Jumaat, 30 Mac 2012

Antibodi Lawan Kanser 2


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.


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 cytotoxiccompounds (eg toxins) or other substances (eg isotopes and drugs) to the cancerous cells. However, due to a number of reasons including poor specificity, poor penetration (ie with solid tumours) and the induced HAMA (ie 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 cancertherapy", 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 (ie the humanised HER2/neuMab marketed under the name Transtuzumab for treatmentof 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 ascytotoxic antibodies nor by Fc mediated inflammatory responses, but rather bind antigen leading to interference in cell signaling and apoptosis. For example, in the case of the HER2/neu Mab, the antibody prevents or "blocks" the binding of a growthfactor 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 autosomalgene 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 a fusion protein of a Pim-1 protein (Friedmann M, et al. "Characterisation of the proto-oncogene pim-1: kinase activityand 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 celllines.


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 ormore 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-1protein 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 celllysate.

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 acolon cancer cell lysate.


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 to Cripto-1protein, 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 the Cripto-1 protein (Bianco C. et al., "Cripto-1 indirectly stimulates the tyrosine phosphorylation of erb B-4 through a novelreceptor", 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 the Cripto-1 protein, Pim-1 protein or colon cell lysate antigen. Theterm "specifically binds" in this context, is to be understood to refer to binding characteristics of a peptide, polypeptide or protein which binds exclusively to the Cripto-1 protein, Pim-1 protein or colon cell lysate antigen or with only negligiblecross reaction with other mammalian proteins.

More preferably, the binding partner is a monodonal antibody or fragment thereof and, particularly, is selected from monoclonal antibodies or fragments thereof which bind to an antigenic determinant of Cripto-1 protein comprising an amino acidsequence substantially corresponding to the amino acid sequence;


and/or an antigen present in a colon cancer cell lysate, wherein said antigen has a molecular weight of 16 Kd or 30 Kd as estimated by SDS-PAGE. The 16 Kd and/or 30 Kd antigen may be a growth factor required for growth of colon cancer cellsand/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')υ, Fab and Fc may be produced by, for example, pepsin and papaincleavage 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 hereinby reference.

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

Monoclonal antibodies and antibody fragment 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 (eg mouse or rat) fused to a human myeloma line (eg Karpas 707 H human myeloma cell line; Karpas A, et al. "A human myeloma cell linesuitable 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., etal. "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 (eg in either tissue culture or serum free using a fermenter) and purified using affinity columns such as protein A (eg for murine Mabs), ProteinG (eg for rat Mabs) or MEP HYPERCEL (eg for IgM and IgG Mabs).

The binding partner of 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 asIdarubicin, Doxorubcin, Daunorubicin and Epirubicin), 5FU, topoisomerase inhibitors (such as Irinotecan), Cisplatin, Carboplatin and Taxol.

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

Further, the binding partner of 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 effectiveagainst colon cancer, lymphoma and breast cancer respectively.

Preferably, the binding partner of 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 includeisotonic 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 (eg antibodyor antibody fragment) 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 toserve only as a guide since a person skilled in the art would be able to readily determine the optimum route of administration and dosage for any particular subject and cancer condition.

The anti-cancer agent 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 combinationwith traditional cancer treatments such as radiotherapy, chemotherapy (eg using anthracyclines, 5FU, topoisomerase inhibitors, Cisplatin and Carboplatin), or hormone therapy or therapies utilising hormone modifiers (eg Catamoxifen).

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 fragmentthereof) 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 coloncancer 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, orthe 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 andcombinations 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 the 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 (eg in an in vitro cell culture, or in an in vivo settingsuch 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 coloncancer cell lysate. The amount of the 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 (eg in an in vitro cell culture, orin 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 effectivesensitising 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 substantiallycorresponding to:


or an antigentic 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 exclusionof any other element, integer or step, or group of elements, integers or steps.

The term "substantially corresponding" as used in relation to an amino add sequence is intended to encompass the specified amino acid sequence as well as related amino add sequences which differ only by the inclusion of one or more amino addsubstitutions, 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 oneor 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, Nα-alkylamino adds.

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 admissionthat 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.


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(Inc.).

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 2×106 of prostate cancer DU145 cells sub-cutaneously 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 2.5×106 of colon cancer Ls174T cells sub-cutaneously 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, 50μg/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 (Austin Research Institute, Heidelberg, Victoria, Australia) by anti-Cripto-1 Mabs (ie C3 and C13), and anti-Pim-1 Mabs (ie P4 and P9).

FIG. 10: Shows graphical results demonstrating the effects of the drug Epirubicin on 3 cell lines: leukemia cell CEM A7, the drug resistant variant CEM A7/R and mouse thyoma cells E3 (A), the effect of Mab C4 on drug resistant leukaemia cell lineCEM/A7R (B) and mouse thyoma 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 (ATCC, USA) by the anti-Cripto-1 Mab, C3 alone, or when combined with the cytotoxic drugs cisplatin (Cis), 5-Fluoricil (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 thyoma E3 cells (Austin Research Institute, Heidelberg, Victoria, Australia) in the presence of anti-Cripto-1 Mabs C3 and C13 and the anti-Pim-1 Mabs 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 (Austin Research Institute, Heidelberg, Victoria, Australia) and the drug Epirubicin (David Bull Laboratories,USA) 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 which shows inhibition of growth of the colon cancer cell line HT 29 (ATCC, USA) by the anti-Cripto-1 Mab, C3 and the 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 and3.60, 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 IFNγ secretion. Mouse spleen cells from immunised and naive mice (normal 1 and 2) were stimulated overnight with and without the 37-mer peptide, and spot forming units (SFU) were counted bydissection 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 cellsrespectively induced by Mab C4. Points, mean of triplicate experiments, bars, SD.



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 patientsin advanced disease (i.e. Dukes B, C, D) where there is local spread through nodes to distant metastases (Dukes D) is poor; in 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-2/neu positive (asit is in approximately 30% of patients), a proportion of patients respond well to the HER-2/neu Mab mentioned above.

There is 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 residues97-113 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 growthfactor-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 oneminute each in phosphate buffered saline containing protease inhibitor.

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

Spleen cells from the immunised rats were isolated and fused with the myeloma NS1 (Xing PX, etal. "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 (ie colon cell lines LS174T and HT29, and breast cancer cell line MCF7) in vitro, using a simple assay involvinggrowing LS174T and MCF7 cells (1×105) in 25 cm2 flasks (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 theculture.

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 colorimetric cytotoxidty assay SRB (sulforhodamine B) (Skehan P, "Newcalorimetric 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.


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

Two of the isolated Mabs (ie C3 and C13) bind to Cripto-1 (a member of EGF family encoded by CR1 in humans, tdgfl in mouse), a soluble or, possibly, cell surface (Mr 36 Kd) GPI-linked protein that appears to be a growth factor which promotes cellsurvival and proliferation and is important in embryonic development and cancer (Brandt R, supra) which has been described in a number of species (eg xenopus, zebrafish, mouse and human). Importantly, in the context of the present invention, theexpression 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 ofamphiregulin 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 inhuman 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 immnunoperoxidase staining withfresh 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 haveobserved 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, theseMabs 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 cytotoxiccompounds 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 areal decrease in cell numbers, indicating that the Mabs induced cancer cell apoptosis.


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 immnunoperoxidase staining to determine binding of the antibody to atarget tissue, b) cell growth inhibition assay eg 3H-thyrnidine 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 1×104 of the cellsin 25 cm2 flasks 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 furtherrelative 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 0.125μg/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 ofcombined use of 5FU, Cisplatin, Epirubicin will be clinically useful.


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 whentested 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 expressionvaries 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.


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 2×106 prostate 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 and10, 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 weightdetermination.


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 wasfurther 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 propidiwA iodide (PI) staining, an indicator of apoptosis. The results showed an increasein PI staining in cells treated with the test Mab.


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 culminatein 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 stresssignals. 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 cellsfollowing 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-Cripto1 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 72hours 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 inducedapoptosis. 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) isaccompanied 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 preliminarysignalling studies clearly show that the anti-Cripto-1 Mabs cause apoptosis through the JNK activation pathway.


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 thisproperty 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 (ie E3). Compared to E3, CEM/A7 and CEM/A7R exhibit around 80 and 40 fold resistance to Epirubicinrespectively (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.


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.


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 FUrespectively as shown in FIGS. 15 and 16.


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).


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 4hours, 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.


Isolation of Anti-Pim-1 Antibodies

Two isolated Mabs (ie P4 and P9) were raised against the product of the Pim-1 oncogene. This gene encodes a protein belonging to the ser-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).


Isolation of Antibodies Against Colon Cell Lysate Antigens

Five of the isolated Mabs (ie 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 were also found to inhibit growth of the colon cancer cell line LS174T andbreast cancer cell line MCF7 in tissue culture (FIG. 23). These antibodies also demonstrated inhibition of the prostate cancer cell line DU 145 especially when used in combination with Cisplatin (FIG. 24).


Humanisation of Antibodies

Fully human anti-Cripto-1 antibody 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


and by testing these in vitro and in vivo in the same manner as that described above for the the production of rat-anti-Cripto-1 Mabs.

The antigens are 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 are used to immunise a Human Ig mice (eg XenoMouse) wherein the mouseimmunoglobulin 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 byidentification 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 are then recovered and used togenerate a panel of suitable recombinant candidate antibody products, each ready for manufacturing scale-up.


Clinical Uses of Antibodies

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


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 witha carrier (eg 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 (eg interferon producing cells, such as ELISPOT or byother 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 EUSPOT IFNγ assay. The mice responded in both antibodyand INFγ productions (as shown in FIGS. 25 and 26).


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. InColo 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.

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.

Antibodi Lawan Kanser

Abstract: An isolated binding partner of a Cripto-1 protein, Pim-1 protein or an antigen present in a colon cancer cell lysate is described. The binding partner inhibits growth of one or more cancer cell types and may be used in an anti-cancer agent for treating cancer in a subject. The binding partner may also be used in a method of inducing apoptosis in a cancer cell, as well as in a method of sensitizing a cancer cell to a cytotoxic compound. In addition, a cancer vaccine is described wherein the vaccine comprises 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, comprises 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. ... http://images2.freshpatents.com/pdf/US20090123470A1.pdf

Shark versus cancer
How Jaws could lend a helping hand in the fight against cancer
By Kelly O'Connor
A breakthrough announcement made recently by a team of researches at La Trobe University in Australia, indicates that antibodies from shark blood could be the next leap forward in cancer treatment. Dr. Stewart Nuttall, one of the lead research scientists for the project, explains it this way: “We're initially interested in sharks because they play a pivotal role in immune evolution. If you look at a shark, anything below them in an invertebrate scale doesn't have an immune system. Sharks and everything above it has an immune system very much like humans. But they also have this unusual type of antibodies.”Shark antibodies are somewhat unusual because of their ability to withstand extreme temperatures, pH levels, and their tiny size; researchers are excited about this because it opens up the possibility of having a pill treatment for cancer rather than having to rely on injections.

Thus far, the major stumbling block with developing an oral treatment for cancer has been the harsh environment of the digestive system. Mick Foley, associate professor of molecular biology at La Trobe University and co-lead researcher, describes the difficulties: “The first step in being able to get an orally available antibody, which really is a bit of a holy grail in therapeutics, is to at least survive the gut. And then you have to get it taken up. And so these molecules seem to be extremely stable in the gut. And therefore there is a good chance that we have at least have got past first base.”

If the antibody survives the inhospitable environment of the digestive system, it then binds on to the surface of the target cancer cells and prevents them from growing. There have already been some promising results from the lab, where the researchers have been testing the efficacy of the shark antibody treatment on breast cancer cells. Dr. Foley describes the results: “In the wells that we've added the shark antibodies you can see that the cells are actually growing less than in the wells where we don't add a shark antibody or we add a completely irrelevant shark antibody.

So this indicates the shark antibody that we have is binding to those cancer cells and for some reason causing them to grow more slowly and perhaps even killing them.”

In order to produce such antibodies, the traditional approach has been to inject sharks with an antigen and wait for a sufficient immune response to develop. Instead, these Australian researchers take genes from the sharks, and modify them in the lab by adding random proteins to cause random mutations in a process which closely mimics how the human immune system works.

This enables the researchers to develop a “library” of antibodies capable of responding to a vast array of diseases and conditions, including malaria and rheumatoid arthritis. Dr. Foley explains: “The aim is to use these shark antibodies as a way of finding high-affinity binding agents to bind to anything we want – such as a molecule on cancer cells, or inflammatory proteins that you could then use in therapy.” Through a slow process of trial and error, the researchers expose antibodies to various target molecules (such as cancer cells), and see if there is any reaction. Dr. Foley summarizes the process: “There is maybe a little bit of gold in there, there's a lot of rubbish, and you have to via a very slow process get rid of the rubbish and make sure that the gold is left. So we do that. We sort of put the library on the target molecule and then wash everything away and then we get back what is important, then we amplify that up and then do it again. And we keep doing it until slowly we get the right ones that come out.”

The advantages (and potential payout) of successfully developing an oral-based antibody treatment for cancer are huge. For patients who must be subjected to constant injections, taking pills would be a dramatic improvement. Antibody-based treatments are also much more specific than the current methods. Although there are already other similar treatments in development, they are all in their early stages with none available for patient use as of yet. This leaves a market potentially worth billions of dollars wide open. So next time you see a shark, try to look past those vicious teeth and occasional man-eating tendencies and just appreciate the irony; one of the newest advances in cancer treatment could come from one of the ocean’s most ancient predators.

This page tells you about the immune system. There is information on
·         What your immune system does
·         In-built immune protection
·         Neutrophils
·         Acquired immunity
·         B cells and T cells
·         What B cells do
·         What antibodies are
·         What T cells do
·         Immunotherapy
·         Monoclonal antibodies

What your immune system does

The immune system protects the body against infection caused by bacteria, viruses, fungi or parasites. It is really a collection of reactions and responses that the body makes to infection. So it is sometimes called the 'immune response'.
The immune system is important to cancer patients in many ways because
·         The cancer can weaken the immune system
·         Cancer treatment can weaken the immune system
·         The immune system may help to fight your cancer
Cancer can weaken the immune system by spreading into the bone marrow where the cells that help fight infection are made. This happens most often in leukaemia or lymphoma. But it can happen with other cancers too. The cancer in the bone marrow stops the bone marrow making so many blood cells.
Chemotherapy and radiotherapy can weaken immunity by causing a drop in the number of white blood cells made in the bone marrow. Apart from bone marrow transplants or stem cell transplants, this effect on the bone marrow is temporary. High doses of steroids can also weaken your immune system temporarily.
Some cells of the immune system can recognise cancer cells as abnormal and kill them. Unfortunately, this is not enough to get rid of a cancer altogether. But some new treatments aim to use the immune system to fight cancer.
There are two main parts of the immune system
·         The inbuilt protection we have from birth
·         The immune protection we develop from being exposed to certain diseases

In-built immune protection

This is also called 'innate immunity'. These immune mechanisms are always ready and prepared to defend the body from infection. They can act immediately (or very quickly). This in-built protection comes from
·         A barrier formed by the skin outside the body
·         Inner linings of the gut and lungs which produce mucus and trap invading bacteria
·         Hairs which move the mucus and trapped bacteria out of the lungs
·         Stomach acid which kills bacteria that have been swallowed
·         Helpful bacteria growing in the bowel which prevent other bacteria from taking over
·         Urine flow which flushes bacteria out of the bladder and urethra
·         White blood cells called 'neutrophils' which can find and kill bacteria and other infectious organisms
The skin forms a waterproof mechanical barrier. But it is also slightly acidic. This helps to keep bacteria out as they don't like acid. Some skin conditions cause loss of this acidity and people are then much more prone to skin infections.
There are several ways that these natural protection mechanisms can be damaged or overcome if you have cancer. For example
·         Something may break the skin barrier (such as having a drip in your arm or a wound from surgery)
·         Chemotherapy may damage to the lining of the gut (severe diarrhoea caused by some chemotherapy drugs can break down the gut lining)
·         A catheter into your bladder can become a route for bacteria to get inside the bladder and cause infection
·         Radiotherapy to the lung can damage the hairs and mucus producing cells that help to remove bacteria
·         Antacids for heartburn may neutralise the stomach acid that kills bacteria
·         Chemotherapy can temporarily reduce the number of neutrophils in the blood (the 'neutrophil count') which means it is more difficult for you to fight off infection


These white blood cells are very important for fighting infection. They can
·         Move to areas of infection in the body
·         Stick to invading bacteria or fungi
·         Swallow up the bacteria, viruses or fungi causing the infection
·         Kill the bacteria they have swallowed with chemicals
Your normal neutrophil count is between 2,000 and 7,500 per cubic millimetre of blood. When you don't have enough neutrophils you are said to be 'neutropaenic'.
Chemotherapy and some radiotherapy treatments can lower your neutrophil count. So, after chemotherapy or radiotherapy you may be more likely to get bacterial or fungal infections (like thrush).
If you are having cancer treatment, it is important for you to know that
·         Infections can become serious more quickly in people with low neutrophil counts
·         Antibiotics could save your life, so if you get a fever or feel ill, phone your cancer centre or go to casualty (Accident and Emergency) straight away
You are most likely to become ill from bugs you carry around with you normally, not from catching someone else's. This means that you don't have to avoid your family, friends or children when you are sent home after chemotherapy.
You can ask your cancer doctor or nurse what precautions you should take against infection. There are also some tips in our cancer drug side effects section.
When your blood counts are low, your cancer specialist may want you to take antibiotics to help prevent severe infection. There is some debate about whether this is useful. In the Significant Trial, some people had an antibiotics during treatment and some had a dummy tablet (placebo). The results showed that fewer people who had antibiotics during treatment had a fever or had to go to hospital for treatment for an infection.

Acquired immunity

This is immune protection the body learns from being exposed to diseases. The body learns to recognise each different kind of bacteria and virus it meets for the first time. The next time that particular bug tries to invade the body, the immune system is ready for it and able to fight it off more easily. This is why you usually only get some infectious diseases once - for example, measles or chicken pox.
Vaccination works by using this 'immune memory'. The vaccine contains a small amount of protein from a disease. This is not harmful, but it allows the immune system to recognise the disease if it meets it again. The immune response can then stop you getting the disease. Some vaccines use tiny amounts of the live bacteria or virus. These are called live attenuated vaccines. Attenuated means that the virus or bacteria has been changed so that it will stimulate the immune system to make antibodies but won't cause the infection. Other types of vaccine use killed bacteria or viruses, or chemicals produced by bacteria and viruses.

B cells and T cells

The white blood cells involved in the acquired immune response are called 'lymphocytes'. There are two main types of lymphocytes - B cells and T cells. B and T lymphocytes are made in the bone marrow, like the other blood cells. They have to fully mature before they can help in the immune response. B cells mature in the bone marrow. But the immature T cells travel through the blood stream to the thymus gland where they become fully developed.
Once they are fully mature, the B and T cells travel to the spleen and lymph nodes ready to fight infection.

What B cells do

B cells react against invading bacteria or viruses by making proteins called antibodies. The antibody made is different for each different type of bug. The antibody locks onto the surface of the invading bacteria or virus. The invader is then marked with the antibody so that the body knows it is dangerous and it can be killed off. Antibodies can also detect (and kill) damaged cells.
The B cells are part of the memory of the immune system. The next time the same bug tries to invade, the B cells that make the right antibody are ready for it. They are able to make their antibody very quickly.

What antibodies are

Antibodies are proteins made by the B cells. They have two ends. One end sticks to proteins on the outside of white blood cells. The other end sticks to the germ (or damaged cell) and helps to kill it. The end of the antibody that sticks to the white blood cell is always the same. So it is called the constant end. The end of the antibody that recognises germs and damaged cells varies depending on the cell it is designed to recognise. So it is called the variable end. Each B cell makes antibodies with a different variable end from other B cells. Cancer cells are not normal cells. So some antibodies with variable ends recognise cancer cells and stick to them.

What T cells do

There are different kinds of T cells called
·         Helper T cells
·         Killer T cells
The helper T cells stimulate the B cells to make antibodies, and help killer cells develop.
Killer T cells kill the body's own cells that have been invaded by the viruses or bacteria. This prevents the bug from reproducing in the cell and then infecting other cells.

Can the immune system cure cancer?

Your immune system is very unlikely to be able to fight off an established cancer completely without help from conventional cancer treatment, although there are very rare documented cases of cancers just disappearing (spontaneous regression). Some treatments use elements of the immune system to help treat cancer.


Immunotherapy is a type of biological therapy. Biological therapies use natural body substances or drugs made from natural body substances to treat cancer. Immunotherapies are treatments that use your immune system. They are used in cancer treatment because cancer cells are different from normal cells and so can be picked up by the immune system.
Many different chemicals that are produced as part of the immune response can now be made in the laboratory. You may have heard of one or two of these
·         Interferon
·         Interleukin 2 (IL2)
·         Monoclonal antibodies
Interferon-alpha and Interleukin 2 act by boosting the immune response to help the body kill off cancer cells.
Scientists are also trying to develop vaccinations against cancer cells. It may be possible for them to help the immune system to be trained to see cancer cells as being invaders and kill them.

Monoclonal antibodies

Monoclonal antibodies are made in the laboratory. The scientists developing them make an antibody with a variable end that recognises cancer cells. 'Monoclonal' just means that all the antibodies are exactly the same type, with the same variable end.
The monoclonal antibodies recognise molecules on the outside of cancer cells. Different antibodies have to be made for different types of cancer, for example
·         Rituximab (Mabthera) recognises CD20 protein on the outside of some lymphoma cells
·         Bevacizumab (Avastin) targets growth factors that help blood vessels grow and is used to treat bowel cancer and breast cancer
·         Trastuzumab (Herceptin) recognises breast cancer cells that produce too much of the protein HER 2 - these cancers are called HER 2 positive
The constant end of cancer treating monoclonal antibodies kills the cancer cells by marking them so other immune system cells pick them out. The job of these other cells is to find antibody labelled cells and kill them. But the scientists can make the monoclonal antibody even better at killing cancer cells by attaching
·         A radioactive atom that delivers radiation directly to the cancer cells
·         A chemotherapy drug that is taken straight to the cancer cells by the monoclonal antibody
Monoclonal antibodies are still being researched but are now used for more and more types of cancer. Look in our clinical trials database for monoclonal antibody trials - type 'antibody' into the free text search box. This is an exciting new area of cancer treatment. There is a lot of research going on into the use of immune system therapies to treat cancers. There is detailed information about this in the biological therapies section of CancerHelp UK. You can also look in the treatment section of the type of cancer you are interested in.

Stress, the immune system and cancer

Many people with cancer believe that they should strengthen their immune systems to help beat the disease. There is a commonly held belief that reducing stress can help to strengthen our immune systems. This is the thinking behind some complementary therapies, using relaxation techniques for instance.
There is some scientific evidence that stress weakens our immunity. Two studies looking at whether stress affected cancer recurrence had conflicting results. While no one knows whether strengthening immunity can help to cure cancer, most doctors and nurses agree that reducing stress is a good thing to do.
While many life stresses cannot be avoided altogether, there are ways of trying not to let things get to you. Many complementary therapies such as meditation, massage and reflexology, can be very relaxing.
You can avoid getting run down and look after yourself by
·         Eating a balanced diet when you are able
·         Trying to eat fresh food whenever possible
·         Getting plenty of rest - even if you find it hard to sleep, you can rest

Ovarian cancer has been fatal for women. More and more women are becoming victims of this cancer. More than 21,000 cases of this disease have been recorded during the year 2009. Statistics reveal that women who have been diagnosed with this cancer when they were less than 30 years have more chances of survival than older women who have crossed 50.
In ovarian cancer, the lethal cancer cells get accumulated in the ovaries. Since ovaries are important for producing eggs for reproduction, this deadly disease affects the female reproductive and sexual organs. Different types of tumor cells like stromal, germ cell and epithelial tumors cause this type of cancer.
A lot of research has gone into fighting the numerous cancer cells that affect the individuals. With all treatments and medications, scientists find it a challenge to treat this disease.
Lately, a new antibody has been found by the researchers in order to fight against the cancer cells that attack the immune system of the body. This has offered new hopes for treating ovarian cancer among women.
This antibody named AD5-10 attaches to the cancer cells weakening the resistance of the tumor cells to the body's immune system. In addition, this antibody is found to be more effective for chemotherapy.
AD5-10 antibody decreases resistance of the cells to carboplatin, a common agent used in chemotherapy. The combined effect of this antibody along with carboplatin was more effective for treating cancer patients. Reports also revealed that the antibody is found to react in this manner only when the cell killers known as lymphocytes NK are present in the tumor cells during the same time.
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http://www.pnl.gov/news/release.aspx?id=796  Silica cages help anti-cancer antibodies kill tumors in mice
RICHLAND, Washington – Packaging anti-cancer drugs into particles of chemically modified silica improve the drugs' ability to fight skin cancer in mice, according to new research. Results published May 3 in the Journal of the American Chemical Society online show the honeycombed particles can help anti-cancer antibodies prevent tumor growth and prolong the lives of mice.
"We are very excited by our preliminary results," said biochemist Chenghong Lei of the Department of Energy's Pacific Northwest National Laboratory, part of the team of PNNL and University of Washington scientists. "We plan to do some additional, larger studies with animals. We hope the results hold up well enough to take it to clinical trials somewhere down the road."
Anti-cancer antibodies are some of the most promising types of cancer therapies. The antibodies target a particular protein on cancer cells and — in a poorly understood way — kill off the cells. Examples include herceptin for one form of breast cancer and cetuximab for colon cancer.
Unlike popping a pill, however, antibody-based treatments require patients to go in for intravenous drips into the arm. These sessions cost time and money, and expose healthy tissue to the antibody, causing side effects.
Packaging antibodies into particles would concentrate them at the tumor and possibly reduce side effects. Other research has shown silicon to be well tolerated by cells, animals and people. So, in collaboration with tumor biologist Karl Erik Hellstrom's group at UW, the scientists explored particles made from material called mesoporous silica against cancer in mice.
"The silica's mesoporous nature provides honeycomb-like structures that can pack lots of individual drug molecules," said PNNL material scientist Jun Liu. "We've been exploring the material for our energy and environmental problems, but it seemed like a natural fit for drug delivery."
In previous work, the team created particles that contain nano-sized hexagonal pores that hold antibodies, enzymes or other proteins. In addition, adorning the silica pores with small chemical groups helps trap proteins inside. But not permanently — these proteins slowly leak out like a time-release capsule.
The researchers wanted to test whether anti-cancer antibodies packaged in modified mesoporous silica would be more effective against tumors than free-flowing antibodies.
To do so, they first chemically modified mesoporous silica particles of about six to 12 micrometers (about 1/10 the diameter of human hair). These particles contained pores of about 30 nanometers in diameter. They found that the extent and choice of chemical modification — amine, carboxylic acid or sulfonic acid groups — determined how fast the antibodies leaked out, a property that can be exploited to fine tune particles to different drugs.
Additional biochemical tests showed that the antibodies released from the silica cages appeared to be structurally sound and worked properly.
They then tested the particles in mouse tumors at UW, filling them with an antibody called anti-CTLA4 that fights many cancers, including melanoma, a skin cancer. The team injected these packaged antibodies into mouse tumors. The team also injected antibodies alone or empty particles in other mouse tumors.
The packaged antibodies slowed the growth of tumors the best. Treatment started when tumors were about 27 cubic millimeters. Untreated tumors grew to 200 cubic millimeters about 5 days post-treatment. Tumors treated with antibodies alone reached 200 cubic millimeters on day 9, showing that antibodies do slow tumor growth. But tumors treated with packaged antibodies didn't reach 200 cubic millimeters until day 30, a significant improvement over antibodies alone.
The team repeated the experiment and found the treatment also prolonged the lives of diseased mice. Of five mice that had been treated with particles alone, all died within 21 days after treatment. But of five mice treated with the packaged antibodies, three were still alive at 21 days, and two at 34 days, when the experiment ended.
The team also measured how much antibody remained in the tumors. Two and four days after injection, the researchers found significantly more antibody in tumors when the antibodies had been encased in the silica particles than when the antibodies had been injected alone.
The team is testing other antibody-cancer pairs in mice, especially other cancers that form solid tumors such as breast cancer. They are also going to explore how the antibodies delivered this way induce the immune system to better fight cancer.
"We want to understand the mechanism, because not much is known about how the slowly leaked antibodies induce changes in the immune system or in the micro-environment of the tumor," said Hellstrom.
Reference: Chenghong Lei, Pu Liu, Baowei Chen, Yumeng Mao, Heather Engelmann, Yongsoon Shin, Jade Jaffar, Ingegerd Hellstrom, Jun Liu, Karl Erik Hellstrom, Local release of highly loaded antibodies from functionalized nanoporous support for cancer immunotherapy, May 3, 2010 J. Am. Chem. Soc., DOI 10.1021/ja102414t (http://pubs.acs.org/doi/full/10.1021/ja102414t). This work was supported by PNNL, Washington Research Foundation, UW Institute of Translational Health Sciences, the NIH, and the U.S. Department of Energy Office of Basic Energy Sciences in the Office of Science.
UW Medicine includes the School of Medicine, Harborview Medical Center, UW Medical Center, Northwest Hospital & Medical Center, UW Medicine Neighborhood Clinics, UW Physicians, Airlift Northwest, and the UW's involvement in the Seattle Cancer Care Alliance. UW Medicine has major academic and service affiliations with Seattle Children's Hospital, Fred Hutchinson Cancer Research Center, and the Veteran's Affairs Puget Sound Health Care System in Seattle and VA Hospital in Boise. The UW School of Medicine is the top public institution in federal funding for biomedical research. Follow us on Twitter - @UWMedicineNews