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Crystal

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Tissue Vaccines for Cancer

Mark A Suckow; Heinrich; Elliot D Rosen

Expert Rev Vaccines. 2007;6(6):925-937. ©2007 Future Drugs Ltd.

Posted 12/13/2007

Abstract and Introduction

Abstract

Most tumors, including prostate carcinoma, are heterogeneous mixtures of

neoplastic cells and supporting stromal matrix. Attempts to vaccinate as a means

to treat or prevent cancer have typically relied on use of a single antigen or

cell type. In the case of whole-cell vaccines, clonal populations of cancer

cells are grown in culture and harvested for vaccine material. However, it is

clear from microarray data that neoplastic cells grown in culture are greatly

different from those found in vivo. Tissue vaccines are harvested directly from

tumors and are used to immunize the animal or the patient. They are

antigenically rich, in that they are comprised of not only neoplastic cells but

also supporting stromal matrix; furthermore, they include antigens that may be

expressed only in vivo and which may be critical to a successful immune response

to the cancer. For these reasons, the idea that tissue vaccines for cancer have

potentially great utility has merit

and should be explored further.

Introduction

Cancer is a group of diseases characterized by uncontrolled growth and spread of

abnormal cells. The clinical outcome of cancer is often significant debilitation

or death. Although great strides have been made in the clinical approach to

cancer, the disease remains a substantial challenge to clinicians and scientists

alike. It is projected that 1,444,920 new cancer cases will be diagnosed and

559,650 people will die from cancer in the USA in 2007.[1] In this paper,

prostate cancer is given particular focus as a solid tissue tumor exemplary of

many other tumor types.

Cancer of the prostate gland is the most commonly diagnosed cancer in men and

the second most common cancer resulting in death of men, on an age-adjusted

basis.[1] In the majority of cases, prostate carcinoma is a disease of older

men, many of whom have comorbid conditions that elevate the risk of cancer

death.[2-10]

Although the precise mechanisms that lead to the development of prostate cancer

are yet to be defined, it is evident that prostate cancer develops as the sum

result of genetic and epigenetic changes, including those that inactivate

tumor-suppressor genes and activate oncogenes.[11,12] For example, the

inheritance of multiple genetic factors in humans, such as ELAC2; a gene in the

HPC1 region encoding for 2'-5'-oligoadenylate-dependent ribonuclease L; and a

gene within a region of linkage on chromosome 8 that encodes for macrophage

scavenger receptor, have all been associated with increased risk for development

of prostate cancer.[13-16]

A variety of risk factors have been identified for prostate cancer, including

advancing age, diet, family history and race.[13,17-19] It is known, for

example, that African-American men and black African men in the UK have

increased susceptibilities to the development of the disease compared with their

Caucasian counterparts.[20] Furthermore, consumption of soy isoflavone-rich

diets have been demonstrated to reduce the incidence of prostate cancer in

native Asian men,[21,22] whereas progeny from east to west developed increasing

incidences of clinical prostate cancer, thereby, linking the change to

environmental factors, possibly related to the change from a soy-based

diet.[23,24] Approximately 20% of all adult human cancers result from chronic

inflammatory processes, presumably triggered by infectious agents or other

environmental factors, and a role for inflammation has also been recently

proposed in prostate carcinogenesis.[25-28]

Current Concepts for Vaccine-mediated Treatment of Prostate Cancer

In 1909, a role for immunologic control of cancer was proposed by Ehrlich

when he predicted that the immune system repressed the growth of carcinomas that

would otherwise occur with much greater frequency.[29] The possibility that the

patient's own immune system might be stimulated in such a way as to effectively

combat cancer has come under renewed interest recently. Although the very

existence of a tumor suggests failure to generate an effective immune response,

a number of studies provide evidence that vaccination is a safe and effective

means to both prevent and treat some cancers.

Immunotherapy is an emerging approach to treatment of prostate cancer. Two main

approaches have met with at least some success: pulsing of dendritic cells with

specific antigens; and vaccination of patients with allogeneic prostate cancer

cells.

The pulsing of dendritic cells with specific antigens is a novel approach to the

treatment of prostate cancer. In general, dendritic cells are harvested from the

patient, pulsed with relevant antigen(s), and returned to the patient. The

APC8015 vaccine (Provenge®; Dendreon, Inc., WA, USA) is constructed of

autologous dendritic cells pulsed with a fusion protein containing human

prostatic acid phosphatase (PAP). Following successful Phase I and II clinical

trials that demonstrated tolerance by patients,[30-34] a Phase III trial showed

a median survival benefit of approximately 4.5 months in Provenge-treated

patients compared with placebo-treated control patients.[35] Most recently, the

US FDA has issued a Complete Response Letter regarding the biologics license

application submitted by Dendreon, Inc. for Provenge (sipuleucel-T) vaccine for

the treatment of asymptomatic, metastatic, androgen-independent prostate

cancer.[201]

The use of whole tumor cells as vaccine components allows a greatly increased

antigenic menu to be presented to the immune system. Although many antigenic

moieties may be unidentified, it is presumed that the rich choice of antigenic

targets facilitates the likelihood of a successful immune response. Moreover,

clinical models of prostate cancer immunotherapy have benefited from adenoviral

vector-mediated in situ gene strategies that upregulate gene expression

specifically within prostate and prostate cancer cells. Expanding and adoptively

transferring prostate antigen-specific immune cells ex vivo has been important

in order to circumvent immunological tolerance established at some tumor sites.

In a Phase I clinical trial, Simons et al. vaccinated post-prostatectomy

patients with metastatic prostate cancer with autologous tumor cells engineered

to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF),[36] a

potent stimulator of systemic

antitumor immunity.[37] Although some minimal side effects were observed, the

authors were encouraged by an immune response characterized by enhanced

delayed-type hypersensitivity (DTH) responses, in which effector cells

characteristic of both Th1 and Th2 responses infiltrated into the DTH sites.

Furthermore, immunoblots containing protein extracts from cultured prostate

cancer cells demonstrated that some patients developed antibody responses to

three extract polypeptides. However, in spite of these encouraging results, the

authors concluded that clinical application of this approach was limited by the

low yield of autologous cancer cells that were needed for production of the

vaccine, and Phase II clinical trials were not pursued for that reason.

Picking up on the potential demonstrated by autologous whole-cell vaccines,

et al. investigated the utility of allogeneic whole-cell prostate cancer

vaccination.[38] They reasoned that because tumor antigens are often conserved

between tumors, allogeneic vaccines might stimulate cross-protective

immunity.[39-41] To test this idea, monthly intradermal administrations of a

vaccine composed of three irradiated allogeneic prostate cancer cell lines were

given for 1 year to patients with progressive disease as defined by two

consecutive increases in prostate-specific antigen (PSA). The treatment did not

produce any signs of toxicity and resulted in decreased PSA velocity, as well as

a cytokine response profile consistent with a Th1 immune response. In addition,

median time to disease progression was 58 weeks compared with 28 weeks for

historical controls. In an extension of this work, two human prostate cancer

cell lines, LN-CaP and PC-3, were

engineered to secrete GM-CSF and irradiated to construct the GVAX® prostate

cancer vaccine (Cell Genesys, Inc., CA, USA). A Phase I clinical trial

demonstrated T- and B-lymphocyte responses against autologous tumor antigens in

patients vaccinated with the GVAX vaccine.[36] In a follow-up Phase I/II trial,

patients demonstrated significantly reduced PSA velocity; dendritic cell and

macrophage infiltrates at vaccination sites; and antibody responses to at least

five antigens present in LN-CaP and PC-3 cells.[42] Furthermore, only mild side

effects associated with vaccination were noted. In multicenter Phase II clinical

trials involving patients with hormone-refractory prostate cancer, median

survivals of 26.2 and 35.0 months in GVAX-treated patients were reported to

compare favorably with a median survival of 18.9 months in patients receiving

the standard of care, docetaxel plus prednisolone.[202] The GVAX prostate cancer

vaccine is presently under

continued evaluation in a Phase III clinical trial.

The use of proapoptotic gene immunotherapy has also provided some promising

results in preclinical models of prostate cancer. Transduction of neoplastic

cells with 'suicide' genes allows for the ex vivo conversion of an inactive

prodrug into toxic metabolites. Gene-directed enzyme prodrugs have been

effective therapeutically against a variety of tumor cell types, with the

directed cytotoxicity being especially practical with respect to the prostate, a

dispensable organ. An important byproduct of the therapy is a localized immune

response, making it a potential vaccination strategy. Several labs have recently

demonstrated that proapoptotic whole tumor-cell vaccines can induce a

significant (superior to traditional irradiated whole-cell vaccines) immune

responses against orthotopic prostate cancer cell tumors in rodent

models.[43-45]

Contribution of Stroma to Tumor Growth & Progression

An emerging body of evidence shows that dynamic epithelial-stromal interactions

in solid tumors may select subsets of stromal cells with the ability to modulate

tumor behavior, and that the local microenvironment promotes emergence of

tumor-associated stromal cells with functions different from those of the normal

stroma.[46-49] For example, fibroblasts derived from breast tumors stimulated

morphogenesis and growth of breast preneoplastic epithelial cells, while

fibroblasts derived from normal breast tissue inhibited this process.[49] Such

functional changes in tumor stroma may be derived partly from the changes in

secretion of growth factors and in the extracellular matrix (ECM).[50,51]

The stroma consists of a mixture of cells, including fibroblasts,

myofibroblasts, glial, epithelial, fat, blood, vascular, and smooth muscle

cells; and the ECM, including extracellular molecules such as cytokines and

growth factors (Figure 1). Of these, the predominant cell type within tumor

stroma is the fibroblast. In some cancers, fibroblasts constitute a greater

proportion of the overall tumor than do the neoplastic cells. Cancer-associated

fibroblasts have been theorized to originate from cancer cells undergoing

epithelial-to-mesenchymal transition; marrow-derived cells that have undergone

migration to, and activation at, the site of the tumor; and resident fibroblasts

that have undergone activation induced by neoplastic cells. In any case,

cancer-associated fibroblasts are functionally and phenotypically distinct from

normal fibroblasts. Fibroblasts engineered to secrete high levels of HGF or

TGF-ß initiated cancer at divergent sites, including

the stomach, prostate and breast in rodents.[52,53] Further, cancer-associated

fibroblasts produce a number of factors that promote proliferation and

progression of cancer. Among these factors are osteonectin, VEGF, and matrix

metalloproteinases (MMPs).[54-56] VEGF, for example, has been implicated in a

number of aspects of cancer growth, including angiogenesis, remodeling of the

ECM, generation of inflammatory cytokines and hematopoietic stem cell

development.[57,58]

Figure 1.

Tumors are complex mixtures of cell types and extracellular factors. Neoplastic

cells elaborate factors that stimulate growth and angiogenesis, and inhibit

apoptosis of supporting stromal tissue. In turn, the stromal tissue, which is

composed of extracellular matrix and a variety of cells, including fibroblasts,

vascular endothelium and smooth muscle, elaborates factors that stimulate growth

of neoplastic cells, tumor vascularization and inflammation, and inhibit

apoptosis.

Of importance to cancer vaccination, tumor stromal cells generate an environment

in which neoplastic cells are exposed to growth factors while avoiding immune

recognition. This is accomplished by elaboration of cytokines that promote

chronic inflammation and events leading to immune tolerance. For example,

thrombospondin-1 is produced by stromal cells and leads to immune suppression

via activation of TGF-ß.[59] There is also an abundance of evidence suggesting

that, during tumor formation, stromal elements 'hide' or protect erroneously

proliferating cells from destruction by the immune system. Via induction of

local hypoxia, deregulated cytokines and a reduction in the local pH, the tumor

microenvironment appears to quiet, adaptive immune responses normally generated

by tumor associated antigens of malignant cells.[60,61] Tumor regression fails

to occur consistently despite upregulated T-cell responses found

peripherally,[62,63] substantiating the

belief that trafficking of T cells directly to malignant cells is inhibited by

aspects of tumor stroma. Other studies have shown that, even when local

trafficking of T cells is functional, the stroma is still capable of limiting

T-cell effector function.[64-66] Finally, regulatory T-cell (CD4+, CD25+)

induction from within the microenvironment can reduce immune recognition of

neoplastic cells and, thus, encourage tumor growth.[67-72]

The Idea Behind Tissue Vaccines

Beyond the neoplastic cells within a tumor, the connective tissue stroma

represents an enormously unexploited reservoir of potentially powerful antigens

for cancer immunotherapy. Indeed, in some carcinomas the stromal compartment may

account for up to 90% of the tumor mass.[73] Tissue vaccines are constructed

directly from harvested tumor material, thus, including not only cancer cells

but connective tissue stroma as well (Figure 2).

Figure 2.

Components of cultured cell vaccines versus tissue vaccines. (A) Whole-cell

tumor vaccines typically consist of one or several clonal, cultured cell lines.

( By contrast, tissue vaccines comprise a vast menu of antigenic targets,

including those offered by neoplastic (cancer) cells, extracellular matrix,

connective tissue cells (such as fibroblasts), inflammatory cells, and

elaborated extracellular factors that promote tumor growth and metastasis.

A major difficulty with cancer vaccination has been the genetic and phenotypic

plasticity of many tumors. Through a number of mechanisms, cancer cells can

develop means to escape immunosurveillance and destruction.[74-76] In contrast

to cancer cells, however, tumor stroma cells are genetically more stable and

should, therefore, represent targets that are less able to escape destruction by

the immune system.[73] Furthermore, because many tumor stroma-associated

antigens are upregulated or expressed only in the tumor microenvironment, they

represent highly unique moieties that are unlikely to be recognized as 'self'

antigens. Against this backdrop, vaccines created from harvested tissue,

including stroma, create an opportunity to overcome problems associated with

immunotolerance and lack of sufficient antigenic choice.[77,78]

Since they are composed of material harvested directly from tumors, an

additional advantage of tissue vaccines is that they include antigens expressed

following in vivo growth versus the more limited antigenic profile of cultured

cells. For example, cultured renal carcinoma cells showed reduced expression of

a variety of genes, including some known to be tumor-associated antigens.[79]

Similarly, gene expression profiling of human A549 lung adenocarcinoma cells

grown in immunodeficient mice demonstrated selective induction and

overexpression of genes important in tumor progression compared with cells grown

in vitro.[80,81] In addition, differential expression of genes in glioma cells

was associated with differences in cell migration in vivo versus in vitro.[82]

The possibility that unique, important antigens can be included offers

intriguing possibilities for the use of tissue vaccines versus vaccines composed

of cancer cells grown or maintained in

culture.

Although a number of investigations have been conducted studying the use of

autologous whole-cell vaccines, such as that described earlier for prostate

cancer,[36] many of those efforts involved the culturing of harvested cells and

thus, such vaccines do not truly constitute tissue vaccines. By contrast, few

studies have examined the use of tissue vaccines for several types of cancer.

Survival and freedom from tumor recurrence benefits were demonstrated for some

patients vaccinated with irradiated cell suspensions from resected stage I lung

tumors, although the authors concluded that the small number of autologous tumor

cells that could be harvested for vaccine production meant that the probable

utility of this approach would be limited.[83] A tissue lysate vaccine was used

in a Phase III trial for the treatment of renal cancer when combined with

nephrectomy.[84-86] In that study, the vaccine was well tolerated and 70-month

progression-free survival was

72% versus 59.3% for the control group. Homogenized, formalin-fixed autologous

tumor tissue was used to vaccinate patients following surgical resection of

hepatocellular carcinoma.[87] Using that approach, 17 out of 24 patients

developed a DTH response against vaccine components, and 1-, 2- and 3-year

recurrence rates were approximately half those of nonvaccinated patients. In a

limited clinical study involving two patients, treatment with alcohol was used

to inactivate resected breast carcinomas prior to reimplantation at the original

site.[88] Neither patient showed tumor recurrence, one at 9 years and the other

at 3 years post-treatment.

Laucius et al. investigated the use of an irradiated tissue vaccine adjuvanted

with bacillus Calmette-Guérin (BCG) as an approach to melanoma.[89] Out of 18

patients who were vaccinated following surgery for tumor removal, four

demonstrated clinical responses to vaccination. Others have further investigated

this approach, although production of the vaccine used involved enzymatic

digestion of the connective tissue stroma; harvested tumor tissue was treated

with collagenase and DNase to produce material that was inactivated by

irradiation and adjuvanted with BCG to produce a vaccine. Out of 40 patients

with measurable metastases, four had complete responses and one had a partial

response to vaccination; and a positive DTH response was observed in patients

exhibiting tumor regression.[90] Vaccination of stage III patients who underwent

surgical removal of tumor and metastases resulted in a 5-year survival rate of

nearly 50%, higher than that of historical

controls for patients treated only by surgery.[91,92] Hapten modification of

this vaccine preparation has been shown to further improve clinical responses of

patients.[93,94]

In contrast to these encouraging data using a melanoma tissue vaccine, great

effort was subsequently given to development of vaccines using cultured

allogeneic melanoma cells. Although results were initially encouraging, two

randomized adjuvant therapy trials of an allogeneic whole-cell melanoma vaccine

adjuvanted with BCG were halted when it became evident that the trials were

unlikely to show a significant benefit in favor of the vaccine arm.[95] This

event underscores the potential efficacy of tissue vaccines versus those

vaccines composed of cultured cell lines, which present a more limited scope of

antigen targets to the immune system.

Tumor Stromal Targets for the Antitumor Immune Response

The presence of stromal components is one feature that distinguishes tissue

vaccines from other whole-cell vaccines. As discussed earlier, it is evident

that the stroma plays an important role in the development and progression of

tumors; and, in contrast to neoplastic cells, stromal elements tend to be far

more genetically stable and less apt to immune evasion. It therefore stands to

reason that immune destruction of the supporting stroma should limit the ability

of a tumor to advance.

Several tumor stroma cell types present attractive targets for immunotherapy.

Among these are tumor fibroblasts, tumor macrophages and tumor endothelial

cells. Many of these cells are initially recruited by the tumor from normal

surrounding tissue early in the tumorigenic process. Once recruited into the

service of the advancing tumor, stromal cells are typically activated relative

to their normal counterparts so that the altered stroma and ECM are conducive to

tumor growth and progression. As part of this process, tumor stromal cells

undergo upregulation or induction of unique antigens that may be employed to

encourage immune recognition. Although data on the precise immunogenicity of

individual stromal antigens are limited, the antigenic complexity offered by the

stromal network is powerful.[77,78] Studies to examine the immunogenic potential

of such antigens are needed to define what, if any, contribution they make to

the protective immune response.

A variety of molecules overexpressed by tumor stromal cells contribute, perhaps

individually and certainly collectively, to the overall utility of tissue

vaccines. For example, fibroblast activation protein-α is a membrane serine

protease, expressed principally in fibroblasts of epithelial tumors and healing

tissue, which promotes formation of tumor stroma and has been found to be a

potential target for specific monoclonal antibody therapy in colon

cancer.[96-100] Neoplastic cells, tumor fibroblasts, tumor endothelial cells and

tumor macrophages all express MMPs, important modulators of the ECM; and MMP

expression is associated with an invasive tumor phenotype and progression of the

tumor.[101-103] Urokinase plasminogen activator (UPA), its associated receptor

(UPAR) and inhibitor (PAI-1) are overexpressed by neoplastic cells, tumor

fibroblasts, tumor endothelial cells and tumor macrophages in a variety of tumor

types, including renal, breast, prostate

and colorectal.[73,104-106] UPA, UPAR, and PAI-1 all play roles in cancer

invasion and metastasis, and the absence of UPA and PAI-1 in genetically

modified mice has been associated with impaired growth of transplanted T241

fibrosarcoma.[107] It can be noted then, that a number of tumor

stroma-associated antigens are expressed exclusively in the micromilieu of the

tumor and are expressed by both neoplastic and tumor stromal cells. In addition

to the examples above, carbonic anhydrase IX is expressed on tumor-associated

fibroblasts and on some cancer cells[108]; and survivin, a protein inhibitor of

apoptosis, is overexpressed in neoplastic and in tumor epithelial cells.[109]

Clearly, the tumor stroma provides an antigenically rich repertoire with which

to engage the immune system. That some of the antigens are expressed by stromal

components, as well as by neoplastic cells, only reinforces the closely

coordinated functions of each. The neoplastic cells of the tumor are reliant

upon the stromal component to modify the ECM and to support the growth and

spread of the tumor. Indeed, to target the stroma is to target the entire tumor.

Tissue Vaccines for Prostate Cancer

While several groups have examined the use of allogeneic whole-cell vaccines or

vaccines based upon pulsing of autologous dendritic cells with antigen for the

treatment of prostate cancer, few have considered the potential utility of

tissue vaccines for prostate cancer. In this regard, we have undertaken studies

in the Lobund-Wistar (LW) rat model of prostate cancer[110] to examine the

possibility that tissue vaccines are effective at both preventing and treating

metastatic prostate cancer.

The LW rat model of prostate adenocarcinoma was originally recognized in a

colony of germfree inbred Wistar strain rats, initially in the 37th generation.

Large adenocarcinomas developed in the prostatic complex spontaneously in male

LW rats at approximately 24 months of age; in approximately 30% of male LW rats

within 12 months following a single intravenous dose of methylnitrosourea (MNU);

and in approximately 80% of male LW rats within 10 months following a single

intravenous dose of MNU and subcutaneous implantation of slow-release

testosterone pellets (Figure 3).[110] In addition, a transplantable cell line,

PAIII, was isolated from a LW rat with spontaneous, metastasized prostate cancer

and can be used to generate large, androgen-independent subcutaneous tumors

following subcutaneous administration to LW rats. In all of these model systems,

tumors will metastasize to the lungs via the lymphatic system. Great homology

exists with the human disease

in that autochthonous tumors undergo transition from an initial

androgen-dependent phase to an androgen-independent phase. Moreover, prostate

tumors in the LW rat express PSA, a marker sometimes associated with prostate

cancer in humans.[111]

Figure 3.

Section of a prostate tumor from a Lobund-Wistar rat. The mass is a typical

adenocarcinoma with scattered acinar structures in an abundant connective tissue

stroma. Section stained with hematoxylin and eosin. Magnified x1000.

The possibility that prostate cancer can be prevented by means of a prophylactic

vaccine would be of tremendous value. Using tissue vaccines composed of lysates

or glutaraldehyde-fixed whole-cell preparations from harvested subcutaneous

PAIII tumors, we demonstrated 50 and 90% reductions, respectively, in the

incidence of MNU-induced autochthonous prostate cancer in the LW rat (Figure

4).[112] When PAIII cells were coincubated with splenocytes from vaccinated LW

rats prior to implantation into naive rats, 80 and 40% reductions in the

incidence of subcutaneous tumors were associated with lysate and whole-cell

tissue vaccination, respectively, indicating that the spleen played an important

role in the observed protective immune response. We have further shown that

vaccination of LW rats bearing autochthonous prostate tumors with a whole-cell

tissue vaccine resulted in complete regression of the primary tumor in 21% of

rats, and a 70% reduction in the

incidence of rats having any evidence of pulmonary metastases. In addition,

when used as an adjunct to external-beam radiation treatment of subcutaneous

PAIII tumors, pretreatment with a tissue vaccine resulted in significant

reduction in tumor size compared with treatment with radiation or tissue vaccine

alone (Suckow MA, unpublished observations).

Figure 4.

Prevention of prostate cancer by tissue vaccines in Lobund-Wistar rats. Prior to

tumor induction with methylnitrosourea, and monthly afterwards, rats were

vaccinated with either media (as a control); a PTE of tumor material harvested

from the tumors of other rats (PTE); or GFT material harvested from other rats.

Compared with controls, PTE vaccination reduced the incidence of prostate cancer

by 50% and GFT cell vaccination reduced the incidence by 90%. GFT:

Glutaraldehyde-fixed tumor; PTE: Potassium thiocyanate extract.

Ideally, a tissue vaccine would include the antigenic complexity associated with

a growing tumor and be adjuvanted by the cytokine environment of such a tumor.

Nonproliferating, live whole tumor-cell vaccines maintain the ability to secrete

cytokines for a short period of time, yet are also processed more

rapidly[113,114] and elicit a weaker immunogenic response than their live

counterparts.[43,113,115-118] As discussed previously, the use of gene therapy

to regulate the onset of an apoptotic death after vaccine administration

approximates some aspects of a tissue vaccine. Still, even though such live,

eventually apoptotic, cells exhibit efficacy, cancer cells that die a necrotic

death may be better able to invoke a systemic antitumor response.[116,119-121]

With this consideration, we developed a strategy that involved administration of

genetically modified PAIII cells engineered to be deficient in tissue factor

(TF).[122] TF is a transmembrane

glycoprotein that is a critical component in the cascade initiating blood

coagulation/hemostasis. Hypercoagulation has been well documented in a variety

of cancers, and TF expression, specifically, has been correlated with the

aggressive growth and spread of cancer.[121-128] Interference with the anti-TF

pathway resulted in increased pulmonary metastasis in an experimental model of

melanoma, indicating an important role for TF in the escape and migration of

neoplastic cells.[129,130] Following subcutaneous administration of TF-deficient

PAIII cells into naive LW rats, small tumors grow which spontaneously regress,

via necrosis rather than apoptosis, within 21 days. When rats were subsequently

challenged with wild-type PAIII cells, the resulting tumors were significantly

smaller in vaccinated versus nonvaccinated rats and the mean number of

metastatic foci in the lungs was reduced by 62.5%.[122]

Xenogeneic Tissue Vaccines

Clinical investigations into the use of autologous vaccines derived from

harvested prostate tumor tissue led Simons et al.[36] to the conclusion that,

even after expansion in culture, there was simply insufficient material for

production of a vaccine quantity that would allow initial and booster doses.

While the authors of that study were encouraged by the immunologic responses

resulting from immunization with their preparation, which was augmented by

manipulation of the autologous cells to secrete GM-CSF, future work focused on

the use of cultured allogeneic prostate cancer cell lines as a source of vaccine

material that could be replenished readily.[42] As a result, attention has

drifted away from the substantial potential that tissue vaccines may hold for

the treatment of cancer.

The obvious conundrum with tissue vaccines is that, typically, the material

harvested from resection or biopsy is insufficient to allow both histopathologic

evaluation and production of vaccine in quantities sufficient to sustain a

complete, and possibly long, course of treatment. Yet, while culture of

harvested tumors might expand the quantity of raw material, by definition this

weakens a main feature of the tissue vaccine: the presence of novel antigens

that might be expressed in vivo but not in vitro.

One possible means of overcoming the limitations to tissue vaccines posed by

insufficient harvests of tumor tissue from patients, might be to use tumor

tissue from other species. If homology exists between even some key antigens,

such xenogeneic tissue vaccines would represent a very novel approach to cancer

treatment. The idea that antigens associated with tumors from one species might

stimulate protective immunity to a tumor in another species is not entirely new.

For example, immunization of mice with human prostate-specific membrane antigen

(PSMA) resulted in an antibody response to native mouse PSMA, suggesting that

the xenogeneic antigen was sufficiently different to overcome immunologic

tolerance, yet sufficiently similar to generate a cross-reactive immune

response.[131] Fong et al. described the administration of mouse PAP-pulsed

dendritic cells to patients with advanced prostate cancer.[132] In that study,

patients developed T-cell

proliferative responses to the homologous self-antigen, and some patients had

stabilization of previously progressing disease. Dogs with stage II-IV melanoma

were vaccinated with human tyrosinase DNA.[133] Of nine vaccinated dogs, three

developed a measurable increase in postvaccine serum antibody to human

tyrosinase; one of these experienced a remission with complete disappearance of

radiographic evidence of pulmonary metastases; and another of the three

responders had complete gross and histopathologic remission of the disease.

Vaccination of stage IV colorectal cancer patients with cultured mouse B16

melanoma and lung carcinoma cells was shown to stimulate a significant

increase in cell-mediated immunoreactivity as evidenced by DTH reactions, as

well as by blood lymphocyte proliferation assays. Furthermore, the mean survival

of vaccinated patients was 17 months versus only 7 months for nonvaccinated

patients.[134]

To investigate the possibility that a xenogeneic prostate tumor tissue vaccine

could prevent the growth of human prostate cancer cells, we vaccinated

immunocompetent BALB/c mice with a glutaraldehyde-fixed tissue vaccine prepared

from PAIII prostate tumors harvested from LW rats.[135] Splenocytes harvested

from vaccinated mice were incubated with PC346C human prostate cancer cells

before orthotopic implantation into the prostates of syngeneic immunodeficient

nude mice. After 10 weeks, we found a 70% reduction in the incidence of prostate

cancer in the mice implanted with PC346C cells that had been coincubated with

splenocytes from vaccinated mice, versus mice implanted with PC346C cells that

had been coincubated with splenocytes from nonvaccinated control mice (Figure

5). Furthermore, cytokine profiles of cultured splenocyte supernatants suggested

that the protective immunity resulted primarily from a Th1 response, with

significant increases in TNF-α,

IL-2 and IFN-γ, although weak serum antibody responses to a lysate of PC346

cells were detected in mice vaccinated with glutaraldehyde-fixed prostate tumor

tissue cells. These data strongly suggest that xenogeneic tissue vaccines could

be used to treat cancer in humans.

Figure 5.

Inhibition of human PC346C prostate cancer cell growth in mice by a xenogeneic

tissue vaccine. Human PC346C prostate cancer cells were coincubated with

splenocytes from immunocompetent BALB/c mice that had been vaccinated with GFT

harvested from Lobund-Wistar rats. After incubation, the cells were

orthotopically implanted into the prostates of syngeneic nude mice. After 10

weeks, prostates were evaluated for tumor growth. The graph shows that GFT

vaccination stimulated immunity sufficient to reduce the incidence of prostate

cancer in the mice by nearly 70% compared with nonvaccinated and

media-vaccinated controls. GFT: Glutaraldehyde-fixed prostate tumor tissue.

The advantages of xenogeneic tissue vaccines are twofold. First, they allow use

of a replenishable source of raw material for vaccine production. The amount of

vaccine available is limited only by the number of animals maintained for tumor

harves. Second, and perhaps most importantly, compared with the use of

autologous vaccines, xenogeneic vaccines present a mechanism by which

immunotolerance can be overcome. Similar to the robust immune response

associated with rejection of transplanted organs from other species, xenogeneic

tissue vaccines will not be recognized as 'self' and should generate vigorous

immunity to homologous antigens.

Expert Commentary

The standard of care for many cancers involves harsh treatments with

chemotherapeutic agents, which often leave the patient clinically frail and

subject to adverse sequelae. By contrast, immunotherapy offers a means to rally

the body's own defenses against the cancer. While most cancer vaccines have been

well tolerated by patients, they have proved to be of inconsistent clinical

value.

One substantial limitation of many cancer vaccines is the limited menu of

antigen targets presented to the immune system. Since cancer can be a plastic,

evolving tissue, the antigenic milieu is not static and vaccines composed of the

broadest possible antigenic repertoires stand the best chance of success. Tissue

vaccines, by their very nature, are antigenically diverse and include antigens

not only from tumor neoplastic cells, but also from the supportive stromal

matrix of the tumor; and they include relevant antigens that are expressed in

vivo, but not in vitro. If administered as a live cell preparation that grows

into a tumor, recruits a stromal matrix and then spontaneously regresses, the

immune system is presented with antigens of a growing, evolving tumor. If

administered as a xenogeneic preparation, the immune system is presented with

antigens that may be homologous but are recognized as foreign, thus, breaking

immunologic tolerance. Together,

these features ascribe an antigenic richness to tissue vaccines that cannot be

achieved by any other means.

In some ways, tissue vaccines are not new. Early vaccines for some infectious

pathogens were constructed as tissue vaccines because the pathogens could only

be grown in sufficient quantities using in vivo systems. For example,

formalin-fixed lung homogenates of moribund guinea pigs infected with

lymphocytic choriomeningitis virus were shown to reduce mortality when given as

a vaccine to naive guinea pigs subsequently challenged with lymphocytic

choriomeningitis virus[136,137]; and vaccines made from formalinized tissues of

either foxes or ferrets infected with distemper virus reduced mortality in naive

foxes subsequently challenged with distemper virus.[138] While most cancer cells

can readily be grown in culture, tumor tissue, with its complex composition of

neoplastic cells and supporting stromal matrix, cannot be precisely replicated

and expanded in vitro. For this reason, tumor material harvested from animal

models may represent a means to rapidly

produce sufficient quantities of tissue vaccine. Although the advantages of

xenogeneic tissue vaccines are obvious, it would also be possible to expand, in

immunodeficient rodents, tumor material harvested from patients either by

resection or biopsy. In this way, then, one could generate quantities of tissue

vaccine sufficient to allow treatment of patients with what would essentially be

autologous tissue vaccines.

In spite of significant potential advantages offered by xenogeneic tissue

vaccines, several hurdles remain. First, the mechanism of action for such

vaccines needs to be established. While studies in mice vaccinated with a

rat-derived tissue vaccine suggest a strong role for Th1 immunity, that response

needs to be characterized in greater detail and the contribution of humoral

immunity to the protective response investigated further. Second, the potential

for autoimmunity needs to be examined. In both rats and mice, immunization with

a rat-derived tissue vaccine did not result in any notable untoward effects

typical of autoimmune disease; however, studies designed to specifically examine

such features need to be conducted. Finally, clinical studies must be undertaken

to determine the utility of xenogeneic tissue vaccines in human patients.

Although the preclinical data are compelling, translation to the clinic will

require careful analysis in human

clinical trials.

Five-year View

We believe that the significant need for novel approaches to cancer may benefit

from the further development of tissue vaccines. Potentially, use of tissue

vaccines or other types of cancer vaccines might be used as part of a standard

immunologic prophylaxis program against those cancers for which an individual

patient may be at greatest risk. That tissue vaccines can be used as autologous

preparations, xenogeneic preparations, or as vaccines that immunize via

induction of a small, spontaneously regressing tumor, supports the tremendous

potential of this approach to cancer.

With proven efficacy in preclinical models, including one which utilized a human

cancer cell line, further research with tissue vaccines should involve

refinement by added adjuvants and use of novel vaccine-delivery systems; and,

ultimately, human clinical testing. Determining optimal administration

conditions will be necessary to avoid encouragement of host immune regulatory

mechanisms and an immunosuppressive tumor environment.[139] While such testing

will need to establish the safety of xenogeneic tissue vaccines and the relative

contribution of antitumor stroma immunity, our demonstration of efficacy

suggests that a relatively unlimited supply of vaccine material obtained from

tumors of other species could overcome the hurdle of insufficient autologous

material described by Simons et al..[36] The possibility that xenogeneic

material could be used to generate vaccines makes Simons' encouraging results a

more translational possibility.

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Sidebar: Key Issues

Most cancer vaccines are based on single proteins or cultured cell lines.

Immunotolerance is an enormous hurdle to successful application of cancer

vaccines.

Tissue vaccines are composed of material harvested directly from tumors.

Tissue vaccines include antigens associated with neoplastic cells and with the

tumor stroma. Furthermore, they include antigens that may be expressed only

following in vivo growth versus growth in culture.

Attempts at autologous cancer vaccines have been limited by finite, typically

insufficient amounts of raw material for vaccine production. By contrast,

xenogeneic tissue vaccines offer a means to generate sufficient quantities of

vaccine raw material, as well as the potential to overcome immunotolerance.

Cancer cells engineered to generate spontaneously regressing tumors following

implantation offer a means to vaccinate the animal using antigens associated

with a growing tumor.

In animal models of prostate cancer, tissue vaccines reduced the incidence by

90% when used prophylactically; resulted in complete tumor regression in 20% and

a 70% reduction in the incidence of metastasis in tumor-bearing animals; and

stimulated immunity associated with a 70% reduction against tumor formation

associated with a human prostate cancer cell line.

Clinical studies are needed to assess the utility of tissue vaccines in

patients.

Acknowledgements

The authors thank Schroeder for technical assistance in work relevant to

this manuscript and for the illustrations in this manuscript.

Reprint Address

Mark A Suckow Associate Research Professor, Biological Sciences, University of

Notre Dame, Freimann Life Science Center, Notre Dame, IN. suckow.1@... .

Mark A Suckow,1 Heinrich,2 Elliot D Rosen3

1Biological Sciences, University of Notre Dame, Freimann Life Science Center,

Notre Dame, IN

2University of Notre Dame, Department of Biological Sciences, Notre Dame, IN

3Indiana University School of Medicine, Department of Medical & Molecular

Genetics, Indianapolis, IN

The Great HPV Vaccine Hoax Exposed

Great Newstarget.com article!! I would cut and paste the whole thing

here but it is 7 pages. This link takes you to page 1 then you can

click NEXT at the bottom of each page to read the whole article.

http://www.newstarg et.com/Report_ HPV_Vaccine_ 1.html

Diane