Patent Application: US-87569807-A

Abstract:
compositions suitable for use as adjuvants in the preparation of vaccines , particularly those vaccines useful in the treatment of cancer , are provided . methods for inhibiting tumor growth in an animal are also disclosed . methods for immunizing an animal against cancer , such as prostate cancer , are also described . the adjuvants described are comprised of an extracellular matrix material , such as small intestinal submucosal tissue . the preparations may take the form of sheets , gels , liquids , trocar , or other solid or semi - solid preparation . the invention provides for enhanced tumor inhibition of 2 - fold or greater , compared to vaccine preparations without the extracellular matrix material , or from 4 - to 5 - fold , compared to preparations without the adjuvant promoting extracellular materials .

Description:
it is advantageous to define several terms before describing the invention . it should be appreciated that the following definitions are used throughout this application . where the definition of terms departs from the commonly used meaning of the term , applicant intends to utilize the definitions provided below , unless specifically indicated . for the purposes of the present invention , the term “ adjuvant ” is defined as a substance which enhances the immune response to an immunogen . for purposes of the present invention , the term , “ adjuvancy ” is defined as the ability of an agent to enhance and / or promote the immune response of animal to a particular antigen . for the purposes of the present invention , the term “ biosynthetic material ” is defined as a material that is in part or whole made up from or derived from a biological tissue . for purposes of the present invention , the term “ biological tissue ” is defined as an animal tissue , including human , or plant tissue that is or that once was ( cadaver tissue , for example ) part of a living tissue or organism . for the purposes of the present invention , the term “ extracellular matrix ” ( hereinafter “ ecm ”) is defined as a tissue derived or bio - synthetic material that is capable of supporting the growth of a cell or culture of cells . by way of examples , some particular ecms include sis , rcm and fem . for the purposes of the present invention , the term “ cancer vaccine ” is defined as any preparation capable of being used as an inoculation material or as part of an inoculation material , that will provide a treatment for , inhibit and / or convey immunity to cancer and / or tumor growth . for the purposes of the present invention , the term “ immunize ” is defined as eliciting an immune response in an animal , both a humoral immune response and a cellular immune response . for the purposes of the present invention , the term “ immune provoking amount ” is defined as an amount of the antigen required to elicit an immune response in the animal . for purposes of the present invention , the term “ facial extracellular matrix ” ( hereinafter “ fem ”) relates to ecm derived from the fascia of porcine or other sources . for purposes of the present invention , the term “ renal capsule material ” ( hereinafter rcm ), relates to ecm derived from the renal capsule of porcine or other sources . the description of the present invention is enhanced by the various examples that follow . the present example provides some examples of materials and methods that may be used in the practice of the present invention . small intestinal submucosa ( sis ) was obtained from cook biotech , inc . ( west lafayette , ind .). the material was provided as a sterile , lyophilized sheet of extracellular matrix . experimental grade material was provided for use in the present studies of an sis preparation that was described as having been prepared by harvesting porcine jejunum and placing 10 - to 20 - cm lengths into saline solution ( 31 - 33 ). following removal of all mesenteric tissues , the jejunal segment was everted and the tunica mucosa abraded using a longitudinal wiping motion with a scalpel handle and moistened gauze . the serosa and tunica muscularis were then gently removed using the same procedure . the remaining tissue was disinfected with peracetic acid , rinsed extensively in high purity water , and sterilized using ethylene oxide prior to implantation . rcm was obtained from cook biotech , inc . ( west lafayette , ind .). briefly , renal capsule was dissected from mature pig kidneys immediately following slaughter . it was thoroughly rinsed under running tap water and disinfected using a dilute solution of peracetic acid in ethanol to remove potential contaminating bacteria and viruses ( 34 ). following disinfection , the rcm was rinsed in high purity water to remove the acid , lyophilized into a sheet form , and subsequently sterilized prior to implantation using ethylene oxide gas . the paiii cell line was derived from an autochthonous prostate tumor of an lw rat . paiii cells have been transplanted into lw rats for many passages with no change in pattern of growth or disease . when paiii cells are transplanted subcutaneously into the flank of lw rats , large , metastasizing adenocarcinomas develop within 40 days , though initial tumors are palpable within 10 days . from the primary tumor , the paiii cells metastasize spontaneously to the lungs . paiii tumors are hormone - independent and refractory to most treatments ( 35 ). gft cell vaccine was a glutaraldehyde - fixed tumor ( gft ) suspension of cells harvested from tumors grown in animals . gft cell vaccine was prepared from tumor tissue ( 36 ). specifically , three grams of a subcutaneous tumor tissue was harvested from a lobund - wistar rat and used in the vaccine preparation . the subcutaneous tumor had been produced by administering prostate adenocarcinoma cells isolated from an autochthonous , metastatic prostate adenocarcinoma in a lw rat ( 37 ). the tissue was finely minced , repeatedly aspirated with a 1 cc syringe , and an aliquot drawn with a 20 - gauge needle to eliminate large aggregates to create a cell suspension in modified eagle &# 39 ; s medium ( mem ). the cell suspension was incubated in 2 . 5 % glutaraldehyde ( v / v ) at 37 ° c . for 120 minutes and then washed thoroughly with media to produce the gft cell preparation . lw rats obtained from a breeding colony maintained at the university of notre dame were used for all studies . in this model , large tumors develop subcutaneously following subcutaneous administration of 1 × 10 6 paiii cells in approximately 99 % of rats . in this model , male , 3 - 4 month old lw rats are administered 1 × 10 6 paiii cells subcutaneously into the flank . after 14 - 21 days , a palpable tumor is present , and by 40 days metastatic foci are present in the lungs . for studies involving resection , the animal is prepared for aseptic surgery . the visible tumor is resected , though the resection is not radical and sufficient tumor bed presumably remains , as tumor re - growth occurs in 100 % of untreated individuals . sheets of single - layer sis or rcm are cut into 2 × 2 cm sections and placed into modified eagle &# 39 ; s medium ( mem ). paiii cells ( 1 × 10 6 ) or cells ( 1 × 10 6 ) harvested directly from a paiii subcutaneous rat tumor are layered on the sis or rcm and incubated at 37 ° c . to create the gft cell vaccine on sis , the sis with attached cells then undergoes glutaraldehyde fixation ( gft ) and washing . glutaraldehyde fixation involves incubating cells in 2 . 5 % glutaraldehyde ( v / v ) for 60 min at 37 ° c ., and then washing with media . alum was purchased as alhydrogel ™, an aluminum hydroxide gel adjuvant ( brenntak biosector , frederikssund , denmark ). statistical analysis — results of survival versus non - survival following challenge with tetanus toxin were compared between groups using the chi - square test with two degrees of freedom . differences were considered significant when p ≦ 0 . 05 . results for mean tumor weight were compared between groups with the wilcoxon rank sum test with significance reached when p ≦ 0 . 05 . the present example demonstrates the utility of the present invention as an effective cancer vaccine adjuvant in vivo . tumor cells were cultured on sis . following three days of growth , the sis with attached cells were fixed with glutaraldehyde . subcutaneous tumors grown in the flank of lobund - wistar rats which had been administered paiii prostate cancer cells 10 days earlier were surgically resected . groups of 5 rats then underwent either no further treatment ; treatment with glutaraldehyde - fixed tumor ( gft ) cells applied directly on the tumor bed ; treatment with glutaraldehyde - fixed ( gf ) sis ( without cells ) applied on the tumor bed ; or treatment with glutaraldehyde - fixed sis ( with cells ) applied on the tumor bed . three weeks later , after tumors had re - grown in most rats , tumors were weighed with the following results : the addition of sis to the gft cell vaccine resulted in a greater than 50 % reduction in mean tumor weight and establishes that sis is an effective adjuvant for cancer ( anti - tumor ) vaccination . the present example demonstrates the utility of the invention for providing a method for expanding a cancer cell population on an extracellular matrix material . the present example also demonstrates the utility of the invention for preparing a highly immunogenic population of cells useful in a cancer vaccine preparation . in the case of cancer , it is likely that many key antigens are expressed by connective tissue matrix and involve interactions of neoplastic cells with the extracellular matrix . cancer cell vaccines grown on an extracellular matrix thus may be prepared according to the present example and used as improved vaccine antigen compositions for vaccination . the present example demonstrates another example of the type of extracellular matrix material that may be used in the practice of the present invention . the present example employs porcine fascia extracellular matrix material ( fem ). studies were conducted as described herein to examine the ability of tumor cells to grow on fem . in these studies , it was demonstrated that tumor cells did grow robustly on the fem material , comparable with that growth supported on the sis and rcm . 2 . expansion of prostate cancer cells on sis and rcm in culture previous investigators have demonstrated the ability of pure cell lines to grow on sis in vitro . for example , badylak et al ( 38 ) showed sis is capable of supporting cultures of nih swiss mouse 3t3 fibroblasts , primary human fibroblasts , keratinocytes , endothelial cells , and an established rat osteosarcoma cell line . the present example demonstrates that an extracellular matrix material preparation as describe herein from sis supports cancer cell growth . in particular , growth of a prostate cancer cell line and a mixed cell population harvested directly from a subcutaneous tumor ( the tumor having been produced by inoculation of rat paiii cells into a lobund - wistar ( lw ) rat ), are shown to grow on the ecm materials under the conditions described here . sheets of single - layer sis and rcm were cut into 2 × 2 cm sections and placed into modified eagle &# 39 ; s medium ( mem ). paiii cells ( 1 × 10 6 ), or cells harvested directly from a paiii subcutaneous rat tumor ( 1 × 10 6 ), were layered on the sis and incubated at 37 ° c . for 72 hours , then fixed in 10 % neutral buffered formalin for 24 h , washed in 70 % ethanol , placed in paraffin and sectioned at 4 - 5 μm . sections were then stained with hematoxylin and eosin stain and examined for cell growth . samples which were incubated with pure paiii cells demonstrated a monolayer of cell growth along the edges of sis and rcm ( fig2 ). in contrast , culture of cells harvested directly from tumors showed growth of cells along the edges of sis and rcm . in addition , in the midsubstance ; vascular structures were re - populated with cells ( fig3 ) compared to control sis which had undergone incubation in media but with no cells added ( fig4 ) of badylak et al ( 38 ) showed that rat osteosarcoma cells and endothelial cells grew only on the edge of the ecm , while fibroblasts populated the ecm midsubstance . when co - cultured , keratinocytes and fibroblasts resulted in a distinct spatial orientation of the two cell types and early epidermal structures were formed . this study demonstrated that prostate cancer cells and mixed cell populations harvested directly from tumors can be grown in culture on three types ( fem , sis and rcm ) of ecm . sis as a vaccine adjuvant to prevent regrowth of tumors following surgical resection in earlier work , the present inventors described the ability of glutaraldehyde - fixed tumor ( gft ) cells harvested directly from a paiii rat tumor to prevent prostate cancer ( 36 ). based upon this , the present example demonstrates that vaccination will inhibit the regrowth of tumors following surgical resection . the present example demonstrates that an sis / whole cell vaccine effectively inhibits tumor regrowth following surgical resection and debulking . studies utilized the lobund - wistar ( lw ) rat prostate cancer model which can be used to induce de novo prostate tumors by chemical induction , or it can be used to grow subcutaneous tumors following implantation of a prostate cancer cell line ( paiii cells ). using the latter system , paiii cells were administered subcutaneously to groups of lw rats . fourteen days after administration of paiii cells , tumors were surgically debulked and vaccines applied as follows : vaccine was prepared by allowing tumor cells harvested from a subcutaneous tumor to grow upon sis in culture for 3 days , after which the material underwent glutaraldehyde fixation ( gft ) and washing ( gft vaccine on sis ). glutaraldehyde fixation involves incubating cells in 2 . 5 % glutaraldehyde ( v / v ) for 60 min at 37 ° c ., and then washing with media . one group of 5 rats underwent only resection ; one group had gft cell vaccine applied to the tumor bed ; one group had sis applied to the tumor bed ; and one group had gft cell vaccine on sis applied to the tumor bed . the results in terms of mean tumor re - growth ( tumor weight in grams ± standard deviation ) after 3 weeks are shown in fig5 and were as follows : gft cell vaccine on sis : 3 . 98 ± 0 . 1 . 37 gm , 2 / 5 with lung metastases the tumors in rats vaccinated with the gft cell vaccine on sis were significantly smaller ( p ≦ 0 . 01 ) than those from rats vaccinated with the gft cell vaccine alone and the control groups . in a second study , cells were cultured on sis for 28 days before implantation . the results from this study are shown in fig6 and are as follows : thus , the result is repeatable and demonstrates that the gft vaccine on sis also inhibited metastasis from the primary tumor to the lungs . these data were not quite significant ( probability of 0 . 053 ) due to the small group size . these data support the idea that efficacy of cancer vaccines is improved by growth of vaccine cells on , or incorporation into , extracellular matrices such as sis . sis gel acts as an adjuvant for a vaccine to prevent cancer because implantation of vaccines incorporated onto solid sis matrix would require incision of tissue , it may not be practical for all applications . thus , the present example demonstrates the utility of the invention to provide a vaccine against cancer in a gel form using an extracellular matrix material , such as sis , and the use of same as a vaccine adjuvant . sis gel is supplied by cook biotech , inc . ( west lafayette , ind .) and is produced from sis material via an acid digestion and purification process . sis gel was diluted 1 : 10 with sterile saline . harvested , glutaraldehyde - fixed cells from paiii tumors were mixed into the sis gel dilution such that each 0 . 25 ml dose of sis gel contained 5 × 10 6 gft cells . groups of ten ( 10 ) male lw rats were administered subcutaneously the following : 0 . 25 ml of sterile saline containing 5 × 10 6 gft cells ; or rats were vaccinated 3 times , 7 days apart . seven days after the last vaccination , all rats were challenged subcutaneously with 1 × 10 6 paiii cells . three weeks after challenge with paiii cells , rats were euthanized and tumors weighed . the results are shown as mean tumor weights (± standard deviation ) in fig7 and are as follows : gft cell vaccine = 0 . 86 g (± 0 . 11 ), 6 / 10 rats with metastases to the lungs gft cell vaccine in sis gel = 0 . 19 (± 0 . 14 ), 1 / 10 rats with metastases to the lungs as can be seen in fig7 , treatment with the gft cells alone resulted in a tumor size of approximately 0 . 86 g .+/− 0 . 11 g ., while treatment with gft cells in the extracellular matrix material ( sis ) in a gel form resulted in a tumor growth of approximately 0 . 19 g .+/− 0 . 14 g ., about one - fourth the size . hence , the addition of the extracellular matrix gel ( sis ) in a 1 : 10 dilution significantly adjuvinated the tumor growth inhibiting activity of the gft cell preparation ( fixed prostate cell vaccine antigen ) about 4 - fold to about 5 - fold . thus , it is demonstrated here that the addition of an extracellular matrix material to a cell - based cancer vaccine will significantly adjuvant a tumor cell preparation used as a vaccine , by 2 - fold or greater . sis gel acts as a vaccine adjuvant for the treatment of cancer the present example demonstrates the utility of the present invention for providing an enhancement of immunity effective both as a preventive measure and as a therapeutic measure . in the present example , groups of six rats were challenged subcutaneously with 1 × 10 6 paiii cells to create tumors . animals were vaccinated 3 times , 7 days apart ; rats underwent surgical resection of tumors ten days after challenge , three days after the first vaccination . an additional group was included in which animals were vaccinated by subcutaneous implantation of gft cell vaccine on a sheet of sis . animals were euthanized 21 days after tumor resection and tumors weighed . the results from this study are shown in fig8 and are summarized as : these studies demonstrate that sis gel has vaccine adjuvant activity and can enhance protective immunity to cancer both before cancer cell challenge and as an adjunct to surgical resection . this means that sis gel enhances immunity effective as a preventative measure ( i . e ., as a vaccine ), and as a therapeutic measure ( fig9 ). the present example demonstrates the utility of the present invention as a clinically acceptable preparation for animal , including human , treatment . in particular , the present example demonstrates that the preparations do not induce tissue damage , and does not result in autoimmune disease . both the gft cell vaccine and sis are safe to use in vivo . the present example demonstrates that repeated administration of the gft cell vaccine failed to induce histopathologic or clinical disease in rats . in addition , the present example demonstrates that sis did not promote tumor growth in vivo , and further demonstrated inherent inhibition of tumor growth in the lw rat tumor model . furthermore , sis is already approved by the u . s . food and drug administration as a medical device for a variety of applications . the present study demonstrates that repeated vaccination with the present preparations does not result in histological evidence of autoimmune disease . groups of 10 three - month - old lw rats were each immunized and boosted monthly for 12 months with either mem or gft cells . freund &# 39 ; s complete adjuvant was used for the initial vaccination , and freund &# 39 ; s incomplete adjuvant was used for booster vaccinations . tissues were then harvested at 15 months of age , fixed in 10 % neutral buffered formalin , sectioned at 3 - 4 μm and stained with hematoxylin and eosin . all rats were clinically normal for the duration of the study . kidney , heart , brain , liver , testis , prostate / seminal vesicle , and spleen were examined and all found to be histologically normal . these results demonstrate that repeated immunization with the gft cell vaccines does not induce tissue damage suggestive of autoimmunity . sis does not promote growth of tumor tissue when placed in vivo the present example demonstrates the utility of the extracellular matrix material preparations as providing an anti - tumor activity with a tumor / cancer cell preparation . the present example also demonstrates that the present preparations do not themselves induce tumor and / or cancer growth . because cancer cells showed an ability to grow on sis and rcm in vitro , it is important to determine if an ecm , such as sis , would promote the growth of residual tumor cells if placed on the bed of a resected tumor in vivo . to evaluate this , groups of 25 male lw rats , age 3 months , underwent induction of subcutaneous paiii tumors as described above . animals were then assigned to one of four different treatment groups : sham surgery control ; physical encasement of the tumor with sis ( tumor was not dissected from the underlying vascular bed ); complete tumor resection ( all grossly visible tumor was removed ); or complete tumor resection followed by overlying the resected tumor bed with sis ( approximately 3 × 3 cm ). three weeks later , rats were euthanized and the tumors weighed . the results ( fig9 ) show that sis did not promote growth of paiii tumors compared to sham surgery or resection alone . overlying of the resected tumor bed with sis led to a significant ( p ≦ 0 . 0009 ) decrease in tumor size versus resection alone ( 39 ). the sis alone , with no cells involved , had a significant - tumor effect . in culture , cancer cell lines and cancer tissue from harvested tumor material both grow rapidly on sis and rcm . when inactivated by glutaraldehyde fixation , cancer cells and tissue grown on sis prevent regrowth of tumors following surgical resection . this effect is observed when cells are grown on sis , and also when glutaraldehyde - fixed tumor cells are mixed into a gel form of sis . furthermore , sis gel is shown to act as a vaccine adjuvant to prevent the development of cancer ; that is , to stimulate protective immunity to challenge with live paiii cells . ecm materials , as demonstrated by sis and rcm , both initiate a robust inflammatory response when implanted in vivo . while not intending to be limited to any particular theory or mechanism of action , it is believed that any antigenic moieties carried along , whether adhered to the ecm or present in a gel or a particulate suspension , will be processed by the immune system , thus possibly accounting for at least one theory by which the ecm may act as a vaccine adjuvant . it is known that sis contains a variety of bioactive species , including tgf - β ( 41 ). while tgf - β can act as a tumor promoter in later stages of tumor progression , it functions as a tumor suppressor in early tumorigenesis ( 42 ). thus , administered at the proper time , such as following resection , the utility discovered herein for sis to inhibit tumor growth may be utilized . the present example is provided to demonstrate the utility of the present vaccines in sis for providing a treatment for cancer and / or to reduce / inhibit tumor growth by use of sis in a sheet - like preparation . while vaccines based on an extracellular matrix have not been described , use of a prostate cancer vaccine comprised of inactivated allogeneic whole prostate cancer cell lines has been described ( michael , et al )( 2005 )( 47 ). in that study , monthly intradermal injections for 12 months of 8 × 10 6 inactivated whole cells were administered , the first two in a standard adjuvant , alum , to patients with hormone - resistant prostate cancer . the adjuvant used in the first two doses administered was bacilli calmette - guerin . the first three doses were given at weekly intervals , and once a month thereafter . this approach led to statistically significant declines in psa ( prostate - specific antigen ) velocity with no evidence of toxicity . further , median time to a defined point of disease progression was increased to 58 weeks from approximately 28 weeks . a whole cell prostate cancer vaccine together with a preparation of the extracellular matrix adjuvant ( diluted 1 × 10 from a commercial preparation , such as that commercially available from a vendor such as cook biotech , inc .) would be used according to the present invention under a clinical regimen wherein the vaccine would be administered intradermally or subcutaneously on a monthly basis for approximately 12 months . vaccine preparations which can be easily injected , such as those including sis gel or a particulate form of sis as adjuvant would be administered by percutaneous injection . a vaccine preparation which includes vaccine fixed on a sheet of sis would be administered either percutaneously by trochar into the subcutaneous space or , in other embodiments , by implantation via a small incision made into the skin . few studies have looked at the utility of vaccination in conjunction with surgical resection of a tumor . pilla et al ( 2006 )( 49 ) administered subcutaneously tumor - derived heat shock protein gp 96 - peptide complex vaccine to advanced stage melanoma patients for up to four treatments , two weeks apart , following surgical resection . that approach resulted in stabilization of disease in 11 / 18 patients post - surgically . berd et al ( 1997 )( 50 ) administered an inactivated autologous whole cell vaccine on a weekly or monthly schedule to melanoma patients with clinically evident lymph node metastases ; this approach resulted in survival rates superior to those resulting from surgery alone . while no studies have looked at the utility of vaccination directly on the tumor bed of a resected prostate tumor , nor the utility of a vaccine incorporated onto a solid - phase adjuvant such as an extracellular matrix , the present examples demonstrate specific clinical use applications of the vaccine . some embodiments of the present invention will provide the vaccine incorporated onto a sheet of extracellular matrix , and will be applied as a sheet directly onto the resected tumor bed at the time of surgery ; or administered intradermally or subcutaneously at a site beyond the tumor bed on a monthly basis . a similar approach used with a different vaccine is described by berd et al ( 1997 ) ( 50 ) using a whole cell vaccine for the treatment of melanoma . in other embodiments , a combination approach may be used in which vaccination is made directly onto the tumor bed , and is applied at the time of resection followed by booster vaccinations given intradermally or subcutaneously . the sheet vaccine would be administered percutaneously by trochar into the subcutaneous space or , possibly , by implantation via a small incision made into the skin . vaccine preparations which can be easily injected , such as those including sis gel or a particulate form of sis as adjuvant , would be administered by direct application of the material onto the tumor bed and / or intradermally or subcutaneously by injection . bell et al . ( 2005 ) ( 65 ). the present example demonstrates the utility of the invention for providing a dermally - applicable formulation of the tissue based adjuvant cancer preparations . while transdermal vaccination has been used for diseases associated with infectious pathogens ( kenney , 2004 ( 59 ); skountzou , 2006 ( 60 ); glenn , 2006 ( 61 ), very few attempts have been made to apply this route of administration to cancer vaccines . transcutaneous immunization was used in mice by administering imiquiod , a cytotoxic t lymphocyte ( ctl ) activator , in an ointment applied to shaved skin ( rechsteiner , 2005 ( 62 )); this approach stimulated ctl activity in general and not against any specific cancer antigen . other investigators described an anti - tumor vaccine by delivery to mice of human carcinoembryonic antigen gene in an adenovirus vector via a thin film of vector placed onto the shaved skin and beneath a patch ( huang , 2005 ( 63 )). this approach resulted in immunologic resistance to challenge with murine mammary adenocarcinoma cells . according to use in the present invention , the cancer antigen of interest , such as a glutaraldehyde fixed preparation of prostate cells , may be prepared in a formulation together with a gel form of the extracellular matrix material , sis . in this formulation , the preparation may be applied to an area to provide the anti - tumor effect . sis is an effective adjuvant for vaccines based on allogeneic cell lines as shown in earlier examples , vaccines utilizing paiii prostate cancer cells or cells directly harvested from prostate tumors in lobund - wistar ( lw ) rats stimulate protective immunity in syngeneic animals . in contrast , allogeneic cells are those which are obtained from a genetically distinct individual of the same species . thus , while the paiii cell line is transplantable between all lw rats and is considered syngeneic , the mt - lu and mat - ly - lu cell lines are derived from the copenhagen rat . these latter two cell lines do not develop into tumors when transplanted into the lw rat . the rfl - 6 cell line is an allogeneic rat fibroblast line which we evaluated to determine if fibroblast antigens enhanced protective immunity against tumor regrowth following resection . groups of 6 lw rats were administered 1 × 10 6 paiii prostate cancer cells subcutaneously to generate tumors . the rats then had the subcutaneous tumors surgically resected . the animals then underwent either no further treatment ( rx ); vaccination with either glutaraldehyde - fixed ( gf ) rfl - 6 cells alone or with gf mat - lu or gf mat - ly - lu cells ; or vaccination with gf rfl - 6 alone or with gf mat - lu or gf mat - ly - lu on sis after 3 days of growth in culture . animals were vaccinated once , directly on the tumor bed . the animals were euthanized 21 days later and results are expressed in mean weight of regrown tumor ( s . d .) and number of lungs positive for metastatic foci out of the total number for the group . the mean weights of re - grown tumors 21 days following resection are in fig1 ( may - lu ) and 11 ( mat - ly - lu ). for fig1 , x = resection of tumor only ; r = resection plus vaccination with gf rfl - 6 cells ; r / s = resection plus vaccination with gf rfl - 6 cells on sis adjuvant ; mlu / r = resection plus vaccination with gf rfl - 6 cells and gf matlu cells ; and mlu / r / s = resection plus vaccination with gf rfl - 6 cells and gf matlu cells on sis adjuvant . for fig1 , x = resection of tumor only ; r = resection plus vaccination with gf rfl - 6 cells ; r / s = resection plus vaccination with gf rfl - 6 cells on sis adjuvant ; mlylu / r = resection plus vaccination with gf rfl - 6 cells and gf matlylu cells ; and mlylu / r / s = resection plus vaccination with gf rfl - 6 cells and gf matlylu cells on sis adjuvant . histologic examination of tumor samples showed chronic inflammation and fibrosis surrounding sis with a zone of acute inflammation at the border of the tumor in some rats treated with gf mat - lylu + rfl on sis in contrast to samples from other treatment groups . in those other groups , tumors were characterized by varying degrees of acute inflammation , primarily at the necrotic center of the tumor , likely due to tissue hypoxia . since the tumor grows from the border , it can be conjectured that gf may - ly - lu + rlf on sis stimulated an inflammatory response at the growing margin sufficient to interfere with tumor growth . these results show that the ecm adjuvant , sis , effectively adjuvantized a vaccine which utilized allogeneic ( mat - lu and matlylu ) cell lines as antigens . sis is an effective adjuvant for vaccines based on xenogeneic cell lines a common problem with cancer immunotherapy is the issue of immunotolerance . through a variety of mechanisms , the host immune system simply fails to effectively respond to the tumor . often , this is because the tumor is recognized as ‘ self ’. thus , antigens which are similar to tumor antigens and which are vigorously recognized as foreign would be of advantage . in this regard , cell lines from another species ( xenogeneic ) would likely be of value . according to the present invention , vaccines based on xenogeneic tumor cells will provide a robust immune response , one capable of attacking the host tumor . in this study , the utility of the human cell lines du145 ( hormone independent prostate carcinoma obtained from a metastatic lesion in the brain of a human patient ) is examined ; and lncap ( hormone dependent prostate carcinoma obtained from lymph node metastasis of a human patient ) as xenogeneic vaccine antigens . cells were grown for three days either on plastic culture vessels or on sis in culture , glutaraldehyde - fixed ( gf ), and then used in the subcutaneous tumor resection model as described above . results are given in mean tumor weight ( s . d .) and number of lungs positive for metastatic foci out of the total number of lungs . rs + gf du145 / gf imr90 on sis : 3 . 2 g ( 1 . 44 ); 1 / 6 lungs positive rs + gf lncap / gf imr90 on sis : 1 . 9 g ( 0 . 92 ); 0 / 6 lungs positive the mean weights of re - grown tumors 21 days following resection are in fig1 . these results indicate that the ecm adjuvant , sis , can serve as an effective adjuvant for xenogeneic cell cancer vaccines . ability of other extracellular matrices ( ecms ) to act as vaccine adjuvants the present example demonstrates the utility of the present invention for providing a vaccine using a variety of different cell - derived matrices . studies using paiii cells grown on the ecms , renal capsule material ( rcm ) and fascia extracellular matrix ( fem ) were conducted using the subcutaneous paiii tumor resection model in the lw rat . briefly , paiii cells were grown in culture for 7 days on either sis , rcm , or fem and then fixed in glutaraldehyde ( gf ) as described above . groups of 6 lw rats were administered 1 × 10 6 paiii prostate cancer cells subcutaneously to generate tumors . after 21 days of tumor growth in the rats , tumors were surgically excised and vaccine applied directly to the tumor bed . groups of rats either underwent no further treatment ( x ); vaccination with gf cells only ( cells ); vaccination with gf cells on sis ( cells / sis ); vaccination with gf cells on fem ; or vaccination with gf cells on rcm . the animals were euthanized 21 days later and results are expressed in mean weight of regrown tumor (± s . d .) as shown in fig1 . rats vaccinated with gf cells grown on either sis , fem , or rcm had mean tumor weights significantly less than rats which were not vaccinated or those vaccinated with gf cells without adjuvant . there were no significant differences between groups vaccinated with gf cells on sis vs . rcm vs . fem , though the group vaccinated with gf cells on rcm had a notably lower mean tumor weight than the other groups . these results demonstrate that a variety of ecms , including sis , rcm , and fem , are effective vaccine adjuvants . proposed preparation of a conditioned ecm tissue material as a cell - free vaccine preparation the present example demonstrates the utility of the present invention for providing an essentially cell - free preparation of an ecm - conditioned vaccine or vaccine adjuvant . this conditioned ecm may be used as a vaccine or vaccine adjuvant . while the current form of sis - adjuvanted cancer vaccine involves the use of inactivated cancer cells grown on , or attached to , the extracellular matrix ( ecm ) may also act as an adjuvant following detachment of such cells . such a ‘ conditioned ’ ecm preparation would reduce potential autoimmune response concerns from residual whole cell material . the conditioned ecm would comprise , for example , growth factors , secreted stromal material , and other factors , but would be essentially free of whole cells . the conditioned ecm would be produced by allowing the tumor / cancer cells to grow on a sheet of sis , for example , as described before . after a period of growth , the cells would be detached or lysed away from the sis , such as by chemical means ( such as by incubation in potassium thiocyanate ) or mechanically ( such as by exposure to ultrasound ). the growth of the cells would create the elaboration from the cells of various growth factors and additional extracellular substance material . the ecm would thus come to contain antigens that serve as targets for immune destruction of tumors . in this way , then , the cell - free conditioned ecm could be used for vaccination in the same ways as ecm with the inactivated cell component as part of the preparation . proposed combination treatments with ecm vaccine and a second active agent the present example is provided to demonstrate the utility of the invention for providing a therapy that includes an ecm vaccine preparation together with another active agent , such as a chemotherapeutic agent . it is anticipated that the inclusion of agents such as the ones named below , either alone or in combination , as well as others of the same class / biological function / biological activity will also be useful in the various applications presented here for clinical treatment . in some cases , the combination is expected to further improve the anti - cancer activity and / or effectiveness of the ecm . in some embodiments , the selected compounds may be admixed with or linked to the ecm , such as by a chemical link . a few examples of what some of these combination agents may include are provided as follows : cyclophosphamide — low dose cyclophosphamide has been shown to inhibit t - regulatory ( suppressor ) cells , thus allowing the immune system to more effectively target the tumor in response to vaccination . ( berraondo p , et al . ( 2007 ), cancer res ., 15 ; 67 ( 18 ): 8847 - 55 . )( lord r , et al . ( 2007 ), j . urol ., 177 ( 6 ): 2136 - 40 ). cytokines , such as il - 21 , have been described as modulating the immune cell population to favor cells capable of generating an effective immune response . ( li y , yee c ., ( 2007 ), blood . 2007 october 5 ), as well as the cytokine , granulocyte / macrophage colony stimulating factor ( gm - csf ). ( chang e y , et al . ( 2000 ), int j . cancer ., 86 ( 5 ): 725 - 30 ). melatonin — melatonin , a neurohormone produced mainly by the pineal gland , is a modulator of haemopoiesis and of immune cell production and function , both in vivo and in vitro . physiologically , melatonin is associated with elaboration of t - helper 1 ( th1 ) cytokines , and its administration favors th1 priming . ( miller s . c ., et al . ( 2006 ), int j exp pathol . 87 ( 3 ): 251 ), ( subramanian p , mirunalini s , dakshayani k b , pandi - perumal s r , trakht i , cardinali d p . prevention by melatonin of hepatocarincinogenesis in rats injected with n - nitrosodiethylamine . j pineal res . 2007 october ; 43 ( 3 ): 305 - 12 . 1 - methyl - tryptophan — a potential reason for failure of cancer vaccines is immune tolerance due to the immunosuppressive enzyme , indolamine - pyrrole 2 , 3 - dioxygenase ( ido ). 1 - methyl - tryptophan inhibits this enzyme . ( ou x , et al . ( 2007 ), j cancer res clin oncol ., oct . 2 , 2007 epub ). cyclooxygenase - 2 ( cox - 2 ) is a rate - limiting enzyme in the synthesis of prostaglandins . it is over - expressed in multiple cancers and has been associated with diminished tumor immunity . celecoxib is a cox - 2 inhibitor and therefore can improve the immune response to anti - cancer vaccination . ( hahn t , et al . ( 2006 ), int j cancer , 118 ( 9 ): 2220 - 31 ). cpg oligonucleotides — cpg oligodeoxynucleotides ( cpg - odns ) affect innate and adaptive immune responses , including antigen presentation , costimulatory molecule expression , dendritic cell maturation , and induction of cytokines enhancing antibody - dependent cell - mediated cytotoxicity ( adcc ). ( lubaroff dm , et al . ( 2007 ), vaccine , 24 ( 35 - 36 ): 6155 - 62 )( kochenderfer j n , et al . ( 2007 ), clin immunol ., 124 ( 2 ): 119 - 30 ). heat shock proteins — the cytosolic members of the heat shock protein 70 ( hsp - 70 ) family have been shown to elicit protective cell mediated immunity in animal tumor models hashemi s m , hassan z m , soudi s , ghazanfari t , kheirandish m , shahabi s . evaluation of anti - tumor effects of tumor cell lysate enriched by hsp - 70 against fibrosarcoma tumor in balb / c mice . int immunopharmacol . 2007 july ; 7 ( 7 ): 920 - 7 . heat shock proteins might either be added to an ecm adjuvant or expression of heat shock proteins induced by cells grown upon an ecm . the present example is provided to demonstrate the utility of the present invention for providing a proposed customized vaccine preparation of the ecm using tissues from a targeted patient to be treated . among other advantages , this approach to vaccine preparation according to the present invention will reduce and / or minimize potential for untoward effects associated with non - self immune responses , as the preparation is actually created using tissue from the intended patient . further , expansion of the harvested tumor tissue on ecm allows the generation of enough material sufficient for continued booster vaccination as dictated by the clinical progression of the patient . this is accomplished while preserving the anti - cancer activities of the preparations described herein . in addition , because the patients own cancer / tumor tissue will be used in the vaccine preparation process , it is envisioned that a customization of the ecm - adjuvanted vaccine to mimic a particular patients cancer and / or tumor cell population will include specialized and patient - specific factors that are excreted from a patients own unique diseased ( i . e ., tumor or cancer ) cell population . this presents the opportunity to supply specific factors in an ecm that are not typically present in a more generalized preparation of tumor cells from an origin other than the intended patient . in this manner , the vaccine is tailored to a particular cancer cell population in the patient . it is expected that this approach will enhance the effectiveness of the preparation as a tumor inhibiting treatment . by way of example , a patients tumor / cancer tissue would be biopsied , and the biopsied material would then be cultured on an ecm material , such as sis . after an appropriate culture time , the tumor / cancer tissue cells would be removed or inactivated . the remaining ecm material would then be processed as described herein to provide a vaccine adjuvant . this adjuvant may then be used in the treatment of the patient . although the present invention has been fully described in conjunction with several embodiments thereof with reference to the accompanying drawings , it is to be understood that various changes and modifications may be apparent to those skilled in the art . such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims , unless they depart therefrom . the references listed below as well as all references cited in the specification are incorporated herein by reference to the extent that they supplement , explain , provide a background for or teach methodology , techniques and / or compositions employed herein . 1 . edwards b k , et al ., ( 2005 ); j natl cancer inst , 97 ( 19 ): 1407 - 27 . 2 . greenlee r t , et al ., ( 2001 ), “ cancer statistics ”, ca cancer j clin , ( 2001 ); 51 : 15 - 36 . 3 . simons j w , sacks n ., ( 2006 ), urol . oncol ., 24 : 419 - 424 . 4 . fukino k , et al ., ( 2004 ), cancer res ., 64 ( 20 ): 7231 - 6 . 5 . bissell m j , et al ., ( 1987 ), j . cell sci . suppl ., 8 ( 3 ): 327 - 43 . 6 . matrisian l m , et al ., ( 2001 ), cancer res ., 61 ( 9 ): 3844 - 6 . 7 . shekhar m p , et al ., ( 2001 ), cancer res ., 61 ( 4 ): 1320 - 6 . 8 . tatenhorst l , et al ., ( 2005 ), brain pathol , 15 ( 1 ): 46 - 54 . 9 . moschella f , et al ., ( 2003 ), oncol res ., 14 ( 3 ): 133 - 45 . 10 . brewer j . m ., immunol lett ., ( 2006 ); 102 ( 1 ): 10 - 5 . 11 . lindblad , e b , ( 2004 ), immunol cell biol ., 82 ( 5 ): 497 - 505 . 12 . barr , t a , et . al ., ( 2006 ), vaccine , 24 ( 17 ): 3399 - 407 . 13 . hodge , j . w ., front biosci , ( 2006 ); 11 : 788 - 803 . 14 . knoll l . d ., ( 2001 ), urology , 57 : 753 - 757 . 15 . knoll l . d ., ( 2002 ), urology , 59 : 758 - 761 . 16 . mantovani f , et al ., ( 2003 ), eur urol ., 44 : 600 - 602 . 17 . o &# 39 ; conner r c , et al ., ( 2001 ), j urology , 165 : 1995 . 18 . o &# 39 ; connor r c , et al ., ( 2002 ), urology , 60 : 697x - 697 xii . 19 . o &# 39 ; connor r c , harding j n , steinberg g d ., urology . 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