AU2013203806B2 - Vaccines for the treatment or prevention of cancer - Google Patents

Vaccines for the treatment or prevention of cancer Download PDF

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AU2013203806B2
AU2013203806B2 AU2013203806A AU2013203806A AU2013203806B2 AU 2013203806 B2 AU2013203806 B2 AU 2013203806B2 AU 2013203806 A AU2013203806 A AU 2013203806A AU 2013203806 A AU2013203806 A AU 2013203806A AU 2013203806 B2 AU2013203806 B2 AU 2013203806B2
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cancer
biological sample
vaccine
cell
exposing
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AU2013203806A1 (en
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Ross Arthur DAVEY
Christopher John WEIR
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Northern Sydney Local Health District
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Northern Sydney Local Health District
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Priority claimed from AU2012905667A external-priority patent/AU2012905667A0/en
Application filed by Northern Sydney Local Health District filed Critical Northern Sydney Local Health District
Priority to AU2013203806A priority Critical patent/AU2013203806B2/en
Priority to PCT/AU2013/001523 priority patent/WO2014100857A1/en
Priority to NZ629700A priority patent/NZ629700A/en
Priority to EP13867300.9A priority patent/EP2934578B1/en
Priority to JP2015549902A priority patent/JP6286445B2/en
Priority to CA2934958A priority patent/CA2934958A1/en
Priority to EP18182531.6A priority patent/EP3446708A1/en
Priority to AU2013370932A priority patent/AU2013370932B2/en
Priority to US14/655,088 priority patent/US10357538B2/en
Publication of AU2013203806A1 publication Critical patent/AU2013203806A1/en
Publication of AU2013203806B2 publication Critical patent/AU2013203806B2/en
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Priority to JP2018017162A priority patent/JP2018111695A/en
Priority to US16/438,703 priority patent/US20190358291A1/en
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Abstract

Abstract The present invention relates to the treatment and prevention of cancer. The present invention relates to vaccines comprising solubilized components of cancer cells or cancer-associated cells. Moreover, the present invention also relates to methods of producing vaccines from 5 biological samples comprising cancer cells or cancer-associated cells and using said vaccines for the treatment or prevention of cancer in subjects. The present invention also relates to methods of producing vaccines, in particular, autologous vaccines.

Description

Vaccines for the Treatment or prevention of cancer Technical Field The present invention relates to the treatment and prevention of cancer. The present 5 invention relates to vaccines comprising solubilized components of cancer cells or cancer associated cells. Moreover, the present invention also relates to methods of producing vaccines from biological samples comprising cancer cells or cancer-associated cells and using said vaccines for the treatment or prevention of cancer in subjects. The present invention also relates to methods of producing vaccines, in particular, autologous 10 vaccines. Background of the Invention Most cancer cells elicit an immune response that is evident by the presence of immune cell infiltrates and inflammation. This response, however, is not strong enough to overcome the cancer cell's defence strategies. Approaches taken to promote an immune is response against cancer cells include stimulating a subject's immune cells, in particular, their dendritic cells, to recognise cancer-cell specific antigens in vitro and then injecting these back into the subject. The molecular and cellular interaction between the immune system and a tumour is complex and it is only relatively recent that the importance of normal cells, tissues and 20 chemokine/cytokine responses in this interaction has been recognised. Blood vessels, connective tissue, stroma and extra-cellular matrix all play a part in supporting tumour growth and the inflammatory environment supplied by the immune system further stimulates growth. The lack of understanding of the complex interactions between tumours and the 25 immune system has hindered the development of cancer immunotherapy. Approaches involving using purified tumour antigens and more complex mixtures of tumour antigens have failed to stimulate adequate immune responses against tumours. The reasons for this are unknown, but may include the genetic instability of tumours and the ability of tumours to evade the immune system by presenting a "normal" appearance or releasing 30 inhibitors. Tumours can respond to an immune response by reducing the amount of targeted antigens, by masking antigens from the immune system or by expressing mutated 2 versions of antigens that are no longer recognised. Such defensive strategies undermine the immune system, making it difficult to maintain an effective immune response at the level required to halt tumour growth and cause regression. Summary of the Invention There would be substantial benefit to being able to direct a cancer subject's immune system to assist in the treatment or prevention of cancer. The inventors have surprisingly found that vaccines based on the solubilized components of cancer cells and normal cells associated with cancer cells, in combination with a non-mammalian polypeptide capable of binding a mammalian protein, are efficient at eliciting an immune response against a cancer cell. Accordingly, in a first aspect of the present invention, there is provided a method for producing a vaccine for the treatment or prevention of cancer, the vaccine comprising solubilized components of a cancer cell or cancer-associated cell, and a non-mammalian polypeptide capable of binding a mammalian protein, the method comprising exposing a biological sample comprising at least one cancer cell or cancer-associated cell to an ionic detergent, a reducing agent, and a non-mammalian polypeptide capable of binding a mammalian protein, to produce a solubilized biological sample comprising components from said cancer cell or cancer-associated cell, and a non-mammalian polypeptide capable of binding a mammalian protein. In an embodiment of the present invention there is provided a method for producing a vaccine for the treatment or prevention of cancer, the method comprising exposing a biological sample comprising at least one cancer cell or cancer-associated cell to an ionic detergent, a reducing agent, and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein, to produce a composition comprising a solubilized biological sample comprising components from said cancer cell or cancer-associated cell and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein, solvent precipitating said composition, followed by resuspension of the resulting precipitate in a suitable liquid, wherein said vaccine comprises said resuspended precipitate. In particular embodiments of the present invention, the biological sample is a biological sample from the subject intended to receive the vaccine. In one embodiment, the ionic detergent is selected from the group consisting of sodium-dodecly-sulphate (SDS), 3-[(3-Cholamidopropyl)dimethylammonio]-1 (1A22)Q14q 119GGGr 3 propanesulfonate (CHAPS), lithium dodecylsulphate, sodium cholate, sodium lauroylsarcosine and cetyltrimethylammonium bromide (CTAB). In another embodiment the biological sample is exposed to an ionic detergent at a concentration of 0.1 to 10 % (w/v). In a preferred embodiment, the ionic detergent is SDS. In a further preferred embodiment, the biological sample is exposed to SDS at a concentration of 0.5 to 1.5 % (w/v). In another embodiment of the first aspect, the reducing agent is selected from the group consisting of 2-mercaptoethanol, 2-mercaptoethanolamine, cysteine-HCl, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), tributylphosphine (TBP) and iodoacetamide. In a further embodiment, the biological sample is exposed to a reducing agent at a concentration of 1 mM to 500 mM. In a preferred embodiment, the reducing agent is TCEP or DTT. In a more preferred embodiment, the biological sample is exposed to TCEP or DTT at a concentration of 1 mM to 100 mM. In a particular embodiment, the biological sample is exposed to an ionic detergent prior to exposure to a reducing agent and a non-mammalian polypeptide capable of binding a mammalian protein. In other embodiments, the biological sample is exposed to an ionic detergent and a reducing agent prior to exposure to a non-mammalian polypeptide capable of binding a mammalian protein. In one embodiment, the non-mammalian polypeptide capable of binding a mammalian protein is a bacterial lectin or adhesin. In another embodiment, the non-mammalian polypeptide capable of binding a mammalian protein is a polypeptide with an RGD or RGD-like motif. In a preferred embodiment, the non-mammalian polypeptide is streptavidin, avidin or Neutravidin. In a particular embodiment, the method further comprises exposing said solubilized biological sample to biotin. In another embodiment, the method further comprises exposing the biological sample to an alkylating reagent. In one embodiment, the solubilized biological sample is partitioned into a soluble fraction and an insoluble fraction. (iq1q215 1hGGG 4 In particular embodiments, the method further comprises solvent precipitation of said solubilized biological sample or a soluble fraction of the solubilized biological sample, followed by resuspension of the resulting precipitate in a suitable liquid. In one embodiment, the solvent is a polar organic solvent. In a preferred embodiment, the polar organic solvent is selected from the group consisting of ethanol, methanol, acetone, isopropanol, propanol and DMF. In a further preferred embodiment, the polar organic solvent is acetone. In a second aspect of the present invention, there is provided a method for producing a vaccine for the treatment or prevention of cancer, the method comprising the steps of: a. exposing a biological sample comprising at least one cancer cell or cancer associated cell to an ionic detergent in a suitable liquid to produce a solubilized biological sample comprising soluble material and insoluble material; b. partitioning the soluble and insoluble material of the solubilized biological sample to produce a soluble fraction and an insoluble fraction; c. exposing the soluble fraction to a reducing agent; d. exposing the soluble fraction to a non-mammalian polypeptide capable of binding a mammalian protein; e. performing a solvent precipitation of the soluble fraction; and resuspending the precipitate in a suitable liquid. In an embodiment of the present invention there is provided a method for producing a vaccine for the treatment or prevention of cancer, the method comprising the steps of: a) exposing a biological sample comprising at least one cancer cell or cancer associated cell to an ionic detergent in a suitable liquid to produce a solubilized biological sample comprising soluble material and insoluble material; b) partitioning the soluble and insoluble material of the solubilized biological sample to produce a soluble fraction and an insoluble fraction; c) exposing the soluble fraction to a reducing agent; d) exposing the soluble fraction to streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein; e) performing a solvent precipitation of the soluble fraction; and f) resuspending the precipitate in a suitable liquid, wherein said vaccine comprises said resuspended precipitate. (n22791 1rGG 5 In a third aspect of the present invention, there is provided a method for producing a vaccine for the treatment or prevention of cancer, the method comprising the steps of: a. exposing a biological sample comprising at one least cancer cell or cancer associated cell to an ionic detergent and a reducing agent in a suitable liquid to produce a solubilized biological sample comprising soluble material and insoluble material; b. partitioning the soluble and insoluble material of the solubilized biological sample to produce a soluble fraction and an insoluble fraction; c. exposing the soluble fraction to a non-mammalian polypeptide capable of binding a mammalian protein; d. performing a solvent precipitation of the soluble fraction; and e. resuspending the precipitate in a suitable liquid. In an embodiment of the present invention there is provided a method for producing a vaccine for the treatment or prevention of cancer, the method comprising the steps of: a) exposing a biological sample comprising at least one cancer cell or cancer associated cell to an ionic detergent and a reducing agent in a suitable liquid to produce a solubilized biological sample comprising soluble material and insoluble material; b) partitioning the soluble and insoluble material of the solubilized biological sample to produce a soluble fraction and an insoluble fraction; c) exposing the soluble fraction to streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein; d) performing a solvent precipitation of the soluble fraction; and e) resuspending the precipitate in a suitable liquid, wherein said vaccine comprises said resuspended precipitate. In particular embodiments, the non-mammalian polypeptide capable of binding a mammalian protein is a bacterial lectin or adhesin In other embodiments, the non-mammalian polypeptide capable of binding a mammalian protein is a polypeptide with an RGD or RGD-like motif. In preferred embodiments, the non-mammalian polypeptide is streptavidin, avidin or Neutravidin. In further embodiments, the method further comprises the step of exposing said soluble fraction to biotin prior to performing said solvent precipitation of the soluble fraction. (10229155 1):GGG 6 In other embodiments, the method further comprises the step of exposing said soluble fraction to an alkylating reagent prior to performing said solvent precipitation of the soluble fraction. In particular embodiments of the present inventions, there is provided the vaccines made by any of the methods herein described. In a fourth aspect of the present invention, there is provided a vaccine for the treatment or prevention of cancer in a subject, wherein the vaccine comprises solubilized and reduced components of a cancer cell or cancer-associated cell, and a non-mammalian polypeptide capable of binding a mammalian protein. In an embodiment of the present invention there is provided a vaccine for the treatment or prevention of cancer in a subject, said vaccine comprising solubilized and reduced components of a cancer cell or cancer-associated cell, and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein. In a fifth aspect of the present invention, there is provided a vaccine for the treatment or prevention of cancer in a subject, wherein the vaccine comprises solubilized, reduced and alkylated components of a cancer cell or cancer-associated cell, and a non mammalian polypeptide capable of binding a mammalian protein. In an embodiment of the present invention there is provided a vaccine for the treatment or prevention of cancer in a subject, said vaccine comprising solubilized, reduced and alkylated components of a cancer cell or cancer-associated cell, and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein. In particular embodiments of the present invention, the cancer cell or cancer associated cell is a cancer cell or cancer-associated cell from said subject. In other embodiments, the non-mammalian polypeptide capable of binding a mammalian protein is a bacterial lectin or adhesin. In further embodiments, the non-mammalian polypeptide capable of binding a mammalian protein is a polypeptide with an RGD or RGD-like motif. In preferred embodiments, the non-mammalian polypeptide is streptavidin, avidin or Neutravidin. M7iO2'; 1WrGC 6a In particular embodiments, the vaccine further comprises biotin. In preferred embodiments, the method of the present invention is performed by a medical practitioner or by a person or persons under the supervision of a medical practitioner, or by a combination thereof. In a sixth aspect of the present invention, there is provided a pharmaceutical composition for the treatment or prevention of cancer comprising any of the vaccines described herein, and a pharmaceutically acceptable carrier. In a preferred embodiment, the pharmaceutical composition is used for the treatment or prevention of cancer in a subject. In a seventh aspect of the present invention, there is provided a method of treatment or prevention of cancer in a subject, the method comprising administering an effective amount of a vaccine of the present invention to a subject. In an eighth aspect of the present invention, there is provided the use of a composition comprising a solubilized biological sample of at least one cancer cell or cancer-associated cell, and a non-mammalian polypeptide capable of binding a mammalian protein, for the manufacture of a medicament for the treatment or prevention of cancer in a subject. In an aspect of the present invention there is provided use of a composition comprising solubilized and reduced components of a biological sample of at least one cancer cell or cancer-associated cell and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein, for the manufacture of a medicament for the treatment or prevention of cancer. In particular embodiments of the present invention, the biological sample is a biopsy sample from a subject intended to be the recipient of the vaccine, medicament or treatment. In a ninth aspect of the present invention, there is provided a method for the treatment or prevention of cancer in a human subject, the method comprising the steps of obtaining a biological sample comprising at least one cancer cell or cancer-associated cell (10229155 1):GGG 7 from said subject, exposing the biological sample to an ionic detergent, a reducing agent, and a non-mammalian polypeptide capable of binding a mammalian protein, to produce a vaccine comprising a solubilized biological sample comprising components from said cancer cell or cancer-associated cell, and a non-mammalian polypeptide capable of binding a mammalian protein, and administering a therapeutically effective amount of said vaccine to said subject, wherein all steps of the method are performed by or under the supervision of a registered medical practitioner having prime responsibility for the clinical care of said subject throughout said method. In an aspect of the present invention there is provided a method for the treatment or prevention of cancer in a human subject, the method comprising the steps of obtaining a biological sample comprising at least one cancer cell or cancer-associated cell from said subject, exposing the biological sample to an ionic detergent, a reducing agent, and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein, to produce a vaccine comprising a solubilized biological sample comprising components from said cancer cell or cancer-associated cell, and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein, and administering a therapeutically effective amount of said vaccine to said subject. In particular embodiments, the method for treatment or prevention is a course of treatment or prevention comprising multiple steps of administering said vaccine to said patient. In a further embodiment, one or more step(s) of the method is conducted by a person or persons under the supervision of said medical practitioner. In one embodiment, the collective steps of the method are performed by a plurality of individuals. In an embodiment, the collective steps of the method are performed at multiple locations. In one embodiment, the step of obtaining a biological sample from said subject is conducted at a different location to said exposing step. In other embodiments, the method for the treatment or prevention of cancer in a human subject further comprises additional steps described herein for the production of said vaccine. In an embodiment the vaccine according to the invention, or the pharmaceutical composition according to the invention, further comprises one or more adjuvant(s), optionally wherein the adjuvant is FIA. In an embodiment the method of treating or preventing cancer in a subject further comprises administering to the subject one or more adjuvant(s), optionally where the adjuvant is FIA.
8 Definitions Throughout this specification, reference to "a" or "one" element does not exclude the plural, unless context determines otherwise. The term "treatment", and the like, in the context of the present specification includes any of the alleviation of the symptoms associated with a cancer, as well as cancer regression and remission. In certain embodiments a treatment will slow, delay or halt the proliferation or metastasis of a cancer, prevent differentiation of a cell line, or reverse the progression of one or more tumours, at least temporarily. The treatment may cure the cancer, or delay morbidity. Hence, in the context of this invention the word "treatment" or derivations thereof when used in relation to a therapeutic application includes all aspects of a therapy, such as the alleviation of pain associated with the cancer being treated, alleviation of the severity of the cancer being treated, improvement in one or more symptoms of the cancer being treated, improvement in the overall well-being of the subject being treated. Use of the word "treatment" or derivatives thereof will be understood to mean that the subject being "treated" may experience any one or more of the aforementioned benefits. The term "prevention", and the like, in the context of the present specification refers to the prevention of the recurrence of all or some of the symptoms associated with a cancer after a remission of said cancer, as well as the prevention of the formation of one or more cancers due to, for example, the metastasis of a cancer. The prevention may prevent morbidity due to one or more cancers, or delay morbidity due to one or more cancers. In the context of this specification, the term "comprising" means including, but not necessarily solely including. Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings. Hence, the term "comprising" and variations thereof is used in an inclusive rather than exclusive meaning such that additional integers or features may optionally be present in a composition, method, etc. that is described as comprising integer A, or comprising integer A and B, etc. In the context of this specification the term "about" will be understood as indicating the usual tolerances that a skilled addressee would associate with the given value. In the context of this specification, where a range is stated for a parameter it will be understood that the parameter includes all values within the stated range, inclusive of the stated endpoints of the range. In the context of this specification, the term "plurality" means any number greater than one. f1A22Qr11 11-GGGr 9 Brief Description of the Drawings Figure 1 shows the results from the initial vaccine trial. A) Tumour growth in control rats (adjuvant only, grey line, n=5) and rats pre-vaccinated (black line, n=3). Rats were challenged with approximately lx1O6 9L tumour cells in the flank at day zero. B) Survival curves for the same rats with solid line for controls (n=5) and dashed line for vaccine treated (n=3). Figure 2 shows the results from the vaccine dosing trial and tumour rechallenge trial. A) Survival of control rats (n=8, Solid line) v rats that received a single vaccination dose (n=8, dashed line). B) Survival of control rats (n=8, Solid line) v rats that received two vaccinations (n=9, dashed line). C) Survival of control rats (n=8, Solid line) v rats that received three vaccination doses (n=9, dashed line). D) Survival of control rats (n=8, Solid line) v rats that received two vaccination doses S.C. (n=8, dashed line). E) Survival of control rats (n=8, Solid line) v rats that were rechallenged (n=4) with tumour cells in the flank. F) Survival of control rats (n=8, Solid line) v rats that were rechallenged (n=5) with tumour in the brain. Figure 3 shows the results obtained from a preliminary analysis of the protein composition of the initial vaccines. A) Vaccinated rat serum reactivity to streptavidin in the vaccine only (no serum reactivity to tumour proteins). B) A silver stained SDS-PAGE gel profile of a typical rat vaccine. Figure 4 shows the survival rates of the rats included in the vaccine components trial. Compares survival time (days) and significant extended survival of the four vaccinated groups. The control group (n=5) had a survival time of 38.4 ± 2.9. Figure 5 summarises an analysis of rat serum after vaccination with a reduced tumour + streptavidin vaccine (Vaccine) and a streptavidin only vaccine (Streptavidin). A) Rat serum antibodies to streptavidin pre- and post-tumour challenge. B) Serum streptavidin serum reactivity during tumour progression. C) C-Reactive protein levels pre- and post-tumour challenge. D) CINC-1 levels 21 days post-tumour challenge. E) ICAM levels 21 days post tumour challenge. F) TNF-a levels 21 and 34 days post tumour challenge. The controls were vaccinated with adjuvant only. Figure 6 shows the results from a flow cytometric blood assay performed with vaccinated and unvaccinated rats. Blood analysis on control rats (Black bars; adjuvant only n=6) and vaccine treated rats (white bar n=6): Pre tumour sample point was taken after vaccination and prior to tumour challenge. Post tumour sampling was performed 21 days after tumour challenge. All rats were bled prior to any treatment to provide baseline control levels of all cell types (Black line, n= 12). (1n227Qf11 110GGG 9a Figure 7 shows the results achieved for a rat vaccine trial using autologous vaccines prepared with SDS extracted tumour proteins, a reducing agent and streptavidin, when compared with unvaccinated subjects.
Figure 8 shows a protein profile of a dog autologous vaccine. A) A silver stained SDS PAGE gel of a typical dog personalised autologous vaccine. B) Streptavidin binding in vaccine. C) Biotin binding in the dog vaccine. Detailed Description of the Invention 5 The present inventors have surprisingly found that vaccines comprising heterologous mixtures of solubilized and reduced self and non-self proteins, polypeptides and cellular components, can generate an enhanced immune response to cancer cells in cancer patients. The invention may be used to produce these vaccines and pharmaceutical 10 compositions for the treatment or prevention of cancer. Accordingly, the present invention provides a method for producing a vaccine for the treatment or prevention of cancer, the vaccine comprising solubilized components of a cancer cell or a cancer associated cell, and a non-mammalian polypeptide capable of binding a mammalian protein, the method comprising exposing a biological sample 15 comprising at least one cancer cell or cancer-associated cell to an ionic detergent, a reducing agent, and a non-mammalian polypeptide capable of binding a mammalian protein, to produce a solubilized biological sample comprising components from said cancer cell or cancer-associated cell, and a non-mammalian polypeptide capable of binding a mammalian protein. 20 A vaccine may be any substance used to stimulate the production of antibodies against one or several cancers, whereby the substance is recognised by the subject's immune system as immunogenic and/or antigenic. The cancer cell may be from any cancer that presents as a solid tumour or a blood (liquid) cancer, including, but not limited to sarcomas, carcinomas, lymphomas, 25 leukemia, myelomas and circulating tumour cells (CTCs). For example, the carcinoma may be that of the bladder, breast, colon, mesothelioma, kidney, liver, lung, including small cell lung cancer, non-small cell lung cancer, head and neck, oesophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate or skin. As further non-limiting examples, the lymphoma may be B-cell lymphoma, T- cell 30 lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, mantle cell lymphoma, myeloma, Burkett's lymphoma, or an extranodal lymphoma of the stomach, breast or brain. The sarcoma may, for example, be fibrosarcoma, rhabdomyosarcoma, chondrosarcoma, leiomyosarcoma, mesothelial sarcoma, angiosarcoma, liposarcoma, 5 tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas, or other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma. The myeloma may, for example, be plasma cell myeloma or Kahler's disease or 10 multiple myeloma. In other examples, the leukemia may be myelogenous leukemia, granulocytic leukemia, lymphatic leukemia, lymphocytic leukemia or lymphoblastic leukemia, polycythemia vera or erythremia. The biological sample may be any sample from a subject which includes at least one cancer cell or cancer-associated cell, including, but not limited to tissue, tissue fluids, 15 blood, blood components, bone marrow, excreta including urine and feces, and secreta including mucus. The biological sample may be more than one type. For example, a biological sample may be comprised of a tissue sample and a blood sample. The biological sample may comprise a tissue sample from one site on a subject and a tissue sample from another site on a subject. The biological sample may comprise more than 20 one sample taken from a subject at different times. For example, a biological sample may comprise two blood samples that are taken from a subject on two separate occasions. In a preferred embodiment, the biological sample comprises a biopsy of a known or suspected cancer or tumour. The biological sample comprising at least one cancer cell or cancer-associated cell may, for example, be a tumour sample. The biological sample will 25 typically comprise cancer cells and non-cancer cells, and non-cellular components such as, for example, plasma, extra-cellular matrix, enzymes, growth factors and cytokines. The biological sample may be collected from a subject under the clinical care of a medical practitioner by, for example, a medical practitioner or a health care professional. A medical practitioner may be any person that is registered, authorized or certified under 30 law to practice medicine independently. A health care professional may be any person that is permitted, authorized, registered or certified to collect a biological sample from a subject either independently or under the supervision of a medical practitioner. For example, the health care professional may be a registered or enrolled nurse, or a medical practitioner's assistant or a clinical assistant. It would be understood that the biological sample may, for example, be collected during routine out-patient procedures that would ordinarily be carried out on a subject with cancer who is under the clinical care of a 5 medical practitioner. In a particular embodiment, the method of the present invention is performed by a medical practitioner or by a person or persons under the supervision of a medical practitioner, or by a combination thereof. A person under the supervision of a medical practitioner may be, for example, a health care professional, a pharmacist, a clinical, 10 medical or pathology laboratory technician, or a scientist. It would be understood that the method of the present invention may be performed in any laboratory by a medical practitioner or by a person or persons under the supervision of a medical practitioner, or by a combination thereof. The biological sample may contain at least one cancer cell from any of the is previously mentioned cancers. The cancer cell may be from one of more of these types of cancers. For example, a blood sample may contain cancer cells that are B-cell lymphoma cells as well as cancer cells that are melanoma cells. Furthermore, a tissue sample may contain cancer cells that are fibrosarcoma cells as well as cancer cells that are liposarcoma cells. 20 The vaccines of the present invention may prevent or delay or retard the development of cancers that, for example, may ordinarily develop from the metastasis of any of the cancers mentioned herein. The vaccines of the present invention may also prevent or delay or retard the recurrence of any of the cancers mentioned herein after treatment. 25 The cancer-associated cell may be any non-cancer cell included in the biological sample due to proximity to a cancer cell. The cancer-associated cell may be from any proximal non-cancerous tissue, including, but not limited to blood vessels, connective tissue, nerves, muscle, brain tissue, stroma, tissue from associated organs and fatty tissues. The cancer-associated cell may be any non-cancer cell, including, but not limited 30 to a white blood cell, a red blood cell, a plasma cell, a fibroblast, or a stem cell.
In one embodiment of the present invention, the biological sample is from the subject that is the intended recipient of the vaccine produced using said biological sample. In this context, the vaccine may be referred to as autologous. The solubilization of a biological sample is understood to mean the disruption of a 5 biological sample in a liquid phase by any appropriate means, typically by chemical, mechanical and/or physical means. The disruption of a biological sample includes, but is not limited to, the disintegration of tissue samples, dissociation of cells from tissues samples, disaggregation of cells, permeabilisation of cell membranes, cell lysis, dissolution of membranes and denaturation of proteins and polypeptides, and the 10 disruption of inter- and intramolecular interactions, including but not limited to disulphide bonds, ionic bonds, hydrogen bonds, hydrophobic bonds and van der Waals. Disruption of the biological sample, and hence solubilization, may be assisted by freeze/thaw cycles, agitation, vortexing, sheering, cutting, grinding, homogenizing, pressure forces, or sonic forces. For example, the solubilization of a biological sample by is exposure to an ionic detergent and a reducing agent may be assisted by passing the material through a syringe and needle, grinding the material in a mortar and pestle, a homogenizer, a French press, a sonicator or a rotary device. A person of skill in the art will understand that a solubilized biological sample will typically include soluble material and insoluble material. Insoluble material may be any 20 material that will form a pellet when a solubilized biological sample is centrifuged at speeds over 1000 rpm (or equivalent) or above. The solubilized biological sample may comprise, for example, 10 % to 99 % (w/w) soluble material. For example, the solubilized biological sample may comprise approximately 20 % (w/w) soluble material and approximately 80 % (w/w) insoluble material, or the solubilized biological sample 25 may comprise approximately 60 % (w/w) soluble material and approximately 40 % (w/w) insoluble material. In preferable circumstances, at least 50 % (w/w) of the solubilized biological sample will be soluble material. It will also be understood by a skilled addressee that the amount of insoluble material and soluble material in the solubilized biological sample will depend on numerous factors including the type of biological 30 sample, the amount of ionic detergent and reducing agent and the type of ionic detergent and reducing agent. For example, a solubilized biological sample derived from a tissue sample may comprise more insoluble material than a solubilized biological sample derived from a blood sample of the same volume. Furthermore, a biological sample that is exposed to a weak ionic detergent may produce a solubilized biological sample with more insoluble material than a solubilized biological sample produced by exposing a biological sample to a strong ionic detergent. 5 The biological sample may be solubilized in any suitable liquid. The liquid may be water or a solution, or salt solution or a buffered solution. The liquid may be a buffered salt solution, including, but not limited to, phosphate-buffered saline (PBS) or tris buffered saline (TBS). In the methods of the present invention, the biological sample is exposed to an ionic 10 detergent. As used herein, an ionic detergent is understood to mean an amphipathic molecule, with a charged polar head group, which aids in solubilization of the components of tissues and cells. Ionic detergents include, but are not limited to the alkyl aryl-sulphonates, the long-chain alcohol-sulphates, the olefine-sulphates and sulphonates, the alpha olefine-sulphates and -sulphonates, the sulphated monoglycerides, 15 the sulphated ethers, the sulphosuccinates, the alkane-sulphonates, the phosphate-esters and the alkyl isethionates. In one embodiment of the present invention, a biological sample is exposed to an ionic detergent selected from the group consisting of sodium-dodecly-sulphate (SDS), 3 [(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), lithium 20 dodecylsulphate, sodium cholate, sodium lauroylsarcosine and cetyltrimethylammonium bromide (CTAB). The biological sample may be exposed to an ionic detergent at any concentration appropriate to assist in achieving solubilization, such as concentrations between 0.1 % (w/v) to 10 % (w/v). For example, the concentration of the ionic detergent may be in the 25 range of 0.1 to 1%, 0.5 to 2%, 1% to 5 %, 2.5 to 7
.
5 % or 5 to 10% (w/v). It will be understood to a person skilled in the art that the type of ionic detergent and final concentration of ionic detergent required to solubilize the biological sample will be influenced by multiple factors, including the amount of biological sample, the suitable liquid chosen, the solubility profile of the detergent, the type of biological sample and the 30 use of adjunct agents or methods to assist solubilization. For example, solubilizing a biological sample that is a tissue sample comprising tumour tissue and tumour-associated connective tissue may require a higher concentration of ionic detergent than solubilizing a biological sample that is a tissue sample comprising tumour tissue with no tumour associated connective tissue. Alternatively, solubilizing a small biological sample in a relatively large volume may require a lower concentration of ionic detergent than solubilizing a large biological sample in a relatively small volume. Furthermore, 5 solubilizing a biological sample with a strong detergent, such as SDS, may require a lower concentration of ionic detergent than solubilizing a biological sample with a weaker detergent, such as sodium lauroylsarcosine. In a preferred embodiment of the present invention, the biological sample is exposed to SDS at a concentration of 0.1 % (w/v) to 10 % (w/v). In a further preferred 10 embodiment of the present invention, the concentration of the SDS is in the range of 0.5 % (w/v) to 1.5 % (w/v). For example, the concentration of the SDS may be in the range of 0.1 to 0.5%, 0.5 to 1%, 0.5 to 0.75%, 0.75% to 1.25, or 1% to 1. 5% (w/v) The method of the present invention comprises exposing the biological sample to a reducing agent. A reducing agent in the context of the present invention is understood to is mean a compound that is capable of reducing disulphide bonds within and between proteins and polypeptides. The skilled addressee will understand that exposing the biological sample to a reducing agent will typically result in a biological sample comprising proteins and polypeptides with reduced disulphide bonds, as well as proteins and polypeptides with disulphide bonds that have not been reduced. The biological 20 sample may, for example, be exposed to the reducing agent at the same time as the biological sample is exposed to the ionic detergent or after the biological sample is exposed to the ionic detergent. In one embodiment of the present invention, the reducing agent is selected from the group consisting of 2-mercaptoethanol, 2-mercaptoethanolamine, cysteine-HCl, 25 dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), tributylphosphine (TBP) and iodoacetamide. In one embodiment of the present invention, the reducing agent is TCEP. In another embodiment, the reducing agent is DTT. In one embodiment, the biological sample is exposed to the reducing agent at a concentration ranging from 1mM to 500mM. For example, the concentration may be 30 1mM to 10 mM, 5mM to 20mM, 10mM to 50mM, 25mM to 100mM, 75mM to 250mM, 200mM to 300mM or 250mM to 500mM. The skilled addressee will understand that the type of reducing agent and concentration of reducing agent used to solubilize the biological sample will typically depend on multiple factors including the type of biological sample, the amount of biological sample, the buffering strength and pH of the suitable liquid chosen, and the strength of the reducing agent. For example, solubilizing a biological sample with a strong reducing agent, such as TCEP, may require a lower 5 concentration of reducing agent than solubilizing a biological sample with a weaker reducing agent, such as DTT. Furthermore, solubilizing a biological sample which is a tissue sample comprising a disulphide-rich connective tissue may require a higher concentration of reducing agent than solubilizing a biological sample which is a blood sample comprising red blood cells, that have less disulphide bonds. 10 In one embodiment, the biological sample is additionally exposed to an alkylating agent. Exposing a biological sample that comprises proteins and polypeptides, which have previously been exposed to a reducing agent, to an alkylating agent may modify some or all of the reduced cysteine residues in proteins and polypeptides by the addition of an alkyl group. This may maintain the oxidised state of some or all of the cysteine is residues and may, for example, prevent the cysteine residues from forming or reforming a disulphide bond with other cysteine residues. Non-limiting examples of alkylating reagents that may be used in the methods of the present invention are iodoacetamide, acrylamide, 4-vinylpyridine, N-ethylmalemide and derivatives thereof. The skilled addressee will understand that the type of alkylating agent and concentration used will 20 typically depend on the reducing agent used, as well as the same multiple factors considered when determining the type of reducing agent and concentration of reducing agent used. For example, a biological sample that has been exposed to the reducing agent may be exposed to an alkylating agent at a concentration that is a molar excess over the concentration of the reducing agent in the biological sample. In another example, a 25 biological sample that has been exposed to the reducing agent TCEP at a concentration of 5mM may then be exposed to iodoacetamide at a concentration of 10 m, whereas a biological sample that has been exposed to the reducing agent DTT at a concentration of 5mM may then be exposed to iodoacetamide at the higher concentration of 15mM, because DTT is known to react with alkylating agents. 30 The methods of the present invention also comprise exposing the biological sample to a non-mammalian polypeptide capable of binding a mammalian protein.
The non-mammalian polypeptide can be any exogenous polypeptide, derived from a non-mammalian organism. A person of skill in the art will understand that, in this instance, an exogenous polypeptide is any polypeptide that is not present in the biological sample when it is taken from the subject. The non-mammalian organism may, for 5 example, be a eukaryotic or prokaryotic, including but not limited to, birds, reptiles, fish, amphibians, bacteria, yeast, virus or fungus. In this context, it will be understood that "derived from a non-mammalian organism" does not require the polypeptide to be a naturally occurring polypeptide that has been extracted from the non-mammalian organism. The polypeptide may be synthetic, recombinant or extracted from an organism. 10 The polypeptide may be a variant of a naturally occurring polypeptide, such as a fragment thereof, or a sequence variant, having the capability of binding a mammalian protein. The polypeptide may be of any appropriate length, such as, for example, 10 to 1000 amino acids. For example, the polypeptide may comprise 10 to 100 amino acids, 50 to 500 amino acids, 25 to 150 amino acids, 250 to 750 amino acids, 500 to 100 amino acids, is 400 to 800 amino acids, 500 to 750 amino acids, 750 to 900 amino acids, or 850 to 1000 amino acids. Many non-mammalian organisms express polypeptides that are capable of binding mammalian proteins. Within the cell, these polypeptides may be utilized as, for example, receptors for cell-cell attachment, cell adhesion, docking and/or communication. A non 20 mammalian polypeptide may, for example, bind a mammalian protein non-specifically through intermolecular interactions, or specifically through mechanisms that rely on specific binding motifs and/or specific receptor-ligand interactions. In particular, microbes display cell-surface polypeptides capable of binding mammalian proteins. Non-limiting examples of categories of these polypeptides are the 25 lectins, the adhesins and the hemagglutinins. These polypeptides can act as receptors for attachment to ligands associated with mammalian cells. In one embodiment of the present invention, the non-mammalian polypeptide is a bacterial lectin or adhesin. In one embodiment of the present invention, the non-mammalian polypeptide capable of binding a mammalian protein is a polypeptide with a tripeptide Arginine 30 Glycine-Aspartic acid (RGD) motif, or RGD-like motif. The RDG-like motif may be a tripeptide Arginine-Tyrosine-Aspartic acid (RYD) motif. As an illustrative example, the RGD and RGD-like motifs are found in many prokaryotic and eukaryotic adhesionrelated proteins. In another embodiment of the present invention, the polypeptide with the RGD or RGD-like motif is streptavidin, avidin or Neutravidin. Streptavidin, avidin and Neutravidin have very similar properties, which each commonly known to, at least, bind biotin with high affinity. Neutravidin is a deglycosylated version of avidin, but may be 5 used interchangeably with avidin or streptavidin. In one embodiment of the method of the present invention, the biological sample is exposed to an ionic detergent prior to exposing the biological sample to a reducing agent, and a non-mammalian polypeptide capable of binding a mammalian polypeptide. In another embodiment of the method present invention, the biological sample is 10 exposed to an ionic detergent and a reducing agent prior to exposing the biological sample to a non-mammalian polypeptide capable of binding a mammalian polypeptide. In one embodiment of the present invention, the method further comprises exposing a solubilized biological sample, which has been exposed to a polypeptide with an RGD or RGD-like motif, to biotin. The biotin may be synthetic or extracted from an organism. It is is believed that the biotin binds to the proteins, carbohydrates and lipids of the solubilized biological sample. Not wishing to be bound by theory, this may result in increased binding between the polypeptide with an RGD or RGD-like motif and the proteins, carbohydrates and lipids of the solubilized biological sample, because biotin binds to RGD and RGD-like motifs. This may assist in presenting the proteins, carbohydrates and 20 lipids of the solubilized components of the cancer cells or cancer-associated cells to the immune system of the subject, which may enhance the immune response. In a further preferred embodiment, the method of the present invention further comprises a solvent precipitation of the solubilized biological sample. Solvent precipitation comprises adding an appropriate volume of a solvent to a sample. In a 25 typical solvent precipitation, approximately 1 to 10 volumes of solvent is added to the sample. By "volume/s" is meant a volume equivalent to that of the sample that is the subject of the solvent precipitation. For example, if the sample is 500 1d, then 2 volumes would be 1000 1d. After adding the solvent, the final volume would be 1500 1d. The solvent precipitation step may comprise adding 1 to 2 volumes, 1 to 4 volumes, 2.5 30 volumes, 2 to 6 volumes, 5 volumes, 4 to 8 volumes, 7.5 volumes or 5 to 10 volumes. In one embodiment of the present invention, the solvent is a polar organic solvent. In a further embodiment, the solvent is selected from the group consisting of ethanol, methanol, acetone, isopropanol, propanol and dimethylformamide (DMF). In a one embodiment, the solvent is acetone. The skilled addressee will understand that the type and volume of solvent to be added will typically be determined by multiple factors including the solvent properties, the concentration of the material to be precipitated and 5 the liquid that the material is in, and the volatility of the solvent. Adding a solvent to a heterogeneous mixture of proteins, carbohydrates and lipids may result in some or all of these molecules forming insoluble aggregates due to the exposure of hydrophobic regions. This insoluble material may be harvested as a precipitate and the precipitate resuspended in any suitable liquid. The precipitate may be harvested by any appropriate 10 method, such as by centrifugation, filtration or sedimentation. It is hypothesized that performing a solvent precipitation may enhance the immunogenicity of the resultant vaccine by making the proteins and carbohydrates appear even more foreign to the immune system. It also provides a simple means of concentrating the vaccine and removing the detergent and reducing agent from the vaccine prior to formulation into a 15 medicament. In one embodiment of the present invention, the method further comprises partitioning the solubilized biological sample into a soluble fraction and an insoluble fraction at any time prior to solvent precipitation. The insoluble fraction may be discarded. For example, if the solubilized biological sample is partitioned prior to 20 exposure of the biological sample to a reducing agent, only the soluble fraction need be exposed to the reducing agent and the non-mammalian polypeptide capable of binding the mammalian protein. The skilled addressee will understand that any appropriate method for partitioning the soluble and insoluble fractions may be used. For example, the soluble and insoluble 25 fractions may be partitioned by centrifugation, filtration or sedimentation. The partitioned fractions may be separated by a physical barrier or may be present in the same container. For example, the solubilized biological sample may be centrifuged to produce a pellet comprising the insoluble fraction and a liquid phase comprising the soluble fraction, but the fractions may be present in the same container in which the solubilized 30 biological sample was centrifuged in. The soluble fraction may be transferred to another container resulting in the fractions being separated by a physical barrier.
The method of the present invention results in a heterologous composition which typically may include denatured, partially denatured and non-denatured proteins, lipids, carbohydrates and nucleic acids, any or all of which may elicit immune responses. The heterologous mixture may contain proteins, lipids, carbohydrates and nucleic acids from 5 cancer cells, as well from cancer-associated cells, such as, for example, non-cancer blood cells and cells from non-cancerous tissues. Not wishing to be bound by theory, it is hypothesized that the methods of the present invention result in a solubilized biological sample with modified proteins, lipids, carbohydrates and nucleic acids that appear foreign to the immune system and elicit an immune response. 10 The inventors believe that exposing the biological sample to a non-mammalian polypeptide capable of binding a mammalian protein during the method of the present invention aids in the efficacy of the vaccine because the polypeptide is exogenous, which will be recognized as foreign by the subject's immune system and stimulate the immune response. It is suggested here that this may facilitate the presentation of the components is of the cancer cells or cancer-associated cells to the immune system, as well as assist in making the components appear more foreign to the immune system, which may enhance the immune response. A particular embodiment of the present invention provides a method of producing a vaccine for the treatment or prevention of cancer comprising exposing a biological 20 sample comprising at least one cancer cell or cancer-associated cell to an ionic detergent in a suitable liquid to produce a solubilized biological sample comprising soluble material and insoluble material, followed by partitioning the soluble and insoluble material of the solubilized biological sample to produce a soluble fraction and an insoluble fraction. The resulting soluble fraction is exposed to a reducing agent, and then exposed to a non 25 mammalian polypeptide capable of binding a mammalian protein. This mixture is solvent precipitated and the precipitate is resuspended in a suitable liquid. In one embodiment of this method, the non-mammalian polypeptide is streptavidin, avidin or Neutravidin. In an alternative embodiment, the partitioning of the solubilized biological fraction is performed after the biological sample is exposed to the ionic detergent and the reducing 30 agent. In a further embodiment, the method also comprises the step of exposing the soluble fraction to biotin prior to solvent precipitation. In yet another embodiment, the method also comprises exposing the soluble fraction to an alkylating agent prior to solvent precipitation.
In embodiments of the present invention, the methods described herein permit the production of a therapeutic product of human origin which satisfies the requirements of for exclusion from regulation by the Australian Therapeutic Goods Administration (TGA) of goods manufactured and used in medical practice. The TGA is part of the Australian 5 government Department of Health and Human Aging and is responsible for regulating medicines and medical devices. Under the relevant provisions, human cells and tissues or therapeutic goods manufactured therefrom, may be excluded from the requirement of inclusion in the Australian Register of Therapeutic Goods (ARTG) and from compliance with TGA legislation. The provision applies to human cells and tissues that are collected 10 from a patient who is under the clinical care and treatment of a medical practitioner registered under a law of a State or an internal Territory; and manufactured by that medical practitioner, or by a person or persons under the professional supervision of that medical practitioner, for therapeutic application of a single indication and in a single course of treatment of that patient by the same medical practitioner, or by a person or 15 persons under the professional supervision of the same medical practitioner. The relevant provision thus requires that the product is for autologous use only. Consistent with those requirements, the present invention provides a method for the treatment or prevention of cancer in a human subject, the method comprising the steps of obtaining a biological sample comprising at least one cancer cell or cancer-associated cell 20 from said subject, exposing the biological sample to an ionic detergent, a reducing agent, and a non-mammalian polypeptide capable of binding a mammalian protein, to produce a vaccine comprising a solubilized biological sample comprising components from said cancer cell or cancer-associated cell, and a non-mammalian polypeptide capable of binding a mammalian protein, and administering a therapeutically effective amount of 25 said vaccine to said subject, wherein all steps of the method are performed by or under the supervision of a registered medical practitioner having prime responsibility for the clinical care of said subject throughout said method. In an embodiment, the method for treatment or prevention is a course of treatment or prevention comprising multiple steps of administering said vaccine to said patient. 30 In a further embodiment, one or more step(s) of the method is conducted by a person or persons under the supervision of said medical practitioner. In an embodiment the collective steps of the method are performed by a plurality of individuals.
In an embodiment, the collective steps of the method are performed at multiple locations. In one embodiment, the step of obtaining a biological sample from said subject is conducted at a different location to said exposing step. In other embodiments, the method for the treatment or prevention of cancer in a 5 human subject further comprises additional steps described herein for the production of said vaccine, or the production of a pharmaceutical composition comprising said vaccine. Compositions, Vaccines and Medicaments The present invention provides vaccines for the treatment or prevention of cancer produced by any of the previously discussed methods. The vaccines may also be used 10 for the manufacture of other medicaments for treatment of prevention of cancer. In another embodiment, the invention also provides pharmaceutical compositions comprising the vaccine of the present invention. The pharmaceutical composition, vaccines and medicaments of the present invention comprise at least solubilized and reduced components of a cancer cell or 15 cancer-associated cell and a non-mammalian polypeptide capable of binding a mammalian protein. The pharmaceutical compositions, vaccines and medicaments of the present invention may further comprise a pharmaceutically acceptable carrier, adjuvant, excipient and/or diluents. For preparing the pharmaceutical compositions, vaccines and 20 medicaments, inert, pharmaceutically acceptable carriers can be either solid or liquid. Liquid form preparations include solutions, suspensions and emulsions, for example water or water-propylene glycol solutions for parenteral injection. Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for either oral or injection administration. Such liquid forms include 25 solutions, suspensions and emulsions. Examples of pharmaceutically acceptable carriers and methods of manufacture for various compositions may be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pennsylvania. The carriers, diluents, excipients and adjuvants must be "acceptable" in terms of 30 being compatible with the other ingredients of the composition, vaccine or medicament, and are generally not deleterious to the subject thereof. Non-limiting examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil; sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile 5 silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxylpropylmethylcellulose; lower alkanols, for example ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, 10 propylene glycol, 1,3- butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from about 10% to about 99.9% by weight of the composition, vaccine or medicament. The pharmaceutical compositions, vaccines and medicaments of the present is invention may be in a form suitable for administration by injection (e.g. for parenteral administration including subcutaneous, intramuscular or intravenous injection) or by oral administration (such as capsules, tablets, caplets, and elixirs, for example). For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered 20 saline, ethanol and 1,2 propylene glycol. Methods for preparing parenterally administrable pharmaceutical compositions, vaccines and medicaments are apparent to those of ordinary skill in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. For oral administration, some examples of suitable carriers, diluents, excipients and 25 adjuvants include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl stearate which delay 30 disintegration. Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.
Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, 5 carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring 10 agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include is magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene 20 glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof. Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate 25 or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono-or di-oleate, -stearate or laurate, polyoxyethylene sorbitan mono-or di-oleate, -stearate or-laurate and the like. Supplementary active ingredients such as adjuvants or biological response modifiers can also be incorporated into the pharmaceutical compositions, vaccines and 30 medicaments of the present invention. Any suitable adjuvant may be included in the pharmaceutical compositions, vaccines and medicaments of the present invention. For example, an aluminium-based adjuvant may be utilised. Suitable aluminium-based adjuvants include, but are not limited to, aluminium hydroxide, aluminium phosphate and combinations thereof. Other specific examples of aluminium-based adjuvants that may be utilised are described in European Patent No. 1216053 and US Patent No. 6,372,223. Other suitable adjuvants include 5 Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminium salts such as aluminium hydroxide gel (alum) or aluminium phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized 10 polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; oil in water emulsions including those described in European Patent No. 0399843, US Patent No. 7,029,678 and PCT Publication No. WO 2007/006939; and/or additional cytokines, such as GM-CSF or interleukin-2, -7, or -12, granulocyte macrophage colony-stimulating factor (GM-CSF), monophosphoryl lipid A (MPL), is cholera toxin (CT) or its constituent subunit, heat labile enterotoxin (LT) or its constituent subunit, toll-like receptor ligand adjuvants such as lipopolysaccharide (LPS) and derivatives thereof (e.g. monophosphoryl lipid A and 3-Deacylated monophosphoryl lipid A), muramyl dipeptide (MDP) and F protein of Respiratory Syncytial Virus (RSV). Dosages and routes of administration 20 The pharmaceutical compositions, vaccines and medicaments of the present invention can be administered to a subject by standard routes including, but not limited to, injection and oral. In some embodiments, they may be administered to a subject in isolation or in combination with other additional therapeutic agent(s). In such embodiments the administration may be simultaneous or sequential. 25 The pharmaceutical compositions, vaccines and medicaments of this invention may also be delivered by intramuscular, subcutaneous and/or intradermal injections. They may be delivered by injection near a lymph node or by injection directly into a tumour. In general, the pharmaceutical compositions, vaccines and medicaments of the present invention can be administered in a manner compatible with the route of 30 administration and physical characteristics of the subject (including health status) and in such a way that the desired effect(s) are induced (i.e. therapeutically effective, immunogenic and/or protective). For example, the appropriate dosage may depend on a variety of factors including, but not limited to, a subject's physical characteristics (e.g. age, weight, sex), whether the composition, vaccine or medicament is being used as single agent or adjuvant therapy, the progression (i.e. pathological state) of the cancer being treated, and other factors readily apparent to those of ordinary skill in the art. 5 Various general considerations when determining an appropriate dosage of compositions, vaccines and medicaments are described, for example, in Gennaro et al. (Eds), (1990), "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, Pennsylvania, USA; and Gilman et al., (Eds), (1990), "Goodman And Gilman's: The Pharmacological Bases of Therapeutics", Pergamon Press. 10 Typically, in treatment applications, the treatment may be for the duration of the cancer. Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages can be determined by the nature and extent of the disease state or condition being treated, the form, route and site of administration, and the nature of the particular subject being treated. Optimum dosages can be determined is using conventional techniques. Some embodiments of the present invention may involve administration of the pharmaceutical composition, vaccine or medicament in multiple, separate doses. Accordingly, the methods for treatment described herein encompass the administration of multiple separated doses to a subject, for example, over a defined period of time. 20 Accordingly, in some embodiments the methods include administering a priming dose, which may be followed by a booster dose. The booster may be for the purpose of re vaccination. In various embodiments, the pharmaceutical composition, vaccine or medicament is administered at least once, twice, three times or more. The pharmaceutical compositions, vaccines and medicaments of this invention may 25 also be useful in combination (administered together or sequentially) with one or more of anti-cancer treatments such as radiation therapy, and/or one or more anti-cancer agents selected from the group consisting of cytostatic agents, cytotoxic agents (such as for example, but not limited to, DNA interactive agents (such as cisplatin or doxorubicin)); taxanes (e.g. taxotere, taxol); topoisomerase Il inhibitors (such as etoposide); 30 topoisomerase I inhibitors (such as irinotecan (or CPT-1 1), camptostar, or topotecan); tubulin interacting agents (such as paclitaxel, docetaxel or the epothilones); hormonal agents (such as tamoxifen); thymidilate synthase inhibitors (such as 5-fluorouracil); antimetabolites (such as methoxtrexate); alkylating agents (such as temozolomide
(TEMODAR(
TM
) from Schering-Plough Corporation, Kenilworth, New Jersey), cyclophosphamide); Farnesyl protein transferase inhibitors (such as, SARASAR(
TM
)(4 [2-[4-[(11 R)-3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1 ,2 5 b]pyridin-11-yl-]-1-piperidinyl]-2-oxoehtyl]-1-piperidinecarboxamide, or SCH 66336 from Schering-Plough Corporation, Kenilworth, New Jersey), tipifamib (Zamestra@ or R115777 from Janssen Pharmaceuticals), L778.123 (a famesyl protein transferase inhibitor from Merck & Company, Whitehouse Station, New Jersey), BMS 214662 (a famesyl protein transferase inhibitor from Bristol-Myers Squibb Pharmaceuticals, 10 Princeton, New Jersey); signal transduction inhibitors (such as, lressa (from Astra Zeneca Pharmaceuticals, England), Tarceva (EGFR kinase inhibitors), antibodies to EGFR (e.g., C225), GLEEVEC(
TM
) (C-abl kinase inhibitor from Novartis Pharmaceuticals, East Hanover, New Jersey); interferons such as, for example, intron (from Schering-Plough Corporation), Peg-lntron (from Schering-Plough Corporation); hormonal therapy 15 combinations; aromatase combinations; ara-C, adriamycin, Cytoxan, and gemcitabine. Subjects The subject is any individual in respect of which any of the methods of treatment or vaccine production or administration are performed. Typically, the subject is an individual having cancer and is under the clinical care of a medical practitioner. The 20 subject may be the same individual from which the biological sample comprising the cancer cells was obtained. The subject may be human or may be a non-human such that reference to a subject or individual means a human or a non-human, such as an individual of any species of social, economic or research importance including but not limited to members of the classifications of ovine, bovine, equine, porcine, feline, canine, primates, 25 rodents, especially domesticated members of those classifications, such as sheep, cattle, horses and dogs. It will be appreciated by persons of ordinary skill in the art that numerous variations and/or modifications can be made to the present invention as disclosed in the specific embodiments without departing from the spirit or scope of the present invention 30 as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
The present invention will now be described with reference to specific examples, which should not be construed as in any way limiting. Examples Example 1 - Preliminary Vaccine Trials and Dosing studies 5 Materials and Methods Cell culture Rat glioma cells (9L) cell were cultured in Basal Medium Eagle (BME) supplemented with 10% (v/v) foetal calf serum and 0.03% (w/v) L-glutamine until approximately 90% confluent. The cells were washed in PBS, trypsinized and collected 10 from the flask. The cells were then washed once with serum free BME, counted and resuspended at a concentration of 1Ox106 cells per ml for injection into rats. Induction and biotin perfusion of tumours for vaccine production Rats were injected with 1x106 9L glioma cells (100 pl) under the skin in the flank and a tumour allowed to establish. Once the tumour reached approximately lcm 3 the rats is were perfused with biotin according to the following method: Rats were anaesthetised and once asleep taped down, and the belly and chest area shaved. An incision was made under the sternum so the heart was visible and 0.5ml of heparin was directly injected into the heart to prevent clotting. A blunt perfusion needle was then inserted into the right atrium of the heart and main blood supply below the heart 20 clamped and cut below. Saline was the pumped through the rat for 20 mins. After this 60 ml of buffer containing 0.05M Tris, 0.15M NaCl, pH 7.6 was pumped through the rat followed by 20 mg of Biotin-ss (Thermo) in PBS. Once the biotin-PBS had passed through, the rat was flushed through with a further 60 ml of buffer containing 0.05M Tris, 0. 15M NaCl, pH 7.6. Biotin perfused tumours were then removed from the animal and 25 later used to prepare the vaccine. Induction of tumours in a donor rat An initial donor rat was injected with 1x10 6 9L glioma cells under the skin in the flank and a tumour allowed to establish. Once a tumour reached a size of approximately 0.5cm 3 , the rat was euthanized and the tumour aseptically removed. The tumour was then 30 chopped up finely to obtain small pieces of tumour approximately 1mm x 1mm in size.
These tumour pieces were kept on ice in serum-free BME media until rats were ready for implantation. Rechallenging rats in the vaccine trial For flank rechallenge experiments, rats were anesthetised and injected with 1x106 5 91 Glioma cells in the flank. For brain rechallenge experiments, a tumour from a donor rat was harvested and cut into small approximately 1mm pieces and stored cold in serum free BME until use. Rats had a small hole drilled in the left side of brain and a small piece of tumour implanted and sealed with bone wax. 10 Rats were monitored for recovery and treated with xylocaine at the site tumour implantation. Rats with brain tumour implants were monitored daily for signs of distress, Initial vaccine trial Every 1 gram of perfused tumour was homogenised and solubilised in 40ml of 0.05M Tris, 0.15M NaCl and 1%SDS buffer (ph7.6) with a protease inhibitor (Roche). is Tumour lysate was then spun down at 10,000 rpm for 30 mins at room temperature. The supernatant was collected and pellet discarded. Supernatant was then run over a pre equilibrated streptavidin (TRIS-NACL-SDS) column (Thermo scientific) at 2 parts supematant:1 part column, and allowed to incubate for 1 hr. The column was washed with 5 x column buffer followed by elution with 1 column volume of Tris-Nacl-SDS 20 buffer with 50mM DTT (incubated for 1 h). 2ml of eluted vaccine proteins were precipitated overnight with 20mls of ice cold acetone and incubated overnight at -20'C. The follow day samples were spun down at 10,000 rpm for 30 mins and supernatant discarded. Pellet was allowed to dry and resuspended in 200pl sterile PBS. Each 200pl batch was used as an individual vaccine for each rat and mixed 1:1 with 25 Freund's Incomplete Adjuvant. Rats received vaccine or FIA with PBS intraperitoneally (i.p), then received a booster shot after 3 weeks. They were then challenged with 1x10 6 9L cells in the flank and this was termed day zero. Animals had tumours measured by calipurs 3 times per week. Tumour size was measured by the equation (width 2X Length)/2. = cm 3 . 30 Vaccine dosing trial Vaccines were prepared by the same method as the initial vaccine trial. A control group of rats (n=8) received 2 vaccinations of PBS/FIA i.p. Vaccine groups of rats were given either 1 (n=8), 2 (n=9) or 3 (n=9) doses of vaccine i.p. A fourth vaccine group of 5 rats (n=8) received 2 doses of vaccine subcutaneously. All groups were challenged with 1x106 9L glioma cells in the flank 2 weeks after the last vaccination. Tumour rechallenge trial Rats from group 1 or 2 (above) that survived the vaccine dosing trial (N=9 total) were split into 2 groups. Group 1 (N=4) were rechallenged with 1x10 6 9L glioma cells in 10 the flank along with untreated controls (n=10). Group 2 (n=5) received a small piece of tumour in the brain along with untreated controls (n=6). Calculation of survival rates and survival times For analysis of the results, a cured rat was one in which the tumour resolved and disappeared. Cure rates were defined by how many per group were cured (e.g. 6/10 = 15 60%). Survival rates were defined in days to euthanasia. For ethical reasons rats were euthanized once the tumours reached an approximate size of 13.5 cm 3 . The mean survival time (days) was calculated for each group. Cured rats are assigned a value of 100 days. Survival curves were plotted to measure for significance between groups. 20 Results Rats in the initial vaccine trial received two vaccinations prior to being rechallenged with 9L cells in the flank (Figure 1A). They showed no adverse effects to the vaccination other than a few lumps which developed at the vaccination site. Average tumour progression time to endpoint in control rats was 35 days, whereas only one rat in the 25 vaccine treated group reached cut off at day 48. The other 2 rats developed tumours however these resolved over time and by approximately day 58 had disappeared. These two rats were termed 'cured' and given a survival time of 100 days and were kept for rechallenge. There was a significant survival advantage in the vaccinated group compared to adjuvant alone (Figure 1 B). 30 After the success of the initial vaccine trial, dosing studies were performed to see if 1, 2 or 3 shots (i.p.) of vaccine was optimal. Remission rates were highest in rats 31 receiving 2 doses compared to 1 or 3 doses (Figure 2). Both 2 and 3 doses gave a significant extended survival time when compared to control, but a single dose did not. There was no extension of mean survival time between 2 doses of vaccine i.p compared to 2 doses of vaccine S.c. Rats which went into remission in the vaccine dosing trial (n=9) were split into 2 groups and rechallenged in the either the brain (n=-4) or the flank (n=5) (Figure 2 E and F). All of these rats showed complete immunity to tumour progression when challenged in both the brain and the flank, suggesting acquired immunity and that the immune system may have been working across the blood brain barrier. Preliminary analysis of vaccines Vaccines used and serum collected from rats in the initial vaccine trials were analysed by SDS-PAGE and western blotting. It was shown that western blot analysis of the vaccines using the serum from the vaccinated rats produced a common 5 or 6 bands between 50 to 75Kda, which were later proven to be fragments of streptavidin (Figure 3A). Experiments using different columns to make the vaccines demonstrated that varying amounts of streptavidin was leeching off the columns. A sample of unperfused tumour lysate was purified using a streptavidin column to produce the complex banding pattern seen in Figure 3B. This suggested that streptavidin may have been selecting the vaccine proteins, and could possibly be doing so by RYD or RGD sites on the tumour proteins. Additional experiments using up to 10% SDS in the tumour extraction buffer still yielded a similar complex vaccine profile when unperfused tumour lysates were purified using a streptavidin column, which suggested a high level of affinity between the tumour proteins and streptavidin, and motivated further analysis. Example 2 - Vaccine components trials Materials and Methods Preparation and vaccination - Streptavidin (50ug) only vaccine A streptavidin vaccine was prepared by solubilising 300 tg streptavidin (Calbiochem) in buffer containing 1% SDS (w/v), 0.05M Tris, 0.15M NaCl ph 7.6. The soluble streptavidin was precipitated overnight with 1 ml acetone at -20'C. The next day, the sample was spun @ 10,000rpm for 30 mins to pellet the precipitate. The precipitated t1Al2797R 1hGGGC 32 streptavidin was then resuspended in 6 00 pl of PBS and mixed with 600pl of FIA (Sigma) for vaccination of 6 rats (0.2 ml per vaccination). Preparation and vaccination - reduced tumour protein vaccine Sections of 6 different induced 9L Glioma tumours were collected, weighed (1 gram) and homogenized in 40 ml of buffer containing 1% SDS (w/v), 0.05M Tris, 0.15M NaCl ph 7.6, and protease inhibitor (Roche). The tumour lysate was spun down at 10,000 rpm for 30 min and the soluble tumour lysate collected. The proteins in 2 ml of this lysate were reduced by adding 20mM TCEP (Sigma) for 2 hours and then precipitated by adding 40ml of acetone and incubating overnight at -20 0 C. The next day, the sample was spun down to precipitate the proteins at 10,000 rpm for 30 mins. The precipitate was resuspended in 1.2 ml of PBS and mixed with 1.2ml of FIA (Sigma) for vaccination of 6 rats (0.3 ml per vaccination). Preparation and vaccination - reduced tumour protein + streptavidin (50,ug) vaccine 2ml of tumour lysate, prepared as per the reduced tumour protein vaccine above, was mixed with 300 tg of streptavidin (Calbiochem) and incubated for another 2 hours, before being precipitated overnight with 40 ml of acetone at -20'C. The next day, the sample was spun down to precipitate the proteins at 10,000 rpm for 30 mins. The precipitate was then resuspended in 1.2 ml of PBS, and mixed with 1.2 ml of FIA (Sigma) for vaccination of 6 rats (0.3 ml per vaccination). A high streptavidin dose vaccine (reduced tumour protein + streptavidin (100p1g) vaccine) was made by the same method as above, but 600ptg of streptavidin was added to 2 ml of the reduced lysate. Control rats received FIA/PBS in a 300pl dose (n=5). All groups received a secondary vaccination 3 weeks later. Streptavidin ELISA The reactivity to streptavidin of serum collected from rats involved in the vaccine components trial was measured via ELISA. Streptavidin (Calbiochem) was coated on ELISA plates (NUNC) at a concentration of 10 gg/ml in 0.1 M NaHCO 3 overnight at 4'C. Plates were blocked the next day in 3% BSA in PBS for 1 hr at 37'C. Rat serum was diluted 1:1000 in 1% BSA/PBS and incubated on plate at 37'C for 1 hr. The plate was then washed 4 times with PBS/0.05 % tween and then plate incubated with a goat anti-rat 11A22Q-7QRZ 1).rGG 33 HRP antibody (Sigma) at a 1:2000 dilution in 1% BSA/PBS for 1 hour at 37'C. Plates were washed again and then substrate added for 10 minutes before being stopped. Absorbance was read at 480 nm. Cytokine analysis Cytokine analysis of serum collected from rats in the vaccine components trial was performed initially using 2 broad screening methods. The rat cytokine bioplex (BioRad) and the Rat proteome profilerTM array (R and D systems) were used according to manufactures instructions for initial rat serum screens to sample a wide range of cytokines. Rat serum samples were also screened using ELISA for rat C-Reactive protein (BD), CINC-2 (R and D systems), ICAM (R and D systems), IL-4(R and D systems), TNF-a (R and D Systems), INF-7 (Bender systems) according to the manufactures instructions. Flow Cytometric Blood assay To assess the levels of Natural Killer (NK), T4, T8, B-cell, Lymphocyte, neutrophil and monocyte levels in the peripheral blood of rats in the vaccine components trial, a flow cytometric assay was developed. A sample of blood from test rats was collected into a 0.5ml EDTA tube to prevent clotting. For each test, 25 tl of blood was added to a TrucountTM tube (BD Pharmingen) and then stained with rat T/B/NK cell cocktail (BD Pharmingen), rat CD8a PE, rat CD4 (domain 1) FITC and rat CD45 PE/Cy7 (Biolegend) for 15 minutes at room temperature. Samples were then lysed using 10mM Tris and ammonium chloride buffer (pH 7.4). Multiple cell populations were analysed using the following gating strategy. All cell subsets were gated as CD45 positive, monocytes, neutrophils and lymphocytes were then analysed by FSC v SSC. T4 cells CD45/CD3/CD4 positive, T8 cells CD45/CD3/CD8 positive, NK cells CD3/CD161a positive and B Cells CD3/CD45 RA positive. Cell numbers per 1d were then calculated by the equation (Cell number/25l) x (Bead number/Bead count). Results In the vaccine components trial, controls rats treated with FIA/PBS survived an average 38 days after being rechallenged. As can be seen in Figure 4, the reduced tumour protein vaccine group average survival time was 43. This was only 5 days more than that the control rats, but this was significant in terms of survival. The streptavidin (50pg) only vaccine group survived an average of 15 days longer than control rats and showed a significant extended survival on curve. The reduced tumour protein + streptavidin (50pg) vaccine induced remission in 2 out of 6 rats. The average survival time in this group was double that of the control rats, and an average 25 days longer survival time than the next 5 best group (streptavidin only). Increasing the dose of streptavidin (100pg) in the reduced tumour protein + streptavidin vaccine decreased the mean survival time dramatically and nullified any remissions. The streptavidin reactivity and cytokine analysis data collected from the vaccine 10 components trial is summarised in Figure 5. Serum antibodies to streptavidin were not evident in any of the reduced tumour protein vaccine groups (see Figure 5A). Interestingly, rats vaccinated with streptavidin alone showed an increase in streptavidin reactivity post tumour challenge (see Figure 5B). Specifically, the streptavidin (50pg) only vaccine group showed an upregulation of 15 cytokines, IL-Ib, IL-13, TNF-a, MIP-3a and VEGF, while M-CSF was down regulated in this group compared to controls. TNF-a, MIP3a and IFN-y showed significant down or up regulation in all groups compared to controls. In cured rats (n=2), TNF-a was upregulated when compared to controls, while IFN-y and MIP-3a were down regulated compared to controls. The cytokine ICAM was down regulated in both the streptavidin 20 (50pg) only vaccine group and the reduced tumour protein + streptavidin (50pg) vaccine group compared to controls, whereas CINC-1 was not significantly elevated in either group. ICAM was significantly reduced at 21 days post tumour challenge in rats treated with any vaccine comprising tumour proteins. Overall, cytokine analysis on serum samples collected at day 20 of the vaccine 25 components trials showed no significant difference between the control groups and the reduced tumour protein vaccine groups (Figures 5C - 5F). In general, C reactive protein levels increased in all groups post tumour challenge but there was no significant difference in levels between groups (see Figure 5C). The cytokine CINC-1 was not significantly different between groups 3 weeks post tumour 30 challenge (see Figure 5D). However, ICAM levels were significantly lower in vaccine treated rats compared to control (Figure 5E). TNF-a levels in vaccine treated rats were significantly increased only in the vaccine treated group 3 weeks post tumour challenge. However, the TNF-a had disappeared in these vaccinated rats 2 weeks later (Figure 6 F). A multicolour flow cytometry assay was developed to analyse multiple peripheral blood cell types in a small peripheral blood sample. The assay detected the number of 5 NK, T4, T8, B Cell, Lymphocytes, monocytes and neutrophils per microliter of blood. Adjuvant only and reduced tumour protein + streptavidin vaccine treated rats had their blood tested one week after secondary vaccination (Pre tumour) and 3 weeks post tumour challenge (Post) (see Figure 6). Significant differences in NK cell levels were seen pre and post-tumour challenge in both adjuvant treated and vaccine treated groups. T4 cells 10 were significantly increased in vaccine treated rats both pre- and post-tumour challenge, but both groups T4 levels dropped significantly post-tumour challenge. The same was seen with T8 cells. Lymphocyte levels were maintained in vaccine treated rats pre- and post tumour challenge however were significantly reduced in the adjuvant only group post-tumour challenge. B cells showed elevation compared to base line levels in both 15 groups, but dropped significantly post tumour challenge. B cell levels returned to normal baseline levels in vaccine treated rats indicating that the response to the tumour may have been cell mediated. Vaccination caused a spike in monocyte levels, which again decreased to normal levels in vaccine treated rats but remained elevated in control groups. NLR was at baseline levels after vaccination but increased significantly after tumour 20 challenge, which is typically seen as a tumours progress. Example 3 - Autologous Vaccine Preparation Materials and Methods Preparation of solubilized biological samples Fresh, healthy pieces of surgically removed tumour tissue (0.1 g of each) were 25 homogenised in buffer containing 1% SDS (v/w), 0.05M Tris, 0.15M NaCl, ph 7.6 and then clarified by centrifugation. For every 0.lg of tumour, 4ml of buffer was added. 1ml fractions of tumour protein extract were reduced for lhr with 20 mM TCEP or 50mM DTT in the 1% SDS buffer. 150pg of streptavidin was then added to each, before incubating for 2hrs with gentle mixing. 30 Vaccine Preparation After incubation the tumour protein-streptavidin mixtures were then precipitated overnight with 5 tolO volumes of ice cold acetone at -20'C. These mixtures were then centrifuged at 10,000 x g for 30 min, before decanting the acetone and allowing the 5 pellets to air-dry. The dry pellets were resuspended in 600 pl of sterile PBS and mixed with 600 pl FIA and emulsified using a 19G needle. The vaccine preparations were divided into three equal doses and subjects vaccinated subcutaneously with single doses at 0 and 3 weeks, followed by assessment of survival in comparison with control, unvaccinated subjects. 10 Results The vaccinated subjects showed an increased mean length of survival of 67 days, while the control group demonstrated a mean length of survival of 38 days (Figure 7). Example 4 - Dog Safety Trial with Autologous Vaccine Materials and Methods 15 Fresh tumour samples comprising cancer cells were obtained by either biopsy of tumour or complete removal of the tumour. Tumour samples were frozen at -20 'C until processing. On day of processing the tumour sample was weighed. For every 0.1 gram of tumour 4mls of buffer containing of 1% SDS (v/w), 0.05M Tris, 0.15M NaCl, ph 7.6 was added, in addition to a protease inhibitor (Roche). The tissue samples were then 20 homogenised and filtered through a sieve to remove larger fibrous material. The resulting lysate was centrifuged at 10,000 rpm for 15 min and the soluble fraction retained. TCEP was added to give a final concentration of 20mM TCEP and the sample then incubated for 1 h at room temperature before adding 100 pl of Biotin-NHS (1mg/ml concentration). After incubation at room temperature for 2 hours, 100 pl of 1 25 mg/ml streptavidin (Sigma) was then added and the mixture incubated for 2 h. The lysate was then precipitated by the addition of at least 5 volumes of cold acetone and an overnight incubation at -20'C. The precipitate was harvested by centrifugation at 10,000 rpm for 30 min at 4'C, the supernatant decanted and the pellet allowed to air-dry until the acetone had evaporated.
37 The pellet was then resuspended in 600 pd of PBS and split into 2 x 0.3 ml aliquots (for 2 vaccinations), before freezing at -20'C. On the day of vaccination, one aliquot of vaccine was thawed and mixed with an equal volume of FIA, and administered subcutaneously. This was repeated 3 weeks later. Results The autologous vaccine prepared for the dog safety trial was analysed by SDS-PAGE and western blot (see Figure 8). The dog safety trial was commenced in March 2011. Despite many of the animals entering the trial in ill-health, there were no adverse reactions to the vaccine other than a localised inflammation at the vaccination site. The results of the trial are presented in Table 1. The vaccine has proven to be safe to deliver which on a wide range of chemotherapies, steroids and other drugs with no adverse reactions (Table 1). It has also been demonstrated to be safe in different breeds and over 10 different tumour types. Table 1: Survival in Dog Safety trial with Autologous Vaccine. AGE/ BREED Tumour Mitotic Expected Actual Other Comments SEX Type/ index survival survival Medications Grade 12 yo Staffodshire Mast cell 18/10 3-4m 3.5 m Prednisone Steroid treatment Male Bull Terrier stage 3 would nullify effect of vaccine 9 yo Staffodshire Haemangio- 6/10 23 months No reoccurance of Male Bull terrier sarcoma (ongoing) haemagiosarcoma, but did present with mast cell tumour 11 months after vaccinations. 12yo Rottweiler Ostero- 12 months 18 months Carboplatin No metastatic disease female Sarcoma (ongoing) prior to or reoccurrence grade 2 vaccine (amputation) 13 yo Kelpie Sarcoma 24 months No reoccurrence or female (liver) (ongoing) metastatic disease 14 yo Labrador Haemangio- 8 weeks Inoperable tumour. male sarcoma Vaccine made from small sample. 12 yo Adeno female Carcinoma (nose) 10 yo German Osteosarcoma < 3 months 9 months Carboplatin Combined female Spitz grade 3 (metastatic and carboplatin and (amputation) disease) vaccinations vaccine safe. Chest mets I Survived longer than ll100OD"A 1bCCCr expected- chest mets. 1 yo Alaskan Osteosacroma 12 months Carboplatin No reoccurrence or riale malamute grade 2 (ongoing) and metastatic disease (amputation) vaccinations 4ax CHOP CHOP relapse: Vaccine made filly Labrador (Biopsy) ,eonar CE yo Cross breed Soft tissue Vaccine after male sarcoma radiation 9 yo Jack Russell Melanoma Progressive disease (oral mass) yo American Melanoma staffordshire 6 yo Silky terrier Melanoma (oral) talph Melanoma 4 yo Golden Retriever yo Cavadoodle yo Sharpei 2 yo Rottweiler 3yo German Connective 4/10 3 months Oral No reoccurrence or /Iale Shepherd tissue sarcoma (ongoing) carboplatin metastatic disease (re-occuting) (prior to vaccine) 1 yo English Lymphoma 3 months Vincrsitine No reoccurrence or riale Setter (re-occuring) (on going) injections and metastatic disease cyclophospha miide 3 yo Labrador Haemangio- 3 weeks Complete removal of bnale sarcoma spleen. Died of (stage 3) internal bleeding spleen before finishing vaccine course 6 yo Rottweiller Malignant 18/10 2 weeks COPD: Pretreated with male cross Lymphoma Doxorubicin COPD protocol, VIncristine however had Cyclophospha relapsed. Vaccinated mide as last resort. Mitrozantrone SASH Labrador Metastatic Lymph node 15 yo Golden Malignant 2/10 2 months No reoccurrence or Female Retriever Melanoma metastatic disease (cheek) 8 yo Miniature Broncho- 15/10 1.5 months No reoccurrence or female Schauzer alveolar metastatic disease carcinoma 13 yo Kelpie cross Mast cell < 4 months 1.5 months No reoccurrence or female tumour predicted metastatic disease High grade 3 10 yo Siberian Connective 5/10 4 weeks No reoccurrence or female Husky tissue sarcoma metastatic disease (grade 2)

Claims (32)

1. A method for producing a vaccine for the treatment or prevention of cancer, the method comprising exposing a biological sample comprising at least one cancer cell or cancer-associated cell to an ionic detergent, a reducing agent, and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein, to produce a composition comprising a solubilized biological sample comprising components from said cancer cell or cancer-associated cell and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein, solvent precipitating said composition, followed by resuspension of the resulting precipitate in a suitable liquid, wherein said vaccine comprises said resuspended precipitate.
2. The method of claim I wherein the biological sample is from the subject intended to receive the vaccine.
3. The method of any one of the preceding claims wherein the ionic detergent is selected from the group consisting of sodium-dodecly-sulphate (SDS), 3-[(3 Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), lithium dodecylsulphate, sodium cholate, sodium lauroylsarcosine and cetyltrimethylammonium bromide (CTAB).
4. The method of any one of the preceding claims wherein the ionic detergent is SDS.
5. The method of claim 4 wherein the biological sample is exposed to SDS at a concentration of 0.5 to 1.5 % (w/v).
6. The method of any one of the preceding claims wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, 2-mercaptoethanolamine, cysteine-HCl, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), tributylphosphine (TBP) and iodoacetamide .
7. The method of any one of the preceding claims wherein the reducing agent is TCEP orDTT. (10229926 1:GGG 40
8. The method of claim 7 wherein the biological sample is exposed to TCEP or DTT at a concentration of 1 mM to 100 mM.
9. The method of any one of claims 1 to 8, wherein the method further comprises exposing said solubilized biological sample to biotin.
10. The method of any one of claims 1 to 9, wherein the method further comprises exposing the biological sample to an alkylating reagent.
11. The method of claim 1 wherein the solvent is a polar organic solvent.
12. The method of claim 11 wherein the polar organic solvent is selected from the group consisting of ethanol, methanol, acetone, isopropanol, propanol and dimethylformamide.
13. The method of claim 12, wherein the polar organic solvent is acetone.
14. A method for producing a vaccine for the treatment or prevention of cancer, the method comprising the steps of: a) exposing a biological sample comprising at least one cancer cell or cancer associated cell to an ionic detergent in a suitable liquid to produce a solubilized biological sample comprising soluble material and insoluble material; b) partitioning the soluble and insoluble material of the solubilized biological sample to produce a soluble fraction and an insoluble fraction; c) exposing the soluble fraction to a reducing agent; d) exposing the soluble fraction to streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein; e) performing a solvent precipitation of the soluble fraction; and 41 f) resuspending the precipitate in a suitable liquid wherein said vaccine comprises said resuspended precipitate.
15. A method for producing a vaccine for the treatment or prevention of cancer, the method comprising the steps of: a) exposing a biological sample comprising at least one cancer cell or cancer associated cell to an ionic detergent and a reducing agent in a suitable liquid to produce a solubilized biological sample comprising soluble material and insoluble material; b) partitioning the soluble and insoluble material of the solubilized biological sample to produce a soluble fraction and an insoluble fraction; c) exposing the soluble fraction to streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein; d) performing a solvent precipitation of the soluble fraction; and e) resuspending the precipitate in a suitable liquid wherein said vaccine comprises said resuspended precipitate.
16. The method of any one of claims 14 or 15, wherein the method further comprises the step of exposing said soluble fraction to biotin at any stage prior to performing said solvent precipitation of the soluble fraction.
17. The method of any one of claims 14 or 15, wherein the method further comprises the step of exposing said soluble fraction to an alkylating reagent at any stage prior to performing said solvent precipitation of the soluble fraction.
18. The method according to claim 14 or 15, wherein the method comprises one or more of (i) the ionic detergent is SDS; or (ii) the biological sample is exposed to SDS at a (1fIAOO2r 2 1.rcCr 42 concentration of 0.5 to 1.5 % (w/v); or (iii) the reducing agent is TCEP or DTT; or (iv) the biological sample is exposed to TCEP or DTT at a concentration of 1 mM to 100 mM; or (v) the solvent is acetone.
19. The method of any one of claims 1 to 18, wherein the method is performed by a medical practitioner or by a person or persons under the supervision of a medical practitioner, or by a combination thereof.
20. A vaccine produced by the method of any one of claims 1 to 19.
21. A vaccine for the treatment or prevention of cancer in a subject, said vaccine comprising solubilized and reduced components of a cancer cell or cancer-associated cell, and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein.
22. A vaccine for the treatment or prevention of cancer in a subject, said vaccine comprising solubilized, reduced and alkylated components of a cancer cell or cancer associated cell, and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein.
23. The vaccine of any one of claims 21 or 22, wherein the cancer cell or cancer associated cell is a cancer cell or cancer-associated cell from said subject.
24. The vaccine of any one of claims 21 to 23, further comprising biotin.
25. A pharmaceutical composition for the treatment or prevention of cancer comprising the vaccine of any one of claims 20 to 24, and a pharmaceutically acceptable carrier.
26. The use of the pharmaceutical composition of claim 25 for the treatment or prevention of cancer in a subject.
27. A method for the treatment or prevention of cancer in a human subject, the method comprising the steps of obtaining a biological sample comprising at least one cancer cell or cancer-associated cell from said subject, exposing the biological sample to an ionic (192492 1(GC 43 detergent, a reducing agent, and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein, to produce a composition comprising a solubilized biological sample comprising components from said cancer cell or cancer associated cell and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein, solvent precipitating said composition, followed by resuspension of the resulting precipitate in a suitable liquid to produce a vaccine, and administering a therapeutically effective amount of said vaccine to said subject.
28. The method according to claim 27, wherein all steps of the method are performed by or under the supervision of a registered medical practitioner having prime responsibility for the clinical care of said subject throughout said method.
29. Use of a composition comprising solubilized and reduced components of a biological sample of at least one cancer cell or cancer-associated cell and streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein, for the manufacture of a medicament for the treatment or prevention of cancer.
30. The method according to claim 1 or 27, wherein said exposing the biological sample to a reducing agent is performed before, after or simultaneously with said exposing the biological sample to an ionic detergent.
31. The method according to claim 1 or 27, wherein said exposing the biological sample or components thereof to streptavidin or a fragment or variant thereof having the capability of binding a mammalian protein is performed after said exposing the biological sample to an ionic detergent and to a reducing agent.
32. The vaccine according to any one of claims 21 to 24, the pharmaceutical composition according to claim 25, or the method according to claim 27, wherein said vaccine or pharmaceutical composition further comprises, or wherein said method further comprises use of, one or more adjuvant(s), optionally wherein the adjuvant is FIA. Northern Sydney Local Health District Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON
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US14/655,088 US10357538B2 (en) 2012-12-24 2013-12-24 Vaccines for the treatment of cancer and compositions for enhancing vaccine efficacy
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WO2005001480A1 (en) * 2003-06-27 2005-01-06 Proteome Systems Intellectual Property Pty Ltd Method of isolating a protein

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