IGF-1R ANTIBODY AND ITS USE FOR THE DIAGNOSIS OF CANCER

Abstract

The present disclosure relates to IGF-IR (insulin like growth factor receptor-1) antibodies characterized by CDR sequences a, to be used in detection methods of IGF-IR expressing tumoral cells.

Claims

1.-15. (canceled)

16. A method for detecting in vitro or ex vivo the presence and/or the location of IGF-1R expressing tumor cells in a subject, the method comprising the steps of: (a) contacting a biological sample from the subject with an IGF-1R antibody, or an antigen-binding fragment thereof, the antibody comprising: i) a heavy chain comprising the following three CDRs, respectively CDR-H1 of sequence SEQ ID No. 1, CDR-H2 of sequence SEQ ID No. 2 and CDR-H3 of sequence SEQ ID No. 3; and ii) a light chain comprising the following three CDRs, respectively CDR-L1 of sequence SEQ ID No. 4, CDR-L2 of sequence SEQ ID No. 5 and CDR-L3 of sequence SEQ ID No. 6; and (b) detecting the binding of the IGF-1R antibody, or antigen-binding fragment thereof, to the biological sample.

17. The method according to claim 16, the antibody comprising a heavy chain variable domain of sequence SEQ ID No. 7, or any sequence with at least 90% of homology with the sequence SEQ ID No. 7; and/or a light chain variable domain of sequence SEQ ID No. 8, or any sequence with at least 90% of homology with the sequence SEQ ID No. 8.

18. The method according to claim 16, wherein the antibody is secreted by the hybridoma deposited at the CNCM, Institut Pasteur, Paris, on Sep. 17, 2014, under number 1-4894.

19. A method for detecting in vitro or ex vivo the percentage of tumor cells expressing IGF-1R in a subject, said method comprising the steps of: (a) contacting a biological sample from the subject with an IGF-1R antibody, or an antigen-binding fragment thereof, the antibody comprising: i) a heavy chain comprising the following three CDRs, respectively CDR-H1 of sequence SEQ ID No. 1, CDR-H2 of sequence SEQ ID No. 2 and CDR-H3 of sequence SEQ ID No. 3; and ii) a light chain comprising the following three CDRs, respectively CDR-L1 of sequence SEQ ID No. 4, CDR-L2 of sequence SEQ ID No. 5 and CDR-L3 of sequence SEQ ID No. 6; and (b) quantifying the percentage of cells expressing IGF-1R in the biological sample.

20. The method according to claim 19, the antibody comprising a heavy chain variable domain of sequence SEQ ID No. 7, or any sequence with at least 90% of homology with the sequence SEQ ID No. 7; and/or a light chain variable domain of sequence SEQ ID No. 8, or any sequence with at least 90% of homology with the sequence SEQ ID No. 8.

21. The method according to claim 19, wherein the antibody is secreted by the hybridoma deposited at the CNCM, Institut Pasteur, Paris, on Sep. 17, 2014, under number 1-4894.

22. A method for determining in vitro or ex vivo the expression level of IGF-1R in tumor cells in a subject, the method comprising the steps of: (a) contacting a biological sample from the subject with an IGF-1R antibody, or an antigen-binding fragment thereof, the antibody comprising: i) a heavy chain comprising the following three CDRs, respectively CDR-H1 of sequence SEQ ID No. 1, CDR-H2 of sequence SEQ ID No. 2 and CDR-H3 of sequence SEQ ID No. 3; and ii) a light chain comprising the following three CDRs, respectively CDR-L1 of sequence SEQ ID No. 4, CDR-L2 of sequence SEQ ID No. 5 and CDR-L3 of sequence SEQ ID No. 6; and (b) quantifying the level of binding of the IGF-1R antibody, or an antigen-binding fragment thereof, to IGF-1R in the biological sample.

23. The method according to claim 22, the antibody comprising a heavy chain variable domain of sequence SEQ ID No. 7, or any sequence with at least 90% of homology with the sequence SEQ ID No. 7; and/or a light chain variable domain of sequence SEQ ID No. 8, or any sequence with at least 90% of homology with the sequence SEQ ID No. 8.

24. The method according to claim 22, wherein the antibody is secreted by the hybridoma deposited at the CNCM, Institut Pasteur, Paris, on Sep. 17, 2014, under number 1-4894.

25. A method for determining in vitro or ex vivo the IGF-1R scoring of tumor cells or of a tumor in a subject, the method comprising the steps of: (a) contacting a biological sample from the subject with an IGF-1R antibody, or an antigen-binding fragment thereof, the antibody comprising: i) a heavy chain comprising the following three CDRs, respectively CDR-H1 of sequence SEQ ID No. 1, CDR-H2 of sequence SEQ ID No. 2 and CDR-H3 of sequence SEQ ID No. 3; and ii) a light chain comprising the following three CDRs, respectively CDR-L1 of sequence SEQ ID No. 4, CDR-L2 of sequence SEQ ID No. 5 and CDR-L3 of sequence SEQ ID No. 6; (b) quantifying by Fluorescence Activated Cell Sorting (FACS) or immunohistochemistry (IHC) the level of binding of the IGF-1R antibody, or an antigen-binding fragment thereof, to IGF-1R in said biological sample; and (c) scoring the tumor cells or the tumor by comparing the quantified level obtained in step (b) to an appropriate scale based on the intensity of the staining and the percentage of positive cells.

26. The method according to claim 25, the antibody comprising a heavy chain variable domain of sequence SEQ ID No. 7, or any sequence with at least 90% of homology with the sequence SEQ ID No. 7; and/or a light chain variable domain of sequence SEQ ID No. 8, or any sequence with at least 90% of homology with the sequence SEQ ID No. 8.

27. The method according to claim 26, wherein the antibody is secreted by the hybridoma deposited at the CNCM, Institut Pasteur, Paris, on Sep. 17, 2014, under number 1-4894.

28. A method for determining whether an oncogenic disorder is susceptible to treatment with an inhibitor targeting the IGF-1R pathway, said method comprising the steps of: (a) determining in vitro or ex vivo the IGF-1R scoring of tumor cells or of a tumor of a subject according to the method of claim 25, and (b) determining that, if the IGF-1R scoring of tumor cells or the tumor is IGF-1R(+), the oncogenic disorder is susceptible to treatment with an antibody drug targeting the IGF-1R pathway.

29. The method of claim 28 wherein said inhibitor is an IGF-1R antibody.

30. The method of claim 29 wherein the IGF-1R antibody is alone, combined or conjugated.

31. A method for determining in vitro or ex vivo the efficacy of a therapeutic regimen designed to alleviate an oncogenic disorder associated with IGF-1R in a subject suffering from the disorder, the method comprising the steps of: (a) measuring a first expression level of IGF-1R according to the method of claim 22 in a first biological sample, the first biological sample corresponding to the first time point of the treatment; (b) measuring a second expression level of IGF-1R according to the method of claim 22 in a second biological sample, the second biological sample corresponding to a second, later time point of the said treatment; (c) calculating the ratio of the first expression level of step (a) to the second expression level of step (b); and (d) determining that the efficacy of the therapeutic regime is high if the ratio of step (c) is greater than 1; or determining that the efficacy of the therapeutic regime is low if the ratio of step (c) is inferior or equal to 1.

32. A method for selecting a cancer patient predicted to benefit or not from the administration of a therapeutic amount of an inhibitor targeting the IGF-1R pathway, the method comprising the steps of: (a) measuring the expression level of IGF-1R according to the method of claim 22 in a biological sample of the patient; (b) comparing the expression level of (a) with a reference expression level; and (c) selecting the patient as being predicted to benefit from a treatment with an antibody drug targeting the IGF-1R pathway, if the ratio of the expression level of (a) to the said reference expression level is greater than 1; or (d) selecting the patient as being not predicted to benefit from a treatment with an inhibitor targeting the IGF-1R pathway, if the ratio of the expression level of (a) to the reference expression level is inferior or equal to 1.

33. The method of claim 32 wherein the inhibitor is an IGF-1R antibody.

34. The method of claim 33 wherein the IGF-1R antibody is alone, combined or conjugated.

35. The method of claim 33 wherein the reference expression level is the expression level of IGF-1R in a biological sample from a healthy subject.

Description

[0215] Other characteristics and advantages of the invention appear in the continuation of the description with the examples and the figures whose legends are represented below.

[0216] FIG. 1: Graphic representation of OD values obtained with 816C12 antibody in the rhIGF1R ELISA. Data fitting and EC.sub.50 determination are determined using Prism application.

[0217] FIGS. 2A-2C: Immunohistochemistry (IHC) patterns of recognition of paraffin embedded tumor MCF-7 with the 816C12 (FIG. 2A), with G11 anti-IGF-1R antibody (Roche Ventana) (FIG. 2B) or AF-305 (R&D system) anti-IGF-1R antibody (FIG. 2C).

[0218] FIG. 3: In vivo activity of an anti-IGF-1R ADC in the MCF-7 xenograft model.

[0219] FIGS. 4A-4C: Immunohistochemistry (IHC) patterns of recognition of paraffin embedded tumor SBC-5 with the 816C12, with G11 anti-IGF-1R antibody (Roche Ventana) (FIG. 4B) or AF-305 (R&D system) anti-IGF-1R antibody (FIG. 4C).

[0220] FIG. 5: In vivo activity of an anti-IGF-1R ADC in the SBC-5 xenograft model.

EXAMPLE 1: 816C12 GENERATION AND SELECTION

[0221] Mabs generated against IGF-1R were produced and selected as described bellow.

[0222] Female Balb/C mice were immunized by subcutaneous injection with 10 g of recombinant human IGF-1R protein (R and D Systems, 391-GR) with Freund Adjuvant. Immunisation was repeated three times at 2 weeks intervals. The fourth injection was made by intraperitoneal injection in presence of adjuvant.

[0223] Three days later spleen cells were fused with SP2OAg14 myeloma cells with PEG 50%. After 14 days of HAT metabolic selection, hybridoma supernatants were tested by FACS using human MCF7 breast cancer cells. Only MCF7 binding antibodies were kept.

[0224] Antibodies of interest were then cloned by limit dilution. Eight days after cloning, supernatants were selected once again by FACS using MCF7 cells. Three positive clones were kept. Isotyping of the secreted antibodies is determined using SBA clonotyping system-HRP kit from Southern Biotechnologies (Cat: 5300-05). Finally, one clone is expanded and frozen.

[0225] Further characterizations of the 816C12 antibody were then performed using hybridoma supernatant such as rhIGF-1R or rmIGF-1R or rhIR ELISA. In all direct ELISAs, proteins of interest were immobilized (1 g/ml) to the bottom of each well. After saturation, hybridoma supernatants were added to the wells. After a 1-hour incubation period and a washing step, a solution of goat anti-mouse IgGHRP labelled polyclonal antibody was used for detection, prior to the addition of the TMB substrate. The reaction was stopped with a 1M H2504 solution before reading the OD with a spectrophotometer at a 450 nm wavelength. Data are presented in Table 6.

TABLE-US-00006 TABLE 6 OD values obtained at 5 g/ml by ELISA rhIGF-1R rmIGF-1R rhIR coating coating coating 816C12 2.622 0.065 0.055 Positive CTRL 2.338 1.293 1.077 Negative CTRL 0.055 0.065 0.048

[0226] The dose response curve for the 816C12 antibody on rhIGF-1R coating is presented in FIG. 1. The values of the EC.sub.50 are determined using Prism application.

[0227] Data showed that the 816C12 antibody only recognizes the rh IGF-1R with an EC.sub.50 of 0.41 nM. It does not bind to the murine form of the IGF-1R nor the human IR.

EXAMPLE 2: EVALUATION OF THE CORRELATION OF THE STAGING WITH THE ANTIBODY OF THE INVENTION AND THE ACTIVITY OF AN ADC TARGETING IGF-1R IN THE MCF-7 XENOGRAFT MODEL

[0228] In order to correlate the grading of tumors with the pharmacology, the tumors have been graded (section 2.1) and then in vivo experiments on MCF-7 xenograft model have been made with an ADC comprising an antibody moiety targeting the IGF-1R known to be internalized and a drug moiety consisting of an auristatin (section 2.2).

[0229] 2.1: Immunohistochemistry Detection of the IGF-1R Expression on the MCF-7 Xenograft Model.

[0230] Sections of tissue from MCF-7 xenograft were deparaffinized, rehydrated, and placed in Target Retrieval Buffer 1 (Dako S1699) in a boiling bath pre-warm at 98 C. for heat-induced epitope retrieval at 98 C. for 40 minutes then 20 additional minutes in the Target Retrieval Buffer. After 3 washes in Tris Buffer Saline-0.05% tween 20 (TBS-T) (Dako 53006) the Endogenous peroxidase activity was blocked using Peroxidase Blocking Reagent (Dako K4007) for five minutes. Sections were washed with TBS-T and incubated a blocking reagent (UltraV block-TA-125UB-LabVision) for 5 minutes before incubation with either the 816C12 monoclonal antibody (at 5 g/ml) or mouse IgG1/kappa (5 g/ml, X0931, Dako) as negative control for 1 hours at room temperature. Sections were washed with TBS-T and incubated with Envision (Dako) for 30 minutes. Diaminobenzidine was used for development of a brown reaction product (Dako K3468). The slides were immersed in hematoxylin for 2 minutes to counterstain (Dako S3309).

[0231] Anti-IGF-1R monoclonal antibody 816C12 of the present invention differentially stains the cell membrane of MCF-7. In this IHC procedure, the brown reaction product correlates to positive staining of the cell membrane and lack of brown reaction product correlates to negative staining and no visualization of the cell membrane. Using membranous algorithm, the scoring for the staining of MCF-7 tumor cells was 3+(FIG. 2A). Using G11 antibody (Roche Ventana) or AF-305 (R&D system) anti-IGF-1R antibodies, section of the same tumor were scored 2+(FIGS. 2B and 2C respectively).

[0232] 2.2: In Vivo Activity of an Anti-IGF-1R ADC in the MCF-7 Xenograft Model.

[0233] Anti-IGF-1R ADC has been evaluated in vivo, in the MCF-7 xenograft model.

[0234] All animal procedures were performed according to the guidelines of the 2010/63/UE Directive on the protection of animals used for scientific purposes. The protocol was approved by the Animal Ethical Committee of the Pierre Fabre Institute. Five millions MCF-7 cells were injected subcutaneous into 7 weeks old Swiss/Nude mice. Prior to cell injection, oestrogen pellets (Innovative Research of America) were implanted to the left flank to mice in order to release estrogens necessary to the in vivo growth of MCF-7 tumors.

[0235] Twenty days after MCF-7 cell implantation, when tumors reached an average size of 120-150 mm.sup.3, the animals were divided into groups of 6 mice according to tumor size and aspect. Anti-IGF-1R ADC was inoculated by intraperitoneal injections for a 6 injection cycle every four days (Q4d4). The health status of animals was monitored daily. Tumor volume was measured twice a week with an electronic caliper until study end. Tumor volume is calculated with the following formula: /6lengthwidthheight. Toxicity was evaluated following the weight of animals three times per week. Statistical analyses were performed at each measure using a Mann-Whitney test.

[0236] Injection of anti-IGF-1R ADC significantly inhibited and even induced a complete tumor growth regression (FIG. 3) as expected for a tumor graded 3+ but not for a tumor graded 2+.

EXAMPLE 3: EVALUATION OF THE CORRELATION OF THE STAGING WITH THE ANTIBODY OF THE INVENTION AND THE ACTIVITY OF AN ADC TARGETING IGF-1R IN THE SBC-5 XENOGRAFT MODEL

[0237] In order to correlate the grading of tumors with the pharmacology, the tumors have been graded (section 3.1) and then in vivo experiments on SBC-5 xenograft model have been made with an ADC comprising an antibody moiety targeting the IGF-1R and a drug moiety consisting of an auristatin (section 3.2).

[0238] 3.1 Immunohistochemistry Detection of the IGF-1R Expression on the SBC-5 Xenograft Model.

[0239] Level of IGF-1R was analyzed using the same protocol described in section 2.1 of the example 2 before.

[0240] When IGF-1R was detected with the 816C12, low levels were detected (1+). (FIG. 4A). When IGF-1R was detected with G11 antibody (Roche Ventana) or AF-305 (R&Dsystem) anti-IGF-1R antibodies, sections from the same tumor were scored 3+ (FIGS. 4B and 4C respectively).

[0241] 3.2: In Vivo Activity of an Anti-IGF-1R ADC in the SBC-5 Xenograft Model.

[0242] Anti-IGF-1R ADC has been evaluated in vivo, in the SBC-5 xenograft model.

[0243] All animal procedures were performed according to the guidelines of the 2010/63/UE Directive on the protection of animals used for scientific purposes. The protocol was approved by the Animal Ethical Committee of the Pierre Fabre Institute. Five millions SBC-5 cells were injected subcutaneous into 7 weeks old Athymic mice. Twelve days after cell implantation, when tumors reached an average size of 150 mm.sup.3, the animals were divided into groups of 6 mice according to tumor size and aspect. Anti-IGF-1R ADC was inoculated by intraperitoneal injections for a 6 injection cycle every four days (Q4d6). The health status of animals was monitored daily. Tumor volume was measured twice a week with an electronic caliper until study end. Tumor volume is calculated with the following formula: /6lengthwidthheight. Toxicity was evaluated following the weight of animals three times per week. Statistical analyses were performed at each measure using a Mann-Whitney test.

[0244] Tumor progression of SBC-5 tumoral cells was not affected by injection of anti-IGF-1R ADC. (FIG. 5) as expected for a tumor graded 1+ but not for a tumor graded 3+.