TREATMENT OF CK8 POSITIVE CANCERS IN RELATION WITH K-RAS GENE STATUS

Abstract

The invention relates to the use of an anti-CK8 antibody alone or in combination therapy (with another antibody and/or chemotherapeutic agent) to (1) treat solid tumours expressing CK8 having wild-type K-Ras (not mutated as disclosed herein) and (2) treat solid tumours expressing CK8 having K-Ras mutation as disclosed herein. The invention also relates to the use of anti-CK8 antibodies having internalizing property, allowing to deliver cytotoxic agent coupled to antibody for the treatment of CK8 positive solid tumours, having wild-type K-Ras (not mutated as disclosed herein) or having K-Ras mutation as disclosed herein.

Claims

1. An anti-CK8 monoclonal antibody or an antigen-binding fragment thereof, wherein said antibody or fragment comprises a heavy chain comprising the following three CDRs, respectively CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 comprises the sequence SEQ ID NO: 49; CDR-H2 comprises the sequence SEQ ID NO: 50; and CDR-H3 comprises the sequence SEQ ID NO: 51; and a light chain comprising the following three CDRs, respectively CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 comprises the sequence SEQ ID NO: 52; CDR-L2 comprises the sequence SEQ ID NO: 47; and CDR-L3 comprises the sequence SEQ ID NO: 53.

2. The antibody or fragment of claim 1, wherein said antibody and fragment has the VH sequence SEQ ID NO: 38 and the VL sequence SEQ ID NO: 36.

3. The antibody of claim 1, further comprising the Heavy constant domain of SEQ ID NO: 18 and/or the Light constant region (kappa) of SEQ ID NO: 20.

4. The antibody of claim 2, further comprising the Heavy constant domain of SEQ ID NO: 18 and/or the Light constant region (kappa) of SEQ ID NO: 20.

5. A method for treating a cancer expressing CK8, the method comprising administering to a patient in need thereof an effective amount of a monoclonal anti-CK8 antibody or an antigen-binding fragment thereof, wherein said monoclonal anti-CK8 antibody and antigen-binding fragment thereof is according to claim 1.

6. The method of claim 5, wherein said antibody and fragment has the VH sequence SEQ ID NO: 38 and the VL sequence SEQ ID NO: 36.

7. The method of claim 5, wherein said antibody further comprises the Heavy constant domain of SEQ ID NO: 18 and/or the Light constant region (kappa) of SEQ ID NO: 20.

8. The method of claim 6, wherein said antibody further comprises the Heavy constant domain of SEQ ID NO: 18 and/or the Light constant region (kappa) of SEQ ID NO: 20.

9. The method of claim 5, wherein the cancer is selected from the group consisting of colorectal (CRC), Head & Neck (HAN), Glioblastoma (GBM), Urinary (U), Prostate (PC), Breast (B), Lung (L), Pancreatic (PRC), and Renal (R) tumor.

10. A pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof, wherein said antibody and fragment is according to claim 1, and a pharmaceutically acceptable vehicle.

11. The composition of claim 10, wherein the antibody and fragment thereof has the VH sequence SEQ ID NO: 38 and the VL sequence SEQ ID NO: 36.

12. An anti-CK8 monoclonal antibody, or an antigen-binding fragment thereof, wherein said antibody or fragment comprises a heavy chain comprising the following three CDRs, respectively CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1 comprises the sequence SEQ ID NO: 43; CDR-H2 comprises the sequence SEQ ID NO: 44; and CDR-H3 comprises the sequence SEQ ID NO: 45; and a light chain comprising the following three CDRs, respectively CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 comprises the sequence SEQ ID NO: 46; CDR-L2 comprises the sequence SEQ ID NO: 47; and CDR-L3 comprises the sequence SEQ ID NO: 48.

13. The antibody or fragment thereof of claim 12, wherein said antibody and fragment has the VH sequence SEQ ID NO: 42 and the VL sequence SEQ ID NO: 40.

14. The antibody of claim 12, further comprising the Heavy constant domain of SEQ ID NO: 18 and/or the Light constant region (kappa) of SEQ ID NO: 20.

15. The antibody of claim 13, further comprising the Heavy constant domain of SEQ ID NO: 18 and/or the Light constant region (kappa) of SEQ ID NO: 20.

16. A method for treating a cancer expressing CK8, the method comprising administering to a patient in need thereof an effective amount of a monoclonal anti-CK8 antibody or an antigen-binding fragment thereof, wherein said monoclonal anti-CK8 antibody and antigen-binding fragment thereof is according to claim 12.

17. The method of claim 16, wherein the antibody or fragment thereof has the VH sequence SEQ ID NO: 42 and the VL sequence SEQ ID NO: 40.

18. The method of claim 16, wherein the antibody further comprises the Heavy constant domain of SEQ ID NO: 18 and/or the Light constant region (kappa) of SEQ ID NO: 20.

19. The method of claim 17, wherein the antibody further comprises the Heavy constant domain of SEQ ID NO: 18 and/or the Light constant region (kappa) of SEQ ID NO: 20.

20. The method of claim 16, wherein the cancer is selected from the group consisting of colorectal (CRC), Head & Neck (HAN), Glioblastoma (GBM), Urinary (U), Prostate (PC), Breast (B), Lung (L), Pancreatic (PRC) and Renal (R), tumor.

21. A pharmaceutical composition comprising an anti-CK8 monoclonal antibody or an antigen-binding fragment thereof, wherein said antibody and fragment is according to claim 12, and a pharmaceutically acceptable vehicle.

22. The composition of claim 21, wherein the antibody and fragment thereof has the VH sequence SEQ ID NO: 42 and the VL sequence SEQ ID NO: 40.

Description

BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS

Target Expression and HzR022 (D-A10) Epitope Detection

[0105] Table 1 illustrates HzR022 (D-A10) cellular staining analyzed by flow cytometry. The mean+ −SD on percentage of labelled cells (%) and Mean of intensity (MFI) are shown on different cell line cancers such as colorectal (CRC), Head & Neck (HAN), Glioblastoma (GBM), Urinary (U), Prostate (PC), Breast (B), Lung (L), Pancreatic (PRC) or Renal (R).

[0106] Table 2 illustrates HzR022 (D-A10) cellular staining analyzed by Immunohistochemistry on Colorectal PDX xenograft models

[0107] Table 3 illustrates HzR022 (D-A10) cellular staining analyzed by Immunohistochemistry on Pancreas PDX xenograft models

In Vivo Proof of Concepts

[0108] FIG. 1 illustrates in vivo inhibition of tumour growth of PDX xenograft CR0029 model CRC K-ras wild type with MAb anti-eCK8/HzR022 (D-A10)-representative experiment.

[0109] FIG. 2 illustrates in vivo inhibition of tumour growth of PDX xenograft CR0455 model CRC K-ras mutated with MAb anti-eCK8/HzR022 (D-A10)-representative experiment.

[0110] Table 4 shows a review of in vivo inhibition of tumour growth of PDX xenograft models CRC K-ras wild type with MAb anti-eCK8/HzR022 (D-A10)-Review on 5 PDX models.

[0111] Table 5 shows a review of in vivo inhibition of tumour growth of PDX xenograft models CRC K-ras mutated with MAb anti-eCK8/HzR022 (D-A10)-Review on 6 PDX models.

[0112] FIG. 3 illustrates In vivo inhibition of tumor growth of Head & Neck cancer CDX model such as Larynx cancer with MAb anti-eCK8/HzR022 (D-A10) in combination with cisplatin from the cell line BICR18.

MAb Ability for Internalisation Related to Antibody Drug Conjugated

[0113] FIG. 4 illustrates D-A10 MAb internalization for solid tumors as Colorectal cancer from the cell line HCT116 (A), Glioblastoma cancer from the cell line U87MG (B), Head & Neck cancer from the cell line BiCR56 (C), Pancreas cancer from the cell line BxPC3 (D), Prostate cancer from the cell line DU145 (E). Fine line: 4° C.—Bold line: 37° C.—Dotted line: untreated.

[0114] FIG. 5 illustrates D-D6 MAb internalization for solid tumors for solid tumors as Colorectal cancer from the cell line HCT116 (A), Glioblastoma cancer from the cell line U87MG (B), Head & Neck cancer from the cell line BiCR56 (C), Pancreas cancer from the cell line BxPC3 (D), Prostate cancer from the cell line DU145 (E). Fine line: 4° C.—Bold line: 37° C.—Dotted line: untreated.

[0115] FIG. 6 illustrates D-F5 MAb internalization for solid tumors as Colorectal cancer from the cell line HCT116 (A), Glioblastoma cancer from the cell line U87MG (B), Head & Neck cancer from the cell line BiCR56 (C), Pancreas cancer from the cell line BxPC3 (D), Prostate cancer from the cell line DU145 (E). .Fine line: 4° C.—Bold line: 37° C.—Dotted line: untreated.

EXAMPLES

[0116] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1: Preparation of Murine Anti-CK8 Antibody

[0117] The murine monoclonal antibodies specific for CK8 were produced using standard hybridoma techniques (Zola et al., Aust J. Exp Biol Med Sci. 1981; 59:303-6). Two different CK8 related peptides were synthesized and used for mice immunization as described in WO 2016/020553. After hybridoma cloning, three murine Mabs were obtained called mD-A10, mD-F5 and mD-D6. Each clone was injected into the peritoneum of nude mice. Protein A chromatography from murine ascitic fluid. The murine ascitic fluid is adjusted at pH 8.3 with the equilibration buffer 0.1 M Tris and 1.5 M Sulfate Ammonium and then loaded onto the rProtein A Sepharose Fast Flow column (GE Healthcare, Saint Cyr au Mont d'or, France). The non-binding proteins are flowed through and removed by several washings with equilibration buffer. The MAb anti-CK8 is eluted off the Protein A column using the elution buffer 0.1 M Citrate Sodium at pH 3.5. After concentration, the PBS solution containing IgG was filtered and the Mab concentration was determined at 280 nm

Example 2: HzR022 (D-A10) MAb Production and Protein A Purification

[0118] Mammalian cells are the preferred hosts for production of therapeutic glycoproteins, due to their capability to glycosylate proteins in the most compatible form for human applications (Jenkins et al., Nat Biotech. 1996; 14:975-81). Mammalian host cells that could be used include, human Hela, 283, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1 African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells. Bacteria very rarely glycosylates proteins, and like other type of common hosts, such as yeasts, filamentous fungi, insect and plant cells yield glycosylation patterns associated with rapid clearance from the blood stream.

[0119] The Chinese hamster ovary (CHO) cells allow consistent generation of genetically stable, highly productive clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media, and permit the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO- and SP2/0-mouse myeloma cells. Production from transgenic animals has also been tested (Jenkins et al., Nat Biotech. 1996; 14:975-81).

[0120] A typical mammalian expression vector contains the promoter element (early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses e.g. RSV, HTLV1, HIV1 and the early promoter of the cytomegalovirus (mCMV, hCMV), which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript (BGH polyA, Herpes thimidine kinase gene of Herpes simplex virus polyA (TKpa), Late SV40 polyA and 3′ UTR_Beta_Globin_polyA). Additional elements include enhancers (Eμ, hIE1), Kozak sequences, signal peptide and intervening sequences flanked by donor and acceptor sites for RNA splicing. Suitable expression vectors for use in practise in practising the present invention include, for examples, vectors such as pcDNA3.1, pcDNA3.3, pOptiVEC, pRSV, pEμMCMV, pMCMVHE-UTR-BG, pHCMVHE-UTR-BG, pMCMV-UTR-BG, pHCMV-UTR-BG, pMCMVHE-SV40, pHCMVHE-SV40, pMCMV-SV40, pHCMV-SV40, pMCMVHE-TK, pHCMVHE-TK, pMCMV-TK, pHCMV-TK, pMCMVHE-BGH, pHCMVHE-BGH, pMCMV-BGH, pHCMV-UTR-BGH).

[0121] The empty CHO Easy C cells (purchased by the CCT collection) were co-transfected with MAb expression vector for light and heavy chains following transient or stable transfection procedure established in our laboratory. Secretion of H and L chains were enabled by the respective human IgH leader sequence. The coding regions for light and heavy chains of MAb anti-CK8 are introduced into the MAb expression vector in the multiple cloning site. The transformants are analysed for correct orientation and reading frame, the expression vector may be transfected into CHO cell line.

[0122] Protein A chromatography from harvested CHO cell culture fluid. The harvested cell culture fluid produced from CHO cells is loaded onto the Hi Trap rProtein A column (GE Healthcare, Saint Cyr au Mont d'Or, France) that is equilibrated with Phosphate buffered saline, pH 7.2. The non-binding proteins are flowed through and removed by several washings with PBS buffer followed. The MAb anti-CK8 is eluted off the Protein A column using a step of elution of 0.1 M Citric acid at pH 3.0. Column eluent is monitored by A280. The anti-CK8 MAb peak is pooled.

Example 3: Cell Culture

[0123] Various tumour-derived cell lines are among the target cells that may be stained with MAb anti-CK8, in such assay procedures.

[0124] Cell lines. The established human neuroglioma cells H4, HS683, U373 or A172 (available from ATCC); the established human colorectal cells HT29; the established human pancreatic cells PANC1 or MIA-PA-CA2; the established human kidney adenocarcinoma cells A704 or ACHN and the established human lung adenocarcinoma cells A549 were grown in Dulbecco's Modified Eagle's Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France). The established human glioblastoma astrocytoma cells U87MG or T98G, the human head and neck cancer cells FaDu or Detroit562, the human urinary cancer cells UM-UC-3, J82, HT1197 or HT1376 and the human prostate cancer cells DU145 were grown in Eagle's Minimum Essential Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France). The established human breast adenocarcinoma cells MDAMB231, MCF-7 or HBL100 and the human colorectal cancer cells HCT116 were grown in Dulbecco's Modified Eagle's Medium Glutamax Low Glucose (Life Technologies, St Aubin, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France).The established human colorectal cells HCT15 or SW480 and the human head and neck cancer cells TR146 were grown in Dulbecco's Modified Eagle's Medium Glutamax High Glucose (Life Technologies, St Aubin, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France). The established human head and neck cancer cells BICR16, BICR18 or BICR56 were grown in Dulbecco's Modified Eagle's Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France), 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France) and 0.4 μg/mL hydrocortisone (Sigma, St Quentin Fallavier, France). The established human head and neck cancer cells SCC9, SCC4 or SCC15 were grown in Dulbecco's Modified Eagle's Medium/F12 (Life Technologies, St Aubin, France) supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France), 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France) and 0,4 μg/mL hydrocortisone (Sigma, St Quentin Fallavier, France). The established human urinary bladder carcinoma cells 5637, the human prostate cancer cells LNCap clone FGC, the established human lung adenocarcinoma cells NCIH1703 or NCIH292, the human pancreatic cancer cells PSN1 or BxPC3 and the human kidney adenocarcinoma cells Caki1 were grown in RPMI-1640 Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France). The established human glioma cells 42MGBA (available from DSMZ) were grown in 80% mixture of RPMI-1640 Medium and Eagle's Minimum Essential Medium at 1:1 (Sigma, St Quentin Fallavier, France) supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France). The established human glioma cells 8MGBA were grown in Eagle's Minimum Essential Medium (Sigma, St Quentin Fallavier, France) supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France). The established human urinary cancer cells TCCSUP and the human pancreatic cancer cells HuPT3 were grown in Eagle's Minimum Essential Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France), 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France), 1% sodium pyruvate (Sigma, St Quentin Fallavier, France) and 1% non-essential amino acid (Sigma, St Quentin Fallavier, France). The human pancreatic cancer cells AsPC1 were grown in RPMI-1640 Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France), 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France) and 1% sodium pyruvate (Sigma, St Quentin Fallavier, France). The human pancreatic cancer cells CFPAC1 were grown in Iscove's Modified Dulbecco's Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France).

Example 4: Antibody Binding Assays by Flow Cytometry

[0125] This example describes methods to investigate on CK8 cellular expression at the cell surface analysed by flow cytometry.

[0126] Flow cytometry experiments for CK8 cellular expression. Briefly, 2.10.sup.5 cells per 96 wells are incubated at 4° C. with a dilution of unconjugated humanised anti CK8 HZMR022/D-A10 MAb at 5 μg/mL then diluted at ½. The negative control MAb used was human IgG1 kappa (Sigma, St Quentin Fallavier, France). Unbound antibodies were washed away with PBS (Life technologies, St Aubin, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). Subsequently, cells are centrifuged (5 min at 400 g) and bound antibody is detected with Phycoerythrin (PE) conjugated Goat anti human IgG (Sigma, St Quentin Fallavier, France) at 4° C. for 30 min. Detection reagent is washed away and cells are centrifuged (5 min at 400 g) and suspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL2 channel, 2000 events per acquisition). During the experiment, the respective isotype controls are included to exclude any unspecific binding events.

[0127] Results of experiments are shown in TABLE 1 (at 5 μg/mL). Various cancer cell lines express CK8 epitope identified with HZMR022/D-A10. Expression patterns varied from cell line to cell lines. In the present study CK8 was expressed on all cell lines tested, among a cell subset.

Example 5: Antibody Binding Assays by Immunohistochemistry

[0128] This example describes methods to investigate on CK8 cellular expression at the cell surface by Immunohistochemistry. The antigen retrieval (AR) was performed following incubation at 95° C. during 30 min in Sodium Citrate (pH6.0). The MAb anti CK8 was incubated 1 hour at room temperature (RT). The secondary antibody as a goat anti human was incubated at RT during 1 hour then with a rabbit polyclonal anti FITC at RT during 30 min. Then the MAb IHC staining was revealed by using an ultravision LP detection system (primary antibody enhancer 10 min, HRP Polymer, 15 min) at RT following by an incubation with DAB at RT during 3 min.

[0129] Eleven CRC PDX models were evaluated as CR0004, CR0012, CR0029, CR0126, CR0196, CR0205, CR0455, CR1530, CR3056, CR3150 and CR6254.

[0130] Results of experiments are shown in TABLE 2. All of CRC PDX models expressed the HzR022 (D-A10) epitope detected by IHC. Different IHC scores were observed as 2+ for strong or 1+ for moderate intensity IHC staining.

[0131] Four PC PDX models were evaluated as PAN-001, PAN-003, PAN-004 and PAN-035.

[0132] Results of experiments are shown in TABLE 3. All of PC PDX models expressed the HzR022 (D-A10) epitope detected by IHC. The IHC score was determined according the membranous and cytoplasmic intensity. The IHC score of 4 was observed for all of them revealing a strong to very strong IHC staining.

Example 6: In Vivo Investigation on PDX Models for CRC

[0133] Each mouse was inoculated subcutaneously at the right flank with one primary human tumour xenograft model (CR00004, CR0012, CR0029, CR0126, CR0196, CR0205, CR0455, CR1530, CR3056, CR3150 or CR6254) tumour fragment (2-3 mm in diameter) for tumour development. When average or individual tumour size reaches 100-250 mm3, mice was randomly (rolling enrollment will be involved if necessary) allocated into 4 groups. Each group contained 1 mouse. The day of grouping and dosing initiation was denoted as day 0. The dosing volume was adjusted for body weight (Dosing volume=5 μL/g). After tumour inoculation, the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for any effects of tumour growth and treatments on normal behavior such as mobility, food and water consumption, body weight gain/loss, eye/hair matting and any other abnormal effect. Death and observed clinical signs were recorded on the basis of the numbers of animals within each subset. Two weeks of dosing-free observation were applied after final treatment. The animals in vehicle group were sacrificed before study termination because of tumour volume (TV) over 3000mm.sup.3. Tumour size was measured by caliper twice weekly in two dimensions. The tumour volume was expressed in mm3 using the formula: TV=0.5 a×b2 where a and b are the long and short diameters of the tumour, respectively. Body weight was measured twice weekly. When individual mouse has a body weight loss ≥15%, the mouse was given dosing holiday(s) until its body weight recovers to body weight loss. Under following conditions, the in-life experiment of individual animal or whole groups was terminated, by human euthanasia, prior to death, or before reaching a comatose state.

Study Design for MAb Impact Regarding CRC Subtypes

[0134]

TABLE-US-00013 Study design of MAb impact- N: animal number per group Dose level Dose Group N Treatment (mg/kg) Route Dosing Frequency 1 1 No treatment NA NA NA 2 1 HzR022 (D-A10) 30 i.v. BIW × 4 MAb anti CK8

K-Ras Wild Type in Vivo Proof of Concept from CRC Patient Derivate (PDX Models)

[0135] 5 HuPrime® CRC K-ras wt xenograft models (selected in CRO cell bank after CK8 IHC positive detection) were selected as CR0004, CR0029, CR0196, CR0205 or CR3056.

[0136] Excepted the CRC PDX K-Ras wt mode CR0205, all of K-Ras wt CRC did not respond to HzR022 (D-A10) MAb. No control of tumour progression was observed at 30 mg/kg among ⅘ CRC PDX K-ras wt models as illustrated in Table 4.

K-Ras Mutated in Vivo Proof of Concept from CRC Patient Derivate (PDX Models)

[0137] 6 HuPrime® CRC K-Ras mutated xenograft models (selected in CRO cell bank after CK8 IHC positive detection) were selected as CR0012, CR0126, CR0455, CR1530, CR3150 or CR6254. HzR022 (D-A10) mediated tumour progression was observed at 30 mg/kg among 6/6 CRC PDX K-Ras mutated models as illustrated in Table 5.

Example 7: In Vivo Investigation on HAN CDX Model

[0138] Human HAN larynx cell line BICR18 was subcutaneously injected in SCID mice, with a concentration of 1.10.sup.6 cells per injection (200 μL). Mice were randomized when the tumours reached a mean volume of about 100 mm.sup.3 for the 9 groups (total 45 mice). All the mice were observed in order to detect any toxic effects of the product. The endpoint was defined by animal ethics as a tumour diameter of >18 mm, significant weight loss or alteration of animal well-being. In order to assess the effectiveness of the compounds on tumourigenesis, tumour volume was measured two times a week. The size of the primary tumours were measured using calipers and the tumour volume (TV) was extrapolated to a sphere using the formula TV= 4/3π×r.sup.3, by calculating the mean radius from the two measurements. The median and standard deviation were also calculated for each group. Median is preferred to mean in order to exclude the extreme values. MAb treatment was administered by intraperitoneal injection twice a week during three weeks at 10 or 1 mg/kg doses. The cisplatin (Myland) treatment was administered by intraperitoneal injection once per week during three weeks at 1, 2.5 or 5 mg/kg doses. The product was prepared in accordance with the sponsor's guidelines, i.e. diluted in PBS. Mice were sacrificed when the tumours reached a maximum volume of 1600 mm.sup.3. The endpoints were defined by clinical trial ethics as a tumour diameter of >18 mm or weight loss of >10% of body weight, or when the tumours are dangerous for mice (necrosis). Statistical analysis was performed with GraphPad Prism software. GraphPad Prism combined scientific graphing, comprehensive curve fitting, understandable statistics, and data organization. The t-test (two-tailed test) was performed on the tumour volume values (mm.sup.3) measured on the day of sacrifice.

Study Design for MAb Impact on HAN CDX

[0139]

TABLE-US-00014 Study design of MAb impact N: animal number per group Dose level Dose Group N Treatment (mg/kg) Route Dosing Frequency 1 5 No treatment — — 2 5 HzR022 (D-A10) 10 i.p. BIW × 3 3 5 Cisplatin 2.5 i.p. QW × 3 4 5 HzR022 (D-A10) + 10 i.p. BIW × 3 Cisplatin 2.5 QW × 3

[0140] Results shown in FIG. 3 that the combination treatment resulted in synergic cytotoxicity of tumour growth of HAN with MAb anti-CK8/HzR022 (D-A10) in combination with cisplatin. Although HzR022 (D-A10) alone did trigger tumour growth control, the combination of HzMR022/D-A10 and cisplatin further reverse the escape of tumour cisplatin sensitivity. In view of rapid cisplatin escape in the cisplatin group, it is highly unexpected that combination of HzMR022/D-A10 and cisplatin demonstrates tumour control over the time.

Example 8: Antibody Internalisation Assays by Flow Cytometry

[0141] This example describes methods to investigate on HzR022 (D-A10) internalisation following CK8 detection at the cell surface analysed by flow cytometry.

[0142] Flow cytometry experiments for MAb internalisation. Briefly, 2.10.sup.5 cells per 96 wells are incubated at 4° C. versus 37° C. during 24 hours with a dilution of unconjugated murine anti CK8 MAb at 50 μg/mL then diluted at ½. The MAb anti CK8 tested were D-A10 (IgG2b), D-F5 (IgG2a) or D-D6 (IgG1), from iDD biotech MAb panel. The isotype matched MAbs used were B-Z1 (IgG1), B-Z2 (IgG2a) or B-E4 (IgG2b) (Diaclone, Besancon, France). Unbound antibodies were washed away with PBS (Life technologies, St Aubin, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). Subsequently, cells are centrifuged (5 min at 400 g) and bound antibody is detected with Fluorescein Isothiocyanate (FITC) conjugated goat (Fab′).sub.2 polyclonal anti mouse Ig (MP Biomedical, Illkirch, France) at 4° C. for 30 min. Detection reagent is washed away and cells are centrifuged (5 min at 400 g) and suspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL1 channel, 3 000 events per acquisition). During the experiment, the respective isotype controls are included to exclude any unspecific binding events

[0143] Results of experiments are shown in FIG. 4 for D-A10 MAb (at 0.78 or 0.39 μg/mL), in FIG. 5 for D-D6 (at 0.78 or 0.39 μg/mL), in FIG. 6 for D-F5 (at 0.78 or 0.39 μg/mL). One example was shown for CRC from HCT116 cell line, GBM from U87MG cell line, HAN from BIRC56 cell line, PC from BxPC3 or PRC from DU145.

[0144] Whatever the MAb D-F5 exhibited a lower internalization potential, a highest internalization potential was observed for D-A10 or D-D6 MAb anti-CK8.

TABLE-US-00015 TABLE 1 eCK8 expression on solid tumours % labelled Cancer Cell line cells MFI Expt (n=) Colon HT29 49 ± 9  203 ± 22 2 HCT116 64 ± 0  283 ± 16 2 SW480 38 ± 3  179 ± 1  2 Head & Neck FaDu 56 ± 8  258 ± 20 4 Detroit562 36 ± 0  203 ± 15 2 BICR16 52 ± 2  206 ± 10 2 BICR18 79 ± 9  257 ± 23 2 BICR56 39 ± 34 123 ± 4  3 SCC9 30 ± 23 193 ± 23 3 SCC4 59 ± 16 222 ± 88 4 SCC 15 41 ± 21 167 ± 13 2 TR146 55 ± 18 140 ± 4  2 GBM H4 58 ± 33 199 ± 40 4 A172 35 ± 23 186 ± 14 4 U373 53 ± 1  161 ± 7  4 8-MG-BA 72 ± 11 261 ± 38 4 42-MG-BA 54 ± 7  247 ± 44 4 T98G 48 ± 25 211 ± 20 4 HS683 54 ± 9  225 ± 24 4 U87-MG 38 ± 23 178 ± 35 4 Urinary 5637 59 ± 20 284 ± 61 4 UM-UC-3 26 ± 23 156 ± 14 2 J82 68 ± 9  319 ± 4  2 TCCSUP 62 ± 10 255 ± 48 2 HT1197 23 ± 7  240 ± 4  2 HT1376 53 ± 3  497 ± 11 2 Prostate DU145 19 ± 11 185 ± 42 4 LNCaP clone FGC 76 ± 9  412 ± 21 2 Breast MCF-7 56 ± 33 250 ± 61 2 MDAMB231 49 ± 14 179 ± 15 3 HBL100 67 ± 8  219 ± 6  2 Lung A549 36 ± 12 193 ± 38 5 NCIH1703 33 ± 10 188 ± 12 4 NCIH292 53 ± 20 272 ± 38 4 Pancreatic HuP-T3 62 ± 17 260 ± 20 2 MIA-Pa-Ca-2 58 ± 14 264 ± 14 4 PANC-1 37 ± 7  198 ± 23 4 PSN-1 15 ± 6  193 ± 16 4 AsPC-1 26 ± 14 181 ± 17 4 BxPC-3 41 ± 17 220 ± 17 4 CFPAC-1 62 ± 4  279 ± 25 4 Renal ACHN 46 ± 20 195 ± 20 4 A704 37 ± 26 184 ± 30 4 Caki1 49 ± 12 267 ± 36 4

TABLE-US-00016 TABLE 2 HzR022 (DA-10) cellular staining analyzed by Immunohistochemistry on Colorectal PDX xenograft models CRC PDX model HzR022 (D-A10) IHC score CR0004 2+ CR0012 2+ CR0029 2+ CR0126 2+ CR0196 2+ CR0205 1+ CR0455 1+ CR1530 2+ CR3056 1+ CR3150 1+ CR6254 2+

TABLE-US-00017 TABLE 3 HzR022 (D-A10) cellular staining analyzed by Immunohistochemistry on Pancreas PDX xenograft models PC PDX Membraneous Cytoplasmic model Intensity Intensity HZMR022/D-A10 IHC score PAN-001 4 4 16 PAN-003 4 4 16 PAN-004 4 4 16 PAN-035 4 4 16

TABLE-US-00018 TABLE 4 In vivo inhibition of tumour growth of PDX xenograft model CRC K-Ras wild type with MAb anti-eCK8/HzR022 (D-A10) - Review on 5 PDX models NR: Non responder to HzR022 (D-A10) MAb R: Responder to HzR022 (D-A10) MAb CRC PDX model K-RAS genomic profile HzR022 (D-A10) activity CR0004 wild type NR CR0029 wild type NR CR0196 wild type NR CR0205 wild type R CR3056 wild type NR

TABLE-US-00019 TABLE 5 In vivo inhibition of tumour growth of PDX xenograft model CRC K-Ras mutated with MAb anti-eCK8/HzR022 (D-A10) - Review on 6 PDX model NR: Non responder to HzR022 (D-A10) MAb R: Responder to HzR022 (D-A10) MAb CRC PDX model K-RAS genomic profil HzR022 (D-A10) activity CR0012 G13D R CR0126 G12V R CR0455 G12D R CR1530 Q61H R CR3150 G12V R CR6254 A146T R