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Definitions

• the present invention relates to vaccine therapy for cancer patients. More specifically, the present invention relates to a vaccine immunotherapy, which immunizes cancer patients, having immune suppression, to both endogenous and exogenous tumor peptides or proteins.
• tumor immunization strategies have been developed. However, all of these strategies are complex and deviate significantly from the conventional immunization strategies used for infectious diseases (Weber J. Tumor, Medscape Anthology 3:2, 2000).
• One such tumor immunization strategy involves Theratope(R), a Sialyl TN polysaccharide mucin antigen conjugated with keyhole limpet hemocyanine and administered with Detox(R) mycobacterium adjuvant and low dose cyclophosphamide (Maclean G D, et al, J Immunother Emphasis Tumor Immunol 19(4):309-316, 1996).
• Prostate specific membrane antigen has been used in patients with metastatic prostate cancer with major clinical responses in a low percentage of patients (Sanda M G, et al, Urology 52:2, 1999; Murphy GP, et al, The prostate. 38:43-78, 1999). Additionally, autologous tumors have been used with low dose cyclophosphamide and BCG to immunize cancer patients with malignant melanoma. However, few clinical responses were reported (Mastrangelo M J, et al, Seminars in Oncology 23(6):773-781 , 1996). Another strategy attempted included using MAGE antigens with a variety of vaccine adjuvants.
• this invention relates to, but not exclusively to, immunizing with endogenous peptide processed and presented by dendritic cells or endogenously administered to an environment (lymph node) where dendritic cells have been prepared and can present them to T-cells effectively.
• This goal is considered by many immunologists to be insurmountable.
• Peptides are much too small to be effective immunogens, their one half life is short they are often non-mutated self antigens to which the patient is immunologically tolerant and gaining a response is tantamount to inducing auto immunity.
• cellular and/or tumoral immunity to tumor-associated antigens has been induced (Weber J.
• a method of immunotherapy to treat cancer by administering an effective amount of a natural cytokine mixture (NCM) including, but not limited to, IL-1 , IL-2, IL-6, IL-8, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF, G-CSF, recombinants thereof, and combinations thereof.
• NCM natural cytokine mixture
• the present invention provides a method of immunotherapy to treat cancer by administering an effective amount of cyclophosphamide (CY) and an effective amount of indomethacin (INDO).
• CY cyclophosphamide
• INDO indomethacin
• Various anti-cancer treatment methods are also provided wherein administration of an effective amount of CY occurs along with an effective amount of a nonsteroidal anti-inflammatory drug (NSAID) including, but not limited to, indomethacin (INDO), Ibuprofen, celecoxib (Celebrex®), rofecoxib (Vioxx®), Coxll inhibitors, and combinations thereof.
• NSAID nonsteroidal anti-inflammatory drug
• the present invention provides a method of immunotherapy to treat cancer by administering an effective amount of a CY in combination with an effective amount of INDO and an effective amount of IFN- ⁇ , IL-2, IL-1 , and TNF- ⁇ .
• the present invention provides a method of immunotherapy to treat cancer by administering an effective amount of a CY in combination with an effective amount of INDO and an effective amount of recombinant IL-2, recombinant IFN- ⁇ , recombinant TFN- ⁇ , and recombinant IL-1.
• the present invention further provides a synergistic anti-cancer treatment by administering an effective amount of CY and INDO in combination with an NCM described herein.
• the present invention provides an anti-metastatic treatment method by promoting differentiation and maturation of immature dendritic cells in a lymph node; allowing presentation by resulting mature dendritic cells of antigen to T- cells to gain immunization of the T-cells to the antigen; and preventing development of metastasis.
• the present invention provides an anti- metastatic method by unblocking immunization at a lymph node; and generating systemic immunity.
• the present invention also provides a skin test and a method of pre-treatment of dendritic cells (DC) by applying an effective amount of CY and INDO in combination with an NCM described herein.
• DC dendritic cells
• the present invention further provides a method of treating monocyte defects characterized by sinus histiocytosis-or a negative NCM skin test by applying an effective amount of CY and INDO in combination with an NCM described herein.
• the present invention provides compositions and methods for eliciting an immune response to endogenous or exogenous tumor antigens.
• Figure 1 is a bar graph showing lymph node size in controls, and cancer controls or IRX-2(NCM) treated populations with squamous cell head & neck cancer (H&NSCC);
• Figure 2 shows two bar graphs, one showing T-cell area and the second showing density in controls and head and neck squamous cancer controls and patients treated with NCM(IRX-2);
• Figure 3 shows two bar graphs showing B-cell area and follicles in the three treatment groups;
• Figure 4 shows a comparison of other cells and sinus histocytosis in the three treatment groups;
• Figure 5 is a graph showing node B&T and Cancer B&T fit plot;
• Figure 6 is a graph illustrating the survival percentage of treated patients at forty-eight months;
• Figure 7 is a graph illustrating the survival of complete and partial responders compared to minor and non-responders;
• Figure 8 is graph
• the present invention provides methods for treating patients utilizing vaccine immunotherapy wherein the patients are immune suppressed.
• immune suppressed it is meant that the patient has reduced cellular immunity and thus impaired capacity to respond to new antigens. More specifically, in blood, T lymphocyte counts are reduced and/or function of these cells is impaired, as shown, e.g. by PHA proliferation assay.
• adjuvant it is meant a composition with the ability to enhance the immune response to a particular antigen. To be effective, an adjuvant must be delivered at or near the site of antigen. Such ability is manifested by a significant increase in immune mediated protection.
• Enhancement of immunity is typically manifested by a significant increase (usually greater than 10 fold) in the titer of antibody raised to the antigen. Enhancement of cellular immunity can be measured by a positive skin test, cytotoxic T-cell assay, ELISPOT assay for ⁇ lFN or IL-2, or T-cell infiltration into the tumor (as described below).
• tumor associated antigen it is meant an analogous protein or peptide (which were previously shown to work by pulsing of dendritic cell ex vivo) or other equivalent antigen.
• the NCM can include recombinant cytokines. Briefly, NCM is prepared in the continuous presence of a 4-aminoquinolone antibiotic and with the continuous or pulsed presence of a mitogen, which in the preferred embodiment is PHA. T lymphocytopenia (low T cell levels in blood) is a diagnostic characteristic of cellular immune deficiency, while impaired function of existing thymphocytes is the other characteristic. There is no generally accepted (clinically approved) way to treat T lymphocytopenia. Bone marrow transplants (+- thymus transplants) have been used in cases of severe combined immunodeficiency (SCID- congenital, irradiation or chemotherapy induced).
• T cells have been tried in AIDS with some effect by much toxicity.
• CD45RO memory T cells
• These new cells are called recent "thymic emigres" and have the surface marker of "na ⁇ ve" T cells i.e., CD45RA.
• NCM therapy results in the production of these new T cells as well as expanding preexisting memory T cells.
• the present invention relates to immunization to provide an immune response to antigens, which is either endogenously or exogenously administered.
• antigens in the past may have been believed to be immunogenic while others used in the present invention may have been thought previously to be non-immunogenic.
• Any antigen can be used with the present invention. Examples of such antigens are EADPTGHSY (melanoma) from MAGE-1 protein, EVDPIGHLY (lung carcinoma) from MAGE-3, EVDPIGHLY (lung carcinoma) from MAGE-3, and many others. (See Bellone, et al, Immunology Today, Vol.
• the present invention is directed towards affecting antigen processing generally; therefore, any antigen can be used with the present invention.
• the present invention can extend to all forms of tumor antigens and haptens including peptides and/or carbohydrates.
• the present invention can extend to areas of applicability as in AIDS virus vaccine in HIV+ patients; other difficult to manage situations; renal transplants, aged individuals, and the like.
• the present invention utilizes several general derived method steps for obtaining immunization in subjects where such immunization was previously thought to be impossible. More specifically, the present invention provides a method for overcoming immune depression by inducing production of naive T cells.
• T cells are meant to mean newly produced T cells, even in adults, wherein these T cells have not yet been exposed to antigen.
• T cells at this stage are non-specific yet capable of becoming specific upon presentation by a mature dendritic cell having antigen, such as tumor peptides, exposed thereon.
• the present invention replenishes or generates new T cells. This is generally accomplished by administering a natural cytokine mixture (NCM).
• NCM includes, but is not limited to, IL1, IL2, IL6, IL8, IL10, IL12, ⁇ lFN, TNF ⁇ , G- and GM-CSF, recombinants thereof, and combinations thereof. The amount and proportions of these constituents are detailed below.
• the NCM is injected around lymphatics that drain into lymph nodes regional to a lesion, such as a tumor or other persistent lesions being treated.
• Peritumoral injection has been associated with little response, even progression and is thus contraindicated.
• a ten (10) day injection scheme is optimal and a twenty (20) day injection protocol, while effective clinically, tends to reduce the TH1 response and shift towards a less desirable TH2 response as measured by lymphoid infiltration into the cancer.
• Bilateral injections are also effective. Where radical neck dissection has occurred, contralaterial injection is effective. It is preferable to block endogenous suppression of T cells, such as caused by various cancer lesions.
• Blocking is effected by the co-delivery of low dose cyclophosphamide (CY) and a non-steroidal anti-inflammatory drug (NSAID).
• NSAID non-steroidal anti-inflammatory drug
• the NSAID of choice is indomethacin (INDO). While INDO is the most effective NSAID, it is also arguably the most toxic.
• Celebrex® and Vioxx®, Cox II NSAIDS are also less effective. Ibuprofen was effective, but the histological responses were characteristic of a TH2 rather than TH1 mediated response, this being less desirable.
• Side effects of NSAIDS are to be aggressively treated with proton inhibitors and a prostaglandin E analog. Zinc and multi-vitamins are useful agents to help restore T cell immunity. Treatment with contrasuppression and zinc without the NCM is ineffective.
• the minimum regimen is perilymphatic treatment with the
• NCM combined with contrasuppression using CY and an NSAID.
• the alternative regimen is the previously mentioned regimen further including zinc and vitamins, possibly including the addition of selenium.
• Preferable dosing of Zinc is 50 to 75 mg.
• a standard multivitamin can be administered.
• the zinc can be an available gluconate.
• Lymphocyte granulocyte or macrophage infiltration of a 90:10 ratio is optimal.
• T and/or B cell infiltration preferably is diffuse and intense and not peripheral. Light infiltration of less than 20% is not associated with a robust clinical response. Tumor reduction and fragmentation in the histological samples is preferred in reflecting a good response.
• Lymph node changes key to good response involve at least five (5) aspects. Lymph node enlargement and not just reversal of tumor induced reduction of size but overall increase in size compared to normal is preferred. Increased T and B cell areas indicate an immunization.
• Sinus histocytosis (SH) is believed to be the accumulation of immature dendritic cells, which have ingested and processed tumor antigens but are unable to mature and present these tumor peptides to naive T cells capable of stimulating TH1 and TH2 effective cells which lead to cytotoxin T cell and B cells. Reversal of SH is preferred.
• the present invention provides for unblocking immunization at a regional lymph node by promoting differentiation and maturation of immature dendritic cells in a regional lymph node and thus allowing presentation by resulting mature dendritic cells of small peptides, generally nine amino acids in length to T cells to gain immunization of the T cells. Additionally, induction of mature dendritic cells is required. Finally, mobilization of peripheral blood T- lymphocytes in T-lymphocytopoenic patients in the presence of induction of naive T cells capable of responding to dendritic cells presenting endogenous tumor peptides is desired. (See, Sprent, et al, Science, Vol 293, July 13, 2001 , pgs 245-248).
• the key mechanistic features of the present invention are the in vivo maturation of dendritic cells resulting in effective peptide antigen presentation.
• increases in CD45 RA positive na ⁇ ve uncommitted T cells have been found. With antigen, this leads to T and B cell clonal expansion, creating immunity in the patient.
• the resulting infiltration into tumors by hematogenous spread leads . to robust tumor destruction.
• the result, as found in the data below, is increased survival due to immunologic memory. (See, Sprent et al, cited above).
• the appearance of an immune T cell means, de facto, that a dendritic cell has been made or allowed to work properly. Also de facto, dendritic cell activation and maturation is to be considered a key factor in cancer immunodeficiency as well as the well-known defects in T cells such as a decreased number and function with anergy and presumed apoptosis.
• the invention utilizes the NCM to immunize patients, such as cancer patients, as well as patients with other lesions or antigen producing disease conditions.
• the present invention utilizes a method of enhancing the immune response of cancer patients to a cancer by administering an effective amount of a composition containing therein the NCM and a tumor-associated antigen, wherein the NCM acts as an adjuvant to produce the immune response.
• the tumor-associated antigen can be either an endogenously processed tumor peptide preparation resident in regional nodes of patients with cancer or in conjunction with an exogenously administered tumor antigen preparation in or near these nodes.
• Tumor peptides, as well as antigens are included herein even though peptides are not expected to be immunogenic where tumor associated protein antigens would more likely be more so since they are complete.
• the composition of the present invention involves the administration of the NCM plus a tumor associated or specific antigen, as defined below with low doses of CY, a cyclooxygenase inhibitor, and other similar compounds which have been shown to further increase the effects of the composition of the present invention.
• NCM that has been previously shown to be effective in promoting T cell development and function in aged, immunosuppressed mice.
• Upon administering this NCM to immunosuppressed patients with head and neck cancer it is demonstrated in this application for the first time that the mobilization of T lymphocytes in the blood of cancer patients treated with the NCM produces an increase in immature, na ⁇ ve T cells bearing both.
• CD2 and CD45 RA are examples of T lymphocytes in the blood of cancer patients treated with the NCM.
• the method of the present invention involves using an NCM with local perilymphatic injections or other injections that are known to those of skill in the art to provide sufficient localization of the immunotherapy compound.
• the injections take place in the neck, but can be applied in other locations as required by the disease to be treated.
• immature dendritic cells that have the morphology of large histocytes. These immature dendritic cells process antigen (endocytosis, phagocytosis and digestion) and evolve into mature dendritic cells, which display peptide fragments (generally nine amino acids) of the digested antigen in the MHC groove for presentation to T cells. T cells, in order to respond, must have antigen presented to them in the MHC groove plus various co-stimulatory signals. Investigators, such as Murphy et al, 1999, have utilized dendritic cells generated in culture and then pulsed with tumor antigens and have achieved a small degree of success in immunizing patients against prostate specific membrane antigen peptides.
• the cells present in the lymph node sinuses which accumulate in cancer, are cells of the lineage of dendritic cells and that following the in vivo treatment with the NCM protocol of the present invention, these cells disappear and the antigen ultimately becomes immunogenic for T cells. They are able then to respond to the tumor. So an aspect of this invention is being able to generate a microenvironment in the regional lymph node, which allows effective antigen processing and presentation.
• the immunization which derives results in T cells able to traffic to the lesion and destroy tumors, is de facto demonstration of adequate antigen processing by dendritic cells.
• the composition of the present invention involves the natural cytokine mixture plus either endogenous or exogenous tumor associated antigen. Additionally, low doses of CY, cyclooxygenase inhibitors, zinc, and other similar compounds have been shown to further increase the effects of the composition of the present invention. Immunization for treatment of patients with cellular immune deficiencies associated with cancer, HIV infection, aging, renal transplants and other such deficiencies can be achieved with the composition of the present invention.
• the present invention has numerous embodiments.
• the present invention provides a method of immunotherapy to treat cancer by administering an effective amount of an NCM including cytokines including, but not limited to, IL-1, IL-2, IL-6, IL-8, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF, G-CSF, recombinants thereof, and combinations thereof.
• the above method further includes administering 75 to 500 units IL-2 equivalence, wherein the administering preferably occurs bilaterally into lymphatics that drain into lymph nodes. Alternatively, the administering can occur unilaterally.
• the NCM is administered for at least one to ten days and up to about twenty days. In one preferred embodiment, administration occurs bilaterally and for about 10 days.
• the NCM can be administered prior to surgery or radiotherapy.
• the NCM can be administered during recurrence of tumors.
• an effective amount of CY can be administered.
• an effective amount of an NSAID can be administered wherein the NSAID can be, but is not limited to, INDO, Ibuprofen, celecoxib (Celebrex®), rofecoxib (Vioxx®), Coxll inhibitors, combinations thereof, and any other similar NSAID known to those of skill in the art.
• a method of immunotherapy to treat cancer by administering an effective amount of CY and an effective amount of INDO.
• Another embodiment of the present invention provides a synergistic anti-cancer treatment method by administering an effective amount of a CY and an effective amount of a NSAID, wherein the NSAID can be, but is not limited to, INDO, Ibuprofen, celecoxib (Celebrex®), rofecoxib (Vioxx®), Coxll inhibitors, combinations thereof, and the like.
• NSAID can be, but is not limited to, INDO, Ibuprofen, celecoxib (Celebrex®), rofecoxib (Vioxx®), Coxll inhibitors, combinations thereof, and the like.
• Another embodiment of the present invention provides a method of immunotherapy to treat cancer by administering an effective amount of CY in combination with an effective amount of INDO and an effective amount of IFN- ⁇ , IL-2, IL-1 , and TNF- ⁇ .
• a further embodiment is directed towards a method of immunotherapy to treat cancer by administering an effective amount of CY in combination with an effective amount of INDO and an effective amount of recombinant IL-2, recombinant IFN- ⁇ , recombinant TFN- ⁇ , and recombinant IL-1.
• a synergistic anti-cancer treatment is also provided by the present invention, wherein the treatment includes the steps of administering an effective amount of CY and INDO in combination with a NCM.
• the NCM can include, but is not limited to, IL-1 , IL-2, IL-6, IL-8, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF, G-CSF, recombinants thereof, combinations thereof, and any other similar cytokine known to those of skill in the art.
• An anti-metastatic treatment method is another embodiment of the present invention wherein the method includes the steps of promoting differentiation and maturation of immature dendritic cells in a lymph node, allowing presentation by resulting mature dendritic cells of antigen to T-cells to gain immunization of the T- cells to the antigen, and preventing development of metastasis.
• a further embodiment provides for an anti-metastatic method including the steps of unblocking immunization at a lymph node, and generating systemic immunity. This method further includes the step of preventing development of metastasis.
• Other embodiments of the present invention provide a method of using a natural cytokine mixture as a diagnostic skin test for predicting treatment outcome by administering an NCM intracutaneously and determining a response to the NCM within 24 hours, wherein a negative skin test indicates unresponsiveness to the NCM and predicts failure of patients to respond to surgery with or without radiotherapy.
• Another embodiment provides a method of pre-treatment of dendritic cells (DC) by applying an effective amount of CY and INDO in combination with an NCM including cytokines such as, but not limited to, IL-1 , IL-2, IL-6, IL-8, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF, G-CSF, recombinants thereof, combinations thereof, and any other similar cytokines known to those of skill in the art.
• the present invention provides a method of treating monocyte defects characterized by sinus histiocytosis or a negative NCM skin test by applying an effective amount of CY and INDO in combination with an NCM.
• the NCM includes, but is not limited to, IL-1 , IL-2, IL-6, IL-8, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF, G-CSF, recombinants thereof, combinations thereof, and any other similar cytokines known to those of skill in the art.
• the present invention provides various methods of eliciting an immune response to endogenous or exogenous tumor antigens by administering an effective amount of an NCM that includes various cytokines including, but not limited to, IL-1 , IL-2, IL-6, IL-8, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF, G-CSF, recombinants thereof, or combinations thereof.
• Another embodiment of the present invention provides administering the NCM described above and an effective amount of CY to elicit an immune response to endogenous or exogenous tumor antigens.
• the method of eliciting an immune response to endogenous or exogenous tumor antigens occurs by administering an effective amount of an NCM; an effective amount of CY; and an effective amount of INDO, wherein the NCM includes cytokines such as, but not limited to, IL-1 , IL-2, IL-6, IL-8, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF, G-CSF, recombinants thereof, and combinations thereof.
• the present invention also provides a composition for eliciting an immune response to endogenous or exogenous tumor antigens.
• the composition includes an effective amount of NCM, wherein the NCM includes cytokines such as, but not limited to, IL-1 , IL-2, IL-6, IL-8, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF, G-CSF, recombinants thereof, and combinations thereof.
• the composition includes an effective amount of CY.
• the composition further includes an effective amount of INDO. For any of the above embodiments, the following administration details and/or protocols for treatment are used.
• the compounds of the present invention (including NCM), and exogenous antigens are administered and dosed to achieve optimal immunization, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, ,body weight.
• the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve immunization including but not limited to improved tumor reduction, fragmentation and infiltration, survival rate or more rapid recovery, or improvement or elimination of symptoms.
• the compounds of the present invention can be administered in various ways.
• the compounds can be administered intra or subcutaneously, or peri or intralymphatically, intranodally or intrasplenically or intramuscularly, intraperitoneally, and intrathorasically. Implants of the compounds can also be useful.
• the patient being treated is a warm-blooded animal and, in particular, mammals including man.
• the pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non- toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.
• the doses can be single doses or multiple doses over a period of several days.
• When administering the compound of the present invention it is generally formulated in a unit dosage injectable form (solution, suspension, emulsion).
• the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
• the carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
• Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, can also be used as solvent systems for compound compositions.
• various additives which enhance the stability, sterility, and isotonicity of the compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
• antimicrobial preservatives for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
• isotonic agents for example, sugars, sodium chloride, and the like.
• Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
• any vehicle, diluent, or additive used would have to be compatible with the compounds.
• Peptides can be polymerized or conjugated to carriers such as human serum albumen as is well known in the art.
• Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.
• a pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres.
• suitable delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres. Examples of delivery systems useful in the present invention include: U.S. Pat. Nos.
• Papilloma virus E6 & E7 obtained commercially Cervical SCC
• the commercially route of antigen administration is preferentially the neck because it is accessible and it contains >30% of the bodies lymph nodes and systemic immunity can be envisioned to result.
• Low Dose CY has been used to augment cellular immunity and decrease suppression by lymphocytes in mice and patients with cancer (Berd D., Progress in Clin Biol Res 288:449-458, 1989; Berd D, et al, Cancer Research 47:3317- 3321 , 1987) and it has been employed in effective immunotherapy of cancer patients (Weber J., Medscape Anthology 3:2, 2000; Murphy GP, Tjoa B A, Simmons S J. The prostate. 38:43-78, 1999; Hadden JW, et al, Arch Otolaryngol Head Neck Surg. 120:395-403,1994).
• Zinc deficiency is associated with improved cellular immunity and treatment with zinc is immunorestorative in mice (Hadden JW., Int'l J Immunopharmacol 17:696-701 , 1995; Saha A., et al. Int'l J Immunopharmacol 17:729-734,1995).
• a cvclooxygenase inhibitor such as INDO: Cancers produce prostaglandins and induce host macrophage production of prostaglandins (Hadden JW. The immunopharmacology ,of head and neck cancer: An update. Int'l J Immunopharmacol 11/12:629-644,1997). Since prostaglandins are known to be immunosuppressive for T cells, inhibition of PG synthesis with cyclooxygenase inhibitors is appropriate.
• Dose and Frequency of Antigens 1-1000 ⁇ g, preferably 10-500; form-soluble (partially polymerized or conjugated to carrier, if necessary). Dosage administration occurs on Day 1 , Day 12, and Day 21. Pre-Rx occurs on Day 12, Day 21 , and Day 31. Site of injections is local (i.e., neck injections).
• Oligopeptides such as PSMA, MAGE fragments, E6, E7 peptides would be poorly immunogenic even pulsing on to dendritic cells. Thus effective immunization would not be expected to occur. Even with effective immunization, tumor regression would be considered surprising by this method, particularly at a distance as with prostate and cervix. Regression of metastastic disease is always a surprising event with immunotherapy. Degree and frequency of clinical responses are a factor in the effectiveness and thus the novelty of this approach. Diagnostic skin tests are another way towards obtaining more effective immunization. Patients can be pretreated with the NCM of the present invention to induce better responses (increase NCM and PHA skin tests and lymphocyte counts and reversal of lymph node abnormalities).
• MATERIALS AND METHODS All steps relating to cell culture are performed under sterile conditions. General methods of cellular immunology not described herein are performed as described in general references for Cellular immunology techniques such as Mishell and Shiigi (Selected Methods in Cellular Immunology, 1981) and are well known to those of skill in the art.
• NCM Natural Cytokine Mixture
• the lymphocytes are washed and distributed in X vivo-10 media (Whittaker Bioproducts) to surface activated cell culture flasks for selection of cell subsets MICROCELLECTORTM T-25 Cell Culture Flasks) in which are immobilized stimulants, i.e. mitogens like PHA.
• immobilized stimulants i.e. mitogens like PHA.
• X vivo-15 and X vivo-20 media were used as indicated.
• the immobilization process for the stimulants is as described by the manufacturer for immobilizing various substances for panning procedures, i.e. separating cells, in the flasks.
• the lymphocytes are exposed to stimulants e.g. PHA for 2-4 hours then washed three times.
• the cells are incubated for 24-48 hours in X vivo-10 media with 80 ⁇ g/ml ciprofloxacin (Miles Lab) at 370 in a CO 2 /air incubator.
• X vivo-10 media 80 ⁇ g/ml ciprofloxacin (Miles Lab) at 370 in a CO 2 /air incubator.
• RPMI 1640 media could be used (Webb et al. 1973).
• HSA Human serum albumin
• HSA Human serum albumin
• Stimulation of lymphocytes The objective was to find a way to stimulate lymphocytes to produce high levels of interleukin-2 in the absence of serum and in a way that did not yield significant quantities of PHA in the supernatant. To do this, the PHA was immobilized on surface activated cell culture flasks for selection of cell subsets (AIS MICROCELLECTORTM T-25 plates) as described in the manufacturer's instructions for "panning" cell separation or pulsed into the cells followed by washing (pulse technique). Media employed in these experiments was X vivo-10 (Whittaker) and is approved for administration to humans by the U.S. Food and Drug Administration for interleukin-2 lymphokine activated killer (LAK) cell protocols.
• LAK interleukin-2 lymphokine activated killer
• Serum-free media capable of supporting human lymphocyte proliferation like minimal essential media (MEM) or RPMI-1640 (Sigma) could also be used.
• PHA HA-16, Murex Diagnostics Ltd., Dartford, UK
• PHA concentration 25 or 50 ⁇ g/ml a high yield of IL-2 in the serum-free supernatant could be obtained.
• Additional detailed information is set forth in International Publication Number WO 03/035004 A2 to Hadden and International Publication Number WO 02/34119 A2 to Hadden, which are incorporated herein by reference in their entirety.
• NCM NCM Preparation of NCM
• pooled human peripheral blood buffy coats obtained from INCAN Blood Bank were incubated with phytohemagglutinin (Murex, Dartford UK) and washed. The cells were incubated in serum-free medium (x-vivo 10, BioWhitaker) for twenty four hours. Batches were prepared from six blood donors and were screened by the INCAN Blood Bank for hepatitis B and C viruses, HTLV 1 and 2, and HIV. Following twenty-four hours of culture, the cultures were centrifuged, the supernatant was filtered through 0.2-micron filters, and the natural cytokine mixture was placed into vials.
• GRAPHPADTM computer software statistics package
• Treatment Regimen Each treatment cycle lasted twenty-one days. Each cycle was initiated with an intravenous infusion of cyclophosphamide (CY) at 300 mg/m 2 and indomethacin (INDO) at 25 mg orally three times a day, and zinc (65 mg elemental zinc as the sulfate) orally once a day. Patients also underwent skin- testing with NCM at 0.1 mL administered intra-dermally to rule out allergic response to components and to show delayed type hypersensitivity (DTH) responses to NCM on day 4. Patients were treated with the NCM at 1.0 mL for ten days or 2.0 mL for twenty days administered intramuscularly at the insertion of the stemocleidomastoid muscle 2.0 cm below the mastoid tip.
• CY cyclophosphamide
• INDO indomethacin
• DTH delayed type hypersensitivity
• a representative biopsy section containing a tumor was selected under low power and the amount of tumor was expressed as a percent of the total area. The remaining stroma was evaluated for the percent of the area having lymphocytes. Tumors were further evaluated for the percent of the specimen area that was solid and the percent fragmented with interspersed leukocyte as infiltration as described. Table II: Patient Characteristics of study in head & neck cancer
• EXAMPLE 1 Local perilymphatic injections in the neck having NCM plus low dose CY, INDO, and zinc have induced clinical regressions in a high percentage of patients with squamous cell head and neck cancer (H&NSCC) (Hadden J W, et al., Arch Otolaryngol Head Neck Surg. 120:395-403, 1994; Meneses A, et al., Arch Pathol Lab Med 122:447-454, 1998; Barrera J, et al., Arch Otolaryngol Head Neck Surg 126:345-351 , 2000; Hadden, et al., 2003; Menesis, et al., 2003) with evidence of improved, recurrence-free survival.
• H&NSCC squamous cell head and neck cancer
• lymph nodes of treated patients were all stimulated and were larger than control nodes with increased T cell area and density. These nodes were thus not only restored to normal but showed evidence of T cell predominance, a known positive correlate with survival in H&NSCC (Hadden JW. Int'l J Immunopharmacol 11/12:629- 644,1997).
• lymph node changes related to B and T cell areas were correlated with the changes in their tumors reflecting T and B cell infiltration, a high degree of correlation was obtained for T cells (p. ⁇ 0.01) and B cells ( ⁇ 0.01) and overall lymphoid presence (p. ⁇ 0.001)(Figure 5). In turn, these changes correlate with tumor reduction by pathological and clinical criteria.
• EXAMPLE 2 Further analysis of the clinical, pathological and survival data of the aforementioned INCAN study offer more insights into the nature of the invention as it relates to immunization of cancer patients to their own autologous tumor antigens and the resulting immune regression of their tumors.
• Figure 6 shows that the treatment with the NCM protocol (IRX-2) is associated with increased survival at 48 months (p ⁇ 0.01).
• Figure 7 shows that clinical responses determine survival in that patients with complete (CR) and partial responses (PR)(> 50% tumor reduction) have better survival than those with minor responses (MR) ( ⁇ 50%, but >25% tumor reduction) or no response (NR)( ⁇ 25%)(p ⁇ 0.01).
• Figure 8 shows that patients with stronger pathological responses (index of 6-9) have better than those with weaker pathological responses ( ⁇ 6) (p ⁇ 0.02).
• Figure 9 shows that lymphoid infiltration into the tumor as a single variable predicts survival (p ⁇ 0.01).
• Chi Square analysis of the relationship of clinical response to the pathological response shows a highly significant relationship (p ⁇ 0.01) indicating that the two are coordinately related to each other as well as to survival and thus providing a statistical triangulation of the data interrelating clinical responses, immune regression parameters, and survival. Such relationships have never been shown for immunotherapy of a human cancer.
• EXAMPLE 3 Two patients were treated with lymphoma of the head and neck. The patients included were those with head and neck cancer who agreed to participate in the protocol. The following scheme was followed. Before treatment, the patients were skin-tested with NCM 0.1 ml subcutaneously in the forearm, the region was marked, and 24 hours later the test was read. The test was considered positive if the induction and erythema was equal or larger than 3mm.
• Case 1 The patient was a 23-year-old male who presented on with a prior history of three months of the presence of a tumor on the left submaxillary region, with no other symptoms. In the emergency room, he was found to have lymph adenopathy of the left submaxillary triangle of approximately 6.5 cm in diameter of a hard consistency, partially fixed at deep levels. The rest of the physical exam was normal. The incisional biopsy showed Hodgkin's lymphoma. The lesion was staged ECIIA. A one-cycle treatment of NCM was given, obtaining a minor response, as the adenopathy reduced in size by 1 cm in diameter.
• the biopsy report obtained after NCM treatment showed 60% of the lesion showed normal lymphocytic infiltration, and the rest of the neoplasia (40%) showed necrosis. No viable tumor cells were found. Following this, the patient received radiation treatment in the neck of 3600 rads. The patient is currently free of disease.
• Case 2 The patient is an 82-year-old male, who presented with a two-month history of a painful mid-neck tumor mass, as well as a 10 kg loss of weight. On physical exam, the patient presented with tumor on the right palatine tonsil, which was enlarged to approximately 4 X 3 cm, with an ulcer in the center of the tonsil.
• a right submaxillary lymph node measured approximately 2 X 2 cm and a lymph node mass at level II and III of approximately 5 X 5 cm.
• the rest of the exam was normal.
• the incisional biopsy of the tonsil and one of the neck's lymph nodes demonstrated defined non-Hodgkin's lymphoma mixed, of intermediate grade.
• the patient was subjected to two cycles of NCM at the end of which a 1 cm reduction in the diameter of the tonsil and neck adenopathy was observed.
• the pathological report post-NCM treatment showed live tumor 20%, fragmented and necrotic 30% and normal lymphocyte infiltration 50%.
• the patient was given chemotherapy (CHOP) for 6 cycles and later external radiotherapy (RT) at a total dose of 4600 rads. He recurred at eight months post RT with adenomegaly at the occipital level. The patient died three months later with evidence of neck disease.
• IB1 , IB2 and IIA were treated with local, perilymphatic injections NCM (10 daily injections) followed by radical hysterectomy at day 21.
• NCM 10 daily injections
• a single IV dose of CY at 300 mg/m.
• oral INDO or ibuprofen and zinc sulfate were administered from days 1 to 21.
• the clinical and pathological response, toxicity and disease-free survival were evaluated. All patients completed NCM treatment and were evaluated for response and toxicity.
• Clinical response was seen in 50% of patients (3 partial response (PR), 2 minor response (MR) (>25% ⁇ 50%reduction)). Seven patients underwent surgery. Pathologically tumor reduction associated with tumor fragmentation was found in five cases.
• EXAMPLE 5 Two patients with liver metastasis from primary hepatocellular carcinoma were treated with intrasplenic NCM (1 or 3 injections). The protocol was otherwise as previously described for the H&NSCC, cervical, or lymphoma cases. One patient with advanced hepatocellular carcinoma had a partial response confirmed by tomography, no histology is available. The other had a partial response confirmed by surgery. Histological exam showed tumor reduction, fragmentation, and lymphoid infiltration.
• EXAMPLE 6 Four patients with squamous cell carcinoma of the penis (human papiloma virus associated) were treated with the NCM protocol as described above; all four had partial responses clinically and the surgical specimen showed tumor reduction and fragmentation and lymphoid infiltration characteristic of the H&NSCC cancer patients. EXAMPLE 7
• the twenty-day injection protocol was effective in terms of clinical response and survival yet the surgical specimen showed less lymphoid infiltration (17% area) than the ten-day unilateral protocol (34% area) or ten day bilateral protocol (33% area) (p ⁇ .05). Less lymphoid infiltration with equivalent tumor reduction responses signifies that antibody- mediated immune responses are involved, since these responses are considered less effective than lymphocyte-mediated responses (i.e., cytotoxic T-cells) and the twenty-day injection protocol involves more labor and expense, a ten-day bilateral injection protocol with 100 units of IL-2 equivalence or site is deemed optimal.
• Figure 10 illustrates a series of doses of the NCM of the present invention on overall survival at twenty-four months. An optimal impact on survival at about 100-233 international units of IL-2 equivalence and no effect at about 16 units and less effect at about 1310 units.
• INDO Nonsteroidal anti Inflammatory Drug
• Newer CoXII inhibitors such as celecoxib(Celebrex®) and rofecoxib (Vioxx®) are thought to have less gastrointestinal toxicity.
• Use of these two agents in place of INDO in a small series of patients gave lesser responses as measured by clinical and pathological criteria and by survival.
• Vioxx® all seven patients had clinical signs of gastritis following a week of therapy.
• Ibuprofen was used as the NSAID and good responses were obtained.
• cytokines corresponding to those present in the NCM singly or in combination are also potentially active.
• cytokines such as IL-1 , IFN- ⁇ , TNF- ⁇ , IL-6, IL-8, GM-CSF, G-CSF, IL-12, and combinations thereof can be used in natural or recombinant form.
• EXAMPLE 8 Role of the Intradermal Skin Test in Prognosis We previously suggested that patients with a negative intradermal skin test to the NCM might show poor clinical responses based upon a single patient. We have now accumulated a series of skin test negative patients and find that they show changes similar to the CY & INDO combination without significant NCM seen in Example 7. Ten patients had negative skin tests (including four from Juarez Hospital) with a NCM of the present invention (i.e., unresponsive to the NCM) and were treated with the NCM plus CY and INDO. These patients had poor clinical responses, smaller tumor reduction and fragmentation, and poor survival (20%)(See, Table V). These observations thus corroborate the conclusion made from Example 7 that INDO and CY have marked activity without NCM.
• the NCM skin test can be usefully employed to predict therapeutic outcome in H & N SCC.
• DNCB dinitroclorobenzene
• the NCM skin test offers a convenient twenty-four hour test. Interestingly, these patients divided into two groups. In one group, Table VI b, the responses were especially poor with no survivors.
• NCM of the present invention was used and was followed by chemotherapy with 5FU and cysplatinum and a second cycle of the NCM of the present invention.
• SCC control patients were isolated and cellular constituents analyzed by flow cytometry using a panel of cell surface markers for dendritic cells (i.e., CD83, CD86, and CD 68).
• Sinus histiocytosis (SH+) is associated with an accumulation of CD68+, CD83+, but CD86-DC's while those without noticeable SH have few CD83+ cells ( Figure 11).
• NCM treatment of the present invention results in a five-times increase in the number of CD86+ (concomitant with CD68+CD83+) cells compared to non-treated cancer controls, indicating a conversion to an "activated" DC phenotype. Controls are untreated H & N SCC compared to NCM treated cancer patients.
• Sinus histiocytosis is characterized by and intrasinusoidal accumulate on of CD68 + CD83 + CD86 " myeloid dendritic cells (DC) and effective use of the NCM of the present invention was associated with a 5-fold increase in CD86 + cells indicative of activated mature DC.
• Sinus histiocytosis is confirmed to represent an accumulation of partially matured DC presumed to be bearing endogenous tumor peptides.
• Full maturation and activation with expression of the co- stimulatory receptor (B7.1 or CD86) reflects use of the NCM of the present invention to correct this defect on maturation and to allow effective antigen presentation to T-cells.
• the NCM of the present invention reverses sinus histiocytosis and lead to effective immunization of "na ⁇ ve" T-cells.
• sinus histiocytosis is characterized by a defect in DC of myeloid origin and the negative NCM skin test predicts a defect in another myeloid cell, the monocyte suggests that these are linked observations (i.e., there exist a myeloid lineage defect that is crucial to host immune response to cancer).
• PSMA T Cell Peptides
• ALF & LLH T Cell Peptides
• KLH Keyhole Limpet Hemocyanin
• Serum Antibody Results Serum was diluted as indicated and added to the wells of a microplate coated with either peptide (ALF or LLH) or ovalbumin. Results are expressed as the average OD at 405 for 5 mice groups. Data are presented in Table VII. Mice immunized with KLH conjugate + NCM were negative for Ovalbumin antibodies, but positive for the peptides. Mice immunized with OVA conjugates + NCM were positive for antibodies for both OVA and the peptides, while those immunized with OVA conjugate + CpG were positive for OVA only. These results indicate that NCM makes conjugated PMSA peptides effective at stimulating both DTH and IgG responses specific for the peptides, while other adjuvants like alum, Ribi-Corixa, and CpG were inactive or poorly active.
• ALF Peptide 1) 0 0.5 2) 0 0.1 3) 1.0 1.0 4) 0 0.4
• Neoadjuvant immunological treatment with IRX-2 in patients with advanced oral cavity squamous cell carcinoma of the head and neck induces clinical and histological responses.
• Huxley et al. "The human HPRT gene on a yeast artificial chromosome is functional when transferred to mouse cells by cell fusion", Genomics, 9:742-750 (1991). Jakobovits et al., "Germ-line transmission and expression of a human-derived yeast artificial chromosome", Nature, Vol. 362, pp. 255-261 (1993).
• vaccinia-PSA can include a prostate-specific immune response in androgen-modulated human prostate cancer. Urology 52:2 (1999).
• Valente G DeStefani A, Jemma C. Infiltrating leukocyte populations and T lymphocyte subsets in H&N SCC from patients receiving perilymphatic injections of rlL-2. Mod Pathol 1990; 3: 702-708
• Verastegui E Hadden EM, Barrera J, Meneses A, Hadden JW.
• IRX-2 A natural cytokine mixture

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• 2006
  • 2006-01-23 US US11/337,358 patent/US20060120996A1/en not_active Abandoned
• 2012
  • 2012-05-07 JP JP2012105898A patent/JP2012144579A/ja active Pending

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