Antibodies Against Cancer

FIELD OF THE INVENTION

The present invention relates to anti-cancer agents and especially agents which inhibit the in vitro and in vivo growth of human colon, prostate and breast cancer cells. The present invention also relates to cancer vaccines.

BACKGROUND TO THE INVENTION

In the early 1980's, there was considerable interest in the development of monoclonal antibodies (Mabs) for use as anti-cancer agents. In some cases, these were designed to be “magic bullets” delivering, by way of conjugation, various cytotoxic compounds (e.g. toxins) or other substances (e.g. isotopes and drugs) to the cancerous cells. However, due to a number of reasons including poor specificity, poor penetration (i.e. with solid tumours) and the induced HAMA (i.e. human anti-mouse antibody) response, these Mab-based anti-cancer agents were unsuccessful and largely abandoned.

In recent times, there has been renewed interest in Mab-based anti-cancer agents and many of the problems previously experienced have been addressed by genetic engineering techniques (Hudson P J, “Recombinant antibody constructs in cancer therapy”, Curr Opin Immunol, 11, pp 548-557 (1999); the disclosure of which is to be considered as incorporated herein by reference). Indeed, there are currently three Mabs (i.e. the humanised HER2/neu Mab marketed under the name Transtuzumab for treatment of HER2/neu positive breast cancer, humanised anti-CD20 Mab known as Rituxan for treatment of Non-Hodgkin lymphoma, and C225 which is an anti-EGFR Mab) which are either being used or are in clinical trials. These antibodies do not act primarily as cytotoxic antibodies nor by Fc mediated inflammatory responses, but rather bind antigen leading to interference in cell signalling and apoptosis. For example, in the case of the HER2/neu Mab, the antibody prevents or “blocks” the binding of a growth factor resulting in the death of HER2/neu positive breast cancer cells.

There is a clear need for more anti-cancer agents to complement existing treatments of cancers. By immunising rats with, or an antigenic portion of, a Cripto-1 protein (Montuori N, et al. “isolation and characterisation of the CRIPTO autosomal gene and its X-linked related sequence”, Am J Hum Genet, 49 (3), pp 555-565 (1991)) known to be expressed in certain cancer cells, or with a fusion protein of a Pim-1 protein (Friedmann M, et al. “Characterisation of the proto-oncogene pim-1: kinase activity and substrate recognition sequence”, Arch Biochem Biophys, 298 (2), pp 594-601 (1992), or a colon cancer cell lysate, the present applicant has produced monoclonal antibodies which have been found, surprisingly, to inhibit growth of various cancer cell lines.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides an isolated binding partner of a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate, wherein said binding partner inhibits growth of one or more cancer cell types.

In a second aspect, the present invention provides an anti-cancer agent comprising a binding partner of a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate, wherein said binding partner inhibits growth of one or more cancer cell types.

In a third aspect, the present invention provides a method of treating cancer in a subject, said method comprising administering to said subject an effective amount of an anti-cancer agent according to the second aspect.

In a fourth aspect, the present invention provides a cancer vaccine comprising a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate or, alternatively, an expressible DNA molecule encoding a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate.

In a fifth aspect, the present invention provides a method of treating cancer in a subject, said method comprising administering to said subject an effective amount of a cancer vaccine according to the fourth aspect.

In a sixth aspect, the present invention provides a method for inducing apoptosis in a cancer cell, said method comprising treating said cell with a binding partner of a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate.

In a seventh aspect, the present invention provides a method of sensitising a cancer cell to a cytotoxic compound, said method comprising treating said cell with a binding partner of a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides graphical results showing inhibition of LS174T colon cancer cells by Mab C4 as well as enhanced sensitivity of the cells to Cisplatin (Cis) caused by Mab C4, after 72 h incubation as measured by ³H-thymidine incorporation.

FIG. 2 provides a bar graph of results demonstrating an inhibitory effect of Mabs C3, C4 and C13 and control Mab BCP7 (anti-mucin1 Mab) on the colon cancer cell line LS174T.

FIG. 3 provides photographs of breast cancer tissue (A) and normal breast tissue (B) samples subjected to immunoperoxidase staining with Mab C4. No staining is seen in the normal breast tissue.

FIG. 4A provides graphical results showing the inhibitory effect of Mab C4 in SCID mice. SCID mice were inoculated with 2×10⁶of prostate cancer DU145 cells subcutaneously and treated with MabC4.

FIG. 4B provides results, in bar graph form, of the effect of Mab C4 on tumour size (by weight) with treated and untreated SCID mice after 24 days following innoculation of prostate cancer DU145 cells and Mab C4.

FIG. 5A provides graphical results showing the inhibitory effect of Mab C13 in SCID mice. SCID mice were inoculated with 2.5×10⁶of colon cancer LS174T cells subcutaneously and treated with Mab C13.

FIG. 5B provides results, in bar graph form, of the effect of Mab C13 on tumour size (by weight) with treated and untreated SCID mice after 25 days following innoculation of colon cancer LS174T cells and Mab C13.

FIG. 6 shows the results of DNA Fragmentation of apoptotic cells induced by the anti-Cripto-1 Mab, C3.

FIG. 7 provides graphical results of FACS assays to determine propidium iodide (PI) staining, an indicator of apoptosis, in colon cancer cells LS174T treated for 72 hours with Mab C4 and the control Mab, Mab BCP7.

FIG. 8A shows the activation of JNK and p38 in LS174T cells which were treated with medium (Lane 1), with C3 (5, 10 μg/ml) (Lanes 2, 3); Cisplatin (25, 50 μg/ml) (Lanes 4, 5); and the combination of C3 (10 μg/ml) and Cisplatin (25, 50 μg/ml) (Lanes 6, 7) for 3 hours. JNK is activated in a dose dependant manner. The combination of C3 and Cisplatin (Cis) further enhanced activation of JNK. P38 was not affected by C3 but was activated by Cisplatin.

FIG. 8B shows the activation of JNK and p38 in LS174T cells which were incubated with medium (m), C4 at 10 μg/ml for 8, 24 or 16 hours.

FIG. 8C shows the activation of JNK and p38 in LS174T cells following 16 hours incubation with medium (1), C3 (10 μg/ml) (2), C4 (10 μg/ml) and Cisplatin (25 μg/ml) (4), Cisplatin (25 μg/ml) (5), and C13 (10 μg/ml) (6).

FIG. 8D shows the activation of JNK and p38 in the LS174T cells following incubation with medium (M), C4 (10 μg/mL) for 24 h, 48 h and 72 h (Lanes 2, 3, 4); C3 (10 μg/ml), C13 for 48 h (Lanes 5, 6) and 72 hours (lane 7, 8); C3 for 48 h (Lane 9). M, medium.

FIG. 9 provides graphical results showing inhibition of growth of CCRF-CEM and CEM/A7R cells (The Burnet Institute, Heidelberg, Victoria, Australia) by anti-Cripto-1 Mabs (i.e. C3 and C13), and anti-Pim-1 Mabs (i.e. P4 and P9).

FIG. 10 shows graphical results demonstrating the effects of the drug Epirubicin on 3 cell lines: leukaemia cell CEM A7, the drug resistant variant CEM A7/R and mouse thymoma cells E3 (A), the effect of Mab C4 on drug resistant leukaemia cell line CEM/A7R (B) and mouse thymoma cells E3 (C) treated with Epirubicin.

FIG. 11 provides graphical results showing inhibition of prostate cancer cell PC3 growth by Mab C3.

FIG. 12 provides tabled and graphical results which show inhibition of growth of prostate cancer cell line DU 145 by the anti-Cripto-1 Mab C3, over time.

FIG. 13 provides tabled and graphical results showing the effects of combining low concentrations of the anti-Cripto-1 Mab, C3 and Cisplatin on the growth of the prostate cancer cell line, PC3.

FIG. 14 shows the effects of combining low concentrations of the anti-Cripto-1 Mab, C3 and Cisplatin on the growth of the prostate cancer cell line, DU 145.

FIG. 15 provides graphical results which show the inhibition of LS174T cell growth by Mab C3 and Epirubicin (7.9-125 μg/ml).

FIG. 16 provides graphical results which show the inhibition of LS174T cell growth by Mab C13 and 5FU (0-3.0 μg/ml).

FIG. 17 provides graphical results showing inhibition of growth of the breast cancer cell line MCF7 (American Type Culture Collection, Manassas, Va., USA) by the anti-Cripto-1 Mab, C3 alone, or when combined with the cytotoxic drugs Cisplatin (Cis), 5-Fluorouracil (5FU) or Carboplatin (Carb) (David Bull Laboratories, USA).

FIG. 18 shows the inhibitory effect of Mab C13 and Epirubicin (E) on breast cancer cell MCF-7.

FIG. 19 provides graphical results showing the inhibitory effect of cross-linked Mab C3 on the breast cancer cell line MCF7.

FIG. 20 provides graphical results from incubation of mouse thymoma E3 cells (The Burnet Institute, Heidelberg, Victoria, Australia) in the presence of anti-Cripto-1 Mabs C3 and C13 and anti-Pim-1 Mabs according to the present invention (P4 and P9) for 24 to 72 hours, showing a reduction in cell numbers compared with cells which have not been exposed to the Mabs, control antibody BC3, an anti-Mucin 1 antibody (The Burnet Institute, Heidelberg, Victoria, Australia) and the drug Epirubicin at an Ic50 concentration of 20 ng (A). FIG. 20(B) provides graphical results from the same experimentation presented in FIG. 20(A), but in this case shows the inhibition of cell growth as a percentage of the control in which no Mabs are present.

FIG. 21 provides graphical results of the inhibition of growth of the colon cancer cell line HT 29 (ATCC, USA) by an anti-Cripto-1 Mab, C3 and an anti-Pim-1 Mab, P4 compared with control antibody BC3.

FIG. 22 provides graphical results which show the inhibition of growth of the colon cancer cell line LS174T by anti-Pim-1 Mab, P4 either alone, or combined with increasing concentrations of Cisplatin.

FIG. 23 provides graphical results showing, by way of percentage change in ³H-thymidine incorporation, the inhibition of growth of the colon cancer cell line LS174T (ATCC, USA) and breast cancer cell line MCF7 by Mabs 1.14, 1.68, 2.20 and 3.60, using anti-Mucin 1 antibody BCP7 as a control.

FIG. 24 shows the effects of combining Mab 1.14 (raised against a colon cancer cell lysate) and Cisplatin on growth of the prostate cancer cell line, DU 145.

FIG. 25 shows the results of titrations of mouse serum tested by ELISA using 37-mer Cripto-1 peptide coated plates.

FIG. 26 shows the results ELISPOT assays for IPNγ secretion. Mouse spleen cells from immunise and naive mice (normal 1 and 2) were stimulated overnight with and without the 37-mer peptide, and spot forming units (SFU) were counted by dissection microscope.

FIG. 27 shows the percentage change in ³H-thymidine incorporation of lung cancer Ben and Colo 338 cells as a function of increasing concentrations of Mab C4 cultured for 72 hours, showing 90% and 60% inhibition in Ben and Colo338 cells respectively induced by Mab C4. Points, mean of triplicate experiments, bars, SD.

FIG. 28 shows the reaction of an anti-Pim-1 Mab (a) showing reaction of Mab P9 tissue culture supernatant with GST-Pim-1 and control recombinant GST-Muc-1 tested by ELISA. Anti-murine Muc-1 Mab M30 (IgM) was used as an isotope matched control. (b) inhibition of cancer cell growth, showing percentage of control in ³H-thymidine incorporation of prostate (DU145, PC3), colon (LS174T), and breast (MCF7) cancer cells in the presence of anti-Pim-1 Mab P3, P4, P5, P6, P8, P9 and P10 at 25 μg/ml for 72 hours. Anti-Muc-1 Mab BC3 was used as a control. (c) Inhibition of cancer cell growth, showing percentage of control in ³H-thymidine incorporation of prostate cancer cell DU145 in the presence of P4, P9 and P10 at concentration of 5-30 μg/ml for 72 hours. Columns show means of triplicate experiments as percentage compared to medium control, bars, SD.

FIG. 29 provides the results of immunohistochemical staining showing reaction of P9 (a) and normal mouse IgM (b) with prostate cancer cells DU145 respectively; reaction of P9 with human (c) and mouse (d) prostate cancer, normal human colon (e) and colon cancer (f); normal lung (g) and lung cancer (h) respectively. (i) and (j) showing the cell surface staining of FITC-P9 with DU145 and TRAMP-C1 (mouse prostate cancer).

FIG. 30 shows flow cytometry showing cell surface staining of Raji cells (A), U937 cells (B), K562 cells (C), PC3 cells (D), DU145 cells (E), and LNCaP cells (F) with P9 labeled with fluorescein isothiocyanate (FITC), BC3-FITC and mIgM-FITC respectively.

FIG. 31 shows the inhibition of DU145 cell growth by combined use of (A) P9 and Cisplatin (Cis) (B) P9 and Epirubicin (Epi) (C) P4 and Epi, and P4 and Cis for 48 hours in tissue culture as measured by percentage of control ³H-thymidine incorporation. Points are means of triplicate experiments. Error bars represent SD in triplicate experiments. The interactions of various concentrations of Pim-1 Mabs with Cis and Epi were subjected to two-way analysis of variance (ANOVA). The p values were marked as *(P<0.05), **(P<0.01), ***(P<0.001).

FIG. 32 shows the detection of apoptosis. Flow cytometry showing apoptosis of CEM/A7R cells after 4 h incubation with (a) medium only or (b) 25 μg/ml P9 tested by Propidium iodide (PI) and Annexin V dual staining; (c) showing cell numbers of CEM/A7R, counted by the trypan blue exclusion method after the cells were cultured in the presence of P9 at 25 μg/ml for 24, 48 and 72 h. (d) showing cell numbers of DU145, PC3 and MCF7, counted by the trypan blue exclusion method after the cells were cultured in the presence of P9 at 25 μg/ml for 72 h. Columns show means of triplicate experiments as percentage compared to control.

FIG. 33 provides Western blots showing in (A) DU145 cell lysates, that P9 reacted with both 44 kDa and 33 kDa molecules of Pim-1, and that both molecules decreased after P9 treatment over time; decrease of phospho-Akt at Ser473 and phosphor-Bad at Ser136, and cleavage of caspase 9 at Asp330 after 1, 3, and 6 h incubation in DU145 cells; (B and C) P9 reacted with both 44 kDa and 33 kDa molecules of Pim-1, and the both molecules decreased; phospho-Akt at Ser473 and phosphor-Bad at Ser112 in PC3 and TRAMP-C1 cell lysates respectively after 3 h of P9 treatment.

FIG. 34 shows the inhibitory effect of anti-Pim-1 Mab P9 in mouse tumour models, (a) showing P9 inhibited human prostate cancer DU145 growing in SCID mice which were inoculated with 1×10⁷of DU145 cells subcutaneously and treated with 0.5 mg P9 per mouse after 8 hours of inoculation of DU145 cells, following 0.25 mg P9 as indicated (arrows). (b) showing P9 inhibited murine prostate cancer cell TRAMP-C1 growing in C57BL/6 mice, which were inoculated with 5×10⁶of TRAMP-C1 cells subcutaneously and treated with 0.5 mg P9 after tumour size reached an average size of 50 mm³, following 0.25 mg P9 as indicated (arrows).

DETAILED DESCRIPTION OF THE INVENTION

The binding partner of the present invention preferably inhibits growth of one or more of colon cancer cells, breast cancer cells, prostate cancer cells, leukemia cells and lung cancer cells, and is characterised in that it binds a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate.

Preferably, the binding partner is an antibody or fragment thereof, but might also be a receptor protein for Cripto-1 protein (Bianco C. et al, “Cripto-1 indirectly stimulates the tyrosine phosphorylation of erb B-4 through a novel receptor”, J Biol Chem, 274(13), pp 8624-8629 (1999)), Pim-1 protein or colon cell lysate antigen or, otherwise, any other peptide, polypeptide or protein which specifically binds to a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate. More preferably, the binding partner is an antibody or fragment thereof which specifically binds to Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate.

The term “specifically binds” in this context, is to be understood to refer to binding characteristics of peptide, polypeptide or protein which binds exclusively to a Cripto-1 protein, Pim-1 protein or a colon cell lysate antigen with only negligible cross reaction with other mammalian proteins.

More preferably, the binding partner according to the present invention is selected from monoclonal antibodies or fragments thereof which bind to an antigenic determinant of Cripto-1 protein comprising an amino acid sequence substantially corresponding to the amino acid sequence:

CPPSFYGRNCEHDVRKE
(SEQ ID No: 1)

and/or an antigenic determinant of a Pim-1 protein and/or an antigen present in a colon cancer cell lysate, wherein said antigen has a molecular weight of 16 kDa or 30 kDa as estimated by SDS-PAGE. The 16 kDa and/or 30 KDa antigen may be a growth factor required for growth of colon cancer cells and/or breast cancer cells.

Monoclonal antibodies according to the present invention may be produced by any of the standard techniques in the art. Fragments of monoclonal antibodies such as F (ab′)₂, Fab and Fc may be produced by, for example, pepsin and papain cleavage as is standard in the art or by recombinant DNA techniques involving expression of antibody genes isolated from a hybridoma cell line or antibody-producing animal cell. Particularly preferred antibody fragments are single chain Fv (scFv) antibody fragments. Methods for producing scFvs are described in Pluckthun A, Bio/Technology, 9, pp 545-551 (1991) and U.S. Pat. No. 4,946,778. It is to be understood that the disclosures contained within these two references are incorporated herein by reference.

It is believed that antibody fragments according to the present invention may provide advantages over monoclonal antibodies and other “large” binding partner types since they may exhibit improved penetration of solid tumours, particularly large tumours.

Monoclonal antibodies and antibody fragments according to the present invention may be humanised in accordance with the technique described in U.S. Pat. No. 5,225,539 (the disclosure of which is incorporated herein by reference).

Monoclonal antibodies and antibody fragments may also be produced by using spleen cells from an immunised animal (e.g. mouse or rat) fused to a human myeloma line (e.g. Karpas 707H human myeloma cell line; Karpas A, et al. “A human myeloma cell line suitable for the generation of human monoclonal antibodies”, Proc Natl Acad Sci USA, 98, pp 1799-1804 (2001)), to produce human antibodies or antibody fragments. Chimeric mouse/human monoclonal antibodies may be made in accordance with Mount P F, et al. “Chimeric (mouse/human) anti-colon cancer antibody c30.6 inhibits the growth of human colorectal cancer xenografts in scid/scid mice”, Cancer Research, 54, pp 6160-6166 (1994), which is also incorporated herein by reference.

Monoclonal antibodies and antibody fragments may be produced in large amounts by standard techniques (e.g. in either tissue culture or serum free using a fermenter) and purified using affinity columns such as protein A (e.g. for murine Mabs), Protein G (e.g. for rat Mabs), Protein L (e.g. for light chain K) or MEP HYPERCEL (e.g. for IgM and IgG Mabs).

The binding partner according to the present invention may be conjugated to a cytotoxic compound or other substances such as those mentioned above. Preferred cytotoxic compounds include first line chemotherapeutics such as anthracyclines (such as Idarubicin, Doxorubicin, Daunorubicin and Epirubicin), 5FU, topoisomerase inhibitors (such as Irinotecan), Cisplatin, Carboplatin and Taxol.

The binding partner according to the present invention may also be conjugated to a first binding protein (e.g. biotin) to enable cross-linking between antibodies or fragments thereof by administering a second binding protein (e.g. avidin) which binds with the first binding protein. In vitro experimentation described hereinafter in Example 11, cross-linking with secondary antibodies achieves an increase in the inhibition of growth or breast cancer cells. Further preliminary experimentation has indicated that a similar result may be achieved with colon cancer cells.

Further, the binding partner according to the present invention may be cross-linked to antibodies such as Panorex (Centacor, Glaxo), Rituxin (Genentech, Roche) or Herceptin (Genentech, Roche). These second antibodies have been shown to be effective against colon cancer, lymphoma and breast cancer respectively.

Preferably, the binding partner according to the present invention is combined with a suitable pharmaceutically-acceptable carrier or diluent to form an anti-cancer agent (which may be for human or animal use). Suitable carriers or diluents include isotonic saline solutions, for example, phosphate-buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration. Typically, the binding partner (e.g. antibody or fragment thereof) may be administered at a dose of from about 0.01 to about 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg body weight. It is to be understood, however, that the routes of administration and dosages mentioned are intended to serve only as a guide since persons skilled in the art would be able to readily determine the optimum route of administration and dosage for any particular subject and cancer condition.

An anti-cancer agent comprising binding partner according to the present invention may be used in a method of treating cancer in a subject. Said method may bring about a reduction in the size of the cancer or, at least, inhibit further growth and/or spread. Said method may also be used in combination with traditional cancer treatments such as radiotherapy, chemotherapy (e.g. using anthracyclines, 5FU, topoisomerase inhibitors, Cisplatin and Carboplatin), or hormone therapy or therapies utilising hormone modifiers (e.g. Tamoxifen).

The present invention also extends to vaccines for cancer and to their use in methods of treating cancer in a subject. Such vaccines may comprise a Cripto-1 protein (or an antigenic fragment thereof), Pim-1 protein (or an antigenic fragment thereof), or an antigen present in a colon cancer cell lysate, or, alternatively, an expressible DNA molecule encoding a Cripto-1 protein (or an antigenic fragment thereof), Pim-1 protein (or an antigenic fragment thereof), or an antigen present in a colon cancer cell lysate.

Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the protein or DNA encapsulated in liposomes. The protein or DNA may also be mixed with excipients or adjuvants which are pharmaceutically acceptable. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. Suitable adjuvants include aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate (alum).

The present invention further extends to a method for inducing apoptosis in a cancer cell, said method comprising treating said cell with a binding partner of a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate. The amount of binding partner used to treat the cancer cell will vary depending upon the nature and identity of the particular binding partner, as well as the environment of the cancer cell (e.g. in an in vitro cell culture, or in an in vivo setting such as a tumour model or a cancer patient). It is however, well within the skill of persons skilled in the art to determine an effective apoptosis-inducing amount of the binding partner.

The present invention still further extends to a method of sensitising a cancer cell to a cytotoxic compound, said method comprising treating said cell with a binding partner of a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate. The amount of binding partner used to sensitise the cancer cell will vary depending upon the nature and identity of the particular binding partner, as well as the environment of the cancer cell (e.g. in an in vitro cell culture, or in an in vivo setting such as a tumour model or a cancer patient), and the nature and identity of the cytotoxic cell to which the cell is to be sensitised. It is however, well within the skill of persons skilled in the art to determine an effective sensitising amount of the binding partner.

Finally, the present invention extends to a method of inducing a CTL response to cancer cells in a subject, said method comprising administering to said subject an effective amount of a peptide comprising an amino acid sequence substantially corresponding to: ELNRTCCLNGGTCMLGSFCACPPSFYGRNCEHDVRKE (SEQ ID NO: 2) or an antigenic fragment thereof.

Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The term “substantially corresponding” as used in relation to an amino acid sequence is intended to encompass the specified amino acid sequence as well as related amino acid sequences which differ only by the inclusion of one or more amino acid substitutions, insertions or additions which do not substantially alter the biological activity of the specified amino acid sequence. In particular, the term is intended to encompass related amino acid sequences which differ only by the inclusion of one or more conservative amino acid substitutions. By conservative amino acid substitutions, the intended combinations are: G, A; V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Na-alkylamino acids.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

The invention will hereinafter be further described by way of the following non-limiting examples and accompanying figures.

EXAMPLES

In colon cancer, there is no response to radiotherapy and little response to drugs such as 5FUDR, levamasole, although recently there has been some improvement with the topoisomerase inhibitor Irinotecan. The prognosis for colon cancer patients in advanced disease (i.e. Dukes B, C and D) where there is local spread through nodes to distant metastases (Dukes D) is poor; in the case of Dukes D, few patients survive a year after diagnosis. For breast cancer, the prognosis is considerably better, other than for those patients with primary disease, and a number of patients do well with cytotoxic/hormonal and radiotherapy treatment. Where the breast cancer is HER-21 neu positive, as it is in approximately 30% of breast cancer patients, a proportion of the patients will respond well to the HER-2/neu Mab mentioned above. There is accordingly a continuing need to identify and develop new treatments for colon and breast cancers.

Production of Antibodies

(1) Lewis rats were immunised in accordance with standard techniques in the art with a KLH-coupled, 17 amino acid peptide derived from Cripto-1 protein having the sequence; CPPSFYGRNCEHDVRKE (SEQ ID NO: 1). This sequence corresponds to residues 97-113 of the human and mouse Cripto-1 protein. It forms part of a modified EGF-like motif that differentiates Cripto-1 from other members of the EGF family (Brandt R, et al. “Identification and biological characterization of an epidermal growth factor-related protein: cripto-1”, J Biol Chem, 269, pp 17320-17328 (1994); Salomon D S. “Cripto: a novel epidermal growth factor (EGF)-related peptide in mammary gland development and neoplasia”, Bioassays, 21, pp 61-70 (1999)).

(2) Balb C mice were immunised in accordance with standard techniques with a colon cell lysate prepared by freeze-drying tumour tissue followed by thawing, repeated three times. The freeze/thaw samples were then homogenised three times for one minute each in phosphate buffered saline containing protease inhibitor.

(3) Balb C mice were immunised in accordance with standard techniques with a 59 kDa fusion protein of Pim-1 with glutathione-S-transferase (GST) (provided by Dr Nancy S Magnuson, Department of Microbiology, Washington State University, United States of America).

Spleen cells from the immunised rats were isolated and fused with the myeloma NS1 (Xing P X, et al. “Monoclonal antibodies to mucin VNTR peptides”, Methods Mol Biol, 125, pp 369-381 (2000)) cells to produce antibody-secreting hybridomas. Hybridomas were initially screened by assessing the ability of antibody-containing supernatants to inhibit growth of cancer cell lines (i.e. colon cell lines LS174T and HT29, and breast cancer cell line MCF7) in vitro, using a simple assay involving growing LS174T and MCF7 cells (1×10⁵) in 25 cm²flasks (in 10 ml of medium) in the presence or absence of 50 μg/ml of anti-Cripto-1 Mabs (C3 and C13). Viable cells were counted by using a phase-contrast microscope on day 6 of the culture.

Other Antibodies

Antibodies against Cripto-1 (Bianco C, et al. “Cripto-1 indirectly stimulates the tyrosine phosphorylation of erb B-4 through a novel receptor”, J Biol Chem, 274(13), pp 8624-8629 (1999)), referred herein as C3 and C13, were raised as described in International patent application No PCT/AU02/00362 (Publication No WO 02/077033) using a KLH-coupled, 17 amino acid Cripto-1 peptide (Salomon DS., “Cripto: a novel epidermal growth factor (EGF)-related peptide in mammary gland development and neoplasia”, Bioassays 21, pp 61-70 (1999)).

Assays for Inhibition of Cancer Cell Growth

Growth inhibition was also assessed by measuring inhibition of uptake of tritiated thymidine, counting cell numbers manually by a trypan blue exclusion assay or by using a colourimetric cytotoxicity assay SRB (sulforhodamine B) (Skehan P, “New colourimetric cytotoxicity assay for anticancer-drug screening”, J Natl Cancer Inst 82, pp 1107-1112 (1990)) which is a rapid and sensitive method for measuring the cellular protein content of the cells.

Example 1
Isolation of Anti-Cripto-1 Antibodies and Summary of Experimental Results

Two of the isolated Mabs (i.e. C3 and C13) bind to Cripto-1 (a member of EGF family encoded by CR1 in humans, tdgxqin mouse), a soluble or, possibly, cell surface (Mr 36 Kd) GPI-linked protein that appears to be a growth factor which promotes cell survival and proliferation and is important in embryonic development and cancer (Brandt R, supra) which has been described in a number of species (e.g. xenopus, zebrafish, mouse and human). Importantly, in the context of the present invention, the expression of Cripto-1 is increased several fold in human colon, gastric, pancreatic, breast and lung cancers and this increase can be detected in premalignant lesions (Brandt R, supra; Saeki T. et al. “Differential immunohistochemical detection of amphiregulin and cripto in human normal colon and colorectal tumours”, Cancer Res, 52, pp 3467-3473 (1992); Salomon D S, supra; Panico L. et al. “Differential immunohistochemical detection of transforming growth factor alpha, amphireguliln and CRIPTO in human normal and malignant tissues”, Int J Cancer, 65, pp 51-56 (1996)). For example, normal colon and breast cells do not contain Cripto-1, whereas it is found in ˜85% of colon and breast cancers.

These anti-Cripto-1 Mabs have yet to be fully characterised with regard to the distribution of tissues to which they bind (especially in developing human mammary gland, lactation and during pregnancy), but using immunoperoxidase staining with fresh or formalin fixed human tissue, indicates that the Mabs are cancer specific and bind to an antigen present in colon cancer (60%) and breast cancer (70%) but which is absent from normal colon tissue. In addition, the present applicants have observed that the anti-Cripto-1 Mabs react with mouse tumours. More importantly, these antibodies showed significant inhibition of the growth of the colon cancer cell line LS174T and breast cancer cell line MCF7 in tissue culture. In addition, these Mabs also showed inhibition of leukemia, lung cancer cells and prostate cancer cells.

In other experimentation, it has been found that by cross linking the antibodies in vitro with a secondary anti-rat antibody an increase in apoptosis can be achieved.

Dose response trials have also been conducted in vitro with cytotoxic compounds including 5FU, Cisplatin and Carboplatin, which showed that substantial increases in the level of inhibition of cancer cell division and growth may be achieved when the Mabs are used in combination with cytotoxic compounds, but also there is a real decrease in cell numbers, indicating that the Mabs induced cancer cell apoptosis.

Example 2
Monoclonal Antibody C4 to Cripto-1

A further anti-Cripto-1 monoclonal antibody, Mab C4 was obtained using the same method as used to raise Mabs C3 and C13. Each Cripto-1 Mab was selected by a) detection of immunoperoxidase staining to determine binding of the antibody to a target tissue, b) cell growth inhibition assay e.g. ³H-thymidine assay in a selected cell line (antibodies showing >60% inhibition by thymidine incorporation) and c) detection of 2-fold decrease in cell no. as determined by trypan blue exclusion. The top line in FIG. 1 shows the inhibition of the LS174T colon cancer cell line by Mab C4 after 72 h of co-culture, whilst FIG. 2 shows the reduction in cell count number by the Mabs C4, C3 and C13 after 7 days of culturing 1×10⁴of the cells in 25 cm²flasks in 10 ml of medium with 30 μg/ml of each Mab. The antibody also enhanced the sensitivity of LS174T to Cisplatin in that addition of the Mab to cells being treated with the drug reduced ³H-thymidine incorporation further relative to incubation with the drug alone at 0.0938 to 0.75 μg/ml.

Similar results were obtained with Epirubicin and 5FU. After 72 h incubation with 0, 10, 20 and 30 μg/ml Mab C4, tritiated thymidine incorporation by LS174T cells was inhibited by 50 to 90% in the presence of 0.04, 0.08, 0.1625 and 0.125 μg/ml Epirubicin. For 5FU, thymidine incorporation was inhibited by 50 to 90% in the presence of 1.5, 1.9, 2.1 and 2.4 μg/ml of the drug. 5FU is a mainstay of treatment for colorectal cancer and is an antimetabolite. The synergistic effect of combined use of 5FU, Cisplatin, Epirubicin will be clinically useful.

Example 3
Testing of Binding of Anti-Cripto-1 Antibodies with Cancer and Normal Tissues

The anti-Cripto-1 Mabs reacted with a number of cancer cell lines, such as LS174T, HT29 (colon cancer), MCF7, T47D (breast cancer), DU145 and PC3 (prostate cancer), Ben and Colo 235 (lung cancer), but not with embryonal kidney cell line 293 when tested by FACS and immunoperoxidase staining. The 3 Mabs also reacted with formalin-fixed tissues, such as colon cancer (7/9), breast cancer (5/7), lung cancer of all types (18/20), stomach (3/4), pancreas (1/2), but did not react with normal breast (0/4), colon (0/8), lung (0/4), stomach (0/2), pancreas (0/2), liver (0/3), and lymphocytes (0/3) by immunoperoxidase staining The intensity and percentage of staining varied from negative to very strong positive, indicating that Cripto-1 expression varies in different cancers. FIG. 3A shows immunoperoxidase staining breast cancer tissue by Mab C4, compared to FIG. 3B in which no staining of normal breast tissue by the antibody is observed.

Example 4
In Vivo Inhibitory Effect of Anti-Cripto-1 Antibodies on Growth of Colon and Prostate Cancer Cells in Mice

SCID mice (6-8 week of age) were inoculated subcutaneously with 2×10⁶prostate cancer cell line DU145 at Day 0 and after 6 h the mice were treated with 500 μg of Mab C4 intraperitoneally, followed by 250 μg at days 2, 4, 7, 9 and 10, and 125 μg at days 14 and 17. Phosphate buffered saline (PBS) (0.5 ml) was used as a control.

Tumours were removed and measured at day 24. The tumour size and weight were significantly reduced by the treatment of Mab C4 (FIGS. 4A and 4B).

Similar results were also demonstrated in the colon cancer model (FIGS. 5A and 5B) using Mab C13,500 μg after 16 h inoculation of LS174T cells, then 250 μg at days 2, 7, 9, 11 and 13. Tumours were excised on day 25 for weight determination.

Example 5
Apoptosis Induced by Anti-Cripto-1 Antibodies

The anti-Cripto-1 monoclonal antibodies stopped cell division as measured by a decrease in ³H-thymidine uptake, and decreased cell numbers (FIGS. 1 and 2 respectively), indicating that the Mabs induced cancer cell apoptosis. This was further demonstrated by DNA fragmentation and FACS assays (FIGS. 6 and 7).

In FIG. 6, soluble DNA was extracted from LS174T cells that had been treated for 72 hours with 50 μg/ml of Mab C3, and electrophoresed on 2% agarose gels. Control samples were from cells treated with cell culture medium.

FIG. 7 shows LS174T cells treated for 72 hours with 30 μg/ml Mab C4 or the control antibody Mab BCP7, then analysed by flow cytometry assay to determine propidium iodide (PI) staining, an indicator of apoptosis. The results showed an increase in PI staining in cells treated with the test Mab.

Example 6
Signal Transduction Mediated by Anti-Cripto-1 Antibodies
(i) Anti-Cripto-1 Mab Induced JNK Activation

Signal transduction pathways controlled by protein kinase modules regulate critical cellular functions including cell growth, differentiation and apoptosis. Three major kinase cascades have been identified in control of apoptosis that culminate in the activation of three different sets of mitogen-activated protein kinases: the extracellular signal-regulated kinase (ERK), JNK/SAPK, and p38. ERK is activated by mitogens and survival factors, while JNK/SAPK and p38 are stimulated by stress signals. The stress-activated kinase cascades including the JNK/SAPK and the p38 pathways are activated in response to different apoptotic stimuli and seem to play a decisive role in apoptosis process.

The role of JNK and p38 activation in anti-Cripto-1 mediated apoptosis was investigated in colon cancer LS17T cell line using different concentration of Mabs and various times of incubation. In particular, JNK/SPAK was activated in LS174T cells following 3 hours incubation with anti-Cripto-1 Mab in a dose dependent manner (FIG. 8A). JNK activation was at the highest level after 24 hours of exposure (FIGS. 8B and 8D), and declined within 48 h, returning to basal level at 72 hours of incubation (FIG. 8D) indicating JNK activation by anti-Cripto-1 antibodies is time dependent.

(ii) JNK Activation by Anti-Cripto-1 Antibodies Preceded p38 Activation

The stress related p38 pathway was also investigated in LS174T cells following anti-Cripto-1 Mab treatment, p38 activation occurred following 48 hours of Mab exposure, when the level of activated JNK declined. p38 was further activated at 72 hours when elevated JNK returned to basal level (FIG. 8D). Thus, JNK activation occurred prior to apoptosis induced by Mab, whilst p38 was activated during the time when apoptosis occurs suggesting that both signals may be involved in the Mab induced apoptosis. In contrast, Cisplatin induced both JNK and p38 MAPK activation (FIGS. 8A and 8C), indicating that the Mabs activated JNK and p38 in a way different from Cisplatin. The potentiation of Cisplatin cytotoxicity by anti-Cripto-1 Mabs (FIG. 1) is accompanied by an increase in JNK phosphorylation (FIG. 8A) and p38 MAPK (FIG. 8B).

Thus, anti-Cripto-1 Mabs induce tumour cell apoptosis through activation of both JNK and p38.

(iii) ERK and Akt Phosphorylation and Cripto-1 Expression

The effect of Mab on the inhibition of ERK and Akt (FIGS. 8B and 8D) survival pathways has not been demonstrated. No changes in the levels of Cripto-1 expression were observed following Mab treatment (FIGS. 8B and 8D). These preliminary signalling studies clearly show that the anti-Cripto-1 Mabs cause apoptosis through the JNK activation pathway.

Example 7
Inhibition of Leukaemia Cells by Anti-Cripto-1 Antibodies

FIG. 9 provides results showing that Mabs C3 and C13 inhibited growth of the T cell lymphoblastic leukaemia cell line CCRF-CEM. The antibodies also inhibited growth of the drug resistant variant of this cell line, CEM/A7R, which acquires this property by over-expression of P-glycoprotein. Thus, this cell line is normally resistant to a variety of naturally derived chemotherapeutic agents.

The Mab C4 showed a similar inhibitory effect on the drug resistant cell lines CEM/A7 and CEM/A7R and on a drug sensitive mouse thymoma cell line (i.e. E3).

Compared to E3, CEM/A7 and CEM/A7R exhibit around 80 and 40 fold resistance to Epirubicin respectively (FIG. 10A). The antibody appears to sensitise the drug resistant cells (FIG. 10B) and drug sensitive cells (FIG. 10C) to Epirubicin. Therefore, C4 can overcome drug resistance which is a common problem in acute leukemia.

Example 8
Effect of Anti-Cripto-1 Antibodies on Prostate Cancer

Cells from the prostate cancer cell line PC3 were cultured with 30, μg/ml Mab C3 for 6 days. Cell numbers were counted at days 2, 3 and 6. FIG. 11 shows that cell numbers were significantly decreased in the presence of the antibodies. Similar effects were also observed in drug resistant DU 145 cells, as shown in FIG. 12.

Mab C3 was also able to sensitise PC3 cells to the drug Epirubicin and DU 145 cells to the drug Cisplatin as shown in FIGS. 13 and 14 respectively.

Example 9
Effects of Anti-Cripto-1 Antibodies and Anti-Cancer Drugs

The ability of Mab C4 to enhance the inhibitory effects of cytotoxic drugs such as Cisplatin in colon cancer cell LS174T is shown in Example 2 above. Similar effects were observed with Mab C3 and 13 with respect to Epirubicin and 5 FU respectively as shown in FIGS. 15 and 16.

Example 10
Anti-Cripto-1 Antibodies and Breast Cancer

As shown in FIG. 17, Mab C3 inhibited growth of breast cancer cells MCF7, and further sensitised the cells to Cisplatin, Carboplatin and 5FU. Similar results were observed with Mab C13 and Epirubicin (FIG. 18).

Example 11
Cross-Linking of Anti-Cripto-1 Antibodies

Mab C3 was cross-linked by anti-rat antibody. The effect of cross-linking the Mab was investigated in breast cancer cell line MCF7, which was incubated with the antibody for 2 hours, and then incubated with rabbit-anti rat antibodies for 4 hours, followed by PI staining BCP7 and MabC3 that had not been cross-linked were used as controls. FIG. 19 shows that cross-linking the Mab resulted in significantly more cell death as determined by flow cytometry using PI staining.

Example 12
Isolation of Anti-Pim-1 Antibodies

Two isolated Mabs (i.e. P4 and P9) were raised against the product of the Pim-1 oncogene. This gene encodes a protein belonging to the serine-threonine kinase class of proteins. The anti-Pim-1 antibodies inhibited growth of mouse thyoma E3 cells (FIG. 20) and, along with the colon (FIGS. 21 and 22) and breast cancer cell lines tested, these antibodies also showed inhibition of leukemia and prostate cancer cell lines (data not shown).

Example 13
Isolation of Antibodies Against Colon Cell Lysate Antigens

Five of the isolated Mabs (i.e. 1.14, 1.68, 2.20, 3.60 and 4.57) were raised against unknown antigens by immunising rats with a lysate of fresh colon cancer tissue. These antibodies were found to inhibit growth of the colon cancer cell line LS174T and breast cancer cell line MCF7 in tissue culture (FIG. 23). These antibodies also demonstrated inhibition of the prostate cancer cell line DU145 especially when used in combination with Cisplatin (FIG. 24).

Example 14
Humanisation of Antibodies

Fully human anti-Cripto-1 antibodies are produced by using 2 peptides coupled to KLH as antigens for immunisation, namely the 17-mer (97-113) peptide (SEQ ID NO: 1) used for the production of the Mabs C3, C4 and C13, and (2) a 37-mer peptide p47 (77-113) containing the 17-mer peptide and the putative binding site of Cripto-1 and its receptor ELNRTCCLNGGTCMLGSFCACPPSFYGRNCEHDVRKE, (SEQ ID NO: 3), and by testing these in vitro and in vivo in the same manner as that described above for the production of rat-anti-Cripto-1 Mabs.

The Cripto-1 protein, Pim-1 protein or colon cancer cell lysate antigens can be used to immunise mice followed by cell fusions with the non-secreting myeloma cell line NSO-bcl 2 (which has no immunoglobulin gene) and screened, or otherwise, can be used to immunise a Human Ig mice (e.g. XenoMouse) wherein the mouse immunoglobulin genes have been “knocked out” and replaced by human genes such that they will only have human antibodies produced (nb. multiple immunisations can be done and the mice screened for the presence of high affinity antibodies) followed by identification of B-cells that produce antibodies with inhibitory functional properties using microplate-based cell growth inhibition assay. The antibody encoding genes of individual B-cells producing inhibitory antibodies can then be recovered and used to generate a panel of suitable recombinant candidate antibody products, each ready for manufacturing scale-up.

Example 15
Clinical Uses of Antibodies

Human Mabs produced in accordance with the procedure described in Example 14 can be administered to patients by intravenous injection at a dose in the range of 0.5 mg-10 mg/kg body weight. The patients can also be administered with a suitable anti-cancer drug.

Example 16
Effect of Cripto-1 Immunisation

In contrast to antibodies which are administered “passively” to the recipient, the Cripto protein or antigenic fragments thereof can be used to “actively” immunise, and produce a vaccine. In such a procedure, the Cripto antigen is combined with a carrier (e.g. alum, mannan, beads or other adjuvants) and used to immunise subjects with cancer as a preventative for cancer. The ensuing immune response can be: a) generation of antibodies including but not limited to those described above; b) production of T cells which recognise the Cripto antigen presented by MHC Class I or II molecules (the ensuing T cell response can be measured as effector cells as either: Cytotoxic T cells, Cytokine (e.g. interferon producing cells, such as ELISPOT or by other means), T cell proliferation, and/or delayed type hypersensitivity reaction in vivo); and/or c) a combination of both antibodies and cellular immunity.

Thus Cripto-1 can be used to produce antibodies which are administered to the recipient or Cripto-1 can be used to “vaccinate” a patient who produces antibodies, T cells or both.

Mice were immunised using the Cripto-1 37-mer peptide mentioned above in Example 14 conjugated with KLH, which was emulsified with CFA. The immune responses were tested by ELISA and ELISPOT IFNγ assay. The mice responded in both antibody and INFγ productions (as shown in FIGS. 25 and 26).

Example 17
Anti-Cripto-1 Antibodies and Lung Cancer

Mab C4 also inhibited, in a dose dependant manner, the incorporation of ³H-thymidine in lung cancer cells-Ben and Colo 38. In Ben cells, incorporation was inhibited by 90% after 72 h incubation with the Mab compared with control cells. In Colo 38 cells, the inhibition was 60% (FIG. 27).

Immunoperoxidase staining of the lung cancer cell line Ben or lung cancer tissues was also shown for Mab C3; both cell surface and cytoplasmic staining of lung cancer cells were observed, whereas no staining was seen in normal lung tissues.

Example 18
Effect of Immunisation with Antigen

In contrast to antibodies which are administered “passively” to a patient, the Pim-1 protein or colon cancer cell lysate antigens or antigenic fragments thereof can be used to “actively” immunise, and produce a vaccine. In such a procedure, the antigen is combined with a carrier (e.g. alum, mannan, beads or other adjuvants) and used to immunise subjects with cancer as a preventative for cancer. The ensuing immune response can be:

- a) the generation of antibodies including but not limited to those described above;
- b) the production of T cells which recognise the antigen presented by MHC Class I or II molecules (the ensuing T cell response can be measured as effector cells as either: Cytotoxic T cells, Cytokine (e.g. interferon producing cells, such as ELISPOT or by other means), T cell proliferation, and/or delayed type hypersensitivity reaction in vivo); and/or
- c) the combination of both antibodies and cellular immunity.

Thus the antigen can be used to produce antibodies which can be administered to the patient or the antigen can be used to “vaccinate” a patient who produces antibodies, T cells or both.

Example 19
Reaction of Anti-Pim-1 Antibodies

Human Pim-1 contains two isoforms of p33 kDa and p44 kDa molecules with distinct subcellular location and function. The p33 kDa molecule is located in the cytosol and the nucleus, while the p44 kDa molecule is found primarily on the plasma membrane (PM), where many signalling proteins localise and thus may interact with Pim-1 in controlling the Pim-1 pathway. Monoclonal antibodies were generated (i.e. P3, P4, P5, P6, P8, P9 and P10) and tested for their binding to recombinant proteins GST-Pim-1 and GST-Muc-1 by ELISA assay (FIG. 28(a)) Inhibition of cancer cell growth in prostate cells (DU145, PC3), colon cells (LS174T) and breast cells (MCF7) by Mabs P3, P4, P5, P6, P8, P9 and P10 was determined by ³H-thymidine incorporation assay (FIG. 28(b & c)). Mabs P4 and P9 were found to be the most effective at suppressing growth of the various cancer cell lines tested and demonstrated increased effectiveness at suppressing cell growth of DU145 cells with increasing concentration (from 5-30 μg/ml) of the Mab used (FIG. 28(c)).

Example 20
Immunohistochemical Staining of Tissues

The anti-Pim-1 Mab P9 was able to detect Pim-1 expression in various tissues derived human prostate cancer (FIG. 29(c)), mouse prostate cancer (FIG. 29(d)), human colon cancer (FIG. 29(f)) and human lung cancer (FIG. 29(h)) but not corresponding normal tissue. Cell surface staining of DU145 cells (prostate cancer cell line) and TRAMP-C1 (mouse prostate cancer cell line) was detected with FITC-labelled P9 Mab (FIG. 29(i) and (j)).

Example 21
Cell Surface Staining of Cell Lines

FITC-labelled P9 antibody was examined for cell surface binding against various cancer cell lines Raji, U937, K562, PC-3, DU145 and LNCaP and compared with binding of a FITC-labelled mouse IgM control antibody and FITC-BC3 antibody used as a positive control (FIG. 30). The anti-Pim-1 Mab P9 bound Pim-1 very effectively on the surface of K562, PC-3, DU145 and LNCaP cells and less effectively on the surface for Raji and U937 cells.

Example 22
Inhibition of DU145 Cell Growth

The Anti-Pim-1 Mabs P9 and P4 synergistically enhanced the cytotoxicity of Cisplatin (cis) and Epirubicin (epi) respectively when used in combination (FIG. 31) as measured by inhibition of cell growth of DU145 cells.

Example 23
Detection of Cell Apoptosis

The anti-Pim-1 Mab P9 induced cancer cell apoptosis shown by trypan blue exclusion, Annexin V and Propidium iodide (PI) staining measured by flow cytometry (FIG. 32). After 4 hours incubation with 25 μg/ml of the P9 Mab, the proportion of apoptotic CEM/A7R cells increased by 32% as measured by the proportion of cells which stained positive for both Annexin V and PI. This demonstrated that the P9 Mab was effective at inhibiting cell growth in vitro. Further, FIG. 32 (C and D) demonstrates the effectiveness of P4 and P9 compared with control BC-3 antibody in causing a reduction in the number of viable cells.

Example 24
Western Blot Analysis

Western Blots showed that anti-Pim-1 Mab P9 reacted with and inhibited both p44 and p33 isoforms of Pim-1 of human and murine prostate cancer cells (FIG. 33). Further, Pim-1 Mab inhibited phosphorylation of Akt at Ser473 and Bad at Ser112 and 136 and activated cleavage of caspase 9 (an indicator of activation of mitochondrial cell death pathway), leading to apoptosis. The results demonstrate that targeting of Pim-1 by the Mab interrupted Pim-1 function, Akt pathways and their downstream effectors.

Example 25
Inhibitory Effect of Anti-Pim-1 Mab P9 in Mouse Tumour Models

In both xenograft and syngeneic prostate cancer models, human DU145 and murine TRAMP-C1, the anti-Pim-1 Mab P9 significantly inhibited the growth of DU145 tumours in SCID mice and established tumour of TRAMP-C1 in C57BL/6 mice (FIG. 34), providing in vivo evidence in targeting Pim-1 kinase for immunotherapy of prostate cancer.

Example 26
Binding of Anti-Pim-1 Mab with Pim-1 on Cancer Cells

The anti-Pim-1 Mabs were examined for both cell surface and intracellular binding to P1m-1 using flow cytometry analysis (Tables 1 and 2). Mab P2, P3, P7 and P8 showed a high percentage (52-93.7%) of intracellular staining in MCF-7, Raji, K562 and NS1 cells. The non-inhibitory Mabs P3, P7 and P8 showed higher percentage of intracellular binding than that of P9 in LOVO (65-84% Vs 30%) or E3 cells (18-27% Vs 14%, Table 1). In contrast, cell surface staining of the anti-Pim-1 Mabs tested by indirect immunofluorescence method is much weaker than that of intracellular staining A greater percentage (27.5%) of K562 bound P9 to the Pim-1 molecule on the cell surface than any other Pim-1 Mab and this was observed for most of the other examined cell lines (Table 2). The results indicate that in addition to cytoplasmic and nuclear expression, Pim-1 is also expressed on the surface of the cancer cell.

TABLE 1

Percentage of intracellular expression of Pim-1 in cancer cell lines

tested by flow cytometry using anti-Pim-1 Mabs

Mabs
MCF7
LOVO
RAJI
K562
E3
NS

P2
52.0
20.0
69.5
70.9
7.7
76.9

P3
62.0
65.0
93.7
88.5
27.0
66.6

P7
65.0
84.0
86.9
83.1
24.0
53.9

P8
60.0
82.0
NT*
NT*
18.0
NT*

P9
66.0
30.0
90.8
91.9
14.0
91.3

BC3
88.0
89.0
98.9
79.8
77.0
94.6

PBS
1.1
0.8
0.7
0.6
0.9
0.3

*Not tested

TABLE 2

Percentage of cell surface expression of Pim-1 in cancer cell lines

tested by flow cytometry using anti-Pim-1 Mabs

Mabs
MCF7
LOVO
RAJI
K562
E3
NS

P2
3.7
1.7
2.4
4.6
1.1
4.0

P3
9.8
3.1
3.9
21.0
4.0
7.9

P7
12.2
3.9
7.5
24.8
9.1
14.6

P8
10.0
2.7
NT*
NT*
7.1
NT*

P9
7.1
4.2
8.5
27.5
10.7
17.6

BC3
96.6
4.9
0.7
8.2
2.2
NT*

PBS
0.5
0.1
0.3
0.54
0.2
0.3

*Not tested

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

	Number	Date	Country
Parent	11943191	Nov 2007	US
Child	12619053		US

	Number	Date	Country
Parent	10470013	Nov 2003	US
Child	11943191		US

Antibodies Against Cancer

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