Classifications

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  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
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  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
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  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
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  • A61K40/19—Dendritic cells
• • A—HUMAN NECESSITIES
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  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/24—Antigen-presenting cells [APC]
• • A—HUMAN NECESSITIES
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  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4271—Melanoma antigens
  • A61K40/4273—Glycoprotein 100 [Gp100]
• • A—HUMAN NECESSITIES
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  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
• • A—HUMAN NECESSITIES
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  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
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  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
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  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55544—Bacterial toxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/57—Skin; melanoma

Definitions

• the present invention relates to molecular immunology and cell biology. Specifically described herein are compositions and methods for treating cancer or infectious diseases using CD4 lymphocyte depleting agents alone or in a combination with any one or more of an immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, or an immune modulating agent.
• the immune system can provide protection against cancers. Effective immune stimulation produces long-lasting memory lymphocytes, capable of rapidly responding to repeat antigen challenge.
• CD4 expressing lymphocytes include both helper T cells and regulatory T cells. T helper cells are critical to mounting an adoptive immune response.
• regulatory T cells Tregs
• CD4 depletion was evaluated as a strategy for removing Treg activity. Although only a small fraction of CD4 lymphocytes are Treg cells, CD4 depletion remains an effective approach for depleting Treg activity, and importantly, it has the potential for rapid translation to clinical use.
• CD4 depleting antibodies have been evaluated as a strategy to inhibit the immune system in clinical trials for autoimmune disorder. However, described herein is the use of CD4 lymphocyte depleting antibody to stimulate the immune system. Provided herein are therapies against cancer and infectious diseases in subjects by combining depletion of CD4+ lymphocytes with any one or more of an immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, and an immune modulating agent, or a combination thereof.
• Various embodiments of the present invention provide methods for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the method includes providing a composition comprising a CD4 lymphocyte depleting agent and administering a therapeutically effective amount of the composition to the subject, thereby treating, preventing, reducing the severity of and/or slowing the progression of the condition in the subject.
• Various embodiments of the present invention provide methods for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the method includes providing a CD4 lymphocyte depleting agent and at least one additional agent selected from the group consisting of an immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, and an immune modulating agent; and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent and a therapeutically effective amount of the at least one of an immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, and an immune modulating agent to the subject, thereby treating, preventing, reducing the severity of and/or slowing the progression of the condition in the subject.
• the methods further comprise administering a therapeutically effective amount of mTOR inhibitor.
• Various embodiments of the present invention provide methods for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the methods include providing a CD4 lymphocyte depleting agent and an adoptive immune therapeutic agent (for example, a dendritic cell (DC) vaccine) and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent and a therapeutically effective amount of the adoptive immune therapeutic agent thereby treating, preventing, reducing the severity of and/or slowing the progression of the condition in the subject.
• an adoptive immune therapeutic agent for example, a dendritic cell (DC) vaccine
• Various embodiments of the present invention provide methods for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the methods include providing a CD4 lymphocyte depleting agent and a checkpoint inhibitor (for example, an anti-PD-1 antibody) and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent and a therapeutically effective amount of the checkpoint inhibitor thereby treating, preventing, reducing the severity of and/or slowing the progression of the condition in the subject.
• Various embodiments of the present invention provide methods for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the methods include providing a CD4 lymphocyte depleting agent, a dendritic cell (DC) vaccine and an mTOR inhibitor and administering an effective amount each of the CD4 lymphocyte depleting agent, dendritic cell vaccine and an mTOR inhibitor thereby treating, preventing, reducing the severity of and/or slowing the progression of the condition in the subject.
• a CD4 lymphocyte depleting agent a dendritic cell (DC) vaccine and an mTOR inhibitor
• compositions comprising a CD4 lymphocyte depleting agent.
• compositions comprising any one of an immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, and an immune modulating agent.
• kits for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject includes a CD4 lymphocyte depleting agent and instructions for using the composition to treat, prevent, reduce the severity of and/or slow the progression of the condition in the subject.
• kits for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject includes a CD4 lymphocyte depleting agent and at least one of an immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, and an immune modulating agent.
• the kit further comprises instructions for using the CD4 lymphocyte depleting agent and the at least one of the immune check point inhibitor, the adoptive immune therapeutic, the immune adjuvant, and the immune modulating agent to treat, prevent, reduce the severity of and/or slow the progression of the condition in the subject.
• the condition is cancer.
• the cancer may be any of kidney cancer, melanoma, prostate cancer, breast cancer, glioblastoma, lung cancer, colon cancer, or bladder cancer.
• the condition is an infectious disease.
• compositions and kits of the present invention find utility in the treatment of cancer or infectious diseases.
• the methods, compositions and kits of the present invention find utility in the treatment of certain subsets of malignant neoplastic cell proliferative disorders or diseases, including but not limited to carcinomas and melanomas.
• carcinomas include renal cell carcinomas.
• Figure 1 depicts, in accordance with various embodiments of the invention, that there are both immune stimulating and inhibiting effects of mTOR inhibition; however the net effect is enhanced anti-tumor immunity
• B16 tumor cells were injected subcutaneous ly in the flank on day 0.
• B16 tumor growth (left) and survival (right) curves are shown.
• results are representative of duplicate experiments.
• Results are representative of duplicate experiments.
• Lymphocytes were characterized with in vitro mixed cultures using pmel-1 lymphocytes and tumor lysate- pulsed, CpG activated DCs treated with temsirolimus for 48 hours. Lymphocytes were stained for CD8, Thy 1.1, Tbet, Eomes and CD4/FoxP3 and analyzed by flow cytometry. Representative result (left) and summary data (right) are provided. Results are representative of duplicate experiments. *p ⁇ 0.05, **p ⁇ 0.01,***p ⁇ 0.005.
• FIG. 2 depicts, in accordance with various embodiments of the invention, that CD4 depletion enhanced the antitumor effect of mTor inhibition
• CD4 lymphocytes were depleted with aCD4 antibody on days 6 and 10. Mice were treated with daily temsirolimus on days 14 to 34. Tumor growth was monitored. Results are representative of triplicate experiments.
• lymphocytes were harvested on day 45, restimulated with CA9 peptide, and stained for CD8 and IFNy.
• spleen, lymph node and blood were collected on days 0, 1, 10.
• Lymphocytes were stained for CD4, CD8 and FoxP3 and analyzed by flow cytometry, (c-e) The percentages of CD4 cells in the spleen, lymph node and blood on days 0, 1, and 10 following CD4 depletion are reported, (d) Following CD4 depletion, percentages of splenocytes that are CD4 or CD8 positive are reported, and percent of CD4 cells that are FoxP3 positive is reported, (e) In the same experiment, the absolute numbers of splenocytes positive for CD4, CD8, and CD4/FoxP3 are reported. *p ⁇ 0.05, **p ⁇ 0.01,***p ⁇ 0.005.
• FIG. 3 depicts, in accordance with various embodiments of the invention, that combination of CD4 depletion and temsirolimus generated anti-tumor immunity that was dependent on memory CD8 cells,
• CD8 cells were depleted by injecting aCD8 antibody on day 36.
• (b) Tumor- bearing mice (n 8) treated with temsirolimus and CD4 depletion, and then rechallenged with RENCA-CA9 35 days after primary tumor implantation.
• CD8 cells were depleted by injecting aCD8 antibody on day 36.
• lymphoctes Lymphocytes were harvested from mice treated with temsirolimus and CD4 depletion. The lymphoctes were cultured in vitro with CA9 peptide and IL2 (lOu/ml ) for 3 days and then adoptively transferred into na ' ive B6 mice, which were challenged 24 hrs later with 2xl0 5 RENCA tumor cells injected i.v. Lungs were collected 30 days after the i.v. tumor challenge. Lung weight (d) and number of lung tumor deposits (e) were determined. IL2, inter leukin-2, *p ⁇ 0.05, **p ⁇ 0.01,***p ⁇ 0.005.
• FIG. 4 depicts, in accordance with various embodiments of the invention, that combination of CD4 depletion and temsirolimus treatment enhanced function of CD8 memory cells
• (b) Splenocytes (n 3 per group) were harvested one day prior to rechallenging with tumor lysate -pulsed DC vaccine, stained with antibodies, and analyzed by flow cytometry.
• Figure 5 depicts, in accordance with various embodiments of the invention, that the combination of FoxP3+ Treg depletion and temsirolimus enhanced CTL function in vivo
• CD4+FoxP3+ cells were assessed by flow cytometry using peripheral lymphocytes obtained before and after treating mice with diphtheria toxin,
• the in vivo CTL results were analyzed by flow cytometry using splenocytes harvested 14 hrs following injection of target cells.
• CTL cytotoxic T lymphocyte, *p ⁇ 0.05, **p ⁇ 0.01,***p ⁇ 0.005.
• FIG. 6 depicts, in accordance with various embodiments of the invention, that adoptive transfer of FoxP3+ Tregs reduced CTL function in vivo in mice treated with the combination of CD4 depletion and temsirolimus.
• B6 received tumor lysate-pulsed DC vaccine, and were treated intraperitoneally with aCD4 antibody on days 6 and 10, and daily temsirolimus on days 14 to 24.
• Tregs sorted from lymphocytes from GFP-FoxP3 mice that received tumor lysate-pulsed DC vaccine, were adoptively transferred on day 20 and in vivo CTL assay was performed on day 35.
• Figure 7 depicts, in accordance with various embodiments of the invention, that after treatment with CD4 depletion, Treg population that recovers is less immunosuppressive
• Mice were treated using the experimental scheme outlined in Figure 4a. On day 45, splenocytes were examined by flow cytometry for CD4+FoxP3+ cells, (b) The splenocytes that recovered after CD4 depletion were used to enrich for CD4+ cells, which were then sorted by CD25 status. For the resulting groups, representative flow cytometry for CD4+CD25+ status and CD4+CD25- status are shown, (c) CD4+CD25- cells were co- cultured with DCs pulsed with B16 tumor lysate.
• CD4+CD25- cells were analyzed by flow cytometry for IFNy or IL4 expression, (d) CD4+CD25+ cells were co-cultured with DCs pulsed with B16 tumor lysate and CD8 cells from mice immunized with DCs pulsed with B16 tumor lysate. CD8 cell proliferation was monitored by flow cytometry using a CFSE dilution assay. *p ⁇ 0.05, **p ⁇ 0.01,***p ⁇ 0.005.
• Figure 8 depicts, in accordance with various embodiments of the invention, that tumor increases Tregs.
• the percent of CD4 cells expressing FoxP3 were examined from lymph nodes, spleen, bone marrow and peripheral blood lymphocytes (PBL) from tumor bearing mice ( ⁇ 1 cm in diameter) and control mice without tumor. Flow cytometry results are shown.
• Figure 9 depicts, in accordance with various embodiments of the invention, that aCD4 antibody depletes effector CD4 subtypes, (a) Splenocytes were collected before (day 0) and 1 or 10 days after administering aCD4 antibody, and analyzed by flow cytometry. The number of total splenocytes staining for CD4 and IL-2, IL-4 or IL-17 is shown, (b) Percent of CD4 cells staining for IL-2, IL-4 or IL-17 is shown.
• Figure 10 depicts, in accordance with various embodiments of the invention, that CD4 depletion prior to immune stimulation prevents formation of CD8 memory cells.
• the experimental scheme is similar to Figure 4.
• An additional group is included where aCD4 antibody is administered on day -1 and +1 (Group A), (a) The percent of CD8 cells expressing Thy 1.1 is shown from splenocytes harvested 5 days following rechallenge of memory cells, (b) The percent of CD8 cells expressing Eomes is shown. *p ⁇ 0.05, **p ⁇ 0.01,***p ⁇ 0.005.
• Figure 11 depicts, in accordance with various embodiments of the invention, that Depletion of Tregs or CD4 cells enhance antitumor effect.
• Figure 12 depicts, in accordance with various embodiments of the invention, that CD4 depletion enhances PD-1 blockade antitumor effect.
• Figure 13 depicts, in accordance with various embodiments of the invention, that CD4 depletion enhances to tumor-specific cellular immunity.
• D B 16 tumor growth curve .
• Figure 14 depicts, in accordance with various embodiments of the invention, that combination of anti-CD4 antibody, Temsirolimus and dendritic cell vaccine exhibited enhanced anti-tumor activity.
• the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).
• the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, reverse, alleviate, ameliorate, inhibit, lessen, slow down or stop the progression or severity of a symptom or condition.
• the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease-state is reduced or halted.
• treatment includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Also, “treatment” may mean to pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented.
• “Beneficial results” or “desired results” may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition, decreasing morbidity and mortality, and prolonging a patient's life or life expectancy.
• "beneficial results” or “desired results” may be alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of cancer progression, delay or slowing of metastasis or invasiveness, and amelioration or palliation of symptoms associated with the cancer.
• administering refers to the placement an agent as disclosed herein into a subject by a method or route which results in at least partial localization of the agents at a desired site.
• a "cancer” or “tumor” as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems, and/or all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Included in this definition are benign and malignant cancers, as well as dormant tumors or micro-metastases.
• cancer or tumor refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems, and/or all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Included in this definition are benign and malignant cancers, as well as dormant tumors or micro-metastases.
• the term “carcinoma” refers to a cancer arising from epithelial cells.
• invasive refers to the ability to infiltrate and destroy surrounding tissue. Melanoma is an invasive form of skin tumor.
• cancer examples include, but are not limited to B-cell lymphomas (Hodgkin's lymphomas and/or non-Hodgkins lymphomas), brain tumor, breast cancer, colon cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer.
• B-cell lymphomas Hodgkin's lymphomas and/or non-Hodgkins lymphomas
• brain tumor breast cancer
• colon cancer lung cancer
• gastric cancer pancreatic cancer
• cervical cancer ovarian cancer
• liver cancer bladder cancer
• cancer of the urinary tract thyroid cancer
• renal cancer carcinoma
• melanoma head and neck cancer
• brain cancer and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer
• Conditions and “disease conditions,” as used herein may include, cancers, tumors or infectious diseases.
• the conditions include but are in no way limited to any form of malignant neoplastic cell proliferative disorders or diseases.
• conditions include any one or more of kidney cancer, melanoma, prostate cancer, breast cancer, glioblastoma, lung cancer, colon cancer, or bladder cancer.
• Immunocell refers to the cells of the mammalian immune system including but not limited to antigen presenting cells, B-cells, basophils, cytotoxic T-cells, dendritic cells, eosinophils, granulocytes, helper T-cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells and T-cells.
• antigen presenting cells B-cells, basophils, cytotoxic T-cells, dendritic cells, eosinophils, granulocytes, helper T-cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells and T-cells.
• Immuno response refers to immunities including but not limited to innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity and/or overactive immunity
• sample or "biological sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a tumor sample from a subject.
• exemplary biological samples include, but are not limited to, a biofluid sample; serum; plasma; urine; saliva; a tumor sample; a tumor biopsy and/or tissue sample etc.
• the term also includes a mixture of the above-mentioned samples.
• sample also includes untreated or pretreated (or pre-processed) biological samples.
• a sample can comprise one or more cells from the subject.
• a sample can be a tumor cell sample, e.g. the sample can comprise cancerous cells, cells from a tumor, and/or a tumor biopsy.
• a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
• Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf.
• patient "individual” and “subject” are used interchangeably herein.
• the subject is mammal.
• the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples.
• the methods described herein can be used to treat domesticated animals and/or pets.
• a subject can be one who has been previously diagnosed with or identified as suffering from or having a disease-state in need of monitoring (e.g., cancer or infectious disease) or one or more complications related to such a disease-state, and optionally, have already undergone treatment for the disease-state or the one or more complications related to the disease/condition.
• a subject can also be one who has not been previously diagnosed as having a disease-state or one or more complications related to the disease/condition.
• a subject can be one who exhibits one or more risk factors for a disease-state or one or more complications related to a disease-state or a subject who does not exhibit risk factors.
• a "subject in need" of treatment for a particular disease-state can be a subject having that disease/condition, diagnosed as having that condition, or at risk of developing that disease.
• statically significant refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p- value.
• CD4 lymphocytes refer to lymphocytes that express CD4, i.e., lymphocytes that are CD4+.
• CD4 lymphocytes may be T cells that express CD4.
• T-cell and "T-lymphocyte” are interchangeable and used synonymously herein. Examples include but are not limited to naive T cells, central memory T cells, effector memory T cells or combinations thereof.
• “Therapeutic agents” as used herein refers to agents that are used to, for example, treat, inhibit, prevent, mitigate the effects of, reduce the severity of, reduce the likelihood of developing, slow the progression of and/or cure, a disease.
• Diseases targeted by the therapeutic agents include but are not limited to infectious diseases, carcinomas, sarcomas, lymphomas, leukemia, germ cell tumors, blastomas, antigens expressed on various immune cells, and antigens expressed on cells associated with various hematologic diseases, and/or inflammatory diseases.
• a CD4 lymphocyte depleting agent is used alone or in combination with various other therapies and agents to target the immune system to treat cancer and/or infectious diseases.
• therapies and agents that can be used in combination with a CD4 lymphocyte depleting agent include but are not limited to immune check point inhibitors, adoptive immune therapies, immune adjuvants, and immune modulating agents.
• the present invention provides a method of treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the method includes providing a composition comprising a CD4 lymphocyte depleting agent and administering a therapeutically effective amount of the composition to the subject, thereby treating, preventing, reducing the severity of and/or slowing the progression of the condition in the subject.
• the condition is cancer or an infectious disease.
• the subject has been diagnosed with cancer.
• the subject has been diagnosed with an infectious disease.
• the subject has had the disease for an amount of time long enough to prime the immune system prior to administration of the composition comprising a CD4 lymphocyte depleting agent.
• the amount of time for which the subject has had the condition is 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 15 days or more, 20 days or more, 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more, or combinations thereof.
• the present invention provides methods for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the methods include providing a CD4 lymphocyte depleting agent and at least one additional agent selected from the group consisting of an immune check point inhibitor, an adoptive immune therapeutic agent, an immune adjuvant, and an immune modulating agent; and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent and a therapeutically effective amount of at least one of an immune check point inhibitor, an adoptive immune therapeutic agent, an immune adjuvant, and an immune modulating agent to the subject, thereby treating, preventing, reducing the severity of and/or slowing the progression of the condition in the subject.
• the methods further comprise administering an mTOR inhibitor to the subject.
• the CD4 lymphocyte depleting agent and the additional agent may be administered sequentially or simultaneously.
• the condition is cancer. In some embodiments, the condition is an infectious disease.
• the present invention provides methods for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the methods include providing a CD4 lymphocyte depleting agent and an adoptive immune therapeutic agent and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent and a therapeutically effect amount of the adoptive immune therapeutic agent to the subject so as to treat, inhibit, prevent, reduce the severity and/or slow the progression of the condition in the subject.
• the CD4 lymphocyte depleting agent is an anti-CD4 antibody or a fragment thereof or a variant thereof and the adoptive immune therapeutic agent is a dendritic cell vaccine.
• the condition is cancer or an infectious disease.
• anti-CD4 antibody and dendritic cell vaccine are administered simultaneously. In some embodiments, the anti-CD4 antibody and the dendritic cell vaccine are administered sequentially. In some embodiments, the methods may further comprise administering an effective amount of an mTOR inhibitor, sequentially or simultaneously with the CD4 lymphocyte depleting agent and the adoptive immune therapeutic agent.
• the present invention provides methods for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the methods include providing a CD4 lymphocyte depleting agent and an immune checkpoint inhibitor and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent and a therapeutically effective amount of the immune checkpoint inhibitor to the subject so as to treat, inhibit, prevent, reduce the severity and/or slow the progression of the condition in the subject.
• the CD4 lymphocyte depleting agent is an anti-CD4 antibody or a fragment thereof or a variant thereof and the checkpoint inhibitor is an anti-PD-1 antibody or a fragment thereof or a variant thereof.
• the condition is cancer or an infectious disease.
• anti-CD4 antibody and anti-PD-1 antibody are administered simultaneously. In some embodiments, the anti-CD4 antibody and the anti-PD-1 antibody are administered sequentially.
• the methods may further comprise administering an mTOR inhibitor. In some embodiments, the methods may further comprise administering an effective amount of an mTOR inhibitor, sequentially or simultaneously with the CD4 lymphocyte depleting agent and the checkpoint inhibitor.
• the present invention provides methods for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the methods include providing a CD4 lymphocyte depleting agent, an adoptive immune therapeutic agent and an mTOR inhibitor and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent, a therapeutically effective amount of the adoptive immune therapeutic agent and a therapeutically effective amount of the mTOR inhibitor to the subject so as to treat, inhibit, prevent, reduce the severity and/or slow the progression of the condition in the subject.
• the CD4 lymphocyte depleting agent is an anti-CD4 antibody or a fragment thereof or a variant thereof.
• the adoptive immune therapeutic agent is a dendritic cell vaccine.
• the mTOR inhibitor is temsirolimus.
• the condition is cancer or an infectious disease.
• anti-CD4 antibody, dendritic cell vaccine and the mTOR inhibitor are administered simultaneously.
• anti-CD4 antibody, dendritic cell vaccine and the mTOR inhibitor are administered sequentially.
• the methods described herein may be used to prevent metastasis of cancer or prevent recurrence of cancer in a subject in need thereof.
• the CD4 lymphocyte depleting agent may be any one or more of small molecule, a peptide, an antibody or a fragment thereof, and a nucleic acid molecule.
• the antibody specifically binds CD4 on CD4- expressing T cells such as regulatory T cells (Treg cells).
• An antibody for example, anti-CD4 antibody or anti-PD-1 antibody
• These antibodies can be from any source, e.g., rat, mouse, guinea pig, dog, cat, rabbit, pig, cow, horse, goat, donkey or human.
• Fragments of antibodies may be any one or more of Fab, F(ab')2, Fv fragments or their fusion proteins.
• the CD4 lymphocyte depleting agent is a humanized anti- CD4 antibody or a fragment thereof.
• the CD4 lymphocyte depleting agents are zanolimumab, keliximab, and OKT4.
• the CD4 lymphocyte depleting agent and the additional agent are administered concurrently. In other embodiments, the CD4 lymphocyte depleting agent and the additional agent are administered sequentially. For example, the CD4 lymphocyte depleting agent is administered before, during or after administering the additional agent. In further embodiments, the the CD4 lymphocyte depleting agent and the additional agent are administered with food or without food. In accordance with the invention, the CD4 lymphocyte depleting agent and/or the additional agent may be used in combination with other agents, including but not limited to cancer vaccines and chemotherapeutic agents. As described herein, the additional agent is any one or more of an immune check point inhibitor, an adoptive immune therapeutic agent, an immune adjuvant, and an immune modulating agent.
• the additional agent is an immune checkpoint inhibitor.
• immune checkpoint inhibitors include but are not limited to anti-PD-1 antibodies such as Lambrolizumab(MK-3475), Nivolumab (BMS-936558) and Pidilizumab (CT-011), anti-PD-Ll antibodies such as MPDL3280A(RG7446), MEDI4736 and BMS- 936559, anti-PD-L2 antibodies, B7-DC-Fc fusion proteins such as AMP-224, anti-CTLA-4 antibodies such as tremelimumab (CP-675,206) and ipilimumab (MDX-010), antibodies against the B7/CD28 receptor superfamily, anti-Indoleamine (2,3)-dioxygenase (IDO) antibodies, anti-IDOl antibodies, anti-ID02 antibodies, tryptophan, tryptophan mimetic, 1- methyl tryptophan (1-MT)), Indoximod (D-l -
• the immune checkpoint inhibitor is selected from the group consisting of an antibody against PD-1, an antibody against PD-L1, an antibody against PD-L2, an antibody against CTLA-4, an antibody against KIR, an antibody against IDOl, an antibody against ID02, an antibody against TIM-3, an antibody against LAG-3, an antibody against OX40R, and an antibody against PS, or a combination thereof.
• the additional agent is an adoptive immune therapeutic.
• the adoptive immune therapeutic is selected from the group consisting of a dendritic cell vaccine, a peptide vaccine, a chimeric T cell antigen-based therapy, T cell- based therapy, an immune cytokine, a heat shock protein-based vaccine, a tumor lysate -based vaccine, a viral vector carrying a tumor antigen, a viral vaccine, a bacterial vaccine, and a fungal vaccine, or a combination thereof. Additional information can be found in Radvanyi et al. Clin Cancer Res. 2012 Dec 15;18(24):6758-70; Epub 2012 Oct 2, which is incorporated herein by reference in its entirety as though fully set forth.
• the additional agent is an immune adjuvant.
• adjuvants include but are not limited to cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, Alhydrogel, ISCOM(s)TM, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, CpG DNA Vaccine Adjuvant, Cholera toxin, Cholera toxin B subunit, Liposomes, Saponin Vaccine Adjuvant, DDA Adjuvant, Squalene-based Adjuvants, Etx B subunit Adjuvant, IL-12 Vaccine Adjuvant, LTK63 Vaccine Mutant Adjuvant, TiterMax Gold Adjuvant, Ribi Vaccine Adjuvant, Montanide ISA 720 Adjuvant, Corynebacterium- derived P40 Vaccine Adjuvant, MPLTM Adjuvant, AS04, AS02, Li
• the immune adjuvant is selected from the group consisting of an aluminum salt, a virosome and an oil-based adjuvant, or a combination thereof.
• the additional agent is an immune modulating agent.
• the immune modulating agent is selected from the group consisting of an mTOR inhibitor, a STAT inhibitor, a TGFP receptor inhibitor, and a tyrosine kinase inhibitor, or a combination thereof.
• the methods may further include administering an mTOR inhibitor to the subject.
• the mTOR inhibitor may be any one or more of a small molecule, a peptide, an antibody or a fragment thereof, a nucleic acid molecule and/or a macrolide compound.
• the antibody specifically binds mTOR so as to inhibit mTOR.
• the antibody may be any one or more of a monoclonal antibody or fragment thereof, a polyclonal antibody or a fragment thereof, a chimeric antibody, a humanized antibody, a human antibody or a fragment thereof, or a single chain antibody.
• antibodies can be from any source, e.g., rat, mouse, guinea pig, dog, cat, rabbit, pig, cow, horse, goat, donkey or human. Fragments of antibodies may be any one or more of Fab, F(ab')2, Fv fragments or their fusion proteins.
• the mTOR inhibitor is a macrolide compound.
• macrolide compounds that may be used with the claimed invention include but are not limited to temsirolimus (CCI-779) or a pharmaceutical equivalent, analog, derivative or a salt thereof, evirolimus (RAD-001) or a pharmaceutical equivalent, analog, derivative or a salt thereof, and/or sirolimus (rapamycin) or a pharmaceutical equivalent, analog, derivative or a salt thereof.
• the additional agent is a STAT inhibitor.
• STAT inhibitors include but are not limited to SOCS (supressors of cytokine singaling), PIAS (protein inhibitors of activated stats) including PIAS1, PIAS2, PIAS3, PIAS4, PIASxa, PIASxb, and PIASy, Nifuroxazide (5-Nitro-2-furaldehyde-p-hydroxybenzoylhydrazone), N- [2-(l, 3, 4-oxadiazolyl)]-4-Quinolinecarboxamide, non-peptidic small molecule inhibitors, Stattic, STA-21, LLL-3, LLL12, XZH-5, S31-201, SF-1066, SF-1087, 17o, Cryptotanshinon, FLL32, C188-9, LY5, BP-1108, BP-1075, Galiellalactone, JQ1, STX-0119, FLLL11, FLLL12, FL
• the additional agent is a JAK inhibitor.
• JAK inhibitors include but are not limited to Tyrphostin AG490, CP-690550, ruxolitinib (INCB018424), TG101348 (SAR 30253), lestaurtinib (CEP701), CYT387, pacritinib (SB 1518), AZD1480, XL019, and LY2784544. Additional information can be found in Mascarenhas et al. (Biology and clinical management of myeloproliferative neoplasms and development of the JAK inhibitor ruxolitinib, Curr Med Chem. 2012; 19(26):4399-413), which is incorporated herein by reference in its entirety as though fully set forth.
• the additional agent is a TGFP receptor inhibitor.
• TGFP receptor inhibitors include but are not limited to dominant negative TGF- ⁇ Type II receptors ( ⁇ ⁇ ), ligand traps, the soluble ⁇ ⁇ ectodomain, the soluble betaglycan ectodomain, recombinant Fc-fusion proteins containing the soluble ectodomain of either TpRII (TpRII-Fc) or the type III receptor/betaglycan, soluble human a2-macroglobulin plasma protein, fully humanized pan-TGF- ⁇ monoclonal neutralizing antibodies including Lerdelimumab (CAT- 152 ), Metelimumab (CAT- 192) and GC-1008 (Fresolimumab), and 1D11; antisense oligonucleotides (ASO) designed to hybridize to their complementary RNA sequence and accelerate mRNA degradation, API 2009 (Trabedersen) and AP-11014; receptor kina
• ASO antisense
• the additional agent is a tyrosine kinase inhibitor.
• tyrosine kinase inhibitors include but are not limited to sunitinib, erlotinib, vandetanib, cediranib, brivanib, foretinib, and dovitinib. Additional information can be found in Huynh H. (Molecularly targeted therapy in hepatocellular carcinoma, Biochem Pharmacol. 2010 Sep l;80(5):550-60. Epub 2010 Apr 4), which is incorporated herein by reference in its entirety as though fully set forth.
• the methods described herein further comprise administering a chemotherapeutic agent to the subject being administered a composition comprising CD4 lymphocyte depleting agent or a composition comprising a CD4 lymphocyte depleting agent and an additional agent selected from the group consisting of an immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, and an immune modulating agent.
• Non-limiting examples of chemotherapeutic agents can include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
• dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, 6-diazo-5-oxo-L-norleu
• cancer examples include but are not limited to, carcinoma, blastoma, and sarcoma. More particular examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin's and non-Hodgkin's lymphoma
• the condition may be a malignant neoplastic cell proliferative disorder or disease. Still in accordance with the invention, the condition may be renal cell carcinoma or melanoma. Diseases targeted by the therapeutic agents include carcinomas, sarcomas, germ cell tumors and/or blastomas.
• infectious diseases are caused by infectious bacteria.
• infectious bacteria include: Helicobacterpyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as M. tuberculosis, M. avium, M. intracellular, M. kansaii, M.
• compositions and methods described herein are contemplated for use in treating infections with these bacterial agents.
• Other infectious organisms include: Plasmodium falciparum and Toxoplasma gondii.
• the compositions and methods described herein are contemplated for use in treating infections with these agents.
• viral antigens may be any antigens present in infectious viruses and that induce an immune response in a subject.
• Retroviridae for example, HIV
• Picornaviridae for example, polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses
• Calciviridae such as strains that cause gastroenteritis
• Togaviridae for example, equine encephalitis viruses, rubella viruses
• Flaviridae for example, dengue viruses, encephalitis viruses, yellow fever viruses
• Coronaviridae for example, coronaviruses
• Rhabdoviridae for example, vesicular stomatitis viruses, rabies viruses
• Filoviridae for example, ebola viruses
• Paramyxoviridae for example, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus
• Orthomyxoviridae for example, influenza viruses
• Bungaviridae for example, Hantaan viruses
• Examples of fungal infections that may be treated with the compositions and methods described herein include but are not limited to: aspergillosis; thrush (caused by Candida albicans); cryptococcosis (caused by Cryptococcus); and histoplasmosis.
• examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.
• the compositions and methods described herein are contemplated for use in treating infections with these fungal agents.
• the subject is a human.
• the subject is a a mammalian including but not limited to human, monkey, ape, dog, cat, cow, horse, goat, pig, rabbit, mouse and rat.
• the composition is administered intravenously, intramuscularly, subcutaneously, intraperitoneally, orally or via inhalation.
• various routes may be utilized to administer the composition of the claimed methods, including but not limited to aerosol, nasal, oral, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsules and/or injections.
• the CD4 lymphocyte depleting agent is administered at 100- 200mg/day, 200-3 OOmg/day, 300-400mg/day, 400-5 OOmg/day, 500-600mg/day, 600- 700mg/day, 700-800mg/day, 800-900mg/day, 900-lOOOmg/day, 1000-11 OOmg/day, 1100- 1200mg/day, 1200- 13 OOmg/day, 1300-1400mg/day, 1400- 15 OOmg/day, 1500-1600mg/day, 1600-1700mg/day, 1700-1800mg/day, 1800-1900mg/day or 1900-2000mg/day.
• the additional agent is administered at 0.1-0.5mg/day, 0.5-1.0mg/day, 1.0- 1.5mg/day, 1.5-2.0mg/day, 2.0-2.5mg/day, 2.5-5mg/day, 5-10mg/day, 10-15mg/day, 15- 20mg/day, 20-25mg/day, 25-30mg/day, 30-35mg/day, 35-40mg/day, 40-45mg/day, 45- 50mg/day, 50-55mg/day, 55-60mg/day, 60-65mg/day, 65-70mg/day, 70-75mg/day, 75- 80mg/day, 80-85mg/day, 85-90mg/day, 90-95mg/day or 95-100mg/day.
• the present invention provides a composition comprising a CD4 lymphocyte depleting agent.
• the present invention provides compositions comprising at least one of an immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, and an immune modulating agent. Examples of immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, and an immune modulating agent are described herein.
• a composition comprising a CD4 lymphocyte depleting agent and a composition comprising an additional agent are two separate compositions.
• the CD4 lymphocyte depleting agents and/or the additional agents useful in the treatment of disease in mammals will often be prepared substantially free of naturally-occurring immunoglobulins or other biological molecules. Preferred CD4 lymphocyte depleting agents and/or the additional agents will also exhibit minimal toxicity when administered to a mammal.
• the pharmaceutical compositions according to the invention can contain any pharmaceutically acceptable excipient. "Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, nontoxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.
• excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
• excipients include but are not limited to starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, wetting agents, emulsifiers, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, setting agents, suspending agents, surfactants, humectants, carriers, stabilizers, and combinations thereof.
• the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration.
• Route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal, parenteral, enteral, topical or local.
• Parenteral refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
• the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection.
• the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release.
• the compositions are administered by injection. Methods for these administrations are known to one skilled in the art.
• compositions according to the invention can contain any pharmaceutically acceptable carrier.
• “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
• the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.
• Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
• compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration.
• Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition.
• Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water.
• Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
• the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
• the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
• a liquid carrier When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension.
• Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
• the pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount.
• the precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
• formulants may be added to the composition.
• a liquid formulation may be preferred.
• these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, bulking agents or combinations thereof.
• Carbohydrate formulants include sugar or sugar alcohols such as monosaccharides, disaccharides, or polysaccharides, or water soluble glucans.
• the saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcellulose, or mixtures thereof.
• “Sugar alcohol” is defined as a C4 to C8 hydrocarbon having an -OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. In one embodiment, the sugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %, more preferable between 2.0 and 6.0 w/v %.
• Amino acids formulants include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added.
• polymers as formulants include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000.
• PVP polyvinylpyrrolidone
• PEG polyethylene glycol
• a buffer in the composition it is also preferred to use a buffer in the composition to minimize pH changes in the solution before lyophilization or after reconstitution.
• Most any physiological buffer may be used including but not limited to citrate, phosphate, succinate, and glutamate buffers or mixtures thereof.
• the concentration is from 0.01 to 0.3 molar.
• Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268,110.
• liposome Another drug delivery system for increasing circulatory half-life is the liposome.
• Methods of preparing liposome delivery systems are discussed in Gabizon et al, Cancer Research (1982) 42:4734; Cafiso, Biochem Biophys Acta (1981) 649: 129; and Szoka, Ann Rev Biophys Eng (1980) 9:467.
• Other drug delivery systems are known in the art and are described in, e.g., Poznansky et al, DRUG DELIVERY SYSTEMS (R. L. Juliano, ed., Oxford, N.Y. 1980), pp. 253-315; M. L. Poznansky, Pharm Revs (1984) 36:277.
• the liquid pharmaceutical composition may be lyophilized to prevent degradation and to preserve sterility.
• Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art.
• the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients.
• a sterile diluent Finger's solution, distilled water, or sterile saline, for example
• the composition is administered to subjects using those methods that are known to those skilled in the art.
• compositions of the invention may be sterilized by conventional, well-known sterilization techniques.
• the resulting solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
• the compositions may contain pharmaceutically-acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and stabilizers (e.g., 1-20% maltose, etc.).
• the present invention provides a kit for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the kit comprises a composition comprising a CD4 lymphocyte depleting agent and instructions for using the composition to treat, prevent, reduce the severity of and/or slow the progression of the condition in the subject.
• the present invention provides a kit for treating, preventing, reducing the severity of and/or slowing the progression of a condition in a subject.
• the kit comprises a composition comprising a CD4 lymphocyte depleting agent and a composition comprising at least one additional agent selected from the group consisting of an immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, and an immune modulating agent; and instructions for using the composition to treat, prevent, reduce the severity of and/or slow the progression of the condition in the subject.
• the kit is an assemblage of materials or components, including at least one of the inventive compositions.
• the kit contains a composition including a drug delivery molecule complexed with a therapeutic agent, as described above.
• the kit is configured particularly for the purpose of treating mammalian subjects. In another embodiment, the kit is configured particularly for the purpose of treating human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.
• Instructions for use may be included in the kit.
• “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to affect a desired outcome.
• the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.
• the materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility.
• the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures.
• the components are typically contained in suitable packaging material(s).
• packaging material refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like.
• the packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment.
• the term "package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components.
• a package can be a glass vial used to contain suitable quantities of a composition containing a CD lymphocyte depleting agent or a composition containing a CD lymphocyte depleting agent and at least one additional agent selected from the group consisting of an immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant, and an immune modulating agent.
• the packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.
• the effective amounts of the CD4 lymphocyte depleting agent in the claimed methods, compositions and/or kits may be in the range of about 100- 200mg/day, 200-3 OOmg/day, 300-400mg/day, 400-5 OOmg/day, 500-600mg/day, 600- 700mg/day, 700-800mg/day, 800-900mg/day, 900-lOOOmg/day, 1000-11 OOmg/day, 1100- 1200mg/day, 1200- 13 OOmg/day, 1300-1400mg/day, 1400- 15 OOmg/day, 1500-1600mg/day, 1600-1700mg/day, 1700-1800mg/day, 1800-1900mg/day or 1900-2000mg/day.
• the CD4 lymphocyte depleting agent is a humanized anti-CD4 antibody (for example zanolimumab).
• the effective amounts of the CD4 lymphocyte depleting agent in the claimed methods, compositions and/or kits may be in the range of about 100- 200mg/week, 200-3 OOmg/week, 300-400mg/week, 400-5 OOmg/week, 500-600mg/week, 600- 700mg/week, 700-800mg/week, 800-900mg/week, 900-lOOOmg/week, 1000-11 OOmg/week, 1100-1200mg/week, 1200- 13 OOmg/week, 1300-1400mg/week, 1400- 15 OOmg/week, 1500- 1600mg/week, 1600-1700mg/week, 1700-1800mg/week, 1800-1900mg/week or 1900- 2000mg/week.
• the CD4 lymphocyte depleting agent is a humanized anti-CD4 antibody (for example zanolimumab). Zanolimumab may be administered at a dose
• the effective amounts of the additional agent in the claimed methods, compositions and/or kits may be in the range of about 0.1-0.5mg/day, 0.5- l .Omg/day, 1.0-1.5mg/day, 1.5-2mg/day, 2.0-2.5mg/day, 2.5-5mg/day, 5-10mg/day, 10- 15mg/day, 15-20mg/day, 20-25mg/day, 25-30mg/day, 30-35mg/day, 35-40mg/day, 40- 45mg/day, 45-50mg/day, 50-55mg/day, 55-60mg/day, 60-65mg/day, 65-70mg/day, 70- 75mg/day, 75-80mg/day, 80-85mg/day, 85-90mg/day, 90-95mg/day, 95-1 OOmg/day, 0.75- lOmg/day or 2-10mg/day.
• the effective amounts of the additional agent in the claimed methods, compositions and/or kits may be in the range of about l-5mg/week, 5- lOmg/week, 10-15mg/week, 15-20mg/week, 20-25mg/week, 25-30mg/week, 30-35mg/week, 35-40mg/week, 40-45mg/week, 45-50mg/week, 50-55mg/week, 55-60mg/week, 60- 65mg/week, 65-70mg/week, 70-75mg/day, 75-80mg/mg, 80-85mg/mg, 85-90mg/week, 90- 95mg/week or 95-100mg/week.
• the additional agents are any of immune check point inhibitor, an adoptive immune therapeutic, an immune adjuvant or an immune modulating agent.
• the at least one additional agent comprises temsirolimus administered at a dose of 25mg over 30-60 minutes per week, evirolimus administered at a dose of 0.75- lOmg per day and/or rapamycin administered at a dose of 2- lOmg per day.
• the CD4 lymphocyte depleting agent and the additional agent may be administered simultaneously at the aforementioned dosages using the appropriate modes of administration, for instance, the modes of administration recommended by the manufacturer for each of the CD4 lymphocyte depleting agent and the additional agent.
• the CD4 lymphocyte depleting agent and the additional agent may be administered sequentially at the aforementioned dosages.
• the additional agent may be administered, for example, daily at the aforementioned dosages and the CD4 lymphocyte depleting agent (for example a humanized anti-CD4 antibody) may be administered for example, daily, weekly, biweekly, every fortnight and/or monthly at the aforementioned dosages.
• the additional agent may be administered, for example, daily, weekly, biweekly, every fortnight and/or monthly, at the aforementioned dosages and the CD4 lymphocyte depleting agent (for example a humanized anti-CD4 antibody) may be administered, for example, daily at the aforementioned dosages.
• each of the additional agent and the CD4 lymphocyte depleting agent may be administered daily, weekly, biweekly, every fortnight and/or monthly, wherein the additional agent is administered at the aforementioned dosages on a day different than the day on which the CD4 lymphocyte depleting agent is administered at the aforementioned dosages.
• the cancer vaccine dose would depend on the vaccine being used.
• the effective dose of the cancer vaccine may be determined by one skilled in the art (such as the physician) or it may be administered per the manufacturers' recommendation.
• the first dose of the cancer vaccine is administered on day 0 and the second dose is administered on day 7.
• the additional agent may be administered on days 2-32, at the aforementioned dosages.
• 2-3 weekly doses of anti-CD4 depleting agent may be administered starting on day 10, at the aforementioned dosages.
• a heat shock protein-based vaccine a heat shock protein (for example, hspl lO or grpl70) may be complexed with a tumor antigen (such as gplOO) and subsequently administered.
• a complex of hspl lO and gplOO at 2.5mg/kg may be administered intradermally.
• Typical dosages of an effective amount of the CD4 lymphocyte depleting agent or the additional agent can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses in cells or in vivo responses in animal models.
• the FDA approved dosage for Temsirolimus is 25mg administered intravenously over 30-60 min every week, for Evirolimus is 0.75mg-10mg per day administered orally, for Rapamycin is about 2-10mg per day administered orally and for Zanolimumab is about 980mg per week administered intravenously.
• the same or similar dosing can be used in accordance with various embodiments of the present invention, or an alternate dosage may be used in connection with alternate embodiments of the invention.
• the actual dosage can depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of relevant cultured cells or histocultured tissue sample, or the responses observed in the appropriate animal models.
• mice Female C57BL/6J, BALB/c mice and Pmel-1 mice, 6-8 week old, were purchased from Jackson laboratory (Bar Harbor, ME) and housed under pathogen-free conditions. FoxP3-GFP transgenic mice express Green Fluorescent Protein under the control of the mouse Foxp3 (forkhead box P3) promoter. DEREG (DEpletion of REGulatory T cells) transgenic mice was generated and described by T.S. Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, et al. J Exp Med. 2007;204:57-63. All experiments involving animals were in compliance with federal and state standards, which include the federal Animal Welfare Act and the NIH guide for the care and use of laboratory animals.
• B16 cells (mouse melanoma cell line) were transduced with human gplOO
• RENCA human melanoma antigen (human melanoma antigen) (B16-gpl00).
• RENCA is a murine renal cell carcinoma (RCC) cell line. The cells were maintained in DMEM or RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies, Grand Island, NY), 2 mmol/L of L-glutamine, 100 units/mL of penicillin, and 100 ⁇ g/mL of streptomycin.
• FBS heat-inactivated fetal bovine serum
• Mouse tumors were generated by subcutaneously injecting 2x10 5 cells into the flank: B16 tumors into C57BL/6 mice and RENCA tumors into Balb/C mice. Tumor diameter was measured with calipers twice a week and tumor volume was calculated (shortest diameter 2 x longest diameter/2). In the lung metastasis model, tumor cells were injected intravenously through the tail vein. Lung metastases were counted using a dissection microscope.
• mAb monoclonal antibodies
• Anti-CD4 GK 1.5 and RM4-5
• anti-CD8 53-6.7
• anti-CD90.1 OX-7
• anti-CD 11c N418)
• anti-Bcl2 BCL/10C4
• anti-T-bet 4B10
• anti-CD62L MEL-14
• anti-CD279 PD-1.29F.1A12
• anti- FoxP3 FJK-16s
• anti-IFN- ⁇ XMG1.2
• anti-IL-2 JES6.5H4
• anti-IL-4 11B11
• IL-17A eBiol787
• CellTrace 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kit was purchased from Molecular Probes (Eugene, OR). Temsirolimus was purchased from LC Laboratory (Woburn,MA).
• CD8 and CD4 T-cells were negatively enriched using mouse CD8 or CD4 recovery column kits (Cedarlane Labs, Burlington, NC). Purity of CD8 and CD4 cells after negative selection was greater than 85%. FoxP3-GFP cells or antibody stained CD4+CD25+ cells were sorted by MoFlo Cell Sorter (Fort Collins, CO). Preparation of DCs and T cell stimulation
• mice were harvested from femurs and tibias and then placed in 12 well plates at a density of lxl 0 6 cells per well with 10% FBS and lOng/ml mouse GM-CSF. The cells were fed every 2 days and harvested 7-9 days later. 75-90 % of cells were CDl lc positive.
• DC vaccine for treatment of mice DCs were pulsed with tumor cell lysate and activated with lOug/ml CpG . DCs were subcutaneously injected into mouse.
• DC was pulsed with lOng/ml mouse gplOO peptide (amino acids 25-33, which is presented by H2-Db class I molecules, Alpha Diagnostic International, San Antonio, TX) and activated with lOug/ml CpG for 2 hours.
• DC was washed with PBS, and co-cultured with CFSE labeled Pmel-1 cells.
• Pmel-1 cells proliferation was analyzed by FACscan.
• Pmel-1 lymphocytes were isolated from lymph nodes and spleen of na ' ive
• mice CD8 lymphocytes were enriched by negative selection using Cedarlane purification column. At least 85% of the resulting cells were CD8+. 5xl0 5 cells were transferred into B57BL/6 mice. The day after adoptive transfer, mice received tumor lysate pulsed DC vaccine. To deplete CD4 cells, aCD4 was administered approximately 7 and 9 days later; mice were inject ip with 250ug of CD4 mAb (clone GK1.5). To deplete CD8 cells, mice received 250ug of CD8 mAb (clone 2.43). To deplete FoxP3 cells in DEREG mice, 5 ⁇ g DT was injected. Flow cytometry was used to confirm depletion of target cells. For mTor inhibitor treatment, 15 ⁇ g temsirolimus was injected i.p. each day for 2 weeks. Flow cytometry was used to analyze memory cells and Treg cells.
• Single-cell suspensions of splenocytes (1 x 10 7 cells/ml) were obtained from na ' ive mice and pulsed with or without 10 ⁇ peptide in DMEM containing 10% FBS for 30 min at 37°C.
• Each cell population was then labeled with a different concentration of CFSE (0.5 or 12.5 ⁇ ) at 2 x 10 7 cells/ml in PBS/0.1%>BSA.
• CFSE labeling was stopped by addition of an equal volume of FBS for 1 min, and washed 3 times with RPMI complete medium. 5 x 10 6 cells from each peptide-pulsed or unpulsed population were mixed and injected i.v.
• Example 2 mTOR inhibition enhances anti-tumor immunity
• Temsirolimus can directly inhibit the growth of some tumors, therefore a tumor prevention study was performed to assess the immune effects of temsirolimus.
• DC vaccine By administering DC vaccine and temsirolimus 13 days prior to tumor challenge, there is no possibility for a direct antitumor effect, and any decrease in tumor growth can be attributed to immune stimulation.
• Administering DC vaccine alone decreased growth of B16 tumor cells in mice, however, most mice eventually died due to tumor growth. In contrast, the combination of DC vaccine and temsirolimus resulted in 100% survival and completely prevented the growth of B16 tumor cells.
• Thy 1.1 Pmel-1 lymphocytes were adoptively transferred into Thy 1.2 B6 mice ( Figure lb).
• Pmel-1 transgenic mice carry a rearranged T cell receptor that recognizes a gplOO epitope (amino acids 25-33) presented by H2-Db MHC class I molecules.
• Lymphocytes were harvested from B6 mice after they were treated with DC vaccine and temsirolimus.
• Temsirolimus had both immune stimulating and immune suppressing effects when administered with the DC vaccine.
• Temsirolimus slightly decreased the percent of CD8 cells that were Pmel-1 lymphocytes (p-value 0.08), however, Pmel-1 lymphocytes had increased expression of Eomes, which is an early marker for memory cell formation.
• Example 3 CD4 depletion enhanced the antitumor effect of mTor inhibition
• Temsirolimus produced a net antitumor immune response despite an increase in Tregs. Furthermore, the presence of tumor itself increased Tregs (Figure 8). Therefore, we hypothesized that the antitumor immunity induced by mTOR inhibition can be further enhanced by targeting Tregs.
• Tregs Currently there is no clinical strategy to selectively remove Tregs; however, it is feasible to deplete all CD4 lymphocytes. However, CD4 effector cells are required for immune activation. Therefore, in our mouse model, CD4 lymphocytes were depleted with aCD4 antibody (aCD4) starting 6 days after immune stimulation by the implanted tumor (Figure 2).
• Figure 1 shows the results in a mouse model of melanoma.
• lymphocytes were harvested on day 45 and assessed for tumor-specific IFN- ⁇ response (Figure 2b).
• IFN- ⁇ response In tumor-bearing mice that received no treatment, there was no IFN- ⁇ response.
• Treatment with either aCD4 or temsirolimus produced some IFN- ⁇ response; however, the combination treatment produced the largest IFN- ⁇ response.
• naive mice were treated with a single dose of aCD4. Nearly all CD4 cells were depleted from the peripheral blood, spleen and lymph nodes by the next day (Figure 2c) while CD8 cells were preserved (Figure 2d).
• FoxP3+CD4+ cells were depleted and remained low even 10 days following administration of aCD4 ( Figure 2e).
• a single dose of aCD4 reduced the population of all CD4 subsets ( Figure 9).
• Example 4 Combination of CD4 depletion and temsirolimus generated anti-tumor immunity that was dependent on memory CD8 cells
• temsirolimus can have a direct antitumor effect, it was important to establish that the combination of aCD4 and temsirolimus was generating an effective antitumor immunity dependent on CD8 lymphocytes.
• Balb/c mice bearing RENCA tumors were treated with temsirolimus alone for 10 days and then challenged with a second RENCA tumor ( Figure 3a).
• Mice injected with aCD8 antibody (aCD8) to deplete CD8 effectors cells had increased growth of the second RENCA tumor, indicating that even temsirolimus alone works at least in part by stimulating an immune response.
• the combination of temsirolimus and aCD4 completely prevented the growth of second RENCA tumors; however, aCD8 removed the antitumor effect on the second tumors, indicating the importance of cellular immunity to tumor control (Figure 3b).
• Example 5 Combination of CD4 depletion and temsirolimus treatment enhanced function of CD8 memory cells
• the expanded CD8 cells from the combination treatment group had the highest expression of Eomes, an early memory marker.
• the combination therapy produced CD 8 lymphocytes with the strongest memory phenotype capable of rapidly expanding in response to a repeat antigen challenge.
• Tregs were present during immune priming since CD4 depletion was initiated at least 6 days after immune stimulation.
• Treg depletion As the underlying mechanism for immune stimulation following CD4 depletion, Tregs were replaced after CD4 depletion ( Figure 6a). Mice treated with aCD4 and temsirolimus developed the best specific immune memory as assessed by in vivo CTL ( Figure 6b). However, when Tregs from mice treated with DC vaccine were adoptively transferred, specific killing decreased to that of control mice that only received the DC vaccine ( Figure 6b, right most panel). These experiments confirm that with aCD4 it is the Treg depletion that enhances specific immune memory formation.
• CD4+CD25+ that recovered after CD4 depletion were less immunosuppressive, possibly because they were less likely to be tumor-specific Tregs (Figure 7d).
• the recovered CD4 effector cells were also less effective as indicated by lower ⁇ and IL4 secretion ( Figure 7c). It is possible that both CD4 effector cells and Tregs were less likely to be tumor- specific.
• Example 8 Combination of CD4 lymphocyte depleting agent and adoptive immune therapeutic agent enhances antitumor affect
• Example 9 Combination of CD4 lymphocyte depleting agent and immune checkpoint inhibitor enhances antitumor affect
• CD4 lymphocytes were depleted with aCD4 antibody on days 6 and 10 ( Figure 13 A). Lymphocytes were harvested on day 45, restimulated with CA9 peptide, and stained for CD8 and IFNy. Results are representative of duplicate experiments ( Figure 13B).
• splenocytes were harvest on day 45 and cultured with IL2, RENCA lysate and CA9 peptide.
• Target cells were prepared by labeling RENCA cells with CFSE.
• Effector and target cells were co-cultured at a ratio of 50: 1 and analyzed by FACS for the percent of CFSE+ cells that were 7-AAD positive and annexin V negative (Figure 13C). Histograms provide mean + SEM. *p ⁇ 0.05.
• B16 tumor growth curve shows that anti-CD4 antibody has anti-tumor effect ( Figure 13D). As shown in Figure 13, CD4 depletion produces tumor- specific CD8 cells capable of activating and tumor cell killing in the presence of tumor antigen.
• Example 11 Combination of CD4 lymphocyte depleting agent, adoptive immune therapeutic agent and mTOR inhibitor enhances anti-tumor affect

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