be obtained by treating F(ab')2, which specifically reacts with CEACAM5, with a reducing agent such as dithiothreitol. Alternatively, the Fab' of an anti-CEACAM5 antibody can be produced by inserting the DNA sequence encoding the Fab' chain of the antibody into a prokaryotic or eukaryotic expression vector, and then introducing that vector into prokaryotic or eukaryotic cells (if necessary) for expression.

[0278] The scFv of an anti-CEACAM5 antibody can be produced by obtaining the sequences of the CDR or VH and VL domains as described above, constructing DNA encoding the scFv fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into prokaryotic or eukaryotic cells (if necessary) to express the scFv. A well-known technique called CDR grafting can be used to produce humanized scFv fragments, which includes selecting the complementarity-determining regions (CDRs) according to the present disclosure and grafting them onto a human scFv fragment framework of a known three-dimensional structure (e.g., International Publication No. 98 / 45322; International Publication No. 87 / 02671; U.S. Patent No. 5,859,205; U.S. Patent No. 5,585,089; U.S. Patent No. 4,816,567; European Patent No. 0173494).

[0279] In one embodiment, the anti-CEACAM5 antibody is tusamitamab (CAS[2349294-95-5]).

[0280] Chemotherapy agents The antibody-drug conjugates for use as disclosed herein typically comprise at least one chemotherapeutic agent (also referred to herein as a cytotoxic agent). As used herein, a chemotherapeutic agent refers to an agent that kills cells, including cancer cells. Such agents preferably halt the division and growth of cancer cells and reduce the size of tumors. The term “chemotherapeutic agent” is used herein interchangeably with the terms “cytotoxic agent,” “growth inhibitor,” or “cell growth suppressant.” As used herein, the term “chemotherapeutic agent” refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction.

[0281] The term “chemotherapeutic agent” is intended to include radioisotopes, enzymes, antibiotics, and toxins such as low-molecular-weight toxins or enzymatically active toxins (including their fragments and / or variants) of bacterial, fungal, plant, or animal origin, as well as various antitumor or anticancer agents disclosed below. In some embodiments, the chemotherapeutic agent is an antimetabolite.

[0282] In further embodiments, the chemotherapeutic agent may be selected from the group consisting of radioisotopes, protein toxins, low molecular weight toxins, and combinations thereof.

[0283] Radioactive isotopes include those suitable for treating cancer. Such radioactive isotopes generally emit primarily beta rays. In further embodiments, the radioactive isotope is At 211 , Bi 212 Er 169 , I 131 , I 125 , Y 90 In 111 , P 32 Re 186 Re 188 Sm 153 Sr 89 The radioactive isotope is selected from the group consisting of radioactive isotopes of Lu and combinations thereof. In one embodiment, the radioactive isotope is an α radioactive isotope, more specifically Th 227 This emits alpha radiation.

[0284] In further embodiments, the low molecular weight toxin is selected from antimetabolites, DNA alkylating agents, DNA crosslinking agents, DNA intercalating agents, antimicrotubule agents, topoisomerase inhibitors, and combinations thereof.

[0285] In further embodiments, the antimicrotubule agent is selected from the group consisting of taxanes, vinca alkaloids, maytansinoids, colchicine, podophyllotoxin, gluceofulvin, and combinations thereof.

[0286] In some embodiments, the cytotoxic agent may be a meitansinoid.

[0287] According to one embodiment, the maytansinoid is selected from maytansinol, maytansinol analogs, and combinations thereof.

[0288] Suitable examples of meitansinol analogs include those having a modified aromatic ring and those having modifications at other positions. Such suitable meitansinoids are listed 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; This information is disclosed in Specification No. 4,362,663; Specification No. 4,364,866; Specification No. 4,450,254; Specification No. 4,322,348; Specification No. 4,371,533; Specification No. 6,333,410; Specification No. 5,475,092; Specification No. 5,585,499; and Specification No. 5,846,545.

[0289] In further embodiments, the cytotoxic conjugates of the present disclosure utilize a thiol-containing maytansinoid (DM1), formally known as N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine, as the cytotoxic agent. DM1 has the following structural formula (I): [ka] It is represented by [this].

[0290] In further embodiments, the cytotoxic conjugates of the present disclosure utilize DM4, a thiol-containing maytansinoid formally known as N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine, as the cytotoxic agent. DM4 has the following structural formula (II): [ka] It is represented by [this].

[0291] In further embodiments of the present disclosure, other maytansins can be used, including thiols and disulfide-containing maytansinoids having mono- or dialkyl substitutions on carbon atoms having sulfur atoms. These include maytansinoids having acylated amino acid side chains having acyl groups having hindered sulfhydryl groups at C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl, wherein the carbon atoms of the acyl groups having thiol functional groups have one or two substituents, and the substituents are linear or branched alkyl or alkenyl groups having CH3, C2H5, 1 to 10 reagents, and any aggregates that may be present in solution.

[0292] Examples of these cytotoxic agents and conjugation methods are further shown in International Publication No. 2008 / 010101, incorporated by reference.

[0293] The immunoconjugates described herein can be prepared as described in International Publication No. 2004 / 091668, the entirety of which is incorporated herein by reference.

[0294] Therefore, in further embodiments, the maytansinoid 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.

[0295] In a further embodiment, in the antibody-drug conjugate, the anti-CEACAM5 antibody is covalently bound to at least one cytotoxic agent via a cleavable or non-cleavable linker.

[0296] In further embodiments, the linker is selected from the group consisting of N-succinimidylpyridyl dithiobutyrate (SPDB), 4-(pyridine-2-yldisulfanyl)-2-sulfobutyrate (sulfo-SPDB), and succinimidyl (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0297] In further embodiments, the linker binds to a lysine or cysteine ​​residue in the Fc region of the anti-CEACAM5 antibody. In even further embodiments, the linker forms a disulfide bond or thioether bond with maytansine.

[0298] In particular, anti-CEACAM5 antibody-drug conjugates are as follows: Formula (III): [ka] Anti-CEACAM5-SPDB-DM4 antibody-drug conjugate; Formula (IV) [ka] Anti-CEACAM5-sulfo-SPDB-DM4-antibody-drug conjugate; and Formula (V): [ka] Anti-CEACAM5-SMCC-DM1 antibody-drug conjugate You can choose from the group consisting of these.

[0299] In equations (III), (IV), and (V) above, "n" corresponds to the number of conjugated chemotherapeutic agent molecules per antibody molecule. This corresponds to the "drug-to-antibody ratio" (or "DAR") as defined below, and can range from 1 to 10.

[0300] In further embodiments, the antibody-drug conjugate of the present disclosure comprises an anti-CEACAM5 antibody comprising the heavy chain (VH) of SEQ ID NO: 8 and the light chain (VL) of SEQ ID NO: 9 (tusamitamab), where tusamitamab is covalently bound to N2'-deacetyl-N-2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4) via N-succinimidylpyridyl dithiobutyrate (SPDB). This yields the antibody-drug conjugate tusamitamaburabutansine (huMAb2-3-SPDB-DM4).

[0301] In one embodiment, the antibody-drug conjugate of the present disclosure is tusamitamablubutansine (CAS[2254086-60-5]).

[0302] As used herein, "linker" means a chemical moiety containing a covalent bond or chain of atoms that covalently binds an antibody to a chemotherapeutic agent moiety (e.g., a cell proliferation inhibitor, a cytotoxic agent, or a growth inhibitor). Suitable linkers are well known in the art and include disulfide groups, thioether groups, acid-unstable groups, photosensitive groups, peptidase-unstable groups, and esterase-unstable groups.

[0303] Conjugates can be prepared by in vitro methods. Conjugate groups are used to link a drug or prodrug to an antibody, such as a chemotherapeutic agent. Suitable conjugate groups are well known in the art and include disulfide groups, thioether groups, acid-unstable groups, photo-unstable groups, peptidase-unstable groups, and esterase-unstable groups. Conjugations of antibodies with chemotherapeutic agents of this disclosure, such as cytotoxic agents, are not limited to, but include, N-succinimidylpyridyl dithiobutyrate (SPDB), 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridine-2-yldisulfanyl)-2-sulfobutyrate (sulfo-SPDB), N-succinimidyl (2-pyridyldithio)propionate (SPDP), succinimidyl (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and iminothiolane (IT These can be prepared using various bifunctional protein coupling agents, including difunctional derivatives of imide esters (such as dimethyladipimidate HCl), active esters (such as diserate subtinimidyl), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies (International Publication No. 94 / 11026).

[0304] The linker may be a "cleavage linker" that facilitates the release of chemotherapeutic agents in cells. For example, acid-unstable linkers, peptidase-sensitive linkers, esterase-unstable linkers, photosensitive linkers, or disulfide-containing linkers (see, for example, U.S. Patent No. 5,208,020) can be used. The linker may also be a "non-cleavage linker" (e.g., an SMCC linker) which may provide better resistance in some cases.

[0305] Generally, conjugates are (i) A step of contacting an aqueous solution of a optionally buffered cell binding agent (e.g., an antibody according to the present disclosure) with a linker and a solution of a chemotherapeutic agent, such as a cytotoxic compound (or drug); (ii) The step of separating the conjugate formed in (i) from the unreacted cell binding agent (e.g., the antibody of the Disclosure) and the unreacted chemotherapeutic agent (e.g., the unreacted cytotoxic compound (or drug)). It can be obtained by a method that includes [a specific method].

[0306] Aqueous solutions of cell-binding agents can be buffered with a buffer such as potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer depends on the properties of the cell-binding agent (e.g., the antibody of this disclosure). Chemotherapy agents, such as cytotoxic compounds (or drugs), are dissolved in organic polar solvents, such as dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).

[0307] The reaction temperature is typically between 20°C and 40°C. The reaction time can vary from 1 to 24 hours. The reaction between the cell conjugate and the chemotherapeutic agent, such as a cytotoxic agent, can be monitored by size exclusion chromatography (SEC) using refractive index measurements and / or a UV detector. If the conjugate yield is too low, the reaction time can be extended.

[0308] To carry out the separation in step (ii), several different chromatographic methods can be used by those skilled in the art. The conjugate can be purified, for example, from the aggregate by mixed support chromatography such as SEC, adsorption chromatography (ion exchange chromatography, IEC, etc.), hydrophobic interaction chromatography (HIC), affinity chromatography, hydroxyapatite chromatography, or high-performance liquid chromatography (HPLC). Purification by dialysis or diafiltration can also be used.

[0309] As used herein, the term “aggregate” means an association that may be formed between two or more cell-binding agents, which may or may not be modified by conjugation. Aggregates can be formed under the influence of numerous parameters, including high concentrations of cell-binding agents in solution (e.g., antibodies of this disclosure), pH of the solution, high shear force, the number of bound dimers and their hydrophobicity, and temperature (see Wang & Gosh, 2008, J. Membrane Sci., 318:311-316 and the references cited therein). It should be noted that the relative effects of some of these parameters have not been clearly established. For proteins and antibodies, those skilled in the art refer to Cromwell et al. (2006, AAPS Journal, 8(3):E572-E579). The content in aggregates can be determined using techniques well known to those skilled in the art, such as SEC (see Walter et al., 1993, Anal. Biochem., 212(2):469-480).

[0310] After step (i) or (ii), the conjugate-containing solution may be subjected to a further step (iii) of chromatography, ultrafiltration and / or diafiltration.

[0311] The conjugate is recovered in an aqueous solution at the end of these steps.

[0312] In further embodiments, the antibody-drug conjugate according to this disclosure is characterized by a “drug-to-antibody ratio” (or “DAR”) in the range of 1 to 10, or 2 to 5, or 3 to 4. This is generally the case for conjugates containing a meitansinoid molecule.

[0313] This DAR number can vary depending on the properties of the antibody and drug used (i.e., chemotherapeutic agents, e.g., cytotoxic agents or growth inhibitors) along with the experimental conditions used in conjugation (such as the ratio of chemotherapeutic agent (e.g., growth inhibitor) to antibody, reaction time, solvent, and properties of any co-solvent). Therefore, contact between an antibody and a chemotherapeutic agent, e.g., a cytotoxic agent or growth inhibitor, can result in a mixture containing several conjugates with different drug-to-antibody ratios, optionally a naked antibody, or optionally, aggregate ratios that differ from one another. Thus, the determined DAR is an average value.

[0314] A method that can be used to determine the DAR consists of measuring the ratio of the absorbances of a substantially purified conjugate solution at λD and 280 nm using a spectrophotometer. 280 nm is a wavelength commonly used to measure protein concentrations, such as antibody concentrations. The wavelength λD is selected so that the drug can be distinguished from the antibody; that is, as is readily known to those skilled in the art, λD is a wavelength at which the drug (i.e., chemotherapeutic agent) has a high absorbance, and λD is a wavelength sufficiently far from 280 nm to avoid substantial overlap of the absorbance peaks of the drug and the antibody. In the case of meitansinoid molecules, λD may be selected as 252 nm. The method for calculating the DAR can be obtained from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science.

[0315] The absorbance of the conjugate at λD (AλD) and 280 nm (A280) is measured using either a monomer peak from size exclusion chromatography (SEC) analysis (enabling the calculation of the "DAR(SEC)" parameter) or a classical spectrophotometer (enabling the calculation of the "DAR(UV)" parameter). The absorbance can be expressed as follows: AλD=(cD x εDλD)+(cA x εAλD) A280=(cD x εD280)+(cA x εA280) (In the formula, cD and cA are the concentrations of the drug (i.e., chemotherapeutic agent) and antibody in solution, respectively. εDλD and εD280 are the molar extinction coefficients of the drug at λD and 280 nm, respectively. εAλD and εA280 are the molar extinction coefficients of the antibody at λD and 280 nm, respectively.

[0316] The resolution of these two equations, which have two unknowns, yields the following equation: cD=[(εA280 x AλD)-(εAλD x A280)] / [(εDλD x εA280)-(εAλD x εD280)] cA=[A280-(cD x εD280)] / εA280

[0317] Next, the average DAR is calculated from the ratio of the antibody concentration to the drug concentration: DAR = cD / cA.

[0318] In some embodiments, the antibody-drug-conjugate is 80 mg / m². 2 ~210 mg / m² 2 , 80 mg / m² 2 ~170mg / m 2 The dosage, or 80 mg / m² 2 ~150mg / m 2 The dosage, or 80 mg / m² 2 ~120 mg / m² 2 The dosage, or 80 mg / m² 2 ~100mg / m 2 It may be administered in the following dose.

[0319] In some embodiments, the antibody-drug conjugate is 80, 100, 120, 150, 170, 180, or 210 mg / m². 2 It can be administered at dose levels of [specify dose level].

[0320] In some embodiments, the administration regimen may include doses administered over a period of about 10 minutes to about 48 hours, or about 1 hour to about 48 hours, for example, 1 hour to 4 hours. In some embodiments, the administration regimen may include doses administered over a period of about 1 hour.

[0321] In some embodiments, the antibody-drug conjugate containing an anti-CEACAM5 antibody may be administered over a period of about 30 minutes to about 3 hours, about 1 hour to about 2 hours, or about 1.5 hours.

[0322] ADC dosage In some embodiments, antibody-drug conjugates are disclosed comprising an anti-CTLA4 antibody and, if present, an anti-CEACAM5 antibody and a chemotherapeutic agent for use in combination with an anti-PD-1 antibody or anti-PD-L1 antibody to treat cancer, wherein the antibody-drug conjugate is distributed at approximately 60 mg / m² of the body surface area of ​​the target requiring it. 2 ~about 210mg / m 2 , or approximately 80 to 170 mg / m² 2 , or approximately 100 to 170 mg / m² 2 , or approximately 120 to 170 mg / m² 2 , or approximately 135 to 170 mg / m² 2 Or approximately 150-170 mg / m² 2 It may be administered in the following dose.

[0323] In some embodiments, the antibody-drug conjugate is approximately 60 to 210 mg / m². 2 , or approximately 80 to 170 mg / m² 2 , or approximately 100 to 150 mg / m² 2 It may be administered in the following dose.

[0324] In various embodiments, the antibody-drug conjugate containing an anti-CEACAM5 antibody is present in an amount of approximately 60, 70, 80, 90, 100, 110, 120, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, or approximately 210 mg / m². 2 It may be administered in the following dose.

[0325] In various embodiments, the antibody-drug conjugate containing an anti-CEACAM5 antibody is administered at approximately 60, 80, 100, 120, 135, 150, 170, 180, 190, or approximately 210 mg / m². 2 It may be administered in the following dose.

[0326] In some embodiments, the ADC is approximately 80 mg / m² 2 ~about 170mg / m 2 At a dose of approximately 80 mg / m², or about 80 mg / m². 2 ~about 150mg / m 2 At a dose of approximately 100 mg / m², or about 100 mg / m². 2 ~about 120mg / m 2 It may be administered in the following dose.

[0327] In some embodiments, the ADC is approximately 80 mg / m² 2 Or approximately 100 mg / m² 2 Or approximately 120 mg / m² 2 Or approximately 150 mg / m² 2 Or approximately 170 mg / m² 2 It may be administered in the following dose.

[0328] According to one embodiment, the antibody-drug conjugate containing an anti-CEACAM5 antibody is approximately 80 mg / m². 2 It may be administered in the following dose.

[0329] In some embodiments, the ADC is approximately 100 mg / m² 2 It may be administered in the following dose.

[0330] In some embodiments, the ADC is approximately 120 mg / m² 2 It may be administered in the following dose.

[0331] In some embodiments, the ADC is approximately 150 mg / m² 2 It may be administered in the following dose.

[0332] In some embodiments, the ADC is approximately 170 mg / m² 2 It may be administered in the following dose.

[0333] According to one embodiment, an antibody-drug conjugate containing an anti-CEACAM5 antibody is administered as a loading dose (or first dose) of approximately 80, 100, 120, 150, or approximately 170 mg / m². 2 It may be administered in the following dose.

[0334] According to one embodiment, an antibody-drug conjugate containing an anti-CEACAM5 antibody is used as a loading dose of approximately 80 mg / m². 2 It may be administered in the following dose.

[0335] According to one embodiment, the antibody-drug conjugate containing an anti-CEACAM5 antibody is used as a loading dose of approximately 100 mg / m². 2 It may be administered in the following dose.

[0336] According to one embodiment, the antibody-drug conjugate containing an anti-CEACAM5 antibody is used as a loading dose of approximately 120 mg / m². 2 It may be administered in the following dose.

[0337] According to one embodiment, the antibody-drug conjugate containing an anti-CEACAM5 antibody is used as a loading dose of approximately 150 mg / m². 2 It may be administered in the following dose.

[0338] According to one embodiment, the antibody-drug conjugate containing an anti-CEACAM5 antibody is used as a loading dose of approximately 170 mg / m². 2 It may be administered in the following dose.

[0339] According to one embodiment, the antibody-drug conjugate containing an anti-CEACAM5 antibody is approximately 80, 100, 120, 150, or approximately 170 mg / m². 2It may be administered as a subsequent dose (or second dose) at the specified dose.

[0340] According to one embodiment, an antibody-drug conjugate containing an anti-CEACAM5 antibody is administered as a subsequent dose of approximately 80 mg / m². 2 It may be administered in the following dose.

[0341] According to one embodiment, an antibody-drug conjugate containing an anti-CEACAM5 antibody is used as a subsequent dose of approximately 100 mg / m². 2 It may be administered in the following dose.

[0342] According to one embodiment, an antibody-drug conjugate containing an anti-CEACAM5 antibody is administered as a subsequent dose of approximately 120 mg / m². 2 It may be administered in the following dose.

[0343] According to one embodiment, an antibody-drug conjugate containing an anti-CEACAM5 antibody is used as a subsequent dose of approximately 150 mg / m². 2 It may be administered in the following dose.

[0344] According to one embodiment, an antibody-drug conjugate containing an anti-CEACAM5 antibody is administered as a subsequent dose of approximately 170 mg / m². 2 It may be administered in the following dose.

[0345] Subsequent doses may be administered on day 1 of the cycle following the first cycle (subsequent or additional cycle).

[0346] According to one embodiment, an antibody-drug conjugate containing an anti-CEACAM5 antibody is administered in the first cycle of treatment, for example on day 1, as a loading dose of about 80, 100, 120, 150, or about 170 mg / m². 2 The dose is given first, and then in an additional cycle, for example on day 1, the subsequent dose is approximately 80, 100, 120, 150, or approximately 170 mg / m². 2 It may be administered in the following dose.

[0347] In certain embodiments, the body surface area (BSA) is 2.2 m². 2For patients with the above condition, the dose of antibody-drug conjugate containing anti-CEACAM5 antibody is 2.2m 2 It can be calculated based on the BSA.

[0348] The antibody-drug conjugate may be tusamitamabrabutansine (huMAb2-3-SPDB-DM4).

[0349] In certain embodiments, ADC is administered approximately once every two weeks. In certain embodiments, ADC is administered approximately once every three weeks. In certain embodiments, ADC is administered approximately once every four weeks. In certain embodiments, ADC is administered approximately once every five weeks. In certain embodiments, ADC is administered approximately once every six weeks.

[0350] Additional medications In certain embodiments, the method and use further include administering to a target an effective amount of at least one additional agent that is effective in treating cancer.

[0351] In certain embodiments, additional agents are selected from the group consisting of immune checkpoint inhibitors (ICIs), platinum-based chemotherapy (e.g., cisplatin or carboplatin), pemetrexed, anti-VEGFR2, FOLFOX, FOLFIRI, TAS-102, anti-EGFR, and any combination thereof.

[0352] In certain embodiments, the ICI is an anti-PD-1 antibody.

[0353] In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, semiprimab, cintilimab, dostallimab, and tislerizumab.

[0354] In certain embodiments, the anti-PD-1 antibody is pembrolizumab.

[0355] In certain embodiments, the ICI is an anti-PD-L1 antibody.

[0356] In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, durvalumab, emvafolimab, BMS-936559, CK-301, CS-1001, SHR-1316 (HTI-1088), CBT-502 (TQB-2450), and any combination thereof.

[0357] In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0358] In certain embodiments, the method includes administering effective doses of tusamitamabrabutansine and pembrolizumab to the target.

[0359] In certain embodiments, the method further comprises administering an effective amount of platinum-based chemotherapy to the target.

[0360] In certain embodiments, platinum-based chemotherapy is selected from cisplatin and carboplatin.

[0361] In certain embodiments, the method further comprises administering an effective amount of pemetrexed to the target.

[0362] In certain embodiments, the method includes administering effective doses of tusamitamabrabutansine, pembrolizumab, and cisplatin to a target.

[0363] In certain embodiments, the method includes administering effective doses of tusamitamabrabutansine, pembrolizumab, cisplatin, and pemetrexed to the target.

[0364] In certain embodiments, the method includes administering effective doses of tusamitamabrabutansine, pembrolizumab, and carboplatin to the target.

[0365] In certain embodiments, the method includes administering effective doses of tusamitamabrabutansine, pembrolizumab, carboplatin, and pemetrexed to the target.

[0366] In one embodiment, the anti-PD-1 antibody or anti-PD-L1 antibody, or a 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 IgG.

[0367] Anti-PD-1 and anti-PD-L1 antibodies, which can interfere with the interaction between PD-1 expressed on the surface of immune cells and PD-L1 expressed on the surface of cancer cells, are useful as immune checkpoint inhibitors. This is because they block pathways that shield tumor cells from immune system components that are capable of fighting cancer and are ready to do so. When PD-1 and PD-L1 interact, they form a biochemical "shield" that protects tumor cells from being destroyed by the immune system. Therefore, blocking either PD-1 or PD-L1, resulting in the disruption of the interaction between PD-1 and PD-L1, interferes with or unmasks the biochemical "shield" that protects tumor cells from being destroyed by the immune system.

[0368] Several anti-PD-1 antibodies have been approved for clinical use in the treatment of cancer. These include pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), semiprimab (LIBTAYO®), cintilimab (TYVYT®), dostallimab (JEMPERLI®), and tislerizumab.

[0369] Similarly, several anti-PD-L1 antibodies have been approved for clinical use in the treatment of cancer. These include atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®).

[0370] In one embodiment, the anti-PD-1 antibody is pembrolizumab or cintilimab.

[0371] In one embodiment, the anti-PD-1 antibody is pembrolizumab, which is a fully human monoclonal IgG1 antibody against human PD-1.

[0372] In one embodiment, an anti-PD-1 antibody or its fragment comprises the light chain and heavy chain CDR of pembrolizumab.

[0373] In one embodiment, the anti-PD-1 antibody or its fragment comprises the heavy chain variable domain (VH) and the light chain variable domain (VL) of pembrolizumab.

[0374] In one embodiment, the anti-PD-1 antibody is cintirimab. In one embodiment, the anti-PD-1 antibody or its fragment comprises the light chain and heavy chain CDR of cintirimab. In one embodiment, the anti-PD-1 antibody or its fragment comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of cintirimab.

[0375] The anti-PD-1 antibody or anti-PD-L1 antibody or its fragment may be a single-domain antibody or its fragment. In particular, a single-domain antibody fragment may consist of a variable heavy chain (VHH) containing CDR1-H, CDR2-H, and CDR3-H of the above antibody. The antibody may also be a heavy-chain antibody, i.e., an antibody lacking a light chain, which may or may not contain the CH1 domain.

[0376] A single-domain antibody or its fragment may include the framework region of a single-domain antibody of a camelid, and optionally the constant domain of a single-domain antibody of a camelid.

[0377] The anti-PD-1 antibody or anti-PD-L1 antibody may be an antibody fragment selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabody, and may also be a humanized antibody fragment.

[0378] The antibody may also be a bispecific or multispecific antibody formed from antibody fragments, wherein at least one antibody fragment is the antibody fragment according to this disclosure. Anti-PD-1 antibodies or anti-PD-L1 antibodies and their fragments can be produced by any technique well known in the art. In particular, the antibodies are produced by techniques already described.

[0379] Anti-PD-1 antibodies or anti-PD-L1 antibodies and their fragments can be isolated (e.g., purified) from a vector such as a membrane or lipid vesicle (e.g., liposome), or used in conjunction with a vector.

[0380] Anti-PD-1 antibodies or anti-PD-L1 antibodies and their fragments can be produced alone or in combination by any technique known in the art, for example, any chemical, biological, genetic, or enzymatic technique, though not limited to these.

[0381] In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody may be administered in doses of 150 mg to 400 mg, or 150 mg to 300 mg.

[0382] In one embodiment, the anti-VEGFR-2 antibody is a monoclonal antibody or a fragment thereof having antagonist activity against VEGFR-2. In one embodiment, the anti-VEGFR-2 antibody is IgG.

[0383] The anti-VEGFR-2 antibody is preferably adapted to the patient. For example, it is preferable to use an anti-mouse VEGFR-2 antibody such as DC-101 in mice and an anti-human VEGFR-2 antibody in humans.

[0384] In one embodiment, the anti-VEGFR-2 antibody is ramucirumab (CAS number 947687-13-0), which is a fully human monoclonal IgG1 antibody against human VEGFR-2.

[0385] In one embodiment, an anti-VEGFR-2 antibody or its fragment comprises the light chain and heavy chain CDR of ramucirumab.

[0386] In one embodiment, the anti-VEGFR-2 antibody or its fragment comprises the heavy chain variable domain (VH) and the light chain variable domain (VL) of ramucirumab.

[0387] The anti-VEGFR-2 antibody or its fragment may be a single-domain antibody or its fragment. In particular, a single-domain antibody fragment may consist of a variable heavy chain (VHH) containing CDR1-H, CDR2-H, and CDR3-H of the antibody described above. The antibody may also be a heavy-chain antibody, i.e., an antibody lacking a light chain, which may or may not contain the CH1 domain.

[0388] A single-domain antibody or its fragment may include the framework region of a single-domain antibody of a camelid, and optionally the constant domain of a single-domain antibody of a camelid.

[0389] Anti-VEGFR-2 antibodies may be antibody fragments selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies, and may particularly be humanized antibody fragments.

[0390] The antibody may also be a bispecific or multispecific antibody formed from antibody fragments, wherein at least one antibody fragment is the antibody fragment according to this disclosure. Anti-VEGFR-2 antibodies and their fragments can be produced by any technique well known in the art. In particular, the antibody can be produced by techniques already described.

[0391] Anti-VEGFR-2 antibodies and their fragments can be isolated (e.g., purified) from or contained within a vector, such as a membrane or lipid vesicle (e.g., liposome).

[0392] Anti-VEGFR-2 antibodies and their fragments can be produced alone or in combination by any technique known in the art, for example, any chemical, biological, genetic, or enzymatic technique, though not limited to these.

[0393] Immunoconjugates containing anti-CEACAM5 antibodies are used in combination with cetuximab for the treatment of cancer.

[0394] Cetuximab (CAS number 205923-56-4) is a chimeric monoclonal IgG1 antibody against the epidermal growth factor receptor (EGFR). Cetuximab itself is used to treat metastatic colorectal cancer, metastatic non-small lung cancer, and head and neck cancer.

[0395] Immunoconjugates containing anti-CEACAM5 antibodies are used in combination with TAS-102 for the treatment of cancer.

[0396] TAS-102 itself is a known chemotherapy regimen approved for human use, involving the combination of triflu-orijen and tipiracil, typically administered in 4-week cycles. TAS-102, in combination with triflu-orijen and tipiracil, is used to treat colorectal cancer.

[0397] As a modified deoxyuridine, trifluorolysine (CAS registry number 70-00-8) is a nucleoside analog that is incorporated into DNA. Modified DNA binds to thymidylate synthase and inhibits the activity of the enzyme. Tipiracil (CAS registry number 183204-74-2) is a thymine analog that prevents the degradation of trifluorozine by thymidine phosphorylase.

[0398] Immunoconjugates containing anti-CEACAM5 antibodies are used in combination with FOLFIRI for cancer treatment.

[0399] FOLFIRI itself is a known chemotherapy regimen approved for human use, comprising the combined administration of folinic acid, 5-fluorouracil, and irinotecan, typically administered in up to 12 two-week cycles. FOLFIRI combines drugs, each with a different mechanism of action and preferably synergistically, to induce cancer cell death.

[0400] 5-Fluorouracil (CAS registry number 51-21-8) is an antimetabolite that primarily inhibits thymidylate synthase and thus blocks thymidine synthesis. 5-Fluorouracil is used in the treatment of colon cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and cervical cancer.

[0401] Folic 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 foliate is L-folic 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 foliate is the calcium salt of L-folic acid. The foliate may also comprise a mixture of two or more stereoisomers.

[0402] Irinotecan (CAS number 97682-44-5) is a semi-synthetic derivative of the alkaloid camptothecin. It is a cytotoxic agent that inhibits topoisomerase I, thereby inhibiting DNA replication and transcription, and is used in the treatment of colon cancer and small cell lung cancer.

[0403] Immunoconjugates containing anti-CEACAM5 antibodies are used in combination with FOLFOX for cancer treatment.

[0404] FOLFOX itself is a known chemotherapy regimen approved for human use, comprising the combined administration of folinic acid, 5-fluorouracil, and oxaliplatin, typically administered in up to 12 two-week cycles. FOLFOX combines drugs, each with a different mechanism of action and preferably synergistically, to induce cancer cell death.

[0405] 5-Fluorouracil (CAS registry number 51-21-8) is an antimetabolite that primarily inhibits thymidylate synthase and thus blocks thymidine synthesis. 5-Fluorouracil is used in the treatment of colon cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and cervical cancer.

[0406] Folic 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 foliate is L-folic 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 foliate is the calcium salt of L-folic acid. The foliate may also comprise a mixture of two or more stereoisomers.

[0407] Oxaliplatin (CAS number 61825-94-3) is known to form crosslinks in DNA strands, preventing DNA replication and transcription, and is used in the treatment of colorectal cancer.

[0408] cancer In one embodiment, cancer is a carcinoma, sarcoma, or blastoma. In a further embodiment, cancer is a carcinoma.

[0409] According to one embodiment, the cancer is a cancer that expresses CEACAM5. A cancer that expresses CEACAM5 may also be called a CEACAM5-positive cancer.

[0410] In some embodiments, the cancer is a CEACAM5-positive cancer.

[0411] CEACAM5-positive cancer is defined as cancer in which the immunohistochemical [IHC] intensity of CEACAM5 is 2+ or higher in 50% or more of cancer cells, or 2+ or higher in 1% or more but less than 50% of cellular tumors (or cancer cells).

[0412] In certain embodiments, cancer has negative or low CEACAM5 expression on tumor cells. Negative or low CEACAM5 expression on tumor cells is defined as a CEACAM5 immunohistochemical [IHC] intensity of 2+ or greater in less than 1% of cells, as measured by immunohistochemistry (IHC).

[0413] In certain embodiments, cancer has moderate CEACAM5 expression on tumor cells. Moderate CEACAM5 expression on tumor cells can be defined as a CEACAM5 immunohistochemical [IHC] intensity of 2+ or higher in 1% to less than 50% of cancer cells, as measured by immunohistochemistry.

[0414] In certain embodiments, cancer has high CEACAM5 expression on tumor cells. High CEACAM5 expression on tumor cells can be defined as a CEACAM5 immunohistochemical [IHC] intensity of 2+ or higher in more than 50% of cancer cells, as measured by immunohistochemistry.

[0415] Immunohistochemical techniques for detecting antigens on cells or tissue sections by immunological and chemical reactions are well known in this field. These techniques are highly sensitive and specific and can detect a wide variety of antigens. The method may include the following steps: a step of binding an antibody to a specific antigen; a step of forming an antibody-antigen complex by incubation with a secondary enzyme-conjugated antibody; and a step of generating a colored deposit at the antibody-antigen binding site in the presence of an enzyme-catalyzed substrate and chromogen.

[0416] CEACAM5 tumor expression can be determined by using immunohistochemistry (IHC) assays. The assay can be performed using an anti-CEACAM5 antibody, such as SANOFI's antibody clone 769. Anti-CEACAM5 clone 769 is a mouse monoclonal antibody with the same specificity as tusamitamablubutansin for the CEACAM5 target. The assay can be performed on a Techmate platformer, a Dako / Agilent Autostainer Link 48 IHC, or any other immunohistochemistry platform. CEACAM5 reactivity is interpreted using a semi-quantitative percentage score (calculated by summing the percentages of intensities 2+ or higher) or H-score for CEACAM5 plasma membrane staining (whole or polar) in tumor cells.

[0417] According to one embodiment, the cancer is selected from hepatocellular carcinoma, colorectal cancer, gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, 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., bile duct cancer), prostate cancer, neuroendocrine cancer, and skin cancer.

[0418] Cancer may be selected from colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, pancreatic cancer, and lung cancer.

[0419] In some embodiments, the cancer may be colorectal cancer.

[0420] In some embodiments, the cancer may be pancreatic cancer.

[0421] In some embodiments, the cancer may be selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, and lung cancer.

[0422] In some embodiments, the cancer is gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, or esophageal cancer.

[0423] According to one embodiment, the cancer is gastric cancer or gastroesophageal junction adenocarcinoma (GEJ).

[0424] According to one embodiment, the cancer is gastric cancer.

[0425] According to one embodiment, the cancer is lung cancer.

[0426] Lung cancer can be non-squamous non-small cell lung cancer (NSQ NSCLC).

[0427] Non-small cell lung cancer (NLC) is a disease in which malignant (cancer) cells form in the lung tissue. Smoking is the main cause of this disease. It is a type of epithelial lung cancer other than small cell lung cancer. There are several types of NLC. Each type of NLC has different types of cancer cells. Each type of cancer cell proliferates and spreads in different ways. The types of NLC are derived from the types of cells found in the cancer and how the cells appear under a microscope: (1) Squamous cell carcinoma: Cancer that begins with squamous epithelial cells (thin, flat cells that look like fish scales). This is also called epidermal carcinoma. (2) Large cell carcinoma: Cancer that can begin with several types of large cells. (3) Adenocarcinoma: Cancer that begins with cells that line the alveoli and produce substances such as mucus.

[0428] In some embodiments, non-squamous non-small cell lung cancer may be advanced or metastatic NSQ NSCLC.

[0429] According to one embodiment, the patient is a patient having a malignant tumor, particularly a malignant solid tumor, more specifically a locally advanced or metastatic solid malignant tumor. A metastatic solid malignant tumor may be a metastatic carcinoma, for example. The carcinoma or cyst may be as described above.

[0430] In certain embodiments, non-squamous non-small cell lung cancer does not have epidermal growth factor receptor (EGFR) sensitization mutations, v-raf mouse sarcoma virus oncogene homolog B1 (BRAF) mutations, or anaplastic lymphoma kinase / c-ros oncogene 1 (ALK / ROS) modifications.

[0431] Pharmaceutical composition or combination In some embodiments, in the uses and methods disclosed herein, ADCs may be administered via parenteral routes. A suitable parenteral route may be intravenous infusion.

[0432] This disclosure also includes combinations relating to ADCs for manufacturing pharmaceuticals for treating cancer.

[0433] In some embodiments, the present disclosure relates to (i) pharmaceutical compositions comprising an ADC disclosed herein and a pharmaceutically acceptable excipient.

[0434] The ADCs of this disclosure can be combined with pharmaceutically acceptable excipients and optionally with a sustained-release matrix (such as a biodegradable polymer) to form therapeutic compositions.

[0435] Therefore, another object of this disclosure relates to pharmaceutical compositions comprising the ADCs of this disclosure and pharmaceutically acceptable carriers or excipients. It also relates to the ADCs or immunoconjugates of this disclosure for use as pharmaceuticals.

[0436] This disclosure also relates to ADCs as disclosed herein for use in treating cancer.

[0437] "Pharmaceutical excipients" or "pharmaceutically acceptable excipients" refer to molecular entities and compositions that, as necessary, do not cause adverse allergic reactions or other undesirable reactions when administered to mammals, particularly humans. Pharmaceutically acceptable carriers or excipients refer to any kind of non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or formulation aids.

[0438] As used herein, “pharmaceutically acceptable carrier or excipient” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial agent, and antifungal agent. Examples of suitable carriers, diluents, and / or excipients include water, amino acids, physiological saline, phosphate-buffered saline, phosphate buffer, acetate, citrate, succinate; amino acids and derivatives such as histidine, arginine, glycine, proline, and glycylglycine; inorganic salts such as NaCl and calcium chloride; sugars or polyalcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, and mannitol; surfactants such as polysorbate 80, polysorbate 20, and poloxamer 188, and one or more combinations thereof. Often, it is preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition, and the formulation may also contain antioxidants, such as tryptamine, and stabilizers, such as Tween 20.

[0439] The form, route of administration, dosage, and regimen of a pharmaceutical composition naturally depend on the condition being treated, the severity of the disease, the patient's age, weight, and sex, etc.

[0440] The pharmaceutical compositions of this disclosure can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration. In one embodiment, the pharmaceutical compositions and combinations of this disclosure are formulated for intravenous administration.

[0441] In particular, pharmaceutical compositions contain pharmaceutically acceptable vehicles or excipients for injectable formulations. These may be dry compositions, especially lyophilized compositions, that enable the formation of an injectable solution when isotonic sterile saline (such as monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, or mixtures of such salts), or optionally sterile water or saline.

[0442] The pharmaceutical composition may be administered through a drug combination device.

[0443] The dosage used for administration can be adapted as a function of various parameters, particularly the mode of use, the associated pathological condition, or the desired duration of treatment.

[0444] To prepare a pharmaceutical composition, an effective amount of antibody-drug conjugate containing an anti-CEACAM5 antibody and / or an anti-CTLA4 antibody and / or an anti-PD-1 antibody or anti-PD-L1 antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0445] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; preparations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and injectable using an appropriate apparatus or system for delivery without decomposition. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi.

[0446] Solutions of the active compound as a free base or a pharmacologically acceptable salt can be prepared in water appropriately mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0447] Antibody-drug conjugates containing anti-CEACAM5 antibodies can be formulated into neutral or salt compositions. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, glycine, histidine, or procaine.

[0448] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by subsequent particle size adjustments in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Sustained absorption of the injectable composition can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin in the composition.

[0449] Sterile injectable solutions are prepared by incorporating the required amount of active compound, along with various other components listed above as needed, into a suitable solvent, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient + any additional desired components from its previously sterilized filtered solution.

[0450] The preparation of larger or higher-concentration solutions for direct injection is also being considered, and the use of DMSO as a solvent is expected to result in extremely rapid penetration, allowing for the delivery of high concentrations of the active agent to small tumor areas.

[0451] Once formulated, the solution is administered in a manner compatible with the administered preparation, in a treatment-effective amount. The preparation can be easily administered in various dosage forms, such as the types of injection solutions mentioned above, but drug-releasing capsules and the like can also be used.

[0452] For parenteral administration in aqueous solutions, for example, the solution should be buffered as needed, and the liquid diluent should first be isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution, or injected into the proposed injection site (see, for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in the dose will inevitably occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for each individual subject.

[0453] Other pharmaceutically acceptable forms include antibody-drug conjugates containing anti-CEACAM5 antibodies formulated for parenteral administration, such as intravenous or intramuscular injection; tablets or other solids for oral administration; time-release capsules; and any other forms currently in use.

[0454] In certain embodiments, the use of liposomes and / or nanoparticles is intended for the introduction of polypeptides into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

[0455] Nanocapsules can generally capture compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles, or biodegradable polylactide or polylactide coglycolide nanoparticles that meet these requirements, are intended for use in this disclosure, and such particles can be readily fabricated.

[0456] Liposomes are formed from phospholipids that disperse in an aqueous medium and spontaneously form multilayer concentric bilayer vesicles (also called multilayer vesicles (MLVs)). MLVs generally have a diameter of 25 nm to 4 μm. Sonic treatment of MLVs results in the formation of smaller monolayer vesicles (SUVs) with a diameter ranging from 200 to 500 Å, containing an aqueous solution in the core. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0457] Method of administration and formulation The methods described herein involve administering a therapeutically effective dose of anti-CEACAM5 ADC to a target. As used herein, “effective dose” or “therapeutic dose” is the dose of a therapeutic agent that results in treatment of a CEACAM5-expressing cancer (e.g., lung cancer, gastric cancer, gastroesophageal junction cancer, or esophageal cancer). As used herein, “to treat” means to cause a detectable improvement in one or more symptoms associated with a CEACAM5-expressing cancer (e.g., lung cancer) or to cause a biological effect (e.g., a decrease in the level of a particular biomarker) that correlates with the underlying pathological mechanism causing the condition or symptom. For example, a dose of anti-CEACAM5 ADC that results in improvement of any of the following symptoms or conditions associated with a CEACAM5-expressing cancer is considered a “therapeutic dose.”

[0458] In another example, if the dose of anti-CEACAM5 ADC did not result in a detectable improvement in one or more CEACAM5-related parameters or symptoms that manifest cancer (e.g., lung cancer, gastric cancer, gastroesophageal junction cancer, or esophageal cancer), or did not produce a biological effect that correlates with the underlying pathological mechanism causing the cancerous condition or symptoms, the treatment was not effective.

[0459] According to some of these embodiments, the anti-CEACAM5 ADC is administered intravenously.

[0460] According to the method disclosed herein, the therapeutically effective dose of anti-CEACAM5 ADC administered to a subject varies depending on the subject's age and size (e.g., body weight or body surface area), the route of administration, and other factors well known to those skilled in the art.

[0461] In certain embodiments, the dosage of the ADC varies according to the body surface area of the subject. In certain embodiments, the dosage of the anti-CEACAM5 ADC administered to the subject is about 1 mg / m 2 ~ about 500 mg / m 2 . In some embodiments, the dosage of the ADC administered to the subject is about 5 mg / m 2 ~ about 300 mg / m 2 . In various embodiments, the dosage of the ADC administered to the subject is about 5 mg / m 2 ~ about 250 mg / m 2 . In various embodiments, the dosage of the ADC administered to the subject is about 60 mg / m 2 ~ about 190 mg / m 2 . In various embodiments, the dosage 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 based on the body surface area of the subject. In certain embodiments, the dosage of the ADC is about 100 mg / m 2 . In certain embodiments, the dosage of the ADC is about 150 mg / m 2 . In certain embodiments, the dosage of the ADC is about 170 mg / m 2 . In certain embodiments, the dosage of the ADC is about 190 mg / m 2 .

[0462] In various embodiments, the dosage of the ADC is 5, 10, 20, 3o, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 210 mg / m 2 based on the body surface area of the subject. In certain embodiments, the dosage of the ADC is 100 mg / m 2 . In certain embodiments, the dosage of the ADC is 150 mg / m 2 . In certain embodiments, the dosage of the ADC is 170 mg / m 2 . In certain embodiments, the dosage of the ADC is 190 mg / m 2 . [Examples]

[0463] The following examples illustrate the most well-known embodiments of the Disclosure. However, it should be understood that these are merely illustrative or descriptive of the application of the principles of the Disclosure. Numerous variations and alternative compositions, methods, and systems can be envisioned by those skilled in the art without departing from the spirit and scope of the Disclosure. Thus, although the Disclosure has been specifically described above, the following examples provide further details in relation to what is currently considered to be the most practical and preferred embodiments of the Disclosure.

[0464] Example 1: Materials and Method Study design and patient Details of the study design for this first-in-human, open-label dose-escalation and expansion study are available elsewhere.

[15]

[0465] Statement on Ethics In short, patients eligible for dose escalation and dose expansion were those aged 18 years or older with locally advanced or metastatic solid tumors for which standard alternative therapies were not available, and with an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 0 or 1.

[0466] The dose-escalation population was enriched with patients having tumor types known to express CEACAM5, but was not limited to those with such tumors; expression was retrospectively confirmed in the central laboratory using IHC on the most recent stored tissue samples.

[0467] The expanded population was limited to separate cohorts of patients with advanced colorectal cancer, NSQ-NSCLC, small cell lung cancer, and gastric adenocarcinoma.

[0468] In this report, the inventors present results for patients with NSQ-NSCLC only. Two independent NSQ-NSCLC expansion-stage populations based on IHC analysis of tumor tissue: CEACAM5 high-expression individuals (defined as patients with CEACAM5 expression at a strength of 2+ or higher in 50% or more of the tumor cell population) and Some patients were found to have moderate CEACAM5 expression (defined as patients with CEACAM5 expression at a strength of 2+ or higher in 1% to less than 50% of the tumor cell population).

[0469] All patients were required to have at least one measurable lesion according to the Solid Tumor Response Assessment Criteria (RECIST) v1.1.

[0470] The main exclusion criteria included: life expectancy of less than 12 weeks; known or symptomatic brain metastases; previous cancer treatment; prior treatment targeting CEACAM5; prior treatment with meitansanoids; decreased bone marrow reserve or organ failure.

[0471] treatment During the blast phase, all patients received intravenous (IV) tusamitamaburabutansine 100 mg / m2 Q2W as determined during the dose escalation phase of this study

[15] . Unless there were signs or symptoms of hypersensitivity response, tusamitamaburabutansine was infused at 2.5 mg / min for 30 minutes, followed by 5 mg / min.

[0472] To prevent hypersensitivity reactions, patients were given an oral antihistamine one hour prior to administration of tusamitamabrabutansine.

[0473] Treatment was continued until the disease progressed, unacceptable toxicity occurred, or the patient requested to stop.

[0474] Outcome During the expansion phase, the primary outcome was assessed by evaluating the objective response rate according to the RECIST v1.1 criteria.

[0475] Important secondary results will be evaluated for safety and will be reported in a separate publication.

[0476] The objectives of the exploratory biomarkers (reported here) included exploring potential associations between CEACAM5 expression characteristics and response (diastolic phase), investigating potential biomarkers (other than CEACAM5) that could predict tussamitamablubutansine activity (diastolic phase), and evaluating the potential of circulating CEA levels as a convenient companion diagnostic for tussamitamablubutansine treatment (by evaluating the correlation between circulating CEA levels and tumor CEACAM5 expression and the correlation between this biomarker and treatment response).

[0477] Tumor sample Tumor samples were obtained using the most recent stored tumor samples (i.e., tumor tissue stored at the time of diagnosis, surgery, or collected before involving the patient in the study and not subjected to anticancer treatment); fresh biopsies at baseline were optional in patients with lesions suitable for biopsy.

[0478] CEACAM5 expression by immunohistochemistry The levels and patterns of CEACAM5 expression in stored tumor samples were analyzed by IHC (intensive clinical hypochlorination) locally at clinical sites and / or intensively in the laboratory using mouse anti-CEACAM5 clone 769, which has the same specificity as tusamitamabrabutansin for the CEACAM5 target.

[0479] For each patient, at least six 5 μm slides and three additional 10 μm slides (or six 5 μm slides) of formalin-fixed paraffin-embedded (FFPE) tissue were provided. CEACAM5 expression was centrally determined by an IHC assay validated using 0.5 μg / mL of anti-CEACAM5 clone 769 antibody.

[0480] CEACAM5 responsiveness was evaluated in tumor cells using a semi-quantitative percentage score (calculated by summing the percentages of intensities of 2+ or higher) or the H score (overall or polarity) of CEACAM5 cell membrane staining; CEACAM5 cytoplasmic staining was also evaluated (center only).

[0481] Tissue RNA / DNA extraction If blocks were provided, FFPE tumor tissue was cut to a thickness of 10 μm using a microtome, and three sections were mounted on adhesive microscope slides. After trimming excess paraffin from the sample slides using a sterile scalpel, the tumor tissue was macro-dissected and collected in individual Eppendorf DNA LoBind tubes. Genomic DNA and total RNA from each human lung cancer FFPE tissue sample were co-extracted using the ALLPREP® DNA / RNA FFPE kit (reference 80234, QIAGEN) according to the manufacturer's instructions, in a final automated extraction step using a QIACUBE automated nucleic acid purifier following an initial manual processing step. Genomic DNA was eluted with 30 μL of ATE buffer, and total RNA was eluted with 20 μL of RNase-free water.

[0482] A total of 71 RNA samples were generated for RNA sequencing analysis.

[0483] Gene expression (RNA-seq) Tissue samples with known CEACAM5 expression levels by IHC were analyzed using RNA-seq. RNA samples from 57 FFPE patient biopsies were sequenced using the KAPA mRNA HYPERPREPKITILLUMINA® Platform. RNA-seq data were processed as follows: Sequencing reads were mapped to the reference genome GRCh 38 using a Spliced ​​Transcripts Alignment to a Reference (STAR) aligner

[25] . Gene expression was first measured in FPKM (Fragments Per Kilobase Million) by CUFFLINK

[26] , and gene-level FPKM was converted to TPM (Transcripts Per Kilobase Million)

[27] . TPM values ​​were log2 transformed and quantile-normalized for downstream analyses, including differential gene expression (DGE) analysis. Samples with fewer than 10,000 detected genes were excluded from downstream analyses. RNA-seq included microenvironment cell population [MCP] counter analysis using publicly available methods [28,29].

[0484] statistical analysis All analyses were performed on a biomarker population defined as patients who had received treatment and undergone at least one evaluable CEACAM5 expression measurement or at least one valid RNA assessment.

[0485] CEACAM5 expression in tumor samples before treatment The percentage of positive tumor cells with an intensity of 2+ or higher across the entire membrane defined CEACAM5 expression in tumor tissue and was analyzed using descriptive statistics.

[0486] The association between CEACAM5 expression characteristics was assessed by calculating p-values ​​from the Mann-Whitney U test (when only two groups exist) or the Kendall-Tau test.

[0487] The Benjamini-Hochberg (BH) multiple correction procedure was used to control the false detection rate, and the adjusted p-value was also calculated.

[0488] Association between baseline biomarkers and tumor response The statistical significance of the association between CEACAM5 expression (moderate vs. high expression) and overall response rate (ORR) was evaluated using a two-sided Fisher exact test.

[0489] Example 2: Results Patients whose biomarkers can be evaluated In this exploratory biomarker analysis of a cohort of non-squamous NSCLC patients who had progressed from the dose escalation phase, the first patient was enrolled on January 2, 2017, and the data cutoff for these analyses was December 2020.

[0490] Of the 888 NSQ-NSCLC patients who underwent pre-screening, 172 (19%) had high CEACAM5 expression and 210 (24%) had moderate CEACAM5 expression.

[0491] Of the 92 patients treated, 64 had high CEACAM5 expression (high expression) and 28 had moderate CEACAM5 expression (moderate expression).

[0492] In summary, the median age was 62.5 years (ranging from 31 to 91 years; 42% were 65 years or older); 51% were male; 72% had an ECOG PS of 1 or higher; and patients had received a median of three prior treatments for progressive disease, including antitubulin (61%) and anti-PD1 / PD-L1 (75%) (range, 1 to 10 lines).

[0493] Clinical findings indicated high CEACAM5 protein expression levels, as measured by immunohistochemistry (IHC), specifically enrichment of clinical responses with an intensity of 2+ or higher in more than 50% of tumor cells as measured by CEACAM5 IHC, in patients (responders). Non-responders were defined as moderate CEACAM5 protein expressors, with CEACAM5 protein expression measured by IHC at an intensity of 2+ or higher in between 1% and less than 50% of the tumor cell population.

[0494] CEACAM5 expression: Staining and distribution patterns in tumors High CEACAM5 expression was superior to total polar membrane expression. In contrast, total membrane and polar membrane expression were similar between moderate CEACAM5 expression levels (Table 1).

[0495] At the time of initial diagnosis, the dominant histological type was adenocarcinoma, and regardless of CEACAM5 expression levels, the majority of tumors expressing CEACAM5 (91.3%) were stage III or higher (Table 1).

[0496] [Table 1]

[0497] Association between CEACAM5 expression and gene expression (RNA-seq) as determined by IHC Differential gene expression analysis identified CEACAM5 mRNA as the gene most associated with high versus moderate CEACAM5 expression induced by IHC (and the only significant adjusted P=0.00265). Expression of other CEACAM family members or other genes was not significantly associated with CEACAM5 IHC after multiple test adjustments (Table 2).

[0498] [Table 2]

[0499] Relationship between CEACAM5 expression, objective response rate, and CEACAM5 mRNA levels A correlation was observed between CEACAM5 mRNA levels, recorded as the sum of the percentages of tumor cells expressing targets of at least 2+ intensity, and CEACAM5 expression as measured by immunohistochemistry, in patients who responded to tusamitamabrabutansine treatment.

[0500] Example 3: Discussion Nearly 20% of patients pre-screened for NSCLC had high CEACAM5 expression as measured by IHC, meaning that more than 50% of tumor cells had a staining intensity of 2+ or higher. A concentration of clinical responses was observed in patients with high CEACAM5 protein expression levels who were treated with tusamitamabrabutansine.

[0501] A correlation was observed between CEACAM5 expression, cCEA, cCEACAM5, and CEACAM5 tumor mRNA levels.

[0502] Furthermore, CEACAM5 mRNA expression was significantly upregulated in patients with high CEACAM5 protein expression versus moderate CEACAM5 protein expression, but other genes, including other CEACAM family genes, were not upregulated (Figure 1 and Table 1).

[0503] Higher levels of CEACAM5 mRNA were observed in CEACAM5 high-expression organisms compared to moderate-expression organisms (P=0.0027) (Figure 1).

[0504] A correlation was observed between CEACAM5 mRNA levels in tumor cells and CEACAM5 IHC staining in tumor cells (Figure 2).

[0505] Furthermore, enrichment of clinical responses was demonstrated in patients treated with ADC and measured by IHC to have high CEACAM5 mRNA levels and high CEACAM5 protein expression (Figures 3 and 4).

[0506] As shown in Figure 5, when examining the mRNA expression of CEACAM family members in response to tusamitamabrabutansine, the median CEACAM5 mRNA level was higher in the responder group compared to the non-responder group. This result indicates enrichment of the clinical response in patients treated with tusamitamabrabutansine according to CEACAM5 mRNA levels. This was not observed for other CEACAM family members, with the exception of a small trend for CEACAM3.

[0507] Clinical findings from the dose escalation and expansion phases of clinical trials showed a concentration of clinical responses in patients with high CEACAM5 protein expression (responders) treated with tusamitamaburabutansine compared to patients treated with ADCs with moderate CEACAM5 protein expression (non-responders).

[0508] Further insights from biomarker analysis support the association between mRNA CEACAM5 expression levels and CEACAM5 protein expression levels.

[0509] Since an association was shown between CEACAM5 protein expression levels and enrichment of clinical response in patients treated with tusamitamaburabutansine, and since an association was shown between CEACAM5 protein expression levels and mRNA CEACAM5 expression levels, mRNA CEACAM5 expression levels are a good biomarker for enrichment of clinical response and are suitable for selecting patients to be treated with an anti-CEACAM5 antibody-drug conjugate, such as tusamitamaburabutansine.

[0510] Furthermore, CEACAM5 mRNA levels are a good biomarker for pre-selecting patients requiring cancer treatment with antibody-drug conjugates (ADCs) containing anti-CEACAM5 antibodies conjugated to cytotoxic agents, for the further step of selecting patients requiring cancer treatment with ADCs, which involves selecting patients with CEACAM5 immunohistochemistry (IHC) staining.

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Claims

1. A method for selecting subjects requiring an antibody-drug conjugate (ADC) for cancer treatment, which includes an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the determined value with a reference value, (iii) If the determined value exceeds the reference value, the step of selecting the subject for cancer treatment. A method that includes at least the following.

2. An antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of cancer in subjects requiring an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent, The aforementioned use, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the determined value with a reference value, (iii) If the determined value exceeds the reference value, the step of administering an effective amount of the ADC to the subject. ADC, including.

3. A method for selecting subjects requiring an antibody-drug conjugate (ADC) for cancer treatment, which includes an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value for the CEACAM5 gene expression level. (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemical (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the intensity determined in step (iv) exceeds the reference intensity, the step of selecting the target for cancer treatment. A method that includes at least the following.

4. An antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of cancer in subjects requiring an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent, The aforementioned use, (i) A step of determining the value of the CEACAM5 gene expression level in the isolated tumor sample obtained from the subject, (ii) A step of comparing the value determined in step (i) with a reference value for the CEACAM5 gene expression level. (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemical (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the determined strength exceeds the standard strength, administer an effective amount of the ADC to the subject. ADC, including.

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 the CEACAM5 gene transcript.

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

7. A method for selecting subjects requiring an antibody-drug conjugate (ADC) for cancer treatment, which includes an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the TPM (Transscripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, after log2 conversion and quantile normalization. (ii) A step of comparing the above value with a reference value, (iii) If the determined value exceeds the reference value, the step of selecting the subject for cancer treatment. A method that includes at least the following.

8. An antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of cancer in subjects requiring an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent, The aforementioned use, (i) A step of determining the TPM (Transscripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, after log2 conversion and quantile normalization. (ii) A step of comparing the above value with a reference value, (iii) If the determined value exceeds the reference value, the step of administering an effective amount of the ADC to the subject. ADC, including.

9. A method for selecting subjects requiring an antibody-drug conjugate (ADC) for cancer treatment, which includes an anti-CEACAM5 antibody conjugated to a cytotoxic agent, (i) A step of determining the TPM (Transscripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, after log2 conversion and quantile normalization. (ii) A step of comparing the value determined in step (i) with a reference value, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemical (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the determined intensity exceeds the standard intensity, the step of selecting the target for cancer treatment. A method that includes at least the following.

10. An antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of cancer in subjects requiring an antibody-drug conjugate (ADC) containing an anti-CEACAM5 antibody conjugated to a cytotoxic agent, The aforementioned use, (i) A step of determining the TPM (Transscripts Per Kilobase Million) value of CEACAM5 mRNA in the isolated tumor sample obtained from the subject, after log2 conversion and quantile normalization. (ii) A step of comparing the value determined in step (i) with a reference value, (iii) If the value determined in step (i) exceeds the reference value in step (ii), the step of selecting the subject for the CEACAM5 immunohistochemistry (IHC) staining test, (iv) A step of determining the intensity of the CEACAM5 protein expression level in the CEACAM5 immunohistochemical (IHC) staining test in the isolated tumor sample obtained from the subject, (v) A step of comparing the strength determined in step (iv) with a reference strength, and (vi) If the determined strength exceeds the standard strength, administer an effective amount of the ADC to the subject. ADC, including.

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

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

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

14. The method according to any one of claims 7, 9, and 11-13, or the antibody-drug conjugate (ADC) for use according to 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 according to any one of claims 7, 9 and 11-14 or the antibody-drug conjugate (ADC) for use according to 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 value for genes that occupy the same rank, and (iii) replacing the values ​​of all genes that occupy the same rank with this mean value.

16. The method according to any one of claims 7, 9, and 11-15, wherein the expression level of the transcript is measured in FPKM (Fragments Per Kilobase Million) and then converted to TPM, or the antibody-drug conjugate (ADC) for use according to any one of claims 8 and 10-15.

17. The method according to claim 16 or an antibody-drug conjugate (ADC) for use, wherein the FPKM (Fragments Per Kilobase Million) can be obtained by counting all 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 according to any one of claims 1 to 17 or an antibody-drug conjugate (ADC) for use, 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 according to any one of claims 1 to 18, or an ADC for use, wherein the antibody-drug conjugate (ADC) is tusamitamabrabutansine.

20. The antibody-drug conjugate (ADC) is at a dose of 80 mg / m ２ or more, particularly 80 mg / m ２ to 210 mg / m ２ , 80 mg / m ２ to 170 mg / m ２ of the dose, or 80 mg / m ２ to 150 mg / m ２ of the dose, or 80 mg / m ２ to 120 mg / m ２ of the dose, or 80 mg / m ２ to 100 mg / m ２ of the dose, particularly 80, 100, 120, 150, 170, 180 or 210 mg / m ２ of the dose, and is administered approximately once every two weeks, or the ADC is administered approximately once every three weeks at a dose of 80 mg / m ２ or more of the surface area of the subject. A method or ADC for use according to any one of claims 1 to 19.

21. The method or antibody-drug conjugate (ADC) for use according to 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 drug effective in treating the cancer.

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

23. The method according to claim 22 or an antibody-drug conjugate (ADC) for use, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, semiprimab, cintilimab, dostallimab, and tislerizumab, or the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

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

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

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

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

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