HK1158090A - Vaccine compositions and methods - Google Patents

Abstract

The present invention relates to pharmaceutical vaccine compositions comprising at least one vaccine antigen together with living immune cells. These immune cells include at least a portion of activated T-cells and act as an adjuvant. Methods for using these pharmaceutical compositions to prevent or treat diseases, such as cancer, infectious diseases and autoimmune disease are also included.

Description

Vaccine compositions and methods

Reference to related applications

This application is based on and claims the rights of U.S. provisional patent application serial No. 61/050,294 filed on 5.5.2008 and U.S. provisional patent application No. 61/049,990 filed on 5.2.2009, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to the field of vaccines, and more particularly, to adjuvanted vaccine compositions.

Background

The use of the strength of the immune system to treat chronic infectious diseases or cancer is a major goal of immunotherapy. Vaccination (aka, active immunotherapy) is designed to activate the immune system to specifically recognize and protect against invading pathogens. Active immunotherapy has been used over the past 200 years to prevent a variety of infectious diseases, such as smallpox, rabies, typhoid, cholera, plague, measles, varicella, mumps, polio, hepatitis b, and tetanus and diphtheria toxins.

The concept of active immunotherapy is now being used to develop therapeutic cancer vaccines, with the aim of treating existing tumors or preventing tumor recurrence, as well as being used to treat and prevent chronic viral infections. However, existing active immunotherapy technologies have not been successful in defending against many modern vaccine targets such as HIV/AIDS, hepatitis b, and cancer. This is due in part to the inability of current vaccination techniques to elicit the correct type of immune response.

The type of immune response generated against infection or other antigenic challenge can generally be distinguished by the subset of T helper (Th) cells involved in the response. Immune responses can be roughly divided into two categories: th1 and Th 2. Th1 immune activation is optimal for intracellular infections such as viruses and involves activation of Natural Killer (NK) cells and cytolytic T Cells (CTLs) that can lyse infected cells, while Th2 immune responses are optimal for humoral (antibody) responses. Th1 immune activation is best expected for cancer therapy, with Th2 immune responses directed more against the secretion of specific antibodies and of relatively low importance for tumor therapy. Prior art vaccine compositions are adept at eliciting a Th2 or humoral immune response, which is ineffective against cancer and most viral diseases.

The use of adjuvants is one strategy to affect the immune response to antigens in vaccine compositions. Oil-in-water emulsions of aluminium salts and squalene (MF59) are the most widely used adjuvants in human vaccines. These adjuvants significantly promoted Th2 humoral (antibody) responses to the antigen and effectively raised serum antibody titers, but did not elicit significant Th1 responses or CTLs. However, vaccines against chronic infections (e.g., Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), tuberculosis, Herpes Simplex Virus (HSV) and cancer cells require the generation of a Th1 cellular immune response to exert both prophylactic and therapeutic effects.

It has been demonstrated that some experimental active immunotherapy techniques and protocols of the prior art successfully elicit Th1 responses against tumor antigens in selected patients, leading to an increased frequency of CTL immune cells in the circulation, which are capable of specifically killing tumor or pathogen infected cells. However, although capable of eliciting a Th1 response, tumor escape mechanisms can overwhelm this immune response, leading to the development of eventual tumors. Viruses have also developed many strategies to avoid immune attack and to maintain targets for the mobile immune system.

Brief Description of Drawings

FIG. 1a is a graph of survival of mice after administration of the composition.

FIG. 1b is a graph of survival of mice after administration of the composition.

Figure 2a is a graph of survival of mice after administration of the indicated compositions.

Figure 2b is a graph of survival of mice after administration of the indicated compositions.

Summary of The Invention

In one aspect, the invention includes pharmaceutical compositions. The composition comprises an adjuvant and one or more antigens, wherein the adjuvant comprises viable immune cells, at least a portion of which are activated T cells. Administration of the composition to a host produces a Th1 response.

In another aspect, the invention includes an adjuvant composition comprising living immune cells, wherein at least a portion of the immune cells are activated T cells. Administration of the adjuvant composition to a host elicits a Th1 immune response.

In yet another aspect, the invention includes a method of making a pharmaceutical composition. The method comprises preparing an adjuvant comprising live immune cells, wherein the immune cells comprise at least a portion of T cells, and combining one or more antigens with the adjuvant, wherein the pharmaceutical composition stimulates an immune response in the host when administered to the host.

In another aspect, the invention includes a method of reducing an antigen associated with or causing a disease in a host. The method comprises administering a pharmaceutical composition comprising an adjuvant and one or more antigens. The adjuvant comprises a living immune cell, wherein the immune cell comprises at least a portion of a T cell, and wherein the pharmaceutical composition stimulates an immune response in a host when administered to the host.

In yet another aspect, the invention includes a method of treating a disease in a patient. The method comprises administering a pharmaceutical composition comprising an adjuvant and one or more antigens. The adjuvant comprises living immune cells, wherein the immune cells comprise at least a portion of T cells, and wherein the pharmaceutical composition stimulates an immune response in the host when administered to a patient.

Detailed description of the preferred embodiments

The present invention provides pharmaceutical vaccine compositions and methods of active immunotherapy that are capable of eliciting prophylactic and therapeutic Th1 immunity against disease in a patient, while also providing a means to overcome immune evasion mechanisms of disease pathogens and tumors. The pharmaceutical compositions of the present invention generally comprise: (1) one or more sources of antigen; and (2) viable activated immune cells, wherein at least a portion are T cells.

In the present invention, a novel vaccine adjuvant for patients is described. The adjuvant may be mixed with one or more vaccine antigens to form a pharmaceutical composition. In some embodiments, the adjuvant may be used alone as an immunostimulant. The novel adjuvant comprises living immune cells, at least a portion of which are T cells. The T cells are preferably of the Th1 phenotype (producing IFN-. gamma.and not IL-4)CD4+ T cells) of memory T cells (CD45RO +, CD 62L)^Lo). The memory Th1 cells were activated at the time of formulation and introduction into the patient. The preferred method of activation is by cross-linking CD3 and CD28 surface molecules on T cells. Other activation methods are also within the scope of the present invention. Activated memory T cells preferably express CD40L when activated and produce large amounts of inflammatory cytokines (e.g., IFN-. gamma., GM-CSF and TNF-. alpha.). These activated Th1 memory cells are preferably allogeneic (allogenic) to the patient.

Pharmaceutical vaccine compositions typically contain at least one antigen in admixture with an adjuvant. According to the prior art, it is well known practice to enhance the immune response induced by the antigen present in a vaccine by means of an adjuvant. Adjuvants as referred to herein are compounds that can increase the inherent immunogenicity of an antigen. The term "adjuvant") is also commonly used as a synonym for "immunostimulant".

Adjuvants for new vaccine targets such as cancer and infectious diseases are not only required to increase immunogenicity to vaccine antigens, but are also often required to shift the existing immune response against vaccine antigens from Th2 to Th 1. In addition, the efficacy of vaccines often requires amplification of this deviating immune response. The vaccine adjuvant compositions of the present invention provide these immunomodulatory and immunopotentiating properties.

Some adjuvants are known to promote Th1 immunity against antigens, including saponins, BCG, liposomes and microparticles, poly I: C, anti-CD 40mAb, co-stimulatory molecules, IC31, TLR9 ligands, KLH, CpG, alpha-galactosylceramide, TLR4 agonists, cholera toxin, cytokines, chemokines, immunostimulatory complexes (ISCOMs), LPS, molecular agonists (e.g., NAIP, CIITA, HET-E, TP-1-leucine rich repeat unit pathway receptors), TNF receptor superfamily (TNFRSF) agonists, alarins, and blockers of immunosuppressive cytokines and Tregs. Each of these adjuvants has the ability to function at one level of the cascade of immunological events necessary for immunomodulation or immunostimulation or for loss of immune evasion ability. However, none of these adjuvants have all of the properties necessary for these effects.

The present invention relates to the following findings: activated immune cells, preferably heterologous Th1 memory cells activated by cross-linking CD3 and CD28 antigens, that produce inflammatory cytokines and express CD40L, can elicit all components of the immune cascade necessary to be effective immunomodulators and immunostimulants. Furthermore, these activated immune cells are able to interfere with inhibitory regulatory mechanisms in order to overcome the ability of pathological organisms and cancers to evade immune attack. This makes these cells ideal adjuvants.

The pharmaceutical composition of one or more vaccine antigens with activated T cells may be used for prophylactic purposes or therapeutic purposes or both. The compositions are administered by all routes conventionally used or recommended for vaccines, including parenteral, intradermal, intramuscular, subcutaneous or mucosal routes. In some embodiments, the composition may also be administered intranodal or intratumoral.

The antigenic component of the pharmaceutical composition includes one or more antigens. If more than one antigen is included in the pharmaceutical composition, the antigens may be from the same source of antigens or different sources of antigens. Any source of antigen may be used in the formulation, for example, the antigen may be from a living cell or organism, and the source material may be freeze-thaw lysate, radiation inactivated (or other inactivation method), used as whole cells or organisms, or a lysate thereof. In some preferred embodiments, tumor cells or tumor cell lysates can be used as the cell source material for the antigens. The cell source material may be from an autologous or heterologous cell source or from a cell line. The antigen may also be derived from naked DNA or RNA encoding the antigen. The nuclear material can be used alone or incorporated into viral vectors. Another example of a source of antigen is an anti-idiotypic antibody or portion thereof that mimics the antigen, or other methods that mimic the structure of the antigen. Antigen pulsed or transfected Dendritic Cells (DCs) may also be the source of antigen in the pharmaceutical composition. The DCs may be pulsed with peptides or intact proteins, recombinant proteins, or mRNA or DNA encoding the antigen, or the DCs may be fused to cells containing the antigen, or the DCs may be transfected with viral vectors containing the antigen, such as retroviruses, lentiviruses, adenoviruses, or these antigen-derived components may be used alone without the DC.

One or more Tumor Associated Antigens (TAAs) may also be used in the pharmaceutical composition, examples of TAAs include: MART-1, gp100, tyrosinase, Melan A, TRP-1, tumor-specific mutant gene products, such as CDK-4, β -catenin, MUM-1, oncogenes such as p53, and ras (K-and H-ras), cancer testis antigens, such as MAGE, GAGE, and NY-ESO1, overexpressed autoantigens, such as MUC1, cyclin B1, Her2-neu, CEA, WT, p53, SART-1, PRAME, p15, and viral antigens, such as HPV E7, EBV-derived antigens, and telomerase.

In a preferred embodiment, the antigenic component may comprise one or more chaperone proteins (also known as heat shock proteins) isolated from a dead infected tissue or tumor. Heat Shock Proteins (HSPs) belong to the main targets of immune responses against bacterial, fungal and parasitic pathogens. Tumor-derived heat shock protein (hsp) -peptide complexes (especially hsp70 and grp94/gp96) have been shown to be useful as effective vaccines for generating anti-tumor immune responses in animals and humans. This approach exploits the peptide binding properties of stress proteins, which are responsible for their function as chaperones in many cellular processes.

Certain chaperones in the extracellular environment are also capable of modulating innate and adaptive immunity due to their ability to chaperone polypeptides and interact with the host's immune system, particularly professional antigen presenting cells. Vaccination with HSP vaccines from tumors can elicit anti-tumor responses and down regulate immunosuppressive mechanisms. The immunogenicity of HSPs may be derived from the antigenic peptides to which they bind.

A preferred method for isolating chaperonin for use as an antigen source is described in us patent No. 6,875,849 to Katsantis. Other methods are described by Srivastava in U.S. patent No. 6,797,480; 6,187,312, respectively; 6,162,436, respectively; 6,139,841, respectively; 6,136,315, respectively; and 5,837,251. HSPs may also be pulsed with antigens, including peptides, whole cells, or cell lysates.

In one embodiment, tumor-derived chaperone-rich cell lysate (CRCL) is used as the antigen source and is obtained by enrichment of the major chaperone proteins from tumor lysate using free solution isoelectric focusing (FS-IEF) techniques as described in the examples below. This technique is a fast and efficient process compared to conventional techniques, yielding up to 5 to 20 times more antigenic material and less time consuming. From a clinical point of view, a multiprotein chaperone complex enriched FS-IEF approach is desirable in terms of high yield from potentially limited tumor sources, and rapid turnaround time from tumor harvest to treatment of patients.

There are many advantages in using CRCL-related peptides as a source of tumor antigens. First, they do not require the identification of tumor-specific peptides. Second, they elicit a polyclonal T lymphocyte response after immunization, preventing the generation of immune evasive variants. Third, they consist of class I MHC-related peptides and class II MHC or helper epitopes, which together induce a more potent and longer lasting immune response. The clear anti-tumor effect of the CRCL vaccine has been demonstrated in a number of animal models, including murine 12B1 leukemia and a20B cell leukemia/lymphoma.

In addition, antigens conventionally used in vaccines can also be used in the compositions of the invention, including whole microorganisms or portions of microorganisms, such as live attenuated whole microorganisms, inactivated microorganisms, recombinant peptides and proteins, glycoproteins, glycolipids, lipopeptides, synthetic peptides or disrupted microorganisms, polysaccharides, used alone or conjugated to a carrier element, such as a carrier protein, can also be used.

In general, any antigen or combination of antigens that can be used to treat or prevent a disease can be used in the pharmaceutical composition. Antigens from infectious pathogens may also serve as a source of antigen and may be referred to herein as disease-causing antigens. Examples of diseases from which antigens can be obtained are: diphtheria, tetanus, poliomyelitis, rabies, pertussis, hepatitis A, hepatitis B and hepatitis C, EBV, CMV, herpes types 1 and 2, yellow fever, typhoid, varicella, pox (smallpox), measles, mumps, German measles, Japanese encephalitis, meningitis, influenza, pneumococcal infections, rotavirus infections, AIDS (HIV1 and 2), cancer, HTLV1 and 2, syphilis, HPV, tuberculosis, Lyme disease, RSV infections, trypanosomiasis, dengue fever, malaria, anthrax, Epbora virus, tularemia, yersinia, West Nile virus, diseases caused by Chlamydia, Neisseria gonorrhoeae (Neisseria NORRhe), Streptococcus pneumoniae (Streptococcus pneoniniae), Moraxella catarrhalis (Moraxella arrhena), Staphylococcus aureus (Staphylococcus aureus) or B (Haemophilus influenzae), malaria, Haemarrhoea disease caused by influenza, Listeriosis, and the like.

The activated Th1 memory cells used in the pharmaceutical composition of the invention are preferably from normal donor blood. Preferred methods of processing and producing a material suitable for use in the present invention are described by Har-Noy in U.S. patent nos. 7,435,592 and 7,402,431 and pending U.S. patent No. 20050191291, which are incorporated herein by reference in their entirety.

The pharmaceutical composition of the invention may be a composition intended to be immunized against a single pathogen or cancer, i.e. it comprises one or more antigens from a single pathogen or cancer. Alternatively, it may be a composition intended to immunize against several different pathogens or cancers, herein referred to as a vaccine combination.

The invention also includes methods of preparing pharmaceutical compositions. The method comprises preparing an adjuvant comprising T cells, preferably activated T cells as described herein. One or more antigens may be combined with an adjuvant to form a pharmaceutical composition. If more than one antigen is included in the composition, the antigens may be from the same source of antigen or different sources of antigen. Administration of the pharmaceutical composition may stimulate an immune response, preferably a Th1 response, in the host.

The adjuvant effect of activated T cells can be obtained when the activated T cells are combined with the antigen of the pharmaceutical composition before administration, i.e. when it is present directly in the pharmaceutical composition. Alternatively, the adjuvant and antigen may be administered separately in sequential steps. For example, the adjuvant may first be administered to the host using any of the techniques described above. Following administration of the adjuvant, the antigen may be administered to the host. Preferably, the adjuvant and antigen are combined to form a pharmaceutical composition prior to administration to the host.

The pharmaceutical compositions of the invention are designed to generate an adaptive Th1 immunity to the antigen in the composition. When administering the pharmaceutical composition of the present invention to a patient suffering from a disease (with existing disease), it may be desirable to stimulate an effective innate immune response in order to control the disease until the adaptive immune response becomes sufficiently robust to have a therapeutic effect. To achieve this, the adjuvant immune cells alone may be administered intravenously at the same time as the vaccine composition is administered or at any time after the vaccine composition is administered.

If the immune response against the vaccine antigens in the composition is not strong enough, an additional booster injection may be administered. Preferably, booster injections may be administered at least 3-7 days apart, more preferably 7-14 days apart. Additional booster injections may be administered monthly or annually, if desired.

To maintain an inflammatory environment that can enable tumors and disease organisms to escape the immune destruction loss, an additional booster injection of activated Th1 memory cells, alone or in formulation with antigen, can be administered. Patients who have been previously vaccinated with a composition comprising heterologous Th1 memory cells can be immunized against the heterologous antigen. Subsequent injection of allogeneic cells can activate the pool of anti-xenoantigen cells, which can release the inflammatory cytokines necessary to disable immune evasion mechanisms.

Examples

Example 1

Mouse

Female BALB/c (H2)^d) Mice were obtained from the national cancer institute (Bethesda, MD) and used at 7 weeks of age.

Preparation of Th-1 cells (CD3/CD28 Cross-linked Th1 cells)

Splenocytes from Balb/c mice were collected and treated with ammonium chloride-potassium (ACK) buffer for lysis of red blood cells. Approximately 7 million to 1 million cells are isolated per spleen. CD4+ T-cells were then purified by positive selection (> 98% purity) on an MS column (Miltenyi Biotec, germany) using CD4 immunomagnetic particles, isolating approximately 800-1200 million CD4 cells at 50-60% yield. Th1 memory cells were generated by expansion of anti-CD 3 and anti-CD 28 coated cis-magnetic beads (CD3/CD 28T-cell expansion beads, Dynal/Invitrogen) at a 3: 1 ratio of primary beads to CD4 cells. Purified CD4 cells were incubated with 20IU/mL recombinant mouse (rm) IL-2, 20ng/mL rmIL-7, and 10ng/mL rmIL-12(Peprotech, New Jersey) and 10. mu.g/mL anti-mouse IL-4mAb (Becton Dickenson) in RPMI 1640 medium (complete medium) containing 10% FBS, penicillin-streptomycin-glutamine, non-essential amino acids (NEAA) (Biological Industries, Israel) and 3.3mM N-acetyl-cysteine (NAC; Sigma). Additional complete medium containing cytokines with rmIL-2 and rmIL-7 was added daily to the CD4 cultures from day 3 to day 6 to maintain the cell concentration at 0.5X10⁶To 1x10⁶Between cells/mL. Additional CD3/CD28 beads were added daily from day 3 to day 6. The number of beads added was calculated to maintain a 1: 1 bead to cell ratio as the cells expanded. After 6 days of culture, CD4 cells expanded approximately 80 to 100 fold and were harvested and debeaded by physical disruption and passage through a magnet. The phenotype of the harvested cells used for the experiment was > 95% CD4+, CD45RB^lo，CD62L^lo，CD44^hiAnd are therefore referred to as "memory cells".

CD3/CD28 Cross-linking

After harvest and before injection, debeaded Th1 memory cells were plated at 2X10⁶Individual cells/ml density cultured in cRPMI 5% CO at 37 ℃₂The microparticles conjugated with CD3/CD28 mAb (T cell expansion, Dynal/Invitrogen) were cultured for 4-6 hours at a 2: 1 bead to cell ratio. After 4 hours, the cells produced IFN-. gamma.and fine was upregulatedExpression of CD40L and FasL on the cell surface. Cross-linked Th1 memory cells used in these experiments expressed FasL and CD40L on the cell surface and produced greater than 2000ng/ml/10⁶Cells/6 h IFN-. gamma.and less than 20pg/ml IL-4/10⁶Cells/6 h.

12B1 cell line

By using human bcr-abl (b)₃a₂) The fusion gene retrovirus transforms BALB/c marrow cells to obtain the murine leukemia cell line 12B 1. These cells express the p210 bcr-abl protein. Cells were cultured at 37 ℃ and 5% CO₂RPMI medium (Gibco/BRL, Gaithersburg, Md.) supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bio-products, Woodland, Calif.). Cells were tested routinely and found to be free of Mycoplasma (Mycoplasma) contamination.

Generation of 12B1 tumors

For injection, 12B1 cells were first washed three times in PBS (Gibco/BRL), then counted and adjusted to 5X10⁴Concentration of individual cells/mL. Female BALB/c mice were injected subcutaneously with 0.1mL (5X 10) in the right groin³Individual cells) and tumor progression was monitored.

Preparation of 12B1 tumor lysate

Tumor cell pellets from 12B1 cell culture were subjected to 6 freeze-thaw cycles in a liquid nitrogen/37 ℃ water bath. Cell lysis was verified by trypan blue exclusion. Protein concentration was determined by BCA assay. The protein was diluted to 25. mu.g/100. mu.l in sterile PBS for immunization of mice.

Preparation of 12B1 chaperone-enriched cell lysate (CRCL)

Tumors from mice with 12B1 were homogenized at a ratio of 1g tumor/5 ml buffer in 10,000g tumor/5 ml buffer in 10mM Tris-Cl (pH 7.4)/10mM NaCl, 0.1% detergent (equal portions of Triton X-100, Triton X-114 and Igepal CA-600, Sigma, St.Louis, Mo.) in a glass-Teflon homogenizer, 0.1% detergent (equal portions of Triton X-100, Triton X-114 and Igepal CA-600, Sigma, St.Louis, Mo.) (including 2. mu.g/ml suppressor peptide, 0.1mg/ml Perfabloc, 0.5mM phenylmethanesulfonate and a complete protease inhibitor cocktail piece (all from Roche Molecular Biochemicals, Indianapolis, Ind.). the homogenate was centrifuged at 10,000g at 4 ℃ for 30 minutes and a sample was taken, called "lysate" (supernatant) which was subsequently dialyzed at 100,000g, 4 ℃ for 60 minutes in a "high speed" reduced concentration "supernatant" BCA "was dialyzed against a double-bundle of gold assay, pierce Endogen, Rockford, il.) protein concentration was determined and free solution-isoelectric focusing (FS-IEF) starting material was frozen in 25mg aliquots. FS-IEF was performed with the following modifications: we replaced ampholytes with Rotolytes (Bio Rad Laboratories, Hercules, Calif.) and used pH ranges of 3.9-5.6, 4.5-6.1 and 5.1-6.8 (5 ml of each a and B reagent for each pH range, 30ml total); we have reduced the detergent concentration to Triton X-100, 0.1% for each of Triton X-114 and Igepal CA-600; we loaded only 25mg of starting material per 60ml total volume of isoelectric focusing (isofocusing) mixture instead of 50mg/60 ml. The urea concentration (6M) remained the same and the isoelectric focusing conditions remained the same (15W constant power), but the length of IEF was extended to 5 hours. SDS-PAGE and Western blot analysis of the fractions was performed and the fractions containing all four major immunogenic chaperones (GRP94/gp96, HSP90, HSP70 and calreticulin) were pooled and dialyzed against 2M urea in 0.1 XPBS, pH 7.4, followed by dialysis into 0.1 XPBS. Protein concentration was determined by BCA assay using bovine serum albumin as a standard, and the protein was diluted to 25. mu.g/100. mu.l in sterile PBS for immunization of mice.

Preparation of liver CRCL

Liver CRCL was prepared from Balb/c mouse liver first used in the experiment using the procedure described above. The protein was diluted to 25. mu.g/100. mu.l in sterile PBS for immunization of mice.

Preparation of TuLy CRCL (12B1 tumor lysate/liver CRCL)

12B1 tumor lysate and liver CRCL were mixed at a ratio of 1: 1. mu.g and the mixture was incubated overnight at 4 ℃. The protein was diluted to 50. mu.g/100. mu.l in sterile PBS for immunization of mice.

Prophylactic vaccination of mice

80 Balb/c mice (8 per group, 10 groups) were used. Mice were vaccinated intradermally (i.d.) in footpads (footpad) on days-14 and-7 prior to tumor cell vaccination. The groups are as follows:

control: PBS 100. mu.l i.d.

12B1 lysate: each mouse was 25. mu.g/100. mu.l i.d.

Liver CRCL: each mouse was 25. mu.g/100. mu.l i.d.

12B1 CRCL: each mouse was 25. mu.g/100. mu.l i.d.

Tuly CRCL (12B1 tumor lysate/liver CRCL): each mouse 50. mu.g/100. mu.l i.d.

Activated Th-1 cells: each mouse 100. mu.l PBS 1X10⁵Individual cells i.d.

Activated Th-1 cells +12B1 lysate: each mouse 100. mu.l PBS 1X10⁵Individual cells +25 μ g lysate i.d.

Activated Th-1 cells + liver CRCL: each mouse 100. mu.l PBS 1X10⁵Individual cells +25 μ g liver CRCL i.d.

Activated Th-1 cells +12B1 CRCL: each mouse 100. mu.l PBS 1X10⁵Individual cells +25 μ g 12B1 CRCL i.d.

Activated Th-1 cells + TuLy CRCL-1X 10 in 100. mu.l PBS per mouse⁵Individual cells +50 μ g TuLy CRCL i.d.

Inoculation of tumor cells and monitoring of tumor volume

Mice from all 10 groups were inoculated under the right inguinal epithelium with 5000 cells of 12B 1/mouse at day 0. Tumor volume was measured every 2 days. When the tumor volume reaches 4000mm³Mice were euthanized at time.

Results

Tumors in the control group became palpable at day 12. The results from the various therapies are shown in fig. 1a and 1 b.

After 6 weeks all mice in the control, CD3/CD28 cross-linked Th1 cells, 12B1 CRCL, liver CRCL, 12B 1-lysate/liver CRCL and CD3/CD28 cross-linked Th1 cells +12B 1-lysate/liver CRCL groups died. 50% of the mice in the combined CD3/CD 28-crosslinked Th1 cells +12B1 CRCL group (best group) had no tumor, 25% in the CD3/CD 28-crosslinked Th1 cells +12B1 lysate group, 12.5% in the 12B1 lysate group, and 12.5% in the CD3/CD 28-crosslinked Th1 cells + liver CRCL group.

There was thus a clear benefit in binding to CD3/CD28 cross-linked Th1 cells and tumor-derived CRCL.

Example 2

In this example, tumor-derived CRCL was used as a source of tumor-specific antigens and combined with activated CD4+ Th-1 cells as adjuvant to treat established leukemias. The procedure is as described in example 1. Animals (8 mice per group) received the indicated treatment.

Preventive setting:

balb/c mice used for the first time in the experiment were treated by injection in the footpad (intradermally) with PBS (control), or 12B 1-derived CRCL (12B1 CRCL, 25 μ g/mouse), or CD3/CD28 cross-linked Th1 cells, or by 12B1 CRCL plus CD3/CD28 cross-linked Th1 cells on days-14 and-7. On day 0, mice were inoculated with 12B1 leukemia cells (5,000 cells/mouse, injected subcutaneously in the left groin). The percent survival is shown in figure 2A.

Treatment setting:

to determine the therapeutic efficacy of the CRCL plus CD3/CD28 cross-linked Th1 cell combination, a 12B1 tumor was established (500012B 1 cells/mouse were inoculated in the left groin on day 0). Mice were treated with PBS, 12B1 CRCL, CD3/CD28 cross-linked Th1 cells alone or CD3/CD28 cross-linked Th1 cells plus CRCL on days 3, 7 and 14. The percent survival of the mice is depicted in fig. 2B.

The results show that the combination of CD3/CD28 crosslinked Th1 cells plus CRCL significantly improved mouse survival compared to CRCL or CD3/CD28 crosslinked Th1 cell monotherapy in both settings.

Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.

Claims (47)

1. A pharmaceutical composition comprising an adjuvant and one or more antigens, wherein the adjuvant comprises living immune cells, at least a portion of which are activated T cells, and administration of the composition to a host produces a Th-1 response.

2. The composition of claim 1, wherein said T cells are memory T cells of Th-1 phenotype.

3. The composition of claim 1, wherein the T cells are activated by cross-linking CD3 and CD28 surface molecules on the T cells.

4. The composition of claim 1, wherein the activated T cells express CD 40L.

5. The composition of claim 1, wherein the T cell is heterologous to the host.

6. The composition of claim 1, wherein at least two antigens are combined with the adjuvant.

7. The composition of claim 1, wherein the one or more antigens are from an antigen source selected from the group consisting of whole cells, organisms, lysates of whole cells or organisms, naked DNA or RNA, nuclear material, peptides or proteins, antibodies, antigen pulsed or transfected dendritic cells.

8. The composition of claim 1, wherein the one or more antigens comprise one or more tumor associated antigens.

9. The composition of claim 1, wherein the one or more antigens comprise one or more heat shock proteins.

10. The composition of claim 1, wherein the source of the one or more antigens is a malignant tumor.

11. The composition of claim 1, wherein the source of the one or more antigens is from a pathogen.

12. An adjuvant composition comprising living immune cells, wherein at least a portion of said immune cells are activated T cells and wherein administration of the adjuvant composition to a host elicits a Th-1 immune response.

13. The composition of claim 12, wherein the T cells are activated by cross-linking CD3 and CD28 surface molecules on the T cells.

14. The composition of claim 12, wherein the T cells express CD 40L.

15. A method of preparing a pharmaceutical composition comprising:

preparing an adjuvant comprising viable immune cells, wherein the immune cells comprise at least a portion of T cells; and

combining one or more antigens with the adjuvant, wherein the pharmaceutical composition stimulates an immune response in the host when administered to the host.

16. The method of claim 15, wherein the adjuvant and the one or more antigens are combined prior to administration to the host.

17. The method of claim 15, wherein the immune response generated is a Th1 response.

18. The method of claim 15, wherein said T cell is a memory T cell of Th-1 phenotype.

19. The method of claim 15, wherein the T cell is an activated T cell.

20. The method of claim 15, wherein the T cells are activated by cross-linking CD3 and CD28 surface molecules on the T cells.

21. The method of claim 15, wherein the activated T cells express CD 40L.

22. The method of claim 15, wherein the T cell is heterologous to the host.

23. The method of claim 15, wherein at least two antigens are combined with an adjuvant.

24. The method of claim 15, wherein the one or more antigens are selected from one or more antigen sources.

25. The method of claim 15, wherein the antigen source is selected from the group consisting of whole cells, organisms, lysates of whole cells or organisms, naked DNA or RNA, nuclear material, antibodies, antigen pulsed or transfected dendritic cells.

26. The method of claim 15, wherein the one or more antigens are tumor associated antigens.

27. The method of claim 15, wherein the one or more antigens are heat shock proteins.

28. The method of claim 15, wherein the one or more antigens are from a malignant tumor.

29. The method of claim 15, wherein the one or more antigens are from a pathogenic agent.

30. A method of reducing an antigen associated with or causing a disease in a host, comprising:

administering a pharmaceutical composition comprising an adjuvant comprising viable immune cells and one or more antigens, wherein the immune cells comprise at least a portion of T cells, and wherein the pharmaceutical composition stimulates an immune response in a host when administered to the host.

31. The method of claim 30, wherein the T cell is an activated T cell.

32. The method of claim 30, wherein the activated T cells express CD 40L.

33. The method of claim 30, wherein the T cell is heterologous to the host.

34. The method of claim 30, wherein the pharmaceutical composition comprises at least two antigens.

35. The method of claim 30, wherein the one or more antigens are tumor associated antigens.

36. The method of claim 30, wherein the one or more antigens are heat shock proteins.

37. The method of claim 30, wherein the one or more antigens are from a malignant tumor.

38. The method of claim 30, wherein the one or more antigens are from a pathogenic antigen.

39. The method of claim 30, further comprising administering a boosting composition.

40. The method of claim 39, wherein the boosting composition comprises living immune cells, at least a portion of which are activated T cells.

41. The method of claim 39, wherein the boosting composition comprises viable immune cells and one or more antigens, wherein at least a portion of the immune cells are activated T cells.

42. The method of claim 30, wherein the adjuvant and the one or more antigens are combined prior to administration to the host.

43. The method of claim 30, wherein the adjuvant and the one or more antigens are administered to the host sequentially.

44. A method of treating a disease in a patient comprising administering a pharmaceutical composition comprising an adjuvant comprising viable immune cells and one or more antigens, wherein the immune cells comprise at least a portion of T cells, and wherein the pharmaceutical composition stimulates an immune response in the host when administered to the patient.

45. The method of claim 44, wherein said T cell is an activated T cell.

46. The method of claim 44, wherein the disease is cancer.

47. The method of claim 44, wherein said disease is caused by an infectious agent.