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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
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  • C12N2501/50—Cell markers; Cell surface determinants
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  • C12N2501/52—CD40, CD40-ligand (CD154)

Definitions

• the present invention relates to the field of vaccines, and more particularly to adjuvanted vaccine compositions.
• Vaccinations are designed to activate the immune system to specifically recognize and protect against invading pathogens.
• active immunotherapy approaches have been used to prevent numerous infectious diseases, including small pox, rabies, typhoid, cholera, plague, measles, varicella, mumps, poliomyelitis, hepatitis B and the tetanus and diphtheria toxins.
• Active immunotherapy concepts are now being applied to develop therapeutic cancer vaccines with the intention of treating existing tumors or preventing tumor recurrence, as well as being applied to the treatment and prevention of chronic viral infections.
• existing active immunotherapy technology has not been successful in protecting against many of the modern vaccine targets such as HIV/AIDS, Hepatitis B and cancer. This is in part due to the inability of current vaccination technology to elicit the correct type of immune responses.
• Th T helper
• ThI immune activation is optimized for intracellular infections such as viruses and involves the activation of Natural Killer (NK) cells and Cytolytic T-cells (CTL) that can lyse infected cells
• Th2 immune responses are optimized for humoral (antibody) responses.
• ThI immune activation is the most highly desired for cancer therapy and Th2 immune responses are directed more at the secretion of specific antibodies and are relatively less important for tumor therapy.
• Prior art vaccine compositions are specialized in eliciting Th2 or humoral immune responses, which are not effective against cancers and most viral diseases.
• adjuvants are a strategy for influencing the immune response to antigens in a vaccine composition.
• Aluminum salts, and squalene oil in water emulsion (MF59) are the most widely used adjuvants in human vaccines. These adjuvants predominantly promote Th2 humoral (antibody) responses to antigens and are effective at elevating serum antibody titers, but do not elicit significant ThI responses or CTLs.
• vaccines against chronic infections e.g., human immunodeficiency virus (HIV), hepatitis C virus (HCV), tuberculosis, herpes simplex virus (HSV)
• HCV herpes simplex virus
• cancer cells require generation of ThI cellular immune responses for protection and therapeutic effect.
• ThI responses Some experimental active immunotherapy techniques and protocols in the prior art have proven to successfully elicit ThI responses against tumor antigens in select patients, resulting in increased frequencies of CTL immune cells in circulation that have the ability to specifically kill tumors or pathogen infected cells. However, despite the ability to elicit ThI responses tumor escape mechanisms can overpower this immune response resulting in eventual tumor progression. Viruses have also developed a number of countermeasures to avoid immune attack and stay moving targets for the immune system. BRIEF DESCRIPTION OF THE DRAWINGS
• Fig. Ia is a graph of the survival of the mice after administration of the stated compositions.
• Fig. Ib is a graph of the survival of the mice after administration of the stated compositions.
• Fig. 2a is a graph of the survival of the mice after administration of the indicated composition.
• Fig. 2b is a graph of the survival of the mice after administration of the indicated composition.
• the present invention includes a pharmaceutical composition.
• the composition comprises an adjuvant and one or more antigens, wherein the adjuvant comprises living immune cells where at least a portion are activated T-cells.
• Administration of the composition to a host generates a Th-I response.
• the present invention includes an adjuvant composition comprising living immune cells wherein at least a portion of the immune cells are activated T-cells.
• the administration of the adjuvant composition to a host elicits a Th-I immune response.
• the present invention includes a method of making a pharmaceutical composition. The method includes preparing an adjuvant comprising living 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, upon administration to a host, stimulates an immune response in the host.
• the present invention includes a method of reducing antigens related to or causing a disease in a host.
• the method includes administering a pharmaceutical composition comprising an adjuvant and one or -A-
• the adjuvant includes living immune cells wherein the immune cells comprise at least a portion of T-cells, and wherein the pharmaceutical composition, upon administration to the host, stimulates an immune response in the host.
• the present invention includes a method of treating a disease in a patient. The method includes administering a pharmaceutical composition comprising an adjuvant and one or more antigens.
• the adjuvant includes living immune cells wherein the immune cells comprise at least a portion of T-cells, and wherein the pharmaceutical composition, upon administration to the patient, stimulates an immune response in the host.
• the present invention provides pharmaceutical vaccine compositions and methods for active immunotherapy that are capable of eliciting protective and therapeutic ThI immunity in patients against diseases while also providing the means to overcome the immunoavoidance mechanisms of the disease pathogens and tumors.
• the pharmaceutical composition of the present invention generally includes: (1) one or more sources of antigen; and (2) living, activated immune cells whereby at least a portion are T-cells .
• a new vaccine adjuvant for patients is described.
• the adjuvant can be mixed together with one or more vaccine antigens to form a pharmaceutical composition.
• the adjuvant can be used alone as an immunostimulant.
• the novel adjuvant comprises living immune cells, where at least a portion are T-cells.
• the T-cells are preferably memory T- cells (CD45RO+, CD62L Lo ) of the ThI phenotype (CD4+ T-cells that produce IFN- ⁇ and not IL-4).
• the memory ThI cells are activated at the time of formulation and introduction to a patient.
• the preferred activation method is by the cross-linking of CD3 and CD28 surface molecules on the T-cells.
• the activated memory T-cells preferably express CD40L upon being activated and produce large amounts of inflammatory cytokines (such as IFN- ⁇ , GM-CSF, and TNF- ⁇ ). These activated ThI memory cells are preferably allogeneic to the patient.
• a pharmaceutical vaccine composition generally contains at least one antigen mixed together with an adjuvant.
• Adjuvants as referred to herein, are compounds that can increase the intrinsic immunogenicity of an antigen.
• the term "adjuvant” is also often used as a synonym for "immuno stimulant”.
• Adjuvants for new vaccine targets such as cancer and infectious diseases are required not only to increase the immunogenicity to vaccine antigens, but are also often required for the deviation of an existing immune response against the vaccine antigen from Th2 to ThI. Additionally, efficacy of the vaccine often requires amplification of this deviated immune response.
• the vaccine adjuvant composition of this invention provides these immunomodulatory and immunopotentiation properties.
• adjuvants are known for promoting ThI immunity to an antigen including: saponins, BCG, liposomes and microparticles, poly LC, anti-CD40 mAbs, co-stimulatory molecules, IC31, TLR9 ligands, KLH, CpG, ⁇ - galactosylceramide, TLR4 agonists, cholera toxin, cytokines, chemokines, immune- stimulating complexes (ISCOMs), LPS, molecular agonists (e.g., agonists for NAIP, CIITA, HET-E, TP-I -leucine -rich repeat pathway receptors), TNF receptor superfamily (TNFRSF) agonists, alarmins and blockers of immunosuppressive cytokines and Tregs.
• saponins e.g., BCG, liposomes and microparticles, poly LC, anti-CD40 mAbs, co-stimulatory molecules, IC31,
• the present invention relates to the discovery that activated immune cells, preferably, allogeneic ThI memory cells activated by cross-linking CD3 and CD28 antigens that produce inflammatory cytokines and express CD40L, can elicit all the components in the immune cascade necessary to act as a potent immunomodulator and immunostimulator.
• activated immune cells can be capable of interfering with suppressive regulatory mechanisms in order to overcome the ability of pathological organisms and cancers to evade immune attack. This makes these cells an ideal adjuvant.
• the pharmaceutical composition of one or more vaccine antigens with activated T-cells cells may be used for prophylactic purposes or therapeutic purposes, or else both.
• the composition may be administered via all the routes conventionally used or recommended for vaccines: including the parenteral, intradermal, intramuscular, subcutaneous or mucosal routes.
• the composition may also be administered intranodally or intratumorally.
• the antigen 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 antigen source or different antigen sources. Any antigen source can be used in the formulation, for example these antigens can be sourced from living cells or organisms, the source material can be freeze/thaw lysates, irradiation inactivated (or other inactivation method), used as whole cells or organisms or lysates therefrom. In some preferred embodiments, tumor cells or tumor cell lysates can serve as the cell source material for the antigens.
• the cell source material can be derived from autologous or allogeneic cell sources or from cell lines.
• Antigens can also be sourced from naked DNA or RNA, which encode for antigens.
• the nuclear material can be used alone or incorporated with viral vectors.
• Another example of antigen source is anti- idiotypic antibodies or portions thereof that mimic antigens, or other methods to mimic the structure of an antigen.
• Antigen-pulsed or transfected dendritic cells (DC) can also be an antigen source in the pharmaceutical composition.
• the DC can be pulsed with peptides or whole proteins, recombinant proteins, or mRNA or DNA encoding for antigen(s), or the DC can be fused with cells containing the antigens, or the DC can be transfected with viral vectors such as retrovirus, lentivirus, adenovirus which contain the antigen, or these antigen source components can be used alone without the DC.
• viral vectors such as retrovirus, lentivirus, adenovirus which contain the antigen, or these antigen source components can be used alone without the DC.
• TAA tumor associated antigens
• examples of TAA include: MART-I, gplOO, tyrosinase, Melan A, TRP-I, tumor-specific mutated gene products, such as CDK- 4, ⁇ -catenin, MUM-I, oncogenes such as p53, and ras (K-and H-ras), cancer testes antigens, such as MAGE, GAGE and NY-ESOl, over-expressed self antigens such as MUCl, cyclin Bl, Her2-neu, CEA, WT, p53, SART-I, PRAME, pl5, and viral antigens such as HPV E7, EBV-derived antigens and telomerase.
• the antigenic component can include one or more chaperone proteins (also known as heat shock proteins) isolated from dead infected tissue or tumors.
• Heat shock proteins HSP
• HSP Heat shock proteins
• Tumor derived heat shock protein (hsp)-peptide complexes have been demonstrated to serve as effective vaccines, producing antitumor immune responses in animals and in man. This approach utilizes the peptide binding properties of stress proteins which are responsible for their functions as molecular chaperones in numerous cellular processes.
• Certain chaperones in extracellular milieu can also be capable of modulating innate and adaptive immunity due to their ability to chaperone polypeptides and to interact with the host's immune system, particularly professional antigen-presenting cells.
• Vaccination with HSP from tumors can elicit an anti-tumor response, and down-regulate immune suppression mechanisms.
• the immunogenicity of HSPs can be derived from the antigenic peptides with which they associate.
• the HSP can also be pulsed with antigens, including peptides, whole cells or cell lysates.
• tumor-derived Chaperone Rich Cell Ly sate is used as an antigen source and is obtained by the enrichment of the major chaperone proteins from tumor lysate using a free solution-isolectric focusing (FS- IEF) technique as described in the Examples below.
• FS- IEF free solution-isolectric focusing
• This technique is a rapid and efficient procedure for obtaining up to 5 to 20 times more antigenic material and in less time compared to conventional techniques.
• the FS-IEF method of multiple chaperone complex enrichment can be desirable from a clinical standpoint in terms of high yield from a potentially limited tumor source, and with a rapid turn- around time from tumor harvest to treatment of the patient.
• CRCL-associated peptides As a source of tumor antigen.
• they do not require the identification of tumor specific peptides.
• they elicit polyclonal T lymphocyte responses following immunization, preventing the outgrowth of immunological escape variants.
• CRCL vaccines have demonstrated a clear anti-tumor effect.
• the antigens conventionally used in vaccines can also be used in the pharmaceutical composition of the present invention, including whole microorganisms or part(s) of the microorganisms such as live attenuated whole microorganisms, inactivated microorganisms, recombinant peptides and proteins, glycoproteins, glycolipids, lipopeptides, synthetic peptides, or ruptured microorganisms, polysaccharides, used alone or conjugated to carrier elements, such as carrier proteins, can also be used.
• whole microorganisms or part(s) of the microorganisms such as live attenuated whole microorganisms, inactivated microorganisms, recombinant peptides and proteins, glycoproteins, glycolipids, lipopeptides, synthetic peptides, or ruptured microorganisms, polysaccharides, used alone or conjugated to carrier elements, such as carrier proteins, can also be used.
• any antigen or combination of antigens that are capable of being used for the treatment or prevention of diseases can be used in the pharmaceutical composition.
• Antigens derived from infectious pathogens can also serve as antigen sources and may be referred to herein as disease causing antigens.
• diseases from which antigens can be sourced are: diphtheria, tetanus, polio, rabies, whooping cough, hepatitis A, B and C, EBV, CMV, herpes 1 and 2, yellow fever, typhoid fever, chicken pox, variola (small pox), measles, mumps, German measles, Japanese encephalitis, meningitis, influenza, pneumococcal infections, rotavirus infections, AIDS (HIVl and 2), cancers, HTLVl and 2, syphilis, HPV, tuberculosis, Lyme disease, RSV infections, Trypanosomiasis, Dengue fever, malaria, anthrax, ebola virus, tularemia, Yersinia, West Nile virus, bacterial ailments caused by Chlamydia, Neisseria gonorrheae, Streptococcus pneumoniae, Moraxella catarrhalis, Staphyloc
• the activated ThI memory cells used in the pharmaceutical compositions of the present invention are preferably derived from normal donor blood. Preferred methods for processing and production of cells suitable for use in the present invention are described by Har-Noy in U.S. patents no. 7,435,592 and 7,402,431 and pending US patent no. 20050191291 which are incorporated herein by reference in their entirety.
• the pharmaceutical composition according to the present invention may be a composition intended for immunization 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 for immunization against several different pathogens or cancers, referred to herein as a vaccine combination.
• the present invention also includes methods of making pharmaceutical compositions. The method includes preparing an adjuvant that includes T-cells, preferably activated T-cells described herein. One or more antigens can be combined with the adjuvant to form the pharmaceutical composition. If more than one antigen is included in the composition, the antigens may be from the same antigenic source or different antigenic sources. Administration of the pharmaceutical composition can stimulate an immune response, preferably a Th-I response in the host.
• the adjuvant action of the activated T-cells can be obtained either when they are combined with the antigen(s) of the pharmaceutical composition prior to being administered, i.e. when it is present directly in the pharmaceutical composition.
• adjuvant and the antigen(s) can be administered separately, in sequential steps.
• the adjuvant may first be administered to the host using any one of the techniques described above. After administration of the adjuvant, the host may be administered the antigen(s). Preferably, the adjuvant and the antigen(s) are combined to form one pharmaceutical composition prior to being administered to the host.
• the pharmaceutical compositions of the present invention are designed to generate adaptive ThI immunity to the antigens in the composition.
• the adjuvant immune cells alone can be administered intravenously at the same time or anytime after the vaccine composition is administered.
• booster injections may be administered.
• the booster injections can be made at least 3-7 days apart, and more preferably 7-14 days apart. Additional booster injections may be administered as needed on a monthly or yearly basis.
• ThI memory cells In order to maintain an inflammatory environment that is capable of disabling the ability of tumors and disease organisms to evade immune destruction, additional booster injections of activated ThI memory cells alone or formulated with antigen can be administered. Patients that have been previously vaccinated with a composition containing allogeneic ThI memory cells can develop anti- alloantigen immunity. Subsequent injections of allogeneic cells can activate the pool of anti-alloantigen cells that can release the inflammatory cytokines necessary for disabling immune avoidance mechanisms.
• mice Female BALB/c (H2 d ) mice were obtained from the National Cancer
• Th-I cells CD3/CD28 cross-linked ThI cells
• Spleen cells from Balb/c mice were harvested and treated with ammonium chloride-potassium (ACK) buffer for lysis of red blood cells. Approximately 70-100 million cells were isolated per spleen. CD4+ T-cells were then purified by positive selection (purity >98%) using CD4 immunomagnetic particles on an MS column (Miltenyi Biotec, Germany), approximately 8-12 million CD4 cells were isolated with a yield of 50-60%. ThI memory cells were generated by expansion with anti-CD3 and anti-CD28- coated paramagnetic beads (CD3/CD28 T-cell expander beads, Dynal/Invitrogen) at an initial bead:CD4 cell ratio of 3:1.
• ACK ammonium chloride-potassium
• the purified CD4 cells were incubated with 20 IU/mL recombinant mouse (rm)IL-2, 20 ng/mL rmIL-7, and 10 ng/mL rmIL-12 (Peprotech, New Jersey) and 10 ⁇ g/mL antimurine IL-4 mAb (Becton Dickenson) in RPMI 1640 media containing 10% FBS, penicillin- streptomycin-glutamine, nonessential amino acids (NEAA) (Biological Industries, Israel) and 3.3 mM N-acetyl-cysteine (NAC; Sigma) (complete media).
• rm recombinant mouse
• rmIL-7 20 IU/mL rmIL-7
• 10 ng/mL rmIL-12 Peprotech, New Jersey
• 10 ⁇ g/mL antimurine IL-4 mAb Becton Dickenson
• RPMI 1640 media containing 10% FBS, penicillin- streptomycin-glut
• CD4 cells Additional cytokine-containing complete media with rmIL-2 and rmlL- 7 was added to the CD4 cultures daily from days 3 to 6 to maintain the cell concentration between 0.5 and 1 x 10 6 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:cell ratio as the cells expanded. After 6 days in culture, the CD4 cells expanded approximately 80 to 100-fold and were harvested and debeaded by physical disruption and passage over a magnet. The phenotype of the harvested cells used in experiments were >95% CD4+, CD45RB 10 , CD62L 10 , CD44 hl and are thus referred to as "memory cells".
• ThI memory cells were cultured at a density of 2 x 10 6 cells/ml in cRPMI for 4-6 hours at 37 0 C in 5% CO 2 with CD3/CD28 mAb conjugated microparticles (T-cell expander, Dynal/Invitrogen) at a 2:1 bead:cell ratio. After 4h, the cells produced IFN- ⁇ and upregulated the expression of CD40L and FasL on the cell surface.
• Cross- linked ThI memory cells used in these experiments expressed FasL and CD40L on the cell surface and produced in excess of 2000ng/ml/10 6 cells/6h IFN- ⁇ and less than 20pg/ml IL-4 per 10 6 cells/6h. 12Bl Cell line
• the murine leukemia cell line 12Bl was obtained by retroviral transformation of BALB/c bone marrow cells with the human bcr-abl (b 3 a 2 ) fusion gene. These cells express the p210 bcr-abl protein. The cells were cultured at 37°C and in 5% CO 2 in RPMI medium (Gibco/BRL, Gaithersburg, MD) supplemented with 10% heat- inactivated fetal bovine serum (Gemini Bio- products, Woodland, CA). Cells were tested routinely and found to be free of Mycoplasma contamination. Generation of 12Bl tumors
• 12Bl cells were first washed 3 times in PBS (Gibco/BRL), then counted and adjusted to a concentration of 5 x 10 4 cells/mL.
• PBS Gibco/BRL
• Female BALB/c mice were injected with 0.1 mL (5 x 10 3 cells) subcutaneously in the right groin and were monitored for tumor development. Preparation of 12Bl Tumor Lysate
• Tumors from 12Bl bearing mice were homogenized in 10 mM Tris-Cl (pH IA)IlO mM NaCl, 0.1% detergents (equal parts Triton X-100, Triton X-114 and Igepal CA-600, Sigma, St. Louis, Mo.), including 2 ⁇ g/ml leupeptin, 0.1 mg/ml Perfabloc, 0.5 mM phenylmethylsulfonate and one complete protease inhibitor cocktail tablet (all from Roche Molecular Biochemicals, Indianapolis, Ind.) in a glass-teflon homogenizer at a ratio of 1 g tumor/5 ml buffer.
• the homogenate was centrifuged at 10,000g, 4 0 C for 30 min, and samples were taken that are referred to as "lysate.”
• the "lysate” (supernatant) was subsequently centrifuged at 100,000g, 4 0 C for 60 min.
• the "high speed” supernatant was dialyzed into sequentially lower concentrations of homogenization buffer, ending in water. Protein concentration was determined using colorimetric bicinchoninic acid assays (BCA Reagent, Pierce Endogen, Rockford, 111.), and the free solution-isoelectric focusing (FS-IEF) starting material was frozen in 25 mg aliquots.
• FS-IEF was performed with the following modifications: we have 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-
• Liver CRCL was prepared form the liver of the na ⁇ ve Balb/c mice using the procedures described above. Proteins were diluted to 25 ⁇ g/100 ⁇ l in sterile
• TuLy CRCL (12Bl tumor lysate/liver CRCL) 12Bl tumor Iy sate and Liver CRCL were mixed at a 1:1 ⁇ g ratio and the mixture was incubated at 4 0 C overnight. Proteins were diluted to 50 ⁇ g/100 ⁇ l in sterile PBS for immunization of mice.
• mice 80 mice per group, 10 groups were used. Mice were vaccinated intra-dermally (i.d.) in the footpad on day -14 and -7 before tumor cell inoculation.
• the groups were as follows:
• TuLy CRCL (12Bl tumor lysate/liver CRCL): 50 ⁇ g/100 ⁇ l per mouse i.d.
• Activated Th-I cells IxIO 5 cells in 100 ⁇ l of PBS per mouse i.d.
• Th-I cells + 12Bl lysate IxIO 5 cells + 25 ⁇ g lysate in 100 ⁇ l of PBS per mouse i.d.
• mice Inoculation of tumor cells and monitoring of tumor volume Mice from all 10 groups were inoculated s.c. on the right groin on day 0 with 5000 12Bl cells/mouse. Tumor volume was determined every 2 days. Mice were euthanized when tumor volume reaches 4000 mm 3 .
• ThI cells 12Bl CRCL, Liver CRCL, 12Bl -Ly sate/liver CRCL and CD3/CD28 cross-linked ThI cells + 12Bl-Lysate/liver CRCL groups.
• 50% of the mice were tumor-free in the combination CD3/CD28 cross-linked ThI cells + 12Bl CRCL group (best group), 25% in the CD3/CD28 cross-linked ThI cells + 12Bl lysate group, 12.5 % in the 12Bl lysate group and 12.5 % in the CD3/CD28 cross-linked ThI cells + liver CRCL group.
• the tumor-derived CRCL are used as a source of tumor- specific antigens and combined with activated CD4+Th- 1 cells as an adjuvant to treat established leukemia.
• the methods are as described above in Example 1. Animals (8 mice per group) received the indicated treatments. Prophylactic setting: Na ⁇ ve Balb/c mice were treated by footpad (intradermal) injection at days -14 and -7 with PBS (control), or 12Bl-derived CRCL (12Bl CRCL, 25 ⁇ g/mouse), or CD3/CD28 cross-linked ThI cells, or by 12Bl CRCL plus CD3/CD28 cross-linked ThI cells. On day 0 mice were inoculated with 12Bl leukemia cells (5,000 cells/mouse, s.c. injection in the left groin). Percent survival is shown in Fig. 2A. Therapeutic setting:

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