THERAPEUTIC AGENTS AND USE THEREOF

Abstract

A therapeutic agent comprising a cell binding agent which binds the Receptor for Advanced Glycation End (RAGE) products linked to an anti-cancer drug, for use in the treatment of gynaecological cancer, endometriosis or polycystic ovary syndrome. Novel cell binding agents, pharmaceutical compositions and methods are also described and claimed.

Claims

1-20. (canceled)

21. A method for treating a proliferative condition selected from gynaecological cancers, endometriosis or polycystic ovary syndrome, wherein the method comprises administering to a patient in need thereof an effective amount of a therapeutic agent comprising a cell binding agent which binds the Receptor for Advanced Glycation End products (RAGE) linked to an anti-cancer drug.

22. The method of claim 21 wherein the cell binding agent is an antibody, a binding fragment thereof or a non-antibody affinity reagent.

23. The method of claim 22 wherein the non-antibody affinity reagent is selected from a peptide, an aptamer, or a nanobody.

24. The method of claim 22 wherein the cell binding agent is a monoclonal antibody.

25. The method of claim 24 wherein the monoclonal antibody is a human or humanised antibody.

26. The method of claim 21 wherein the cell binding agent binds a region of RAGE comprising SEQ. ID. NO. 24.

27. The method of claim 21 wherein the anti-cancer drug is a cytotoxin; a hormone; a cytokine, chemokine, or other cell signaling molecule; or a nucleic acid.

28. The method of claim 21 wherein the cell binding agent is linked to the anti-cancer drug by way of a chemical linking group.

29. The method of claim 21 wherein the ratio of drug to cell binding agent molecules is from about 1:1 to 1:8.

30. The method of claim 29 wherein the ratio of drug to cell binding agent molecules is from about 1:1.5 to 1:3.5.

31. The method of claim 21 wherein the proliferative condition is a gynaecological cancer selected from endometrial cancer or ovarian cancer.

32. A cell binding agent which specifically binds to SEQ. ID. NO. 24, or to a V-region, or to a region for which MAB11451 binding is specific.

33. The cell binding agent of claim 32 which is an antibody or a binding fragment thereof.

34. The cell binding agent of claim 32 linked to an anti-cancer drug thereby forming a therapeutic agent.

35. The cell binding agent of claim 34 combined with a pharmaceutically acceptable carrier thereby forming a pharmaceutical composition.

36. A method for preparing the therapeutic agent of claim 34 whereby the method comprises linking a cell binding agent that binds a Receptor for Advanced Glycation End (RAGE) product to an anticancer agent.

37. The method of claim 36 wherein, in a preliminary step, a linking group is added to the anticancer agent, then a resulting product is reacted with the cell binding agent.

38. The method of claim 21 wherein the therapeutic agent is administered together with an anti-hormonal agent that upregulates RAGE in gynaecological cancer.

39. The method of claim 38 wherein the anti-hormonal agent is taxoxifen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0046] The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings in which

[0047] FIG. 1 is a graphical representation of the multiligand transmembrane receptor of the immunoglobulin superfamily, RAGE and some of its variant forms;

[0048] FIG. 2 is a series of images showing RAGE protein expression in biopsies from the endometrium and ovary of a healthy patient and patients with endometrial or ovarian cancer.

[0049] FIG. 3 is a series of graphs showing (A) the expression of AGER mRNA in four endometrial epithelial cell lines derived from two well-differentiated type I and type II adenocarcinomas; HEC1 (HEC1A, HEC1B, HEC50) and Ishikawa respectively; (B) the results of an immunohistochemistry study showing that endometrial RAGE is overexpressed in hyperplasia and Endometrial cancer; and (C) immunohistochemistry results for RAGE staining in healthy ovary or ovarian cancer (OC) biopsies.

[0050] FIG. 4 is a series of Western blots showing RAGE protein expression in the cell lines of FIG. 3;

[0051] FIG. 5 is representative Western blots showing expression of RAGE protein in six ovarian cancer cell lines: TOV21G, TOV112D, UWB1.289, UACC-1598, COV644 and SKOV3, and one normal ovarian cell line: HOSEpiC;

[0052] FIG. 6 is a series of graphs showing RAGE expression scoring (intensity and distribution: H-score) in endometrial biopsy samples, taken during the proliferative phase of the menstrual;

[0053] FIG. 7 is a series of graphs showing RAGE expression scoring (intensity and distribution: H-score) in endometrial biopsy samples, taken during the secretory phase of the menstrual cycle;

[0054] FIG. 8 is a series of graphs showing AGER mRNA expression in endometrial biopsy samples taken from polycystic ovary syndrome patients during the proliferative phase of the menstrual cycle;

[0055] FIG. 9 is a series of graphs showing AGER mRNA expression in endometrial biopsy samples taken from polycystic ovary syndrome patients, during the secretory phase of the menstrual cycle;

[0056] FIG. 10 is a series of confocal microscopy images showing the internalisation of anti-RAGE antibody in HEC 1A cells;

[0057] FIG. 11 is a series of graphs illustrating how delivering cytotoxins in the form of RAGE targeting ADC improves drug potency in endometrial cancer cells;

[0058] FIG. 12 is a series of graphs showing how delivering cytotoxins in the form of RAGE targeting ADC improves drug potency in ovarian cancer cells;

[0059] FIG. 13 is a series of graphs showing that RAGE targeting ADCs are more potent killers of endometrial cancer cells than cytotoxin or antibody treatment alone;

[0060] FIG. 14 is a graph illustrating that RAGE targeting ADCs induce apoptosis of endometrial cancer cells.

[0061] FIG. 15 is a series of graphs showing that RAGE targeting ADCs are more potent killers of ovarian cancer cells than cytotoxin or antibody treatment alone;

[0062] FIG. 16 is a graph showing that RAGE targeting ADCs induce apoptosis of ovarian cancer cells;

[0063] FIG. 17 is a graph illustrating how using a non-cleavable linker improves ADC potency in endometrial (Ishikawa) and ovarian (TOV112D) cancer cells.

[0064] FIG. 18 is a series of confocal microscopy images showing antibody internalisation in ovarian (B-F) and endometrial cancer cells (G-K) that have been treated with 5 different anti-RAGE antibodies;

[0065] FIG. 19 is a series of graphs showing cell survival rates in HEC 1A cells when treated with ADCs in accordance with the invention. (A) IC50 curves at 96 h, and (B) a time-course graph of cells treated with ADCs (5 μg/ml);

[0066] FIG. 20 is a series of graphs showing cell survival data for a range of cell lines when treated with ADCs in accordance with the invention;

[0067] FIG. 21 shows the results of experiments revealing the effect of tamoxifen (Tx) on endometrial expression of RAGE.

EXAMPLE 1

Expression of RAGE in Gynaecological Cancers and Non Oncological Proliferative Conditions

[0068] Endometrial biopsies were collected from the endometrium of a healthy patient (FIG. 2A), and patients with endometrial cancer (FIG. 2B), endometrial hyperplasia (FIG. 2C), or endometriosis (FIG. 2D). Biopsies were fixed and paraffin embedded for analysis of RAGE expression by immunohistochemistry.

[0069] Further biopsy images show RAGE expression in a healthy ovary (FIG. 2E) and ovarian cancer (endometrioid adenocarcinoma; FIG. 2F). Positive staining was observed in the epithelial cells of the ovarian cystic masses whereas healthy tissue did not express the target.

[0070] The expression of AGER mRNA in four endometrial epithelial cell lines derived from two well-differentiated type I and type II adenocarcinomas; HEC1 (HEC1A, HEC1B, HEC50) and Ishikawa respectively, was measured. Epithelial cells were cultured in 6-well plates in control medium. Total RNA was extracted once cells reached confluence for analysis of AGER mRNA expression by quantitative PCR. Data are presented as box plots showing the median (line), 25.sup.th and 75.sup.th percentiles (box) and 10.sup.th and 90.sup.th percentile (whiskers), n=5, in FIG. 3A.

[0071] In a further experiment, RAGE protein expression was measured in the endometrial biopsies from patients diagnosed with hyperplasia, endometrial cancer Type I or Type II and postmenopausal (PM) controls by immunohistochemistry. Endometrial biopsy samples were grouped as follows: PM (n=25, median=0.2), Hyperplasia (n=21, median=5.5), type I EC (n=18, median=1.5), type II EC (n=17, median=2). IHC samples were scored blind by three independent observers. Values shown are median IHC scores and statistical analysis was performed using a Mann-Whitney test *p<0.05, **p<0.01, compared to PM control.

[0072] The results are shown in FIG. 3B. RAGE expression was noted in the membrane and cytoplasm of the tumour cells as well as endometrial cells obtained from hyperplasia patients. PM staining was almost negative. Statistically significant differences in RAGE expression were observed between PM control and all study groups.

[0073] RAGE protein expression was also measured by Immunohistochemistry in ovarian biopsies from patients diagnosed with ovarian cancer (n=19) and healthy control patients (n=8). IHC samples were scored blind by three independent observers. The results are shown graphically in FIG. 3C. Values shown are median IHC scores and statistical analysis was performed using a Mann-Whitney test, **p<0.01, compared to healthy control.

[0074] RAGE protein expression in the four endometrial cancer epithelial cell lines (HEC1A, HEC1B, HEC50 and Ishikawa), six ovarian cancer epithelial cell lines (TOV21G, TOV112D, UWB1.289, UACC-1598, COV644, SKOV3) and a non-cancerous ovarian cell line (HOSEpiC) were determined by Western blot. Epithelial cells were cultured in 6-well plates in control medium. Protein was extracted once cells reached confluence for analysis of RAGE protein expression. Data are presented as representative Western blots for endometrial and ovarian cell lines, FIGS. 4 and 5, respectively.

[0075] These results clearly show that RAGE is upregulated in these gynaecological cancers.

[0076] In further experiments, endometrial biopsies were collected from patients during the proliferative phase (n=32) of the menstrual cycle, and subdivided into four groups: fertile (n=9), endometriosis (n=11), ovulatory PCOS (n=12) or anovulatory PCOS (n=14). Biopsies were fixed and paraffin embedded for analysis of RAGE expression by immunohistochemistry. RAGE expression scoring (intensity and distribution: H-score) in glandular epithelium (A), luminal epithelium (B) and stroma (C) was performed blind, by three independent reviewers. The results are shown in FIG. 6. Data are presented as box plots showing the median (line), 25.sup.th and 75.sup.th percentiles (box) and 10.sup.th and 90.sup.th percentile (whiskers), and analysed by Mann-Whitney U test, values differ from fertile: * P<0.05.

[0077] In a separate test, endometrial biopsies were collected from patients during the secretory phase (n=41) of the menstrual cycle, and, as before, subdivided into four groups: fertile (n=12), endometriosis (n=18), ovulatory PCOS (n=11) or anovulatory PCOS (n=14). Biopsies were fixed and paraffin embedded for analysis of RAGE expression by immunohistochemistry.

[0078] RAGE expression scoring (intensity and distribution: H-score) in glandular epithelium (A), luminal epithelium (B) and stroma (C) was performed blind, by three independent reviewers. The results are shown in FIG. 7. Data are presented as box plots showing the median (line), 25.sup.th and 75.sup.th percentiles (box) and 10.sup.th and 90.sup.th percentile (whiskers), and analysed by Mann-Whitney U test, values differ from fertile: * P<0.05.

[0079] In another set of experiments, endometrial biopsies were collected from patients suffering from polycystic ovary syndrome during the proliferative phase and secretive phase (n=32) of the menstrual cycle, and subdivided into three groups: fertile (n=2), endometriosis (n=6) or anovulatory PCOS (n=7). Total RNA was extracted from whole endometrial biopsies (A) and endometrial epithelial biopsies (B) for analysis of AGER mRNA expression by quantitative PCR. The results are shown in FIGS. 8 and 9, respectively. Data are presented as box plots showing the median (line), 25.sup.th and 75.sup.th percentiles (box) and 10.sup.th and 90.sup.th percentile (whiskers), and analysed by Mann-Whitney U test, values differ from fertile: * P<0.05.

[0080] These data show that expression of AGER mRNA and its protein product RAGE is increased in endometrial and ovarian cancers, as well as endometriosis, hyperplasia and polycystic ovary syndrome patients during the proliferative and secretive phase of the menstrual cycle. AGER mRNA expression is also increased in endometrial epithelial cells during the proliferative and secretive phases of the menstrual cycle, and RAGE protein expression is increased in endometrial epithelium during the proliferative phase, and in the endometrial epithelium and stroma during the secretive phase of the menstrual cycle.

EXAMPLE 2

Efficacy of RAGE as a Carrier

[0081] HEC 1A cells derived from an endometrial adenocarcinoma were cultured on 8-well chamber slides to 80% confluence. Cells were treated with murine, anti-human RAGE (MAB11451; Clone 176902) for the times shown. Cells were fixed and permeabalised, before staining with anti-murine FITC-labelled secondary antibody. Representative images were acquired on a Zeiss 710 confocal microscope and examples are shown in FIG. 10.

[0082] This showed that Anti-RAGE antibody is rapidly internalised in cells, making it a good carrier for drugs.

EXAMPLE 3

Preparation of Antibody-Drug Conjugates

[0083] A murine IgG2B antibody against recombinant human RAGE (R&D Systems Cat No. MAB11451) was reconstituted to 1.59 mg/mL in 10 mM Tris/Cl, 2 mM EDTA pH 8.0. The antibody was reduced with 3.5 molar equivalents of 10 mM TCEP:Ab in water for 2 h at 37° C. Without purification the reduced antibody was split in two one each half alkylated with 6.5 molar equivalents of 10 mM vcMMAE or mcMMAF:Ab in DMA (final DMA concentration in the alkylation mixture was 5% v/v) for 2 h at 22° C. Following alkyation N-acetyl cysteine was used to quench any unreacted toxin linker. The conjugates were purified using a HiTrap G25 column equilibrated in 5 mM histidine/Cl, 50 mM trehalose, 0.01% w/v olysorbate 20, pH 6.0. The conjugates were analysed by size exclusion chromatography for monomeric content and concentration (using a calibration curve of naked antibody) using size exclusion chromatography. Running conditions: Agilent 1100 HPLC, TOSOH TSKgel G3000SWXL 7.8 mm×30 cm, 5 μm column, 0.5 mL/min in, 0.2 M Potassium Phosphate, 0.25 M Potassium Chloride, 10% IPA, pH 6.95. Drug loading of the conjugates was confirmed using a combination of HIC and reverse phase chromatography. HIC was carried out using a TOSOH Butyl-NPR 4.6 mm×3.5 cm, 2.5 μm column run at 0.8 mL/min with a 12 min linear gradient between A—1.5M (NH4)2SO4, 25 mM NaPi, pH 6.95±0.05 and B—75% 25 mM NaPi, pH 6.95±0.05, 25% IPA. Reverse phase analysis was performed on a Polymer Labs PLRP 2.1 mm×5 cm, 5 μm column run at 1 mL/min at 80° C. with a 25 min linear gradient between 0.05% TFA/H2O and 0.04% TFA/CH3CN. Samples were first reduced by incubation with DTT at pH 8.0 at 37° C. for 15 min. Due to the complex disulphide structure of an IgG2B and hence potential conjugation site variability both the HIC and PLRP chromatographic patterns were too complex to provide an accurate estimation of average drug loading but did confirm a significant level of drug conjugation.

[0084] The resulting RAGE ADC was designated ‘SNIPER’.

EXAMPLE 4

Effects of ADC on Human Gynaecological Cancer Cells

[0085] The cytotoxicity of the SNIPER ADC prepared in Example 3 was tested in a direct comparison to treatment with drug alone or anti-RAGE antibody alone.

[0086] Endometrial (Ishikawa) or ovarian (TOV112D) cancer cells were cultured in 96-well plates and treated with an extended concentration range of MMAE, MMAF, RAGE MMAE or RAGE MMAF for 24 or 48 h. Data was analysed by non-linear regression and IC50 concentrations determined for each treatment. After 24 h treatment, RAGE MMAE (FIG. 11E: IC50=31.02 μg/ml=0.65 as MMAE μM MMAE) was twice as potent as MMAE alone (FIG. 11A: IC50=1.4 μM), whilst RAGE MMAF (FIG. 11G: IC50=16.66 μg/ml=0.32 μM MMAF) was four times more potent as MMAF alone (FIG. 11C: IC50=1.3 μM). After 48 h treatment, RAGE MMAE (FIG. 11F: IC50=9.54 μg/ml=0.2 as MMAE μM MMAE) was again twice as potent as MMAE alone (FIG. 11B: IC50=0.46 μM), and RAGE MMAF (FIG. 11H: IC50=6.48 μg/ml=0.12 μM MMAF) was five times more potent as MMAF alone (FIG. 11D: IC50=0.63 μM).

[0087] IC50 concentrations in ovarian (TOV112D) cancer cells after 24 h treatment were 16.67 μg/ml 0.65 μM MMAE) for RAGE MMAE (FIG. 12C) and 2.5 μg/ml 0.05 μM MMAF) for RAGE MMAF (FIG. 12D). It was not possible to determine IC50 values for the MMAE or MMAF treatments (FIGS. 12A & B, respectively) alone in these cells (i.e. the IC50 was greater than the top concentration tested).

[0088] These data demonstrate that delivering cytotoxic agents in the form of a RAGE targeting ADC increases the potency of the drug.

[0089] In separate experiments, Ishikawa (A) or HEC1A (B) cells were seeded into 96-well plates and treated with control medium or medium containing MMAE, MMAF, anti-RAGE antibody, SNIPER MMAE or SNIPER MMAF (shown as RAGE MMAE and RAGE MMAF respectively in FIG. 13) for 24 h. After treatment, cell viability in both cell lines (FIG. 13), and cell apoptosis in Ishikawa cells (caspase activation; FIG. 14) were determined by a fluorescence-based cell viability assay (Apotox Glo Triplex assay, Promega) according to the manufacturer's instructions. Data are presented as box plots showing the median (line), 25.sup.th and 75.sup.th percentiles (box) and 10.sup.th and 90.sup.th percentile (whiskers), n=4. Data were analysed by ANOVA and Dunnett's pairwise multiple comparison t-test. Values differ from control: * P<0.05. Cell killing and the induction of apoptosis was significantly increased following treatment with ADCs compared to treatment with the drug or antibody alone.

[0090] In separate experiments, TOV112D, UWB1.289 or UACC-1595 cells were seeded into 96-well plates and treated with control medium or medium containing MMAE, MMAF, anti-RAGE antibody, SNIPER MMAE or SNIPER MMAF for 24 h. After treatment, cell viability in TOV112D, UWB1.289 and UACC-1595 cells (FIG. 15) and the degree of apoptosis in TOV112D cells (caspase activation; FIG. 16) were determined by a fluorescence-based cell viability assay (Apotox Glo Triplex assay, Promega) according to the manufacturer's instructions. Data are presented as box plots showing the median (line), 25.sup.th and 75.sup.th percentiles (box) and 10.sup.th and 90.sup.th percentile (whiskers), n=4. Data were analysed by ANOVA and Dunnett's pairwise multiple comparison t-test. Values differ from control: * P<0.05. Cell killing and the induction of apoptosis was significantly increased following treatment with ADCs compared to treatment with the drug or antibody alone.

[0091] These data demonstrate that treating cancerous cells with ADCs targeting RAGE is an effective killing strategy that significantly improves the efficacy of the conjugated cytotoxin.

EXAMPLE 5

Comparison of Cleavable and Non-Cleavable Linkers

[0092] The linkers used in Examples 3 & 4 were directly compared. Ishikawa or TOV112D cells were seeded into 96-well plates and treated with control medium or medium containing MMAE, MMAF, SNIPER MMAE or SNIPER MMAF for 24 h. After treatment, cell viability (FIG. 17) was determined by a fluorescence-based cell viability assay (Apotox Glo Triplex assay, Promega) according to the manufacturer's instructions. Data are presented as box plots showing the median (line), 25.sup.th and 75.sup.th percentiles (box) and 10.sup.th and 90.sup.th percentile (whiskers), n=4. Data were analysed by ANOVA and Dunnett's pairwise multiple comparison t-test. Values differ between groups: * P<0.05. SNIPER ADCs were used at 20 μg/ml and drug alone treatments were at equivalent molar concentrations. Cell killing was increased following treatment with ADCs comprising the non-cleavable linker, MMAF, compared to the cleavable linker, MMAE.

[0093] These data demonstrate the importance of the correct antibody-linker-drug combination for effective cancer cell killing.

EXAMPLE 6

Internalisation of Anti-RAGE Antibodies in Ovarian and Endometrial Cells

[0094] Using conventional methods as described for example in Köhler, G. & Milstein, C. Nature 256, 495-497 (1975 and Köhler, G. & Milstein, C. Eur. J. Immun. 6, 511-519 (1976), a series of anti-RAGE antibodies were developed. These were designated AA4, HG6 and DF6. The VH protein sequence of AA4 was as shown in SEQ ID NO 25 and the VL protein sequence of AA4 was as shown in SEQ ID NO 26. The VH protein sequence of HG6 was as shown in SEQ ID NO 25 and the VL protein sequence of HG6 was as shown in SEQ ID NO 26. The VH protein sequence of DF6 was as shown in SEQ ID NO 25 and the VL protein sequence of DF6 was as shown in SEQ ID NO 26.

[0095] TOV112D ovarian (B-F) or HEC 1A endometrial (G-K) cancer cells were cultured on 8-well chamber slides to 80% confluence. Cells were treated with different anti-human RAGE antibodies for 1 h. The antibodies used were MOL403, MOL405, AA4, HG6 and DF6, which bind to the following regions of RAGE, respectively: V-type domain, stub region (SEQ ID No. 24), C-type domain 1, C-type domain 1 and stub region (SEQ ID No. 24). Cells were fixed and permeabilised, before staining with FITC or Alexfluor 488 labelled secondary antibody. Representative images were acquired on a Zeiss 710 confocal microscope and the results are shown in FIG. 18.

[0096] All antibodies were internalised in the cells, but internalisation of the MOL403 (V-type domain binding) antibody was assessed as being significantly greater than the other antibodies tested.

EXAMPLE 7

Effects of ADC on Healthy and Cancer Cells Over 96 Hours

[0097] The methodology of Example 4 was repeated over a 96 h period, using a range of cell lines including endometrial cancer cell lines, Ishikawa, HEC1A, HEC1B, HEC50 and ovarian cancer cells TOV112D as well as healthy endometrial and ovarian cells. The antibody construct used was the SNIPER construct of Example 3.

[0098] Results are shown in Table 1 hereinafter. The results show that ADCs are more efficacious after 96 h. In addition, it is clear from Table 1 that the SNIPER-ADC kills endometrial/ovarian cancer cells more effectively than the healthy control cells.

EXAMPLE 8

Relative Efficacy of RAGE ADCs Against Gynaecological Cancer Cells

[0099] Analysis of the cell killing abilities of ADCs comprising the antibody clones AA4, HG6 and DF6 with MMAE or MMAF, revealed that they were less efficacious than the SNIPER ADC. Antibodies were conjugated to MMAE or MMAF as previously described, and cell viability over a period of 24 to 96 h was determined, also as previously described. Within the concentration ranges tested, 0.01 to 100 μg/ml; it was not possible to determine IC50 values for any of the new antibody clones at the 24, 48 or 72 h time points. After 96 h exposure, IC50 values were determined, showing that the ADCs were less efficacious than the SNIPER ADC at 96 h. An example IC50 comparison graph is shown in FIG. 19A. In addition, comparison of cell killing during the course of the experiment demonstrated that the SNIPER ADC was significantly more effective than the other ADCs (a comparison between AA4 MMAE and SNIPER MMAE is shown in FIG. 19B).

[0100] Comparisons of the AA4, HG6 and DF6 ADCs to the SNIPER ADC were made within normal ovarian (HOSEpic) and ovarian cancer (TOV112D and SKOV3) cells, and normal endometrial (Healthy) and endometrial cancer (HEC1A, HEC1B and Ishikawa) cells. Cells were treated for 96 h with 5 μg/ml of each of the ADCs, and cell health monitored as previously described. Within the ovarian cell lines, the SNIPER MMAE ADC was more efficacious compared to the other MMAE ADCs in SKOV3 cells, whilst the SNIPER MMAF ADC was more efficacious in TOV112D and SKOV3 cells (FIG. 20A, B). Data are presented as mean (SEM), and were analysed by ANOVA and Dunnett's pairwise multiple comparison t-test. Values differ from the antibody only control: * P<0.05, ** P<0.01, *** P<0.001.

[0101] Within the endometrial cells, the SNIPER MMAE and the SNIPER MMAF ADCs were both significantly more efficacious compared to the other ADCs in HEC1A, HEC1B and Ishikawa cells. There was no significant effect on healthy endometrial cells by any of the ADCs tested (FIG. 20C, D).

EXAMPLE 9

Tamoxifen Upregulates Endometrial RAGE Expression.

[0102] RAGE protein expression was measured by Immunohistochemistry in endometrial biopsies from patients diagnosed with endometrial hyperplasia, Type I or Type II endometrial cancer (EC), postmenopausal controls as well as breast cancer patients taking tamoxifen as part of their treatment that have developed, or not, endometrial cancer. 138 patients were grouped as follows: PM (n=25, median=0.2), Hyperplasia (n=21, median=5.5), type I EC (n=18, median=1.5), type II EC (n=17, median=2), TX no EC (n=19, median=4), type I EC plus TX (n=21, median=4) and type II EC plus TX (n=17, median=0.2).

[0103] IHC samples were scored blind by three independent observers. Values shown are median IHC scores and statistical analysis was performed using a Mann-Whitney test *p<0.05, **p<0.01, ***p<0.001, compared to PM control. Table number 2 below shows between group comparisons.

[0104] The results are shown in FIG. 21. RAGE expression was noted in the membrane and cytoplasm of tumour cells and endometrial cells obtained from hyperplasia patients. PM staining was almost negative. RAGE expression was also observed in the epithelium and stromal cells of the endometrium from breast cancer patients taken tamoxifen that have not developed Endometrial cancer (Tx no EC). Tamoxifen upregulation of RAGE was also observed in endometrium from EC patients compared to endometrium of EC not taking tamoxifen.

[0105] Estrogen receptor a (ER) expression was also measured and was found to be expressed in all groups. Its expression was used as control for tamoxifen action in EC patients.

TABLE-US-00001 TABLE 1 IC50 SNIPER-MMAE (μg/ml) SNIPER-MMAF [Drug only equivalent, (μg/ml) [Drug only Cell μM] equivalent, μM] Tissue line 24 h 48 h 96 h 24 h 48 h 96 h Endometrium Healthy ND 10.72 15.19 ND 7.25 4.17 [0.22] [0.31] [0.14] [0.08] HEC1A 10.34 4.69 1.02 24.11 0.81 0.74 [0.22] [0.1] [0.02] [0.46] [0.02] [0.02] HEC1B 29.04 8.65 5.67 ND 1.96 1.27 [0.61] [0.18] [0.12] [0.04] [0.02] HEC50 ND 7.64 2.18 17.82 0.86 0.94 [0.16] [0.05] [0.33] [0.02] [0.02] Ishikawa 31.02 9.54 3.86 16.7 6.48 2.42 [0.65] [0.2] [0.08] [0.32] [0.12] [0.04] Ovary Healthy ND ND 41.02 ND 14.36 4.87 [0.86] [0.27] [0.09] TOV112D 22.6 16.17 0.54 ND 2.51 0.59 [0.47] [0.34] [0.01] [0.05] [0.01] ND = not determined within the ADC concentration range used (0.01 to 100 μg/ml)

TABLE-US-00002 TABLE 2 EC type Comparisons II RAGE plus EC EC type TX no Hy- expression Tx type I II EC perplasia PM EC type I 0.0320 0.0500 0.0003 0.5419 0.0093 0.0002 plus Tx EC type II 0.3572 0.4442 0.0074 0.0007 0.0450 plus Tx EC type I 0.8008 0.2476 0.0015 0.0301 EC type II 0.0003 0.0014 0.0072 TX no EC 0.0011 0.0003 Hyperplasia 0.0011

Sequences Referred to Herein

[0106]

TABLE-US-00003 SEQ ID NO  1 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQS ELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGTVTLTCEVPAQPSPQIHWMKDGVPLPLPPSPV LILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGSVGGSGLGTLALALGILGGLGTAALLIGVILWQRRQRRGEERKAPENQE EEEERAELNQSEEPEAGESSIGGP  2 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLTRRHPETGLFTLQSELMVTPARGGDP RPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEIGPQDQG TYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGEGFDKVREAEDSPQHM  3 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVVEESRRSRKRPCEQEVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVK EQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQI HWMKDGVPLPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGSVGGSGLGTLALALGILGGLGTAALLIGVILWQ RRQRRGEERKAPENQEEEEERAELNQSEEPEAGESSTGGP  4 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKET KSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRP TFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEIGPQDQGTY SCVATHSSHGPQESRAVSISIIEPGEEGPTAGSVGGSGLGTLALALGILGGLGTAALLIGVILWQRRQRRGEERKAPENQEEEEERAELNQSEEP EAGESSTGGP  5 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVVEESRRSRKRPCEQEVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVK EQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQI HWMKDGVPLPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGEGFDKVREAEDSPQHM  6 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQS ELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDGLRTREPTAVW PPIPATGPRKAVLSASASSNQARRGQLQVRGLIKSGKQKIAPNTCDWGDGQQERNGRPQKTRRKRR  7 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQS ELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDGVPLPLPPSPV LILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGEGFDKVREAEDSPQHM  8 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKET KSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRP TFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDVSDLERGAGRTRRGGANCRLCGRIR AGNSSPGPGDPGRPGDSRPAHWGHLVAKAATPRRGEEGPRKPGGRGGACRTESVGGT  9 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQS ELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDNQARRGQLQVR GLIKSGKQKIAPNTCDWGDGQQERNGRPQKTRRKRRSVQN 10 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQS ELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDGVPLPLPPSPV LILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGEGFDKVREAEDSPQHM 11 MAAGTAVGACASGGGPIGGGARRWSSSSWWNRNPDL 12 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNWWWSQKVEQ 13 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG ILGGLGTAALLIGVILWQRRQRRGEERKAPENQEEEEERAELNQSEEPEAGESSTGGP 14 MVTPARGGDPRPTFSCSFSPGPPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDGVPLPLPPSPVLI LPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGEGFDKVREAEDSPQHM 15 MERRPSPITESVSTSLRTFTITASDWIFPPSEIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPE TGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDGVP LPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGEGFDKVREAEDSPQHM 16 MNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTP ARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGVVAPGGIVTLICEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEI GPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGEGFDKVREAEDSPQHM 17 MERRPSPITESVSTSLRTFTITASDWIFPPSEIPGKPEIVDSASELTAGVPHKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPE TGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDGVP LPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGEGFDKVREAEDSPQHM 18 MGSPWCLMRRGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGI VTLICEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGSVGGSGLGTLALALGILG GLGTAALLIGVILWQRRQRRGEERKAPENQEEEEERAELNQSEEPEAGESSTGGP 19 MNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTP ARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEI GPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGEGFDKVREAEDSPQHM 20 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQS ELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWGEHRWGGPQAHVSTFWKSDP 21 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNWWWSQKVEQ 22 MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEG IFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQS ELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGIVTLICEVPAQPSPQIHWMKDGVPLPLPPSPV LILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGSVGGSGLGTLALALGILGGLGTAALLIGVILWQRRQRRAELNQSEEPEA GESSTGGP 23 MNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGICVSEGSYPAGILSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTP ARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWGEHRWGGPQAHVSTFWKSDP 24 SISIIEPGEEGPTAGSVGGSGLGTLALA 25 QVQLQQSGAELVKPGASVKLSCKTSGYTFTNYYIYWVIQRPGHGLEWIGEINPSNGGINFSERFKSRAKLTVDKPSSTAYMQLSSLTSDDSAVYY CTINFDYWGQGSTLTVSS 26 DVLMTQTPLSLPVSLGDQASMSCRSSQNIVHNNGNTYLQWYLQKPGQSPKLLIYQVSNRFFGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQ GSHLPLTFGAGTKLELK 27 QVQLLQPGAELVRPGASVRLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYY CAREGYWGQGTLVTVSA 28 ELVMTQSPLTLSVTIGQPASISCKSGQSLLYSNGKTYLYWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQ GTHFPYTFGGGTKLEIK

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