EP4684034A1 - Ceacam5 mrna assay for patient selection in cancer therapy - Google Patents

Ceacam5 mrna assay for patient selection in cancer therapy

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Publication number
EP4684034A1
EP4684034A1 EP24716660.6A EP24716660A EP4684034A1 EP 4684034 A1 EP4684034 A1 EP 4684034A1 EP 24716660 A EP24716660 A EP 24716660A EP 4684034 A1 EP4684034 A1 EP 4684034A1
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EP
European Patent Office
Prior art keywords
ceacam5
antibody
cancer
subject
adc
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24716660.6A
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German (de)
French (fr)
Inventor
Anne-Laure BAUCHET
Cécile Combeau
Brigitte Demers
Christophe Henry
Cintia PALU
Nils TERNES
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Sanofi SA
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Sanofi SA
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Publication of EP4684034A1 publication Critical patent/EP4684034A1/en
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6857—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from lung cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5752—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the lungs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure relates to the field of cancer therapy.
  • N-NSCLC non-squamous non-small cell lung cancer
  • CEACAMs Carcinoembryonic antigen-related cell adhesion molecules
  • CEACAMs are cell-surface glycoproteins involved in cell adhesion, cell signaling, and promotion of cancer progression and metastasis [6]. Consequently, they are promising targets for novel anticancer agents.
  • CEACAMs Although different CEACAMs show specific expression patterns across tissues, they demonstrate similarity with respect to structural homology and amino acid sequences [7], This requires that molecules targeting CEACAMs must specifically bind to individual CEACAMs to limit cross-reactivity and dose-limiting toxicity.
  • 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 ). High levels of CEACAM5 expression have since been observed in several epithelial tumors, whereas in normal adult tissue, its expression is limited to few tissues (Hammarstrom S., Semin Cancer Biol. 1999;9(2) :67-81 ; Thompson JA., Tumor Biol. 1995;16(1 ):10-16).
  • CEACAM5 is overexpressed in many epithelial tumors [8], which facilitates tumorigenesis and metastasis [9].
  • CEACAM5 is expressed at higher levels in NSCLC but is not expressed in normal lung tissue [7, 10].
  • higher expression of CEACAM5 in human NSCLC tissue is correlated with worse histological grade [10], and higher expression of CEACAM5 is associated with poor survival in patients with NSCLC.
  • ADC antibodydrug conjugate
  • Tusamitamab ravtansine (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 is composed of an anti-CEACAM5 humanized monoclonal antibody conjugated to a potent cytotoxic maytansinoid N2’- deacetyl-N-2’(4-methyl-4-mercapto-1 -oxopentyl)-maytansine (DM4) payload via a cleavable linker [7], The antibody component of tusamitamab ravtansine binds to the CEACAM5 extracellular domain, which is followed by internalization of the ADC and subsequent release of DM4 into the tumor cell [14], DM4 inhibits microtubule assembly, resulting in cell cycle arrest and apoptosis [14
  • tusamitamab ravtansine demonstrated a promising objective response rate of 20.3% and a favorable safety profile with the most common treatment-emergent adverse events being asthenia, reversible corneal events, peripheral neuropathy, dyspnea, and diarrhea, with infrequent hematological toxicities compared with those reported with docetaxel [16]; in addition, among patients who achieved a partial response, 47% were treated for longer than 1 year, suggesting that response to tusamitamab ravtansine was durable and frequently sustained [17].
  • the expression “an enrichment of clinical responses in patients” with regard to a treatment with tusamitamab ravtansine intends to refer to an increase of the overall response rate (ORR) following administration of the given treatment.
  • the ORR is defined as the proportion of patients who have a partial or complete response to therapy; it does not include stable disease and is a direct measure of drug tumoricidal activity.
  • a correlation was found between the CEACAM5 protein expression level in the tumor (measured by immunohistochemistry (IHC) staining) and the CEACAM5 tumor mRNA level. The CEACAM5 mRNA expression was significantly upregulated in patients with high vs moderate CEACAM5 protein expression.
  • the mRNA CEACAM5 expression level was shown to be a good biomarker, predictive of an enrichment of clinical responses, in patients to be treated with an anti-CEACAM5 antibody-drug conjugate, such as tusamitamab ravtansine; alternatively the results show that patients can be selected based on CEACAM5 mRNA level instead of IHC.
  • the Example section therefore supports the use of CEACAM5 mRNA level as a biomarker for selecting and treating patients in need of a cancer treatment with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • ADC antibody-drug conjugate
  • Example section supports the use of CEACAM5 mRNA level as a biomarker for selecting patients in need of a cancer treatment with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for a further selection step with a CEACAM5 immunohistochemistry (IHC) staining and subsequent treatment of patients in need of a cancer treatment cancer with the ADC.
  • ADC antibody-drug conjugate
  • IHC CEACAM5 immunohistochemistry
  • CEACAM5 gene expression level for example of CEACAM5 gene transcript level, for example of CEACAM5 mRNA level, for example of Iog2-transformed, quantile- normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA may be useful as a biomarker for selection of patients for treatment of cancers which typically express CEA Cell Adhesion Molecule 5 (CEACAM5) on their tumoral cells.
  • TPM Transcripts Per Kilobase Million
  • the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • a subject in need thereof is a subject having a cancer.
  • An isolated sample of a tumor of said cancer may be used.
  • the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the samples tumor of step (i) and step (iv) may be the same sample or different samples.
  • the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody- drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibodydrug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
  • ADC antibody-drug conjugate
  • the use comprising: [0053] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
  • the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
  • ADC antibody-drug conjugate
  • the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for characterizing said tumor as a CEACAM5-high expresser tumor.
  • the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test.
  • IHC immunohistochemistry
  • the present disclosure relates to a use of a measure of a value of CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a treatment of a cancer with a CEACAM5 targeting therapeutic agent, for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • a CEACAM5 targeting therapeutic agent for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • ADC antibody-drug conjugate
  • the present disclosure relates to a use of a measure of a value of CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test, said staining test being for selecting said subject for a treatment of a cancer with a CEACAM5 targeting therapeutic agent, for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent
  • a CEACAM5 targeting therapeutic agent for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent
  • the value of the CEACAM5 gene expression level may be a measure of a CEACAM5 gene transcript.
  • the CEACAM5 gene transcript may be a mRNA.
  • methods and uses of the disclosure comprise a step of determining a value of a CEACAM5 mRNA level in an isolated sample of a tumor.
  • the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the value of the CEACAM5 gene expression level may be a measure of a CEACAM5 gene transcript.
  • the CEACAM5 gene transcript may be a mRNA.
  • the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of: [0078] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
  • ADC antibody-drug conjugate
  • TPM Transcripts Per Kilobase Million
  • the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the cancer may be treated.
  • the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
  • ADC antibody-drug conjugate
  • the use comprising (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject, (ii) comparing said value with a reference value, and (iii) administering to said subject an effective amount of said ADC if the determined value is above the reference value.
  • TPM Transcripts Per Kilobase Million
  • the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
  • ADC antibody-drug conjugate
  • RNA ribonucleic acid
  • TPM log-transformed quantile-normalized transcripts per kilobase million
  • antibody-drug conjugate comprising an anti-CEACAM5- antibody conjugated to a cytotoxic agent
  • ADC antibody-drug conjugate
  • the ADC may be used in an effective amount.
  • the use may be in a subject in need thereof.
  • the biomarker CEACAM5 mRNA is measured in an isolated biological sample.
  • the anti-CEACAM5 antibody may comprise a HCDR1 having the amino acid sequence of SEQ ID NO: 1 , a HCDR2 having the amino acid sequence of SEQ ID NO: 2, a HCDR3 having the amino acid sequence of SEQ ID NO: 3, a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence NTR, and a LCDR3 having the amino acid sequence of SEQ ID NO: 5.
  • the anti-CEACAM5 antibody may comprise a variable domain of a heavy chain (VH) consisting of SEQ ID NO: 6 and a variable domain of a light chain (VL) consisting of SEQ ID NO: 7.
  • the anti-CEACAM5 antibody may be tusamitamab.
  • the cytotoxic agent may be selected from the group consisting of radioisotopes, protein toxins, small molecule toxins, and any combination thereof.
  • the small molecule toxin may be selected from antimetabolites, DNA-alkylating agents, DNA-cross-linking agents, DNA-intercalating agents, anti-microtubule agents, topoisomerase inhibitors, and any combination thereof.
  • the anti-microtubule agent may be selected from taxanes, vinca alkaloids, maytansinoids, colchicine, podophyllotoxin, griseofulvin, and any combination thereof.
  • the cytotoxic agent may be maytansinoid or maytansinoid analog.
  • the maytansinoid may be selected from N2’-deacetyl- N2’-(3-mercapto-1 -oxopropyl)-maytansine (DM1 ), N2’-deacetyl-N2’-(4-methyl-4-mercapto- 1 -oxopentyl)-maytansine (DM4), and combinations thereof.
  • the anti-CEACAM5 antibody may be covalently attached via a cleavable or non-cleavable linker to the at least one chemotherapeutic agent.
  • linker may be selected from N-succinimidyl pyridyldithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and succinimidyl(N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC).
  • SPDB N-succinimidyl pyridyldithiobutyrate
  • sulfo-SPDB 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid
  • SMCC succinimidyl(N-maleimidomethyl) cyclohexane-1 -carboxylate
  • the anti-CEACAM5 antibody may be covalently attached via a cleavable linker to the at least one chemotherapeutic agent, the linker being selected from N-succinimidyl pyridyldithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2- sulfo-butyric acid (sulfo-SPDB), and succinimidyl(N-maleimidomethyl) cyclohexane-1 - carboxylate (SMCC).
  • SPDB N-succinimidyl pyridyldithiobutyrate
  • sulfo-SPDB 4-(pyridin-2-yldisulfanyl)-2- sulfo-butyric acid
  • SMCC succinimidyl(N-maleimidomethyl) cyclohexane-1 - carboxylate
  • 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), and which may be covalently linked to N2’-deacetyl-N-2’(4-methyl-4- mercapto-1 -oxopentyl)-maytansine (DM4) via N-succinimidyl pyridyldithiobutyrate (SPDB).
  • VH heavy chain
  • VL light chain
  • SPDB N-succinimidyl pyridyldithiobutyrate
  • the antibody-drug conjugate may be characterized by a drug-to-antibody ratio (DAR) ranging from 1 to 10.
  • DAR drug-to-antibody ratio
  • the antibody-drug conjugate may be tusamitamab ravtansine.
  • the reference value may be a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA.
  • the reference value may be at least from about 7 to about 13.
  • the reference value may be at least about 7, or may be at least about 8, or at least about 9, or at least about 10, or at least about 11 , or at least about 12, or at least about 13.
  • the quantile normalization may be obtained by (i) ranking the transcripts of the sample by level of expression, (ii) calculating an average value for genes occupying a same rank, and (iii) substituting the values of all genes occupying said same rank with this average value.
  • the level of expression of the transcripts may be measured in Fragments Per Kilobase Million (FPKM) before being converted in TPM.
  • FPKM Fragments Per Kilobase Million
  • the Fragments Per Kilobase Million may be obtained by counting the total transcripts in said sample, dividing the obtained transcripts counts by 1 ,000,000, and dividing the obtained values by the length of the gene, in kilobases.
  • the cancer may be selected from the group consisting of hepatocellular carcinoma, colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer, uterine cervix 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.
  • GEJ gastroesophageal junction adenocarcinoma
  • the cancer may be selected from the group consisting of colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer, uterine cervix 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.
  • GEJ gastroesophageal junction adenocarcinoma
  • esophageal cancer esophageal cancer
  • lung cancer uterine cervix cancer
  • pancreatic cancer ovarian cancer
  • thyroid cancer bladder cancer
  • endometrial cancer breast cancer
  • liver cancer biliary tract cancer
  • prostate cancer neuroendocrine cancer
  • the cancer may be selected from a colorectal cancer, a gastric cancer, a gastroesophageal junction adenocarcinoma (GEJ), an esophageal cancer, a pancreatic cancer and a lung cancer.
  • GEJ gastroesophageal junction adenocarcinoma
  • esophageal cancer a pancreatic cancer and a lung cancer.
  • the cancer may be a gastric cancer, a gastroesophageal junction (GEJ) adenocarcinoma, or an esophageal cancer.
  • GEJ gastroesophageal junction
  • the cancer may be gastric cancer. [0138] In some embodiments, the cancer may be colorectal cancer.
  • the cancer may be pancreatic cancer.
  • the cancer may be gastroesophageal junction adenocarcinoma (GEJ).
  • GEJ gastroesophageal junction adenocarcinoma
  • the cancer may be lung cancer.
  • the lung cancer may be a non-squamous non-small cell lung cancer (NSQ NSCLC).
  • NSCLC non-squamous non-small cell lung cancer
  • the non-squamous non-small cell lung cancer may be an advanced or metastatic NSQ NSCLC.
  • the non-squamous non-small cell lung cancer has no epidermal growth factor receptor (EGFR) sensitizing mutation or v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation or anaplastic lymphoma kinase/c-ros oncogene 1 (ALK/ROS) alterations.
  • EGFR epidermal growth factor receptor
  • BRAF v-raf murine sarcoma viral oncogene homolog B1
  • ALK/ROS anaplastic lymphoma kinase/c-ros oncogene 1
  • the anti-CEACAM5 antibody may be tusamitamab.
  • the ADC may be tusamitamab ravtansine.
  • the ADC may be administered in a dose of > 80 mg/m 2 , relative to the surface body area of said subject, about once every two weeks, or the ADC may be administered in a dose of > 80 mg/m 2 , relative to the surface body area of said subject about once every three weeks.
  • the ADC may be administered in a dose of 80 mg/m 2 to 210 mg/m 2 , 80 mg/m 2 to 170 mg/m 2 , or at a dose of 80 mg/m 2 to 150 mg/m 2 , or at a dose of 80 mg/m 2 to 120 mg/m 2 , or at a dose of 80 mg/m 2 to 100 mg/m 2 .
  • the ADC may be administered in a dose of 80, 100, 120, 150, 170, 180, or 210 mg/m 2 .
  • the method or the use as described herein may further comprise administering to the subject an effective amount of at least one additional agent effective to treat the cancer.
  • the additional agent may be selected from the group consisting of an immune checkpoint inhibitor (ICI), a platinum-based chemotherapy, pemetrexed, anti-VEGFR2, FOLFOX, FOLFIRI, TAS-102, anti-EGFR, and any combination thereof.
  • ICI immune checkpoint inhibitor
  • the ICI may be an anti-PD-1 antibody or an anti-PD- L1 antibody.
  • the anti-PD-1 antibody may be selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sintilimab, dostarlimab, and tislelizumab.
  • the anti-PD-L1 antibody may be selected from the group consisting of atezolizumab, avelumab, and durvalumab.
  • the subject an effective amount of tusamitamab ravtansine and pembrolizumab.
  • the method or the use as described herein may further comprise administering to the subject an effective amount of a platinum-based chemotherapy.
  • the platinum-based chemotherapy may be selected from cisplatin and carboplatin.
  • the method or the use as described herein may further comprise administering to the subject an effective amount of pemetrexed.
  • Figure 1 shows CEACAM5 mRNA transcripts per million are elevated in high vs moderate CEACAM5 expression.
  • Figure 2 shows CEACAM5 mRNA correlation with CEACAM5 IHC H-score.
  • Figure 3 Shows CEACAM5 mRNA level correlation with CEACAM5 expression by immunohistochemistry recorded as the sum of the percentage of the tumoral cells expressing the target at least 2+ intensity according to patient response to tusamitamab ravtansine treatment.
  • Figure 4 Shows CEACAM5 mRNA level correlation with CEACAM5 expression by immunohistochemistry recorded as the sum of the percentage of the tumoral cells expressing the target at least 2+ intensity in patients who responded to tusamitamab ravtansine treatment.
  • Figure 5 Shows a box plots of CEACAMs family members expression according to clinical responses in patients treated with tusamitamab ravtansine. [BRIEF DESCRIPTION OF THE SEQUENCES]
  • SEQ ID NO: 1 -5 show the sequences CDR-H1 , CDR-H2, CDR-H3, CDR-L1 and CDR-L3 of the anti-CEACAM5-antibody (huMAb2-3).
  • SEQ ID NO: 6 shows the sequence of the variable domain of the heavy chain (VH) of the anti-CEACAM5-antibody (huMAb2-3).
  • SEQ ID NO: 7 shows the sequence of the variable domain of the light chain (VL) of the anti-CEACAM5-antibody (huMAb2-3).
  • SEQ ID NO: 8 shows the heavy chain sequence of the anti-CEACAM5- antibody (huMAb2-3).
  • SEQ ID NO: 9 shows the light chain sequence of the anti-CEACAM5- antibody (huMAb2-3).
  • a or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • the term indicates deviation from the indicated numerical value by ⁇ 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%.
  • “about” indicates deviation from the indicated numerical value by ⁇ 10%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 3%.
  • “about” indicates deviation from the indicated numerical value by ⁇ 2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.9%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.8%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.7%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.6%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.3%.
  • “about” indicates deviation from the indicated numerical value by ⁇ 0.1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.05%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.01 %.
  • an “antibody” may 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 a disulfide bond.
  • the light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH).
  • VH variable domain
  • CH constant domains
  • the variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen.
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.
  • the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and hence the combining site.
  • Complementarity Determining Regions or CDRs therefore refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
  • the light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1 -L, CDR2-L, CDR3-L and CDR1 -H, CDR2-H, CDR3-H, respectively.
  • a conventional antibody antigenbinding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • antibody intends to refer to conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular variable heavy chain of single domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies. Fragments of antibody considered herein are antigen-binding fragments.
  • FRs Framework Regions
  • the light and heavy chains of an immunoglobulin each have four FRs, designated FR1 -L, FR2-L, FR3-L, FR4-L, and FR1 -H, FR2-H, FR3-H, FR4-H, respectively.
  • a human framework region is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.
  • CDR/FR definition in an immunoglobulin light or heavy chain is to be determined based on IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1 ):55-77; www.imgt.org).
  • antibody or immunoglobulin also includes “single domain antibodies” which have been more recently described and which are antibodies whose complementary determining regions are part of a single domain polypeptide.
  • single domain antibodies include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered single domain antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine.
  • Single domain antibodies may be naturally occurring single domain antibodies known as heavy chain antibody devoid of light chains.
  • camelidae species for example camel, dromedary, llama, alpaca and guanaco, produce heavy chain antibodies naturally devoid of light chain.
  • VHH variable heavy chain of these single domain antibodies devoid of light chains
  • VHHs Similar to conventional VH domains, VHHs contain four FRs and three CDRs. VHH have advantages over conventional antibodies: they are about ten times smaller than IgG molecules, and as a consequence properly folded functional VHH can be produced by in vitro expression while achieving high yield. Furthermore, VHH are very stable, and resistant to the action of proteases. The properties and production of VHH have been reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov;77(1 ):13-22).
  • the term "monoclonal antibody” or “mAb” as used herein refers to an antibody molecule of a single amino acid sequence, which is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method.
  • a monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e., produced by protein engineering.
  • humanized antibody refers to an antibody which is wholly or partially of non-human origin, and which has been modified to replace certain amino acids, in particular in the framework regions of the VH and VL domains, in order to avoid or minimize an immune response in humans.
  • the constant domains of a humanized antibody are most of the time human CH and CL domains.
  • “Fragments” of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody.
  • 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.
  • a fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.
  • Fab denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of the heavy chain and the entire light chain are bound together through a disulfide bond. It is usually obtained among fragments by treating IgG with a protease, such as papain.
  • F(ab')2 refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than 2 identical Fab fragments bound via a disulfide bond of the hinge region. It is usually obtained among fragments by treating IgG with a protease, such as pepsin.
  • Fab'“ refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.
  • a single chain Fv (“scFv”) polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker.
  • the human scFv fragment of the disclosure includes CDRs that are held in appropriate conformation, in particular by using gene recombination techniques.
  • Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.
  • dsFv is a VH::VL heterodimer stabilized by a disulphide bond.
  • (dsFv)2” denotes two dsFv coupled by a peptide linker.
  • BsAb denotes an antibody which combines the antigen-binding sites of two antibodies within a single molecule. Thus, BsAbs are able to bind two different antigens simultaneously. Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions as described for instance in EP 2 050 764 A1 .
  • multispecific antibody denotes an antibody which combines the antigen-binding sites of two or more antibodies within a single molecule.
  • diabodies refers to small antibody fragments with two antigenbinding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigenbinding sites.
  • amino acid sequence “at least 85% identical to a reference sequence” is a sequence having, on its entire length, 85%, or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of the reference amino acid sequence.
  • a percentage of “sequence identity” between amino acid sequences may be determined by comparing the two sequences, optimally aligned over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Optimal alignment of sequences for comparison is conducted by global pairwise alignment, e.g., using the algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970).
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge, size or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • Examples of groups of amino acids that have side chains with similar chemical properties include 1 ) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine.
  • Conservative amino acids substitution groups can also be defined on the basis of amino acid size.
  • purified and “isolated” it is meant, when referring to a polypeptide (i.e., the antibody of the disclosure) or a nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • purified as used herein in particular means at least 75%, 85%, 95%, or 98% by weight, of biological macromolecules of the same type are present.
  • nucleic acid molecule which encodes a particular polypeptide refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
  • the term “subject” or “patient” denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • a subject according to the disclosure is a human.
  • administering refers to delivering to a subject a composition described herein.
  • the composition can be administered to a subject using methods known in the art.
  • the composition can be administered intravenously, subcutaneously, intramuscularly, intradermally, or via any mucosal surface, e.g., orally, sublingually, buccally, nasally, rectally, vaginally or via pulmonary route.
  • the administration is intravenous.
  • the administration is subcutaneous.
  • treat or “treatment” or “therapy” refers to the administration of a compound or a composition according to the disclosure with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a disorder, the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, or otherwise arrest or inhibit further development of the disorder in a statistically significant manner. More particularly, “treating” or “treatment” includes any approach for obtaining beneficial or desired results in a subject’s cancer condition.
  • Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more cancer symptoms or conditions, diminishment or reduction of the extent of a cancer disease or of a cancer symptom, stabilizing, i.e., not worsening, the state of a cancer disease or of a cancer symptom, prevention of a cancer disease or of a cancer symptom’s spread, delay or slowing of cancer disease or cancer symptom progression, amelioration or palliation of the cancer disease state, diminishment of the reoccurrence of cancer disease, and remission, whether partial or total and whether detectable or undetectable.
  • treatment as used herein includes any cure, amelioration, or reduction of a cancer disease or symptom.
  • a “reduction” of a symptom or a disease means decreasing of the severity or frequency of the disease or symptom, or elimination of the disease or symptom.
  • the terms “effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes considered.
  • the specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner and may vary depending on factors such as the type and stage of pathological processes considered, the patient’s medical history and age, and the administration of other therapeutic agents.
  • the unit “mg/m 2 ” indicates the amount of compound in mg per m 2 of subject body surface administered per dose.
  • the person skilled in the art is aware how to determine the required amount of compound for the subject to be treated based on his body surface, which in turn may be calculated based on height and body weight.
  • the unit “mg/kg” indicates the amount of compound in mg per kg of subject body administered per dose. The person skilled in the art is aware how to determine the required amount of compound for the subject to be treated based on his body weight. [0202] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.
  • Biomarker intends to refer a biological molecule, for example a protein or a metabolite, that is differentially present, increased or decreased, in a biological sample obtained from a subject or a group of subjects having a first phenotype, e.g., having a disease such as a cancer, as compared to a biological sample from a subject or a group of subjects having a second phenotype, e.g., not having the disease. In use, the biomarker is isolated from the subject.
  • sample or “biological sample” intends to refer to a biological material isolated from a subject.
  • the biological sample may contain any biological material suitable for detecting the biomarker, i.e., CEACAM5 mRNA, and may comprise cellular and/or non- cellular material from the subject.
  • the sample may be isolated from any suitable biological tissue or fluid such as, for example, kidney tissue, blood, blood plasma (plasma), blood serum (serum), urine, or cerebral spinal fluid (CSF).
  • a biological sample is a blood plasma (plasma) or a blood serum (serum) sample,
  • a “value of reference” or a “threshold value” intends to refer to a level of CEACAM5 mRNA that is indicative of a particular disease state, phenotype, such as cancer, or lack thereof, as well as combinations of disease states, phenotypes, or lack thereof, in the concerned subject.
  • Referenced herein may be trade names for components including various ingredients utilized in the present disclosure.
  • the inventors herein do not intend to be limited by materials under any particular trade name. Equivalent materials (e.g., those obtained from a different source under a different name or reference number) to those referenced by trade name may be substituted and utilized in the descriptions herein.
  • the present disclosure relates to methods and uses for selecting a subject in need thereof for a treatment of a cancer with a therapeutic agent targeting CEACAM5.
  • a therapeutic agent targeting CEACAM5 may be an anti- CEACAM5 antibody conjugated to a cytotoxic agent.
  • the methods and uses of the disclosure may comprise a step of determining the amount of CEACAM5 gene expression (gene transcript or RNA) in a tumor sample.
  • the methods and uses of the disclosure may comprise a step of determining the amount of CEACAM5 gene expression such as CEACAM5 mRNA level in a tumor sample.
  • the amount of CEACAM5 gene expression may be expressed as an amount relative to the total gene expression in the tumor sample and relative to the total length of expressed DNA or any other known method.
  • the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of: [0216] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
  • the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the samples tumor of step (i) and step (iv) may be the same sample or different samples.
  • the present disclosure relates to a method for diagnosing a subject in need thereof as being eligible for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to a method for diagnosing a subject in need thereof as being eligible for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibodydrug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody- drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
  • ADC antibody-drug conjugate
  • the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
  • ADC antibody-drug conjugate
  • the use comprising: [0258] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
  • the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for characterizing said tumor as a CEACAM5-high expresser tumor.
  • the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test.
  • IHC immunohistochemistry
  • the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a treatment of a cancer with a CEACAM5 targeting therapeutic agent, for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • a CEACAM5 targeting therapeutic agent for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • ADC antibody-drug conjugate
  • the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a treatment of a cancer with a CEACAM5 targeting therapeutic agent, for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • a CEACAM5 targeting therapeutic agent for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • ADC antibody-drug conjugate
  • the value of the CEACAM5 gene expression level may be a measure of a CEACAM5 gene transcript.
  • the CEACAM5 gene transcript may be a mRNA.
  • methods and uses of the disclosure comprise a step of determining a value of a CEACAM5 mRNA level in an isolated sample of a tumor.
  • the amount of CEACAM5 gene expression may be expressed as a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value.
  • TPM Transcripts Per Kilobase Million
  • the determined value or level may be compared to a threshold value or value of reference.
  • a deviation from the value of reference may be indicative of a tumor expressing CEACAM5 at a level sufficient to make it responsive to a treatment targeting CEACAM5.
  • the determined intensity or protein expression level may be compared to a threshold intensity or intensity of reference.
  • a deviation from the intensity of reference may be indicative of a tumor expressing CEACAM5 at a level sufficient to make it responsive to a treatment targeting CEACAM5 expressing cancer.
  • the methods and uses of the disclosure may be for characterizing a subject in need thereof as being responsive to a treatment targeting CEACAM5, for example an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • the methods and uses of the disclosure may be for selecting a subject in need thereof as being responsive to a treatment targeting CEACAM5, for example an anti- CEACAM5 antibody conjugated to a cytotoxic agent.
  • the methods and uses of the disclosure may be for monitoring a responsiveness of a subject in need thereof to a treatment targeting CEACAM5, for example an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • the methods and uses of the disclosure may be for selecting a treatment targeting CEACAM5, for example an anti-CEACAM5 antibody conjugated to a cytotoxic agent, according to a CEACAM5 gene expression level in a tumor of a subject in need thereof.
  • the methods and uses of the disclosure may be for selecting a treatment targeting CEACAM5, for example an anti-CEACAM5 antibody conjugated to a cytotoxic agent, according to a CEACAM5 protein expression intensity in a tumor of a subject in need thereof.
  • the methods and uses of the disclosure may be for characterizing a tumor in a subject in need thereof as being responsive to a treatment targeting CEACAM5, for example an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • An isolated sample may be a sample isolated from a tumor. A sample is isolated prior to the implementation of the methods and uses as disclosed herein.
  • the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to a method for diagnosing a subject in need thereof as being eligible for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to a method for diagnosing a subject in need thereof as being eligible for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
  • the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof, [0320] the use comprising (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject, (ii) comparing said value with a reference value, and (iii) administering to said subject an effective amount of said ADC if the determined value is above the reference value.
  • ADC antibody-drug conjugate
  • TPM Transcripts Per Kilobase Million
  • the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
  • ADC antibody-drug conjugate
  • the reference value may be at least from about 7 to about 13.
  • the reference value may be at least about 7, or may be at least about 8, or at least about 9, or at least about 10, or at least about 11 , or at least about 12, or at least about 13.
  • the quantile normalization may be obtained by (i) ranking the transcripts of the sample by level of expression, (ii) calculating an average value for genes occupying a same rank, and (iii) substituting the values of all genes occupying said same rank with this average value.
  • the level of expression of the transcripts may be measured in Fragments Per Kilobase Million (FPKM) before being converted in TPM.
  • the Fragments Per Kilobase Million may be obtained by counting the total transcripts in said sample, dividing the obtained transcripts counts by 1 ,000,000, and dividing the obtained values by the length of the gene, in kilobases.
  • a step of the methods and uses disclosed herein is to determine the relative expression of the CEACAM5 gene relative to other genes in a tumor sample. Many methods may be used for such determination.
  • the relative amount of CEACAM5 gene transcript is then compared to a value of reference. A significant deviation from the value of reference may be indicative of a tumor expressing CEACAM5 which may be responsive to a therapeutic agent targeting specifically CEACAM5.
  • genes transcripts may be measured by any methods known in the art, such as microarrays, large-scale real-time reverse transcription PCR, RNA- Sequencing (RNA-Seq), Next Generation Sequencing (NGS)
  • RNA-seq The gene expression (RNA-seq) of tumor sample may be obtained by any methods known in the art.
  • RNA-Seq is a sequencing method used to determine gene expression levels. The number of reads determined to have originated from each transcript (usually by alignment) are proportional to their expression level. RNA-Seq may be used to generate a gene expression profile for tumor samples across many cancer types and to determine which gene expression levels are responsible for tumor development. The RNA-Seq data may be harmonized by aligning raw RNA reads to the GRCh38 reference genome build and calculating gene expression levels with standardized protocols. RNA-Seq data may be available as aligned reads (BAM) and expression levels as: raw counts and normalized with TPM, FPKM, or FPKM-UQ.
  • BAM aligned reads
  • the gene expression (RNA-seq) may be obtained as follows.
  • RNA-seq Genes transcripts (RNA) from an isolated tumor sample may be sequenced by using KAPA mRNA HYPERPREPKITILLUMINA® Platform. RNA-seq data may be processed as follows: sequencing reads may be mapped to the reference genome GRCh 38 using, for example, Spliced Transcripts Alignment to a Reference (STAR) aligner [25]. Gene expression may be first measured in FPKM (Fragments Per Kilobase Million) by CUFFLINK [26] and the gene-level FPKM may be converted into TPM (Transcripts Per Kilobase Million) [27], TPM values may be Iog2-transformed and quantile-normalized for the downstream analysis, including differential gene expression (DGE) analysis. In some embodiments, samples with a number of genes detected below 10,000 may be excluded from the downstream analysis. RNA-seq may include microenvironment cell populations [MCP] counter analysis, according to published methods [28, 29].
  • MCP microenvironment cell populations
  • the measure of CEACAM5 gene expression level may be normalized.
  • the measure of CEACAM5 gene expression level (mRNA or measure of gene transcript level) may be Iog2-transformed.
  • the CEACAM5 gene expression level (mRNA) in a tumor sample is expressed as a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value.
  • the normalization of the measure of CEACAM5 gene expression level may be obtain with the steps of:
  • TPM Transcripts Per Kilobase Million
  • the Iog2-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 is above the reference value, then the tumor may be qualified as being responsive to a CEACAM5 targeting treatment. A subject in need thereof for whom the determined value is above the reference value may be selected for a CEACAM5 targeting treatment.
  • TPM Transcripts Per Kilobase Million
  • the reference value may be at least from about 7 to about 13. [0350] In some embodiments, the reference value may be at least about 7, or may be at least about 8, or at least about 9, or at least about 10, or at least about 11 , or at least about 12, or at least about 13.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >7.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >7.5.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >8.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >8.5.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >9.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >9.5.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >10.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >10.5.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >11.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >11.5.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >12.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >12.5
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >13.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >13.5.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >14.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >14.5.
  • the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >15.
  • CEACAM5 protein expression level (or intensity) may be measured by immunohistochemistry (IHC).
  • the level and pattern of CEACAM5 protein expression in a tumor sample isolated from a patient in need thereof may be analyzed by immunohistochemistry (IHC), for example as disclosed 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 by Blumenthal etal.(BMC Cancer. 2007;7:2. Published 2007 Jan 3. doi:10.1186/1471 -2407-7- 2).
  • IHC immunohistochemistry
  • CEACAM5 reactivity may be assessed in tumor cells using semi-quantitative Percent Scores (calculated by summing the percentages of intensities >2+) or H-score for CEACAM5 plasma membrane staining (whole or polarized).
  • CEACAM5 high expressers may be defined as patients with CEACAM5 protein expression at an intensity of > 2+ in > 50% of the tumor cell population of a tumor sample.
  • CEACAM5 moderate expressers may be defined as patients with CEACAM5 protein expression at an intensity of > 2+ in > 1% to ⁇ 50% of the tumor cell population of a tumor sample.
  • a reference intensity of a CEACAM5 protein expression level measured with a CEACAM5 immunohistochemistry (IHC) staining test may be an intensity of > 2+ in > 50% of the tumor cell population.
  • a patient in need of a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent and having a CEACAM5 expression at an intensity of > 2+ in > 50% of the tumor cell population may be selected for said treatment.
  • ADC antibody-drug conjugate
  • a patient in need of a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent and having a CEACAM5 expression at an intensity of > 2+ in > 50% of the tumor cell population may be administered said treatment.
  • ADC antibody-drug conjugate
  • a patient in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent may be first subjected to a determination of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said patient. If the determined value of CEACAM5 gene expression level in an isolated sample of a tumor obtained from said patient is above a reference value, then the patient may be selected for a subsequent testing for determination of an intensity of a CEACAM5 protein expression level with a CEACAM5 immunohistochemistry (IHC) staining test.
  • IHC immunohistochemistry
  • the patient may be selected for the treatment with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • ADC antibody-drug conjugate
  • the patient may be administered with the treatment with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
  • ADC antibody-drug conjugate
  • Antibody-drug conjugate comprising an anti-CEACAM5-antibody
  • the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5-antibody, or a fragment thereof.
  • ADC antibody-drug conjugate
  • the antibody-drug conjugate typically comprises 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.
  • the anti-CEACAM5-antibody may be covalently attached via a cleavable or non-cleavable linker to at least one chemotherapeutic agent.
  • the antibody-drug conjugate comprises an anti-CEACAM5-antibody or a fragment thereof.
  • the antibody-drug conjugate comprises a humanized anti-CEACAM5-antibody or a fragment thereof.
  • the anti-CEACAM5-antibody or a fragment thereof comprises a CDR-H1 consisting of SEQ ID NO: 1 , CDR-H2 consisting of SEQ ID NO: 2, CDR-H3 consisting of SEQ ID NO: 3, CDR-L1 consisting of SEQ ID NO: 4, CDR-L2 consisting of amino acid sequence NTR, and CDR-L3 consisting of SEQ ID NO: 5.
  • the anti-CEACAM5-antibody or a fragment thereof comprises a variable domain of a heavy chain (VH) having at least 90% identity to SEQ ID NO: 6, and a variable domain of a light chain (VL) having at least 90% identity to SEQ ID NO: 7, wherein CDR1 -H consists of SEQ ID NO: 1 , CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1 -L consists of SEQ ID NO: 4, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.
  • the anti-CEACAM5-antibody or a fragment thereof comprises a variable domain of a heavy chain (VH) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO: 6, and a variable domain of a light chain (VL) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO: 7, wherein CDR1 -H consists of SEQ ID NO: 1 , CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1 -L consists of SEQ ID NO: 4, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.
  • VH heavy chain
  • VL variable domain of a light chain
  • the anti-CEACAM5-antibody or a fragment thereof comprises a variable domain of a heavy chain (VH) consisting of SEQ ID NO: 6 and a variable domain of a light chain (VL) consisting of SEQ ID NO: 7.
  • the anti-CEACAM5-antibody or a fragment thereof comprises in a further embodiment:
  • variable domain of heavy chain consisting of sequence EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGIT YAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLV TVSS (SEQ ID NO: 6, with CDRs shown in bold characters) in which FR1 -H spans amino acid positions 1 to 25, CDR1 -H spans amino acid positions 26 to 33 (SEQ ID NO: 1 ), FR2- H spans amino acid positions 34 to 50, CDR2-H spans amino acid positions 51 to 58 (SEQ ID NO: 2), FR3-H spans amino acid positions 59 to 96, CDR3-H spans amino acid positions 97 to 109 (SEQ ID NO: 3), and FR4-H spans amino acid positions 1 10 to 120, and
  • the anti-CEACAM5-antibody or a fragment thereof comprises a heavy chain (HC) having at least 90% sequence identity to SEQ ID NO: 8 and a light chain (LC) having at least 90% sequence identity to SEQ ID NO: 9, wherein CDR1 - H consists of SEQ ID NO: 1 , CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1 -L consists of SEQ ID NO: 4, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.
  • HC heavy chain
  • LC light chain
  • the anti-CEACAM5-antibody or a fragment thereof comprises a heavy chain (HC) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO: 8 and a light chain (LC) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO: 9, wherein CDR1 -H consists of SEQ ID NO: 1 , CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1 -L consists of SEQ ID NO: 4, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.
  • HC heavy chain
  • LC light chain
  • the anti-CEACAM5-antibody or a fragment thereof comprises a heavy chain (HC) consisting of SEQ ID NO: 8 and a light chain (LC) consisting of SEQ ID NO: 9.
  • the anti-CEACAM5-antibody may also be a single domain antibody or a fragment thereof.
  • a single domain antibody fragment may consist of a variable heavy chain (VHH) which comprises the CDR1 -H, CDR2-H and CDR3-H of the antibodies as described above.
  • VHH variable heavy chain
  • the antibody may also be a heavy chain antibody, i.e., an antibody devoid of light chain, which may or may not contain a CH1 domain.
  • the single domain antibody or a fragment thereof may also comprise the framework regions of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody.
  • the anti-CEACAM5-antibody may also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies.
  • the antibody may also be a bispecific or multispecific antibody formed from antibody fragments, at least one antibody fragment being an antibody fragment according to the disclosure.
  • Multispecific antibodies are polyvalent protein complexes as described for instance in EP 2 050 764 A1 or US 2005/0003403 A1 .
  • the anti-CEACAM5-antibody and fragments thereof can be produced by any technique well known in the art. In particular, said antibodies are produced by techniques as hereinafter described.
  • the anti-CEACAM5-antibody and fragments thereof can be used in an isolated (e.g., purified) from or contained in a vector, such as a membrane or lipid vesicle (e.g., a liposome).
  • a vector such as a membrane or lipid vesicle (e.g., a liposome).
  • the anti-CEACAM5-antibody and fragments thereof may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic, or enzymatic technique, either alone or in combination.
  • anti-CEACAM5-antibody and fragments thereof can readily produce by standard techniques for production of polypeptides. For instance, they can be synthesized using well- known solid phase method, in particular using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions. Alternatively, anti-CEACAM5-antibody and fragments thereof can be synthesized by recombinant DNA techniques as is well-known in the art.
  • these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.
  • Anti-CEACAM5-antibody and fragments thereof are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, the technique disclosed in the application W02009/032661 , CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991 ); Studnicka GM et al.
  • the Fab of the anti-CEACAM5-antibody can be obtained by treating an antibody which specifically reacts with CEACAM5 with a protease, such as papain. Also, the Fab of the anti-CEACAM5-antibody can be produced by inserting DNA sequences encoding both chains of the Fab of the anti-CEACAM5-antibody into a vector for prokaryotic expression, or for eukaryotic expression, and introducing the vector into prokaryotic or eukaryotic cells (as appropriate) to express the Fab of the anti-CEACAM5-antibody.
  • the F(ab')2 of the anti-CEACAM5-antibody can be obtained treating an antibody which specifically reacts with CEACAM5 with a protease, pepsin. Also, the F(ab')2 of the anti-CEACAM5-antibody can be produced by binding Fab' described below via a thioether bond or a disulfide bond.
  • the Fab' of the anti-CEACAM5-antibody can be obtained treating F(ab')2 which specifically reacts with CEACAM5 with a reducing agent, such as dithiothreitol. Also, the Fab' of the anti-CEACAM5-antibody can be produced by inserting DNA sequences encoding Fab' chains of the antibody into a vector for prokaryotic expression, or a vector for eukaryotic expression, and introducing the vector into prokaryotic or eukaryotic cells (as appropriate) to perform its expression.
  • the scFv of the anti-CEACAM5-antibody can be produced by taking sequences of the CDRs or VH and VL domains as previously described, constructing a DNA encoding an scFv fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into prokaryotic or eukaryotic cells (as appropriate) to express the scFv.
  • a well- known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) according to the disclosure and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, e. g., W098/45322; WO 87/02671 ; US5,859,205; US5,585,089; US4, 816,567; EP0173494).
  • the anti-CEACAM5 antibody is tusamitamab (CAS [2349294-95-5],
  • the antibody-drug conjugate for the use according to the present disclosure typically comprises at least one chemotherapeutic agent (also referred herein to cytotoxic agent).
  • a chemotherapeutic agent as used herein refers to an agent that kills cells, including cancer cells. Such agents favorably stop cancer cells from dividing and growing and cause tumors to shrink in size.
  • the expression “chemotherapeutic agent” is used herein interchangeably with the expressions “cytotoxic agent”, “growth inhibitory agent” or “cytostatic drug”.
  • chemotherapeutic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • chemotherapeutic agent is intended to include radioisotopes, enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below.
  • the chemotherapeutic agent is an antimetabolite.
  • the chemotherapeutic agent is selected from the group consisting of radioisotopes, protein toxins, small molecule toxins, and combinations thereof.
  • Radioisotopes include radioactive isotopes suitable for treating cancer. Such radioisotopes generally emit mainly beta-radiation. In a further embodiment, the radioisotopes are selected from the group consisting of At 211 , Bi 212 , Er 169 , I 131 , I 125 , Y 90 , In 111 , P 32 , Re 186 , Re 188 , Sm 153 , Sr 89 , radioactive isotopes of Lu, and combinations thereof. In an embodiment, the radioactive isotope is alpha-emitter isotope, more specifically Th 227 , which emits alpha-radiation.
  • the small molecule toxins are selected from antimetabolites, DNA-alkylating agents, DNA-cross-linking agents, DNA-intercalating agents, anti-microtubule agents, topoisomerase inhibitors, and combinations thereof.
  • the anti-microtubule agent is selected from the group consisting of taxanes, vinca alkaloids, maytansinoids, colchicine, podophyllotoxin, gruseofulvin, and combinations thereof.
  • a chemotherapeutic agent may be a maytansinoid.
  • maytansinoids are selected from maytansinol, maytansinol analogs, and combinations thereof.
  • suitable maytansinol analogues include those having a modified aromatic ring and those having modifications at other positions.
  • Such suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331 ,598; 4,361 ,650; 4,362,663; 4,364,866; 4,450,254;
  • the cytotoxic conjugates of the present disclosure utilize the thiol-containing maytansinoid (DM1 ), formally termed A/2’-deacetyl-/ ⁇ /2’-(3- mercapto-1 -oxopropyl)-maytansine, as the cytotoxic agent.
  • DM1 is represented by the following structural formula (I):
  • the cytotoxic conjugates of the present disclosure utilize the thiol-containing maytansinoid DM4, formally termed A/2’-deacetyl-/V-2’(4-methyl- 4-mercapto-1 -oxopentyl)-maytansine, as the cytotoxic agent.
  • DM4 is represented by the following structural formula (II):
  • maytansines including thiol and disulfide-containing maytansinoids bearing a mono or di-alkyl substitution on the carbon atom bearing the sulfur atom
  • maytansines including thiol and disulfide-containing maytansinoids bearing a mono or di-alkyl substitution on the carbon atom bearing the sulfur atom
  • maytansines including thiol and disulfide-containing maytansinoids bearing a mono or di-alkyl substitution on the carbon atom bearing the sulfur atom.
  • These include a maytansinoid having, at C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl, an acylated amino acid side chain with an acyl group bearing a hindered sulfhydryl group, wherein the carbon atom of the acyl group bearing the thiol functionality has one or two substituents, said substituents being CH 3 , C2H5, linear or branched alkyl or alkenyl having from 1 to 10
  • immunoconjugates according to the present disclosure can be prepared as described in the application WO 2004/091668, the entire content of which is incorporated herein by reference.
  • the maytansinoids are selected from the group consisting of A/2’-deacetyl-/ ⁇ /2’-(3-mercapto-1 -oxopropyl)-maytansine (DM1 ) or A/2’-deacetyl-/V-2X4-methyl-4-mercapto-1 -oxopentyl)-maytansine (DM4), and combinations thereof.
  • the anti-CEACAM5- antibody is covalently attached via a cleavable or non-cleavable linker to the at least one chemotherapeutic agent.
  • the linker is selected from the group consisting of N-succinimidyl pyridyldithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and succinimidyl(N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC).
  • SPDB N-succinimidyl pyridyldithiobutyrate
  • sulfo-SPDB 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid
  • SMCC succinimidyl(N-maleimidomethyl) cyclohexane-1 -carboxylate
  • the linker binds to a lysine or cysteine residue in the Fc region of the anti-CEACAM5 antibody. In a further embodiment, the linker forms a disulfide bond or a thioether bond with the maytansine.
  • the anti-CEACAM5-antibody-drug conjugate may be selected from the group consisting of:
  • n corresponds to the number of molecules of chemotherapeutic agent conjugated per molecule of antibody. It corresponds to the “drug-to-antibody ratio” (or “DAR”) defined below and may range from 1 to 10.
  • the antibody-drug conjugate of the present disclosure comprises an anti-CEACAM5-antibody, which comprises a heavy chain (VH) of SEQ ID NO: 8 and a light chain (VL) of SEQ ID NO: 9 (tusamitamab), wherein tusamitamab is covalently linked to N2’-deacetyl-N-2’(4-methyl-4-mercapto-1 -oxopentyl)-maytansine (DM4) via N-succinimidyl pyridyldithiobutyrate (SPDB).
  • SPDB N-succinimidyl pyridyldithiobutyrate
  • the antibody-drug conjugate of the present disclosure is tusamitamab ravtansine (CAS [2254086-60-5]).
  • Linker means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches the antibody to the chemotherapeutic agent moiety (e.g., a cytostatic agent, a cytotoxic agent or a growth inhibitory agent). Suitable linkers are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups.
  • the conjugates may be prepared by in vitro methods.
  • a linking group is used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups.
  • Conjugation of an antibody with a chemotherapeutic agent of the disclosure may be made using a variety of bifunctional protein coupling agents including but not limited to N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5- nitro-2-pyridinyl)dithio]-2,5-dioxo-1 -pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2- yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl (N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL),
  • SPDB N-succin
  • a ricin immunotoxin can be prepared as described in Vitetta et al (1987).
  • Carbon labeled 1 -isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (WO 94/1 1026).
  • the linker may be a "cleavable linker" facilitating release of the chemotherapeutic agent in the cell.
  • a "cleavable linker” facilitating release of the chemotherapeutic agent in the cell.
  • an acid-labile linker, a peptidase-sensitive linker, an esterase labile linker, a photolabile linker or a disulfide-containing linker See e.g., U.S. Patent No. 5,208,020
  • the linker may be also a "non-cleavable linker” (for example SMCC linker) that might led to better tolerance in some cases.
  • the conjugate can be obtained by a process comprising the steps of:
  • the aqueous solution of cell-binding agent can be buffered with buffers such as, e.g., potassium phosphate, acetate, citrate or N-2-Hydroxyethylpiperazine-N’-2- ethanesulfonic acid (Hepes buffer).
  • the buffer depends upon the nature of the cell-binding agent (e.g., antibody of the disclosure).
  • the chemotherapeutic agent such as the cytotoxic compound (or agent) is in solution in an organic polar solvent, e.g., dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).
  • DMSO dimethyl sulfoxide
  • DMA dimethylacetamide
  • the reaction temperature is usually comprised between 20°C and 40°C.
  • the reaction time can vary from 1 hour to 24 hours.
  • the reaction between the cell-binding agent and the chemotherapeutic agent, such as the cytotoxic agent, can be monitored by size exclusion chromatography (SEC) with a refractometric and/or UV detector. If the conjugate yield is too low, the reaction time can be extended.
  • SEC size exclusion chromatography
  • the conjugate can be purified, for example from aggregates, e.g., by SEC, adsorption chromatography (such as ion exchange chromatography, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed-support chromatography such as hydroxyapatite chromatography, or high-performance liquid chromatography (HPLC). Purification by dialysis or diafiltration can also be used.
  • adsorption chromatography such as ion exchange chromatography, IEC
  • HIC hydrophobic interaction chromatography
  • HPLC high-performance liquid chromatography
  • the term “aggregates” means the associations which can be formed between two or more cell-binding agents, said agents being modified or not by conjugation.
  • the aggregates can be formed under the influence of a great number of parameters, such as a high concentration of cell-binding agent (e.g., antibody of the disclosure) in the solution, the pH of the solution, high shearing forces, the number of bonded dimers and their hydrophobic character, the temperature (see Wang & Gosh, 2008, J. Membrane Sci., 318: 311 -316, and references cited therein); note that the relative influence of some of these parameters is not clearly established.
  • a high concentration of cell-binding agent e.g., antibody of the disclosure
  • step (i) or (ii) the conjugate-containing solution can be submitted to an additional step (iii) of chromatography, ultrafiltration and/or diafiltration.
  • the conjugate is recovered at the end of these steps in an aqueous solution.
  • the antibody-drug conjugate according to the disclosure is characterized by a “drug-to-antibody ratio” (or “DAR”) ranging from 1 to 10, or from 2 to 5, or from 3 to 4. This is generally the case of conjugates including maytansinoid molecules.
  • DAR drug-to-antibody ratio
  • This DAR number can vary with the nature of the antibody and of the drug (i.e. the chemotherapeutic agent, such as a cytotoxic agent or a growth-inhibitory agent) used along with the experimental conditions used for the conjugation (like the ratio chemotherapeutic agent (e.g., growth-inhibitory agent)/antibody, the reaction time, the nature of the solvent and of the cosolvent if any).
  • the contact between the antibody and the chemotherapeutic agent such as a cytotoxic agent or a growth-inhibitory agent, leads to a mixture comprising several conjugates differing from one another by different drug-to-antibody ratios; optionally the naked antibody; optionally aggregates.
  • the DAR that is determined is thus a mean value.
  • a method which can be used to determine the DAR consists in measuring spectrophotometrically the ratio of the absorbance at of a solution of substantially purified conjugate at AD and 280 nm.
  • 280 nm is a wavelength generally used for measuring protein concentration, such as antibody concentration.
  • the wavelength AD is selected so as to allow discriminating the drug from the antibody, i.e., as readily known to the skilled person, AD is a wavelength at which the drug (i.e., chemotherapeutic agent) has a high absorbance and AD is sufficiently remote from 280 nm to avoid substantial overlap in the absorbance peaks of the drug and antibody.
  • AD may be selected as being 252 nm in the case of maytansinoid molecules.
  • a method of DAR calculation may be derived from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science:
  • the absorbances for the conjugate at AD (AAD) and at 280 nm (A280) are measured either on the monomeric peak of the size exclusion chromatography (SEC) analysis (allowing to calculate the “DAR(SEC)” parameter) or using a classic spectrophotometer apparatus (allowing to calculate the “DAR(UV)” parameter).
  • SEC size exclusion chromatography
  • AAD (cD x eDAD) + (cA x eAAD)
  • A280 (cD x eD280) + (cA x eA280)
  • cD and cA are respectively the concentrations in the solution of the drug (i.e., chemotherapeutic agent) and of the antibody
  • eDAD and eD280 are respectively the molar extinction coefficients of the drug at AD and 280 nm
  • eAAD and eA280 are respectively the molar extinction coefficients of the antibody at AD and 280 nm.
  • the antibody-drug conjugate may be administered at a dose of 80 mg/m 2 to 210 mg/m 2 , 80 mg/m 2 to 170 mg/m 2 , or at a dose of 80 mg/m 2 to 150 mg/m 2 , or at a dose of 80 mg/m 2 to 120 mg/m 2 , or at a dose of 80 mg/m 2 to 100 mg/m 2 .
  • the antibody-drug conjugate may be administered at a dose level of 80, 100, 120, 150, 170, 180, or 210 mg/m 2 .
  • the dosage regimen may comprise administration of a dose over a period of about 10 minutes to about 48 hours, or of about 1 h to about 48h, such as over a period of 1 h to 4h. In some embodiments, the dosage regimen may comprise administration of a dose over a period of about 1 h.
  • the antibody-drug conjugate comprising an anti- CEACAM5-antibody may be administered over about 30 min to about 3 hours, over about 1 hour to about 2hours, or over about 1 .5 hours.
  • an antibody-drug conjugate comprising an anti-CEACAM5-antibody and a chemotherapeutic agent for use for treating a cancer in combination with an anti-CTLA4 antibody and, when presents, an anti-PD-1 antibody or anti-PD-L1 antibody, wherein the antibody-drug conjugate may be administered at a dose of about 60 mg/m 2 to about 210 mg/m 2 , or from about 80 to about 170 mg/m 2 , or from about 100 to about 170 mg/m 2 , or from about 120 to about 170 mg/m 2 , or from about 135 to about 170 mg/m 2 , or from about 150 to about 170 mg/m 2 of body surface area of a subject in need thereof.
  • the antibody-drug conjugate may be administered at a dose of from about 60 to about 210 mg/m 2 , or from about 80 to about 170 mg/m 2 , or from about 100 to about 150 mg/m 2 .
  • the antibody-drug conjugate comprising the anti- CEACAM5 antibody may be administered at a dose of about 60, 70, 80, 90, 100, 110, 120, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, or about 210 mg/m 2 .
  • the antibody-drug conjugate comprising the anti- CEACAM5 antibody may be administered at a dose of about 60, 80, 100, 120, 135, 150, 170, 180, 190 or about 210 mg/m 2 .
  • the ADC may be administered at a dose of about 80 mg/m 2 to about 170 mg/m 2 , or at a dose of about 80 mg/m 2 to about 150 mg/m 2 , or at a dose of about 100 mg/m 2 to about 120 mg/m 2 .
  • the ADC may be administered at a dose of about 80 mg/m 2 or about 100 mg/m 2 or about 120 mg/m 2 or about 150 mg/m 2 or about 170 mg/m 2 .
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80 mg/m 2 .
  • the ADC may be administered at a dose of about 100 mg/m 2 .
  • the ADC may be administered at a dose of about 120 mg/m 2 .
  • the ADC may be administered at a dose of about 150 mg/m 2 .
  • the ADC may be administered at a dose of about 170 mg/m 2 .
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80, 100, 120, 150 or about 170 mg/m 2 , as a loading dose (or first dose).
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80 mg/m 2 , as a loading dose.
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 100 mg/m 2 , as a loading dose.
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 120 mg/m 2 , as a loading dose.
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 150 mg/m 2 , as a loading dose.
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 170 mg/m 2 , as a loading dose.
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80, 100, 120, 150 or about 170 mg/m 2 , as a subsequent dose (or second dose).
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80 mg/m 2 , as a subsequent dose.
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 100 mg/m 2 , as a subsequent dose.
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 120 mg/m 2 , as a subsequent dose.
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 150 mg/m 2 , as a subsequent dose.
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 170 mg/m 2 , as a subsequent dose.
  • a subsequent dose may be administered on day 1 of cycle(s) subsequent to the first cycle (the subsequent or additional cycles).
  • the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80, 100, 120, 150 or about 170 mg/m 2 , as a loading dose, on a first cycle of treatment, e.g., at day 1 , and then at a dose of about 80, 100, 120, 150 or about 170 mg/m 2 , as a subsequent dose, e.g., at day 1 , on additional cycle(s).
  • the dose of the antibody-drug conjugate comprising an anti-CEACAM5-antibody may be calculated based on a BSA of 2.2 m 2 .
  • the antibody-drug conjugate may be tusamitamab ravtansine (huMAb2-3- SPDB-DM4).
  • the ADC is administered about once every two weeks. In certain embodiments, the ADC is administered about once every three weeks. In certain embodiments, the ADC is administered about once every four weeks. In certain embodiments, the ADC is administered about once every five weeks. In certain embodiments, the ADC is administered about once every six weeks.
  • the methods and uses further comprise administering to the subject an effective amount of at least one additional agent effective to treat the cancer.
  • the additional agent is selected from the group consisting of an immune checkpoint inhibitor (ICI), a platinum-based chemotherapy (e.g., cisplatin or carboplatin), pemetrexed, anti-VEGFR2, FOLFOX, FOLFIRI, TAS-102, anti- EGFR, and any combination thereof.
  • ICI immune checkpoint inhibitor
  • platinum-based chemotherapy e.g., cisplatin or carboplatin
  • the ICI is an anti-PD-1 antibody.
  • the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sintilimab, dostarlimab, and tislelizumab.
  • the anti-PD-1 antibody is pembrolizumab.
  • the ICI is an anti-PD-L1 antibody.
  • the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, durvalumab, envafolimab, BMS-936559, CK-301 , CS-1001 , SHR-1316 (HTI-1088), CBT-502 (TQB-2450), and any combination thereof.
  • the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.
  • the method comprises administering to the subject an effective amount of tusamitamab ravtansine and pembrolizumab. [0518] In certain embodiments, the method further comprises administering to the subject an effective amount of a platinum-based chemotherapy.
  • the platinum-based chemotherapy is selected from cisplatin and carboplatin.
  • the method further comprises administering to the subject an effective amount of pemetrexed.
  • the method comprises administering to the subject an effective amount of tusamitamab ravtansine, pembrolizumab, and cisplatin.
  • the method comprises administering to the subject an effective amount of tusamitamab ravtansine, pembrolizumab, cisplatin, and pemetrexed.
  • the method comprises administering to the subject an effective amount of tusamitamab ravtansine, pembrolizumab, and carboplatin.
  • the method comprises administering to the subject an effective amount of tusamitamab ravtansine, pembrolizumab, carboplatin, and pemetrexed.
  • the anti-PD-1 antibody or the anti-PD-L1 antibody, or a fragment thereof is a monoclonal antibody having interfering activity with interaction between PD-1 and PD-L1.
  • the anti-PD-1 antibody or the anti-PD-L1 antibody is an IgG.
  • Anti-PD-1 antibodies and anti-PD-L1 antibodies capable of interfering with interaction between PD-1 , which is expressed on the surface of immune cells, and PD-L1 , which is expressed on the surface of cancer cells, are useful as immune checkpoint inhibitors, thereby blocking a pathway that shields tumor cells from immune system components able and poised to fight cancer.
  • PD-1 and PD-L1 interact, they form a biochemical “shield” protecting tumor cells from being destroyed by the immune system.
  • blockade of either PD-1 or PD-L1 leading to blockade of interaction between PD-1 and PD-L1 prevents or unmasks the biochemical “shield” protecting tumor cells from being destroyed by the immune system.
  • a number of anti-PD-1 antibodies have been approved for clinical use in the treatment of cancer. These include pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cemiplimab (LIBTAYO®), sintilimab (TYVYT®), dostarlimab (JEMPERLI®), and tislelizumab.
  • KEYTRUDA® pembrolizumab
  • OPDIVO® nivolumab
  • LIBTAYO® cemiplimab
  • SIBTAYO® sintilimab
  • JEMPERLI® dostarlimab
  • tislelizumab tislelizumab.
  • anti-PD-L1 antibodies have been approved for clinical use in the treatment of cancer. These include atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®).
  • the anti-PD-1 antibody is pembrolizumab or sintilimab.
  • the anti-PD-1 antibody is pembrolizumab. It is a fully human monoclonal lgG1 antibody against human PD-1.
  • the anti-PD-1 antibody or a fragment thereof, comprises the light chain and heavy chain CDRs of pembrolizumab.
  • the anti-PD-1 antibody or a fragment thereof comprises the variable domain of heavy chain (VH) and the variable domain of light chain (VL) of pembrolizumab.
  • the anti-PD-1 antibody is sintilimab. In one embodiment, the anti-PD-1 antibody, or a fragment thereof, comprises the light chain and heavy chain CDRs of sintilimab. In one embodiment, the anti-PD-1 antibody or a fragment thereof comprises the variable domain of heavy chain (VH) and the variable domain of light chain (VL) of sintilimab.
  • the anti-PD-1 antibody or the anti-PD-L1 antibody, or a fragment thereof may also be a single domain antibody or a fragment thereof.
  • a single domain antibody fragment may consist of a variable heavy chain (VHH) which comprises the CDR1 - H, CDR2-H and CDR3-H of the antibodies as described above.
  • VHH variable heavy chain
  • the antibody may also be a heavy chain antibody, i.e., an antibody devoid of light chain, which may or may not contain a CH1 domain.
  • the single domain antibody or a fragment thereof may also comprise the framework regions of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody.
  • the anti-PD-1 antibody or the anti-PD-L1 antibody may also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies.
  • the antibody may also be a bispecific or multispecific antibody formed from antibody fragments, at least one antibody fragment being an antibody fragment according to the disclosure.
  • the anti-PD-1 antibody or the anti-PD-L1 antibody and fragments thereof can be produced by any technique well known in the art. In particular, said antibodies are produced by techniques as already described.
  • the anti-PD-1 antibody or the anti-PD-L1 antibody and fragments thereof can be used in an isolated (e.g., purified) from or contained in a vector, such as a membrane or lipid vesicle (e.g., a liposome).
  • the anti-PD-1 antibody or the anti-PD-L1 antibody and fragments thereof may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic, or enzymatic technique, either alone or in combination.
  • the anti-PD-1 antibody or the anti-PD-L1 antibody may be administered at a dose of 150 mg to 400 mg, or at a dose of 150 mg to 300 mg.
  • the anti-VEGFR-2 antibody is a monoclonal antibody, or a fragment thereof having antagonist activity to VEGFR-2. In one embodiment, the anti- VEGFR-2 antibody is an IgG.
  • the anti-VEGFR-2 antibody is preferably adapted to the patient.
  • an anti-mouse VEGFR-2 antibody such as DC-101 is preferably used on mice, and an anti-human VEGFR-2 antibody on humans.
  • the anti-VEGFR-2 antibody is ramucirumab (CAS number 947687-13-0). It is a fully human monoclonal lgG1 antibody against human VEGFR-2.
  • the anti-VEGFR-2 antibody or fragment thereof comprises the light chain and heavy chain CDRs of ramucirumab.
  • the anti-VEGFR-2 antibody or fragment thereof comprises the variable domain of heavy chain (VH) and the variable domain of light chain (VL) of ramucirumab.
  • the anti-VEGFR-2 antibody or fragment thereof may also be a single domain antibody or a fragment thereof.
  • a single domain antibody fragment may consist of a variable heavy chain (VHH) which comprises the CDR1 -H, CDR2-H and CDR3-H of the antibodies as described above.
  • VHH variable heavy chain
  • the antibody may also be a heavy chain antibody, i.e., an antibody devoid of light chain, which may or may not contain a CH1 domain.
  • the single domain antibody or a fragment thereof may also comprise the framework regions of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody.
  • the anti-VEGFR-2 antibody may also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies.
  • the antibody may also be a bispecific or multispecific antibody formed from antibody fragments, at least one antibody fragment being an antibody fragment according to the disclosure.
  • the anti-VEGFR-2 antibody and fragments thereof can be produced by any technique well known in the art. In particular, said antibodies are produced by techniques as already described.
  • the anti-VEGFR-2 antibody and fragments thereof can be used in an isolated (e.g., purified) from or contained in a vector, such as a membrane or lipid vesicle (e.g., a liposome).
  • a vector such as a membrane or lipid vesicle (e.g., a liposome).
  • the anti-VEGFR-2 antibody and fragments thereof may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic, or enzymatic technique, either alone or in combination.
  • the immunoconjugate comprising an antiCEACAM5-antibody is to be used in combination with cetuximab for the treatment of cancer.
  • Cetuximab (CAS number 205923-56-4) is a chimeric monoclonal lgG1 antibody against epidermal growth factor receptor (EGFR). Cetuximab itself has been used for the treatment of metastatic colorectal cancer, metastatic non-small lung cancer and head and neck cancer.
  • the immunoconjugate comprising an antiCEACAM5-antibody is to be used in combination with TAS-102 for the treatment of cancer.
  • TAS-102 itself is a known chemotherapy regimen approved for human use comprising the combined administration of trifluoridine and tipiracil and which is typically administered in 4-week cycles. TAS-102 combines trifluoridine and tipiracil and has been used in the treatment of colorectal cancer.
  • trifluoridine As a modified deoxyuridine, trifluoridine (CAS registry number 70-00-8) is a nucleoside analogue which is incorporated into DNA. The modified DNA binds to thymidiylate synthase, inhibiting the enzyme’s activity. Tipiracil (CAS registry number 183204-74-2) is a thymine analogue, which prevents the degradation of trifluoridine by thymidine phosphorylase.
  • the immunoconjugate comprising an antiCEACAM5-antibody is to be used in combination with FOLFIRI for the treatment of cancer.
  • FOLFIRI itself is a known chemotherapy regimen approved for human use comprising the combined administration of folinic acid, 5-fluoro-uracil and irinotecan and which is typically administered in up to 12 two-week cycles.
  • FOLFIRI combines drugs, each with a different mechanism of action and favorably with synergistic effects, causing the death of cancer cells.
  • 5-Fluoro-uracil (CAS registry number 51 -21 -8) is an anti-metabolite, which principally inhibits thymidylate synthase and thus blocks the synthesis of thymidine.
  • 5- Fluoro-uracil has been used the treatment of colon cancer, esophageal cancer, stomach cancer, pancreatic cancer, breast cancer, and cervical cancer.
  • Folinic acid also known as leucovorin (CAS registry number 58-05-9), stabilizes the complex between 5-fluoro-uracil and thymidylate synthase, increasing the cytotoxicity of 5-fluoro-uracil.
  • folinic acid is L-folinic acid (N-[4-[[[(6S)- 2-amino-5-formyl-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl]methyl]amino]benzoyl]-L- glutamic acid).
  • folinic acid is the calcium salt of L-folinic acid.
  • Folinic acid may also comprise a mixture two or more stereoisomers.
  • Irinotecan (CAS Number 97682-44-5) is a cytotoxic which is a semi-synthetic derivative of the alkaloid camptothecin and inhibits topoisomerase I resulting in inhibition of DNA replication and transcription and which has been used in the treatment of colon cancer and small cell lung cancer.
  • the immunoconjugate comprising an antiCEACAM5-antibody is to be used in combination with FOLFOX for the treatment of cancer.
  • FOLFOX itself is a known chemotherapy regimen approved for human use comprising the combined administration of folinic acid, 5-fluoro-uracil and oxaliplatin and which is typically administered in up to 12 two-week cycles. FOLFOX combines drugs, each with a different mechanism of action and favorably with synergistic effects, causing the death of cancer cells.
  • 5-Fluoro-uracil (CAS registry number 51 -21 -8) is an anti-metabolite, which principally inhibits thymidylate synthase and thus blocks the synthesis of thymidine.
  • 5- Fluoro-uracil has been used in the treatment of colon cancer, esophageal cancer, stomach cancer, pancreatic cancer, breast cancer, and cervical cancer.
  • Folinic acid also known as leucovorin (CAS registry number 58-05-9), stabilizes the complex between 5-fluoro-uracil and thymidylate synthase, increasing the cytotoxicity of 5-fluoro-uracil.
  • folinic acid is L-folinic acid (N-[4-[[[(6S)- 2-amino-5-formyl-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl]methyl]amino]benzoyl]-L- glutamic acid).
  • folinic acid is the calcium salt of L-folinic acid.
  • Folinic acid may also comprise a mixture two or more stereoisomers.
  • Oxaliplatin (CAS Number 61825-94-3) is known to form cross-links in DNA- strands, preventing DNA replication and transcription and has been used in the treatment of colorectal cancer.
  • the cancer is a carcinoma, a sarcoma or a blastoma. In a further embodiment, the cancer is a carcinoma.
  • the cancer is a cancer expressing CEACAM5.
  • a cancer expressing CEACAM5 may also be named CEACAM5 positive cancer.
  • the cancer is a CEACAM5-positive cancer.
  • a CEACAM5-positive cancer is defined as cancer for which a CEACAM5 immunohistochemical [IHC] intensity is > 2+ in > 50% of cancer cells or > 2+ intensity in > 1% and ⁇ 50% of the cells tumors (or cancer cells).
  • IHC immunohistochemical
  • the cancer has a negative or low CEACAM5 expression on tumor cells.
  • a negative or low CEACAM5 expression on tumor cells defined as being a CEACAM5 immunohistochemical [IHC] intensity > 2+ in ⁇ 1% of cells, as measured by immunohistochemistry (IHC).
  • the cancer has a moderate CEACAM5 expression on tumor cells.
  • a moderate CEACAM5 expression on tumor cells may be defined as being a CEACAM5 immunohistochemical [IHC] intensity > 2+ in > 1 % and in ⁇ 50% of cancer cells, as measured by immunohistochemistry.
  • the cancer has a high CEACAM5 expression on tumor cells.
  • a high CEACAM5 expression on tumor cells may be defined as being a CEACAM5 immunohistochemical [IHC] intensity > 2+ intensity in > 50% of cancer cells, as measured by immunohistochemistry.
  • IHC immunohistochemical
  • Immunohistochemical techniques for detecting antigens on cells or in tissue sections by means of immunological and chemical reactions are well-known in the field. Those techniques are highly sensitive and specific and can detect a wide variety of antigens.
  • Immunohistochemistry methods comprise the following steps: binding of an antibody to a specific antigen; formation of an antibody-antigen complex by incubation with a secondary, enzyme-conjugated antibody, and generation of colored deposits at the sites of antibodyantigen binding in presence of substrate and chromogen catalyzed by the enzyme.
  • the CEACAM5 tumor expression may be determined by using Immunohistochemistry (IHC) assay.
  • An assay may be done using anti-CEACAM5 antibody such as SANOFI’s antibody clone 769.
  • Anti-CEACAM5 clone 769 is a murine monoclonal antibody with the same specificity as tusamitamab ravtansine to the CEACAM5 target.
  • the assay may be run on Techmate platformer or on a Dako/Agilent Autostainer Link 48 IHC or any other immunohistochemistry platforms.
  • CEACAM5 reactivity is to be performed using semi-quantitative Percent Scores (calculated by summing the percentages of intensities >2+) or H-score for CEACAM5 plasma membrane staining (whole or polarized) in tumor cells.
  • the cancer is selected from hepatocellular carcinoma, colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer (e.g., non-squamous non-small cell lung cancer), uterus cervix cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer (for instance cholangiocarcinoma), prostate cancer, neuroendocrine cancer and skin cancer.
  • GEJ gastroesophageal junction adenocarcinoma
  • the cancer may be selected from a colorectal cancer, a gastric cancer, a gastroesophageal junction adenocarcinoma (GEJ), an esophageal cancer, a pancreatic cancer and a lung cancer.
  • GEJ gastroesophageal junction adenocarcinoma
  • esophageal cancer a pancreatic cancer and a lung cancer.
  • the cancer may be a colorectal cancer.
  • the cancer may be a pancreatic cancer.
  • the cancer may be selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, and lung cancer.
  • GEJ gastroesophageal junction
  • the cancer may be a gastric cancer, a gastroesophageal junction (GEJ) adenocarcinoma, or an esophageal cancer.
  • GEJ gastroesophageal junction
  • the cancer is gastric cancer or gastroesophageal junction adenocarcinoma (GEJ).
  • GEJ gastroesophageal junction adenocarcinoma
  • the cancer is a gastric cancer.
  • the cancer is a lung cancer.
  • a lung cancer may be a non-squamous non-small-cell lung cancer (NSQ NSCLC).
  • Non-small cell lung cancer is a disease in which malignant (cancer) cells form in the tissues of the lung. Smoking is the major cause of the disease. This is a type of epithelial lung cancer other than small cell lung carcinoma.
  • the non-squamous non-small cell lung cancer may be an advanced or metastatic NSQ NSCLC.
  • the subject is a patient with malignant tumor, in particular with a malignant solid tumor, and more specifically with locally advanced or metastatic solid malignant tumor.
  • a metastatic solid malignant tumor may be a metastatic cancer, for example a metastatic carcinoma.
  • a cancer or a carcinoma may be as above indicated.
  • the non-squamous non-small cell lung cancer has no epidermal growth factor receptor (EGFR) sensitizing mutation or v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation or anaplastic lymphoma kinase/c-ros oncogene 1 (ALK/ROS) alterations.
  • EGFR epidermal growth factor receptor
  • BRAF v-raf murine sarcoma viral oncogene homolog B1
  • ALK/ROS anaplastic lymphoma kinase/c-ros oncogene 1
  • the administration of the ADC may be carried out by parenteral route.
  • a suitable parenteral route may be intravenous infusion.
  • the present disclosure also relates to an ADC for the manufacture of a medicament for the treatment of cancer, the combination comprising.
  • the present disclosure relates to a pharmaceutical composition comprising (i) an ADC as disclosed herein, and a pharmaceutically acceptable excipient.
  • the ADC of the disclosure may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • Another object of the disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an ADC of the disclosure and a pharmaceutically acceptable carrier or excipient.
  • ADC or an immunoconjugate according to the disclosure for use as a medicament.
  • the disclosure also relates to an ADC according to the disclosure, for use as for treating cancer.
  • “Pharmaceutical excipient” or “pharmaceutically acceptable excipient” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • “pharmaceutically-acceptable carriers or excipients” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible.
  • suitable carriers, diluents and/or excipients include one or more of water, amino acids, saline, phosphate buffered saline, buffer phosphate, acetate, citrate, succinate; amino acids and derivates such as histidine, arginine, glycine, proline, glycylglycine; inorganic salts NaCI, calcium chloride; sugars or polyalcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as Polysorbate 80, polysorbate 20, poloxamer 188; and the like, as well as combination thereof.
  • isotonic agents such as sugars, polyal
  • compositions The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and gender of the subject, etc.
  • compositions of the disclosure can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.
  • pharmaceutical compositions and combinations of the disclosure are formulated for intravenous administration.
  • the pharmaceutical compositions contain vehicles or excipients, which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles or excipients which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical composition can be administrated through drug combination devices.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • an effective amount of antibodydrug conjugate comprising an anti-CEACAM5-antibody and/or of an anti-CTLA4 antibody and/or of an anti-PD-1 antibody or anti-PD-L1 antibody may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and injectable with the appropriate device or system for delivery without degradation. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the antibody-drug conjugate comprising an anti-CEACAM5-antibody can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, glycine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the subsequent of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions for parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the antibody-drug conjugate comprising an anti-CEACAM5-antibody formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used.
  • liposomes and/or nanoparticles are contemplated for the introduction of polypeptides into host cells.
  • the formation and use of liposomes and/or nanoparticles are known to those of skill in the art.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles, or biodegradable polylactide or polylactide co glycolide nanoparticules that meet these requirements are contemplated for use in the present disclosure, and such particles may be easily made.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)).
  • MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
  • an “effective amount” or “therapeutically effective amount” is a dose of the therapeutic that results in treatment of CEACAM5 expressing cancer (e.g. lung cancer, gastric cancer, gastroesopageal junction cancer or esophageal cancer).
  • “treating” refers to causing a detectable improvement in one or more symptoms associated with CEACAM5 expressing cancer (e.g. lung cancer ) or causing a biological effect (e.g., a decrease in the level of a particular biomarker) that is correlated with the underlying pathologic mechanism(s) giving rise to the condition or symptom(s).
  • a dose of anti- CEACAM5 ADC which causes an improvement in any of the following symptoms or conditions associated with CEACAM5 expressing cancer is deemed a "therapeutically effective amount”:
  • a treatment has not been effective when a dose of anti- CEACAM5 ADC does not result in a detectable improvement in one or more parameters or symptoms associated with a CEACAM5 expressing cancer (e.g., lung cancer, gastric cancer, gastroesophageal junction cancer or esophageal cancer) or which does not cause a biological effect that is correlated with the underlying pathologic mechanism(s) giving rise to the condition or symptom(s) of cancer.
  • a CEACAM5 expressing cancer e.g., lung cancer, gastric cancer, gastroesophageal junction cancer or esophageal cancer
  • the anti-CEACAM5 ADC is administered intravenously.
  • a therapeutically effective amount of anti-CEACAM5 ADC that is administered to the subject will vary depending upon the age and the size (e.g., body weight or body surface area) of the subject as well as the route of administration and other factors well known to those of ordinary skill in the art.
  • the dose of the ADC varies depending on the body surface area of the subject.
  • the dose of anti-CEACAM5 ADC administered to the subject is from about 1 mg/m 2 to about 500 mg/m 2 .
  • the dose of the ADC administered to the subject is from about 5 mg/m 2 to about 300 mg/m 2 .
  • the dose of the ADC administered to the subject is from about 5 mg/m 2 to about 250 mg/m 2 .
  • the dose of the ADC administered to the subject is from about 60 mg/m 2 to about 190 mg/m 2 .
  • the dose is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 210 mg/m 2 based on the body surface area of the subject.
  • the dose of the ADC is about 100 mg/m 2 .
  • the dose of the ADC is about 150 mg/m 2 .
  • the dose of the ADC is about 170 mg/m 2 .
  • the dose of the ADC is about 190 mg/m 2 .
  • the dose of the ADC is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 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 dose of the ADC is 100 mg/m 2 . In certain embodiments, the dose of the ADC is 150 mg/m 2 . In certain embodiments, the dose of the ADC is 170 mg/m 2 . In certain embodiments, the dose of the ADC is 190 mg/m 2 .
  • the dose escalation phase population was enriched for, but not restricted to, patients with tumor types known to express CEACAM5, and expression was confirmed retrospectively in a central laboratory using IHC on the most recent archival tissue samples.
  • the expansion phase population was restricted to separate cohorts of patients with advanced colorectal cancer, NSQ-NSCLC, small-cell lung cancer, and gastric adenocarcinoma.
  • CEACAM5 high expressers defined as patients with CEACAM5 expression at an intensity > 2+ in > 50% of the tumor cell population
  • CEACAM5 moderate expressers defined as patients with CEACAM5 expression at an intensity > 2+ in > 1% to ⁇ 50% of the tumor cell population.
  • IV intravenous
  • Tusamitamab ravtansine 100 mg/m2 Q2W as determined during the dose escalation phase of this study [15].
  • Tusamitamab ravtansine was infused at 2.5 mg/min for 30 minutes, then 5 mg/min thereafter, provided there were no signs or symptoms of a hypersensitivity reaction.
  • T reatment was continued until disease progression, unacceptable toxicity, or patient willingness to stop.
  • the primary outcome during expansion phase assessed the objective response rate per RECIST v1 .1 criteria.
  • Exploratory biomarker objectives included exploring the potential link between CEACAM5 expression characteristics and response (expansion phase), investigating potential biomarkers (other than CEACAM5) that might predict tusamitamab ravtansine activity (expansion phase), and evaluating the potential for circulating CEA levels to be a convenient companion diagnosis for tusamitamab ravtansine treatment (by evaluating correlation between circulating CEA levels and tumor CEACAM5 expression and correlation between this biomarker and treatment response).
  • Tumor samples were obtained using the latest archival tumor samples (ie, tumor tissue archived at diagnosis, at surgery, or taken before patient inclusion in the study and not undergoing anticancer treatment); fresh biopsy at baseline was optional in patients who had a lesion amenable for biopsy.
  • CEACAM5 reactivity was assessed in tumor cells using semi-quantitative Percent Scores (calculated by summing the percentages of intensities >2+) or H-score for CEACAM5 plasma membrane staining (whole or polarized); CEACAM5 cytoplasmic staining was also evaluated (centrally only).
  • FFPE tumor tissues were cut at 10 pm thickness using a microtome, and 3 sections were mounted on adhesive microscope slides. After trimming excess paraffin off the sample slide using a sterile scalpel, the tumor tissue was macrodissected and collected in individual Eppendorf DNA LoBind tubes. Genomic DNA and Total RNA from each human lung cancer FFPE tissue sample was co-extracted using the ALLPREP® DNA/RNA FFPE kit (ref. 80234, QIAGEN), according to the manufacturer’s instructions, with initial manual processing steps followed by final automated extraction steps using the QIACUBE automated nucleic acid purification instrument. Genomic DNA was eluted in 30 pL ATE buffer and Total RNA eluted in 20 pL RNase-free water.
  • RNA-seq Gene expression
  • RNA samples with known CEACAM5 expression level by IHC were analyzed using RNA-seq.
  • RNA samples from 57 FFPE patient biopsies were sequenced by using KAPA mRNA HYPERPREPKITILLUMINA® Platform.
  • RNA-seq data was processed as follows: sequencing reads were mapped to the reference genome GRCh 38 using Spliced Transcripts Alignment to a Reference (STAR) aligner [25].
  • RNA-seq included microenvironment cell populations [MCP] counter analysis, according to published methods [28, 29],
  • median age was 62.5 years (range, 31-91 years; 42% were >65 years of age); 51 % were male; 72% had ECOG PS >1 ; patients had received a median of 3 prior lines of therapy (range, 1-10 lines) for advanced disease, including antitubulin agents (61%) and anti-PD1/PD-L1 agents (75%).
  • CEACAM5 expression pattern of staining and distribution in tumors
  • High CEACAM5 expressers showed a predominance of whole over polarized membrane expression. In contrast, whole and polarized membrane expression was similar among moderate CEACAM5 expressers. (Table 1).
  • CEACAM5 expression pattern of staining and distribution in tumors
  • Adenocarcinoma 91 (98.9) 63 (98.4) 28 (100)
  • CEACAM5 carcinoembryonic antigen-related cell adhesion molecule 5
  • NSCLC non-small cell lung cancer
  • SD standard deviation.
  • the other CEACAM family member expression or other gene expression are not significantly associated with CEACAM5 IHC after multiple testing correction (Table 2).
  • Table 2 RNA-seq gene expression fold changes in high versus moderate CEACAM5 expression by IHC: The top 10 ranking genes, including the closest ranking other CEACAM family member.
  • CEACAM carcinogembryonic antigen-related cell adhesion molecule
  • CL confidence limit
  • logFC log fold change
  • EXAMPLE 3 Discussion [0662] Almost 20% of prescreened patients with NSCLC had high CEACAM5 expression as measured by IHC, i.e., staining intensity > 2+ in > 50% of tumor cells. An enrichment of clinical responses in patients having a high CEACAM5 protein expression level and being treating with tusamitamab ravtansine was observed.
  • CEACAM5 mRNA expression was significantly upregulated in patients with high vs moderate CEACAM5 protein expression, but other genes, including other CEACAM family genes were not ( Figure 1 and Table 1).
  • CEACAM5 mRNA level is a good biomarker for pre-selecting patients in need of a cancer treatment cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for a further step of selection with CEACAM5 immunohistochemistry (IHC) staining and treating patients in need of a cancer treatment cancer with the ADC.
  • ADC antibody-drug conjugate
  • IHC immunohistochemistry

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Abstract

The disclosure relates to methods and uses for selecting and treating patients with cancer, where the cancer expresses CEACAM5. The methods include determining a log2- transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject, (ii) comparing said value with a reference value, and (iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value. The agent for treating selected subjects can be tusamitamab ravtansine. Such agent can be used in combination with one or more additional agents to treat the cancer. In certain embodiments the cancer is non-squamous non- small cell lung cancer (NSQ NSCLC)

Description

[TITLE]
CEACAM5 mRNA ASSAY FOR PATIENT SELECTION IN CANCER THERAPY
[REFERENCE TO SEQUENCE LISTING]
[0001] The instant application contains a Sequence Listing which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy, created on March 12, 2024, is named PI022916.WO SANOFI (S327) LISTING. xml.
[TECHNICAL FIELD]
[0002] The present disclosure relates to the field of cancer therapy.
[TECHNICAL BACKGROUND]
[0003] Despite recent advances in the treatment of cancer, there remains a need for effective new treatment at the time of disease progression after first-line therapy. Current therapeutic approaches for subsequent systemic options combining an inhibitor of angiogenesis with a systemic cytotoxic agent such as docetaxel entail serious hematological and other toxicities. Docetaxel, pemetrexed, or gemcitabine used as single cytotoxic agents offer very limited other options. Therefore, targeted cytotoxic therapies may offer an improvement in safety and tolerability as well as efficacy.
[0004] Lung cancer is the leading cause of cancer-related mortality globally [1]. In the United States, non-squamous non-small cell lung cancer (NSQ-NSCLC) comprises 45.4% of new lung cancer diagnoses, and among these, 47.7% have distant metastases [2] with an associated poor five-year relative survival of 5-8% [3].
[0005] While some lung cancers respond to therapies targeting a variety of oncogene drivers [4] or to immune checkpoint inhibitors ( ICIs) [5], a continuing unmet need exists for novel therapies for patients whose lung cancers are without actionable targets or are non-responsive to ICIs.
[0006] Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are cell-surface glycoproteins involved in cell adhesion, cell signaling, and promotion of cancer progression and metastasis [6]. Consequently, they are promising targets for novel anticancer agents. [0007] Although different CEACAMs show specific expression patterns across tissues, they demonstrate similarity with respect to structural homology and amino acid sequences [7], This requires 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 ). High levels of CEACAM5 expression have since been observed in several epithelial tumors, whereas in normal adult tissue, its expression is limited to few tissues (Hammarstrom S., Semin Cancer Biol. 1999;9(2) :67-81 ; Thompson JA., Tumor Biol. 1995;16(1 ):10-16).
[0009] Among CEACAMs, therapies targeting CEACAM5 are of particular interest in lung cancer. CEACAM5 is overexpressed in many epithelial tumors [8], which facilitates tumorigenesis and metastasis [9]. CEACAM5 is expressed at higher levels in NSCLC but is not expressed in normal lung tissue [7, 10]. In addition, higher expression of CEACAM5 in human NSCLC tissue is correlated with worse histological grade [10], and higher expression of CEACAM5 is associated with poor survival in patients with NSCLC. [11 , 12] Taken together, these findings suggest that CEACAM5 is a promising target for antibodydrug conjugate (ADC) therapy.
[0010] Antibody-drug conjugates (ADCs) have shown promise in improving outcomes in patients with lung cancer [13]. Tusamitamab ravtansine (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 is composed of an anti-CEACAM5 humanized monoclonal antibody conjugated to a potent cytotoxic maytansinoid N2’- deacetyl-N-2’(4-methyl-4-mercapto-1 -oxopentyl)-maytansine (DM4) payload via a cleavable linker [7], The antibody component of tusamitamab ravtansine binds to the CEACAM5 extracellular domain, which is followed by internalization of the ADC and subsequent release of DM4 into the tumor cell [14], DM4 inhibits microtubule assembly, resulting in cell cycle arrest and apoptosis [14], Both DM4 and its active metabolite, S- methyl-DM4, cross the cell membrane, meaning that the cytotoxic effect of tusamitamab ravtansine is both direct via specific binding to CEACAM5-expressing tumors cells and indirect via local diffusion of DM4 and S-methyl-DM4 (the bystander effect) [14],
[0011] Encouraging preliminary anti-tumor activity of tusamitamab ravtansine in participants heavily pre-treated for NSQ NSCLC has been demonstrated in an ongoing study (TED13751). [0012] In the dose escalation phase of a first-in-human Phase 1/2 study of tusamitamab ravtansine among patients with advanced solid tumors for which no standard alternative therapy was available (ClinicalTrials.gov NCT02187848), the primary doselimiting toxicity was reversible keratopathy, and the maximum tolerated dose was 100 mg/m2 every 2 weeks. [15].
[0013] In the expansion phase of the same study among patients with advanced NSQ-NSCLC expressing high levels of CEACAM5, defined as at least 50% of tumor cells with 2+ or 3+ staining intensity on immunohistochemistry (IHC), tusamitamab ravtansine demonstrated a promising objective response rate of 20.3% and a favorable safety profile with the most common treatment-emergent adverse events being asthenia, reversible corneal events, peripheral neuropathy, dyspnea, and diarrhea, with infrequent hematological toxicities compared with those reported with docetaxel [16]; in addition, among patients who achieved a partial response, 47% were treated for longer than 1 year, suggesting that response to tusamitamab ravtansine was durable and frequently sustained [17].
[0014] While these clinical trial data show promise, in clinical practice, potential barriers for the pre-screening of patients with cancer, e.g., NSQ-NSCLC, likely to respond to CEACAM5 targeted therapies include the availability of archival tumor biopsies for measurement of CEACAM5 expression by immunohistochemistry.
[0015] From the aforementioned Phase 1/2 study, we explored biomarker associations with: 1 ) tumor CEACAM5 expression by immunohistochemistry, with tumor CEACAM5 mRNA levels ; and 2) whether CEACAM5 mRNA predicted tumor objective response rate.
[SUMMARY]
[0016] As shown in the Examples, there was an enrichment of clinical responses in patients having a high CEACAM5 protein expression level and being treated with tusamitamab ravtansine (tusamitamab ravtansine responders).
[0017] Within the disclosure, the expression “an enrichment of clinical responses in patients” with regard to a treatment with tusamitamab ravtansine intends to refer to an increase of the overall response rate (ORR) following administration of the given treatment. The ORR is defined as the proportion of patients who have a partial or complete response to therapy; it does not include stable disease and is a direct measure of drug tumoricidal activity. [0018] Further, a correlation was found between the CEACAM5 protein expression level in the tumor (measured by immunohistochemistry (IHC) staining) and the CEACAM5 tumor mRNA level. The CEACAM5 mRNA expression was significantly upregulated in patients with high vs moderate CEACAM5 protein expression. Further, there was an enrichment of clinical responses in patients with high CEACAM5 tumor mRNA level and high CEACAM5 immunohistochemistry (IHC) staining (i.e., at > 2+ intensity in > 50% of tumor cells) in tusamitamab ravtansine responders.
[0019] Since an enrichment of clinical responses with high CEACAM5 protein expression was shown and an association between the CEACAM5 protein expression and the mRNA CEACAM5 expression level was shown, then the mRNA CEACAM5 expression level was shown to be a good biomarker, predictive of an enrichment of clinical responses, in patients to be treated with an anti-CEACAM5 antibody-drug conjugate, such as tusamitamab ravtansine; alternatively the results show that patients can be selected based on CEACAM5 mRNA level instead of IHC.
[0020] The Example section therefore supports the use of CEACAM5 mRNA level as a biomarker for selecting and treating patients in need of a cancer treatment with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
[0021] Further, the Example section supports the use of CEACAM5 mRNA level as a biomarker for selecting patients in need of a cancer treatment with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for a further selection step with a CEACAM5 immunohistochemistry (IHC) staining and subsequent treatment of patients in need of a cancer treatment cancer with the ADC.
[0022] It is of general knowledge for a skilled person that the protein expression level does not necessary correlate with the expression level of the corresponding mRNA.
[0023] The disclosure is based at least in part on the observation that particular values of CEACAM5 gene expression level, for example of CEACAM5 gene transcript level, for example of CEACAM5 mRNA level, for example of Iog2-transformed, quantile- normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA may be useful as a biomarker for selection of patients for treatment of cancers which typically express CEA Cell Adhesion Molecule 5 (CEACAM5) on their tumoral cells.
[0024] In some embodiments, the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0025] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0026] (ii) comparing said determined value with a reference value, and
[0027] (iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value.
[0028] Within the disclosure, a subject in need thereof is a subject having a cancer. An isolated sample of a tumor of said cancer may be used.
[0029] Herein the terms “subject”, “patient”, “subject in need thereof” and “patient in need thereof” are used interchangeably.
[0030] In some embodiments, the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0031] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0032] (ii) comparing said value determined at step (i) with a reference value of a CEACAM5 gene expression level,
[0033] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0034] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0035] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0036] (vi) selecting said subject for a treatment of a cancer if the intensity determined at step (iv) is above the reference intensity.
[0037] The samples tumor of step (i) and step (iv) may be the same sample or different samples.
[0038] In some embodiments, the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody- drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0039] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0040] (ii) comparing said determined value with a reference value,
[0041 ] (iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value, and
[0042] (iv) administering to said selected subject an effective amount of said ADC.
[0043] In some embodiments, the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibodydrug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0044] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0045] (ii) comparing said value determined at step (i) with a reference value of a CEACAM5 gene expression level,
[0046] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0047] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0048] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0049] (vi) selecting said subject for a treatment of a cancer if the intensity determined at step (iv) is above the reference intensity, and
[0050] (vii) administering to said selected subject an effective amount of said ADC.
[0051 ] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
[0052] the use comprising: [0053] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0054] (ii) comparing said determined value with a reference value, and
[0055] (iii) administering to said subject an effective amount of said ADC if said determined value is above the reference value.
[0056] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
[0057] the use comprising:
[0058] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0059] (ii) comparing said value determined at step (i) with a reference value of a CEACAM5 gene expression level,
[0060] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0061 ] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0062] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0063] (vi) administering to said subject an effective amount of said ADC if said determined intensity is above the reference intensity.
[0064] In some embodiments, the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for characterizing said tumor as a CEACAM5-high expresser tumor.
[0065] In some embodiments, the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test.
[0066] In some embodiments, the present disclosure relates to a use of a measure of a value of CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a treatment of a cancer with a CEACAM5 targeting therapeutic agent, for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
[0067] In some embodiments, the present disclosure relates to a use of a measure of a value of CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test, said staining test being for selecting said subject for a treatment of a cancer with a CEACAM5 targeting therapeutic agent, for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent
[0068] In some embodiments, the value of the CEACAM5 gene expression level may be a measure of a CEACAM5 gene transcript.
[0069] In some embodiments, the CEACAM5 gene transcript may be a mRNA.
[0070] In some embodiments, methods and uses of the disclosure comprise a step of determining a value of a CEACAM5 mRNA level in an isolated sample of a tumor.
[0071 ] According to one of its objects, the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0072] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0073] (ii) comparing said value with a reference value, and
[0074] (iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value.
[0075] In some embodiments, the value of the CEACAM5 gene expression level may be a measure of a CEACAM5 gene transcript.
[0076] In some embodiments, the CEACAM5 gene transcript may be a mRNA.
[0077] According to one of its objects, the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of: [0078] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0079] (ii) comparing said value determined at step (i) with a reference value,
[0080] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0081 ] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0082] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0083] (vi) selecting said subject for a treatment of a cancer if the determined intensity is above the reference intensity.
[0084] According to another of its objects, the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0085] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0086] (ii) comparing said value with a reference value,
[0087] (iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value, and
[0088] (iv) administering to said selected subject an effective amount of said ADC.
[0089] Thereby the cancer may be treated.
[0090] According to another of its objects, the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0091] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject, [0092] (ii) comparing said value determined at step (i) with a reference value,
[0093] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0094] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0095] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0096] (vi) selecting said subject for a treatment of a cancer if the determined intensity is above the reference intensity, and
[0097] (vii) administering to said selected subject an effective amount of said ADC.
[0098] According to another of its objects, the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
[0099] the use comprising (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject, (ii) comparing said value with a reference value, and (iii) administering to said subject an effective amount of said ADC if the determined value is above the reference value.
[0100] According to another of its objects, the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
[0101 ] the use comprising :
[0102] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0103] (ii) comparing said value determined at step (i) with a reference value,
[0104] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0105] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject, [0106] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0107] (vi) administering to said subject an effective amount of said ADC if the determined intensity is above the reference intensity.
[0108] As shown in the Examples section, using ribonucleic acid (RNA) sequencing, expression of around 15,000 genes, including CEACAM5, was evaluated. The RNA data were filtered and defined as log-transformed quantile-normalized transcripts per kilobase million (TPM). It has been observed that differential gene expression analysis has identified CEACAM5 mRNA to be the most associated gene (and the only significant one adjusted P = 0.00265) with CEACAM5 high vs moderate expression by IHC. CEACAM5 mRNA transcripts per million are elevated in high vs moderate CEACAM5 expression; and (C) CEACAM5 mRNA correlates with CEACAM5 IHC H-score. Accordingly, the gene expression level of CEACAM5 (RNA) may be used to identify and select patients responsive to CEACAM5 targeting treatment, such as an antibody-drug conjugate comprising an anti- CEACAM5-antibody conjugated to a cytotoxic agent.
[0109] In the disclosure, “antibody-drug conjugate comprising an anti-CEACAM5- antibody conjugated to a cytotoxic agent”, “antibody-drug conjugate” and “ADC” are used interchangeably.
[0110] In some embodiments, the ADC may be used in an effective amount.
[0111] In some embodiments, the use may be in a subject in need thereof.
[0112] In a use as disclosed herein, the biomarker CEACAM5 mRNA is measured in an isolated biological sample.
[0113] In some embodiments, the anti-CEACAM5 antibody may comprise a HCDR1 having the amino acid sequence of SEQ ID NO: 1 , a HCDR2 having the amino acid sequence of SEQ ID NO: 2, a HCDR3 having the amino acid sequence of SEQ ID NO: 3, a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence NTR, and a LCDR3 having the amino acid sequence of SEQ ID NO: 5.
[0114] In some embodiments, the anti-CEACAM5 antibody may comprise a variable domain of a heavy chain (VH) consisting of SEQ ID NO: 6 and a variable domain of a light chain (VL) consisting of SEQ ID NO: 7.
[0115] In some embodiments, the anti-CEACAM5 antibody may be tusamitamab. [0116] In some embodiments, the cytotoxic agent may be selected from the group consisting of radioisotopes, protein toxins, small molecule toxins, and any combination thereof.
[0117] In some embodiments, the small molecule toxin may be selected from antimetabolites, DNA-alkylating agents, DNA-cross-linking agents, DNA-intercalating agents, anti-microtubule agents, topoisomerase inhibitors, and any combination thereof.
[0118] In some embodiments, the anti-microtubule agent may be selected from taxanes, vinca alkaloids, maytansinoids, colchicine, podophyllotoxin, griseofulvin, and any combination thereof.
[0119] In some embodiments, the cytotoxic agent may be maytansinoid or maytansinoid analog.
[0120] In some embodiments, the maytansinoid may be selected from N2’-deacetyl- N2’-(3-mercapto-1 -oxopropyl)-maytansine (DM1 ), N2’-deacetyl-N2’-(4-methyl-4-mercapto- 1 -oxopentyl)-maytansine (DM4), and combinations thereof.
[0121] In some embodiments, the anti-CEACAM5 antibody may be covalently attached via a cleavable or non-cleavable linker to the at least one chemotherapeutic agent.
[0122] In some embodiments, linker may be selected from N-succinimidyl pyridyldithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and succinimidyl(N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC).
[0123] In some embodiments, the anti-CEACAM5 antibody may be covalently attached via a cleavable linker to the at least one chemotherapeutic agent, the linker being selected from N-succinimidyl pyridyldithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2- sulfo-butyric acid (sulfo-SPDB), and succinimidyl(N-maleimidomethyl) cyclohexane-1 - carboxylate (SMCC).
[0124] 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), and which may be covalently linked to N2’-deacetyl-N-2’(4-methyl-4- mercapto-1 -oxopentyl)-maytansine (DM4) via N-succinimidyl pyridyldithiobutyrate (SPDB).
[0125] In some embodiments, the antibody-drug conjugate may be characterized by a drug-to-antibody ratio (DAR) ranging from 1 to 10.
[0126] In some embodiments, the antibody-drug conjugate may be tusamitamab ravtansine. [0127] In some embodiments, the reference value may be a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA.
[0128] In some embodiments, the reference value may be at least from about 7 to about 13.
[0129] In some embodiments, the reference value may be at least about 7, or may be at least about 8, or at least about 9, or at least about 10, or at least about 11 , or at least about 12, or at least about 13.
[0130] In some embodiments, the quantile normalization may be obtained by (i) ranking the transcripts of the sample by level of expression, (ii) calculating an average value for genes occupying a same rank, and (iii) substituting the values of all genes occupying said same rank with this average value.
[0131] In some embodiments, the level of expression of the transcripts may be measured in Fragments Per Kilobase Million (FPKM) before being converted in TPM.
[0132] In some embodiments, the Fragments Per Kilobase Million (FPKM) may be obtained by counting the total transcripts in said sample, dividing the obtained transcripts counts by 1 ,000,000, and dividing the obtained values by the length of the gene, in kilobases.
[0133] In some embodiments, the cancer may be selected from the group consisting of hepatocellular carcinoma, colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer, uterine cervix cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer, prostate cancer, neuroendocrine cancer, and skin cancer.
[0134] In some embodiments, the cancer may be selected from the group consisting of colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer, uterine cervix cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer, prostate cancer, neuroendocrine cancer, and skin cancer.
[0135] In some embodiments, the cancer may be selected from a colorectal cancer, a gastric cancer, a gastroesophageal junction adenocarcinoma (GEJ), an esophageal cancer, a pancreatic cancer and a lung cancer.
[0136] In some embodiments, the cancer may be a gastric cancer, a gastroesophageal junction (GEJ) adenocarcinoma, or an esophageal cancer.
[0137] In some embodiments, the cancer may be gastric cancer. [0138] In some embodiments, the cancer may be colorectal cancer.
[0139] In some embodiments, the cancer may be pancreatic cancer.
[0140] In some embodiments, the cancer may be gastroesophageal junction adenocarcinoma (GEJ).
[0141] In some embodiments, the cancer may be lung cancer.
[0142] In some embodiments, the lung cancer may be a non-squamous non-small cell lung cancer (NSQ NSCLC).
[0143] In some embodiments, the non-squamous non-small cell lung cancer may be an advanced or metastatic NSQ NSCLC.
[0144] In some embodiments, the non-squamous non-small cell lung cancer has no epidermal growth factor receptor (EGFR) sensitizing mutation or v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation or anaplastic lymphoma kinase/c-ros oncogene 1 (ALK/ROS) alterations.
[0145] In some embodiments, the anti-CEACAM5 antibody may be tusamitamab.
[0146] In some embodiments, the ADC may be tusamitamab ravtansine.
[0147] In some embodiments, the ADC may be administered in a dose of > 80 mg/m2, relative to the surface body area of said subject, about once every two weeks, or the ADC may be administered in a dose of > 80 mg/m2, relative to the surface body area of said subject about once every three weeks.
[0148] In some embodiments, the ADC may be administered in a dose of 80 mg/m2 to 210 mg/m2, 80 mg/m2 to 170 mg/m2, or at a dose of 80 mg/m2 to 150 mg/m2, or at a dose of 80 mg/m2 to 120 mg/m2, or at a dose of 80 mg/m2 to 100 mg/m2.
[0149] In some embodiments, the ADC may be administered in a dose of 80, 100, 120, 150, 170, 180, or 210 mg/m2.
[0150] In some embodiments, the method or the use as described herein may further comprise administering to the subject an effective amount of at least one additional agent effective to treat the cancer.
[0151] In some embodiments, the additional agent may be selected from the group consisting of an immune checkpoint inhibitor (ICI), a platinum-based chemotherapy, pemetrexed, anti-VEGFR2, FOLFOX, FOLFIRI, TAS-102, anti-EGFR, and any combination thereof. [0152] In some embodiments, the ICI may be an anti-PD-1 antibody or an anti-PD- L1 antibody.
[0153] In some embodiments, the anti-PD-1 antibody may be selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sintilimab, dostarlimab, and tislelizumab.
[0154] In some embodiments, the anti-PD-L1 antibody may be selected from the group consisting of atezolizumab, avelumab, and durvalumab.
[0155] In some embodiments, the subject an effective amount of tusamitamab ravtansine and pembrolizumab.
[0156] In some embodiments, the method or the use as described herein may further comprise administering to the subject an effective amount of a platinum-based chemotherapy.
[0157] In some embodiments, the platinum-based chemotherapy may be selected from cisplatin and carboplatin.
[0158] In some embodiments, the method or the use as described herein may further comprise administering to the subject an effective amount of pemetrexed.
[DESCRIPTION OF THE FIGURES]
[0159] Figure 1 : shows CEACAM5 mRNA transcripts per million are elevated in high vs moderate CEACAM5 expression.
[0160] Figure 2: shows CEACAM5 mRNA correlation with CEACAM5 IHC H-score.
[0161] Figure 3: Shows CEACAM5 mRNA level correlation with CEACAM5 expression by immunohistochemistry recorded as the sum of the percentage of the tumoral cells expressing the target at least 2+ intensity according to patient response to tusamitamab ravtansine treatment.
[0162] Figure 4: Shows CEACAM5 mRNA level correlation with CEACAM5 expression by immunohistochemistry recorded as the sum of the percentage of the tumoral cells expressing the target at least 2+ intensity in patients who responded to tusamitamab ravtansine treatment.
[0163] Figure 5: Shows a box plots of CEACAMs family members expression according to clinical responses in patients treated with tusamitamab ravtansine. [BRIEF DESCRIPTION OF THE SEQUENCES]
[0164] SEQ ID NO: 1 -5 show the sequences CDR-H1 , CDR-H2, CDR-H3, CDR-L1 and CDR-L3 of the anti-CEACAM5-antibody (huMAb2-3).
[0165] SEQ ID NO: 6 shows the sequence of the variable domain of the heavy chain (VH) of the anti-CEACAM5-antibody (huMAb2-3).
[0166] SEQ ID NO: 7 shows the sequence of the variable domain of the light chain (VL) of the anti-CEACAM5-antibody (huMAb2-3).
[0167] SEQ ID NO: 8 shows the heavy chain sequence of the anti-CEACAM5- antibody (huMAb2-3).
[0168] SEQ ID NO: 9 shows the light chain sequence of the anti-CEACAM5- antibody (huMAb2-3).
[DETAILED DESCRIPTION]
Definitions
[0169] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, may provide one of skill with a general dictionary of many of the terms used in this disclosure. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well- known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. [0170] Units, prefixes, and symbols are denoted in their International System Units (Systeme International des Unites (SI)) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
[0171] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
[0172] Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of" and/or "consisting essentially of" are also provided.
[0173] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence," is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.
[0174] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: 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).
[0175] The term “approximately” or "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some embodiments, the term indicates deviation from the indicated numerical value by ±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%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±10%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.9%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.05%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.01 %.
[0176] An “antibody” may 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 a disulfide bond. There are two types of light chain, lambda (I) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs therefore refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1 -L, CDR2-L, CDR3-L and CDR1 -H, CDR2-H, CDR3-H, respectively. A conventional antibody antigenbinding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
[0177] As used herein, the term “antibody” intends to refer to conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular variable heavy chain of single domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies. Fragments of antibody considered herein are antigen-binding fragments.
[0178] “Framework Regions” (FRs) refer to amino acid sequences interposed between CDRs, i.e., to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1 -L, FR2-L, FR3-L, FR4-L, and FR1 -H, FR2-H, FR3-H, FR4-H, respectively. A human framework region is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.
[0179] In the context of the disclosure, CDR/FR definition in an immunoglobulin light or heavy chain is to be determined based on IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1 ):55-77; www.imgt.org).
[0180] As used herein, antibody or immunoglobulin also includes “single domain antibodies” which have been more recently described and which are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. Single domain antibodies may be naturally occurring single domain antibodies known as heavy chain antibody devoid of light chains. In particular, camelidae species, for example camel, dromedary, llama, alpaca and guanaco, produce heavy chain antibodies naturally devoid of light chain. Camelid heavy chain antibodies also lack the CH1 domain. [0181] The variable heavy chain of these single domain antibodies devoid of light chains are known in the art as “VHH” or “Nanobody®”. Similar to conventional VH domains, VHHs contain four FRs and three CDRs. VHH have advantages over conventional antibodies: they are about ten times smaller than IgG molecules, and as a consequence properly folded functional VHH can be produced by in vitro expression while achieving high yield. Furthermore, VHH are very stable, and resistant to the action of proteases. The properties and production of VHH have been reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 Nov;77(1 ):13-22).
[0182] The term "monoclonal antibody" or “mAb” as used herein refers to an antibody molecule of a single amino acid sequence, which is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e., produced by protein engineering.
[0183] The term "humanized antibody" refers to an antibody which is wholly or partially of non-human origin, and which has been modified to replace certain amino acids, in particular in the framework regions of the VH and VL domains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most of the time human CH and CL domains.
[0184] “Fragments” of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.
[0185] The term “Fab” denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of the heavy chain and the entire light chain are bound together through a disulfide bond. It is usually obtained among fragments by treating IgG with a protease, such as papain.
[0186] The term “F(ab')2” refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than 2 identical Fab fragments bound via a disulfide bond of the hinge region. It is usually obtained among fragments by treating IgG with a protease, such as pepsin. [0187] The term “Fab'“ refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.
[0188] A single chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. The human scFv fragment of the disclosure includes CDRs that are held in appropriate conformation, in particular by using gene recombination techniques. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. “dsFv” is a VH::VL heterodimer stabilized by a disulphide bond. “(dsFv)2” denotes two dsFv coupled by a peptide linker.
[0189] The term “bispecific antibody” or “BsAb” denotes an antibody which combines the antigen-binding sites of two antibodies within a single molecule. Thus, BsAbs are able to bind two different antigens simultaneously. Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions as described for instance in EP 2 050 764 A1 .
[0190] The term “multispecific antibody” denotes an antibody which combines the antigen-binding sites of two or more antibodies within a single molecule.
[0191] The term "diabodies" refers to small antibody fragments with two antigenbinding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigenbinding sites.
[0192] An amino acid sequence “at least 85% identical to a reference sequence” is a sequence having, on its entire length, 85%, or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of the reference amino acid sequence.
[0193] A percentage of “sequence identity” between amino acid sequences may be determined by comparing the two sequences, optimally aligned over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison is conducted by global pairwise alignment, e.g., using the algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970). The percentage of sequence identity can be readily determined for instance using the program Needle, with the BLOSUM62 matrix, and the following parameters gap-open=10, gap-extend=0.5.
[0194] A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge, size or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include 1 ) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups can also be defined on the basis of amino acid size.
[0195] By "purified" and "isolated" it is meant, when referring to a polypeptide (i.e., the antibody of the disclosure) or a nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein in particular means at least 75%, 85%, 95%, or 98% by weight, of biological macromolecules of the same type are present. An “isolated” nucleic acid molecule which encodes a particular polypeptide refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
[0196] As used herein, the term “subject” or “patient" denotes a mammal, such as a rodent, a feline, a canine, and a primate. In particular, a subject according to the disclosure is a human.
[0197] "Administer" or "administering," as used herein refers to delivering to a subject a composition described herein. The composition can be administered to a subject using methods known in the art. In particular, the composition can be administered intravenously, subcutaneously, intramuscularly, intradermally, or via any mucosal surface, e.g., orally, sublingually, buccally, nasally, rectally, vaginally or via pulmonary route. In some embodiments, the administration is intravenous. In some embodiments, the administration is subcutaneous.
[0198] The terms “treat” or “treatment” or “therapy” refers to the administration of a compound or a composition according to the disclosure with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a disorder, the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, or otherwise arrest or inhibit further development of the disorder in a statistically significant manner. More particularly, “treating” or “treatment” includes any approach for obtaining beneficial or desired results in a subject’s cancer condition. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more cancer symptoms or conditions, diminishment or reduction of the extent of a cancer disease or of a cancer symptom, stabilizing, i.e., not worsening, the state of a cancer disease or of a cancer symptom, prevention of a cancer disease or of a cancer symptom’s spread, delay or slowing of cancer disease or cancer symptom progression, amelioration or palliation of the cancer disease state, diminishment of the reoccurrence of cancer disease, and remission, whether partial or total and whether detectable or undetectable. In other words, "treatment" as used herein includes any cure, amelioration, or reduction of a cancer disease or symptom. A “reduction” of a symptom or a disease means decreasing of the severity or frequency of the disease or symptom, or elimination of the disease or symptom.
[0199] As used herein, the terms “effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes considered. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner and may vary depending on factors such as the type and stage of pathological processes considered, the patient’s medical history and age, and the administration of other therapeutic agents.
[0200] The unit “mg/m2” indicates the amount of compound in mg per m2 of subject body surface administered per dose. The person skilled in the art is aware how to determine the required amount of compound for the subject to be treated based on his body surface, which in turn may be calculated based on height and body weight.
[0201] The unit “mg/kg” indicates the amount of compound in mg per kg of subject body administered per dose. The person skilled in the art is aware how to determine the required amount of compound for the subject to be treated based on his body weight. [0202] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.
[0203] “Biomarker” intends to refer a biological molecule, for example a protein or a metabolite, that is differentially present, increased or decreased, in a biological sample obtained from a subject or a group of subjects having a first phenotype, e.g., having a disease such as a cancer, as compared to a biological sample from a subject or a group of subjects having a second phenotype, e.g., not having the disease. In use, the biomarker is isolated from the subject.
[0204] “Sample” or “biological sample” intends to refer to a biological material isolated from a subject. The biological sample may contain any biological material suitable for detecting the biomarker, i.e., CEACAM5 mRNA, and may comprise cellular and/or non- cellular material from the subject. The sample may be isolated from any suitable biological tissue or fluid such as, for example, kidney tissue, blood, blood plasma (plasma), blood serum (serum), urine, or cerebral spinal fluid (CSF). In some embodiments, a biological sample is a blood plasma (plasma) or a blood serum (serum) sample,
[0205] A “value of reference” or a “threshold value” intends to refer to a level of CEACAM5 mRNA that is indicative of a particular disease state, phenotype, such as cancer, or lack thereof, as well as combinations of disease states, phenotypes, or lack thereof, in the concerned subject.
[0206] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0207] The list of sources, ingredients, and components as described hereinafter are listed such that combinations and mixtures thereof are also contemplated and within the scope herein.
[0208] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
[0209] All lists of items, such as, for example, lists of ingredients, are intended to and should be interpreted as Markush groups. Thus, all lists can be read and interpreted as items “selected from the group consisting of’ the list of items “and combinations and mixtures thereof.”
[0210] Referenced herein may be trade names for components including various ingredients utilized in the present disclosure. The inventors herein do not intend to be limited by materials under any particular trade name. Equivalent materials (e.g., those obtained from a different source under a different name or reference number) to those referenced by trade name may be substituted and utilized in the descriptions herein.
CEACAM mRNA measures, methods and uses
[0211 ] According to one of its objects, the present disclosure relates to methods and uses for selecting a subject in need thereof for a treatment of a cancer with a therapeutic agent targeting CEACAM5. A therapeutic agent targeting CEACAM5 may be an anti- CEACAM5 antibody conjugated to a cytotoxic agent.
[0212] The methods and uses of the disclosure may comprise a step of determining the amount of CEACAM5 gene expression (gene transcript or RNA) in a tumor sample.
[0213] The methods and uses of the disclosure may comprise a step of determining the amount of CEACAM5 gene expression such as CEACAM5 mRNA level in a tumor sample.
[0214] The amount of CEACAM5 gene expression may be expressed as an amount relative to the total gene expression in the tumor sample and relative to the total length of expressed DNA or any other known method.
[0215] In some embodiments, the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of: [0216] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0217] (ii) comparing said determined value with a reference value, and
[0218] (iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value.
[0219] In some embodiments, the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0220] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0221] (ii) comparing said value determined at step (i) with a reference value of a CEACAM5 gene expression level,
[0222] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0223] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0224] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0225] (vi) selecting said subject for a treatment of a cancer if the intensity determined at step (iv) is above the reference intensity.
[0226] The samples tumor of step (i) and step (iv) may be the same sample or different samples.
[0227] In some embodiments, the present disclosure relates to a method for diagnosing a subject in need thereof as being eligible for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0228] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0229] (ii) comparing said determined value with a reference value, and [0230] (iii) selecting said subject as being eligible for a treatment of a cancer if the determined value is above the reference value.
[0231 ] In some embodiments, the present disclosure relates to a method for diagnosing a subject in need thereof as being eligible for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0232] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0233] (ii) comparing said value determined at step (i) with a reference value of a CEACAM5 gene expression level,
[0234] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0235] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0236] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0237] (vi) selecting said subject for a treatment of a cancer if the intensity determined at step (iv) is above the reference intensity.
[0238] In some embodiments, the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibodydrug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0239] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0240] (ii) comparing said determined value with a reference value,
[0241 ] (iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value, and
[0242] (iv) administering to said selected subject an effective amount of said ADC.
[0243] In some embodiments, the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody- drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0244] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0245] (ii) comparing said value determined at step (i) with a reference value of a CEACAM5 gene expression level,
[0246] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0247] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0248] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0249] (vi) selecting said subject for a treatment of a cancer if the intensity determined at step (iv) is above the reference intensity, and
[0250] (vii) administering to said selected subject an effective amount of said ADC.
[0251] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
[0252] the use comprising:
[0253] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0254] (ii) comparing said determined value with a reference value, and
[0255] (iii) administering to said subject an effective amount of said ADC if said determined value is above the reference value.
[0256] In some embodiments, the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
[0257] the use comprising: [0258] (i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
[0259] (ii) comparing said value determined at step (i) with a reference value of a CEACAM5 gene expression level,
[0260] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0261 ] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0262] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0263] (vi) administering to said subject an effective amount of said ADC if said determined intensity is above the reference intensity.
[0264] In some embodiments, the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for characterizing said tumor as a CEACAM5-high expresser tumor.
[0265] In some embodiments, the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test.
[0266] In some embodiments, the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a treatment of a cancer with a CEACAM5 targeting therapeutic agent, for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
[0267] In some embodiments, the present disclosure relates to a use of a measure of a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from a subject in need thereof for selecting said subject for a treatment of a cancer with a CEACAM5 targeting therapeutic agent, for example an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
[0268] In some embodiments, the value of the CEACAM5 gene expression level may be a measure of a CEACAM5 gene transcript. [0269] In some embodiments, the CEACAM5 gene transcript may be a mRNA.
[0270] In some embodiments, methods and uses of the disclosure comprise a step of determining a value of a CEACAM5 mRNA level in an isolated sample of a tumor.
[0271 ] In some embodiments, the amount of CEACAM5 gene expression may be expressed as a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value.
[0272] The determined value or level may be compared to a threshold value or value of reference.
[0273] A deviation from the value of reference may be indicative of a tumor expressing CEACAM5 at a level sufficient to make it responsive to a treatment targeting CEACAM5.
[0274] In case of the CEACAM5 immunohistochemistry (IHC) staining test, the determined intensity or protein expression level may be compared to a threshold intensity or intensity of reference.
[0275] A deviation from the intensity of reference may be indicative of a tumor expressing CEACAM5 at a level sufficient to make it responsive to a treatment targeting CEACAM5 expressing cancer.
[0276] The methods and uses of the disclosure may be for characterizing a subject in need thereof as being responsive to a treatment targeting CEACAM5, for example an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
[0277] The methods and uses of the disclosure may be for selecting a subject in need thereof as being responsive to a treatment targeting CEACAM5, for example an anti- CEACAM5 antibody conjugated to a cytotoxic agent.
[0278] The methods and uses of the disclosure may be for monitoring a responsiveness of a subject in need thereof to a treatment targeting CEACAM5, for example an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
[0279] The methods and uses of the disclosure may be for selecting a treatment targeting CEACAM5, for example an anti-CEACAM5 antibody conjugated to a cytotoxic agent, according to a CEACAM5 gene expression level in a tumor of a subject in need thereof.
[0280] The methods and uses of the disclosure may be for selecting a treatment targeting CEACAM5, for example an anti-CEACAM5 antibody conjugated to a cytotoxic agent, according to a CEACAM5 protein expression intensity in a tumor of a subject in need thereof.
[0281 ] The methods and uses of the disclosure may be for characterizing a tumor in a subject in need thereof as being responsive to a treatment targeting CEACAM5, for example an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
[0282] The methods and uses of the disclosure are carried on isolated biological sample. An isolated sample may be a sample isolated from a tumor. A sample is isolated prior to the implementation of the methods and uses as disclosed herein.
[0283] The methods and uses of the disclosure are carried out in vitro.
[0284] According to one of its objects, the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0285] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0286] (ii) comparing said value with a reference value, and
[0287] (iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value.
[0288] According to one of its objects, the present disclosure relates to a method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0289] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0290] (ii) comparing said value determined at step (i) with a reference value,
[0291 ] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0292] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject, [0293] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0294] (vi) selecting said subject for a treatment of a cancer if the determined intensity is above the reference intensity.
[0295] According to one of its objects, the present disclosure relates to a method for diagnosing a subject in need thereof as being eligible for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0296] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0297] (ii) comparing said value with a reference value, and
[0298] (iii) selecting said subject as being eligible for a treatment of a cancer if the determined value is above the reference value.
[0299] According to one of its objects, the present disclosure relates to a method for diagnosing a subject in need thereof as being eligible for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0300] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0301] (ii) comparing said value determined at step (i) with a reference value,
[0302] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0303] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0304] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0305] (vi) selecting said subject for a treatment of a cancer if the determined intensity is above the reference intensity. [0306] According to another of its objects, the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0307] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0308] (ii) comparing said value with a reference value,
[0309] (iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value, and
[0310] (iv) administering to said selected subject an effective amount of said ADC.
[0311 ] According to another of its objects, the present disclosure relates to a method for selecting and treating a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
[0312] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0313] (ii) comparing said value determined at step (i) with a reference value,
[0314] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0315] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0316] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0317] (vi) selecting said subject for a treatment of a cancer if the determined intensity is above the reference intensity, and
[0318] (vii) administering to said selected subject an effective amount of said ADC.
[0319] According to another of its objects, the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof, [0320] the use comprising (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject, (ii) comparing said value with a reference value, and (iii) administering to said subject an effective amount of said ADC if the determined value is above the reference value.
[0321 ] According to another of its objects, the present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof,
[0322] the use comprising:
[0323] (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
[0324] (ii) comparing said value determined at step (i) with a reference value,
[0325] (iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
[0326] (iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
[0327] (v) comparing said intensity determined at step (iv) with a reference intensity, and
[0328] (vi) administering to said subject an effective amount of said ADC if the determined intensity is above the reference intensity.
[0329] In some embodiments, the reference value may be at least from about 7 to about 13.
[0330] In some embodiments, the reference value may be at least about 7, or may be at least about 8, or at least about 9, or at least about 10, or at least about 11 , or at least about 12, or at least about 13.
[0331 ] In some embodiments, the quantile normalization may be obtained by (i) ranking the transcripts of the sample by level of expression, (ii) calculating an average value for genes occupying a same rank, and (iii) substituting the values of all genes occupying said same rank with this average value. [0332] In some embodiments, the level of expression of the transcripts may be measured in Fragments Per Kilobase Million (FPKM) before being converted in TPM.
[0333] In some embodiments, the Fragments Per Kilobase Million (FPKM) may be obtained by counting the total transcripts in said sample, dividing the obtained transcripts counts by 1 ,000,000, and dividing the obtained values by the length of the gene, in kilobases.
[0334] A step of the methods and uses disclosed herein is to determine the relative expression of the CEACAM5 gene relative to other genes in a tumor sample. Many methods may be used for such determination.
[0335] The relative amount of CEACAM5 gene transcript is then compared to a value of reference. A significant deviation from the value of reference may be indicative of a tumor expressing CEACAM5 which may be responsive to a therapeutic agent targeting specifically CEACAM5.
[0336] The amounts of genes transcripts may be measured by any methods known in the art, such as microarrays, large-scale real-time reverse transcription PCR, RNA- Sequencing (RNA-Seq), Next Generation Sequencing (NGS)
[0337] The gene expression (RNA-seq) of tumor sample may be obtained by any methods known in the art.
[0338] RNA-Seq is a sequencing method used to determine gene expression levels. The number of reads determined to have originated from each transcript (usually by alignment) are proportional to their expression level. RNA-Seq may be used to generate a gene expression profile for tumor samples across many cancer types and to determine which gene expression levels are responsible for tumor development. The RNA-Seq data may be harmonized by aligning raw RNA reads to the GRCh38 reference genome build and calculating gene expression levels with standardized protocols. RNA-Seq data may be available as aligned reads (BAM) and expression levels as: raw counts and normalized with TPM, FPKM, or FPKM-UQ.
[0339] In some embodiments, the gene expression (RNA-seq) may be obtained as follows.
[0340] Genes transcripts (RNA) from an isolated tumor sample may be sequenced by using KAPA mRNA HYPERPREPKITILLUMINA® Platform. RNA-seq data may be processed as follows: sequencing reads may be mapped to the reference genome GRCh 38 using, for example, Spliced Transcripts Alignment to a Reference (STAR) aligner [25]. Gene expression may be first measured in FPKM (Fragments Per Kilobase Million) by CUFFLINK [26] and the gene-level FPKM may be converted into TPM (Transcripts Per Kilobase Million) [27], TPM values may be Iog2-transformed and quantile-normalized for the downstream analysis, including differential gene expression (DGE) analysis. In some embodiments, samples with a number of genes detected below 10,000 may be excluded from the downstream analysis. RNA-seq may include microenvironment cell populations [MCP] counter analysis, according to published methods [28, 29].
[0341] In some embodiments, the measure of CEACAM5 gene expression level (mRNA or measure of gene transcript level) may be normalized.
[0342] In some embodiments, the measure of CEACAM5 gene expression level (mRNA or measure of gene transcript level) may be Iog2-transformed.
[0343] In some embodiments, the CEACAM5 gene expression level (mRNA) in a tumor sample is expressed as a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value.
[0344] The normalization of the measure of CEACAM5 gene expression level may be obtain with the steps of:
[0345] - Log2 transformation of the RNA-seq raw count values,
[0346] - Quantile normalization of the RNA-seq data by matching the distribution of expression values across samples by ranking the values and adjusting them to have the same distribution, and
[0347] - expression of the data as Transcripts Per Kilobase Million (TPM) by scaling the normalized expression values by the length of the gene and the total number of reads sequenced, and then scaling to a million to make the values more interpretable.
[0348] In some embodiments, the Iog2-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 is above the reference value, then the tumor may be qualified as being responsive to a CEACAM5 targeting treatment. A subject in need thereof for whom the determined value is above the reference value may be selected for a CEACAM5 targeting treatment.
[0349] In some embodiments, the reference value may be at least from about 7 to about 13. [0350] In some embodiments, the reference value may be at least about 7, or may be at least about 8, or at least about 9, or at least about 10, or at least about 11 , or at least about 12, or at least about 13.
[0351] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =7.
[0352] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =7.5.
[0353] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =8.
[0354] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =8.5.
[0355] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =9.
[0356] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =9.5.
[0357] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =10.
[0358] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =10.5.
[0359] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =1 1.
[0360] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =1 1.5. [0361] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =12.
[0362] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =12.5
[0363] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =13.
[0364] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =13.5.
[0365] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =14.
[0366] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =14.5.
[0367] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about =15.
[0368] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >7.
[0369] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >7.5.
[0370] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >8.
[0371] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >8.5. [0372] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >9.
[0373] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >9.5.
[0374] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >10.
[0375] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >10.5.
[0376] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >11.
[0377] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >11.5.
[0378] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >12.
[0379] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >12.5
[0380] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >13.
[0381] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >13.5.
[0382] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >14. [0383] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >14.5.
[0384] In some embodiments, the Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) reference value for CEACAM5 mRNA may be about >15.
[0385] The CEACAM5 protein expression level (or intensity) may be measured by immunohistochemistry (IHC).
[0386] The level and pattern of CEACAM5 protein expression in a tumor sample isolated from a patient in need thereof may be analyzed by immunohistochemistry (IHC), for example as disclosed 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 by Blumenthal etal.(BMC Cancer. 2007;7:2. Published 2007 Jan 3. doi:10.1186/1471 -2407-7- 2).
[0387] CEACAM5 reactivity may be assessed in tumor cells using semi-quantitative Percent Scores (calculated by summing the percentages of intensities >2+) or H-score for CEACAM5 plasma membrane staining (whole or polarized).
[0388] CEACAM5 high expressers may be defined as patients with CEACAM5 protein expression at an intensity of > 2+ in > 50% of the tumor cell population of a tumor sample.
[0389] CEACAM5 moderate expressers may be defined as patients with CEACAM5 protein expression at an intensity of > 2+ in > 1% to < 50% of the tumor cell population of a tumor sample.
[0390] In some embodiments, a reference intensity of a CEACAM5 protein expression level measured with a CEACAM5 immunohistochemistry (IHC) staining test may be an intensity of > 2+ in > 50% of the tumor cell population.
[0391] A patient in need of a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent and having a CEACAM5 expression at an intensity of > 2+ in > 50% of the tumor cell population may be selected for said treatment.
[0392] A patient in need of a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent and having a CEACAM5 expression at an intensity of > 2+ in > 50% of the tumor cell population may be administered said treatment.
[0393] A patient in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent may be first subjected to a determination of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said patient. If the determined value of CEACAM5 gene expression level in an isolated sample of a tumor obtained from said patient is above a reference value, then the patient may be selected for a subsequent testing for determination of an intensity of a CEACAM5 protein expression level with a CEACAM5 immunohistochemistry (IHC) staining test.
[0394] If the determined intensity is above a reference intensity, then the patient may be selected for the treatment with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
[0395] If the determined intensity is above a reference intensity, then the patient may be administered with the treatment with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent.
Antibody-drug conjugate comprising an anti-CEACAM5-antibody
[0396] The present disclosure relates to an antibody-drug conjugate (ADC) comprising an anti-CEACAM5-antibody, or a fragment thereof.
[0397] The antibody-drug conjugate typically comprises 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. In the antibody-drug conjugate, the anti-CEACAM5-antibody may be covalently attached via a cleavable or non-cleavable linker to at least one chemotherapeutic agent.
Antl-CEA CAM5-antibody
[0398] According to an embodiment, the antibody-drug conjugate comprises an anti-CEACAM5-antibody or a fragment thereof.
[0399] According to an embodiment, the antibody-drug conjugate comprises a humanized anti-CEACAM5-antibody or a fragment thereof. [0400] According to an embodiment, the anti-CEACAM5-antibody or a fragment thereof comprises a CDR-H1 consisting of SEQ ID NO: 1 , CDR-H2 consisting of SEQ ID NO: 2, CDR-H3 consisting of SEQ ID NO: 3, CDR-L1 consisting of SEQ ID NO: 4, CDR-L2 consisting of amino acid sequence NTR, and CDR-L3 consisting of SEQ ID NO: 5.
[0401] In a further embodiment, the anti-CEACAM5-antibody or a fragment thereof comprises a variable domain of a heavy chain (VH) having at least 90% identity to SEQ ID NO: 6, and a variable domain of a light chain (VL) having at least 90% identity to SEQ ID NO: 7, wherein CDR1 -H consists of SEQ ID NO: 1 , CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1 -L consists of SEQ ID NO: 4, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.
[0402] In a further embodiment, the anti-CEACAM5-antibody or a fragment thereof comprises a variable domain of a heavy chain (VH) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO: 6, and a variable domain of a light chain (VL) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO: 7, wherein CDR1 -H consists of SEQ ID NO: 1 , CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1 -L consists of SEQ ID NO: 4, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.
[0403] In a further embodiment, the anti-CEACAM5-antibody or a fragment thereof comprises a variable domain of a heavy chain (VH) consisting of SEQ ID NO: 6 and a variable domain of a light chain (VL) consisting of SEQ ID NO: 7.
[0404] The anti-CEACAM5-antibody or a fragment thereof comprises in a further embodiment:
[0405] - a variable domain of heavy chain consisting of sequence EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGIT YAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLV TVSS (SEQ ID NO: 6, with CDRs shown in bold characters) in which FR1 -H spans amino acid positions 1 to 25, CDR1 -H spans amino acid positions 26 to 33 (SEQ ID NO: 1 ), FR2- H spans amino acid positions 34 to 50, CDR2-H spans amino acid positions 51 to 58 (SEQ ID NO: 2), FR3-H spans amino acid positions 59 to 96, CDR3-H spans amino acid positions 97 to 109 (SEQ ID NO: 3), and FR4-H spans amino acid positions 1 10 to 120, and
[0406] - a variable domain of light chain consisting of sequence DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPS FSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK (SEQ ID NO: 7, with CDRs shown in bold characters) in which FR1 -L spans amino acid positions 1 to 26, CDR1 - L spans amino acid positions 27 to 32 (SEQ ID NO: 4), FR2-L spans amino acid positions 33 to 49, CDR2-L spans amino acid positions 50 to 52, FR3-L spans amino acid positions 53 to 88, CDR3-L spans amino acid positions 89 to 97 (SEQ ID NO: 5), and FR4-L spans amino acid positions 98 to 107.
[0407] In a further embodiment, the anti-CEACAM5-antibody or a fragment thereof comprises a heavy chain (HC) having at least 90% sequence identity to SEQ ID NO: 8 and a light chain (LC) having at least 90% sequence identity to SEQ ID NO: 9, wherein CDR1 - H consists of SEQ ID NO: 1 , CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1 -L consists of SEQ ID NO: 4, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.
[0408] In a further embodiment, the anti-CEACAM5-antibody or a fragment thereof comprises a heavy chain (HC) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO: 8 and a light chain (LC) having at least 92%, at least 95%, at least 98% identity to SEQ ID NO: 9, wherein CDR1 -H consists of SEQ ID NO: 1 , CDR2-H consists of SEQ ID NO: 2, CDR3-H consists of SEQ ID NO: 3, CDR1 -L consists of SEQ ID NO: 4, CDR2-L consists of amino acid sequence NTR, and CDR3-L consists of SEQ ID NO: 5.
[0409] In a further embodiment, the anti-CEACAM5-antibody or a fragment thereof comprises a heavy chain (HC) consisting of SEQ ID NO: 8 and a light chain (LC) consisting of SEQ ID NO: 9.
[0410] The anti-CEACAM5-antibody may also be a single domain antibody or a fragment thereof. In particular, a single domain antibody fragment may consist of a variable heavy chain (VHH) which comprises the CDR1 -H, CDR2-H and CDR3-H of the antibodies as described above. The antibody may also be a heavy chain antibody, i.e., an antibody devoid of light chain, which may or may not contain a CH1 domain.
[0411] The single domain antibody or a fragment thereof may also comprise the framework regions of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody.
[0412] The anti-CEACAM5-antibody may also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies.
[0413] The antibody may also be a bispecific or multispecific antibody formed from antibody fragments, at least one antibody fragment being an antibody fragment according to the disclosure. Multispecific antibodies are polyvalent protein complexes as described for instance in EP 2 050 764 A1 or US 2005/0003403 A1 . [0414] The anti-CEACAM5-antibody and fragments thereof can be produced by any technique well known in the art. In particular, said antibodies are produced by techniques as hereinafter described.
[0415] The anti-CEACAM5-antibody and fragments thereof can be used in an isolated (e.g., purified) from or contained in a vector, such as a membrane or lipid vesicle (e.g., a liposome).
[0416] The anti-CEACAM5-antibody and fragments thereof may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic, or enzymatic technique, either alone or in combination.
[0417] Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce the anti-CEACAM5-antibody and fragments thereof, by standard techniques for production of polypeptides. For instance, they can be synthesized using well- known solid phase method, in particular using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions. Alternatively, anti-CEACAM5-antibody and fragments thereof 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 incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.
[0418] Anti-CEACAM5-antibody and fragments thereof are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
[0419] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (See, e. g., Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art including, for example, the technique disclosed in the application W02009/032661 , CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991 ); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5, 565, 332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).
[0420] The Fab of the anti-CEACAM5-antibody can be obtained by treating an antibody which specifically reacts with CEACAM5 with a protease, such as papain. Also, the Fab of the anti-CEACAM5-antibody can be produced by inserting DNA sequences encoding both chains of the Fab of the anti-CEACAM5-antibody into a vector for prokaryotic expression, or for eukaryotic expression, and introducing the vector into prokaryotic or eukaryotic cells (as appropriate) to express the Fab of the anti-CEACAM5-antibody.
[0421] The F(ab')2 of the anti-CEACAM5-antibody can be obtained treating an antibody which specifically reacts with CEACAM5 with a protease, pepsin. Also, the F(ab')2 of the anti-CEACAM5-antibody can be produced by binding Fab' described below via a thioether bond or a disulfide bond.
[0422] The Fab' of the anti-CEACAM5-antibody can be obtained treating F(ab')2 which specifically reacts with CEACAM5 with a reducing agent, such as dithiothreitol. Also, the Fab' of the anti-CEACAM5-antibody can be produced by inserting DNA sequences encoding Fab' chains of the antibody into a vector for prokaryotic expression, or a vector for eukaryotic expression, and introducing the vector into prokaryotic or eukaryotic cells (as appropriate) to perform its expression.
[0423] The scFv of the anti-CEACAM5-antibody can be produced by taking sequences of the CDRs or VH and VL domains as previously described, constructing a DNA encoding an scFv fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into prokaryotic or eukaryotic cells (as appropriate) to express the scFv. To generate a humanized scFv fragment, a well- known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) according to the disclosure and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, e. g., W098/45322; WO 87/02671 ; US5,859,205; US5,585,089; US4, 816,567; EP0173494).
[0424] In an embodiment, the anti-CEACAM5 antibody is tusamitamab (CAS [2349294-95-5],
Chemotherapeutic agents
[0425] The antibody-drug conjugate for the use according to the present disclosure typically comprises at least one chemotherapeutic agent (also referred herein to cytotoxic agent). A chemotherapeutic agent as used herein refers to an agent that kills cells, including cancer cells. Such agents favorably stop cancer cells from dividing and growing and cause tumors to shrink in size. The expression “chemotherapeutic agent” is used herein interchangeably with the expressions “cytotoxic agent”, “growth inhibitory agent” or “cytostatic drug”.
[0426] The term "chemotherapeutic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term “chemotherapeutic agent" is intended to include radioisotopes, enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. In some embodiments, the chemotherapeutic agent is an antimetabolite.
[0427] In a further embodiment, the chemotherapeutic agent is selected from the group consisting of radioisotopes, protein toxins, small molecule toxins, and combinations thereof.
[0428] Radioisotopes include radioactive isotopes suitable for treating cancer. Such radioisotopes generally emit mainly beta-radiation. In a further embodiment, the radioisotopes are selected from the group consisting of At211 , Bi212, Er169, I131, I125, Y90, In111 , P32, Re186, Re188, Sm153, Sr89, radioactive isotopes of Lu, and combinations thereof. In an embodiment, the radioactive isotope is alpha-emitter isotope, more specifically Th227, which emits alpha-radiation.
[0429] In a further embodiment, the small molecule toxins are selected from antimetabolites, DNA-alkylating agents, DNA-cross-linking agents, DNA-intercalating agents, anti-microtubule agents, topoisomerase inhibitors, and combinations thereof.
[0430] In a further embodiment, the anti-microtubule agent is selected from the group consisting of taxanes, vinca alkaloids, maytansinoids, colchicine, podophyllotoxin, gruseofulvin, and combinations thereof.
[0431] In some embodiment a chemotherapeutic agent may be a maytansinoid.
[0432] According to an embodiment, maytansinoids are selected from maytansinol, maytansinol analogs, and combinations thereof.
[0433] Examples of suitable maytansinol analogues include those having a modified aromatic ring and those having modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331 ,598; 4,361 ,650; 4,362,663; 4,364,866; 4,450,254;
4,322,348; 4,371 ,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.
[0434] In a further embodiment, the cytotoxic conjugates of the present disclosure utilize the thiol-containing maytansinoid (DM1 ), formally termed A/2’-deacetyl-/\/2’-(3- mercapto-1 -oxopropyl)-maytansine, as the cytotoxic agent. DM1 is represented by the following structural formula (I): [0435] In a further embodiment, the cytotoxic conjugates of the present disclosure utilize the thiol-containing maytansinoid DM4, formally termed A/2’-deacetyl-/V-2’(4-methyl- 4-mercapto-1 -oxopentyl)-maytansine, as the cytotoxic agent. DM4 is represented by the following structural formula (II):
(II).
[0436] In further embodiments of the disclosure other maytansines, including thiol and disulfide-containing maytansinoids bearing a mono or di-alkyl substitution on the carbon atom bearing the sulfur atom, may be used. These include a maytansinoid having, at C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl, an acylated amino acid side chain with an acyl group bearing a hindered sulfhydryl group, wherein the carbon atom of the acyl group bearing the thiol functionality has one or two substituents, said substituents being CH3, C2H5, linear or branched alkyl or alkenyl having from 1 to 10 reagents and any aggregate which may be present in the solution.
[0437] Examples of these cytotoxic agents and of methods of conjugation are further given in the application WO 2008/010101 which is incorporated by reference.
[0438] The immunoconjugates according to the present disclosure can be prepared as described in the application WO 2004/091668, the entire content of which is incorporated herein by reference.
[0439] Accordingly, in a further embodiment, the maytansinoids are selected from the group consisting of A/2’-deacetyl-/\/2’-(3-mercapto-1 -oxopropyl)-maytansine (DM1 ) or A/2’-deacetyl-/V-2X4-methyl-4-mercapto-1 -oxopentyl)-maytansine (DM4), and combinations thereof.
[0440] In a further embodiment, in the antibody-drug conjugate, the anti-CEACAM5- antibody is covalently attached via a cleavable or non-cleavable linker to the at least one chemotherapeutic agent.
[0441] In a further embodiment, the linker is selected from the group consisting of N-succinimidyl pyridyldithiobutyrate (SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and succinimidyl(N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC).
[0442] In a further embodiment, the linker binds to a lysine or cysteine residue in the Fc region of the anti-CEACAM5 antibody. In a further embodiment, the linker forms a disulfide bond or a thioether bond with the maytansine.
[0443] In particular, the anti-CEACAM5-antibody-drug conjugate may be selected from the group consisting of:
[0444] the anti-CEACAM5-SPDB-DM4-antibody-drug conjugate of formula (III):
[0446] and
[0447] an anti-CEACAM5-SMCC-DM1 -antibody-drug conjugate of formula (V): anti-CEACAM5-SMCC-DM1 (V).
[0448] In formulas (III), (IV) and (V) above, “n” corresponds to the number of molecules of chemotherapeutic agent conjugated per molecule of antibody. It corresponds to the “drug-to-antibody ratio” (or “DAR”) defined below and may range from 1 to 10.
[0449] In a further embodiment, the antibody-drug conjugate of the present disclosure comprises an anti-CEACAM5-antibody, which comprises a heavy chain (VH) of SEQ ID NO: 8 and a light chain (VL) of SEQ ID NO: 9 (tusamitamab), wherein tusamitamab is covalently linked to N2’-deacetyl-N-2’(4-methyl-4-mercapto-1 -oxopentyl)-maytansine (DM4) via N-succinimidyl pyridyldithiobutyrate (SPDB). Thereby, the antibody-drug conjugate tusamitamab ravtansine (huMAb2-3-SPDB-DM4) is obtained.
[0450] In an embodiment, the antibody-drug conjugate of the present disclosure is tusamitamab ravtansine (CAS [2254086-60-5]).
[0451] “Linker”, as used herein, means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches the antibody to the chemotherapeutic agent moiety (e.g., a cytostatic agent, a cytotoxic agent or a growth inhibitory agent). Suitable linkers are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups.
[0452] The conjugates may be prepared by in vitro methods. In order to link a drug or prodrug to the antibody, e.g., a chemotherapeutic agent, a linking group is used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Conjugation of an antibody with a chemotherapeutic agent of the disclosure, such as a cytotoxic agent, may be made using a variety of bifunctional protein coupling agents including but not limited to N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5- nitro-2-pyridinyl)dithio]-2,5-dioxo-1 -pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2- yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl (N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4- dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al (1987). Carbon labeled 1 -isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (WO 94/1 1026).
[0453] The linker may be a "cleavable linker" facilitating release of the chemotherapeutic agent in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase labile linker, a photolabile linker or a disulfide-containing linker (See e.g., U.S. Patent No. 5,208,020) may be used. The linker may be also a "non-cleavable linker" (for example SMCC linker) that might led to better tolerance in some cases.
[0454] In general, the conjugate can be obtained by a process comprising the steps of:
[0455] (i) bringing into contact an optionally-buffered aqueous solution of a cellbinding agent (e.g., an antibody according to the disclosure) with solutions of a linker and a chemotherapeutic agent, such as a cytotoxic compound (or agent);
[0456] (ii) then optionally separating the conjugate which was formed in (i) from the unreacted cell-binding agent (e.g., antibody of the disclosure) and unreacted chemotherapeutic agent, such as unreacted cytotoxic compound (or agent).
[0457] The aqueous solution of cell-binding agent can be buffered with buffers such as, e.g., potassium phosphate, acetate, citrate or N-2-Hydroxyethylpiperazine-N’-2- ethanesulfonic acid (Hepes buffer). The buffer depends upon the nature of the cell-binding agent (e.g., antibody of the disclosure). The chemotherapeutic agent, such as the cytotoxic compound (or agent), is in solution in an organic polar solvent, e.g., dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA). [0458] The reaction temperature is usually comprised between 20°C and 40°C. The reaction time can vary from 1 hour to 24 hours. The reaction between the cell-binding agent and the chemotherapeutic agent, such as the cytotoxic agent, can be monitored by size exclusion chromatography (SEC) with a refractometric and/or UV detector. If the conjugate yield is too low, the reaction time can be extended.
[0459] A number of different chromatography methods can be used by the person skilled in the art in order to perform the separation of step (ii): the conjugate can be purified, for example from aggregates, e.g., by SEC, adsorption chromatography (such as ion exchange chromatography, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed-support chromatography such as hydroxyapatite chromatography, or high-performance liquid chromatography (HPLC). Purification by dialysis or diafiltration can also be used.
[0460] As used herein, the term “aggregates” means the associations which can be formed between two or more cell-binding agents, said agents being modified or not by conjugation. The aggregates can be formed under the influence of a great number of parameters, such as a high concentration of cell-binding agent (e.g., antibody of the disclosure) in the solution, the pH of the solution, high shearing forces, the number of bonded dimers and their hydrophobic character, the temperature (see Wang & Gosh, 2008, J. Membrane Sci., 318: 311 -316, and references cited therein); note that the relative influence of some of these parameters is not clearly established. In the case of proteins and antibodies, the person skilled in the art will refer to Cromwell et al. (2006, AAPS Journal, 8(3): E572-E579). The content in aggregates can be determined with techniques well known to the skilled person, such as SEC (see Walter et al., 1993, Anal. Biochem., 212(2): 469- 480).
[0461] After step (i) or (ii), the conjugate-containing solution can be submitted to an additional step (iii) of chromatography, ultrafiltration and/or diafiltration.
[0462] The conjugate is recovered at the end of these steps in an aqueous solution.
[0463] In a further embodiment, the antibody-drug conjugate according to the disclosure is characterized by a “drug-to-antibody ratio” (or “DAR”) ranging from 1 to 10, or from 2 to 5, or from 3 to 4. This is generally the case of conjugates including maytansinoid molecules.
[0464] This DAR number can vary with the nature of the antibody and of the drug (i.e. the chemotherapeutic agent, such as a cytotoxic agent or a growth-inhibitory agent) used along with the experimental conditions used for the conjugation (like the ratio chemotherapeutic agent (e.g., growth-inhibitory agent)/antibody, the reaction time, the nature of the solvent and of the cosolvent if any). Thus the contact between the antibody and the chemotherapeutic agent, such as a cytotoxic agent or a growth-inhibitory agent, leads to a mixture comprising several conjugates differing from one another by different drug-to-antibody ratios; optionally the naked antibody; optionally aggregates. The DAR that is determined is thus a mean value.
[0465] A method which can be used to determine the DAR consists in measuring spectrophotometrically the ratio of the absorbance at of a solution of substantially purified conjugate at AD and 280 nm. 280 nm is a wavelength generally used for measuring protein concentration, such as antibody concentration. The wavelength AD is selected so as to allow discriminating the drug from the antibody, i.e., as readily known to the skilled person, AD is a wavelength at which the drug (i.e., chemotherapeutic agent) has a high absorbance and AD is sufficiently remote from 280 nm to avoid substantial overlap in the absorbance peaks of the drug and antibody. AD may be selected as being 252 nm in the case of maytansinoid molecules. A method of DAR calculation may be derived from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science:
[0466] The absorbances for the conjugate at AD (AAD) and at 280 nm (A280) are measured either on the monomeric peak of the size exclusion chromatography (SEC) analysis (allowing to calculate the “DAR(SEC)” parameter) or using a classic spectrophotometer apparatus (allowing to calculate the “DAR(UV)” parameter). The absorbances can be expressed as follows:
[0467] AAD = (cD x eDAD) + (cA x eAAD)
[0468] A280 = (cD x eD280) + (cA x eA280)
[0469] wherein:
[0470] cD and cA are respectively the concentrations in the solution of the drug (i.e., chemotherapeutic agent) and of the antibody
[0471 ] eDAD and eD280 are respectively the molar extinction coefficients of the drug at AD and 280 nm
[0472] eAAD and eA280 are respectively the molar extinction coefficients of the antibody at AD and 280 nm.
[0473] Resolution of these two equations with two unknowns leads to the following equations:
[0474] cD = [(eA280 x AAD) - (eAAD x A280)] / [(eDAD x eA280) - (eAAD x eD280)] [0475] cA = [A280 - (cD x eD280)] / eA280
[0476] The average DAR is then calculated from the ratio of the drug concentration to that of the antibody: DAR = cD / cA.
[0477] In some embodiments, the antibody-drug conjugate may be administered at a dose of 80 mg/m2 to 210 mg/m2, 80 mg/m2 to 170 mg/m2, or at a dose of 80 mg/m2 to 150 mg/m2, or at a dose of 80 mg/m2 to 120 mg/m2, or at a dose of 80 mg/m2 to 100 mg/m2.
[0478] In some embodiments, the antibody-drug conjugate may be administered at a dose level of 80, 100, 120, 150, 170, 180, or 210 mg/m2.
[0479] In some embodiments, the dosage regimen may comprise administration of a dose over a period of about 10 minutes to about 48 hours, or of about 1 h to about 48h, such as over a period of 1 h to 4h. In some embodiments, the dosage regimen may comprise administration of a dose over a period of about 1 h.
[0480] In some embodiments, the antibody-drug conjugate comprising an anti- CEACAM5-antibody may be administered over about 30 min to about 3 hours, over about 1 hour to about 2hours, or over about 1 .5 hours.
ADC dosages
[0481 ] In some embodiments, it is disclosed an antibody-drug conjugate comprising an anti-CEACAM5-antibody and a chemotherapeutic agent for use for treating a cancer in combination with an anti-CTLA4 antibody and, when presents, an anti-PD-1 antibody or anti-PD-L1 antibody, wherein the antibody-drug conjugate may be administered at a dose of about 60 mg/m2 to about 210 mg/m2, or from about 80 to about 170 mg/m2, or from about 100 to about 170 mg/m2, or from about 120 to about 170 mg/m2, or from about 135 to about 170 mg/m2, or from about 150 to about 170 mg/m2 of body surface area of a subject in need thereof.
[0482] In some embodiments, the antibody-drug conjugate may be administered at a dose of from about 60 to about 210 mg/m2, or from about 80 to about 170 mg/m2, or from about 100 to about 150 mg/m2.
[0483] In various embodiments, the antibody-drug conjugate comprising the anti- CEACAM5 antibody may be administered at a dose of about 60, 70, 80, 90, 100, 110, 120, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, or about 210 mg/m2. [0484] In various embodiments, the antibody-drug conjugate comprising the anti- CEACAM5 antibody may be administered at a dose of about 60, 80, 100, 120, 135, 150, 170, 180, 190 or about 210 mg/m2.
[0485] In some embodiments, the ADC may be administered at a dose of about 80 mg/m2 to about 170 mg/m2, or at a dose of about 80 mg/m2 to about 150 mg/m2, or at a dose of about 100 mg/m2 to about 120 mg/m2.
[0486] In some embodiments, the ADC may be administered at a dose of about 80 mg/m2 or about 100 mg/m2 or about 120 mg/m2 or about 150 mg/m2 or about 170 mg/m2.
[0487] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80 mg/m2.
[0488] In some embodiments, the ADC may be administered at a dose of about 100 mg/m2.
[0489] In some embodiments, the ADC may be administered at a dose of about 120 mg/m2.
[0490] In some embodiments, the ADC may be administered at a dose of about 150 mg/m2.
[0491 ] In some embodiments, the ADC may be administered at a dose of about 170 mg/m2.
[0492] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80, 100, 120, 150 or about 170 mg/m2, as a loading dose (or first dose).
[0493] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80 mg/m2, as a loading dose.
[0494] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 100 mg/m2, as a loading dose.
[0495] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 120 mg/m2, as a loading dose. [0496] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 150 mg/m2, as a loading dose.
[0497] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 170 mg/m2, as a loading dose.
[0498] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80, 100, 120, 150 or about 170 mg/m2, as a subsequent dose (or second dose).
[0499] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80 mg/m2, as a subsequent dose.
[0500] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 100 mg/m2, as a subsequent dose.
[0501] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 120 mg/m2, as a subsequent dose.
[0502] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 150 mg/m2, as a subsequent dose.
[0503] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 170 mg/m2, as a subsequent dose.
[0504] A subsequent dose may be administered on day 1 of cycle(s) subsequent to the first cycle (the subsequent or additional cycles).
[0505] According to an embodiment, the antibody-drug conjugate comprising the anti-CEACAM5 antibody may be administered at a dose of about 80, 100, 120, 150 or about 170 mg/m2, as a loading dose, on a first cycle of treatment, e.g., at day 1 , and then at a dose of about 80, 100, 120, 150 or about 170 mg/m2, as a subsequent dose, e.g., at day 1 , on additional cycle(s). [0506] In certain embodiments, for subjects with a body surface area (BSA) >2.2 m2, the dose of the antibody-drug conjugate comprising an anti-CEACAM5-antibody may be calculated based on a BSA of 2.2 m2.
[0507] The antibody-drug conjugate may be tusamitamab ravtansine (huMAb2-3- SPDB-DM4).
[0508] In certain embodiments, the ADC is administered about once every two weeks. In certain embodiments, the ADC is administered about once every three weeks. In certain embodiments, the ADC is administered about once every four weeks. In certain embodiments, the ADC is administered about once every five weeks. In certain embodiments, the ADC is administered about once every six weeks.
Further agents
[0509] In certain embodiments, the methods and uses further comprise administering to the subject an effective amount of at least one additional agent effective to treat the cancer.
[0510] In certain embodiments, the additional agent is selected from the group consisting of an immune checkpoint inhibitor (ICI), a platinum-based chemotherapy (e.g., cisplatin or carboplatin), pemetrexed, anti-VEGFR2, FOLFOX, FOLFIRI, TAS-102, anti- EGFR, and any combination thereof.
[0511] In certain embodiments, the ICI is an anti-PD-1 antibody.
[0512] In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sintilimab, dostarlimab, and tislelizumab.
[0513] In certain embodiments, the anti-PD-1 antibody is pembrolizumab.
[0514] In certain embodiments, the ICI is an anti-PD-L1 antibody.
[0515] In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, durvalumab, envafolimab, BMS-936559, CK-301 , CS-1001 , SHR-1316 (HTI-1088), CBT-502 (TQB-2450), and any combination thereof.
[0516] In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.
[0517] In certain embodiments, the method comprises administering to the subject an effective amount of tusamitamab ravtansine and pembrolizumab. [0518] In certain embodiments, the method further comprises administering to the subject an effective amount of a platinum-based chemotherapy.
[0519] In certain embodiments, the platinum-based chemotherapy is selected from cisplatin and carboplatin.
[0520] In certain embodiments, the method further comprises administering to the subject an effective amount of pemetrexed.
[0521] In certain embodiments, the method comprises administering to the subject an effective amount of tusamitamab ravtansine, pembrolizumab, and cisplatin.
[0522] In certain embodiments, the method comprises administering to the subject an effective amount of tusamitamab ravtansine, pembrolizumab, cisplatin, and pemetrexed.
[0523] In certain embodiments, the method comprises administering to the subject an effective amount of tusamitamab ravtansine, pembrolizumab, and carboplatin.
[0524] In certain embodiments, the method comprises administering to the subject an effective amount of tusamitamab ravtansine, pembrolizumab, carboplatin, and pemetrexed.
[0525] In one embodiment, the anti-PD-1 antibody or the anti-PD-L1 antibody, or a fragment thereof, is a monoclonal antibody having interfering activity with interaction between PD-1 and PD-L1. In one embodiment, the anti-PD-1 antibody or the anti-PD-L1 antibody is an IgG.
[0526] Anti-PD-1 antibodies and anti-PD-L1 antibodies capable of interfering with interaction between PD-1 , which is expressed on the surface of immune cells, and PD-L1 , which is expressed on the surface of cancer cells, are useful as immune checkpoint inhibitors, thereby blocking a pathway that shields tumor cells from immune system components able and poised to fight cancer. When PD-1 and PD-L1 interact, they form a biochemical “shield” protecting tumor cells from being destroyed by the immune system. Thus, blockade of either PD-1 or PD-L1 leading to blockade of interaction between PD-1 and PD-L1 prevents or unmasks the biochemical “shield” protecting tumor cells from being destroyed by the immune system.
[0527] A number of anti-PD-1 antibodies have been approved for clinical use in the treatment of cancer. These include pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cemiplimab (LIBTAYO®), sintilimab (TYVYT®), dostarlimab (JEMPERLI®), and tislelizumab. [0528] Similarly, a number of anti-PD-L1 antibodies have been approved for clinical use in the treatment of cancer. These include atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®).
[0529] In one embodiment, the anti-PD-1 antibody is pembrolizumab or sintilimab.
[0530] In one embodiment, the anti-PD-1 antibody is pembrolizumab. It is a fully human monoclonal lgG1 antibody against human PD-1.
[0531] In one embodiment, the anti-PD-1 antibody, or a fragment thereof, comprises the light chain and heavy chain CDRs of pembrolizumab.
[0532] In one embodiment, the anti-PD-1 antibody or a fragment thereof comprises the variable domain of heavy chain (VH) and the variable domain of light chain (VL) of pembrolizumab.
[0533] In one embodiment, the anti-PD-1 antibody is sintilimab. In one embodiment, the anti-PD-1 antibody, or a fragment thereof, comprises the light chain and heavy chain CDRs of sintilimab. In one embodiment, the anti-PD-1 antibody or a fragment thereof comprises the variable domain of heavy chain (VH) and the variable domain of light chain (VL) of sintilimab.
[0534] The anti-PD-1 antibody or the anti-PD-L1 antibody, or a fragment thereof, may also be a single domain antibody or a fragment thereof. In particular, a single domain antibody fragment may consist of a variable heavy chain (VHH) which comprises the CDR1 - H, CDR2-H and CDR3-H of the antibodies as described above. The antibody may also be a heavy chain antibody, i.e., an antibody devoid of light chain, which may or may not contain a CH1 domain.
[0535] The single domain antibody or a fragment thereof may also comprise the framework regions of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody.
[0536] The anti-PD-1 antibody or the anti-PD-L1 antibody may also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies.
[0537] The antibody may also be a bispecific or multispecific antibody formed from antibody fragments, at least one antibody fragment being an antibody fragment according to the disclosure. The anti-PD-1 antibody or the anti-PD-L1 antibody and fragments thereof can be produced by any technique well known in the art. In particular, said antibodies are produced by techniques as already described. [0538] The anti-PD-1 antibody or the anti-PD-L1 antibody and fragments thereof can be used in an isolated (e.g., purified) from or contained in a vector, such as a membrane or lipid vesicle (e.g., a liposome).
[0539] The anti-PD-1 antibody or the anti-PD-L1 antibody and fragments thereof may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic, or enzymatic technique, either alone or in combination.
[0540] In some embodiments, the anti-PD-1 antibody or the anti-PD-L1 antibody may be administered at a dose of 150 mg to 400 mg, or at a dose of 150 mg to 300 mg.
[0541] In one embodiment, the anti-VEGFR-2 antibody is a monoclonal antibody, or a fragment thereof having antagonist activity to VEGFR-2. In one embodiment, the anti- VEGFR-2 antibody is an IgG.
[0542] The anti-VEGFR-2 antibody is preferably adapted to the patient. For example, an anti-mouse VEGFR-2 antibody such as DC-101 is preferably used on mice, and an anti-human VEGFR-2 antibody on humans.
[0543] In one embodiment, the anti-VEGFR-2 antibody is ramucirumab (CAS number 947687-13-0). It is a fully human monoclonal lgG1 antibody against human VEGFR-2.
[0544] In one embodiment, the anti-VEGFR-2 antibody or fragment thereof comprises the light chain and heavy chain CDRs of ramucirumab.
[0545] In one embodiment, the anti-VEGFR-2 antibody or fragment thereof comprises the variable domain of heavy chain (VH) and the variable domain of light chain (VL) of ramucirumab.
[0546] The anti-VEGFR-2 antibody or fragment thereof may also be a single domain antibody or a fragment thereof. In particular, a single domain antibody fragment may consist of a variable heavy chain (VHH) which comprises the CDR1 -H, CDR2-H and CDR3-H of the antibodies as described above. The antibody may also be a heavy chain antibody, i.e., an antibody devoid of light chain, which may or may not contain a CH1 domain.
[0547] The single domain antibody or a fragment thereof may also comprise the framework regions of a camelid single domain antibody, and optionally the constant domain of a camelid single domain antibody. [0548] The anti-VEGFR-2 antibody may also be an antibody fragment, in particular a humanized antibody fragment, selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabodies.
[0549] The antibody may also be a bispecific or multispecific antibody formed from antibody fragments, at least one antibody fragment being an antibody fragment according to the disclosure. The anti-VEGFR-2 antibody and fragments thereof can be produced by any technique well known in the art. In particular, said antibodies are produced by techniques as already described.
[0550] The anti-VEGFR-2 antibody and fragments thereof can be used in an isolated (e.g., purified) from or contained in a vector, such as a membrane or lipid vesicle (e.g., a liposome).
[0551] The anti-VEGFR-2 antibody and fragments thereof may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic, or enzymatic technique, either alone or in combination.
[0552] The immunoconjugate comprising an antiCEACAM5-antibody is to be used in combination with cetuximab for the treatment of cancer.
[0553] Cetuximab (CAS number 205923-56-4) is a chimeric monoclonal lgG1 antibody against epidermal growth factor receptor (EGFR). Cetuximab itself has been used for the treatment of metastatic colorectal cancer, metastatic non-small lung cancer and head and neck cancer.
[0554] The immunoconjugate comprising an antiCEACAM5-antibody is to be used in combination with TAS-102 for the treatment of cancer.
[0555] TAS-102 itself is a known chemotherapy regimen approved for human use comprising the combined administration of trifluoridine and tipiracil and which is typically administered in 4-week cycles. TAS-102 combines trifluoridine and tipiracil and has been used in the treatment of colorectal cancer.
[0556] As a modified deoxyuridine, trifluoridine (CAS registry number 70-00-8) is a nucleoside analogue which is incorporated into DNA. The modified DNA binds to thymidiylate synthase, inhibiting the enzyme’s activity. Tipiracil (CAS registry number 183204-74-2) is a thymine analogue, which prevents the degradation of trifluoridine by thymidine phosphorylase. [0557] The immunoconjugate comprising an antiCEACAM5-antibody is to be used in combination with FOLFIRI for the treatment of cancer.
[0558] FOLFIRI itself is a known chemotherapy regimen approved for human use comprising the combined administration of folinic acid, 5-fluoro-uracil and irinotecan and which is typically administered in up to 12 two-week cycles. FOLFIRI combines drugs, each with a different mechanism of action and favorably with synergistic effects, causing the death of cancer cells.
[0559] 5-Fluoro-uracil (CAS registry number 51 -21 -8) is an anti-metabolite, which principally inhibits thymidylate synthase and thus blocks the synthesis of thymidine. 5- Fluoro-uracil has been used the treatment of colon cancer, esophageal cancer, stomach cancer, pancreatic cancer, breast cancer, and cervical cancer.
[0560] Folinic acid, also known as leucovorin (CAS registry number 58-05-9), stabilizes the complex between 5-fluoro-uracil and thymidylate synthase, increasing the cytotoxicity of 5-fluoro-uracil. In one embodiment, folinic acid is L-folinic acid (N-[4-[[[(6S)- 2-amino-5-formyl-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl]methyl]amino]benzoyl]-L- glutamic acid). In another embodiment folinic acid is the calcium salt of L-folinic acid. Folinic acid may also comprise a mixture two or more stereoisomers.
[0561 ] Irinotecan (CAS Number 97682-44-5) is a cytotoxic which is a semi-synthetic derivative of the alkaloid camptothecin and inhibits topoisomerase I resulting in inhibition of DNA replication and transcription and which has been used in the treatment of colon cancer and small cell lung cancer.
[0562] The immunoconjugate comprising an antiCEACAM5-antibody is to be used in combination with FOLFOX for the treatment of cancer.
[0563] FOLFOX itself is a known chemotherapy regimen approved for human use comprising the combined administration of folinic acid, 5-fluoro-uracil and oxaliplatin and which is typically administered in up to 12 two-week cycles. FOLFOX combines drugs, each with a different mechanism of action and favorably with synergistic effects, causing the death of cancer cells.
[0564] 5-Fluoro-uracil (CAS registry number 51 -21 -8) is an anti-metabolite, which principally inhibits thymidylate synthase and thus blocks the synthesis of thymidine. 5- Fluoro-uracil has been used in the treatment of colon cancer, esophageal cancer, stomach cancer, pancreatic cancer, breast cancer, and cervical cancer.
[0565] Folinic acid, also known as leucovorin (CAS registry number 58-05-9), stabilizes the complex between 5-fluoro-uracil and thymidylate synthase, increasing the cytotoxicity of 5-fluoro-uracil. In one embodiment, folinic acid is L-folinic acid (N-[4-[[[(6S)- 2-amino-5-formyl-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl]methyl]amino]benzoyl]-L- glutamic acid). In another embodiment folinic acid is the calcium salt of L-folinic acid. Folinic acid may also comprise a mixture two or more stereoisomers.
[0566] Oxaliplatin (CAS Number 61825-94-3) is known to form cross-links in DNA- strands, preventing DNA replication and transcription and has been used in the treatment of colorectal cancer.
Cancer
[0567] In an embodiment, the cancer is a carcinoma, a sarcoma or a blastoma. In a further embodiment, the cancer is a carcinoma.
[0568] According to an embodiment, the cancer is a cancer expressing CEACAM5. A cancer expressing CEACAM5 may also be named CEACAM5 positive cancer.
[0569] In some embodiments, the cancer is a CEACAM5-positive cancer.
[0570] A CEACAM5-positive cancer is defined as cancer for which a CEACAM5 immunohistochemical [IHC] intensity is > 2+ in > 50% of cancer cells or > 2+ intensity in > 1% and < 50% of the cells tumors (or cancer cells).
[0571] In certain embodiments, the cancer has a negative or low CEACAM5 expression on tumor cells. A negative or low CEACAM5 expression on tumor cells defined as being a CEACAM5 immunohistochemical [IHC] intensity > 2+ in < 1% of cells, as measured by immunohistochemistry (IHC).
[0572] In certain embodiments, the cancer has a moderate CEACAM5 expression on tumor cells. A moderate CEACAM5 expression on tumor cells may be defined as being a CEACAM5 immunohistochemical [IHC] intensity > 2+ in > 1 % and in < 50% of cancer cells, as measured by immunohistochemistry.
[0573] In certain embodiments, the cancer has a high CEACAM5 expression on tumor cells. A high CEACAM5 expression on tumor cells may be defined as being a CEACAM5 immunohistochemical [IHC] intensity > 2+ intensity in > 50% of cancer cells, as measured by immunohistochemistry. [0574] Immunohistochemical techniques for detecting antigens on cells or in tissue sections by means of immunological and chemical reactions are well-known in the field. Those techniques are highly sensitive and specific and can detect a wide variety of antigens. Immunohistochemistry methods comprise the following steps: binding of an antibody to a specific antigen; formation of an antibody-antigen complex by incubation with a secondary, enzyme-conjugated antibody, and generation of colored deposits at the sites of antibodyantigen binding in presence of substrate and chromogen catalyzed by the enzyme.
[0575] The CEACAM5 tumor expression may be determined by using Immunohistochemistry (IHC) assay. An assay may be done using anti-CEACAM5 antibody such as SANOFI’s antibody clone 769. Anti-CEACAM5 clone 769 is a murine monoclonal antibody with the same specificity as tusamitamab ravtansine to the CEACAM5 target. The assay may be run on Techmate platformer or on a Dako/Agilent Autostainer Link 48 IHC or any other immunohistochemistry platforms. Interpretation of CEACAM5 reactivity is to be performed using semi-quantitative Percent Scores (calculated by summing the percentages of intensities >2+) or H-score for CEACAM5 plasma membrane staining (whole or polarized) in tumor cells.
[0576] According to an embodiment, the cancer is selected from hepatocellular carcinoma, colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer (e.g., non-squamous non-small cell lung cancer), uterus cervix cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer (for instance cholangiocarcinoma), prostate cancer, neuroendocrine cancer and skin cancer.
[0577] The cancer may be selected from a colorectal cancer, a gastric cancer, a gastroesophageal junction adenocarcinoma (GEJ), an esophageal cancer, a pancreatic cancer and a lung cancer.
[0578] In some embodiments, the cancer may be a colorectal cancer.
[0579] In some embodiments, the cancer may be a pancreatic cancer.
[0580] In some embodiments, the cancer may be selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, and lung cancer.
[0581] In some embodiments, the cancer may be a gastric cancer, a gastroesophageal junction (GEJ) adenocarcinoma, or an esophageal cancer.
[0582] According to an embodiment, the cancer is gastric cancer or gastroesophageal junction adenocarcinoma (GEJ). [0583] According to an embodiment, the cancer is a gastric cancer.
[0584] According to an embodiment, the cancer is a lung cancer.
[0585] A lung cancer may be a non-squamous non-small-cell lung cancer (NSQ NSCLC).
[0586] Non-small cell lung cancer is a disease in which malignant (cancer) cells form in the tissues of the lung. Smoking is the major cause of the disease. This is a type of epithelial lung cancer other than small cell lung carcinoma. There are several types of non- small cell lung cancer. Each type of non-small cell lung cancer has different kinds of cancer cells. The cancer cells of each type grow and spread in different ways. The types of non- small cell lung cancer are named for the kinds of cells found in the cancer and how the cells look under a microscope: (1 ) squamous cell carcinoma: Cancer that begins in squamous cells, which are thin, flat cells that look like fish scales. This is also called epidermoid carcinoma. (2) large cell carcinoma: Cancer that may begin in several types of large cells. (3) adenocarcinoma: Cancer that begins in the cells that line the alveoli and make substances such as mucus.
[0587] In some embodiments, the non-squamous non-small cell lung cancer may be an advanced or metastatic NSQ NSCLC.
[0588] According to an embodiment, the subject is a patient with malignant tumor, in particular with a malignant solid tumor, and more specifically with locally advanced or metastatic solid malignant tumor. A metastatic solid malignant tumor may be a metastatic cancer, for example a metastatic carcinoma. A cancer or a carcinoma may be as above indicated.
[0589] In some embodiments, the non-squamous non-small cell lung cancer has no epidermal growth factor receptor (EGFR) sensitizing mutation or v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation or anaplastic lymphoma kinase/c-ros oncogene 1 (ALK/ROS) alterations.
Pharmaceutical compositions or combinations
[0590] In some embodiments, in the uses and methods as disclosed herein, the administration of the ADC may be carried out by parenteral route. A suitable parenteral route may be intravenous infusion.
[0591] The present disclosure also relates to an ADC for the manufacture of a medicament for the treatment of cancer, the combination comprising. [0592] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising (i) an ADC as disclosed herein, and a pharmaceutically acceptable excipient.
[0593] The ADC of the disclosure may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
[0594] Thus, another object of the disclosure relates to a pharmaceutical composition comprising an ADC of the disclosure and a pharmaceutically acceptable carrier or excipient. ADC or an immunoconjugate according to the disclosure, for use as a medicament.
[0595] The disclosure also relates to an ADC according to the disclosure, for use as for treating cancer.
[0596] "Pharmaceutical excipient" or "pharmaceutically acceptable excipient" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
[0597] As used herein, “pharmaceutically-acceptable carriers or excipients” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents and/or excipients include one or more of water, amino acids, saline, phosphate buffered saline, buffer phosphate, acetate, citrate, succinate; amino acids and derivates such as histidine, arginine, glycine, proline, glycylglycine; inorganic salts NaCI, calcium chloride; sugars or polyalcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as Polysorbate 80, polysorbate 20, poloxamer 188; and the like, as well as combination thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition, and formulation may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20.
[0598] The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and gender of the subject, etc.
[0599] The pharmaceutical compositions of the disclosure can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like. In an embodiment, the pharmaceutical compositions and combinations of the disclosure are formulated for intravenous administration.
[0600] In particular, the pharmaceutical compositions contain vehicles or excipients, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
[0601] The pharmaceutical composition can be administrated through drug combination devices.
[0602] The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of used, of the relevant pathology, or alternatively of the desired duration of treatment.
[0603] To prepare pharmaceutical compositions, an effective amount of antibodydrug conjugate comprising an anti-CEACAM5-antibody and/or of an anti-CTLA4 antibody and/or of an anti-PD-1 antibody or anti-PD-L1 antibody may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
[0604] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and injectable with the appropriate device or system for delivery without degradation. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
[0605] Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0606] The antibody-drug conjugate comprising an anti-CEACAM5-antibody can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, glycine, histidine, procaine and the like.
[0607] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the subsequent of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
[0608] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0609] The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
[0610] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
[0611] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
[0612] The antibody-drug conjugate comprising an anti-CEACAM5-antibody formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used.
[0613] In certain embodiments, the use of liposomes and/or nanoparticles is contemplated for the introduction of polypeptides into host cells. The formation and use of liposomes and/or nanoparticles are known to those of skill in the art.
[0614] Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles, or biodegradable polylactide or polylactide co glycolide nanoparticules that meet these requirements are contemplated for use in the present disclosure, and such particles may be easily made.
[0615] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
Methods of Administration and Formulations
[0616] The methods described herein comprise administering a therapeutically effective amount of an anti-CEACAM5 ADC to a subject. As used herein, an “effective amount” or "therapeutically effective amount" is a dose of the therapeutic that results in treatment of CEACAM5 expressing cancer (e.g. lung cancer, gastric cancer, gastroesopageal junction cancer or esophageal cancer). As used herein, “treating” refers to causing a detectable improvement in one or more symptoms associated with CEACAM5 expressing cancer (e.g. lung cancer ) or causing a biological effect (e.g., a decrease in the level of a particular biomarker) that is correlated with the underlying pathologic mechanism(s) giving rise to the condition or symptom(s). For example, a dose of anti- CEACAM5 ADC which causes an improvement in any of the following symptoms or conditions associated with CEACAM5 expressing cancer is deemed a "therapeutically effective amount”:
[0617] In another example, a treatment has not been effective when a dose of anti- CEACAM5 ADC does not result in a detectable improvement in one or more parameters or symptoms associated with a CEACAM5 expressing cancer (e.g., lung cancer, gastric cancer, gastroesophageal junction cancer or esophageal cancer) or which does not cause a biological effect that is correlated with the underlying pathologic mechanism(s) giving rise to the condition or symptom(s) of cancer.
[0618] According to some of these embodiments, the anti-CEACAM5 ADC is administered intravenously.
[0619] In accordance with the methods of the present disclosure, a therapeutically effective amount of anti-CEACAM5 ADC that is administered to the subject will vary depending upon the age and the size (e.g., body weight or body surface area) of the subject as well as the route of administration and other factors well known to those of ordinary skill in the art.
[0620] In certain embodiments, the dose of the ADC varies depending on the body surface area of the subject. In certain embodiments, the dose of anti-CEACAM5 ADC administered to the subject is from about 1 mg/m2 to about 500 mg/m2. In some embodiments, the dose of the ADC administered to the subject is from about 5 mg/m2 to about 300 mg/m2. In various embodiments, the dose of the ADC administered to the subject is from about 5 mg/m2 to about 250 mg/m2. In various embodiments, the dose of the ADC administered to the subject is from about 60 mg/m2 to about 190 mg/m2. In various embodiments, the dose is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 210 mg/m2 based on the body surface area of the subject. In certain embodiments, the dose of the ADC is about 100 mg/m2. In certain embodiments, the dose of the ADC is about 150 mg/m2. In certain embodiments, the dose of the ADC is about 170 mg/m2. In certain embodiments, the dose of the ADC is about 190 mg/m2. [0621 ] In various embodiments, the dose of the ADC is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 210 mg/m2 based on the body surface area of the subject. In certain embodiments, the dose of the ADC is 100 mg/m2. In certain embodiments, the dose of the ADC is 150 mg/m2. In certain embodiments, the dose of the ADC is 170 mg/m2. In certain embodiments, the dose of the ADC is 190 mg/m2.
[EXAMPLES]
[0622] The following examples illustrate the embodiments of the disclosure that are presently best known. However, it is to be understood that the following are only exemplary or illustrative of the application of the principles of the present disclosure. Numerous modifications and alternative compositions, methods, and systems may be devised by those skilled in the art without departing from the spirit and scope of the present disclosure. Thus, while the present disclosure has been described above with particularity, the following examples provide further detail in connection with what are presently deemed to be the most practical and preferred embodiments of the disclosure.
EXAMPLE 1 : Materials & Methods
Study Design and Patients
[0623] Study design details from this phase 1/2 first-in-human, open-label, dose escalation and expansion trial of tusamitamab ravtansine have been published elsewhere. [15]
Ethics statement
[0624] Briefly, patients eligible for the dose escalation and expansion phases were >18 years old with locally advanced or metastatic solid tumors for which no standard alternative therapy was available, and Eastern Cooperative Oncology Group performance status (ECOG PS) 0 or 1 .
[0625] The dose escalation phase population was enriched for, but not restricted to, patients with tumor types known to express CEACAM5, and expression was confirmed retrospectively in a central laboratory using IHC on the most recent archival tissue samples.
[0626] The expansion phase population was restricted to separate cohorts of patients with advanced colorectal cancer, NSQ-NSCLC, small-cell lung cancer, and gastric adenocarcinoma.
[0627] In this report, we present results for patients with NSQ-NSCLC only. There were 2 independent NSQ- NSCLC expansion phase populations based upon IHC analysis of tumor tissue:
[0628] CEACAM5 high expressers, defined as patients with CEACAM5 expression at an intensity > 2+ in > 50% of the tumor cell population and [0629] CEACAM5 moderate expressers, defined as patients with CEACAM5 expression at an intensity > 2+ in > 1% to < 50% of the tumor cell population.
[0630] All patients were required to have at least one measurable lesion by Response Evaluation Criteria in Solid Tumors (RECIST) v1 .1 .
[0631] Key exclusion criteria included: a life expectancy < 12 weeks; known or symptomatic brain metastases; in receipt of other cancer treatment; having received previous therapy targeting CEACAM5; having received prior maytansanoid treatments; and poor bone marrow reserve or organ dysfunction.
Treatments
[0632] During expansion phase, all patients received intravenous (IV) tusamitamab ravtansine 100 mg/m2 Q2W as determined during the dose escalation phase of this study [15]. Tusamitamab ravtansine was infused at 2.5 mg/min for 30 minutes, then 5 mg/min thereafter, provided there were no signs or symptoms of a hypersensitivity reaction.
[0633] To prevent hypersensitivity reactions, patients were premedicated with an oral antihistamine 1 hour before receiving tusamitamab ravtansine.
[0634] T reatment was continued until disease progression, unacceptable toxicity, or patient willingness to stop.
Outcomes
[0635] The primary outcome during expansion phase assessed the objective response rate per RECIST v1 .1 criteria.
[0636] Key secondary outcomes assessed safety, which will be reported in a separate publication.
[0637] Exploratory biomarker objectives (reported here) included exploring the potential link between CEACAM5 expression characteristics and response (expansion phase), investigating potential biomarkers (other than CEACAM5) that might predict tusamitamab ravtansine activity (expansion phase), and evaluating the potential for circulating CEA levels to be a convenient companion diagnosis for tusamitamab ravtansine treatment (by evaluating correlation between circulating CEA levels and tumor CEACAM5 expression and correlation between this biomarker and treatment response). Tumor samples
[0638] Tumor samples were obtained using the latest archival tumor samples (ie, tumor tissue archived at diagnosis, at surgery, or taken before patient inclusion in the study and not undergoing anticancer treatment); fresh biopsy at baseline was optional in patients who had a lesion amenable for biopsy.
CEACAM5 expression by immunohistochemistry
[0639] The level and pattern of CEACAM5 expression in archival tumor samples was analyzed by IHC locally at clinical sites and/or centrally in a laboratory using mouse anti-CEACAM5 clone 769, which has the same specificity as tusamitamab ravtansine for the CEACAM5 target; .
[0640] At least six 5-pm slides plus either three additional 10-pm slides (or six 5-pm slides) of formalin-fixed paraffin-embedded (FFPE) tissue was to be provided for each patient. CEACAM5 expression was determined centrally thanks to a validated IHC assay using anti-CEACAM5 clone 769 antibody at 0.5pg/mL.
[0641] CEACAM5 reactivity was assessed in tumor cells using semi-quantitative Percent Scores (calculated by summing the percentages of intensities >2+) or H-score for CEACAM5 plasma membrane staining (whole or polarized); CEACAM5 cytoplasmic staining was also evaluated (centrally only).
Tissue RNA/DNA extraction
[0642] If blocks were provided, FFPE tumor tissues were cut at 10 pm thickness using a microtome, and 3 sections were mounted on adhesive microscope slides. After trimming excess paraffin off the sample slide using a sterile scalpel, the tumor tissue was macrodissected and collected in individual Eppendorf DNA LoBind tubes. Genomic DNA and Total RNA from each human lung cancer FFPE tissue sample was co-extracted using the ALLPREP® DNA/RNA FFPE kit (ref. 80234, QIAGEN), according to the manufacturer’s instructions, with initial manual processing steps followed by final automated extraction steps using the QIACUBE automated nucleic acid purification instrument. Genomic DNA was eluted in 30 pL ATE buffer and Total RNA eluted in 20 pL RNase-free water.
[0643] A total of 71 RNA samples were generated for RNA sequencing analysis. Gene expression (RNA-seq)
[0644] Tissue samples with known CEACAM5 expression level by IHC were analyzed using RNA-seq. RNA samples from 57 FFPE patient biopsies were sequenced by using KAPA mRNA HYPERPREPKITILLUMINA® Platform. RNA-seq data was processed as follows: sequencing reads were mapped to the reference genome GRCh 38 using Spliced Transcripts Alignment to a Reference (STAR) aligner [25]. Gene expression was initially measured in FPKM (Fragments Per Kilobase Million) by CUFFLINK [26] and the gene-level FPKM was converted into TPM (Transcripts Per Kilobase Million) [27], TPM values were Iog2-transformed and quantile-normalized for the downstream analysis, including differential gene expression (DGE) analysis. Samples with the number of genes detected below 10,000 were excluded from the downstream analysis. RNA-seq included microenvironment cell populations [MCP] counter analysis, according to published methods [28, 29],
Statistical Analysis
[0645] All analyses were conducted in the biomarkers population, defined as patients who received treatment and who had at least one evaluable CEACAM5 expression measurement or at least one valid RNA assessment.
[0646] The percentage of positive tumor cells at intensity >2+ on the overall membrane defined CEACAM5 expression in tumor tissue, which was analyzed using descriptive statistics.
[0647] Associations between CEACAM5 expression characteristics were evaluated by calculating P values from Mann-Whitney U tests (when only 2 groups were present) or Kendall tau.
[0648] Adjusted P values were also calculated using the Benjamini-Hochberg (BH) multiple correction procedure to control the false discovery rate. Association between baseline biomarkers and tumor response
[0649] Statistical significance of the association between CEACAM5 expression (moderate versus high expression) and overall response rate (ORR) was evaluated with a two-sided Fisher’s exact test.
EXAMPLE 2: Results
Biomarker-evaluable patients
[0650] In this exploratory biomarker analysis of the cohort of patients with advanced non-squamous NSCLC from the dose expansion phase, the first patient was enrolled on January 2, 2017, and the data cutoff for these analyses was December 2020.
[0651] Of 888 prescreened patients with NSQ-NSCLC, 172 (19%) had high CEACAM5 expression and 210 (24%) had moderate CEACAM5 expression.
[0652] 92 patients were treated: 64 with high CEACAM5 expression (high expressors) and 28 with moderate CEACAM5 expression (moderate expressors)
[0653] In summary, median age was 62.5 years (range, 31-91 years; 42% were >65 years of age); 51 % were male; 72% had ECOG PS >1 ; patients had received a median of 3 prior lines of therapy (range, 1-10 lines) for advanced disease, including antitubulin agents (61%) and anti-PD1/PD-L1 agents (75%).
[0654] The clinical findings showed that there was an enrichment of clinical responses in patients (responders) with high CEACAM5 protein expression level measured by immunohistochemistry (IHC), i.e., at an intensity of > 2+ intensity in > 50% of tumor cells measured by CEACAM5 IHC. Non-responders were CEACAM5 protein moderate expressers, defined with a CEACAM5 protein expression measured by IHC at an intensity of > 2+ in > 1 % to < 50% of the tumor cell population.
CEACAM5 expression: pattern of staining and distribution in tumors
[0655] High CEACAM5 expressers showed a predominance of whole over polarized membrane expression. In contrast, whole and polarized membrane expression was similar among moderate CEACAM5 expressers. (Table 1).
[0656] At initial diagnosis, the predominant histological type was adenocarcinoma and the majority (91.3%) of tumors expressing CEACAM5 were stage III or more , irrespective of CEACAM5 expression level (Table 1). Table 1. CEACAM5 expression: pattern of staining and distribution in tumors
Percentage of positive cells Pooled High and High CEACAM5 Moderate CEACAM5 with CEACAM5 > 2+ intensity Moderate CEACAM5 expressers expressers expressers (N = 92) (N = 64) (N = 28)
Pattern of staining, mean ± SD
Whole membrane 51 .95 ± 37.43 6.96 ± 9.02
Polarized 25.08 ± 31 .69 a 6.89 ± 12.04
Histology type, n (%)
Adenocarcinoma 91 (98.9) 63 (98.4) 28 (100)
Other 1 (1.1) 1 (1.6) 0
Staging, n (%)
Stage 1 6 (6.5) 5 (7.8) 1 (3.6)
Stage II 2 (2.2) 2 (3.1) 0
Stage III 20 (21.7) 14 (21.9) 6 (21.4)
Stage IV 64 (69.6) 43 (67.2) 21 (75.0)
[0657] aN = 63 for polarized membrane.
[0658] CEACAM5, carcinoembryonic antigen-related cell adhesion molecule 5; NSCLC, non-small cell lung cancer; SD, standard deviation.
Associations between CEACAM5 expression by IHC and gene expression (RNA-seq)
[0659] Differential gene expression analysis has identified CEACAM5 mRNA to be the most associated gene (and the only significant one adjusted P = 0.00265) with CEACAM5 high vs moderate expression by IHC. The other CEACAM family member expression or other gene expression are not significantly associated with CEACAM5 IHC after multiple testing correction (Table 2). Table 2. RNA-seq gene expression fold changes in high versus moderate CEACAM5 expression by IHC: The top 10 ranking genes, including the closest ranking other CEACAM family member.
Rank Gene logFC Lower CL Upper CL AveExpr Pvalue Adjusted P value
1 CEACAM5 0.46 0.28 0.63 7.1 3.2e-06 0.05
2 TACR1 -0.96 -1 .4 -0.51 4.2 6.8e-05 0.38
3 HOXB5 1 0.54 1.5 5.3 7.3e-05 0.38
4 RIOX2 -0.15 -0.22 -0.071 6.5 0.00024 0.92
5 SGCE -0.22 -0.33 -0.1 6.4 0.00034 1
6 HOXB6 0.82 0.38 1.3 5.2 0.00045 1
7 ENTPD8 1 .2 0.53 1.9 4.8 0.00069 1
8 MNX1 0.99 0.44 1.5 4.6 0.00072 1
9 CYP4Z1 -0.93 -1 .4 -0.41 4.2 0.00076 1
10 RASL1 1 A -0.55 -0.86 -0.24 5.5 0.00077 1
CEACAM3 0.65 0.13 1.2 6.3 0.016 1
[0660] CEACAM, carcinogembryonic antigen-related cell adhesion molecule; CL, confidence limit; logFC, log fold change.
Associations between CEACAM5 expression, objective response rate and
CEACAM5 mRNA level
[0661] A correlation between CEACAM5 mRNA level and CEACAM5 expression measured by immunohistochemistry recorded as the sum of the percentage of the tumoral cells expressing the target at least 2+ intensity was observed in patients who responded to tusamitamab ravtansine treatment.
EXAMPLE 3: Discussion [0662] Almost 20% of prescreened patients with NSCLC had high CEACAM5 expression as measured by IHC, i.e., staining intensity > 2+ in > 50% of tumor cells. An enrichment of clinical responses in patients having a high CEACAM5 protein expression level and being treating with tusamitamab ravtansine was observed.
[0663] An association was found between CEACAM5 expression, cCEA, cCEACAM5, and CEACAM5 tumor mRNA levels.
[0664] Further, CEACAM5 mRNA expression was significantly upregulated in patients with high vs moderate CEACAM5 protein expression, but other genes, including other CEACAM family genes were not (Figure 1 and Table 1).
[0665] Higher levels of CEACAM5 mRNA were observed in CEACAM5 high expressors vs moderate expressor (P=0.0027) (Figure 1).
[0666] An association was found between CEACAM5 mRNA level in tumor cells and CEACAM5 IHC staining in tumor cells (Figure 2).
[0667] Further, an enrichment of clinical responses in patients treated with the ADC and having a high CEACAM5 mRNA level and a high CEACAM5 protein expression as measured by IHC was shown (Figures 3 and 4).
[0668] As shown on Figure 5, illustrating the CEACAMs family members mRNA expression according to clinical responses to tusamitamab ravtansine, the median of CEACAM5 mRNA level was higher in responders group compared to non-responders group. This result shows an enrichment of clinical responses in patients treated with tusamitamab ravtansine according to the CEACAM5 mRNA levels. This was not observed for other CEACAM family members, except for a small trend for CEACAM3.
[0669] The clinical findings from the dose escalation and expansion phases of the clinical study showed an enrichment of clinical responses in patients with high CEACAM5 protein expression (responders) and treated with tusamitamab ravtansine versus patients treated with the ADC and with moderate CEACAM5 protein expression (non-responders).
[0670] Further findings from the biomarker analysis support the association between mRNA CEACAM5 expression level and CEACAM5 protein expression level.
[0671] Since an association between the CEACAM5 protein expression level and the enrichment of clinical responses was shown in patients treated with tusamitamab ravtansine and since an association between the CEACAM5 protein expression level and the mRNA CEACAM5 expression level was shown, then the mRNA CEACAM5 expression level was shown to be a good biomarker of the enrichment of clinical responses and for selecting patients to be treated by an anti-CEACAM5 antibody-drug conjugate such as tusamitamab ravtansine. [0672] Furthermore, CEACAM5 mRNA level is a good biomarker for pre-selecting patients in need of a cancer treatment cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent for a further step of selection with CEACAM5 immunohistochemistry (IHC) staining and treating patients in need of a cancer treatment cancer with the ADC.
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Claims

[CLAIMS]
1 . A method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
(i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
(ii) comparing said determined value with a reference value, and
(iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value.
2. An antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof, the use comprising:
(i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
(ii) comparing said determined value with a reference value, and
(iii) administering to said subject an effective amount of said ADC if said determined value is above the reference value.
3. A method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
(i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
(ii) comparing said value determined at step (i) with a reference value of a CEACAM5 gene expression level,
(iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
(iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject, (v) comparing said intensity determined at step (iv) with a reference intensity, and
(vi) selecting said subject for a treatment of a cancer if the intensity determined at step (iv) is above the reference intensity.
4. An antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof, the use comprising:
(i) determining a value of a CEACAM5 gene expression level in an isolated sample of a tumor obtained from said subject,
(ii) comparing said value determined at step (i) with a reference value of a CEACAM5 gene expression level,
(iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
(iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
(v) comparing said intensity determined at step (iv) with a reference intensity, and
(vi) administering to said subject an effective amount of said ADC if said determined intensity is above the reference intensity.
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 a CEACAM5 gene transcript.
6. The method according or the antibody-drug conjugate (ADC) for use according to claim 3, wherein the CEACAM5 gene transcript is a mRNA.
7. A method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
(i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
(ii) comparing said value with a reference value, and (iii) selecting said subject for a treatment of a cancer if the determined value is above the reference value.
8. An antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof, the use comprising:
(i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
(ii) comparing said value with a reference value, and
(iii) administering to said subject an effective amount of said ADC if the determined value is above the reference value.
9. A method for selecting a subject in need thereof for a treatment of a cancer with an antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, the method comprising at least the steps of:
(i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
(ii) comparing said value determined at step (i) with a reference value,
(iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
(iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
(v) comparing said intensity determined at step (iv) with a reference intensity, and
(vi) selecting said subject for a treatment of a cancer if the determined intensity is above the reference intensity.
10. An antibody-drug conjugate (ADC) comprising an anti-CEACAM5 antibody conjugated to a cytotoxic agent, for use in the treatment of a cancer in a subject in need thereof, the use comprising: (i) determining a Iog2-transformed, quantile-normalized, Transcripts Per Kilobase Million (TPM) value for CEACAM5 mRNA in an isolated sample of a tumor obtained from said subject,
(ii) comparing said value determined at step (i) with a reference value,
(iii) selecting said subject for a CEACAM5 immunohistochemistry (IHC) staining test if the value determined at step (i) is above the reference value of step (ii),
(iv) determining an intensity of a CEACAM5 protein expression level with said CEACAM5 immunohistochemistry (IHC) staining test in an isolated sample of a tumor obtained from said subject,
(v) comparing said intensity determined at step (iv) with a reference intensity, and
(vi) administering to said subject an effective amount of said ADC if the determined intensity is above the reference intensity.
1 1 . The method according to any one of claims 1 , 3, 5-7, and 9 or the antibody-drug conjugate (ADC) for use according to any one of claims 2, 4-6, 8 and 10, wherein the anti- CEACAM5 antibody comprises a HCDR1 having the amino acid sequence of SEQ ID NO: 1 , a HCDR2 having the amino acid sequence of SEQ ID NO: 2, a HCDR3 having the amino acid sequence of SEQ ID NO: 3, a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence NTR, and a LCDR3 having the amino acid sequence of SEQ ID NO: 5.
12. The method according to any one of claims 1 , 3, 5-7, 9, and 11 or the antibodydrug conjugate (ADC) for use according to any one of claims 2, 4-6, 8 and 10-1 1 , wherein the cytotoxic agent is maytansinoid or maytansinoid analog.
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 from 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 is 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 level of expression, (ii) calculating an average value for genes occupying a same rank, and (iii) substituting the values of all genes occupying said same rank with this average value.
16. The method according to any one of claims 7, 9 and 11 -15 or the antibody-drug conjugate (ADC) for use according to any one of claims 8 and 10-15, wherein the level of expression of the transcripts is measured in Fragments Per Kilobase Million (FPKM) before being converted in TPM.
17. The method or the antibody-drug conjugate (ADC) for use according to claim 16, wherein the Fragments Per Kilobase Million (FPKM) is obtained by counting the total transcripts in said sample, dividing the obtained transcripts counts by 1 ,000,000, and dividing the obtained values by the length of the gene, in kilobases.
18. The method or the antibody-drug conjugate (ADC) for use according to any one of claims 1 -17, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma (GEJ), esophageal cancer, lung cancer, uterine cervix cancer, pancreatic cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, biliary tract cancer, prostate cancer, neuroendocrine cancer, and skin cancer.
19. The method or the antibody-drug conjugate (ADC) for use according to any one of claims 1 -18, wherein the ADC is tusamitamab ravtansine.
20. The method or the antibody-drug conjugate (ADC) for use according to any one of claims 1 -19, wherein the ADC is administered in a dose of > 80 mg/m2, in particular in a dose of 80 mg/m2 to 210 mg/m2, 80 mg/m2 to 170 mg/m2, or at a dose of 80 mg/m2 to 150 mg/m2, or at a dose of 80 mg/m2 to 120 mg/m2, or at a dose of 80 mg/m2 to 100 mg/m2, and in particular in a dose of 80, 100, 120, 150, 170, 180, or 210 mg/m2, relative to the surface body area of said subject, about once every two weeks, or the ADC is administered in a dose of > 80 mg/m2, relative to the surface body area of said subject about once every three weeks.
21 . The method or the 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 agent effective to treat the cancer.
22. The method or the antibody-drug conjugate (ADC) for use according to claim 21 , wherein the additional agent is selected from the group consisting of an immune checkpoint inhibitor (ICI), in particular an anti-PD-1 antibody or an anti-PD-L1 antibody, a platinum-based chemotherapy, pemetrexed, anti-VEGFR2, FOLFOX, FOLFIRI, TAS-102, anti-EGFR, and any combination thereof.
23. The method or the antibody-drug conjugate (ADC) for use according to claim
22, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, sintilimab, dostarlimab, and tislelizumab or wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.
24. The method or the antibody-drug conjugate (ADC) for use according to claim
23, comprising administering to the subject an effective amount of tusamitamab ravtansine and pembrolizumab.
25. The method or the antibody-drug conjugate (ADC) for use according to claim
22, further comprising administering to the subject an effective amount of a platinum-based chemotherapy.
26. The method or the antibody-drug conjugate (ADC) for use according to claim 25, wherein the platinum-based chemotherapy is selected from cisplatin and carboplatin.
27. The method or the antibody-drug conjugate (ADC) for use according to claim
25 or 26, further comprising administering to the subject an effective amount of pemetrexed.
EP24716660.6A 2023-03-23 2024-03-22 Ceacam5 mrna assay for patient selection in cancer therapy Pending EP4684034A1 (en)

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