HK1068913B - Antigen presenting cells, method for their preparation and their use for cancer vaccines - Google Patents

Description

Antigen presenting cells, method for the production thereof and use thereof for cancer vaccines

The present invention relates to the field of medicine, in particular to products, substances and compositions for use in a method of treatment of the human or animal body, more particularly to products, substances and compositions for use in the diagnosis, treatment and prevention of cancer. The present invention relates to cancer vaccines and methods of making the same.

Background

Several Tumor Associated Antigens (TAAs) that are constitutively expressed by transformed cells of different tissue types have recently been identified (Renkvist N. et al. Cancer Immunol. Immunother.50: 3-15, 2001).

Many of these TAAs are capable of providing a variety of dominant immunoantigenic peptides recognized by CD8+ Cytotoxic T Lymphocytes (CTLs) under specific HLA class I homospecific (Renkvist n. et al. Cancer immunol. immunother.50: 3-15, 2001); furthermore, selected TAAs, such as MAGE (Jager E. et al, J.exp.Med., 187: 265. sub.270, 1998), NY-ESO-1(Jager E. et al, J.exp.Med., 187: 265. sub.270, 1998), SSX (Tureci O et al, Cancer Res; 56 (20): 4766. sub.721996), tyrosinase (Topalian S.L. et al, J.exp.Med., 1965. sub.1971, 1996), Melan-A/MART-1(Zarour H.M. et al, Proc.Natl.Acad.Sci.USA, 97: 400. sub.405, 2000) simultaneously include epitopes recognized by CD4+ T lymphocytes under specific HLA type II isotypes, thus are capable of inducing humoral immune responses directed to TAA (Wang R. 276. Trend F., 22, Immun. sub.22).

Different classes of TAAs have been identified that may play an important role as therapeutic targets:

i) cancer-testis antigens (CTA) expressed in various tissue tumors but not in normal tissues other than testis and placenta, such as MAGE, GAGE, SSX SART-1, BAGE, NY-ESO-1, XAGE-1, TRAG-3 and SAGE, some of which represent multiple families (Traversari c., Minerva biotech, 11: 243-253, 1999);

ii) differentiation-specific antigens expressed in normal and neoplastic melanocytes, such as tyrosinase, Melan-a/MART-1, gp100/Pme117, TRP-1/gp75, TRP-2(Traversari c., Minerva biotech, 11: 243-253, 1999);

iii) antigens which are overexpressed in malignant tissue of different tissues but are also present in their benign counterparts, such as PRAME (Ikeda H. et al, Immunity, 6: 199-: 243-: 635-640, 1999), alpha-fetoprotein (Meng w.s. et al, mol. immunol., 37: 943-950, 2001);

iv) antigens derived from point mutations in genes encoding ubiquitously expressed proteins, such as MUM-1, β -catenin, HLA-A2, CDK4 and caspase 8(Traversari C., Minerva Biotech., 11: 243-253, 1999);

v) viral antigens (Traversari c., Minerva biotech, 11: 243-253, 1999).

In addition to TAAs, cellular elements critical for effective immunogenicity and for efficient recognition by the host's T lymphocytes include HLA class I and HLA class II antigens as well as costimulatory/helper molecules (e.g., CD40, CD54, CD58, CD80, CD81) (Fleuren G.J. et al, Immunol. Rev., 145: 91-122, 1995).

In the known class of TAAs, CTA is a particularly suitable therapeutic target among active specific immunotherapy for cancer patients due to its limited expression in normal tissues and its known in vivo immunogenicity in vivo, particularly in mammals, including humans (Jager E. et al, J.Exp. Med., 187: 265. sub.270, 1998; Rejnolds S.R. et al, int.J.cancer, 72: 972. sub.976, 1997). However, the heterogeneous expression of a particular CTA in a neoplastic lesion in different patients limits its biological suitability for therapeutic vaccination directed against CTA. Indeed, malignant lesions of different cancer patients may often express only selective CTA (Sahin U. et al, Clin. cancer Res., 6: 3916-B. Etc., Melanoma res, 7: S83-S88, 1997) and/or heterogeneous (dos Santos n.r. et al, Cancer res., 60: 1654-1662, 2000) expression (Jungbluth A.A., et al, Br.J.cancer, 83: 493-497, 2000). These events, which may occur separately or simultaneously in vivo, may also contribute to the constitutive immunogenicity of malignant cells, contributing to disease progression (Speiser D.E. et al, J.Exp.Med., 186: 645-. Thus, because of the loss or possible down-regulation of target CTA expression in neoplastic lesions, immunotherapy focusing on the immunological targeting of different immunogenic epitopes of a single CTA cannot be administered to a large number of cancer patients; furthermore, in vivo immune targeting of a single CTA may result in CTA-deprived tumor variants that effectively evade the therapy-induced/amplified CTA-specific immune response. Other limitations of therapeutic approaches to targeting single CTA arise from their heterogeneous expression within the lesion (Schultz-Thater e. et al, br.j. cancer, 83: 204-208, 2000), and presentation of different immunogenic epitopes of single CTA by a specific HLA type I or HLA type II allospecificity only allows treatment of patients with certain defined HLA phenotypes.

To partially obviate these limitations, recent treatment regimens have employed more than one immunogenic epitope of either single or multiple CTAs, or the entire CTA protein, as a vaccination agent (Conference on Cancer Vaccines, editors Ferrantini M. and Belladeli F., Roman-Italy, 11.15-16.1999; http:// www.cancerresearch.org).

Therefore, there is a need for a cancer vaccine that overcomes the deficiencies of the prior art, and in particular that overcomes the poor immunogenicity and in vivo immunoselection, thereby enabling the application of the cancer vaccine to a wide range of cancer patients, without being limited to a specific single targeting CTA or TAA, whereby the cancer vaccine is not "restricted" to selected HLA type I and/or HLA type II antigens.

Recent evidence from in vitro experiments suggests that expression in tumor cells of different tissues is induced or up-regulated after exposure of all of the CTA genes studied among the currently known CTA genes to DNA hypomethylating agents (dos Santos N.R. et al, Cancer Res., 60: 1654-1662, 2000; Weber J. et al, Cancer Res., 54: 1766-1771, 1994). CTA induction was found to continue until several weeks after treatment was completed and was still detectable. These findings support the idea that CTA belongs to a class of TAAs that are synthetically regulated by DNA methylation. Furthermore, treatment of neoplastic cells with DNA hypomethylating agents can induce simultaneous and continuous up-regulation of their expressed HLA type I antigens and of the HLA type I allospecificity of interest, and also up-regulation of the expression of the co-stimulatory/accessory molecules CD54 and CD58 (Coral S. et al, J.Immunother., 22: 16-24, 1999).

Despite the therapeutic promise of CTA, CTA still exhibits many deficiencies, such as the heterogeneous expression of specific CTA studied to date in different neoplastic lesions where CTA-positive and CTA-negative malignant cells coexist; only selected CTAs among the CTAs identified so far can be expressed on different neoplastic lesions, not restricted by tissue origin; threshold levels of expression of a particular CTA on neoplastic cells are necessary for its recognition by CTA-specific CTLs and vaccines directed against a particular CTA require a suitable HLA type I phenotype and, for selective CTAs, a patient's HLA type II phenotype.

Due to their unique biological properties, selected CTAs are used in different clinical trials with the aim of inducing or enhancing CTA-specific immune responses in patients with different tissue malignancies. As is known to experts in the field, different protocols are currently used for clinically in vivo administration of therapeutic CTA or for creating more effective vaccination tools at the preclinical level (dos Santos N-R. et al, Cancer Res., 60: 1654-. Notably, due primarily to the limitations of various technical and practical conditions, only a limited number of immunogenic epitopes of a particular CTA or a single intact CTA protein are currently used clinically for therapeutic purposes. The following list includes the main strategies currently employed or assumed to date for administering CTA to cancer patients; it should also be emphasized that the same protocol is used to administer TAAs belonging to other types of currently known TAAs to the patient, and that different adjuvants and/or carriers are sometimes used to boost the immunogenicity of the therapeutic agent.

● represents a synthetic peptide of an immunogenic epitope of a single CTA or of multiple CTAs recognized by CD8+ T cells (Conference on Cancer Vaccines, compiled by Ferrantini m. and belanderli f. roman-italy, 11 months 15-16 days 1999;http://www.cancerresearch.org)。

● represents a synthetic peptide encapsulated in liposomes of a single CTA or of immunogenic epitopes of multiple CTAs (Steller M.A. et al, Clin. cancer Res., 4: 2103-2109, 1998).

● Whole synthetic protein of a single CTA (Conference on Cancer Vaccines, authored by Ferrantini M. and Belladelli F., Roman-Italy, 11 months 15-16 days 1999;http://www.cancerresearch.org)。

● express epitopes of a single CTA or multiple CTAs recognized by CD8+ T cells (Jene L. et al., Trends immunol., 22: 102-107, 2001).

● naked DNA shooting (Park J.H. et al, mol.cells, 9: 384-391, 1999)

● ex vivo autologous PBMC/macrophages loaded with synthetic peptides representing epitopes for either single or multiple CTAs recognized by CD8+ T cells (Conference on cancer vaccines, edited by Ferrantini M. and Belladeli F., Roman-Italy, 11 months-16 days 1999;http://www.cancerresearch.org)。

● is loaded ex vivo with a synthetic peptide representing a single CTA or an epitope of multiple CTAs recognized by CD8+ T cells or loaded with a single CTAEither intact synthetic proteins of CTA or autologous dendritic cells loaded with preparations of intact tumor cells (Conference on Cancer Vaccines, authored by Ferrantini m. and belandelli f. roman-italy, 11 months to 16 days 1999;http://www.cancerresearchorg; jenne l, et al, Trends immunol, 22: 102-107, 2001).

● transfected or transduced DNA/RNA ex vivo to express full-length CTA or autologous dendritic cells fused to intact tumor cells (Jene L. et al, Trends immunol., 22: 102-107, 2001).

● autologous T lymphocytes transfected or transduced with DNA/RNA ex vivo to express full-length CTA.

For autologous cancer vaccines, which are the main object of the present invention, a number of patent references may be cited. WO99/42128 discloses methods for determining HLA transcription or expression profile of solid tumors, for selecting appropriate treatments and/or for monitoring tumor progression. The purpose of this reference is to inhibit certain isotypes of HLA-G to enhance natural anti-tumor responses. The method comprises extracting cells from a tumor sample, lysing and reacting the lysate with an antibody directed against an HLA class I antigen.

DE29913522 provides a device for preparing cancer vaccines by subjecting tumor cells extracted from a patient to a pressure of 200-9000 bar in order to kill or destroy the tumor cells while keeping their surface intact, and then re-injecting said cells into the patient.

WO00/02581 discloses a telomerase protein or peptide capable of inducing a T cell response against an oncogene or a mutated tumor suppressor protein or peptide. The peptides are used in cancer vaccines.

WO00/18933 discloses DNA constructs which cause the expression of functionally inactive modified antigens which are not altered in terms of efficiency of DNA, RNA transcription and translation or in terms of the production of antigenic peptides. Patients suffering from cancer are treated by administering RNA or plasmid DNA encoding a modified human cancer associated antigen, particularly the PSMA antigen. In a different embodiment, autologous dendritic cells that have been contacted in vitro with RNA or plasmid DNA are used as vaccines.

WO00/20581 discloses a cancer vaccine comprising a novel isolated MAGE-A3 Human Leukocyte Antigen (HLA) type II-binding peptide. The peptides can also be used to selectively enrich a population of T lymphocytes, wherein the CD4+ T lymphocytes are specific for the peptides. The enriched lymphocytes are also useful as cancer vaccines.

WO00/25813 discloses universal tumor-associated antigens (TAAs) in combination with major histocompatibility complex molecules. The method of treatment comprises administering a nucleic acid molecule encoding a TAA, the antigen being processed by an antigen presenting cell capable of activating cytotoxic lymphocytes and killing cells expressing the TAA. In addition to the specific hTERT peptide, the identification of different TAAs was achieved by complex computer-assisted methods of synthesizing computer-designed peptides and biological analysis to confirm the usefulness of the peptides.

WO00/26249 discloses fragments of the human WT-1 protein or the human gata-1 protein. These peptide fragments are used in cancer vaccines by activating Cytotoxic T Lymphocytes (CTLs).

US6077519 provides cancer vaccines comprising a composition of T cell epitopes recovered by acid elution of epitopes derived from tumor tissue.

WO00/46352 provides a cancer vaccine comprising human T lymphocytes expressing a functional CD86 molecule. The T lymphocytes are obtained by subjecting T cells to at least two successive stimulations, each involving at least one activator (an antibody against CD2, 3 or 28) and a cytokine (interleukin) stimulating the proliferation of T cells.

Coral S et al Journal of immunothery 22 (1): 16-24, 1999 teaches that the potential immunogenicity of melanoma cells and their recognition by host cytotoxic cells depends on the presence and expression levels of Human Leukocyte Antigen (HLA) type I antigens, co-stimulatory molecules and melanoma-associated antigen (MAA) on neoplastic cells. There may be a suggestion that 5-AZA-CdR, which is an active and/or passive specific immunotherapy for human melanoma, could enhance the recognition of melanoma by cytotoxic cells by systemic administration.

Momparler, Anticancer Drugs Apr; 8(4): 358-68, 1997, mentions that 5-AZA-CdR is useful as a chemotherapeutic agent.

Shichijo S et al, Jpn.J. cancer Res.87, 751- & 756, 1996, 7 months investigated whether the demethylating agent 5-AZA-CdR could induce the production of MAGE1, 2, 3 and 6 in normal and malignant lymphoid cells in order to better understand its expression mechanism in the cells. The authors showed induction of the CTA studied in selective test samples and discussed that demethylation was insufficient to stimulate induction of MAGE genes in all cases, and the results enabled one to better understand the mechanism of expression of MAGE genes in cells. There is no suggestion that treatment may be involved.

Summary of the invention

It has now been found a method of producing antigen presenting cells, the method comprising:

a) collecting the cells from the body of the subject;

b) activating the collected cells

c) Culturing and optionally expanding the activated cells ex vivo;

d) treating said cultured and optionally expanded cells with DNA hypomethylating agents such that said cells simultaneously express multiple tumor associated antigens.

The cells obtainable according to the method of the invention and their cellular components, alone or in combination with said cells, are used for the prevention and treatment of malignancies of different tissue types, in particular of mammals including humans, wherein said tumors constitutively express one or more of a plurality of tumor associated antigens expressed in said cells.

In the context of the present invention, the cells are referred to simply as ADHAPI-cells.

Most conveniently, the cells obtainable by the above method are used in the form of a cancer vaccine.

The invention will be further described in detail hereinafter by way of examples and the accompanying drawings, in which:

FIG. 1 shows the proliferation of autologous (aMLR) PBMC (R) stimulated with ADHAPI-cells/B-EBV or control B-EBV cells (S);

FIG. 2 shows the proliferation of autologous (aMLR) PBMC (R) stimulated with ADHAPI-cells/PWM-B or control PWM-B cells (S);

FIG. 3 shows the proliferation of autologous (aMLR) PBMC (R) stimulated by ADHAPI-cells/CD 40L-B or control CD40L-B cells (S);

FIG. 4 shows the proliferation of autologous (aMLR) PBMC (R) stimulated with ADHAPI-cells/PWM-PBMC or control PWM-PBMC cells (S);

FIG. 5 shows the proliferation of autologous (aMLR) PBMC (R) stimulated with ADHAPI-cells/PHA-PBMC and control PHA-PBMC;

FIG. 6 shows proliferation of autologous (aMLR) PBMC (R) stimulated with ADHAPI-cells/PHA- + PWM-PBMC or control PHA- + PWM-PBMC (S).

Detailed description of the invention

According to the present invention, there is virtually no limitation on the types of cells that can be treated to generate antigen-presenting cells, as long as the cells of the type are appropriately activated and treated with a hypomethylating agent.

According to the present invention, cells are collected from a subject, particularly a mammal, more particularly a human. In one possible embodiment of the invention, the human is a cancer patient.

In a first preferred embodiment of the present invention, the antigen-presenting cells obtainable by the above-described method are immune cells.

In a second preferred embodiment of the present invention, the antigen-presenting cells obtainable by the above method are non-immune cells.

The cells obtainable according to the invention are capable of expressing a common dominant immune cancer antigen or are capable of expressing a common non-dominant immune cancer antigen.

In certain particular embodiments of the invention, the cells suitable for use in the methods described herein are:

● EB Virus-an immortalized, DNA hypomethylating agent treated B-lymphoblastoid cell line (ADHAPI-cells/B-EBV) produced from cells of Peripheral Blood Mononuclear (PBMC) from cancer patients or healthy subjects in advanced disease.

● pokeweed mitogen (PWM) activated, DNA hypomethylating agent treated B-lymphocytes (ADHAPI-cells/PWM-B), which cells were generated from B lymphocytes purified from PBMCs of cancer patients or healthy subjects in advanced stages of disease.

● CD40 activated, DNA hypomethylating agent treated B-lymphocytes (ADHAPI-cells/CD 40-B) generated from purified B-lymphocytes from PBMCs of cancer patients or healthy subjects in advanced disease.

● pokeweed mitogen (PWM) activated, DNA hypomethylating agent treated PBMC (ADHAPI-cells/PWM-PBMC) produced from purified PBMC from cancer patients or healthy subjects in advanced disease.

● Phytohemagglutinin (PHA) + recombinant human interleukin-2 (rhIL-2) activated, DNA hypomethylating agent treated PBMC (ADHAPI-cells/PHA-rhIL 2-PBMC) produced from purified PBMC of cancer patients or healthy subjects in advanced disease.

● Phytohemagglutinin (PHA) + recombinant human interleukin-2 (rhIL-2) + poke-land mitogen (PWM) -activated, DNA hypomethylating agent treated PBMCs (ADHAPI-cells/PHA-rhIL 2-PWM-PBMCs), which cells were produced from purified PBMCs of cancer patients or healthy subjects at an advanced stage of the disease.

● dendritic cells, monocytes, macrophages.

● CD34+ cells, fibroblasts, stem cells, fibroblasts, and keratinocytes (keratinocytes).

The cells obtainable by the method of the invention are suitable for use as a medicament for the prevention and treatment of malignancies of different histotypes, wherein the tumors constitutively express one or more cancer antigens that are either immunodominant or non-immunodominant.

Another possible embodiment of the invention can be applied in those cases where the direct antigen presenting capacity of the vaccinated cells is not desired or necessary. In such embodiments, the vaccinated cells or cellular fractions thereof obtainable by the method of the invention may be used as a "reservoir" for mixed (pooled) cancer antigens to vaccinate a patient.

In a preferred embodiment of the invention, the TAA selected is CTA.

This embodiment of the invention provides the skilled person with the following advantages:

CTA is immunogenic since it contains an epitope recognized by HLA type I-restricted CTA-specific CD8+ CTL.

CTA is immunogenic since it contains epitopes recognized by HLA class II-restricted CTA-specific CD4+ T lymphocytes.

The selected CTA contains epitopes presented by both HLA class I and HLA class II antigens; thus, selected CTAs induced both CD8+ CTL and CD4+ T lymphocyte responses.

CTA is not expressed in benign tissues other than testis and placenta.

Different CTAs can be expressed simultaneously in neoplastic cells of both solid and hematopoietic malignancies, providing multiple therapeutic targets co-expressed on transformed cells.

The different CTAs are expressed homogeneously in both simultaneous and sequential metastatic lesions in a given patient.

Different CTAs can be expressed in malignant tissues of different tissue origin, providing a common therapeutic target shared by human tumors regardless of their specific tissue type.

Different CTAs can encode a variety of immunogenic peptides presented with the same specificity for different HLA types I and II.

In another embodiment of the invention, histone deacetylase inhibitors can cooperate with DNA hypomethylating agents to induce/up-regulate the expression of CTA, HLA antigens and co-stimulatory/accessory molecules on neoplastic cells of different tissues. In fact, DNA methylation and histone deacetylation act as a synergistic layer of epigenetic gene silencing in cancer (Fuks F. et al, nat. Gene., 24: 88-91, 2000), with preliminary minimal demethylation of DNA followed by treatment with histone deacetylase inhibitors, resulting in the observation that selected hypermethylated genes are strongly reactivated in colorectal cancer cells with tumor suppressor function (Cameron E. et al, nat. Gene., 21: 103-107, 1999).

The activation step in the method of the invention is carried out according to common general knowledge, in any case compiled by current protocols in Immunology, Coligan j.e., et al, Wiley.

Demethylation in the process of the invention is well known and reported in the literature and for further information reference may be made to Santini v, et al, ann. 573-586, 2001.

The hypomethylating agents, also known in the art as demethylating agents, for the purposes of the present invention are well known in the art. DNA demethylating agents are widely disclosed in the literature, see for example WO 01/29235, US 5851773. One preferred DNA demethylating agent is 5-AZA-cytidine, or more preferably 5-AZA-2' -deoxycytidine (5-AZA-CdR).

The antigen-presenting cells of the present invention are suitable for the preparation of cancer vaccines. In a preferred embodiment of the invention, the vaccine is an autologous vaccine.

In another preferred embodiment of the invention, the vaccine is an allogeneic vaccine. In this embodiment, the cells obtainable according to the above-described method can be used both as antigen presenting cells and as "reservoir" form of mixed cancer antigens, which can be cells or cell components.

In another embodiment of the invention, the cells and/or cellular components may be used in a method of producing effector immune cells for use in the preparation of products for use in well-known adoptive immunotherapy. In another embodiment of the invention, the vaccine disclosed herein may be used in combination with a systemic pretreatment of cancer patients with a hypomethylating agent such as decitabine. This embodiment may be carried out using a commercial product, for example a kit comprising a vaccine of the invention and a pharmaceutical composition suitable for systemic administration of a hypomethylating agent such as decitabine.

Vaccines can be prepared according to techniques well known to those skilled in the art, following only common general knowledge. For example, patent references mentioned in the present specification are fully disclosed for the preparation of cancer vaccines, see for example WO00/25813 or WO 00/46352.

The skilled person is able to establish without any difficulty a suitable way of using the vaccine of the invention, in particular a dosing regimen.

The following examples further illustrate the invention.

Example 1

ADHAPI-cell/B-EBV

PBMC purification

PBMCs were purified from heparinized peripheral blood of cancer patients or healthy subjects at advanced disease by standard Ficoll-Hypaque density gradient centrifugation.

Immortalization of PBMCs using EB virus (EBV) to generate autologous B-lymphoblastoid cells Cell line

The B-EBV + lymphoblastoid cell line was prepared by the following method: at 37 ℃ and 5% CO₂In RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (or human AB serum) and 2mM L-glutamine, PBMC were cultured with supernatant from the B95.8 marmoset cell line.

Preparation of ADHAPI-cells/B-EBV and control B-EBV cells

At 37 ℃ and 5% CO₂In a humidified environment, B-EBV + lymphoblastoid cell line (7.5X 10) was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (or 10% heat-inactivated human AB serum) and 2mM L-glutamine⁵Cells/ml) and pulsed four times with 1 μ M5-AZA-2' -deoxycytidine (5-AZA-CdR) every 12 hours; then, half of the medium was replaced with fresh medium and cultured for another 48 hours. The cells are then used for experimental procedures and/or frozen under viable conditions. Control cells (B-EBV cells) were cultured under similar experimental conditions but without the 5-AZA-CdR pulse.

Final recovery of ADHAPI-cells/B-EBV and control B-EBV cells

The results are shown in Table I.

Autologous mixed lymphocyte reaction (aMLR) and MLR

ADHAPI-cells/B-EBV and control B-EBV cells (stimuli ═ S) were collected, washed twice with Hanks' balanced salt solution (HBSS) and X-ray treated (75 Gy). Scalar concentrations (from 1x 10) were used for aMLR and MLR⁶Cells/ml to 6X10⁴Cells/ml) of ADHAPI-cells/B-EBV or control B-EBV cells were added to autologous or allogeneic PBMC (1X 10) in Basal Iscove's medium⁶Cells/ml) (reactant ═ R) and plated onto 96-well U-bottom plates to reach a final volume of 200 μ L/well, with 10% heat-inactivated human AB serum, 2mM L-glutamine, 100U/ml penicillin, 100 μ g/ml streptomycin sulfate added to the Basal Iscove's medium. At 37 ℃ and 5% CO₂After 24 hours in a humid environment, 100. mu.l of culture supernatant was collected and immediately stored at-80 ℃ until used for cytokine analysis. Then, 100. mu.l of fresh medium was added to each well and cultured for another 5 days, at this time³H-TdR (1. mu. LCi/well) pulsed O/N on the culture; the plates were then harvested and incorporation into R cells was determined using a beta counter³H-TdR。

Stimulation by ADHAPI-cells/B-EBV or control B-EBV cells (S) in aMLR Proliferation of autologous PBMC (R)

See fig. 1.

Phenotypic profiles of ADHAPI-cells/B-EBV and control B-EBV cells

The results are shown in Table II.

RT-PCR analysis of expression by ADHAPI-cells/B-EBV and control B-EBV cells CTA

The experimental conditions and primers used to assess the expression of CTA on the cells studied are as follows:

for MAGE-1, -2, -3, -4, Brasseur, f, et al, int.j.cancer 63: 375-; for GAGE 1-6, Van den Eynde, b. et al, j.exp.med.182: 689 698, 1995; for NY-ESO-1, Stockert, e, et al, j.exp.med.187: 265, 270, 1998; for SSX-2; sahin, u, et al, clin. 3916-3922, 2000.

5-AZA-CdR	-	+
			MAGE-1	0/4	4/4
MAGE-2	N/T	NT
			MAGE-3	0/4	4/4
MAGE-4	NT	NT
			NY-ESO-1	0/4	4/4
GAGE-1-6	0/4	4/4
			SSX-2	2/4	4/4

^aPositive/tested; NT, not tested;

ELISA evaluation by ADHAPI-cells/B-EBV or control B-EBV in aMLR IFN-gamma released from cell (S) -stimulated PBMC (R)

The results are shown in Table III.

Example 2

ADHAPI-cell/PWM-B

Purification of B-lymphocytes

PBMCs were purified from heparinized peripheral blood of cancer patients or healthy subjects at advanced disease by standard Ficoll-Hypaque density gradient centrifugation, and purified B lymphocytes were obtained by conventional E rosetting techniques using neuraminidase-treated sheep blood erythrocytes.

Preparation of PWM-activated B cells

To purified B-lymphocytes (1.5X106 cells/ml) PWM (3. mu.g/ml) was added and the mixture was incubated at 37 ℃ and 5% CO₂In basal Iscove's medium supplemented with 10% heat-inactivated human AB serum, 2mM L-glutamine, 100U/ml penicillin, 100. mu.g/ml streptomycin sulfate for 48 hours.

Preparation of ADHAPI-cells/PWM-B and control PWM-B cells

Pulse PWM-activated B-lymphocytes four times every 12 hours with 1 μ M5-AZA-2' -deoxycytidine (5-AZA-CdR); then, half of the medium was replaced with fresh medium and cultured for another 48 hours. The cells are then used for experimental procedures and/or frozen under viable conditions. Control cells (PWM-B cells) were cultured under similar experimental conditions but without the 5-AZA-CdR pulse.

Final recovery of ADHAPI-cells/PWM-B and control PWM-B cells

The results are shown in Table I.

Autologous mixed lymphocyte reaction (aMLR) and MLR

ADHAPI-cells/PWM-B and control PWM-B cells (stimulator ═ S) were collected, washed three times with Hanks' balanced salt solution (HBSS) supplemented with 0.5% α -methyl mannopyranoside and X-ray treated (30 Gy). Scalar concentrations (from 1x 10) were used for aMLR and MLR⁶Cells/ml to 6X10⁴Cells/ml) of ADHAPI-cells/PWM-B or control PWM-B cells were added to autologous or allogeneic PBMC (from 1X 10) in Basal Iscove's medium⁶Cells/ml) (reactant ═ R) and plated onto 96-well U-bottom plates to reach a final volume of 200 μ L/well, with 10% heat-inactivated human AB serum, 2mM L-glutamine, 100U/ml penicillin, 100 μ g/ml streptomycin sulfate added to the Basal Iscove's medium. At 37 ℃ and 5% CO₂After 6 days of incubation in a moist environment, 100. mu.l of culture supernatant was collected from each well and immediately presented at-80 ℃ until used for cytokine analysis. Then, 100. mu.l of fresh medium was added to each well and used³H-TdR (1. mu. Ci/well) the cultures were pulsed O/N; the plates were then harvested and incorporation into R cells was determined using a beta counter³H-TdR。

Phenotypic profiles of ADHAPI-cells/PWM-B and control PWM-B cells

The results are shown in Table II.

RT-PCRAnalysis of expression by ADHAPI-cells/PWM-B and control PWM-B cells CTA of

5-AZA-CdR	-	+
			MAGE-1	0/4	4/4
MAGE-2	0/4	4/4
			MAGE-3	0/4	4/4
MAGE-4	0/4	4/4
			NY-ESO-1	0/4	4/4
GAGE-1-6	0/4	4/4
			SSX-2	1/4	4/4

^aPositive/tested; NT, not tested;

stimulation by ADHAPI-cells/PWM-B or control PWM-B cells (S) in aMLR Proliferation of stimulated autologous PBMC (R)

The results are shown in FIG. 2.

ELISA evaluation by ADHAPI-cells/PWM-B or control PWM-B cells(S)Stimulated allogeneic (MLR) and autologous (aMLR) PBMC (R) released IFN γThe results are shown in Table III.

Example 3

ADHAPI-cell/CD 40L-B

PBMC purification

PBMCs were purified from heparinized or Acid Citrate Dextrose (ACD) -anticoagulated peripheral blood of cancer patients or healthy subjects at advanced disease by standard Ficoll-Hypaque density gradient centrifugation.

Preparation of NIH3T3-CD 40L-activated PBMC

At 37 ℃ and 5% CO₂In a humid environment with the addition of 10% heat-inactivated human AB serum, 2mM L-glutamine, 2ng/ml recombinant human (rh) interleukin 4(rhIL-4), 50. mu.g/ml human transferrin, 5. mu.g/ml rh insulin, 5.5x10^-7M Cyclosporin A (CsA), 100U/ml penicillin, 100. mu.g/ml streptomycin sulfate Basal Iscove's Medium (complete Medium) PBMC (2X 10)⁶Cell/ml) with half confluency, X-ray treated(75Gy) NIH3T3-CD40L were co-cultured. After 6 days of culture, PBMCs were harvested and washed twice with HBSS at 1X10⁶Cell/ml concentrations were resuspended in complete medium and incubated with freshly prepared NIH3T3-CD40L as described above at 37 ℃ and 5% CO₂Co-culturing for 3 days in a humid environment. This process is repeated every 2-3 days until a maximum incubation time of 16-18 days.

Generation of ADHAPI-cells/CD 40L-B and control CD40L-B cells

After 16-18 days of culture, activated PBMCs were harvested and restimulated with NIH3T3-CD40L as described above; at 37 ℃ and 5% CO₂After O/N incubation in a humid environment, the cultures were pulsed four times every 12 hours with 1. mu.M 5-AZA-2' -deoxycytidine (5-AZA-CdR); then, the cells were harvested and restimulated with NIH3T3-CD40L and cultured for an additional 48 hours as described above. The cells are then used for experimental procedures and/or frozen under viable conditions. Control cells (CD40L-B cells) were cultured under similar experimental conditions without the 5-AZA-CdR pulse.

Final recovery of ADHAPI-cells/CD 40L-B and control CD40L-B cells

The results are shown in Table I.

Autologous mixed lymphocyte reaction (aMLR) and MLR

ADHAPI-cells/CD 40L-B and control CD40L-B cells (stimuli ═ S) were collected, rinsed three times with Hanks' balanced salt solution (HBSS) and X-ray treated (50 Gy). Scalar concentrations (from 1x 10) were used for aMLR and MLR⁶Cells/ml to 6X10⁴Cells/ml) of ADHAPI-cells/CD 40L-B or control CD40L-B cells were added to autologous or allogeneic PBMCs (1x 10) in basalscove's medium⁶Cells/ml) (reactant ═ R) and plated onto 96-well U-bottom plates to reach a final volume of 200 μ L/well, with 10% heat-inactivated human AB serum, 2mM L-glutamine, 100U/ml penicillin, 100 μ g/ml streptomycin sulfate added to the Basal Iscove's medium. At 37 ℃ and 5% CO₂Of (2)After 24 hours of incubation in a wet environment, 100. mu.l of culture supernatant was collected and immediately stored at-80 ℃ until used for cytokine analysis. Then, 100. mu.l of fresh medium was added to each well and cultured for another 5 days, at this time³H-TdR (1. mu. Ci/well) the cultures were pulsed O/N; the plates were then harvested and incorporation into R cells was determined using a beta counter³H-TdR。

Phenotypic profiles of ADHAPI-cells/CD 40L-B and control CD40L-B cellsThe results are shown in Table II.

RT-PCR analysis was performed by ADHAPI-cells/CD 40L-B and control CD40L-B cells Expressed CTA

5-AZA-CdR	-	+
			MAGE-1	0/10	10/10
MAGE-2	0/10	9/10
			MAGE-3	0/11	10/11
MAGE-4	0/11	11/11
			NY-ESO-1	0/14	14/14
GAGE-1-6	0/14	14/14
			SSX-2	0/14	13/14

^aPositive/tested.

In aMLR by ADHAPI-cells/CD 40L-B or control CD40L-B cells (S) Stimulated proliferation of autologous PBMC (R)

The results are shown in FIG. 3.

ELISA evaluation by ADHAPI-cells/CD 40L-B or control in aMLR CD40L-B cell (S) -stimulated PBMC (R) -released IFN-gamma

The results are shown in Table III.

Example 4

ADHAPI-cell/PWM-PBMC

PBMC purification

PBMCs were purified from heparinized peripheral blood of cancer patients or healthy subjects at advanced disease by standard Ficoll-Hypaque density gradient centrifugation.

Preparation of PWM-activated PBMC

To PBMC (1.5X 10)⁶Cells/ml) was added PWM (3. mu.g/ml) and incubated at 37 ℃ with 5% CO₂In Basal Iscove's medium supplemented with 10% heat-inactivated human AB serum, 2mM L-glutamine, 100U/ml penicillin, 100. mu.g/ml streptomycin sulfate for 48 hours.

Preparation of ADHAPI-cell/PWM-PBMC and control PWM-PBMC cells

PWM-activated PBMC were pulsed four times with 1 μ M5-AZA-2' -deoxycytidine (5-AZA-CdR) every 12 hours; then, half of the medium was replaced with fresh medium and cultured for another 48 hours. The cells are then used for experimental procedures and/or frozen under viable conditions. Control cells (PWM-PBMC cells) were cultured under similar experimental conditions but without the 5-AZA-CdR pulse.

Final recovery of ADHAPI-cells/PWM-PBMC and control PWM-PBMC cellsHarvesting machine

The results are shown in Table I.

Autologous mixed lymphocyte reaction (aMLR) and MLR

ADHAPI-cells/PWM-PBMC and control PWM-PBMC cells (stimuli ═ S) were collected, washed three times with Hanks' balanced salt solution supplemented with 0.5% α -methyl mannopyranoside and X-ray treated (30 Gy). Scalar concentrations (from 1x 10) for aMLR and MLR⁶Cells/ml to 6X10⁴Cells/ml) of ADHAPI-cells/PWM-PBMC or control PWM-PBMC cells were added to autologous or allogeneic PBMC (1X 10) in Basal Iscove's medium⁶Cells/ml) (reactant ═ R) and plated onto 96-well U-bottom plates to reach a final volume of 200 μ L/well, with 10% heat-inactivated human AB serum, 2mM L-glutamine, 100U/ml penicillin, 100 μ g/ml streptomycin sulfate added to the Basal Iscove's medium. At 37 ℃ and5％CO₂after 6 days of incubation in a moist environment, 100. mu.l of culture supernatant was collected from each well and immediately stored at-80 ℃ until used for cytokine analysis. Then, 100. mu.l of fresh medium was added to each well and used³H-TdR (1. mu. Ci/well) the cultures were pulsed O/N; then harvested and assayed for incorporation into R cells using a beta counter³H-TdR。

Phenotypic profiles of ADHAPI-cell/PWM-PBMC and control PWM-PBMC cellsThe results are shown in Table II.

RT-PCR analysis by ADHAPI-cells/PWM-PBMC and control CTA expressed by PWM-PBMC cells

5-AZA-CdR	-	+
			MAGE-1	0/4	4/4
MAGE-2	0/4	3/4
			MAGE-3	0/4	4/4
MAGE-4	1/4	3/4
			NY-ESO-1	0/4	4/4
GAGE-1-6	0/4	3/4
			SSX-2	0/4	3/4

^aPositive/tested; NT, not tested;

ADHAPI-cell/PWM-PBMC or control PWM-PBMC in aMLR Cell (S) -stimulated proliferation of autologous (aMLR) PBMC (R)

The results are shown in FIG. 4.

ELISA evaluation by ADHAPI-cells/PWM-PBMC in aMLR or controls PWM-PBMC cell (S) -stimulated autologous PBMC (R) -released IFN-gammaThe results are shown in Table III.

Example 5

ADHAPI-cell/PHA-PBMC

PBMC purification

PBMCs were purified from heparinized peripheral blood of cancer patients or healthy subjects at advanced disease by standard Ficoll-Hypaque density gradient centrifugation.

Preparation of PHA-activated PBMC

To PBMC (1.5X 10)⁶Cells/ml) was added PHA (10. mu.g/ml) and 100UI/ml rhIL-2 at 37 ℃ and 5% CO₂In RPMI 1640 medium (complete medium) supplemented with 10% heat-inactivated fetal bovine serum (or in Basal Iscove's medium supplemented with 10% heat-inactivated human AB serum), 2mM L-glutamine, 100U/ml penicillin, 100. mu.g/ml streptomycin sulfate for 48 hours.

Preparation of ADHAPI-cell/PHA-PBMC and control PHA-PBMC

PHA-activated PBMC were pulsed four times with 1 μ M5-AZA-2' -deoxycytidine (5-AZA-CdR) every 12 hours; then, half of the medium was replaced with fresh complete medium without PHA-M and cultured for another 48 hours. The cells are then used for experimental procedures and/or frozen under viable conditions. Control cells (PHA-PBMC) were cultured under similar experimental conditions but without the 5-AZA-CdR pulse.

Final recovery of ADHAPI-cell/PHA-PBMC and control PHA-PBMC

The results are shown in Table I.

Autologous mixed lymphocyte reaction (aMLR) and MLR

ADHAPI-cells/PHA-PBMC and control PHA-PBMC (stimuli ═ S) were collected, washed three times with Hanks' balanced salt solution supplemented with 0.5% α -methyl mannopyranoside and X-ray treated (50 Gy). Scalar concentrations (from 1x 10) were used for aMLR and MLR⁶Cells/ml to 6X10⁴Cells/ml) of ADHAPI-cells/PHA-PBMC or control PHA-PBMC were added to autologous or allogeneic PBMC (1X 10) in Basal Iscove's medium⁶Cells/ml) (reactant ═ R) and plated onto 96-well U-bottom plates to a final volume of 200 μ L/well, with 10% heat-inactivated human AB serum, 2mM L-glutamine, 10 mM L-glutamine, added to Basal Iscove's medium0U/ml penicillin, 100. mu.g/ml streptomycin sulfate. At 37 ℃ and 5% CO₂After 24 hours in a humid environment, 100. mu.l of culture supernatant was collected from each well and immediately stored at-80 ℃ until used for cytokine analysis. Then, 100. mu.l of fresh medium was added to each well and cultured for another 5 days, at this time³H-TdR (1. mu. Ci/well) the cultures were pulsed O/N; the plates were then harvested and incorporation into R cells was determined using a beta counter³H-TdR。

Phenotypic profiles of ADHAPI-cell/PHA-PBMC and control PHA-PBMC

The results are shown in Table II.

RT-PCR analysis with ADHAPI-cell/PHA-PBMC and control PHA-PBMC

Expressed CTA

5-AZA-CdR	-	+
			MAGE-1	0/12	12/12
MAGE-2	0/3	3/3
			MAGE-3	0/12	12/12
MAGE-4	0/4	4/4
			NY-ESO-1	0/6	6/6
GAGE-1-6	0/4	4/4
			SSX-2	0/6	6/6

^aPositive/tested; NT, not tested;

stimulated by ADHAPI-cells/PHA-PBMC and control PHA-PBMC in aMLR Proliferation of stimulated autologous PBMC (R)

The results are shown in FIG. 5.

ELISA evaluation by ADHAPI-cell/PHA-PBMC or control PHA-PBMC (S) -stimulated allogeneic (MLR) and autologous (aMLR) PBMC (R) release IFN-gamma of

The results are shown in Table III.

Example 6

ADHAPI-cell/PHA + PWM-PBMC

PBMC purification

PBMC were purified from heparinized or ACD-anticoagulated peripheral blood of cancer patients or healthy subjects at advanced disease by standard Ficoll-Hypaque density gradient centrifugation.

Preparation of PHA + PWM-activated PBMC

To PBMC (1.5X 10)⁶Cells/ml) was added PHA-M (10. mu.g/ml), PWM (3. mu.g/ml) and 100UI/ml rhIL-2 and incubated at 37 ℃ and 5% CO₂In Basal Iscove's medium (complete medium) supplemented with 10% heat-inactivated human AB serum (or with 10% heat-inactivated autologous serum), 2mM L-glutamine, 100U/ml penicillin, 100. mu.g/ml streptomycin sulfate for 48 hours.

ADHAPI-cell/PHA + PWM-PBMC and control PHA + PWM-PBMC Preparation of

PHA + PWM-activated PBMC were pulsed four times with 1 μ M5-AZA-2' -deoxycytidine (5-AZA-CdR) every 12 hours; then, half of the medium was changed to fresh complete medium without PHA or PWM and cultured for another 48 hours. The cells are then used for experimental procedures and/or frozen under viable conditions. Control cells (PHA + PWM-PBMC) were cultured under similar experimental conditions but without the 5-AZA-CdR pulse.

ADHAPI-cell/PHA + PWM-PBMC and control PHA + PWM-PBMC Final recovery of

The results are shown in Table I.

Autologous mixed lymphocyte reaction (aMLR) and MLR

ADHAPI-cells/PHA + PWM-PBMC and control PHA + PWM-PBMC (stimuli ═ S) were collected, washed three times with Hanks' balanced salt solution supplemented with 0.5% α -methyl mannopyranoside and X-ray treated (50 Gy). Scalar concentrations (from 1x 10) were used for aMLR and MLR⁶Cells/ml to 6X10⁴Cells/ml) of ADHAPI-cells/PHA-rhIL 2- + PWM-PBMC or control PHA + PWM-PBMC cells were added to autologous or allogeneic PBMC (1x 10) in Basal Iscove's medium⁶Cells/ml) (reactant ═ R) and plated onto 96-well U-bottom plates to reach a final volume of 200 μ L/well, with 10% heat-inactivated human AB serum, 2mM L-glutamine, 100U/ml penicillin, 100 μ g/ml streptomycin sulfate added to the Basal Iscove's medium. At 37 ℃ and 5% CO₂After 6 days of incubation in a moist environment, 100. mu.l of culture supernatant was collected from each well and immediately stored at-80 ℃ until used for cytokine analysis. Then, 100. mu.l of fresh medium was added to each well and used³H-TdR (1. mu. Ci/well) the cultures were pulsed O/N; the plates were then harvested and incorporation into R cells was determined using a beta counter³H-TdR。

ADHAPI-cell/PHA + PWM-PBMC and control PHA + PWM-PBMC Phenotype profile of

The results are shown in Table II.

RT-PCR analysis by ADHAPI-cells/PHA + PWM-PBMC and control CTA expressed by PHA + PWM-PBMC

5-AZA-CdR	-	+
			MAGE-1	0/7	7/7
MAGE-2	0/7	7/7
			MAGE-3	0/7	7/7
MAGE-4	0/7	7/7
			NY-ESO-1	0/7	7/7
GAGE-1-6	0/7	7/7
			SSX-2	0/7	7/7

^aPositive/tested.

ADHAPI-cell/PHA + PWM-PBMC or controls in aMLR PHA + PWM-PBMC stimulated proliferation of autologous (aMLR) PBMC (R)

The results are shown in FIG. 6.

ELISA evaluation by ADHAPI-cells/PHA + PWM-PBMC or controls PHA + PWM-PBMC (S) -stimulated allogeneic (MLR) and autologous (aMLR) PBMC (R) Released IFN-gamma

The results are shown in Table III.

ADHAPI-cell in vivo tumorigenicity

180 days after ADHAPI-cell administration, a single subcutaneous xenograft of live ADHAPI cells/PHA-rhIL 2-PWM-PBMC (12X 10)⁶) And its control cell (14X 10)⁶) ADHAPI-cell/CD 40L-B (8X 10)⁶) And its control cell (8X 10)⁶) Or x-ray-treated (30Gy) ADHAPI-cell/PHA-rhIL 2 PWM-PBMC (12x 10)⁶) And its control cell (14X 10)⁶) X-ray treated (50Gy) ADHAPI-cells/CD 40L-B (15X 10)⁶) And its control cell (18X 10)⁶) Neither induced tumor formation at the injection or remote (clinically detectable) site nor affected the overall health and body weight of BALB/c nu/nu mice. Repeated subcutaneous xenografts live ADHAPI-cells/B-EBV 180 days after the first administration (5X 10)⁶/l^stInjecting; 1x10⁷/2^ndAnd subsequent injections) and control B-EBV cells (5x 10)⁶/l^stInjecting; 1X10⁷/2^ndAnd subsequent injections) or X-ray treated ADHAPI-cells/B-EBV (75Gy) (5X 10)⁶/l^stInjecting; 1x10⁷/2^ndAnd subsequent injections) and x-ray treated (75Gy) control B-EBV cells (5X 10)⁶/l^stInjecting; 1x10⁷/2^ndAnd subsequent injections) neither induced tumor formation at the injection or distant (clinically detectable) site nor affected the overall health and body weight of BALB/c nu/nu mice on days 0, 33, 63 and 96. The overall health and body weight of ADHAPI-cell-treated animals were comparable to control animals (untreated or transplanted B-EBV cells).

ADHAPI-advantage of cells as multivalent cellular CTA vaccine

ADHAPI-cells represent a completely new and improved approach and have many prominent/significant advantages over the main protocols that have been utilized, or assumed to date, to most effectively administer known CTAs to cancer patients. Among these:

ADHAPI-cellular versus non-genetically modified cellular CTA vaccine

ADHAPI-cells are novel and unique APC vaccines due to the ability to simultaneously express multiple/all methylation-regulated CTAs; being endogenously synthesized, CTA is able to enter both HLA type I and HLA type II antigen processing pathways in ADHAPI-cells (Jenne L et al, Trends immunol., 22: 102-107, 2001).

Thus, ADHAPI-cells are able to simultaneously present immunogenic epitopes of endogenously synthesized CTA to CD8+ and CD4+ autologous T lymphocytes due to their constitutive cell membrane expressing HLA type I and HLA type II antigens; thus, ADHAPI-cells can simultaneously induce/amplify CTL and humoral immune responses directed to CTA. In addition, ADHAPI-cells can express and present to host T cells methylation-regulated CTAs (as well as known and yet unknown non-immunodominant epitopes of CTAs) that have not been identified and characterized.

In contrast to ADHAPI-cells, the main limitations shared by synthetic CTA peptide-pulsed, synthetic CTA whole protein-pulsed or whole tumor cell preparations-pulsed autologous APC vaccines (e.g., dendritic cells, PBMC), and electrofusion-generated tumor cell-dendritic cell hybrids (Kugler A. et al, nat. Med., 6: 332. sub. 336, 2000. TureiO. et al, Cancer Res., 56: 4766. sub. 4772, 1996. ed.) include: i) the fate of ex vivo loaded synthetic CTA peptides, intact synthetic CTA proteins, or tumor-derived CTA in vivo is unknown, which can significantly affect the duration of antigen presentation to the host immune system; II) limited amounts of synthetic CTA peptides, intact synthetic CTA proteins or tumor-derived CTA that can be loaded ex vivo onto HLA type I and/or HLA type II antigens of a cellular vaccine can significantly hinder the immunogenicity of the administered CTA; iii) known HLA class I antigens that are restricted by the HLA phenotype of the patient and the number of CTAs identified to date are still relatively limited-and even more limited HLA class II antigen-restricted immunogenic epitopes; iv) a sufficient amount of fresh tumor tissue is available, which also well indicates that different CTAs expressed in neoplastic lesions (Jenne l, et al, Trends immunol., 22: 102-107, 2001).

ADHAPI-cells express endogenously synthesized CTA for a long period of time; thus, unlike autologous APC vaccines that are pulsed with ex situ synthetic CTA peptides or with synthetic CTA intact protein or with intact tumor cell preparations, ADHAPI-cells are able to continue to stimulate the host's immune response in vivo with fewer administrations to the patient. Since ADHAPI-cells have no long-term in vivo tumorigenic effects, this hypothesis is further supported by the possibility of anticipating the administration of ADHAPI-cells in the form of live, non-x-ray treated cell vaccines. And once ADHAPI-cells undergo physiological death in vivo, they will still act as a "repository" of endogenously synthesized CTA peptides and proteins, which will further and effectively enhance the presentation of HLA type I-restricted CTA epitopes to CD8+ T cells by dendritic cells of patients through cross-primed immune mechanisms, as well as the presentation of HLA type II-restricted CTA epitopes to CD4+ T cells through well-defined exogenous CTA pathways of antigen processing.

ADHAPI-cells retain their APC function; in fact, they effectively stimulate the proliferation and release of IFN- γ from autologous and allogeneic PBMC; moreover, ADHAPI-cells are in most cases more potent stimulators than their respective control cells. In this regard, it is relevant that ADHAPI cells can simultaneously express higher levels of HLA class I antigen and/or different co-stimulatory/helper molecules in addition to CTA than their respective control cells. These evidence clearly demonstrates the great advantage of ADHAPI-cells as autologous cell vaccines compared to autologous tumor cells that are poorly immunogenic and cannot constitutively express several co-stimulatory/helper molecules. Moreover, in contrast to autologous dendritic cells generated and expanded ex vivo, the ADHAPI-cell vaccine is generated by fully mature and immunocompetent APCs; this overcomes potential limitations imposed by the dendritic cell maturation stage used to prepare the cell vaccine, which may affect its tolerogenicity without affecting its immunogenicity.

Compared to other cell vaccines, ex vivo production of ADHAPI-cell vaccines expressing simultaneously multiple/all methylation-regulated CTAs is simple, in most cases rapid, does not require cumbersome in vitro cell manipulation, does not involve genetic manipulation, does not require autologous tumor tissue, and is highly reproducible by PBMCs of healthy individuals and cancer patients.

Furthermore, nearly 100% of the ADHAPI-cell preparations expressed all of the CTAs studied, which were able to induce demethylation in the APC. With these characteristics, the preparation of ADHAPI-cell vaccines is easier to standardize and control quality (e.g., flow cytometry analysis of selected cell surface molecules and RT-PCR of selected CTAs) and efficacy (e.g., quantitative RT-PCR of selected CTAs). Furthermore, in contrast to other cellular vaccines that have heretofore had to be freshly prepared at each administration to a patient, thus producing significant inter-agent differences (e.g., cell viability, phenotype profile of the vaccinating cells, amount of synthetic CTA peptide or synthetic CTA intact protein or intact tumor cell preparation loaded, efficacy of generating tumor cell-dendritic cell hybrids by electrofusion), ADHAPI-cellular vaccines, once made and tested for viability, quality and efficacy, can be aliquoted, suitably frozen and stored in viable conditions until used for therapeutic purposes. Moreover, the ADHAPI-cell vaccines provide a virtually unlimited source of therapeutic agent for each patient, since they do not require the acquisition of autologous tumor tissue to pulse autologous cell vaccines or prepare tumor cell-dendritic cell hybrids ex vivo, and they can be rapidly prepared in large quantities from duplicate leukaphereses.

Whereas ADHAPI-cells can simultaneously express multiple/all methylation-regulated CTAs, wherein the CTA is endogenously synthesized and is capable of entering both the HLA type I and HLAII antigen processing pathways, since ADHAPI-cells have the potential to express and present to the host's T cells methylation-regulated CTA (as well as known and yet unknown non-immunodominant epitopes of CTA) that has not yet been identified and characterized, and since the known HLA class I antigen-and HLA class II antigen-restricted immunogenic epitopes of CTAs identified to date are still limited in number (the CTA epitopes can thus be used for therapeutic purposes depending on the HLA phenotype of the patient), an additional advantage of ADHAPI-cells is that they are likely to be able to simultaneously present known and yet unknown immunogenic epitopes of different CTAs in any and many HLA class I and HLA class II homospecificities. Thus, treatment with the ADHAPI-cell vaccine is not limited to patients with a defined HLA phenotype, as compared to synthetic CTA peptide pulsed or synthetic CTA whole protein pulsed cell vaccines; thus all cancer patients expressing one or more CTAs with neoplastic lesions can be candidates for ADHAPI-cell vaccine therapy regardless of their HLA phenotype. In this regard, among the CTAs known to date, one or more of them are commonly expressed in most of the different histological malignancies studied; thus, inoculation of ADHAPI-cells is appropriate for the vast majority of cancer patients. One piece of meaningful information is that MAGE, GAGE or NY-ESO-1 is expressed in 96% of human tumors (Cancer Immunol. Immunother.50: 3-15, 2001).

Compared to synthetic CTA peptide pulsed or synthetic CTA whole protein pulsed cellular vaccines, where a limited amount of protein can be loaded ex vivo onto HLA type I and/or HLA type II antigens of the cellular vaccine, significantly hindering the immunogenicity of the administered CTA, and since ADHAPI-cells express multiple/all methylation-regulated CTAs simultaneously, ADHAPI-cellular vaccines are able to overcome the immunoselection of CTA-negative tumor variants that arise during treatment against a single or several CTAs, and overcome the constitutively heterogeneous and sometimes down-regulated expression of different CTAs in a particular neoplastic lesion.

ADHAPI-cell vaccines consist of autologous functional APCs that simultaneously express multiple/all known methylation-regulated CTAs and most likely yet unidentified CTAs whose expression is regulated by DNA methylation; moreover, ADHAPI-cell vaccines can be used for patients with CTA-positive tumors of different tissue types. These functional and phenotypic characteristics have distinct advantages over currently employed allogeneic tumor cell vaccines (e.g., lysates of intact mixed neoplastic cell lines or unpurified extracts thereof, antigens shed from mixed neoplastic cell lines), which may, in fact, contain insufficient amounts of known and yet unknown immunologically relevant CTAs, contain non-relevant cellular components that can compete with CTAs for immune responses, may be increasingly toxic due to being allogeneic, require efficient processing by the patient's immune system, and can only be used in patients with malignancies of the same tissue type.

ADHAPI-cellular versus genetically modified cellular CTA vaccine

The preparation of ADHAPI-cells does not include ex vivo genetic manipulation of autologous dendritic cells or other autologous APCs that is required for the production of genetically modified cell vaccines that express the selected CTA after transfection or transduction. Moreover, many limiting factors affect genetically modified cellular vaccines compared to ADHAPI/cell; among these limiting factors are i) the low efficiency of the transfection methods available; ii) inducing a cellular immune response against the antigen of the viral vector used for cell transduction, resulting in destruction of the genetically modified vaccinated cells; iii) the presence of neutralizing antibodies pre-existing or vaccination-induced to interfere with vaccine administration; iv) direct effect of viral vectors on survival, maturation and antigen-presenting capacity of transduced cells (Jenne l. et al, Trends immunol., 22: 102-107, 2001).

TABLE I

Recovery of ADHAPI-cells and control cells

Cell type	ADHAPI-cells	Control cells
			B-EBV	114±25	175±51
PWM-B	16±5	38±17
			CD40L-B	75±27	96±5
PWM-PBMC	26±11	45±16
			PHA-PBMC	23±10	63±25
PHA+PWM-PBMC	35±28	63±36

^aData represent the mean% +/-of cells recovered compared to the number of cells used for their production (100%) in 3(a), 4(b), 4(c), 4(d), 7(e) and 5(f) independent experimentsSD。

TABLE II

Phenotypic profile of ADHAPI-cells compared to autologous control cells

^*Data were obtained by Student's paired t-test comparing the mean of mean fluorescence intensities obtained using flow cytometry in 6(a), 6(b), 4(c), 4(d), 6(e) and 2(f) independent experiments. Statistically significant differences invariably represent upregulation of the expression of the studied antigen on ADHAPI-cells compared to autologous control cells.

^fIs not significant;

^ga value of p;

^hnot detected;ADHAPI-cell/CD 40L-B82-100% CD20 +; control CD40L-B cells were 87-99% CD20 +.

TABLE III

Enzyme-linked immunosorbent assay (ELISA) was used to assess IFN-. gamma.release from autologous (R) (aMLR) and allogeneic (MLR) PBMC (R) stimulated by ADHAPI-cells (S) or control cells (S).^*

^*Data represent mean + -SD (pg/ml) of IFN- γ release in 2(a), 4(b), 4(c), 4(d), 3(e) and 4(f) independent experiments. The S/R ratio is: 3: 1(a), 1: 1(b), 1: 2(e), 1: 1(d), 1: 1(e), 1: 2 (f). In thatMeasuring IFN- γ release at 24 hours (a), 6 days (b), 24 hours (c), 6 days (d), 24 hours (e) and 6 days (f) after the start of the culture;^gp-value obtained by Student's paired t-test relative to control cells;^hnot detected.

Claims (18)

1. A method of generating cancer testis antigen presenting cells comprising:

a) collecting PBMC cells from a subject;

b) activating the collected PBMC cells;

c) culturing and optionally expanding the activated cells ex vivo;

d) the cultured and optionally expanded cells were pulsed four times with 1 μ M5-AZA-2' -deoxycytidine (5-AZA-CdR) every 12 hours, then half of the medium was changed to fresh medium and cultured for another 48 hours to allow the cells to simultaneously express multiple cancer testis antigens.

2. The method of claim 1, wherein the subject is a cancer patient.

3. The method of any one of claims 1-2, wherein the cell is an epstein barr virus-immortalized B-lymphoblastoid cell line.

4. The method of any one of claims 1-2, wherein the cell is a pokeweed mitogen (PWM) -activated B-lymphocyte.

5. The method of any one of claims 1-2, wherein the cell is a CD 40-activated B-lymphocyte.

6. The method of any one of claims 1-2, wherein the cells are Phytohemagglutinin (PHA) + recombinant human interleukin-2 (rhIL-2) -activated PBMCs.

7. The method of any one of claims 1-2, wherein the cells are Phytohemagglutinin (PHA) + recombinant human interleukin-2 (rhIL-2) + poke-weed mitogen (PWM) -activated PBMCs.

8. The method of any one of claims 1-2, wherein the cell is a dendritic cell, monocyte, macrophage.

9. A cell obtained by the method of any one of claims 1-8, which simultaneously expresses cancer testis antigens MAGE-1, MAGE-2, MAGE-3, MAGE-4, NY-ESO-1, GAGE-1-6 and SSX-2.

10. Use of the cells and/or cellular fractions thereof of claim 9 for the preparation of a medicament for the prevention and treatment of malignancies of different tissue types that constitutively express one or more cancer antigens.

11. The use of claim 10, wherein the cells are stored as a repository of mixed antigens.

12. The use of claim 11, wherein the medicament is a cancer vaccine.

13. A cancer vaccine comprising the cell of claim 9.

14. The vaccine of claim 13, which is autologous.

15. The vaccine of claim 13, which is allogeneic.

16. The vaccine of claim 13 or 15, wherein the cells are used as a reservoir for mixed antigens.

17. The vaccine of claim 15 or 16, wherein a cellular fraction of the cells is used.

18. Use of the cell of claim 9 and/or a cellular component thereof in a method for producing an effector immune cell for the preparation of a product useful in adoptive immunotherapy.