WO1996025664A1

WO1996025664A1 - Immunotherapy screening, prognosis, and treatment methods and compositions

Info

Publication number: WO1996025664A1
Application number: PCT/US1996/001876
Authority: WO
Inventors: Avi Eisenthal; Meir Shinitzky; Amnon Gonenne
Original assignee: Immunotherapy, Inc.; Yeda Research And Development Corporation, Ltd.
Priority date: 1995-02-17
Filing date: 1996-02-12
Publication date: 1996-08-22
Also published as: IL117159A0; AU4977896A

Abstract

The invention describes an immunogen or immunogenic preparation derived from cells, cell membranes or proteins therefrom, that have been modified by treatment with a 2',3'-nucleoside or nucleotide dialdehyde crosslinking agent and by exposure to hydrostatic pressure. The pressure and crosslinker treatment of cells performed in accordance with the invention is termed 'PCL-modification'. Improved immunogenicity is obtained if the cells are treated with pressure at the same time that they are subjected to the protein crosslinking compounds. The cells suitable for PCL-modification in accordance with the invention and for use as immunogens may be tumor or cancer cells, transformed cells, virus-infected or microorganism-infected cells, e.g., bacteria, parasites, yeast, and the like. The PCL-modified tumor or infected cells are especially capable of inducing and eliciting a specific and potent immune response against the respective tumor cells, infected cells, or otherwise altered cells in both animals and human patients. Human peripheral blood mononuclear cells (PBMC) are sensitized and specifically stimulated to immunoreact against PCL-modified tumor or infected cells as determined by in vitro sensitization assays. Such assays, alone and in combination with PBMC cytokine analyses, serve as prognostic means to determine or identify those patients who will either respond or will not respond to PCL-immunotherapies and treatments, both in vitro and in vivo.

Description

IMMUNOTΗERAPY SCREENING, PROGNOSIS, AND TREATMENT METHODS AND COMPOSITIONS

FIELD OF THE INVENTION

The present invention is generally in the field of immunotherapy and treatment of cancer and infection, and relates to novel immunogens, anti-tumor and anti-infected or transformed cell immunogenic preparations, and vaccines for use in animals, including humans. The invention also relates to processes for the preparation of the immunogens, and methods of utilizing the immunogenic preparations as anti-tumor cell and anti-infected or transformed cell immunogens both in vitro and in vivo.

The immunogens of the present invention may be derived from tumor cells which are treated as described herein to increase their specific immunogenicity. The immunogen may be whole cells treated as described, plasma membranes derived from these cells, or tumor-specific immunogenic proteins obtained from the cells or cell membranes. In addition, the immunogens may be derived from cells infected and/or transformed with viruses or other microorganisms or agents that are treated as described herein to increase their specific in vivo and in vitro immunogenicity.

BACKGROUND OF THE INVENTION

As is generally known in the art, cancer and/or tumor cells frequently display on their external surfaces specific neo-antigens which are foreign to the immune system and immune cells of the host. Nevertheless, for reasons which are not entirely clear, tumor or cancer cells often escape immune surveillance, and the immune system fails to develop an effective immune reaction against these cells. Attempts have been made to immunize cancer patients with preparations that will stimulate their immune systems to develop a reaction against the neo-antigens, with the hope that such an immune reaction will destroy the residing cancer or tumor.

U.S. Patent No. 4,931,275 discloses anti-tumor vaccines which contain as an active ingredient tumor cells, plasma membranes, or specific membrane proteins obtained from these cells or membranes, which have been treated to augment their immunogenic properties. The treatment described to augment cells' immunogenicity in accordance with this patent consists of either treatment with cholesteryl hemisuccinate, which rigidifies the lipid layer of the plasma membrane, or application of hydrostatic pressure up to about 1500 atmospheres (atm), or a combination of the two treatments.

The development of tumors and the transformation of normal cells to malignancy can result from a variety of different causes. Transforming events can occur spontaneously by random mutations, by gene rearrangement, or, they may be induced by chemical, physical, viral, or microorganismal agents. Major classes of chemical carcinogens are known to include polycyclic aromatic hydrocarbons, such as found in tar and soot, and aromatic amines, such as found in certain dyes. Examples of physical carcinogens are X-rays and ionizing and ultraviolet radiation. In addition, both RNA and DNA viruses and viral oncogenes, are capable of transforming cells. When viral genes are introduced into cells, the infected cells are then triggered to express on the cell surface virus-associated antigens that can be recognized by the immune system. Moreover, the abnormal maintenance of certain viral oncogenes in a transcriptionally active state can result in transformation. In a similar manner, the infection of cells by bacteria or parasites or other microorganisms may lead to the expression of antigens at the cell surface and to recognition by immune cells.

The expression of at least some oncogenic, neo-antigenic, or "non- self protein products, particularly those resulting from the mutation of a normal protein, should ideally render tumor, transformed, and infected cells sufficiently distinct from normal, unaffected cells so that the former can be detected by the immune system. However, as mentioned above, tumor and transformed cells, including cells infected with microorganisms that can affect the composition of the plasma membrane of the infected cells, often have the ability to avoid immunologic surveillance and detection.

In the case of tumors, the task of host immune surveillance is especially formidable because tumor cells and other abnormal cells have many similarities to normal cells, in spite of their abnormal propensities to fail to respond to normal regulatory signals, to proliferate and spread throughout the host, and to interfere with normal organ function. Further, although foreign antigens or mutated proteins may be expressed on the surfaces of transformed cells and tumor cells, the cells of the immune system frequently develop an ineffective or weak immune response against such tumor cells. Alternatively, the emergence of tumors in an animal may reflect the failure or inefficiency of immune surveillance and the virtual absence of an immune response during progressive growth of the tumor. Thus, important problems and goals in tumor immunology and for the practitioner reside in determining why such immune responses are ineffective or nonexistent and what can be done to stimulate the cells of the immune system to develop a reaction against tumor or non-self antigens with the end result of eradicating the tumor, cancer, or otherwise abnormal cells. Many human tumors and tumors of other animals express antigens which do not elicit a strong cellular and humoral immune response in the host, but instead result in a ineffective and undetectable host immune response. This could be because cancer cells produce certain proteins that are either not expressed at all, or are present in much lower quantities in and on the surfaces of normal cells. The relevant tumor antigens, or proteins, generally fall into two major categories: unique tumor specific antigens, which are found only on tumor cells and not on other host cells; and tumor associated antigens ("TAA") or determinants, which are found on tumor cells and also on some normal cells, but have qualitative and quantitative differences in expression on tumor and normal cells.

The best-studied unique tumor antigens are the neoantigens expressed on tumors induced in inbred mice by oncogenic viruses and chemical carcinogens. In contrast, spontaneous tumors, such as those induced by exposure to environmental carcinogens, have no predictable antigenic markers. However, it is pointed out that even if unique antigens are not found on human tumor cells, it may not be because such antigens do not exist, but because such antigens are difficult to detect, given the methods available in the art at the present time. Accordingly, needed in the art are ways to stimulate a strong and sustained immune response against tumor cells, which may have great potential for being highly immunogenic, but can successfully evade immune destruction.

The expression of MHC proteins on tumor cells is believed to be critical for the immunologic recognition and destruction of the tumor cells. This is especially true if T lymphocytes are required for the cognitive and/or effector stages of specific anti-tumor immune responses, since T cells can recognize antigens only in association with MHC molecules (Goodman, J.W. , 1994.. "Antigen Presentation & the Major Histocompatibility Complex" , In: Basic and Clinical Immunology. Eds. D.P. Stites, A.I. Terr, and T.G. Parslow, Appleton & Lange, Norwalk, Connecticut, pp.58-65). Thus, it is possible that tumors which can stimulate protective immune responses express adequate amounts of MHC molecules, while other tumors which are not immunogenic fail to express enough MHC proteins, or fail to express any MHC proteins. The lack of expression of sufficient amounts of MHC molecules may be particularly important in the case of certain chemically- and virally-induced tumors. In addition, in a number of murine and human spontaneously arising tumors, including neuroblastomas, basal cell carcinomas, small-cell lung tumors, choriocarcinomas, and B cell lymphomas, the expression of class I MHC antigens may be significantly reduced or virtually absent (Ramakrishna, V. et al. 1993. "Increased projection of MHC and tumor antigens in murine B16-BL6 melanoma induced by hydrostatic pressure and chemical crosslinking", Cancer Immunol. Immunother. , 36:293-299). It is noted here that the elucidation of the processing pathways for the presentation of protein antigens in association with MHC molecules to T cells has shown that T cells recognize small peptides derived from intracellular degradation of proteins that are inserted into a peptide-binding cleft in the MHC molecule and are then transported with the MHC molecule to the cell surface (Matsumura, M. et al. , 1992. "Emerging principles for the recognition of peptide antigens by MHC class I molecules", Science, 251:921; Neefjes, J.J. et al., 1993. "Selective and ATP- dependent translocation of peptides by the MHC-encoded transporter", Science, 261:769). Thus, any abnormal cellular protein, and not just proteins derived from the cell membrane, is capable of being a potential immunogen for presentation to the immune system. In view of this, the presence in a tumor cell of a nonfunctional or truncated protein product of a mutated allele could potentially result in the immunogenicity of that product. In a related fashion, the presence in a virally-transformed or infected cell of a viral protein product in conjunction with MHC molecules may also potentially result in the immunogenicity of that viral product expressed by the cell. The goal of therapies to treat and eradicate cancers and other types of transformed cells is to provide efficient and safe methods by which to increase the host's anti-tumor or foreign cell response against weakly immunogenic cell types, such as tumors and transformed cells.

Tumor antigens elicit both humoral (antibody or B cell-mediated) and cell mediated immune responses in vivo, and virtually all of the effector components of the immune system have the potential to contribute to the eradication of tumor cells. However, the T cell response is a most important host response for the control of growth of antigenic tumor cells, and transformed or infected cells, via cell-mediated immunity. The T cell response is effective for both the direct killing of tumor cells or infected (e.g. , virus- or bacteria-infected) cells (by cytotoxic T cells) and the activation of other components of the immune system.

T cell immunity to tumors and infected cells involves the function of two T cell subsets: MHC class H-restricted T cells, which largely represent CD4 helper T cells (i.e., T_H) that mediate their effect by the secretion of lymphokines to activate other effector cells and to induce inflammatory responses; and MHC class I-restricted T cells, which represent CD8 cytotoxic T (T_c) cells that also secrete lymphokines, but mediate their effect primarily by the direct lysis or killing of tumor cells. Because most tumor cells express class I, but not class π MHC molecules, the T_H cell subset cannot directly recognize these tumor cells. As a result, most T_H cell responses are dependent upon antigen-presenting cells or APCs, such as macrophages, B lymphocytes and dendritic cells, to present the relevant tumor antigens in the context of class II MHC molecules for cell activation (Germain, R.N. et al., 1993. "The biochemistry and cell biology of antigen processing and presentation", Ann. Rev. Immunol. , 11:403; and Brodsky, F. et al., 1991. "The cell biology of antigen processing and presentation", Ann. Rev. Immunol. , 9:707). Antigen presenting cells capture, process, and present most proteinaceous immunogens to the CD4 helper T cell subset. Following antigen- specific triggering, activated T_H cells secrete lymphokines that, in turn, activate T_c cells, macrophages, natural killer (NK) cells, and B cells; activated T_H cells also produce other lymphokines, such as lymphotoxin or tumor necrosis factor (TNF) which may also be directly lytic to tumor cells.

Recent findings have shown that two functional subsets of T_H cells exist. The first subset, the type 1 or T_H1 subset, appears to facilitate and then to reinforce primarily a cell-mediated immune response by cytotoxic T cells, i.e. , T_c cells (see below); the second subset, the type 2 or T_H2 subset, appears to help B lymphocytes to mature and then to produce antibodies (C. Ezzell, 1993, The J. of NIH Research, 5:59-64). The two T cell subsets are also distinguished by the types of cytokines that they produce. It has been demonstrated by in vitro studies that T_H1 cells release both interleukin-2 (IL-2) and interferon-7 (IFN-7), while T_H2 cells release a combination of interleukin-4 (IL-4), interleukin-5 (I -5), interleukin- 6 (IL-6), and interleukin-10 (IL-10). Both IL-4 and IL-10 has been shown to shut down the cell-mediated immune response. Conversely, IFN-7 produced by T_H1 cells promotes cytotoxic T cell proliferation and inhibits antibody production. Thus, the various cytokines elaborated by the these two T cell subsets are cross- regulatory and can, when manipulated by a pathogen (e.g. , virus, parasite or tumor cell) seriously suppress an individual's effective immune response status to the particular pathogen. (IcL at page 59). With particular regard to HIV infection, disease, and pathogenesis, it has been observed that as patients progress toward AIDS disease, there is a change in the patient's immune response to the virus, such that they move away from a T_Hl-type response (i.e., predominantly cell-mediated) to a less effective T_H2-type response (i.e., primarily antibody production). Thus, a "T_H1 to T_H2 switch" might be involved in the progression toward AIDS (C. Ezzell, 1993, The J. ofNIH Research, 5:59-64).

In contrast, the action of T_H cells and subsets thereof, class I- restricted T_c cells are capable of directly recognizing and killing tumor target cells by disrupting the target cell membrane and nucleus (Bjorkman, P. et al., 1990. "Structure, function, and diversity of class I major histocompatibility complex molecules", Ann. Rev. Biochem. , 59:253). Only a minor fraction of class I- restricted T cells is capable of providing helper functions; thus, effective T_c cell responses are generally dependent upon class H-restricted T_H cell responses to provide the necessary helper factors to activate and promote the proliferation of T_c cells. The T cell receptor of an antigen-specific T_c cell clone recognizes class I MHC -peptide complexes which, after mtracellular processing of viral or tumor antigens appear, at the surface of virally infected or transformed cells, for example. Cytotoxic T cells become activated to eradicate foreign cells by releasing toxins or inducing the target cell to commit suicide, perhaps by physical contact with the foreign target cell. The activated T_c cells proliferate and give rise to additional T_c cells having the same antigen specificity. As mentioned above, the role of antigen-presenting cells is to offer antigenic peptides complexed with MHC molecules to the available repertoire of T cells. Thus, MHC molecules are obligatory components of the immunogenic complex recognized by T cells and play a central role in the immune response to foreign and tumor antigens. The ability of T cells to recognize specific features of MHC proteins is crucial for the immune system to function properly and to discriminate "self from "non-self".

Although the host's immune system may often be inadequate to control tumor growth, thus causing tumor cells to escape immune surveillance and destruction by the appropriately stimulated immune cells, a potential solution to this problem is to manipulate and amplify the immune system and its cellular components in order to promote tumor eradication. This solution is also conducive for eliminating cells infected or transformed by other exogenous agents, e.g., drugs, carcinogens, viruses, other microorganisms.

Several distinct approaches to immunotherapy are being studied in the hope of developing important strategies for the treatment of tumors and cells expressing foreign antigens. Some of these techniques are more successful than others. For example, immunizing tumor-bearing hosts with isolated tumor cells or tumor antigens has generally been ineffective due, in part, to immunoincompetence and the universal suppressive stated commonly found in these individuals. Therefore, there is a need in the art for novel and successful ways to modify or modulate the host-tumor relationship to induce an increased response to the tumor or tumor load, to infected cells, and to cells displaying non-self antigens.

There is also a need in the art for successful immunotherapeutics and immunotherapy techniques which result in reduction of tumor mass, and, ultimately, in the complete eradication of tumor or infected cells. Currently there is a great disparity between the availability of potential new immunotherapeutics and the relative lack of diagnostic assays by which to select and monitor patients. For example, a clinical study with immunotherapy for a particular malignancy or tumor may demonstrate that only a small percentage of patients actually respond. The practical use of this new immunotherapeutic treatment would be much improved if the physician could prospectively identify those patients who are most likely to respond to particular immunotherapy treatments. In addition, treatment with some types of immunotherapy can cause severe toxicity and there has been some concern expressed about whether such treatments can or should be applied to the broad population. The unfavorable risk-benefit ratio that argues against applying immunotherapies indiscriminately to every patient with cancer would be significantly improved if the therapy could be targeted at the "likely-to-respond" population of patients who could be previously identified through a suitable prognostic and/or diagnostic assay. Because immunotherapy offers medical treatment of great precision and specificity, it demands equally customized and precise prognostic and/or diagnostic assays that can describe the specific pathophysiology in an individual patient.

The present invention provides immunotherapy methods and applications which are prognostic, therapeutic, and prophylactic. The invention fulfills the grave needs of ameliorating and augmenting the immune response to foreign or non-self antigens in and/or on cells in both a therapeutic and a prophylactic manner, as detailed hereinbelow.

SUMMARY OF THE INVENTION

The present invention provides a method and treatment to modify tumor or infected or otherwise transformed cells to augment the immune response so that the cells of the immune system are stimulated toward more efficient recognition and eradication of the tumor or infected or otherwise transformed cells, particularly after immunotherapy involving the use of such modified cells. Cells to be modified or treated are non-normal or pathogenic cells, for example, tumor cells or infected or transformed cells. Transformed cells may also include some virus- infected cells, bacteria-infected cells, parasite-infected cells, or cells otherwise altered away from their normal state due to cancer or to tumorigenic or malignant transformation by a foreign pathogen or other endogenous or exogenous transformation-inducing agents. The cells treated in accordance with the invention may be used as immunogens to elevate the immune response to the nonmodified counterparts of the modified cells both in vitro and in vivo.

The modification resulting in heightened immune response is the treatment of cells with a crosslinking effect amount of a crosslinking agent and with hydrostatic pressure in a particular pressure range, preferably at the same time, as described herein. For convenience, the term "PCL-treated" or "PCL- modified" cells is used herein and means "pressure and crosslinker treatment or modification" of cells in accordance with the invention. The invention also provides the above-described modification process to render tumor or infected cells more recognizable and immunogenic to the cells of the immune system.

Another object of the invention is to provide modified whole cells, or components thereof, i.e., plasma membranes and membrane proteins, to increase and stimulate the immune response to tumor antigens and other foreign antigens presented by cells at the cell surface. The immune cell stimulation may occur ex vivo; the effector immune cells may be isolated from an individual, incubated with the modified cells, or components thereof, to activate the effector immune cell populations, and the activated effector cells may be re-introduced into the individual to carry out an immune response in vivo.

It is yet another object of the invention to provide novel immunogens for therapeutic use in augmenting the immune response against tumors, transformed cells, and the like, to allow for the destruction or alleviation of the tumor or foreign cells in the host.

A further object of the invention is to provide a method for treating a tumor in a tumor-bearing individual, comprising sensitizing immune cells to the cell surface antigens presented by the tumor and introducing the sensitized immune cells to the individual to eradicate the tumor.

Yet another of the invention is to provide a method for treating a tumor in a tumor-bearing individual, comprising immunizing the individual with cells modified in accordance with the invention to augment the individual's immune response to the tumor cells.

Another object of the invention is to use the above-described immunogen comprising modified whole tumor cells, immunogenic plasma membranes or proteins derived therefrom, as a prophylactic or a therapeutic vaccine capable of inducing a specific anti-tumor immune response.

A further object of the invention is to screen and monitor patients by the pressure-crosslinking technique in combination with in vitro sensitization assays. In this manner, candidates for in vivo immunotherapy can be screened, if desired, for the ability of their PCL-modified tumor cells or infected cells or transformed cells to stimulate autologous mononuclear blood cells in an in vitro sensitization assay, prior to immunoadoptive therapy or vaccination.

Thus, it is also an object of the invention to determine a "likely-to- respond" population of individuals whose reasonable likelihood of positive and successful response to their tumors or infected or transformed cells during the course of immunotherapy can be assessed in an objective and reliable manner. In this way, it can be determined or predicted which individuals will ultimately benefit from in vivo immunotherapy treatments and will produce a cell mediated immune respond to reduce or eradicate tumor cells, cancer cells, or infected cells.

Yet another object of the invention is to use the results obtained from the in vitro sensitization assays combined with cytokine secretion by a patient's peripheral blood mononuclear cells as a method of determining or resolving whether a cancer or tumored patient should undergo chemotherapy or immunotherapy treatment regimens to eradicate the cancer or tumor.

Another object of the invention is to provide a reliable and objective clinical application of PCL-modified tumor or infected cells in cancer or infected patients by determining the immunogenicity of PCL-modified human tumor or infected cells in a functional test performed ex vivo.

It is another object of the present invention to provide a novel immunogen capable of inducing an anti-tumor immune response. More specifically, it is an object of the present invention to provide such an immunogen which is derived from modified whole tumor cells, membranes thereof, or immunogenic proteins obtained from the modified cells and/or cell membranes.

It is another object of the present invention to provide a process for preparing said immunogen. It is a further object of the present invention to provide a vaccine comprising said immunogen.

It is a still further object of the present invention to provide a method of treating a tumor comprising immunizing a tumor patient with such an anti-tumor vaccine.

Further objects and advantages afforded by the invention will be apparent from the detailed description hereinbelow.

DESCRIPTION OF THE DRAWINGS

In describing the invention, reference will at times be made to the accompanying drawings in which:

Fig. la-le depicts histology of the delayed-type hypersensitivity reaction (DTH) in the ear of mice challenged with 10³ irradiated syngeneic EL4 leukemia cells in the ear following priming with an immunogenic preparation: a. unprimed: b. primed with unmodified EL4 cells; c. primed with AdA treated EL4 cells; d. primed with pressure treated Ε1Λ cells; and e. primed with AdA and pressure treated EL4 cells. Fig. 2 is a graphic representation of an experiment in which the cytotoxicity of anti-EL4 effector lymphocytes against EL4 target cells at various effector: target (E:T) ratios has been tested, using various anti-EL4 effector lymphocyte preparations obtained from mice treated as follows: non-immunized mice (Treatment I); mice immunized with untreated EL4 cells (Treatment II): mice immunized with AdA-treated EL4 cells (Treatment HI); and mice immunized with pressure and AdA-treated EL4 cells (Treatment IV).

Fig. 3 is a graphic representation of a similar experiment to that shown in Fig 2. in which the cytotoxicity of anti- ARadLV 136 effector lymphocytes against ARadLV 136 target cells at various effector: target (E:T) ratios has been tested using various anti-ARadLV 136 effector lymphocyte preparations obtained from mice treated as follows: non-immunized mice (Treatment I): mice immunized with untreated ARadLV 136 cells (Treatment II); mice immunized with AdA-treated ARadLV 136 cells (Treatment HI); and mice immunized with pressure and AdA-treated ARadLV 136 cells.

Fig. 4 is a graphic representation of results of a reciprocal assay in which the cross- reactivity of anti-tumor effector lymphocytes has been tested: effector cells: from mice immunized with pressure and AdA-treated ARadLV 136 cells; target cells: EL4 cells (filled circle). Effector cells: obtained from mice immunized with untreated EL4 cells; target cells: ARadLV 136 cells (empty triangle). Effector cells: obtained from mice immunized with AdA-treated EL4 cells; target cells: ARadLV 136 cells (filled triangle). Effector cells: AdA-treated ARadLV 136 cells: target cells: Ε1A cells (filled squares). Effector cells: AdA and pressure-treated EL4 cells; target cells: ARadLV 136 cells (empty squares).

Fig. 5 is a graphic representation of an experiment in which the survivability of mice challenged with tumor cells after immunization with one of the following preparations was tested: EL4 cells treated with various levels of hydrostatic pressure in the presence of 40 mM AdA (filled squares); EL4 cells treated with increasing level of hydrostatic pressure and then with 40 mM AdA (empty squares); B16 melanoma cells treated with various levels of hydrostatic pressure in the presence of 40 mM AdA (filled circles) and B16 melanoma cells treated with hydrostatic pressure and then with 40 mM AdA.

Fig. 6 shows the survivability of mice challenged with tumor cells following immunization with one of the following preparations: EL4 leukemia cells treated for 10 min with hydrostatic pressure of 1350 atm in the presence of various concentrations of AdA (filled squares); B16 melanoma cells treated in the same manner (filled circles); B16 melanoma cells treated in the same manner with AMPdA (empty circles).

Fig. 7a and 7b show a three-dimensional overlay of antigen expression on B16-BL6 melanoma cell plasma membrane surface as analyzed on FACScan by indirect immunofluorescence: A & B - negative controls (A - autofluorescence; B -cells reacted only with secondary antibody); C - unmodified cells; D & X - cells exposed to 20 Mm AdA; E & Z - cells exposed to hydrostatic pressure of 1 ,200 atm for 15 minutes; F & Y - cells exposed simultaneously to both 20 Mm AdA and 1 ,200 atm pressure. Fig. 7a: fluorescence results using anti-MHC class I (Kb + Db) antibody; Fig. 7b: fluorescence results using anti-B16 antibody. Fig. 8 shows the effect (as per cent of positive cells counted by FACScan instrument out of 10,000 events) of graded levels of hydrostatic pressure (applied for 15 minutes) combined with a constant dose of 20 Mm AdA on antigen expression in B16-BL6 melanoma cells; filled bars - class I antigen, gray bars -B16 antigen.

Fig. 9 shows the effect (per cent positive as determined on FACScan out of 10,000 events) of varying concentrations of AdA combined with a constant level of hydrostatic pressure (1200 atm, 15 minutes) on antigen expression in B16- BL6 melanoma cells: black bars - MHC class I antigen, gray bars - B16 antigen.

Fig. 10 shows the results of delayed type hypersensitivity assays performed in humans using both PCL-modified and unmodified autologous tumor cells as immunogens.

Fig. 11 shows the results of delayed type hypersensitivity assays performed in humans using both PCL-modified and unmodified allogeneic tumor cells as immunogens.

DESCRIPTION OF THE INVENTION

In accordance with the present invention it was found that the immunogenicity of an immunogen derived from treated tumor cells was augmented by exposing tumor cells to the crosslinking action of a chemical crosslinking agent, which is a 2' , 3'- nucleoside or nucleotide dialdehyde (referred to herein as "crosslinker" or "crosslinking agent" or "crosslinking compound").

In addition, it was found in accordance with the invention that tumor-specific immunogenicity was augmented by subjecting the cells to a combined treatment of exposure to both crosslinking agent and hydrostatic pressure. It was also found that this immunogenicity was even further augmented if exposure of cells to crosslinking agent and to hydrostatic pressure was done simultaneously. Simultaneous refers to the crosslinking of cells at the time that they are exposed to hydrostatic pressure.

One aspect of the present invention provides an immunogen derived from modified tumor cells and capable of inducing an anti-tumor immune response, wherein the modified tumor cells have been prepared by exposing tumor cells to the crosslinking agent at a concentration and for a time sufficient to cause crosslinking of proteins in the cells' plasma membranes.

In a preferred embodiment of the present invention, the modified tumor cells are prepared by exposing tumor cells to both the crosslinking agent and to hydrostatic pressure at a level and for a time sufficient to cause displacement of proteins in the cells' plasma membranes.

In a more preferred embodiment of the present invention, exposure to the crosslinking agent and to hydrostatic pressure is done at the same time. The concentration of the crosslinking agent is in the range of about 1 Mm to about 40 Mm, preferably about 5 Mm to 20 Mm, and more preferably about 10 Mm to about 15 Mm. The hydrostatic pressure is within the range of about 800 to about 1400 atmospheres (atm), preferably about 1000 atm to about 1200 atm.

Suφrisingly, pressure above about 1400-1500 atm or greater yields an immunogen having a far inferior anti-cancer immunization potency. The application and release of pressure is preferably gradual, e.g. , over a period of about 5 to 15 minutes.

In addition, the crosslinking agent is preferably a 2', 3' - dialdehyde of a natural nucleotide or nucleoside, since non-naturally occurring, i.e. synthetic, nucleosides or nucleotides are very often highly toxic.

The preferred crosslinking agents are represented by the following formula I:

CH HC

//

0 0

wherein, R is H, or a mono-, di- or tri-phosphate group, and

B is a nucleotide base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil. Examples of such crosslinking agents are 2', 3'- adenosine dialdehyde (AdA) and 2', 3'-adenosine monophosphate dialdehyde

(AMPdA).

The compound of formula I may be prepared by reacting a nucleoside or a nucleotide of the following formula II:

wherein R and B have the meanings given above for formula I, with an oxidizing agent, e.g. an alkali periodate.

The immunogen may consist of the whole modified tumor cells, membranes derived from such cells, as well as proteinaceous material (e.g. , membrane proteins and fragments thereof) obtained from such cells or membranes which substantially retain the capability of the modified tumor cells to induce the anti-tumor immune response.

Preferably, after the treatment in accordance with the invention, the modified tumor cells are exposed to high intensity radiation in order to destroy their genetic material. This is particularly important where the whole modified tumor cells are used for immunization and may not necessarily be required where said immunogen consists of cell membrane preparations or proteins or membrane and protein fragments.

In another aspect, the present invention provides a process for preparing an immunogen derived from modified tumor cells and capable of inducing an anti-tumor immune response, said process comprising the steps of: a) providing tumor cells; b) incubating the cells with said crosslinking agent at a concentration and for a time sufficient to obtain crosslinking between membrane proteins; and c) removing the crosslinking agent from the treated cells.

It will be appreciated that the above-described method applies as well to cells which are otherwise non-normal due to viral or microorganismal infection, or to another type of change or cell transformation.

The above process preferably comprises also exposing the tumor cells to hydrostatic pressure at a level and for a time sufficient to cause displacement of proteins in the plasma membranes of the cells. The hydrostatic pressure is preferably applied during the incubation step of the cells with said crosslinking agent (step b).

Where the desired immunogen consists of the whole modified tumor cells, the product of the above process may be used per se or after several purification treatments, e.g. , consisting of centrifugation and removal of the supernatant.

Where the preparation consists of membranes of such modified tumor cells, the modified tumor cells are subjected to further treatment in which the cells are disrupted, e.g. by exposure to a hypotonic medium or by sonication. and then the membrane fragments are collected e.g. by centrifugation in a sucrose gradient, as known in the art and as generally described in Example 11.

Where the desired immunogen consists of protein material (e.g., proteins or protein fragments), the whole modified cells or the plasma membranes are subjected to further treatment, for example, dissolving or solubilizing the membranes using detergents, separating the proteinaceous material by one of various methods conventionally known, e.g., gel filtration, and then determining which of the separated proteins and/or proteinaceous material fragments possesses the desired immunogenicity.

The immunogen may be used for the immunization of cancer patients against their tumor or may be used for the sensitization and proliferation of immune cells in vitro (i.e. , in in vitro sensitization or "IVS" assays). For immunization, the immunogen may be injected into a patient together with a pharmaceutically acceptable carrier or adjuvant in an amount sufficient to achieve an anti-cancer or tumor immune response.

For in vitro sensitization, peripheral blood mononuclear cells, including immune cells, e.g., leukocytes or lymphocytes, are withdrawn from the patient by known methods and are then cultured together with the immunogen until a population of such immune cells reactive against said immunogen is obtained (see Examples 6 and 14). Such a stimulated population of immune cells may then be reinjected into a cancer patient in order to treat his/her tumor.

In another aspect, the present invention thus provides a vaccine composition comprising the immunogen and a pharmaceutically acceptable carrier. By a still further aspect, the present invention provides a method of treatment of a cancer or tumor comprising injecting a cancer or tumored patient with the immunogen or with the sensitized immune cells as described. While the immunization of patients in accordance with the present invention can be performed by the use of an allogeneic immunogen, it is preferably performed by the use of an autologous immunogen. The use of an autologous immunogen provides significant advantages in that the immune response which occurs is primarily directed against the neo-antigen of the tumor. When an allogeneic immunogenic preparation is used, the resulting immune response will be against all of the "non-self or foreign antigens of such an immunogen. The use of an autologous immunogen has the further advantage in that the neo-antigens associated with a specific tumor may differ from one patient to another. However, allogeneic immunogens provide significant immune responses against PCL- modified allogeneic cells in humans as demonstrated in Example 7 and shown in Fig. 10.

Where an autologous preparation is used, the method comprises the steps of: a) withdrawing tumor growth from a patient by biopsy or surgery; b) dissociating intact tumor cells by mechanical or enzymatic means; c) dispersing the cells in a medium; d) incubating the cells with 2' , 3'-adenosine dialdehyde (AdA) in a concentration and for a time sufficient to cause crosslinking of proteins in the cells' plasma membranes; e) removing the AdA and preparing a tumor-specific immunogen derived from the modified cells obtained; and f) injecting the immunogen into the patient, whereby an anti-tumor immune response in the patient is induced.

In a preferred embodiment, step d) also includes exposing the cells to hydrostatic pressure between about 800 and about 1400 atmospheres, preferably about 900 and about 1200 atmospheres, more preferably, about 1000 atmospheres, at the same time that the cells are exposed to the 2', 3 '-nucleoside or nucleotide dialdehyde crosslinker. As an alternative, step f) may involve providing the modified tumor cells in an in vitro sensitization assay with immune cells to generate stimulated, sensitized immune cells, i.e., leukocytes and lymphocytes, which will react against and ultimately destroy the tumor cells following injection in vivo.

Where an allogeneic immunogen is used, an immunogen derived from modified tumor cells obtained from a defined tumor cell line may be used. In addition, modified tumor cells obtained from the same tumor type from another source or donor individual may be used with equal success.

As described and in accordance with other aspects of the invention, the PCL-modification technique addresses a formidable problem in the art concerning ways in which to stimulate, increase, and improve a host animal's immune response to tumor cells or otherwise transformed, infected, or foreign cells, by providing modified cells which present tumor and foreign antigens to a host in the context of MHC molecules for appropriate recognition and destruction by the effector cells of the immune system. The modified cells, or membranes or proteins derived from the cells, are used as immunogens or in immunogenic preparations to immunize both naϊve animals, including humans, as well as to treat tumored, infected, or diseased animals, including humans, which have previously encountered the tumor or foreign antigens. When used as immunogens in vivo, the modified cells or cell preparations may include pharmaceutically acceptable carriers, excipients, or formulations, such as normal or buffered saline and the like; 'classical' adjuvants and emulsions, such as alum, incomplete Freund's adjuvant, and the like; and additional immunostimulants, such as 3-deacylated monophosphoryl lipid A (3-D-MPL), should these be needed or desired.

Monophosphoryl lipid A can be suspended in either saline or in oil- water emulsion. Additional components of the immunogenic preparations can include, for example, sugars such as lactose, dextrose, and the like, and merthiolate, provided that the immunogenicity of the modified cells is not adversely affected.

In addition to the use of the above-described classical adjuvants, another aspect of the invention comprises the use of other, 'non-classical' adjuvants which are co-injected in vivo or are formulated into the immunogenic preparation or vaccine comprising PCL-modified cells, cell preparations, or plasma membranes to enhance the immune response (see Examples 16 and 17). Nonlimiting examples of such non-classical adjuvants, or mixtures and combinations thereof, that can also be injected with PCL-modified cells or membranes include human growth hormone (hGH), hematopoietic cell stimulating factors such as granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G- CSF), and the like, Bacillus calmette guerin or BCG, saponins, T cell stimulating or activating factors, such as OKT3, TNF-or, and the like, interleukins, e.g., IL-1 to IL-16, and interferons, e.g. , alpha, beta, and gamma interferons. Those skilled in the art will appreciate that the use of such non-classical adjuvants, e.g. , GM- CSF or hGH, will ultimately allow conservation of the number of PCL-treated cells that are used as immunogens in accordance with the invention. In particular, the use of adjuvants is likely to allow a reduction in the number of tumor or infected cells required for use in immunogenic preparations, especially in combination with

10 one or more adjuvants in amounts determined to provide an effective and enhanced immune response, for example, as deteπnined by DTH assay, Example 17. It will also be understood by those skilled in the art that combinations or mixtures of adjuvants (e.g. , GM-CSF together with hGH, as a nonlimiting example) may be

15 used in conjunction with PCL-based immunogens to augment the immune response. In addition, for those compounds which may affect cells by different biochemical or biological pathways, for example, GM-CSF and hGH, the use of a combination of adjuvants may result in a synergistic effect, thereby providing a more

-,„ pronounced augmentation of the immune response to the immunizing preparation. Non-classical adjuvants may be administered in doses which one skilled in the art may determine by known methods and protocols to be efficacious in augmenting the immune response. As an exemplary guide, non-classical adjuvants may be administered in the range of about 1 g to 1 mg per injection, preferably about 100

25 μg to about 400 μg, and more preferably about 50 μg to 200 μg. GM-CSF in particular may be administered in the range of about 1 to 500 μg per injection, preferably about 100 to 200 μg per injection, and more preferably about 50 to 100 μg per injection. As another example, human growth hormone may be

30 administered in the range of about 0.01 to 0.3 mg/kg of body weight, preferably about 0.05 to 0.1 mg/kg.

As will be apparent to those of skill in the art, the doses or amounts of modified cells for use as immunogens will be determined by the skilled 5 - 22 -

practitioner and will depend upon the types of cells used, the tumor load, if applicable, and the overall condition and immune status of the patient. As a general guide, an immunogenic formulation is prepared comprising PCL-modified cells in the range of at least about 1.0 x 10^s to 1.0 x 10⁶ or more (e.g., to about 20.0 x 10°) per immunizing dose, in admixture with saline or other excipients known in the art. Although fewer and more than about 10° cells may be used, a suitable number of cells is about 1-2 x 10⁶ for an immunizing dose. Those skilled in the art can routinely determine the appropriate cell number for an immunizing dose, depending on the type of tumor or infection under treatment. A greater number of modified cells formulated in the immunogenic preparation may increase or heighten an individual's immune response to his/her tumor or infection. Normal modes of administration, e.g., intravenous, subcutaneous, intradermal, intramuscular, sublingual, intraperitoneal, percutaneous, intrathecal, intracutaneous, or enteral, may be used with the immunogenic compositions afforded by the invention; the preferred routes of immunization are intradermal and intravenous. In addition, local administration to the afflicted site may be accomplished through means known in the art, including injection and implantation. As explained supra, the term modified as used herein means cells treated in accordance with the methods of the invention. The terms tumor cell, transformed cell, cancer cell, and infected cell refer to cells which contain, display or present on their surfaces foreign protein or peptide antigens to the cells of the immune system, regardless of whether the presented antigens are autologous or allogeneic to the host. The term antigen can refer to a protein or peptide structure, molecule, complex, or component thereof, that is generally recognized as foreign or non-self by cells of the immune system. It will be appreciated by those in the art that an antigen may be an epitope or determinant comprised of a series of amino acid residues, e.g., comprising from about 3 to about 7 residues, or from about 5 to about 10 or more residues, that are recognized or bound by immune cells due to their particular configuration and/or conformation characteristics. The method of the invention encompasses cells which have in some way become distinct from normal cells due to genetic or in vivo events, or to exogenous events or agents, resulting in cancer cells, tumor cells, non-normal or non-self cells, or cells causing another type of pathogenic or disease condition in an animal, including humans.

Examples of cells which may be modified by the PCL methods of the invention include, but are not limited to, all types of tumor or cancer cells of various origin, including, but not limited to, cells derived from pancreatic tumors, ovarian tumors, melanomas, sarcomas, breast tumors, colon cancers, lung cancers (e.g., mesotheliomas), small-cell lung carcinomas, non-small cell lung carcinomas, liver or renal cancers, bladder cancers, prostate cancers, tumors and cancers of hematopoietic origin (e.g. , lymphomas and leukemias), and the like; as well as virally-infected cells of different origins, including those of the lymphoid and hematopoietic system, including virally-infected T and B cells, macrophages, neutrophils, and the like. Viruses include DNA and RNA viruses which infect and/or reside in cells of various types and which may be oncogenic. Non-limiting examples of the types of virally-infected cells that are suitable for PCL modification and used in the invention are human immunodeficiency virus (HIV)- infected cells of various strains (e.g., HIV-1 and HIV-2), human T-lymphotropic virus-infected cells, Herpes simplex virus-infected cells, Epstein-Barr virus-infected cells, Hepatitis A, B, C, D, and E-infected cells, and the like. The invention is not limited to cells at any particular stage in development. The various cell types may also be transformed, infected, or otherwise altered by a variety of endogenous or exogenous means, such as by genetic mutations or abnormalities, exposure to chemical or physical carcinogens, infection by viruses, intracellular bacteria, parasites, or other disease-causing agents, thereby resulting in cells, e.g. , tumor cells or cancer cells or infected cells, that are transformed or moφhologically changed away from their normal cell counteφarts. Examples of the types of bacterially- infected cells for use in the invention include, but are not limited to cells infected with the mycobacteria that cause leprosy, E. co/Hnfected cells, S. αurews-infected cells, Streptococcus-infected cells, Shigella dysenteriae-infected cells, Borrelia burgdorferi-infected cells, Bacillus tuberculosis-infected cells, meningococcal-infected cells and the like. Non-limiting examples of types of parasites that can infect cells and that can be treated and used in accordance with the invention are malaria, leishmania, and schistosomes, and the like.

Tumor cells, cancer cells, and otherwise transformed cells may lose their regulated proliferative state due to the acquisition of means by which to cause disease, grow uncontrollably, and invade distant tissue. Such cells may also display unique or characteristic tumor associated antigens on the cell surface and the processed antigens may be complexed with class I or class π MHC structures for presentation to the effector cells of the immune system. Similarly, cells transformed by viruses, bacteria, parasites, and the like, may also present processed antigens, peptides, proteins, or structures of the foreign pathogen, microorganism, or other infectious agent (e.g. , prions), complexed with class I or class π MHC molecules for presentation to the effector cells of the immune system. In an optimal situation, such foreign antigens are presented by or complexed or associated with MHC class I structures on the surfaces of infected or transformed cells for recognition and efficient killing by cytotoxic T cells. In another aspect of the invention, human tumor cells exposed to crosslinker and pressure in accordance with the invention were found to have increased and specific immunogenicity, as tested and monitored by novel in vitro sensitization (TVS) assays using human peripheral blood mononuclear cells (PBMCs or PBM cells). PBMCs are comprised of approximately 90% lymphocytes and are also referred to herein as peripheral blood lymphocytes or PBLs. In particular, mononuclear blood cells taken from the same patient whose tumor cells were previously excised and PCL-treated served as the responders in the IVS assay (see Examples 2, 6, and 14). These results showed that PCL-modified human tumor cells could sensitize and stimulate autologous human lymphocytes in an IVS assay. In accordance with this aspect of the invention, autologous cells which respond to tumor cells, and autologous cells which do not respond to tumor cells (i.e. , responder PBM cells versus nonresponder PBM cells) can be determined by PCL- modification combined with the use of the IVS assay.

Thus, this aspect of the invention provides a functional ex vivo assay for determining the immunogenicity of PCL-modified human tumor cells and the ability of an individual's mononuclear blood cells to respond to or immunoreact against the modified tumor cells. As will be described further hereinbelow, a correlative parameter that is used to determine an individual's immune response status is the cytokine profile or pattern secreted by stimulated versus unstimulated mononuclear cells. The cytokine profile as determined by measuring the levels of cytokine secreted by PBMCs indicates if an individual has produced a cell mediated immune response or a T_H2 to T_H1 switch. Based on a successful immune cell response to PCL-modified tumor cells (or to other foreign cells that have been PCL-modified) relative to unmodified cells, it can be reasonably predicted that when such modified cells are used in immunogenic preparations to immunize patients whose PBM cells were assayed in accordance with the invention, a positive immunoreactive response to the corresponding tumor or foreign cells will be also mounted and observed in vivo. It will also be appreciated by those in the art that, in addition to determining the levels of cytokines that are secreted by PBMCs, the cytokine pattern can also be determined by analyzing the cytokine messenger RNA levels by methods known in the art, i.e. , PCR and RT-PCR.

By a successful or positive in vitro immune cell response is meant that immune cells are sensitized and are immunoreactive against the PCL-modified target cells (e.g., PCL-modified tumor cells or infected cells) in an IVS assay, such that the immune cells proliferate or otherwise react against the modified cells. Accordingly, and as described further hereinbelow, a high stimulation or response index is achieved (i.e. , greater than or equal to about 1.5 to 2.0, or above, preferably greater than or equal to 2.0). A successful or positive immune cell response is also determined by the ability of sensitized immune cells to react against the appropriate target cells in a cytotoxic assay. Thus, this aspect of the invention provides an assay or protocol the results of which are predictive of in vivo efficacy and are indicative of the likelihood of success in using PCL-modified cells as immunogens in vivo in human patients who will be most likely to mount an immune response to their tumor or infection. This is an essential step toward the clinical application of PCL-modified tumor cells in cancer patients presenting with various tumor types, for example, or in infected patients presenting with infections caused by a variety of viruses or microorganisms.

In a related aspect, the invention provides a prognostic application and allows the evaluation of potential selection criteria for determining those individuals who may be responders and those who may be non-responders to the PCL-modification treatment, IVS assay, and eventual immunotherapy or immunoadoptive therapy using PCL-modification of tumors, and the stimulation, sensitization, and immunoreactivity of human immune cells (or PBMCs) in vivo. The term "PCL- immunotherapy" as used herein refers to the immunization or vaccination of a patient with PCL-modified cells, e.g. , tumor cells or infected cells, to elicit a specific immune response (i.e., an infected cell- or tumor cell- inhibiting or reducing response) against the tumor cells or the infected cells.

The technique of immunoadoptive therapy is known among those having skill in the art; a description of the technique is found in U.S. Patent No. 5,192,537 to Michael E. Osband. In brief, in immunoadoptive therapy, the peripheral blood mononuclear cells of a patient afflicted with a cancer or tumor or infection are treated to activate their responsiveness to one or more antigens associated with the tumor, cancer, or infecting agent. Mononuclear cells are first obtained from the patient's blood, e.g., peripheral blood. If necessary or desired, suppressor T lymphocytes are then removed and the remaining cells are suspended in a tissue culture medium containing a non-specific lymphocyte activator (e.g., phytohemagglutinin). The mononuclear cells may also be incubated with an extract of the patient's tumor or infected cells, preferably tumor or infected cells that have been PCL-modified in accordance with the invention, and with the patient's own serum. The cells are then incubated, preferably under hyperthermic conditions (e.g. , about 38°C to about 41°C) for a period of time so that they are activated against the patient's modified autologous tumor or infected cells (i.e. , in an IVS assay). If desired, after in vitro sensitization, the activated mononuclear cells may be subjected to one or more additional procedures to re-deplete various kinds of suppressor cells, including macrophages, to additionally boost their activity. Such depletion methods are known in the art and include the use of gamma radiation (e.g., in the range of 50-400 rad) to remove radiosensitive suppressor cells. The activated cells are then re-infused or administered to the patient using established procedures known to the skilled practitioner in order to reduce or eliminate the tumor or infected cells, to inhibit tumor growth, or to reduce or eliminate recurrences of cancer or infection. Additional anti-T cell suppressor drugs or agents (e.g. , cimetidine) may be used in carrying out the immunoadoptive therapy protocol, if necessary or desired.

Another embodiment of the invention provides selection criteria for the screening and monitoring of patients for immunocompetence and immuno- reactivity to tumors, infection, and to foreign or pathogenic cells using a combination of PCL-modification of cells and IVS techniques. In accordance with this embodiment, candidates for PCL-immunotherapy undergo screening for the ability of their PCL-modified tumor cells to stimulate their autologous peripheral blood mononuclear cells to become sensitized and activated against, and to proliferate in response to, PCL-modified antigens in an IVS assay. It is to be understood that PCL-modified autologous and allogeneic cells, e.g., tumor cells or infected cells, may be used to stimulate an individual's PBMCs in the IVS assay. If the allogeneic cells are tumor cells, they should ideally be of the same tumor type as the patient's tumor. Similarly, if the allogeneic cells are infected cells, they should infected with the same infective agent as the patient's infected cells, and preferably be of the same cell type.

A criterion discovered and employed for determining those individuals who would be most likely to respond to PCL modification and immunotherapy techniques employing PCL-modified cells as immunogens or vaccines is termed the IVS response or stimulation ratio, also called the response or stimulation index (RT or SI) herein (see Example 14). In accordance with the invention, the response ratio is determined from the quantitative measurement of a patient's PBMC proliferative response to PCL-modified tumor cells (numerator) in relation to the measurement of the patient's PBMC proliferative response to non- modified or "native" tumor cells (denominator). As an exemplary guide, patients whose IVS response ratio is less than about 1.5 to about 2.0 are less likely to respond to subsequent PCL-immunotherapy than patients whose response ratio is equal to or exceeds about 1.5 to about 2.0. Patients who have a response or stimulation index value greater than or equal to about 1.5 to about 2.0 represent a patient population showing a high level or positive response to the PCL-modified tumor or infected cells.

Accordingly, in a preferred embodiment, an SI value of 2.0 or above generally correlates with an exceptionally good immune activation response, and an SI value of less than 1.5 to 2 generally correlates with a poor immune activation response. In addition, the ability to determine whether an individual will have a good or a poor cellular immune response and/or a switch from T_H2 to T_H1 cell production is improved upon by also analyzing the corresponding cytokine synthesis and secretion of the peripheral blood cells in the IVS assay. Thus, in general, those patients having a low stimulation or response ratio and a cytokine profile indicating a lack of a T_H2 to T_H1 switch using PCL-modified and unmodified cells as stimulators are considered to be poor responders and, consequently, such patients are not likely to benefit from subsequent PCL- immunotherapy, based on the determined response or stimulation ratio and the cytokine pattern. By contrast, patients having a high ratio and a cytokine pattern that is indicative of a T_H2 to T_H1 switch are considered to be good responders to the tumor or infected cells and are determined to be likely to benefit from subsequent immunotherapy, particularly PCL-based immunotherapy. Additional control studies to determine the general immune responsiveness or immune status of a patient's PBMCs can be performed in accordance with the method using mononuclear cells isolated from the patient's blood. In these control IVS assays, which are optimally performed in accordance with the invention, the responsiveness of a patient's PBM cell population to stimulation by mitogenic agents, nonspecific activators, or cytokines such as phytohemagglutinin (PHA), pokeweed mitogen, or anti-CD3 antibody (e.g., OKT3) is evaluated (see Example 14).

In accordance with a method of the invention particular to a patient beset with a tumor, a baseline IVS analysis is performed as described herein using a patient's PBMCs and his or her autologous tumor cells within about one to two weeks following tumor surgery (see Example 14). Based upon the immune response level of the patient's own PBMCs to his or her PCL-modified tumor cells in the IVS assays performed as described, reliable and objective determinations can be made concerning whether or not an individual patient would be likely to benefit from subsequent PCL-immunotherapy, and whether or not an individual will or will not be enrolled in subsequent PCL-immunotherapy procedures. As an example, a protocol used for tumor patient assessment in accordance with the invention is as follows: after tumor surgery, a patient may undergo either chemotherapy or radiation regimens, or combinations thereof. During this time, the IVS assays as described herein are carried out using as stimulators the tumor cells processed from the patient's tumor, and as responders the patient's PBMCs. About a month after recovery from either the chemotherapy or irradiation post- surgery adjunct treatments, PCL-immunotherapy is begun, if the IVS assays which were performed following surgery showed a high level of response by the patient's PBMCs toward the autologous PCL-modified tumor cells.

Examples of selection criteria used for determining those patients to include in tumor treatment involving PCL-modification of tumors, IVS assay, and PCL-immunotherapy or adoptive immunotherapy techniques include, but are not limited to, selecting patients having certain cancers or tumors, such as colorectal carcinomas, non-small cell lung carcinomas, renal carcinomas, and ovarian carcinomas, who undergo tumor resection surgery. Nonlimiting exclusion criteria for excluding patients from subsequent PCL-immunotherapy or immunoadoptive therapies include those patients undergoing chemotherapy or radiotherapy for up to four weeks prior to surgery; patients undergoing any experimental therapy known or intended to improve immune status; and patients greater than or equal to 80 years of age.

The results obtained using the IVS assay and shown in Example 14 indicate that, unlike unmodified native tumor cells, PCL-modified tumor cells are potent stimulators of immunoreactivity by an individual's autologous lymphocytes. Those skilled in the art will also appreciate that PCL-modified cells, e.g., tumor cells, in combination with IVS assays, can be used to clonally expand discrete and specifically immunoreactive populations of peripheral blood mononuclear cells, in particular, lymphocytes, e.g., T cells, which can be harvested and used in immunoadoptive therapy protocols. For example, for clonal T cell expansion, the following exemplary procedure can be performed: following the IVS assay as described, activated T cell populations are enriched and/or separated, and expanded from the patient's mononuclear cells using methods known to those skilled in the art. T cell enrichment and separation procedures include, but are not limited to, flow cell cytometry or cell sorter methods, nylon wool column separation, antibody or lectin column chromatography designed to isolate or separate discrete T cell populations based on surface antigen or receptor specificities, and B lymphocyte removal using specific antibodies and complement. Following the T cell enrichment procedures, the activated T cell population can then be cultured in the presence of T cell specific or nonspecific cytokines or activators, or mixtures thereof, (and additional modified tumor cells as in vitro "boosting" antigen) to cause the proliferation and growth of the so-selected immunoreactive and enriched activated T cell population. Thereafter, the proliferating and immunoreactive T cell population can be used to immunize a patient against his or her tumor in immunoadoptive therapy techniques. It is to be understood that the above- described methods, with modifications known to those in the art, may be used to enrich for or separate other types or populations of specifically immunoreactive mononuclear blood cells.

For the purpose of monitoring patients' progress during PCL- immunotherapy employing another method of the invention, patients who receive such immunotherapy should optimally demonstrate a progressive increase in their IVS response ratios as a function of their immunization with PCL-modified, autologous cells as immunogens or vaccines, such that an increase in the IVS response ratio correlates with a patient's therapeutic outcome using the PCL- immunotherapy technique. For example, as the patient is monitored during immunotherapy using modified cells (e.g. tumor cells or infected cells, and the like), the mean IVS response ratio should remain at a value of at least about 1.5 to 2.0, or above, and should optimally increase during the progression of the immunotherapy schedule or immunizing regimen. As a guide, those individuals who do not exhibit a steadily high response ratio or who do not demonstrate an increase in their IVS response ratios after about 4 to 6 weeks of monitoring via the IVS assay are not likely to benefit from the immunotherapy afforded by PCL technology as described. Accordingly, patients undergoing PCL-immunotherapy can be removed from the therapy i) if their response ratios do not stay at or progress to about 1.5 to 2 or above, ii) if their low or negative response ratios do not convert to IVS positivity, or iii) if their response ratios progressively decline. Another aspect of the invention relates to a method of determining whether or not a cancer or tumored patient should receive immunotherapy or other treatment regimens, such as chemotherapy or radiation therapy, following surgery and excision of his or her tumor. The invention also relates to a method for determining whether or not a tumored or cancer patient will have a successful clinical outcome after immunotherapy, particularly, PCL immunotherapy, and whether or not that patient should enter an immunotherapy or adoptive immunotherapy protocol as a treatment regimen for his or her cancer or tumor. These methods are based upon the results of the IVS analysis as detailed above, in particular, the stimulation or response index outcome (RT), combined with the results of in vitro cytokine analysis, to detect whether or not a patient's PBMCs are synthesizing and secreting the appropriate cytokines to generate, support, or augment a cell mediated immune response to immunoreact against and to fight off his or her tumor.

The ability of a patient undergoing PCL immunotherapy to generate and maintain a cytotoxic T cell (i.e., CD8+ cells) and/or a T_H1 immune response (i.e. , cell-mediated immunity) against his or her tumor, or against infected or altered cells, is crucial to the ability of that patient to respond immunologically to the tumor or cancer or infection by producing activated cytotoxic T cells that will ultimately kill the tumor cells or virus-infected cells. As mentioned hereinabove, T_H1 cells produce IL-2, which causes the continued proliferation of specific T lymphocytes or CD8-I- lymphocytes, and also produce IFN-7, which activates the anti-tumor and anti-virus properties of cytotoxic T lymphocytes. In contrast, T_H2 cells (i.e., CD4+ cells) produce IL-4, IL-5. IL-6, and IL-10, which stimulate the proliferation and maturation of antibody-producing B lymphocytes, which are active participants in the humoral immune response to antigen. In addition, the synthesis and release of IFN-7 by T_H1 cells during a strong cell-mediated response may suppress T_H2 activation of B lymphocytes that are responding to the same immunogenic stimulus. Similarly, IL-4 and IL-10 released by T_H2 cells during a strong humoral immune response may actually suppress the ability of T_H1 cells to carry out a successful cell mediated immune response.

In view of the above, it is possible that such cross-regulatory interactions during a patient's in vivo immune response may actually suppress an effective, cell-mediated response to a tumor or viral infection after antibody production is triggered. Thus, the generation and maintenance of a strong cell mediated immune response, a T_H1 response, or a T_H2 to T_H1 switch in patients receiving PCL treatment for their tumor or infection is an optimum situation and endpoint for those patients who will benefit from PCL immunotherapy and/or who will best respond to PCL immunotherapy and treatment as a clinical therapeutic protocol. Such a cell-mediated immune response is best orchestrated by the production of levels of secreted T_H1 cytokines that are stimulatory for or directly linked to a cell-mediated immune response (e.g. , IL-2 or IFN-7) and the production of levels of secreted T_H2 cytokines that are inhibitory for a cell- mediated immune response, or are stimulatory for a humoral immune response (e.g., IL-4, IL-5, IL-6, and IL-10). For example, high levels of T_H1 cytokines secreted by PBLs in response to PCL-modified stimulator cells relative to the levels of these cytokines secreted by PBLs in response to unmodified stimulator cells are indicative of a cell mediated immune response. Also, low or virtually no levels of T_H2 cytokines that are inhibitory for a cell-mediated immune response, or that are stimulatory for a humoral immune response secreted by PBLs in response to PCL- modified stimulator cells relative to the levels of these cytokines secreted by PBLs in response to unmodified stimulator cells are indicative of a cell mediated immune response.

Alternatively, a patient may also convert from primarily a T_H2 immune response to a T_H1 response (i.e. , a T_H2 to T_H1 "switch" or conversion) during the course of in vivo or in vitro PCL treatment involving immunization with PCL-modified cells (e.g., tumors or virus-infected cells). Such a T_H2 to T_H1 switch is accompanied by a cytokine secretion pattern that reflects a T_H1 response and is indicative that the patient will respond appropriately (i.e. , by producing a cell mediated immune response) to destroy tumor cells or virus-infected cells during the course of PCL immunotherapy.

In view of the foregoing, it is clear that in addition to monitoring a patient's IVS response and determining the patient's cell proliferation or response index, it is also a beneficial improvement to determine and measure concomitantly the levels of particular cytokines that are being synthesized and secreted by the patient's PBMCs (responders) as a result of stimulation by PCL-modified cells (stimulators) relative to unmodified cells. As a particular example, given that high levels of IL-10 synthesis and production may suppress the T_H1 response, an optimum finding, which indicates that a patient will respond to tumor or infection in a positive way by generating a cell mediated response, is that the patient is producing low (or virtually no) levels of the IL-10 cytokine in response to PCL- modified stimulator cells (e.g. , tumor or infected cells); thus, the T_H1 response is not likely to be affected or suppressed. Further, since IFN-7 is released by T_H1 cells and is indicative that a cell mediated reponse has been generated against a tumor or infected cell, the finding that a patient is producing high levels of IFN- 7cytokine in response to PCL-modified stimulator cells (e.g. , tumor or infected cells) is also indicative that a positive and successful clinical response to PCL treatment and immunotherapy has been achieved by the patient. The results presented in Tables 12. 13 and 14 of Example 15 demonstrate that in vitro determinations of an individual's IVS response index and the analysis of the individual's cytokine profile parameters after assaying the response of a patient's PBMC cell populations to PCL-modified tumor or infected stimulator cells relative to a response to unmodified stimulator cells offer to the practitioner a way to predict, determine, or support a decision concerning whether or not a patient should undergo (or continue) immunotherapy, in particular. PCL immunotherapy; whether or not a patient will have a positive or successful clinical outcome as a result of PCL treatment and immunotherapy; and whether or not an alternative type of treatment regimen, e.g., chemotherapy or radiation therapy, should be instituted for a patient instead of PCL immunotherapy.

As will be apparent to those in the art based upon the foregoing, a positive PBMC proliferative response, as measured by IVS assays (i.e. , a positive response or stimulation index), is necessary to determine whether or not an individual is responding to vaccination or immunogens comprising PCL-modified cells by producing a cell mediated response and/or a T_H1 response against tumors or infected cells, and the like. However, what is also optimally required to predict if a patient will have a successful clinical outcome involving immunotherapy, particularly PCL immunotherapy, is information concerning both the PBMC proliferative response against PCL-modified tumor or infected cell immunogens and an indication that the appropriate cell types are activated and proliferating in response to the modified tumor or infected cells in the IVS assay, and/or that a T_H2 to T_H1 switch has occurred.

As described hereinabove, the determination of the cytokine profile or pattern that is produced by an individual's activated PBMCs provides the indication and is capable of demonstrating that an appropriate immune response is occurring and is also indicative of a T_H2 to T_H1 pattern or conversion. In addition, the proliferating PBMCs or PBLs cells can be phenotyped using methods known in the art (e.g., B and/or T cell specific antibody markers and assays such as cell sorting, immunolabeling, or immunochemistry protocols) to distinguish the discrete populations and/or subsets of lymphocytes that are responding in the IVS assays. Several commercially-available antibody preparations directed against cell surface markers specific for T cells, e.g.. monoclonal anti-CD8 antibody and monoclonal anti-CD4 antibody preparations, can be used in the immunophenotyping analyses. In this way, immunophenotyping of the proliferating cells can determine if a CD4+ or a CD8+ cell population is being stimulated to respond to PCL-modified tumor or infected cells. In addition, in vitro cytotoxic T cell assays (e.g., see Example 8) can be performed on the proliferating cells in the IVS assays to determine if cytotoxic T cells (CTLs) are proliferating and responding to PCL- modified tumor or infected cells. Accordingly, the present invention allows the determination and prediction of a positive or negative clinical outcome against tumors or infection by using the IVS analysis, the resulting stimulation or response index, and the correlation of these parameters with cytokine profiles and patterns as detailed in Example 15 hereinbelow.

The invention provides a method of determining if an individual beset with a cancer, a tumor, or an infection will or will not be likely to respond to in vivo immunotherapy treatment by mounting a cell mediated response to reduce, inhibit, or destroy the tumor or infection, comprising: a) crosslinking autologous or allogeneic cancer, tumor or infected cells with a 2', 3' nucleoside or nucleotide d.aldehyde crosslinker at a concentration and for a time effective to crosslink proteins in the cells' plasma membranes and treating the cells with hydrostatic pressure for a time sufficient to cause a modification of proteins in the cells' plas ma membranes, thereby resulting in a crosslinked and pressure-treated modified cell preparation; b) measuring the ability of the modified cells of step a) to stimulate the proliferation and immunoreactivity of the individual's mononuclear blood cells compared with the ability of native or unmodified cells to stimulate the proliferation and immunoreactivity of the mononuclear blood cells; and c) determining the pattern or profile of cytokines secreted by the mononuclear blood cells by measuring the levels of secreted T_H1 stimulatory cytokines and the levels of secreted T_H2 cytokines that are (i) inhibitory for a cell-mediated immune response or (ii) stimulatory for a humoral immune response, to determine if the individual will or will not respond to immunotherapy treatment. It is noted that when allogeneic cells are used, they are obtained from another individual or donor having the same type of cancer, tumor, or infection.

The cytokine pattern as mentioned above in step c) and used in conjunction with the response or stimulation index obtained from step b) as disclosed herein can be used to determine that an individual is likely to respond in a positive manner to immunotherapy treatment (i.e. , by mounting a cell mediated immune response and/or a T_H1 response) based on the following exemplary parameters: 1) a high level of mononuclear blood cell proliferation as determined in accordance with step c), and 2) at least one or more of: (i) a high level of stimulatory T_H1 cytokine secretion; (ii) a low level of T_H1 inhibitory cytokine secretion; (iii) a low level of stimulatory T_H2 cytokine secretion; or (iv) a low level of cytokines stimulatory for the humoral immune response, as determined according to step c). The determination that an individual will respond in a positive manner to immunotherapy treatment by mounting a cell mediated immune response can also be concluded based on 1) a low level of mononuclear blood cell proliferation as determined in accordance with step b) (indicated by a response or stimulation index of less than about 1.5), and 2) at least one or more of: (i) a high level of stimulatory T_H1 cytokine secretion; (ii) a low level of T_H1 inhibitory cytokine secretion; (iii) a low level of stimulatory T_H2 cytokine secretion; or (iv) a low level of cytokines stimulatory for the humoral immune response, as measured according to step c). In this case, the cytokine pattern indicates that a T_H2 to T_H1 switch has occurred in the individual.

The determination that an individual will not respond in a positive manner to immunotherapy treatment and is not likely to mount a successful cell mediated immune response can be concluded based on 1) a low level of mononuclear blood cell proliferation as determined in accordance with step b) (indicated by a response or stimulation index of less than about 1.5), and 2) at least one or more of: (i) a low level of stimulatory T_H1 cytokine secretion; (ii) a high level of T_H1 inhibitory cytokine secretion; (iii) a high level of stimulatory T_H2 cytokine secretion; and/or (iv) a high level of cytokines stimulatory for the humoral immune response, as measured according to step c). This cytokine pattern indicates that a T_H2 to T_H1 switch has not occurred in the individual.

Moreover, the determination that an individual will not respond in a positive manner to immunotherapy treatment and is not likely to mount a successful cell mediated immune response can also be concluded based on 1) a high or relatively high level of mononuclear blood cell proliferation as determined in accordance with step b) (indicated by a response or stimulation index of about 1.5 to 2 or greater), and 2) at least one or more of: (i) a low level of stimulatory T_H1 cytokine secretion; (ii) a high level of T_H1 inhibitory cytokine secretion; (iii) a high level of stimulatory T_H2 cytokine secretion; and/or (iv) a high level of cytokines stimulatory for the humoral immune response, as measured according to step c). This cytokine pattern combined with a positive proliferation index indicates that a T_H2 to T_H1 switch has not occurred.

More particularly, this aspect of the invention allows several clinically-relevant determinations to be made with respect to the results of performing the IVS assays, including determining a stimulation index or response index, determining the associated cytokine profile or pattern, and if desired or necessary, immunophenotyping the PBMCs to determine which cell subsets are stimulated and proliferating in the in vitro IVS assays in response to PCL-modified cells, using a tumored or infected patient's PBMCs as responders, PCL-modified tumor or infected cells as stimulators, and unmodified tumor or infected cells as control stimulators.

In accordance with the invention, a given patient's clinical outcome or prognosis with respect to immunotherapy treatment, particularly, PCL immunotherapy, can be directly correlated with the results of the above-mentioned determinations and can be concluded to be either positive (i.e. , a high likelihood of success) or negative (i.e., a low likelihood of success) based on the following four exemplary conditions:

(1) a positive or high proliferative response to PCL-modified stimulator cells in the IVS assay (e.g., a response index greater than 1.5 or 2) combined with a positive cytokine profile or pattern determined by the types of cytokines secreted by responder PBMCs is a predictor of a positive or successful clinical outcome or a positive prognosis for immunotherapy treatment, particularly PCL immunotherapy, for the patient. In general, a positive cytokine profile or pattern is one in which the tested PBMCs show a production of high levels of secreted T_H1 cytokines that are stimulatory for, directly linked to, or associated with a cell-mediated immune response (e.g., IL-2 or IFN-7), and/or a production of low (or virtually no) levels of secreted T_H2 cytokines that (i) are inhibitory for a cell-mediated immune response, or (ii) are stimulatory for a humoral immune response (e.g., IL-4, IL-5, IL-6, and IL-10 and the like). The levels of secreted cytokines are determined by comparing the stimulation or response values obtained in the in vitro proliferation assays for PBMCs cultured in the presence of PCL-modified stimulator cells (e.g. , tumor or infected cells) versus PBMCs cultured in the presence of unmodified stimulator cells (e.g., tumor or infected cells). As a particular example, a positive or successful prognosis for immunotherapy treatment is based upon results showing secretion of low levels of IL-10 (and/or IL-4) and secretion of high levels of IFN- 7(and/or IL-2), thus indicating a T_H1 profile or a T_H2 to a T_H1 profile) of a patient's PBMCs in response to stimulation by PCL-modified stimulator cells (e.g. , Example 15 and the IVS(+) panels of Tables 12 and 13, and Table 14); (2) a negative or low proliferative response to PCL-modified stimulator cells in the IVS assay (e.g. , a response index of less than 1.5 or 2) combined with a positive cytokine profile or pattern as determined by the secretion of T_H1 stimulatory cytokines by responder PBMCs, detailed in (1) above, is also a predictor of a positive or successful clinical outcome or a positive prognosis for immunotherapy treatment, particularly PCL immunotherapy, for a patient. That the patient is secreting T_H1 cytokines which indicate the production of a cell mediated immune response against tumor or infected cells, in spite of the low proliferation of PMBCs, serves as a positive indicator of successful immunotherapy. In particular, Example 15, and the IVS(-) panel of Table 12 demonstrate that after stimulation by PCL-modified stimulator cells, the secretion of IFN-7 by responder PBLs increases and is indicative that the PCL-modification protocol has forced a cytokine profile change and a corresponding T_H2 to T_H1 pattern change; thus, the patient is likely to respond to PCL immunotherapy during the course of treatment. In addition, repeated immunizations or vaccinations of a patient with PCL-modified cell- containing immunogenic preparations may increase the patient's response index and/or may aid in maintaining the T_H1 pattern or the T_H2 to T_H1 pattern or can result in the conversion from a T_H2 to a T_H1 response in a patient. The T_H2 to T_H1 conversion then allows a corresponding positive response to immunotherapy treatment regimens and a positive clinical outcome during future PCL- immunotherapy treatment; (3) a negative or low proliferative response to PCL-modified stimulator cells in the IVS assay (e.g. , a response index of less than 1.5 or 2) combined with a negative cytokine profile or pattern determined by the types of cytokines secreted by responder PBMCs is a predictor of a negative or unsuccessful clinical outcome or a negative prognosis for immunotherapy treatment, particularly PCL immunotherapy, for the patient. In general, a negative cytokine profile or pattern is one in which there is a production of high levels of secreted T_H2 cytokines that are either inhibitory for a cell-mediated immune response or stimulatory for a humoral immune response (e.g., IL-4, IL-5, IL-6, and IL-10). As a particular example, a negative or unsuccessful prognosis for immunotherapy treatment is based upon results showing secretion of higher levels of IL-10 (and/or IL-4) and secretion of no or low levels of IFN-7 (and/or IL-2). thus indicating a sustained T_H2 profile or a T_H1 to a T_H2 conversion pattern) of a patient's PBMCs in response to stimulation by PCL-modified stimulator cells (e.g., Example 15 and the IVS(-) panel of Table 13). Such results are predictive of a negative clinical outcome or a negative prognosis of PCL immunotherapy for the patient, with the effect that the patient is not likely to respond to PCL immunotherapy; and

(4) a positive or high proliferative response of a patient's PBMCs to PCL-modified stimulator cells in the IVS assay relative to that observed in response to unmodified stimulator cells, combined with a negative cytokine profile or pattern indicative of one or more of the following: that a cell mediated response has not occurred, that a T_H2 to T_H1 conversion has not occurred, that a T_H1 to T_H2 conversion has occurred; or that a humoral response has occurred is predictive that the patient is unlikely to respond to immunotherapy and will have a negative prognosis and clinical outcome for this type of treatment. The negative cytokine profile may be due to several factors that may be tested, if necessary or desired, such as the stimulation and proliferation of suppressor T cells. However, despite the positive proliferation response, combined with the poor cytokine pattern, the fourth condition prognoses a negative clinical outcome of immunotherapy treatment for the patient. Thus, the present invention provides the clinician or skilled practitioner with the tools and methods to predict and determine the clinical outcome of immunotherapy, particularly PCL immunotherapy, for a given patient based on the in vitro analyses performed using PCL-modification technology. In addition, the skilled practitioner is able to make informed and substantiated decisions regarding further and future treatment for cancer patients and patients harboring microbial or viral infections that can dramatically impact on the outcome of their diseases in a beneficial and humane manner.

In a related aspect of the invention, cytokine treatment may be combined with the PCL-modification methodology of the invention to further augment or improve the directed response of immune cells against tumor cells, infected cells, and transformed cells. Although cytokines are not completely specific for anti-tumor directed effector cells (e.g. , PBMCs), these factors have the ability to augment and enhance one or more components of cellular immune function. The treatment of cells with cytokines, such as gamma interferon, or other activators, such as phytohemagglutinin, pokeweed mitogen, or combinations thereof, prior to crosslinking and pressure modification as described herein is likely to cause a further increase or enhancement in the expression of MHC antigens, in addition to a stabilization of the presentation of MHC structures as a consequence of crosslinking. The cytokines may be purified or recombinantly produced; highly purified and recombinant cytokines are also commercially available. Examples of suitable cytokines include, but are not limited to, interleukins, e.g., interleukin-2 (IL-2) and interleukin-12 (IL-12), tumor necrosis factor (TNF), alpha interferon (IFN-α), beta interferon (IFN-β), gamma interferon (IFN-7), and hematopoietic factors such as granulocyte-macrophage stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF). Cells may be incubated with cytokine for 24 to 72 hours in order to achieve augmented expression of MHC structures at the cell surface. Another approach to the use of cytokines in the invention is the transfection of tumor cells in vitro with genes encoding cytokines, and modifying the tumor cells in accordance with the invention. The transfected, modified tumor cells, when used as immunogens to stimulate the immune response after immunization in vivo are thus capable of producing immunostimulatory cytokines in abundance, particularly at the site of tumor growth. Genes encoding IL-2, GM- CSF, and IFN-7 are particularly useful for stimulating a particular immune effector function when the cytokine is produced and secreted by the modified and transfected cells. In addition, T cell activator compounds which cause stimulation or proliferation, such as anti-T cell receptor compounds, anti-CD3 compounds, or OKT3, can be used in conjunction with PCL-modified tumor cells or infected cells in an immunogenic preparation or vaccine to augment or enhance a patient's immune response to the tumor or infected cells (for example, see Example 15,

Table 12, in which OKT3 caused a dramatic increase in the secretion of IFN-7 by the IVS(+) individual's PMBCs (i.e. , from 571 to greater than 4400 pg/ml). EXAMPLES

The examples as set forth herein are meant to exemplify the various aspects of carrying out the invention and are not intended to limit the invention in any way.

Example 1

Cells and reagents

Cells

Cells from an EL4 tumor, which is a chemically induced T-leukemia

(Gorrer, 1961. In: Harris RJG, ed., Biological Approach to Cancer Chemotherapy.

Academic Press, New York) were maintained in ascites form in the peritoneal cavity of 6-8 week old C57B1/6J female mice. About 10⁵ cells were inoculated intraperitoneally (i.p.) and 10 days later the cells (approximately 5xl0⁸ cells per animal) were harvested.

Cells of ARadLV 136, which is a radiation-induced leukemogenic variant of ARadLV, were maintained in vitro as described previously (Haran-Ghere et al. , 1977, J. Immunol. 118:600). B16-BL6 melanoma tumor cells syngeneic (i.e. , autologous) to

C57BL/6 mice were serially passaged in mice by subcutaneous inoculation of 2-5 x

10° cells in 1.0 mL Hanks Balanced Salt Solution (HBSS).

2' . 3' - Adenosine dialdehyde (AdA)

AdA, which is a biologically compatible chemical crosslinker, was synthesized by a modification of the procedure previously described (Hansske et al. , 1974, Bioorg. Chem. 3:367). The procedure was as follows: Adenosine

(Sigma Chemical Co., St. Louis, MO) and sodium metaperiodate (Fluka Chemie AG, Buchs. FRG) were mixed in 100 mL aqueous solution to a final concentration of 10 mM of each of these substances, stirred in the dark, and cooled with ice water for 1 hour, and then concentrated to 5 mL under vacuum at 30° C. The resulting concentrate was then incubated for 12 hours at 4°C and the crystalline product which was obtained was separated and found to be homogeneous in thin- layer chromatography (silica gel G plate, 0.2 mm thickness, Merck Darmstadt; running solvent; acetonitrile/water, 4: 1 v/v, R_F 0.80). The crystals were filtered, washed three times with cold water and dried over silica gel in vacuum (12 mm Hg: 1.6 kPa).

The yield of the above preparation procedure was found to be approximately 90%. The obtained product had a melting point of 110°C and melting was accompanied by decomposition, this being in agreement with previous reports (Hansske et al., 1974, supra). The final AdA product as prepared and used in the PCL modification of the invention should be active in crosslinking membrane proteins in accordance with the invention, should be free of iodate impurities, and should be soluble at the pH in which crosslinking is performed (i.e. , about neutral) in accordance with the invention. Those skilled in the art will appreciate that the molar concentration of AdA can be determined by measuring the adenosine concentration or the dialdehyde concentration of the AdA preparation. It is noted, for example, that an AdA concentration of about 20 mM, as determined by measurement of the adenosine concentration of the AdA preparation is equivalent to an AdA concentration of about 10-13 mM, as determined by measurement of the dialdehyde concentration of the same AdA preparation. Accordingly, in vitro and in vivo results obtained using about 20 mM AdA as measured by its adenosine content and those obtained using about 10-13 mM AdA as measured by its dialdehyde content are essentially equivalent.

The 2', 3 '-nucleoside and nucleotide dialdehydes have several features which make them advantageous for use in the invention. These crosslinkers are biocompatible and are virtually non-toxic to cells as used in accordance with the invention; they are membrane impermeable; they have a slow rate of uptake into the cell, and thus are retained longer in the cell membrane where they can effect their crosslinking functions; they do not interfere with solubilizing plasma membranes prior to membrane isolation; they are non- immunogenic by themselves; they also possess a relatively long shelf life. Example 2

Preparation of human tumor cells from resected human tumors

Freshly resected tumors (usually 1-3 x 1-3 x 1-2 cm in size) were transferred in the cold (approximately 4°C) within 1 hour from the operating room (OR). Tumors were transferred to a 100 mm petri dish, rinsed 3-5 times in 10 mL cold PBS, and then transferred to a second petri dish where necrotic and fatty tissue were removed. The tumor tissue was cut into small pieces (approximately 1- 5 mm² each), transferred to a 500 mL plastic flask containing: 100 mL of RPMI medium, 50 mg collagenase (Sigma), 1500 units DNase type IV, 5 mg Hyaluronidase type V, 0.01 M Hepes (Biological Industries, Israel), 0.03% L- glutamine (Biological Industries, Israel), Pen 5000 units/Strep 5 mg (Biological Industries, Israel), 1 :20 dilution of Fungizone (Biomycin-2, Biological Industries, Israel), and 25 mg gentamycin (Biological Industries, Israel). After stirring the tumor in the enzymatic solution for 2-3 hours (depending on the tumor size) , the tumor cell suspension was transferred to a 50 mL plastic centrifuge tube through a 120 micron nylon mesh, and washed one time in phosphate buffered saline (PBS). The cell pellet was then resuspended in 35 mL PBS and layered gently on top of 15 mL of Ficoll gradient (1.077 g/cm³). After centrifuging for 20 minutes at 450 x g at room temperature, cells in the inteφhase were collected, washed twice in PBS by centrifuging at room temperature at 400 x g for 10 minutes, counted for viability using trypan blue exclusion, and diluted or concentrated to obtain the desired cell number.

One of skill in the art will appreciate that in patients bearing a tumor, the tumor may be wholly or partially excised by biopsy or surgery employing techniques and practices known to the skilled practitioner. Appropriate precautions are taken for safety and sterility. Individual tumor cells may be dissociated into single cell suspensions or dispersions using conventional enzymatic, chemical, or mechanical means.

It is also noted that the tumor cell suspension obtained as described herein from freshly resected tumor tissue and used in accordance with the invention represents a heterogeneous population of cells of which about 20% to about 70% is comprised of tumor cells and about 30% to about 80% is comprised of a mixture of mononuclear cell types from peripheral blood, including accessory cells (e.g., macrophages, monocytes, or antigen-presenting cells) and lymphocytes, that reside within the tumor tissue (for example, see Table 5). When such freshly obtained tumor cell preparations are used for PCL modification, the accessory cells resident in the tumor are also capable of being modified. It is envisioned that such PCL modification of the non-tumor accessory cells or antigen-presenting cells and lymphocytes, which are part of the tumor preparation, may affect or enhance the presentation of antigenic molecules, either alone or in combination with MHC proteins and the like, when these PCL-modified cells are used in association with PCL-modified tumor cells in an immunogenic preparation, thereby allowing a better presentation of immunogenic structures by these cells to the lymphocytes, particularly, the T lymphocyte subsets, of the immune system.

Example 3

Anti-CD3 and PHA stimulation of PBMCs In the wells of a 96-well microtiter plate, 2x10^s cells were incubated in 0.2 mL tissue culture medium, e.g. , RPMI (Gibco), supplemented to contain 20 μL of either (a) anti-CD3 monoclonal antibody or PHA in a 96-well microplate. Anti-CD3 antibody was obtained in culture supernatant prepared from hybridoma cell line number 454 (Dr. J. Lawrence, Cornell Medical Center, N.Y.). Other sources of anti-human CD3 antibody can be used. For example, OKT 3, a hybridoma cell line which produces anti-CD3 monoclonal antibody directed against human peripheral T cells, is available through the ATCC, e.g. , ATCC CRL 8001. Another anti-human CD3 monoclonal antibody-producing hybridoma cell line is also available through the ATCC, ATCC HB 231. For these assays, both anti- CD3 monoclonal antibody and PHA were diluted 1 :20 in culture medium. Example 4

Preparation of cells from solid tumor or ascites

A subcutaneous tumor was carefully excised, minced with scissors into fragments 1-2 mm in size, and stirred in a triple-enzyme mixture of hyaluronidase, deoxyribonuclease and collagenase (Sigma Chemical Co. , St. Louis, Mo.) for 30-60 min in HBSS (Ca²⁺-and Mg²⁺-free) as described by Lafreniere, R. and S.A. Rosenberg. 1986. "A novel approach to the generation and identification of experimental hepatic metastases in a murine model", JNCI, 76:309. The suspension was then collected and passed through 100-μm nylon mesh, washed three times in HBSS, and resuspended at the appropriate concentration. The cells were either frozen in aliquots at -70° C for 24 h and then transferred to liquid nitrogen and stored for later use, or serially passaged in vitro every 3-4 days. Cells were cultured as adherent monolayers in tissue-culture flasks (Falcon 3024), seeded at approximately 3x10^s cells/75-cm² flask in 20 mL complete medium containing RPMI-1640, heat-inactivated fetal calf serum (10% v/v), penicillin (100 U/mL), streptomycin (100 μg/mL), 0.03% fresh L-glutamine, 0.1 mM non- essential amino acids, 0.1 μM sodium pyruvate, 50 μg gentamicin/mL, 0.5 μg solubilized amphotericin B (Sigma Chemical Co., St. Louis, Mo.) and 50 μM 2- mercaptoethanol. Cultures were incubated at 37°C in a humidified atmosphere of 5 % CO , and passaged every 3-4 days. Cells were harvested from confluent B16- BL6 monolayers by draining the medium, washed twice with HBSS and then were overlayed with 1-2 mL trypsin/versene (Gibco) for 2-3 min while the bottom of the flask was tapped occasionally. Digestion was stopped by the addition of 10 mL complete medium. Detached cells were washed twice in HBSS and resuspended at the desired concentration.

Example 5

Modification of tumor cells

After harvesting tumor cells, the cells were washed twice with PBS (pH 7.4) and then were subjected to one of the modification treatments described hereinbelow. The viability of the modified tumor cells was assessed by trypan blue dye exclusion.

Modification I: Crosslinking of proteins on the tumor cell surface. About 10⁸ cells/mL were inoculated into a 50 mL tube (Falcon, Becton Dickinson Labware, N.J.) holding a PBS solution containing 0.5 % AdA (about 20 mM to 40 mM, preferably less than 40 mM) and were incubated in this solution for 1 hour at room temperature with occasional mixing. Unbound AdA was removed by three cycles of centrifugation at 1500 φ for 5 minutes, followed by gentle resuspending of the pellet in PBS (in the final resuspension, PBS was added to obtain the desired cell concentration).

Modification II: Application of hydrostatic pressure. This modification was performed in a manner similar to that previously described (Richert et al. , 1986, Cancer Immunol. Immunother. 22:119). Cells were dispersed in PBS at a concentration of 10⁸ cells/mL in a capped Eppendorf plastic tube (1.5 mL, Netheler and Hinz GmbH, Hamburg, FRG) and filled to the brim. A 0.5 inch (1.27 cm) 18G needle was inserted through the cap and served as a vent for pressure equalization. Both the needle and the tube were filled with PBS and the cap was pressed down without entrapping any air bubbles. Removal of all air bubbles is important, as bubbles may cause cell disruption upon release of pressure. The tubes were then placed inside a pressure bomb of 40 mL capacity (Aminco, American Instruments Co. , Md.), filled with PBS and sealed.

Pressure was gradually applied to reach a level of about 1200 atm within 7-8 minutes and this pressure was maintained for about 15 minutes. Thereafter, the bomb was unlocked and allowed to decompress gradually to ambient pressure in about 8-15 minutes. The cells were then transferred to a 50 mL tube in 10 mL of PBS and were centrifuged at 1500 φm for 5 minutes. The pellet was then gently resuspended in PBS to the desired cell concentration.

Modification IH: Hydrostatic pressure and crosslinking in sequence. Cells were pressurized and then immediately crosslinked by the two modification procedures outlined above. Modification IV: Hydrostatic pressure and crosslinking simultaneously. Cells were pressurized and crosslinked at the same time by the two modification treatments described above. The exposure of cells to the crosslinking compound and to hydrostatic pressure at the same time in accordance with the invention is a preferred PCL-modification.

Example 6

In vitro sensitization (TVS) assay

The proliferative response of effector cells, i.e., PBMCs or lymphocytes, in the presence of tumor cells was carried out as follows: For human experiments using human PBMCs. 2 x 10^s viable PBM cells in 0.1 mL of conditioned medium were co-cultured with irradiated tumor cells (10,000 R) in the wells of a 96-well microtiter plate (final concentration of tumor cells in 0.1 mL was 5 x 10* 10 5 x 10^s). Tumor cells were either PCL-treated or PCL-untreated. Tumor cells were prepared by cutting the tumors into small pieces (approximately 1 mm), followed by enzymatic digestion for 2-3 hours, and then separating viable cells on a cell separation gradient (450 x g for 20 minutes at room temperature), or such as is described in Examples 2 and 4. PBMC were isolated from 20 mL of blood taken from the patient, followed by dilution (1 : 1) in PBS and separation on a cell separation gradient as described for tumor cells. PCL-modification was carried out by exposing 5 x 10° to 1 x 10⁷ cells (either tumor cells or PBMCs) to 1200 atmospheres of hydrostatic pressure in the presence of 40 mM AdA. Thereafter, cells were irradiated at 10,000 R. The endpoint of the IVS assay was the measurement of cell proliferation which correlates directly with the extent of stimulation. Cell proliferation was measured by adding [³H]-thymidine for the final 6 hours of incubation of a 5 day IVS assay. Cells were then harvested using a cell harvester (Packard) and radioreactivity retained on the filters was counted using a beta counter (Packard).

For mouse experiments using B16-BL6 cells, samples containing 2 x 10^s viable cells, i.e. , splenocytes, were co-cultured with 1 x 10^s irradiated (50 Gy) PCL-treated or untreated B16-BL6 cells in a 96-well flat-bottomed microplate (Nunc Denmark), for 48 h at 37°C in a humidity-controlled incubator under a 5 % CO₂ atmosphere. The culture medium consisted of RPMI-1640 medium plus 10% heat-inactivated fetal calf serum supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL). Cultures of effector cells and "stimulator" tumor cells were pulsed with [methyl-³H]thymidine (Amersham) after about 42 h of culture, and after 6 h, the cells were harvested and the incorporated radioactivity was measured by conventional methods.

It is pointed out that in vitro sensitization assays may also be described as mixed lymphocyte culture (MLC) assays. In MLC, T cells respond to foreign histocompatibility antigens on unrelated lymphocytes or monocytes. The test may be performed as either a "one way" or a "two way" assay. In a one way MLC assay, the stimulating cells are treated with either irradiation (approximately 1500-2000 R) or with mitomycin to prevent DNA synthesis without killing the cells. The magnitude of the response is the result of DNA synthesis measured in the non-irradiated or non-mitomycin treated cells. In a two way MLC, DNA synthesis of both stimulating and responding cells represents the net response of both sets of cells. The individual contributions of each cell population cannot be discerned. Controls include co-culture of syngeneic irradiated and nonirradiated pairs (to determine baseline DNA synthesis) and co-culture of allogeneic irradiated pairs (to determine adequate inactivation by irradiation).

More specifically, for a one way MLC, responder peripheral blood lymphocytes are mixed 1 : 1 with irradiated stimulator cells and incubated at 37°C in a humidified atmosphere with 5 % CO₂. After 5 days, the culture is pulsed with [³H]-thymidine to label the nucleic acid in the responder cells. After 18 hours, the cells are harvested and counted for internalized radioactivity. For example, if the MHC or HLA antigens of the stimulator cells differ from those of the responder cells, the responder cells undergo blastogenesis, synthesize DNA, and proliferate; increased sample radioactivity is the result. If there are no MHC or HLA difference, the cells remain quiescent and no increase in radioactivity is measured at the end of the assay.

Example 7

Delayed-tvpe hypersensitivity (DTH) assay Animal model studies and assessment

For non-human animals, the DTH response was measured by skin reaction in the ear as described by Vadas et al., 1975. Int. Arch. Allergy Appl. Immunol. 49:670. For immunization, unmodified or modified tumor cells were irradiated (10,000 rad) and then injected i.p. into C57BL/6J female mice at a dose of 10⁷ viable cells in 1 mL of PBS/mouse (cell counts were determined before irradiation). After 8 days, immunization was repeated as above with a fresh batch of unmodified or modified tumor cells.

A sample of 10^s unmodified and irradiated tumor cells (an empirically determined optimal dose) in 10 μL PBS was injected intradermally 8 days later in the right ear (0.5 in. , 1.27 cm, 30G needle, Becton Dickinson, N.J.). The left ear (control ear) was injected with 10 μL PBS. After 10 hours, the mice were injected i.p. with 0.1 mL of a 1.0 mM 5-fluoro-2' deoxyuridine (FdUrd, Sigma) solution. 30 minutes later, the mice were injected i.v. with 2 μCi of 5-^12sI- labeled 2 '-deoxyuridine (^12SIdUrdR, sp. act. 5 Ci/ng, Amersham, UK) in the lateral tail vein.

Mice were sacrificed after 24 h from the time of challenge with tumor cells. The ears were then cut out carefully at the rims and the amount of radioactivity determined in a gamma counter (Gammamatic, Kontron). The results were expressed as the ratio of radioactivity in the right ear to that in the left ear (R/L ^I2SIdUrd index). Five mice were included in each group. Control groups included unprimed mice, as well as those primed with unmodified tumor cells.

Histology of the ear

For histological purposes, the ears were fixed with Bouin's fixative for 48 h at room temperature. Excess fixative was removed thereafter by extensive washing with 70% ethanol. Any hair present was carefully shaved off and the ears were cut to uniform size for embedding and subsequent sectioning on a rotary microtome (Spencer model No. 820; American Optical Co.). The slides were stained with eosin and in some cases with toluidine blue (Vadas et al., 1975. , supra).

Human studies and assessment

In human patients, to determine the immunogenicity of autologous or allogeneic tumor cells PCL-modified in accordance with the invention and used as immunogens, patients were evaluated at baseline using the DTH skin test. The patients were given two separate subcutaneous or intradermal injections, side by side, of unmodified (i.e. , no PCL treatment) and of PCL-modified tumor cells (1 x 10^s to 1 x 10° cells/injection). The cells were irradiated by a dose of 10,000 rad. so that they lost their ability to proliferate, and yet still maintained their immunogenic potential. In particular, 25 patients received immunogens comprising autologous tumor cells modified with crosslinking and pressure treatment in accordance with the invention.

The development of the skin test reaction or immune reaction in humans (i.e. , DTH) was scored at 24, 36, 48, and 72 hours after injection of the cells. 24-48 hours were frequently optimal and normal for observing the peak of a reaction. An immune reaction elicited by the injected cells was evidenced by a swollen, red area which appeared in the skin at the inoculation site. The degree of a patient's skin reaction was determined by the diameter of the redness (erythema) and the degree of swelling (induration), as described by Scornick et al. 1981. Cancer Immunother. Immunol. , 11 :93.

DTH skin reactions were scored as follows: "-", " + ", " + + ", or " + + + ", where "-" indicates no reaction and " + + + " indicates maximal redness and swelling reactions. Alternatively, DTH responses can be scored numerically, such that a score of "3" indicates a very strong response (and corresponds to the above-described " + + + " response); a score of "2" indicates a moderate response, corresponding to " + + "; and a score of "0-1 " indicates a minimal or poor DTH response, corresponding to " + ". above.

As an example, in people who have a history of exposure to tuberculosis, tuberculosis infection, or previous vaccination, a standard maximal reaction is obtained after injection with tuberculin (a lipoprotein of Mycobacterium tuberculosis). Thus, the DTH test allowed the determination of the immunogenic potential of the tested cells. A high immunogenic potential correlated with a severe DTH reaction. As shown in Fig. 10, 23 out of 25 patients had DTH responses of "2" and above when PCL-modified cells were used to as immunogens in DTH analyses, compared with unmodified cells.

To test the immunogenicity of modified allogeneic cells in humans, essentially the same procedure as described above for autologous cells was carried out. However, each patient was treated with an PCL-treated or untreated allogeneic tumor preparation which corresponded to his or her tumor type (e.g. , a melanoma patient was treated with a donor's allogeneic PCL-modified melanoma tumor cells, and a lung cancer patient was treated with a donor's allogeneic PCL- modified lung tumor cells, and so forth, depending on the type of cancer with which a given patient was afflicted). The patients were given two separate subcutaneous injections, side by side, of unmodified (i.e., no PCL treatment) and of PCL-modified allogeneic tumor cells at 1 x 10^s cells per injection. For PCL modification, the tumor cells were treated with AdA crosslinker at about 40 mM at the same time that hydrostatic pressure in the range of between 1200 to 1400 atm was being applied. The development of the DTH skin test reaction was scored at 48 hours after injection. In Fig. 11 , patient #13 and patient #14 had lung cancer; patient #15 and patient #20 had melanoma; and patient #17 and patient #18 had colorectal cancer. All six cancer patients had a demonstrably significant increase in their DTH responses to the PCL-modified allogeneic tumor cells relative to the unmodified tumor cells, consistent with the results obtained using PCL-modified autologous tumor cell vaccines.

The results of these allogeneic cell experiments in human patients indicated that while non-treated (i.e. , non-modified) cells caused a minimal, ("-" or " +."), DTH reaction, the injection of PCL-modified autologous or allogeneic cells resulted in a very strong immune reaction, (" + + " to " + + + ") in all patients tested. Maximal reactions appeared within 36-48 hours after the injection of cells, as expected by the normal progress of a DTH reaction and response in vivo.

Example 8 ^lChromium-release cytotoxicity assay

To determine the cytotoxic potential of T cells primed with cell surface antigens presented to them, chromium release cytotoxicity assays were performed. A five hour ⁵ 'chromium release cytotoxicity assay was carried out as described by Brunner et al. 1976. "The ⁵¹Cr release assay as used for the quantitative measurement of cell-mediated cytolysis in vitro", In: In vitro methods in cell mediated immunity. Eds. B.R. Bloom and J.R. David, Academic Press, London, p. 423. Target cells used in these assays were EL4 (EL4 originated from a chemically-induced T cell leukemia; Gorer, P. A. 1961. "The isoantigens of malignant cells", In: Biological approaches to cancer immunotherapy. Ed. R.J. Harris, Academic Press, New York, p.219) and were maintained in ascites form in the peritoneal cavity of 6-8 week old C57B1/6J mice. ARadLV 136 is a radiation- induced leukemogenic variant of ARadLV and is maintained in vitro as described in N. Haran-Ghera et al. 1977. J. Immunol. , 118:600.

Briefly, EL4 as well as ARadLV 136 target cells were washed once in RPMI-1640 medium and the supernatant was aspirated to leave 0.1 mL with the cell pellet. The pellet was gently dispersed and resuspended in PBS with 5 % fetal calf serum and centrifuged at low speed (1000 φm for 5 min). 0.1 mL Na₂ ⁵¹CrO₄ solution (Amersham, UK; 1 mCi/mL, sp. act. 200 mCi/mg) per 2x10^s target cells was added to the pellet in 0.1 mL buffer and the suspension was gently vortexed. Incubation was done in small petri dishes in a humidified incubator, flushed with 5 % CO₂ and set at 37°C, for 1 hour with occasional swirling. Thereafter, the labeled cells were washed three times with PBS and their concentration was finally adjusted to 0.5xl0⁶ cells/mL. Cytotoxic effector cells were obtained from spleens of C57BL/6J mice previously sensitized with PCL-modified or unmodified EL4 cells as well as ARadLV 136 cells (see above). Spleens were transferred to petri dishes in a sterile manner in PBS and washed three times with cold PBS. All adhering adipose tissues were teased away with forceps and the spleens were finally minced and crushed with a hub of a 10 mL sterile disposable plastic syringe. Cell suspensions were then transferred to 15 mL conical tubes through 100 μm nylon mesh. Erythrocytes were lysed with ammonium chloride lysing buffer (9 parts 0.16 M NH₄C1 + 1 part 0.17 M TRIS pH 7.6; 2 mL/spleen for 1-2 min), and macrophages were depleted by incubating cells in dishes for 60 min at 37° C. The cells were then washed, centrifuged, and resuspended as before in PBS containing 5 % fetal calf serum. Enrichment of T cells was done by incubation of cells with nylon fibers (Fenwall Laboratories, Deerfield, 111.) Aliquots of 0.2 mL ⁵¹Cr-labeled target cell suspension containing 10^s cells were pipetted into round-bottomed plastic tubes (12x55 mm, Falcon); equal volumes of various dilutions of spleen effector cells were then added to the target cells to yield ratios of lymphocytes to target cells of 50, 10, 2.5 and 1.25. The tubes were then centrifuged at 1000 φm for 2 min before incubation at 37°C in a humidified incubator flushed with 5 % CO₂. After 5 hours, 0.6 mL of PBS were added, the tubes were centrifuged at 1000 φm for 5 min. , and 0.5 mL of supernatant was collected for counting in a well-type gamma counter (Gammamatic, Kontron). Maximum release was determined by adding 0.6 mL 0.5 % NP-40, and spontaneous release was counted from tubes containing labeled target cells alone. The percentage of specific lysis was defined and calculated by the following formula:

Specific lysis (%) = experimental ⁵¹Cr release - spontaneous release maximal release - spontaneous release x 100

The results from the cytotoxicity tests as described are presented in Figs. 2 and 3. As seen in these figures, the cytotoxic ability of anti-EL4 effector cells, isolated from spleens of mice and primed with AdA-treated, or pressure -I- AdA-treated tumor cells, to lyse ^slCr-EI_4 targets remained high (about 60%) at all effector: target ratios. In contrast, the ability of anti-(ARadLV 136) effector cells to lyse ^5ICr-ARadLV 136 target cells was generally low (about 15 %) and varied at the different lymphocyte-to-target cell ratios.

However, it can be seen that with the exception of the 10: 1 E:T ratio in the experiment with the ARadLV 136 cells shown in Fig. 2, the cytotoxic effect after immunization with either AdA-treated cells or AdA and pressure-treated tumor cells was much larger than immunization with the other preparations.

In order to investigate the specific nature of tumor cell lysis, the effector cells, anti-EL4 cells as well as anti- ARadLV 136 cells, were tested in a reciprocal manner, i.e. anti-El_4 vs. ⁵¹CrARadLV 136 and vice versa. The results are shown in Fig. 4. Target cell lysis in these experiments was low and insignificant (about 4% - 8%), compared with the direct assays in either tumor system as shown in Figs. 2 and 3. These results suggest that a high degree of specific recognition is achieved.

Example 9 DTH response in mice immunized with PCL-modified EL4 cells

The maximal DTH response to EL4 cells was achieved in mice immunized, i.e. vaccinated, with tumor cells modified by both AdA crosslinking and the application of hydrostatic pressure. A marked increase in DTH reactivity was also seen in mice vaccinated with tumor cells treated with AdA alone. The

DTH response after priming with cells modified by pressure treatment only, was essentially the same as that observed after priming with unmodified tumor cells.

Against the relatively strong DTH response obtained with EL4 cells, only a very weak response, compared with control, was obtained with the ARadLV 136 cells, although the priming with cells modified by exposure to both pressure and AdA showed the strongest response. The mild activity elicited by ARadLV

136 cells is presumably a result of an unidentified non-specific activity.

The results of the DTH assay are shown in the following Table 1 : TABLE 1

Delayed-type hypersensitivity response to 10^s irradiated tumor cells implanted in the right ear of mice primed with treated tumor cells of the same kind

Prevaccination PJL ^,23IdUrd uptake*

EL4 ARadLV 136

Unprimed 0.76±0.27 0.95±0.09

Primed:

Tumor unmodified 0.94±0.08 1.2 ±0.12

Tumor + AdA 1.4±0.12 1.2 ±0.2

Tumor + pressure 0.94±0.02 0.9 ±0.2

Tumor + pressure + AdA 2.92± 1.60 1.3 ±0.3

^* Results are expressed as mean values ±SEM of ratio of ^12SIdUrd uptake in the right vs the left ear (R/L) obtained in five separate experiments.

Histological examination of the ears is depicted in Fig. la-le and as can be seen in this figure, there is a predominant infiltration of monocytes or macrophages 24 hours after the challenge. In ear sections stained with toluidine blue, it was not possible to detect many cells with granules typical of basophils. This indicated that the inflammatory reaction was distinct from cutaneous basophil hypersensitivity.

Example 10

Lymphocyte proliferation assay The assay was modified from a procedure described earlier (Vanky et al. ,

1976, In: Bloom and David, eds. , supra). Tumor cells (5x10^s), EL4 cells as well as ARadLV 136 cells, were heavily irradiated (10,000 rad) prior to incubation with an equal number of effector cells. Effector cells were prepared from spleen, cleared of erythrocytes, and enriched for T cells. The proliferative responses of effector lymphocytes in the presence of inactivated stimulators (tumor cells) were assayed in triplicate in round-bottomed 96-well microtiter plates (Greimer,

Labortecknik, FRG). The above mixed cultures were maintained in RPMI-1640 medium containing 5 % fetal calf serum, penicillin (100 U/mL). streptomycin (100 μg/mL), sodium pyruvate (1 mM), non-essential amino acids (1 %) and 2- mercaptoethanol (5x10^s M) for 120 h in a humidified air incubator flushed with 5 % CO₂. Wells containing stimulator cells alone served as controls. [³H]Thymidine was pulsed in the last 18 hours with 2 μCi[methyl-³H] thymidine (Amersham, UK). At the end of the incubation period, the plates were centrifuged at 1500 φm for 10 minutes and washed once with cold PBS. 0.2 mL ice-cold 10% trichloroacetic acid was then added to the cell pellets. The cells were then harvested using a cell harvester (Titertek, Flow Laboratories, UK) and automatically transferred to glass-fiber filters. Excess acid was aspirated and filters were washed with 70% ethanol. Washing was repeated twice and the discs punched out in the machine were placed at the bottom of scintillation vials and left to dry overnight at room temperature. Scintillation counting was performed by adding 5 mL of scintillation fluid (Instamix/xylene, 4: 1) to the vials. Incoφorated [³H]thymidine was expressed as cpm ± SEM, and values that were at least double the controls were considered to be positive.

The results of [³H]thymidine uptake in the 5-day mixed cultures are presented in Tables 2 and 3. The results show that the proliferative capacity of anti-EL4 effector cells against syngeneic and allogeneic targets results in high incoφoration of the label when cells were grown in the presence of the syngeneic target EL4, while moderate proliferation was seen for the irrelevant, H-2-identical (ARadLV 136), and H-2-disparate (P815) target cells. Similar responses were noted for anti- ARadLV 136 effector cell proliferation in the presence of syngeneic ARadLV 136, H-2-identical (EL4), and H-2-disparate (P815) target cells. In addition, the results clearly show that proliferation of anti-tumor effector cells in the presence of the relevant tumor cells was considerably enhanced in the groups treated with AdA, with pressure, or with AdA plus pressure treatments.

° Example 11

Preparation of plasma membranes from cells and isolation of cytosolic and membrane-associated or shed protein and protein complexes from cells

Plasma membranes were prepared from cells, e.g., tumor cells, essentially as described by Maeda, T. et al. 1983. Biochim. Biophys. Acta. , 731 : 115.

Briefly, unmodified or PCL-modified tumor cells were homogenized in cold

Hank's Balanced Salt Solution (HBSS) containing EDTA, PMSF, and DNAse using a polytron homogenizer with three cycles of homogenization, at 5 seconds per cycle. The crude homogenate was first centrifuged at low speed (i.e. , about 800 0 φ ) to remove debris and nuclei. The supernatant was collected and layered on top of a 41 % sucrose solution and centrifuged at 91 ,000 x g for 60 minutes. The interface band was carefully aspirated using a pasteur pipet and was centrifuged at 100,000 x g for 90 minutes. The pellet was then resuspended in a small volume of , HBSS. Protein content was determined by Lowry's Folin-Ciocalteau assay (Lowry, O.H. et al. 1951. J. Biol. Chem. , 193:265).

Methods to prepare cytosolic and membrane proteins from cells. Cells are centrifuged in cell medium and the pelleted cells are washed once in PBS. For tumor isolates, tumor cells are dispersed in cell medium by mincing with a scalpel 0 prior to centrifuging. The washed cells are resuspended in a hypotonic buffer A (Buffer A: 10 mM KCl, 10 mM HEPES, pH 8.0, 1 mM EDTA/EGTA, protease inhibitors and phosphatase inhibitors) at a final cell density of about 10 million per mL in buffer A for about 15 to 30 minutes on ice. Thereafter, 63 μL of 10% NP- 5 40 or Triton-X^® 100 is added per mL of cell suspension (i.e. , about 0.6% nonionic detergent final concentration). The cell and detergent mixture is vortexed for about 15-30 seconds, and centrifuged in an microcentrifuge (Eppendorf) for about 1 minute. The resulting cell pellet contains cell debris (i.e. , connective tissue) and _Q nuclei. The resulting cell supernatant contains cytosolic proteins and solubilized plasma membrane proteins.

To isolate the cytosolic cell protein fraction only, cells are resuspended in buffer A, quick frozen in liquid nitrogen, thawed, and centrifuged for about 30 minutes in a microcentrifuge (Eppendorf). The resulting supernatant contains 5 predominantly cytosolic proteins. To isolate the plasma membrane protein fraction, the cell pellet resulting from the above-described 30 minute centrifugation is extracted on ice for about 30 minutes in buffer A containing 0.5% NP-40 or Triton-X^® 100 and is then centrifuged for 5 minutes in a microcentrifuge (Eppendorf). The soluble fraction contains predominantly plasma membrane proteins and residual cytosol.

In another method, cells are treated with crosslinker and pressure in accordance with the invention (i.e. , about 10 to 20 mM 2', 3' nucleoside or nucleotide dialdehyde, at the same time that the cells are exposed to about 800 to 1400 atm hydrostatic pressure; preferably 10 mM crosslinker and 1200 atm pressure). The PCL-treated cells are then subjected to hydrostatic pressure of greater than or equal to about 1600 atm and the cells are centrifuged to pellet cell debris. The resulting cell supernatant is applied to a G 100 or G250 column, whereby the fractions of crosslinked proteins are isolated. Such a high pressure method allows the collapse of the cell membrane structure and the corresponding release and isolation of soluble proteins or protein complexes, some or all of which have their hydrophilic portions in association with membrane lipids.

Example 12

Protection of animals immunized with modified tumor cells and challenged with tumor

In the in vivo experiments described in this example, the viability of C57BL mice challenged with 1 x 10⁵ viable untreated tumor cells was tested following pretreatment with various immunogenic preparations modified in accordance with the invention. Survival after challenge was scored at day 30.

The pretreatment consisted of two vaccinations (or immunizations), one at three weeks and the other at one week prior to the tumor cell challenge. The immunizations were performed using an immunogenic preparation comprising cells subjected to one of the following modification treatments: exposure to AdA, application of hydrostatic pressure, or a combination of the two treatments, following essentially the same procedure as described in Example 1 modifications. The cells used for vaccination were of the same kind as the cells used to challenge the mice.

The cells used in this experiment were either EL-4 or BL6 melanoma cells which are a very invasive variant of the B16 cell line (Hart 1979, Am. J. Pathology, 97:587). The B16-BL6 tumor was serially passaged in syngeneic C57BL mice by subcutaneous (s.c.) inoculation of 2 - 5 x 10° cells. Three test were performed as described: Test No. 1 :

Four groups of mice were used, each pretreated with one of following preparations. Molar concentrations of AdA were based on adenosine concentration.

Group 1 : EL-4 leukemia cells treated for 10 minutes with various levels of hydrostatic pressure, in the presence of 40 mM AdA. Group 2: EL-4 leukemia cells treated for 10 minutes with various levels of hydrostatic pressure and then with 40 mM AdA. Group 3: B16 melanoma cells treated as described for Group 1 cells. Group 4: B16 melanoma cells treated as described for Group 2 cells. The results are shown in Fig. 5 (each point represents an average of 10 animals): Group 1 - filled squares; Group 2 - empty squares; Group 3 - filled circles; Group 4 - empty circles. These results demonstrate that maximal survivability of animals was obtained after immunization or vaccination of animals with an immunogenic preparation in which the cells had been modified by exposure to hydrostatic pressure of 1200-1400 atm. Suφrisingly far inferior results were obtained using an immunogenic preparation of cells that had been modified by exposure to hydrostatic pressure of 1500 atm. Further, these results show that simultaneous exposure of cells used as immunogens to both hydrostatic pressure and AdA leads to a higher survivability of the mice, compared with the use of cells that had been exposed to these two treatments in sequence. Test No. 2:

Several groups of C57BL mice (10 mice in each group) were pretreated by one of the following preparations:

Treatment 1: EL-4 leukemia cells were treated for 10 minutes with hydrostatic pressure of 1350 atm in the presence of various increasing concentrations of AdA; Treatment 2: B16 melanoma cells treated as described for Treatment 1. Treatment 3: B16 melanoma cells treated for 10 minutes with hydrostatic pressure of 1350 atm in the presence of various concentrations of AMPdA.

The results of these experiments are shown in Fig. 6 (each point represents an average of 10 animals): Treatment 1 - filled squares; Treatment 2 - filled circles; Treatment 3 - empty circles. As can be clearly observed from this test, the maximal survival rate was obtained using AdA or AMPdA at a concentration of about above 30 mM. Test No. 3:

Five groups of C57BL mice (6 mice in each group) were immunized subcutaneously and then challenged with B16-BL6 cells. The preparation used for immunization comprised plasma membranes isolated from untreated or treated cells by discontinuous sucrose gradient centrifugation (Maeda et al., 1983, Biochim. Biophys. Acta 731 : 115). Details of the immunizing preparation are provided in Table 4.

The following parameters were tested for each group of animals: survival rate; mean tumor diameter, measured using standard callipers in three orthogonal directions and presented by the mean value; and metastatic nodules in lungs, scored after removal of the lungs, fixation with Bouin's fixative, and then washing with 70% ethanol. The results of this experiment are also shown in the following Table 4:

These results demonstrate that immunization with an immunizing preparation comprising plasma membranes isolated from B16-BL6 cells treated with pressure and AdA in accordance with the invention (e.g., Treatments 4 and 5 as described above) resulted in a very high survival rate; the mean tumor diameter was minimal, and no metastatic nodules were observed in the lungs. Accordingly, these results prove the unexpectedly high potency of the vaccination/immunization treatment of the invention.

Example 13

10

High-level expression of MHC class I and B16-BL6 melanoma-specific antigens on B16-BL6 cells modified by exposure of cells to AdA crosslinker at the same time as exposure to hydrostatic pressure

Flow cytometry was employed to analyze the presence of MHC class I

, c antigens (H-2k^b) and the tumor- specific retroviral antigen on B16-BL6 melanoma cells, derived from s.c. tumors, either directly after obtaining single cell suspensions, or after passaging the cells in culture 6 to 8 times over a period of 3-4 weeks.

About 1 x 10⁶ PCL-modified and unmodified B16-BL6 tumor cells were 0 incubated as a first step with unlabelled primary antibodies. In this incubation, either 30 μL (25 μg) of anti-class I monoclonal antibody (clone 28-8-6, obtained from Dr. D. Sachs, National Cancer Institute, U.S.A.) or 10 μL (8 μg) of monoclonal antibody against a retroviral antigen on the surface of B16 cells (i.e. , ⁵ MM2.9B6), (Leong et al. 1988, Cancer Res. 48:4954) was added to the cell preparation in a final volume of 50 μL of buffer containing 1 % FCS (fetal calf serum) and 0.01 % sodium azide, and the cell and antibody mixture was incubated for 45 minutes at 4°C Cells were washed twice in HBS (Hepes Buffered Saline) 0 and after centrifugation, the cell pellet was resuspended in a solution of a labeled secondary antibody: 50 μL of a solution containing 40 μg of the F(ab')₂ fragment of FITC-GAMIG (fluorescein isothiocyanate labeled goat anti-mouse IgG) in HBS containing 1 % FCS and 0.1 % sodium azide, and incubated for an additional 45 minutes at 4°C in the dark. Thereafter, the cells were washed as before and fixed 5 in 1 % freshly prepared paraformaldehyde for 20 minutes. Fixed cells were washed three times in HBS, resuspended in 0.5 mL HBS, and passed through lOOμ nylon mesh prior to flow cytometric analysis. Samples were stored for no longer than 24 hours in the dark at 4°C, as samples stored for longer periods gave rise to high autofluorescence.

Non-reacted B16-BL6 cells and B16-BL6 cells which reacted directly with FITC-GAMIG, and 2% BSA (bovine serum albumin) served as negative controls. PCL-modified and unmodified cells were labeled and analyzed on either FACS 440 (Becton-Dickinson, Mountainview, CA) or on the FACScan instrument (Becton-Dickinson). Cell populations that were determined to be positive on dual parameter (forward and orthogonal light scatter) analysis were gated, and data were acquired on live gates. Histograms were generated using either Consort 40 software on FACS 440 or consort 30 with LYSYS Software available with FACScan. Appropriate controls were introduced in order to apply logic threshold values. Amelanotic cells which appeared in the population were gated out in the present analysis by light scatter gating. Approximately 10,000 events were tested in each sample. Exposure to AdA, AMPdA and/or to hydrostatic pressure was carried out in a manner similar to that described in Example 5.

As can be seen in Figs. 7a and 7b, maximum expression of both class I and B16-BL6 melanoma- specific antigen was observed with B16-BL6 cells which had been modified by 1 ,200 atmospheres of hydrostatic pressure and simultaneous crosslinking with 20 mM AdA.

In another set of experiments, B16-BL6 cells were exposed to hydrostatic pressure in the range of 600 to 1 ,200 atm and to a constant concentration of AdA of 20 mM. The results are shown in Fig. 8. In a further experiment, B16-BL6 cells were exposed to a constant level of 1,200 atm of hydrostatic pressure during incubation with AdA at concentrations from 0.002 mM to 20 mM, and the results are shown in Fig. 9. As can be seen from these figures, maximum fluorescent intensity for both class I and B16-BL6 tumor antigen was observed following PCL- modification with a combination of a level of pressure of about 1 ,200 atm and an AdA protein crosslinker concentration of about 20 mM.

Example 14

5 Increased in vitro immunogenicity of human tumor cells modified by 2'. 3'- nucleoside or nucleotide dialdehyde protein crosslinker and hydrostatic pressure

PCL-modification studies were extended to human tumors. In general, tumors from 23 patients ranging in age from about 47-80 were processed as described above. In particular, the human tumor types employed were colon

10 carcinomas (primary and metastatic), renal cell carcinomas (primary), non- small cell lung carcinomas (primary), lung carcinomas (primary), and ovarian carcinomas. Tumor cells were obtained and PCL-modified as described in Examples 2 and 5 shortly after tumor resection. Tumor cells may be freshly

J5 prepared and PCL-modified (i.e., within one hour after surgery) or they may be kept in the cold (i.e., 4°C) for up to three days prior to PCL treatment. The resected tumor may be frozen for further or subsequent use or PCL treatment of the tumor cells. It was also discovered that cells such as murine melanoma (i.e., B16 cells) can be successfully stored in the cold for at least one month prior to 0 PCL, especially while data collection via experimentation is ongoing.

The immunogenicity of the human tumor cells was measured in a 5-day IVS assay (see Example 6) in which irradiated PCL-modified or unmodified tumor cells served as stimulators and autologous, PBMCs obtained from the patient about 7 to ^$ 10 days post surgery served as the responders. Autologous, irradiated, PCL- modified PBMCs served as control stimulators. In addition, the non-specific stimulation of PBMCs with anti-CD3 antibody and PHA was used to assess the patient's general immune status. As seen hereinbelow, the proliferative response to 0 CD3 and PHA indicated that all patients were equally immunoresponsive, thus suggesting that the lack of response in a minority of patients (about 25 % or fewer) is not a consequence of immunosuppression or abnormal functioning of immune cells.

The IVS assay results showed that in 5 out of 7 (5/7; 70%) colon 5 carcinomas studied, PCL-modified colon carcinoma cells induced a proliferative response (i.e. , greater than 2-fold) by the patient's autologous PBMCs with a mean response or stimulation index in the responding patients of 6.2 ±_ 1.8, as shown in Table 5:

TABLE 5

Colon Carcinoma - IVS Assay

Tumor Histology Age %V* %τ** RT RP

1 AC 68 50 20 9.0 1.3

2 AC 67 30 40 2.3 0.6

3 AC 60 20 40 6.1 3.1

4 AC 53 50 37 1.2 0.8

5 AC 68 40 40 2.3 1.5

6 AC (Mets) 59 33 27 11.1 1.9

7 AC 49 78 39 0.4 0.6

60.5 ± 43 ± 35 ± 4.6± 1.4±

2.9 7 3 1.6 0.3

*: percent viable cells (total) after Ficoll gradient separation as assessed by Trypan

Blue exclusion.

**: percent tumor cells after Ficoll gradient separation as assessed by cytopathological analysis.

AC: Adenocarcinoma

Responders (RT≥2): 5/7 (71 %), mean SI: 6.2± 1.8

Non-responders : 2/7 (29%), mean : 0.8 ±0.4

The calculation of the mean response ratios are as follows:

(cpm responders + modified tumor) - (cpm modified tumor only)

RT=

(cpm responders + unmodified tumor) - (cpm unmodified tumor),

where RT indicates the proliferation index of responder PBMCs, with PCL- modified tumor cells serving as stimulators in the IVS assay. RT signifies the ratio between the proliferation of responder cells (PBMCs) in the presence of PCL- modified tumor cells and in the presence of unmodified tumor cells.

_ (cpm responders + modified PBMC) - (cpm modified PBMC only)

RP (cpm responders + unmodified PBMC) - (cpm unmodified PBMC),

where RP indicates the proliferation index of responder PBMCs, with PCL- modified autologous PBMCs serving as stimulators. RP signifies the ratio between the proliferation of responder cells (PBMCs) in the presence of PCL-modified autologous PBMCs and unmodified PBMCs.

A comparison of RT/RP shows the specificity of the response of the PBM cells against the PCL-modified tumor cells and is correlated with eventual outcome of PCL-immunotherapy. The RP value serves as a control value, which shows that specific foreign or non-self antigen(s), and not autoantigens, causes specific stimulation and immunoreactivity of the PBM cells in the IVS assay.

The results in Table 5 demonstrate that there is no correlation among the age, %V, %T, and the stimulation response defined by RT> 2. As discussed above, the actual RP value obtained in studies involving colon carcinoma cells was close to 1 , thus indicating that the PBMCs in the assays did not express significant autoantigens which caused nonspecific proliferation. By contrast, the RT values showed that 71 % of the cases had stimulation or response ratios _≥ 2, demonstrating that PCL-modified tumor cells induced specific cell proliferation by the autologous responder PBMC population.

IVS assay results also showed that in 3 out of 4 (3/4; 75 %) lung carcinomas studied, PCL-modified lung carcinoma cells induced a proliferative response (i.e. , greater than 2-fold) by autologous PBMCs with a mean stimulation index of 3.0 0.6, as shown in Table 6. All of the tumors tested in Table 6 are lung tumors

(carcinomas). Adenocarcinoma (AC) is a histological type of non-small cell lung carcinoma (NSCC). Thus, histologically different PCL-modified lung carcinomas were able to stimulate PBMCs in the IVS assay. TABLE 6

Lung NSCC - IVS Assay

Tumor histology Age %V* %T** RT RP

1 AC 63 50 50 2.9 0.8

2 NSCC 76 86 20 1.0 1.0

3 SCC 66 90 25 2.0 1.2

4 AC 64 90 30 4.2 0.8

67.2± 79 ± 31 ± 2.5± 0.9±

3.0 10 7 0.7 0.1

Responders (RT≥2): 3/4 (75 %), mean SI: 3.0±0.6 Non-responders : 1/7 (25 %), mean : 1.0

Table 7 presents the PHA and CD3 (assayed using monoclonal anti-CD3 antibody) responses of the PBMCs of patients whose lung carcinomas were analyzed (as presented in Table 6 above). As discussed above, such assays using non-specific cell stimulating agents (such as PHA and/or anti-T cell antibody CD3) serve as controls for the general status of an individual's immune responsiveness, independent of stimulation with specific, PCL-modified cells (e.g. , tumor cells) used as stimulators in the IVS assays performed in accordance with the invention. Further, the results of these studies showed that the response of PBMCs to either PHA or OKT 3 cannot necessarily be used alone to predict whether or not a patient's PBMCs will be stimulated to react against PCL-modified tumor cells. TABLE 7

Lung NSCC - PHA. OKT3 responses

Patient PHA-pre OKT3-pre PHA-post OKT-post

PHA-control OKT3-control PHA-pre OKT3-pre

1 0.82 1.86 1.47 1.03

2 ND

3 0.09 0.32 1.01 1.43

4 0.49 0.18 0.17 0.19

Mean±SEM 0.5±0.2 0.8±0.5 0.9±0.4 0.9±0.4

It was further demonstrated that 4 out of 4 (4/4; 100%) PCL-modified renal cell carcinomas studied induced a proliferative response (i.e., greater than 2-fold) by autologous PBMCs with a mean stimulation index of 6.3 _+ 3.3, as shown in Table 8:

TABLE 8

RCC - IVS Assay

Tumor Histology Age %V* %T** RT RP

1 RCC 73 82 34 16.1 1.0

2 RCC 69 65 32 3.6 0.6

3 RCC 72 70 46 2.5 0.6

4 RCC 57 90 38 _ 2.9 _ 0.7

67.8± 77± 37± 6.3± 0.7±

3.7 6 3 3.3 0.1

RCC: Renal cell carcinoma

Responders (RT>2): 4/4 (100%), mean : 6.3±3.3

Table 9 presents the PHA and OKT3 responses of the PBMCs of patients whose renal cell carcinomas were analyzed (see Table 8 above): TABLE 9

RCC - PHA and OKT3 responses

Patient PHA-pre OKT3-pre PHA-post OKT3-post

PHA-control OKT3-control PHA-pre OKT3-pre

1 1.29 0.48 0.12 0.14

2 0.12 0.43 3.60 0.81

3 0.29 <0.1 2.03 29.10

4 1.5 0.82 0.22 0.83

Mean ± SEM 0.8±0.3 0.5±0.1 1.5±0.8 0.6±0.2

In addition, assays similar to those presented using tumor tissue and cells from colon, lung, and renal cell carcinomas were performed using tumor cells derived from tumor tissue and cells from one patient's ovarian carcinoma. The response ratio was less than one, indicating that this individual was a nonresponder (i.e. , under the conditions of the IVS assays described herein, the individual's PBMCs were unable to mount an immunoreactive response against autologous PCL-modified ovarian tumor cells) and would not be likely to respond in a positive fashion to PCL-modification of cells and subsequent PCL-immunotherapy.

In another analysis using colon tumor tissue and PBMCs from patients over 80 years of age, nonresponse status was observed (Table 10), thus leading to the conclusion that such individuals should be excluded from the PCL-modification technology and immunotherapy approaches as described for the invention.

TABLE 10

Colon Patients > 80 Years of Age

Tumor Histology Age %V %T RT RP

Colon AC 83 45 42 1.0 1.5 AC 80 72 42 1.0 1.1

81.5 58 42 1.0 1.3

Responders (RT> : 0/2 (0%), mean : 1.0 TABLE 11

PHA and OKT3 responses - Patients > 80 years old

Patient PHA-pre OKT3-pre PHA-post OKT3-post PHA-control OKT3-control PHA-pre OKT3-pre

1 0.23 0.07 1.86 1.31

2 0.27 0.20 1.28 0.71

Mean ± SEM 0.3±0.1 0.2±0.1 1.6±0.3 1.0±0.3

Using various tumor types in IVS assays with histologically different tumors revealed that, in the majority of cases, a stimulation ranging from about 250 to about 630% was induced by the PCL-modified tumor cells. In addition, when PCL-modified normal cells, i.e. , PBMCs, were used as sensitizing cells in the IVS assay, minimal stimulation, ranging from 0 to about 40% , was observed. The highest extent of stimulation was noted for renal cell carcinoma cells (and also melanoma cells). Although a lower extent of stimulation was noted for non-small cell lung carcinoma cells and colon carcinoma cells, in all of these cases, without any exception, a stimulatory effect was noted. It is probable that renal cell carcinomas and melanomas may be more immunogenic types of tumors than non- small cell lung carcinomas and colon carcinomas; however, response ratios greater than or equal to 2 were demonstrated against all of the tumor types presented in Tables 5, 6, and 8. In addition, the highest response ratio values determined by the novel methods of the invention may be directly correlated with the highest response rates (or more aggressive immunoreactivity) against the relevant tumor types in vivo.

Example 15

IVS assay and cytokine analysis results for the determination of the course of treatment and the prediction of the clinical outcome of PCL immunotherapy

Experiments were performed further to those described in Example 14 in which cancer patients were screened as described for their responsiveness to PCL immunotherapy and to determine whether or not a patient was likely to have a positive clinical outcome from PCL-immunotherapy and/or adoptive immunotherapy treatments. As mentioned in the preceding example, a positive clinical outcome of PCL immunotherapy potentially correlates highly with a patient's stimulation or response index of greater than about 1.5 or 2, obtained via IVS assays using the patient's peripheral blood mononuclear cells (PBMCs) as responders and PCL-modified or control (unmodified) tumor cells (or infected cells) as stimulators in these in vitro stimulation and proliferation assays.

The experiments set forth in this example utilized a correlation between the stimulation or response index and cytokine synthesis and secretion by a patient's PBMCs to determine whether or not a patient should undergo an alternative treatment regimen (i.e., chemotherapy), or whether or not a patient would be a successful or an unsuccessful candidate for prolonged immunotherapy protocols. The secretion of cytokines by PBMCs was tested by in vitro protocols such as enzyme linked immunosorbent assays (ELISAs). The cytokine ELISAs were carried out as follows:

Primary antibody was prepared by diluting the anti-cytokine capture antibody in carbonate buffer (0.1 M) at pH 9.5. The antibodies used were 1) IL-4: Mouse anti-human IL-4 monoclonal antibody (at a concentration of 2 μg/mL); IL- 10: 2) Rat anti-human IL-10 monoclonal antibody (at a concentration of 4 g/mL); 3) IFN-γ: rabbit polyclonal anti-human IFN-γ (at a concentration of 1 :250); and 4) IL-2: Mouse anti-human IL-2 monoclonal antibody (at a concentration of 2.5 μg/mL). 50 μL of antibody were added to the wells of a 96-well microtiter plate (Nunc). The plates were incubated overnight at 2-8°C or for 8 hours at room temperature and then washed three times with phosphate buffered saline (PBS) containing 0.05 % Tween, pH 7.4.

Next, the wells of the plates were blocked with PBS supplemented to contain 10% fetal calf serum (FCS) (250 μL/well); the plates were incubated for 1 - 2 hours at room temperature; and then were washed two times with PBS containing 0.05 % Tween. To the blocked wells of the plates were added 40 μL per well of the standards (i.e.. recombinant human IL-4 at concentrations of 15 pg/mL-2 ng/mL: recombinant human IL-10 at concentrations of 15 pg/mL-2 ng/mL; recombinant human IFN-γ at concentrations of 60 pg/mL-4 ng/mL; and recombinant human IL-2 at concentrations of 15 pg/mL-1 ng/mL), or about 40 to 100 μL of the samples to be tested (i.e. , the culture supernatants from the IVS assays), diluted in complete cell culture medium. The plates containing the standards and test samples were incubated for 1 hour at 37 °C and then were washed three to four times with PBS containing 0.05% Tween.

The anti-cytokine secondary monoclonal antibodies (mAb) were diluted in PBS containing 10% FCS. The monoclonal antibodies used were as follows: for IL-4 detection, biotinylated rat anti-human IL-4 mAb at a concentration of 1 μg/mL; for IL-10 detection, biotinylated rat anti-human IL-10 mAb at a concentration of 2 μg/mL; for IFN-γ detection, mouse anti-human IFN-γ mAb at a concentration of 2 μg/mL; and for IL-2 detection, biotinylated mouse anti-human IL-2 mAb at a concentration of 1.25 μg/mL. 10 μL of the anti-cytokine monoclonal antibodies were added to the appropriate wells; the plates were incubated for 1 hour at 37 °C and then washed three to four times with PBS containing 0.05 % Tween. To detect bound antibody-cytokine complexes, a detection reagent was used comprising avidin-peroxidase diluted 1 :500 in PBS containing 10% FCS (1 mg/mL solution; Jacson ImmunoResearch Lab. , Inc.) for detection of the IL-2, IL-4 and IL-10 cytokines. In the case of IFN-γ, dilute peroxidase-conjugated goat anti- mouse IgG (H+L) (Jacson ImmunoResearch Lab., Inc.) was diluted 1 :500 in the same buffer. The detection antibodies (100 μL) were added to the wells of the plates and were incubated at room temperature for 1 hour. Thereafter, the plates were washed four times with PBS containing 0.05 % Tween.

The substrate for the peroxidase reaction development was prepared by dissolving 1 mg of tetramethyl benzidine (TMB) (Sigma) in 1 mL DMSO and adding 9 mL of 0.05 M phosphate-citrate buffer, pH 5.0. Immediately prior to use, 2 μL of 30% hydrogen peroxide (Sigma) were added per 10 mL of substrate buffer solution. All components for human IL-4 and IL-10 cytokine detection were obtained from PharMgen; the Duoset ELISA Development System for human IL-2 detection and all components used for human IFN-γ were purchased from Genzyme Diagnostics (Cambridge, MA).

Patients with colon tumors, renal cell carcinomas, and non-small cell lung carcinomas were tested. The results of these studies are shown in Tables 12, 13, and 14. Tables 12 and 13 are divided into two panels each: one panel shows the results of a patient whose PBMCs responded positively to PCL-modified colon carcinoma cells in the IVS assay (i.e., a positive responder, IVS(+)); the other panel shows the results of a patient whose PBMCs did not proliferate in response to PCL-modified colon carcinoma cells in the IVS assay (a negative responder, IVS(-)). Table 14 shows the results of a positive responder to PCL-modified non- small cell lung carcinoma cells in the IVS assay. In Tables 12, 13, and 14, the proliferation and cytokine secretion of tumor sensitizer cells, responder PBMCs, and sensitizer and responder cells cultured together are presented. For controls, the sensitizer cells were either unmodified or PCL-modified PBLs or tumor cells. Other controls included PBL responder cells cultured together with only complete medium or with the T cell stimulators PHA or OKT3. The PBL responders (R) + tumor cell sensitizers (S) were cultured together and tested in the in vitro assay and included unmodified PBLs with PBL responder cells (i.e. , PBL + R); PCL- modified PBLs + PBL responder cells (i.e. , PBL-PCL 4- R); unmodified tumor cells + PBL responder cells (i.e. , Tu + R); and PCL-modified tumor cells -I- PBL responder cells (i.e., Tu-PCL + R). In these studies, the levels of IL-4 that were secreted were below detection and IL-2 levels were not assessable.

Notwithstanding, the data demonstrating the effect of PCL treatment of tumor cells used as stimulations on the secretion of the cytokines IL-10 and IFN-γ by responder PBLs supports the applicability of this procedure to determine T_H1 and τ_H2 patterns, thus allowing the determination of a positive or a negative clinical outcome of PCL immunotherapy for a patient as described hereinabove.

TABLE 14 rvs (+)

IVS RT: 8.3 RP: 1.9

Prolif. IL-10 IFN-γ

Groups (cpm) (pg/ml) (pg/ml)

Sensitizers (S)

PBL 1571 ±822 0 0

PBL-PCL 1 ±43 0 1359

Tu 3726 ±867 0 5340

Tu-PCL 2645 ±217 0 3150

Responders CR)

-fcompl. med 1947 ±673 0 0

+PHA 18243 ±2646 1047 0

+OKT3 8857±2238 357 0

R + S

PBL+R 2500 ±344 102 1641

PBL-PCL+R 2930 ±8 0 1641

Tu+R 4359 ±884 0 1527

Tu-PCL+R 7993 ±824 0 2349

Specifically, in all of the IVS(+) individuals, an increase in the level of IFN-γ that was secreted by responder PBLs that had been stimulated with PCL- modified tumor cells was observed versus the level of this cytokine that was secreted by PBLs stimulated with unmodified tumor cells (i.e., 0 versus 2174 pg/mL in Table 12; 0 versus 540 pg/mL in Table 13; and 1527 versus 2349 pg/mL in Table 14). More particularly, in Table 12, it is seen that there was a complete shut off of IL-10 secretion (i.e., from 1109 to 0 pg/mL) in the IVS(+) patient, in conjunction with a complete "turn on" of IFN-γ secretion (i.e., from 0 to 2174 pg/mL). Also, the IVS(-I-) patient in Table 14 showed a high response index (RT: 8.3), no IL-10 production, and a boost in IFN-γ production (i.e. , from 1527 to 2349 pg/mL). These results indicate that the patients have produced a cell mediated response to their tumors, that a T_H1 response was produced, and/or a T_H2 to T_H1 conversion had occurred. These results are also indicative and predictive of a positive clinical response or outcome to PCL immunotherapy (i.e. , the patient can be expected to maintain a cell mediated response to fight and destroy his or her tumor cells during the course of immunotherapy).

With regard to the IVS(-) individuals shown in Tables 12 and 13, two different results (which correlate with at least two different clinical outcomes of PCL immunotherapy) are observed. In Table 12, PCL-modification of the IVS (-) patient's tumor cells and the use of these cells as stimulators in the IVS assay caused an increase in the level of IFN-γ that was secreted by responder PBLs (0 versus 370 pg/mL). In this case, the production of IFN-γ after stimulation by PCL-modified tumor cells in this IVS(-) patient correlates with a pattern of T_H2 to T_H1 conversion and is predictive of a potential clinical response or outcome to PCL immunotherapy, despite the low response index found for this patient. In addition, the response by this patient may be boosted by repeated vaccinations or immunizations with PCL-modified tumor cells to maintain the T_H1 response and/or to activate and stimulate more of the appropriate cell types to respond. By contrast, in Table 13, the IVS(-) patient shows secretion of IL-10 and a complete turn off of IFN-γ secretion after culture and stimulation of PBLs by PCL-modified tumor cells. This indicates that a T_H2 to T_H1 conversion has not occurred; therefore, in the absence of a proliferation response and no production of IFN-γ, it can be determined that this patient will have neither a successful cell mediated immune response nor a successful clinical response, and thus is unlikely to benefit from immunotherapy. It is also noted that decreases in the production of an IVS(-) patient's IFN-γ and IL-10 secretion can be indicative of a suppressor T cell response which provides a poor prognosis for that individual in that he or she does not have the ability to produce an adequate cell mediated immune response and therefore is predicted to have a negative or adverse clinical outcome involving immunotherapy.

The invention also encompasses a more rapid or quick screening assay for determimng the cytokine pattern secreted by a patient's peripheral blood cells and/or a T_H2 to T_H1 conversion in response to PCL modified stimulator cells (e.g., tumor or infected cells) relative to nonmodified stimulator cells. The more rapid assay method comprises the use of the tumor preparation containing both tumor cells and resident PBMC populations as described in Example 2. In this version of analyzing the cytokine pattern, the cytokines secreted by the PBLs localized within the tumor can be assayed as described to determine the T_H profile; the results obtained are expected to be representative of the response determined by assaying the patient's PBLs isolated from blood. By analyzing the profile of cytokines elicited by the PBLs comprising the tumor preparation prior to and after PCL modification of the cells in the tumor preparation, the cytokine pattern of the tumor-associated PBLs can be analyzed without the need to rely on a patient's freshly drawn blood sample. In this manner, the determination of the cytokine pattern can be determined more rapidly, thereby increasing the efficiency of these types of analyses. In addition, the cytokine pattern analysis can still be performed using PMNs isolated from a patient's blood sample using the disclosed IVS assays, as necessary or desired.

Moreover, it will be appreciated that the tumor cell preparation containing the various types of mononuclear blood cells as described in Example 2 can also be used to analyze the proliferation of a patient's mononuclear blood cells against tumor cells having particular surface molecules, as well as to detect the presence or absence of particular cell surface molecules which are on these PBMCs and which are associated with the generation of an appropriate immune response against antigens, e.g. , tumor and infected cell surface molecules, and the like. Accordingly, the suspended admixture of tumor cells and peripheral blood cells derived from the tumor tissue preparation may ultimately used in the IVS assays as described herein to measure proliferation, cytokine secretion, and the like, and also to substitute for or to supplement the use of a patient's freshly drawn blood sample and the cells obtained therefrom in these types of assays.

In a related aspect, the determinations as described above can be used to decide if an individual should undergo chemotherapy or radiation treatment(s) rather than immunotherapy treatment(s) . If a patient has a low or negative response index and shows no indication that his or her cytokine pattern supports a cell mediated or a T_H1 response, the medical provider can offer chemotherapy or radiation treatment regimens to the patient which will ultimately benefit the patient and impact in a significant way on his or her survival outcome.

Example 16

In vivo immunization and survival studies of animals vaccinated with an immunogenic preparation comprising modified B16/B16 melanoma cells and non- classical adjuvant to enhance the immune response

Experiments similar to those as described in Example 12 are performed to test the survivability of C57BL 6 mice immunized prophylactically with immunogenic preparations comprising B16/BL6 tumor cells that are PCL- modified by exposing the tumor cells to AdA crosslinker at about 10 mM at the time that the cells are also subjected to hydrostatic pressure of about 1200 atmospheres. The immunogenic preparations also contain either GM-CSF or hGH as a non-classical adjuvant. Unmodified or untreated cells are used as controls as described. As described in Example 12, BL6/BL6 melanoma cells are a very invasive variant of the B16 cell line (Hart 1979, Am. J. Pathology, 97:587) and are obtained from B16/BL6 tumors that are serially passaged in syngeneic C57BL/6 mice by subcutaneous (s.c.) inoculation of 2 - 5 x 10⁶ cells.

In the in vivo experiments described in this example, the viability of C57BL mice challenged with 1 x 10⁵ viable non-PCL-treated B16/BL6 tumor cells is tested following immunizations with the immunogenic preparations comprising B16/BL6 cells, either unmodified or PCL-modified in accordance with the invention. The cells used for vaccination are of the same kind as the cells used to challenge the mice.

After the immunizations with the immunogen preparations, the immunized animals are challenged by injection with viable B16/BL6 tumor cells and the ability of immunized animals to survive the challenge is assessed over a period of about 45 days or longer post-challenge.

The immunization protocol comprises two vaccinations, i.e. , injections of an immunogenic preparation comprising about 20 x 10⁶ PCL-modified 83

B16/BL6 tumor cells formulated with either hGH or GM-CSF at concentrations of 1 μg/mL to 100 μg/mL per injection, administered one week apart. The dose concentrations used for each adjuvant are typically 1, 5, 10, 20, 50, and 100 μg/mL. One week after the last vaccination, animals are challenged with tumor cells. Control immunogens contain unmodified cells (e.g., in medium such as Hank's Balance Salt Solution, HBSS) with and without the presence of adjuvant, as well as PCL-modified cells without adjuvant. In general, 6-10 mice are immunized to test the controls and each immunogenic preparation containing PCL-modified cells and the various doses of adjuvant.

The following parameters are tested for each group of animals described in this example: survival rate and the tumor number and mean tumor diameter (mm²) measured using standard callipers in three orthogonal directions and presented by the mean value.

Example 17

DTH response in a melanoma patient treated with allogeneic PCL-treated melanoma cells and GM-CSF as non-classical adjuvant

DTH analyses were performed in a human patient to assess the DTH response to PCL-immunogens administered in combination with non-classical adjuvants, such as GM-CSF, as described hereinabove and in Example 16. A melanoma patient was immunized with an immunogen comprising PCL-modified allogeneic melanoma cells in conjunction with non-classical adjuvant, i.e. , GM- CSF. It is to be understood that autologous melanoma cells may also be used.

Prior to the immunization protocol using PCL-modified immunogen and adjuvant, the patient was screened or pretested in a baseline DTH assay to select the optimal adjuvant dose of GM-CSF to use during the immunization protocol. In the DTH baseline screening assay, 1 x 10^s PCL-treated allogeneic melanoma cells were injected subcutaneously (SC) at sites 2 inches apart in the patient's forearm. PCL- treated cells were injected alone (0.5 cc) or mixed with 20 μg (low dose) or 100 μg (high dose) of GM-CSF (Leukine, available from Immunex Corp.) in 0.1 cc. Cells for immunization were PCL modified as described using 10 mM AdA and 1200 atm pressure. The GM-CSF injections were repeated using the respective low and high doses at 24 and 48 hours at the DTH immunization sites. The development of the DTH response was scored at 24 and 48 hours after immunization by measuring the diameter or size of the area of the area of erythema at the immunization site using callipers and as known by those in the art (Table 15). For example, if the DTH response area was essentially circular, a single diameter was measured and reported; if the response area was non-circular or irregular in shape, the area was measured in two dimensions (at two independent positions) and the mean of the measurements was determined. Based on the patient's response to the DTH baseline assay, a given dose of GM-CSF (i.e., 100 μg) was selected for use in the PCL immunization protocol.

The immunization protocol was generally carried out about 48 hours following the baseline DTH assay and comprised a course of three subcutaneous injections, most preferably at sites near a draining lymph node. For the initial injection (day 0), the patient was immunized SC (e.g. , in the forearm or in the thigh) with 1 x 10⁷ PCL-treated cells, together with 100 μg of GM-CSF (0.6 cc total). In general, the immunizing dose of PCL-modified cells as immunogen was on the order of about, or greater than, ten times the number of cells used in the DTH screening assay (e.g. , 1 x 10⁷ cells versus 1 x 10⁵ cells in the DTH screen). The second injection at 24 hours comprised GM-CSF adjuvant alone (100 μg) at the same site and the third injection at 48 hours comprised adjuvant alone (100 μg). As described herein, IVS assays were performed at several intervals (e.g., at four and six weeks) following the immunization protocol to assess the patient's immune response status and potency level. At the end of the protocol, DTH assays were again performed as described to evaluate the patient's immune response to both PCL modified and unmodified cells. The use of GM-CSF as adjuvant revealed a clear and significant augmentation of the patient's DTH immune response in a dose-dependent manner, as evidenced by the size of the area of the erythema (i.e. , the DTH response area) at the immunization site after injections with 10⁵ PCL-treated melanoma cells and either 20 or 100 μg of GM-CSF administered as adjuvant as described. The results 85

are presented in Table 15:

TABLE 15

Size of DTH Response Area at Immunization Site

Immunogen Adjuvant 24 hours after 48 hours after immunization immunization

1x10^s PCL- treated allogeneic — 0.5 cm — melanoma cells

1x10^s PCL- treated allogeneic GM-CSF (20 μg) 2.5 cm 2.1 cm melanoma cells

1x10^s PCL- treated allogeneic GM-CSF 3.5 cm 2.9 cm melanoma cells (100 μg)

Example 18

PBMC proliferation associated with cell surface molecule presentation (e.g.. co- stimulatory molecules and cell adhesion molecules)

Experiments are performed to analyze cell surface molecules present on tumor cells, infected cells, or on PBMCs in order to determine the immunogenic potential of antigen presenting cells and the ability of PBLs to respond to antigen. The analysis of cell surface molecules complements the proliferation and cytokine secretion measurements as described and offers additional markers for determining and predicting if a patient will produce an effective immune response and have a successful clinical outcome of immunotherapy treatment.

In these experiments, IVS assays are performed to determine the potential immune responsiveness of a patient's mononuclear blood cells to PCL- modified versus unmodified tumor cells by testing for the ability of the PBMCs to proliferate in response to co-stimulatory molecules, such as B7-1 and B7-2, as well as other co-stimulatory molecules, which are present on the surfaces of some tumor cells (see, for example, June, CH. et al. , 1994, Immunol. Today, 15:321-331 ; Freeman, G.J. et al. , 1993, Science, 262:909-911; Nabavi, N. et al., 1992, Nature, 36Q:266-268; Freeman, G.J. et al. , 1991 , /. Exp. Med. , 174:625-631; and Gimmi, CD. et al., 1991, Proc. Natl. Acad. Sci. USA, 8 : 6575-6579). In addition to the proliferation analyses, the presence or absence Of B7-1 and B7-2 is assayed using conventional techniques, for example, ELISA and monoclonal antibody detection. It has been reported that the B7-1 and B7-2 glycoprotein molecules are not present on many types of tumor cells. Further, a tumor cell may have the proper MHC or HLA presentation, but it also requires the proper co- stimulatory signals provided by molecules such as B7-1 or B7-2 to stimulate an appropriate immune response. Thus, tumor cells lacking these co-stimulatory molecules may not stimulate an appropriate immune response by a patient's lymphocytes.

In accordance with the invention, tumor cells are subjected to PCL modification and are tested in IVS assays with a patient's PBMCs. along with unmodified tumor cell controls. The presence or absence of B7-1 or B7-2 co- stimulatory molecules on tumor cells is determined by ELISA assay using commercially-available anti-B7 monoclonal antibodies (e.g., Valle, A. et al. , 1990, Immunology, 69:531-535) and protocols similar to those described for the cytokine analyses. Proliferation in response to PCL-modified tumor cells that are shown to have B7-1 or B7-2 co-stimulatory molecules in these assays relative to unmodified tumor cells is indicative that a patient will be likely to respond in a positive manner to immunotherapy to reduce or eradicate the tumor. In accordance with the invention, PCL-modification of tumor cells may enhance the presentation of and/or force the appearance of B7-1 and B7-2 molecules on the tumor cell surface, if the levels of these molecules are low or suboptimal on the native or unmodified cells. As described above, these types of analyses can also be performed on cells from surgically-removed tumor tissue preparations containing PBMCs and tumor cells before and after PCL treatment. Similarly, the presentation of cell adhesion molecules (CAM), for example, ICAM-1, ICAM-2, ICAM-3 (de Fougerolles, A.R. et al. , 1994, J. Exp. Med. , 179:619-629; Kirchhausen, T. et al. , 1993, J. Leukoc. Biol. , 53:342-346) which are displayed on the surfaces of B cells, T cells, and macrophages, and which interact with the LFA-1, 2, or 3 ligand molecules present on T cells, may be augmented and improved by PCL modification in accordance with the invention. PCL-modified PBMCs may have increased or augmented ICAM presentation as a result of the crosslinking and pressure treatment of the invention, and thus, will be expected to interact more successfully with (or to adhere more effectively to) target cells to effect their ultimate destruction. To determine if a patient's PBMCs display ICAM molecules that may impact in a positive way on the patient's ability to generate a successful immune response against tumor or infected cells or foreign antigens, the presence of ICAM molecules is assessed by antibody assays, e.g. ELISA, following co-culture of PBLs with PCL-modified tumor or infected cells to measure the proliferative response (i.e. , in an IVS assay). In addition, commercially-available anti-ICAM antibodies (monoclonal or polyclonal) are used in E ISAs to determine the levels of ICAM molecules on the cell surfaces of the PBMCs in these assays. Alternatively, anti-ICAM antibodies are added to the

IVS assay cell samples and the levels of PBMC proliferation is measured. Control samples receive no anti-ICAM antibody. If the addition of anti-ICAM antibodies results in a decrease in cell proliferation relative to the controls without antibody in these assays, it is concluded that ICAM molecules are present on the responding cells and that the added anti-ICAM antibody has inhibited proliferation by binding to ICAM molecules on the cell surface. The presence of detectable ICAM molecules on a patient's PBMCs is indicative that the patient is likely to respond positively to tumor or infected cells and will be able to mount an effective cell mediated immune response against the specific target cells. These types of analyses can also be performed on cells obtained from surgically-removed tumor tissue preparations containing PBMCs and tumor cells before and after PCL treatment. The contents of all patents, patent applications, published articles, books, and abstracts cited herein are hereby incorporated by reference in their entirety.

As various changes can be made in the above-described subject matter without departing from the scope and spirit of the invention, it is intended that all subject matter contained in the above description, shown in the accompanying drawings, or defined in the appended claims will be inteφreted as illustrative, and not in a limiting sense.

Claims

HAT IS CLAIMED IS:

1. A method of determining if an individual beset with a cancer, a tumor, or an infection is likely to respond to in vivo immunotherapy treatment by mounting a cell mediated response to reduce, inhibit, or destroy the tumor or infection, comprising: a) exposing autologous or allogeneic cancer, tumor or infected cells to a 2', 3' nucleoside or nucleotide dialdehyde crosslinking compound at a concentration and for a time effective to crosslink proteins in the cells' plasma membranes and subjecting the cells to hydrostatic pressure for a time sufficient to 0 cause a modification of proteins in the cells' plasma membranes, thereby resulting in a crosslinked and pressure-treated modified cell preparation; b) measuring the ability of the modified cells of step a) to stimulate the proliferation and immunoreactivity of the individual's mononuclear 5 blood cells compared with the ability of native or unmodified cells to stimulate the proliferation and immunoreactivity of the mononuclear blood cells; and c) determining the cytokine pattern secreted by the mononuclear blood cells, said cytokine pattern comprising the levels of secreted T_H1 cytokines o that are stimulatory for a cell mediated immune response, and the levels of secreted T_H2 cytokines that are (i) inhibitory for a cell-mediated immune response or (ii) stimulatory for a humoral immune response, to ascertain if the individual will or will not respond to immunotherapy treatment.

5

2. The method according to claim 1 , wherein a response or stimulation index is obtained from the measuring step b).

3. The method according to claim 2, wherein the response or 0 stimulation index is about 1.5 to 2 or greater and indicates a high level of proliferative response of the peripheral blood mononuclear cells to modified stimulator cells.

4. The method according to claim 2, wherein the response or stimulation index is less than about 1.5 to 2 and indicates a low level of proliferative response of the peripheral blood mononuclear cells to modified stimulator cells.

5. The method according to claim 3, wherein the high level of proliferative response combined with at least one or more of: (i) a high level of stimulatory T_H1 cytokine secretion; (ii) a low level of T_H1 inhibitory cytokine secretion; (iii) a low level of stimulatory T_H2 cytokine secretion; or (iv) a low level of cytokines stimulatory for the humoral immune response, indicate that the individual will respond to immunotherapy treatment by mounting a cell mediated immune response against the cancer, tumor or infected cells.

6. The method according to claim 4, wherein the low level of proliferative response combined with at least one or more of: (i) a high level of stimulatory T_H1 cytokine secretion; (ii) a low level of T_H1 inhibitory cytokine secretion; (iii) a low level of stimulatory T_H2 cytokine secretion; or (iv) a low level of cytokines stimulatory for the humoral immune response indicate that the individual will respond to immunotherapy treatment by mounting a cell mediated immune response against the cancer, tumor or infected cells.

7. The method according to claim 3, wherein the high level of proliferative response combined with at least one or more of: (i) a low level of stimulatory T_H1 cytokine secretion; (ii) a high level of T_H1 inhibitory cytokine secretion; (iii) a high level of stimulatory T_H2 cytokine secretion; or (iv) a high level of cytokines stimulatory for the humoral immune response indicate that the individual will not respond to immunotherapy treatment by mounting a cell mediated immune response against the cancer, tumor or infected cells.

8. The method according to claim 4, wherein the low level of proliferative response combined with at least one or more of: (i) a low level of stimulatory T_H1 cytokine secretion; (ii) a high level of T_H1 inhibitory cytokine secretion; (iii) a high level of stimulatory T_H2 cytokine secretion; or (iv) a high level of cytokines stimulatory for the humoral immune response indicate that the individual will not respond to immunotherapy treatment by mounting a cell mediated immune response against the cancer, tumor or infected cells.

9. The method according to claim 5 or claim 6, wherein the cytokine pattern indicates that a cell mediated immune response or a T_H2 to T_H1 switch has occurred in the individual.

10. The method according to claim 7 or claim 8, wherein the cytokine pattern indicates (i) that a sustained T_H2 response has occurred or (ii) that a T_H2 to T_H1 switch has not occurred in the individual.

11. The method according to claim 1 , further comprising the step of determining the phenotypes of mononuclear blood cell populations that are proliferating in response to the modified stimulator cells by immunophenotyping or by cytotoxic T cell assay.

12. The method according to claim 11 , wherein the cell populations are CD4+ T cells or CD8+ T cells.

13. The method according to any of claims 1 to 4, wherein the mononuclear blood cells of step b) are obtained from a patient afflicted with a tumor.

14. The method according to any of claims 1 to 4, wherein the mononuclear blood cells of step b) are obtained from a patient afflicted with an infection.

15. The method according to claim 13, wherein the tumor cells are pancreatic tumor cells, ovarian tumor cells, melanoma cells, carcinoma cells. breast tumor cells, colon cancer cells, renal cancer cells, lung tumor cells, non- small cell lung carcinoma cells, small cell lung carcinoma cells, bladder cancer cells, hematopoietic cancer cells, or prostate cancer cells.

16. The method according to claim 15, wherein the tumor cells are melanoma cells, colon cancer cells, renal carcinoma cells, or non-small cell lung carcinoma cells.

17. The method according to claim 14, wherein the infected cells are cells infected with bacteria, viruses, parasites, or yeast.

18. The method according to claim 17, wherein the infected cells are virus-infected cells.

19. The method according to claim 1 , wherein the 2', 3' nucleoside or nucleotide dialdehyde crosslinker is 2' , 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde.

20. The method according to claim 19, wherein the 2', 3'-adenosine dialdehyde or 2' , 3 '-adenosine monophosphate dialdehyde is used at a concentration of about 1 mM to about 20 mM.

21. The method according to claim 20, wherein the 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde is used at a concentration of about 5 mM to about 15 mM.

22. The method according to claim 1 , wherein the hydrostatic pressure is in the range of about 800 to about 1400 atmospheres.

23. The method according to claim 22, wherein the hydrostatic pressure is in the range of about 900 to about 1200 atmospheres.

24. The method according to claim 1 , wherein exposure of the cells to the 2', 3' nucleoside or nucleotide dialdehyde crosslinker in step a) occurs at the same time that the cells are subjected to hydrostatic pressure.

25. A method for improving the ability to select patients who are likely to produce a cell mediated immune response to a cancer, tumor, or infection during in vivo immunotherapy treatment, comprising: a) obtaining tumor cells or infected cells from the tumored or infected patient, or from another individual having the same type of tumor or infection; b) exposing the tumor or infected cells to i) a 2', 3' nucleoside or nucleotide dialdehyde crosslinker at a concentration and for a time sufficient to cause crosslinking of the proteins in the cells' plasma membranes, and ii) to hydrostatic pressure for a time sufficient to cause a modification of the proteins in the cells' plasma membranes, thereby resulting in a modified tumor or infected cell preparation; c) incubating mononuclear blood cells from the blood of a patient afflicted with the tumor or infection in admixture with the crosslinker- and pressure-modified cells of step b), under conditions to provide stimulated and immunoreactive cells specifically immunoreactive against the modified tumor or infected cells; d) measuring the level of specific stimulation or immunoreactivity of the mononuclear blood cells of step c) against the modified tumor or infected cells relative to the level of stimulation or immunoreactivity of the mononuclear blood cells against unmodified tumor or infected cells to obtain a response or stimulation index; and _e) determining the cytokine pattern secreted by the stimulated mononuclear blood cells of step d), said cytokine pattern comprising the levels of secreted T_H1 cytokines that are stimulatory for a cell mediated immune response, and the levels of secreted T_H2 cytokines that are (i) inhibitory for a cell-mediated immune response or (ii) stimulatory for a humoral immune response, to select those patients who are likely to produce a cell mediated immune response to a cancer, tumor, or infection during in vivo immunotherapy treatment.

26. The method according to claim 25, wherein the response or stimulation index of step d) is above about 1.5 to 2, indicating a high level of proliferative response and is combined with at least one or more of: (i) a high level of stimulatory T_H1 cytokine secretion; (ii) a low level of T_H1 inhibitory cytokine secretion; (iii) a low level of stimulatory T_H2 cytokine secretion; or (iv) a low level of cytokines stimulatory for the humoral immune response, thereby indicating that 0 the patient is likely to produce a cell mediated immune response to a cancer, tumor, or infection during immunotherapy treatment.

27. The method according to claim 25, wherein the response or 5 stimulation index of step e) is below about 1.5 to 2, indicating a low level of proliferative response and is combined with at least one or more of: (i) a high level of stimulatory T_H1 cytokine secretion; (ii) a low level of T_H1 inhibitory cytokine secretion; (iii) a low level of stimulatory T_H2 cytokine secretion; or (iv) a low level _Q of cytokines stimulatory for the humoral immune response, thereby indicating that the patient is likely to produce a cell mediated immune response to a cancer. tumor, or infection during immunotherapy treatment.

28. The method according to claim 25, wherein the response or 5 stimulation index of step e) is above about 1.5 to 2, indicating a high level of proliferative response and is combined with at least one or more of: (i) a low level of stimulatory T_H1 cytokine secretion; (ii) a high level of T_H1 inhibitory cytokine secretion; (iii) a high level of stimulatory T_H2 cytokine secretion; or (iv) a high 0 level of cytokines stimulatory for the humoral immune response, thereby indicating that the patient is not likely to produce a cell mediated immune response to a cancer, tumor, or infection during immunotherapy treatment.

₅ 29. The method according to claim 25, wherein the response or stimulation index of step e) is below about 1.5 to 2, indicating a low level of proliferative response and is combined with at least one or more of: (i) a low level of stimulatory T_H1 cytokine secretion; (ii) a high level of T_H1 inhibitory cytokine secretion; (iii) a high level of stimulatory T_H2 cytokine secretion; or (iv) a high level of cytokines stimulatory for the humoral immune response, thereby indicating that the patient is not likely to produce a cell mediated immune response to a cancer, tumor, or infection during immunotherapy treatment.

30. The method according to claim 26 or claim 27, wherein the cytokine pattern indicates that a cell mediated immune response or a T_H2 to T_H1 switch has occurred in the patient.

31. The method according to claim 28 or claim 29, wherein the cytokine pattern indicates (i) that a sustained T_H2 response has occurred or (ii) that a T_H2 to

T_H1 switch has not occurred in the patient.

32. The method according to claim 25, further comprising the step of determining the phenotypes of mononuclear blood cell populations that are proliferating in response to the modified stimulator cells by immunophenotyping or by cytotoxic T cell assay.

33. The method according to claim 32, wherein the cell populations are CD4+ T cells or CD8+ T cells.

34. The method according to claim 25, wherein, in said cell obtaining step b), the tumor or infected cells are autologous to the patient.

35. The method according to claim 25, wherein, in said cell obtaining step b), the tumor or infected cells are allogeneic to the patient, the allogeneic cells deriving from the same type of tumor or infection.

36. The method according to claim 25. wherein the 2', 3' nucleoside or nucleotide dialdehyde crosslinker is 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde.

37. The method according to claim 36, wherein the 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde is used at a concentration of about 1 mM to about 20 mM.

38. The method according to claim 37, wherein the 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde is used at a concentration of about 5 mM to about 15 mM.

39. The method according to claim 25, wherein the hydrostatic pressure is in the range of about 800 to about 1400 atmospheres.

40. The method according to claim 39, wherein the hydrostatic pressure is in the range of about 900 to about 1200 atmospheres.

41. The method according to claim 25, wherein exposure of the cells to the 2', 3' nucleoside or nucleotide dialdehyde crosslinker in step a) occurs at the same time that cells are exposed to the hydrostatic pressure.

42. A method of monitoring the continued immunoresponsiveness of a patient afflicted with a tumor or infection and receiving immunotherapy, the immunotherapy comprising immunizing the patient with an immunogenic preparation of tumor cells or infected cells that have been exposed to a 2', 3'- nucleoside or nucleotide dialdehyde crosslinking agent and to hydrostatic pressure, comprising: a) treating tumor cells or infected cells obtained from the patient with the 2', 3 '-nucleoside or nucleotide dialdehyde crosslinking agent and hydrostatic pressure in amounts and for a time sufficient to crosslink and modify 97

the cells' plasma membrane proteins; b) culturing the modified tumor cells or infected cells of step a) with mononuclear blood cells from the patient, under conditions to provide stimulated and immunoreactive mononuclear blood cells having specific immunoreactivity against the modified tumor or infected cells; c) measuring the level of specific stimulation or immunoreactivity of the mononuclear blood cells of step b) against the modified tumor or infected cells in relation to the level of stimulation or immunoreactivity of the mononuclear blood cells against unmodified tumor or infected cells to obtain a response or stimulation index; d) determining the secreted cytokine pattern by measuring the levels of secreted T_H1 cytokines that are stimulatory for a cell-mediated immune response and the levels of secreted T_H2 cytokines that are (i) inhibitory for a cell- mediated immune response or that are (ii) stimulatory for a humoral immune response, said cytokines being secreted by the stimulated mononuclear blood cells of step d); and e) repeating steps a) through d) during the course of the patient's immunotherapy to monitor an increase in the patient's response or stimulation index, and to determine that a cell mediated immune response, a T_H1 response, or a T_H2 to T_H1 conversion has occurred as a function of the immunization of the patient with the crosslinked and pressure-treated modified tumor or infected cells used as immunogens during the course of the immunotherapy.

43. The method according to claim 42, wherein the response or stimulation index is greater than or equal to about 2.

44. The method according to claim 42, wherein the tumor cells are pancreatic tumor cells, ovarian tumor cells, melanoma cells, carcinoma cells, breast tumor cells, colon cancer cells, renal cancer cells, lung tumor cells, non- small cell lung carcinoma cells, small cell lung carcinoma cells, bladder cancer cells, hematopoietic cancer cells, or prostate cancer cells.

° 45. The method according to claim 44, wherein the tumor cells are melanoma cells, colon cancer cells, renal carcinoma cells, or non-small cell lung carcinoma cells.

46. The method according to claim 42, wherein the infected cells are cells infected with bacteria, viruses, parasites, or yeast.

47. The method according to claim 46, wherein the infected cells are virus- infected cells.

48. The method according to claim 42, wherein the 2' , 3' nucleoside or nucleotide dialdehyde crosslinker is 2'. 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde.

49. The method according to claim 48, wherein the 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde is used at a concentration of about 1 mM to about 20 mM.

50. The method according to claim 49, wherein the 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde is used at a concentration of about 5 mM to about 15 mM.

51. The method according to claim 42, wherein the hydrostatic pressure is in the range of about 800 to about 1400 atmospheres.

52. The method according to claim 51 , wherein the hydrostatic pressure is in the range of about 900 to about 1200 atmospheres.

53. The method according to claim 42, wherein exposure of the cells to the 2', 3' nucleoside or nucleotide dialdehyde crosslinker in step a) occurs at the same time that the cells are subjected to hydrostatic pressure.

54. A method of determining if a patient should undergo anti-cancer or anti-tumor treatment regimens comprising chemotherapy, radiation therapy, or immunotherapy, comprising the steps of: a) collecting mononuclear blood cells from the patient afflicted with a cancer or tumor; b) obtaining autologous cancer cells or tumor cells from the patient or allogeneic cells from a donor having the same type of tumor or infection; c) exposing the cancer or tumor cells to i): a 2', 3' nucleoside or nucleotide dialdehyde crosslinker at a concentration and for a time sufficient to 0 cause crosslinking of the proteins in the cells' plasma membranes, and ii) to hydrostatic pressure for a time sufficient to cause a modification of the proteins in the cells' plasma membranes, thereby resulting in a crosslinked and pressure- treated modified cancer or tumor cell preparation; 5 d) incubating the mononuclear blood cells from step a) in admixture with the modified cells of step c) under conditions to provide immunoreactive, sensitized mononuclear blood cells specific to the modified cancer or tumor cells; _Q e) measuring the level of proliferation of the mononuclear blood cells in response to the modified cancer or tumor cells relative to the level of proliferation of the mononuclear cells in response to native or unmodified tumor cells or infected cells; f) determining the pattern of cytokines secreted by the 5 mononuclear blood cells by measuring the levels of secreted T_H1 cytokines that are stimulatory for a cell-mediated immune response and the levels of secreted T_H2 cytokines that are inhibitory for a cell-mediated immune response or that are stimulatory to a humoral immune response; and 0 g) assessing whether the patient should undergo chemotherapy or radiation therapy, based on the mononuclear blood cell proliferation level of step e) and at least one of: (i) the levels of stimulatory T_H1 cytokines, (ii) the levels of the inhibitory T_H2 cytokines, or (iii) the levels of the humoral response stimulatory 5 cytokines according to step f).

55. The method according to claim 54, wherein a low proliferation level of mononuclear cells combined with low stimulatory T_H1 cytokine levels or high inhibitory T_H2 cytokine levels or high humoral response stimulatory cytokine levels indicate that the patient should not undergo immunotherapy treatment.

56. The method according to claim 54, wherein the tumor cells are pancreatic tumor cells, ovarian tumor cells, melanoma cells, carcinoma cells, breast tumor cells, colon cancer cells, renal cancer cells, lung tumor cells, non- small cell lung carcinoma cells, small cell lung carcinoma cells, bladder cancer cells, hematopoietic cancer cells, or prostate cancer cells.

57. The method according to claim 54, wherein the tumor cells are melanoma cells, colon cancer cells, renal carcinoma cells, or non-small cell lung carcinoma cells.

58. The method according to claim 54, wherein the 2', 3' nucleoside or nucleotide dialdehyde crosslinker is 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde.

59. The method according to claim 58, wherein the 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde is used at a concentration of about 1 mM to about 20 mM.

60. The method according to claim 59, wherein the 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde is used at a concentration of about 5 mM to about 15 mM.

61. The method according to claim 54, wherein the hydrostatic pressure is in the range of about 800 to about 1400 atmospheres.

62. The method according to claim 61 , wherein the hydrostatic pressure is in the range of about 900 to about 1200 atmospheres.

63. The method according to claim 54, wherein the exposure of the cells to the 2', 3' nucleoside or nucleotide dialdehyde crosslinker in step c) occurs at the

^ same time as the exposure of the cells to hydrostatic pressure.

64. The method according to claim 54, wherein the 2', 3' nucleoside or nucleotide dialdehyde crosslinker is present at a concentration of about 10 mM and the hydrostatic pressure is 1200 atmospheres. 0

65. A vaccine preparation capable of inducing an immune response against tumor cells or infected cells, said preparation comprising an anti-tumor cell or anti-infected cell effective amount of an immunogenic preparation comprising 5 modified tumor or infected cells or plasma membranes obtained therefrom; wherein said modified cells are prepared by exposing isolated tumor or infected cells to a crosslinking effective concentration of a 2 ',3 '-nucleoside or nucleotide dialdehyde crosslinking agent and to hydrostatic pressure of from about 800 to 1 ,200 0 atmospheres for a time sufficient to cause modification of the plasma membranes of said tumor or infected cells, and further comprising an adjuvant selected from the group consisting of hematopoietic factors, lymphokines, cytokines, cell growth and proliferation compounds, growth hormones, immunopotentiating compounds, and immunostimulatory compounds and a pharmaceutically acceptable carrier, diluent 5 or excipient.

66. The vaccine preparation according to claim 65, wherein said cells are exposed to said 2 ',3 '-nucleoside or nucleotide dialdehyde crosslinking agent at the same time that said cells are subjected to said hydrostatic pressure.

67. The vaccine preparation according to claim 65 or claim 66, wherein said hydrostatic pressure is from about 900 to 1200 atmospheres.

68. The vaccine preparation according to claim 65 or claim 66, wherein said adjuvant is selected from the group consisting of interleukins 1 to 16, interferons, human growth hormone, saponin, granulocyte-macrophage colony stimulating factor, granulocyte stimulating factor, anti-CD3, saponin, and BCG.

69. The vaccine preparation according to claim 65 or claim 66, wherein the 2', 3 '-nucleoside or nucleotide dialdehyde crosslinker is represented by the following formula I:

I R0-CH ₂ ° R

CH HC // ^ o 0

wherein R is H or a mono-, di-, or tri-phosphate group; and B is a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

70. An immunogen derived from modified tumor cells or infected cells or plasma membranes thereof, wherein said modified tumor or infected cells are capable of inducing an anti-tumor or anti-infected cell immune response and are prepared by 1) exposing isolated tumor or infected cells to a crosslinking effective concentration of a 2', 3 '-nucleoside or nucleotide dialdehyde crosslinking agent and 2) subjecting said tumor or infected cells to hydrostatic pressure of from about 900 to 1 ,200 atmospheres for a time sufficient to cause modification of the plasma membranes of said tumor cells, said immunogen further comprising an adjuvant selected from the group consisting of hematopoietic factors, lymphokines, cytokines, cell growth and proliferation compounds, growth hormones, immunopotentiating compounds, and immunostimulatory compounds and a pharmaceutically acceptable carrier, diluent or excipient.

71. The immunogen according to claim 70, wherein said cells are exposed to said 2 ',3 '-nucleoside or nucleotide dialdehyde crosslinking agent at the same time that said cells are subjected to said hydrostatic pressure.

72. The immunogen according to claim 70 or claim 71, wherein said hydrostatic pressure is from about 1000 to 1200 atmospheres.

73. The immunogen according to claim 70 or claim 71 , wherein said adjuvant is selected from the group consisting of interleukins 1 to 16, interferons, human growth hormone, saponin, granulocyte-macrophage colony stimulating factor, granulocyte stimulating factor, anti-CD3, saponin, and BCG.

74. The immunogen according to claim 70 or 71 , wherein the 2', 3'- nucleoside or nucleotide dialdehyde crosslinker is represented by the following formula I:

I

75. A method of enhancing a patient's immune response to tumor or infected cells and of increasing survival over the tumor or infection, comprising: a) preparing said immunogen according to any one of claims 70 to 74; b) immunizing the patient with said immunogen prepared in step a); and c) boosting the patient with the immunogen prepared in step b).

76. A method of screening a tumored or infected patient for the ability of his or her autologous mononuclear blood cells to specifically immunoreact or proliferate in response to tumor or infected cells in an in vitro sensitization assay, whereby the ability of the mononuclear blood cells to immunoreact or proliferate in vitro serves as an indicator that the patient's mononuclear blood cells will be immunoreactive against the tumor or infected cells in vivo, comprising: a) exposing tumor or infected cells to i) a 2', 3' nucleoside or nucleotide dialdehyde crosslinker at a concentration and for a time sufficient to cause crosslinking of the proteins in the cells' plasma membranes, and ii) to hydrostatic pressure for a time sufficient to cause a modification of the proteins in the cells' plasma membranes, thereby resulting in a modified tumor or infected cell preparation; b) incubating mononuclear blood cells from the infected or tumored individual in admixture with the crosslinker- and pressure-modified cells of step a), under conditions to provide immunoreactive, sensitized cells specific for the modified tumor or infected cells; and c) measuring the level of specific stimulation or immunoreactivity of the mononuclear blood cells of step d) against the modified tumor or infected cells in relation to the level of stimulation or immunoreactivity of the mononuclear blood cells against unmodified tumor or infected cells to obtain a response or stimulation index, wherein the response or stimulation index of greater than or equal to about 1.5 to 2 indicates that the individual will mount an effective tumor or infected cell-reducing or inhibiting response against the tumor or infected cells, when crosslinked- and pressure-modified tumor or infected cells are used as immunogens in in vivo immunotherapy techniques.

77. A method for determining the level of immune responsiveness of a tumored or infected patient to a tumor cell, transformed cell, or infected cell, comprising: a) obtaining tumor cells or infected cells from the patient afflicted with a tumor or infection, or from another individual having the same type of tumor or infection; b) exposing the tumor or infected cells of step b) to i) a 2', 3' nucleoside or nucleotide dialdehyde crosslinker at a concentration and for a time sufficient to cause crosslinking of the proteins in the cells' plasma membranes, and ii) to hydrostatic pressure in the range of about 900 to about 1200 atmospheres for a time sufficient to cause a modification of the proteins in the cells' plasma membranes, thereby resulting in a modified tumor or infected cell preparation; c) incubating mononuclear blood cells obtained from the blood of the tumored or infected patient in admixture with the crosslinker- and pressure- modified cells of step c), under conditions to provide stimulated and immunoreactive cells specifically immunoreactive against the modified tumor or infected cells; and d) measuring the level of specific stimulation or immunoreactivity of the mononuclear blood cells of step d) against the modified tumor or infected cells in relation to the level of stimulation or immunoreactivity of the mononuclear blood cells against unmodified tumor or infected cells to obtain a response or stimulation index, wherein a response or stimulation index of greater than or equal to about 1.5 to about 2 indicates that the individual will mount an effective tumor or infected cell-reducing or inhibiting response when the crosslinked- and pressure- modified tumor or infected cells are used as immunogens in in vivo immunotherapy techniques.

78. The method according to claim 77, further wherein, in the measuring step e), a response or stimulation index of less than about 1.5 to 2 indicates that the individual will not mount an effective tumor or infected cell- reducing or inhibiting response when the crosslinked- and pressure-modified tumor or infected cells are used as immunogens in in vivo immunotherapy techniques.

79. The method according to claim 77, further comprising the step of isolating discrete populations of stimulated T lymphocytes specifically immunoreactive against the tumor or infected cells.

80. A method for clonally expanding a patient's populations of T lymphocytes specifically immunoreactive against tumor or infected cells for use in immunoadoptive therapy protocols, comprising: a) obtaining tumor cells or infected cells from the patient afflicted

10 with a tumor or infection, or from another individual having the same type of tumor or infection; b) exposing the cells from step a) to i) a 2' , 3' nucleoside or nucleotide dialdehyde crosslinker at a concentration and for a time sufficient to

1 cause crosslinking of the proteins in the cells' plasma membranes, and ii) to hydrostatic pressure of from about 900 to about 1200 atmospheres for a time sufficient to cause a modification of the proteins in the cells' plasma membranes, thereby resulting in a modified tumor cell preparation;

20 c) incubating mononuclear blood cells obtained from the blood of the tumored or infected patient in admixture with the crosslinker- and pressure- modified tumor cells of step b), under conditions to provide immunoreactive T lymphocytes specific for the modified tumor or infected cells; d) separating and enriching the specifically immunoreactive T

25 lymphocyte population from the other mononuclear blood cells; and e) culturing the separated immunoreactive T lymphocytes with T lymphocyte activators.

'if

81. The method according to claim 80, further comprising the step g) of immunizing the patient from whom the T lymphocytes were enriched with an immunogenic preparation of the immunoreactive and activated T lymphocytes of step e).

35

82. The method according to claim 77 or claim 80, wherein, in said cell obtaining step b), the tumor or infected cells are autologous to the patient.

83. The method according to claim 77 or claim 80, wherein, in said cell obtaining step b), the tumor or infected cells are allogeneic to the patient, the allogeneic cells deriving from the same type of tumor or infection.

84. The method according to claim 77 or claim 80, wherein the mononuclear blood cells are collected from a patient afflicted with a tumor.

85. The method according to claim 84, wherein the tumor cells are pancreatic tumor cells, ovarian tumor cells, melanoma cells, carcinoma cells, breast tumor cells, colon cancer cells, renal cancer cells, lung tumor cells, non- small cell lung carcinoma cells, small cell lung carcinoma cells, bladder cancer cells, or prostate cancer cells.

86. The method according to claim 77 or claim 80, wherein the mononuclear blood cells are collected from a patient afflicted with an infection.

87. The method according to claim 86, wherein the infected cells are cells infected with bacteria, parasites, viruses, or yeast.

88. The method according to any one of claims 1, 25, 42, 54, 76, 77 or 80, wherein the 2', 3 '-nucleoside or nucleotide dialdehyde crosslinker is represented by the following formula I:

89. The method according to claim 88, wherein the 2', 3' nucleoside or nucleotide dialdehyde crosslinker is 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde.

90. The method according to claim 99, wherein the 2', 3 '-adenosine dialdehyde or 2', 3 '-adenosine monophosphate dialdehyde is used at a concentration of about 5 mM to about 20 mM.

91. The method according to any one of claims 76, 77 or 80, wherein the cells are exposed to crosslinker at the same time that they are subjected to hydrostatic pressure.