Methods and compositions for treating cancer and infectious diseases

Abstract

To provide compositions comprising a CD4 lymphocyte-depleting agent and use methods thereof for treating, preventing, ameliorating the severity and / or slowing the progression of diseases in subjects in need thereof.SOLUTION: Disclosed is a method for treating, preventing, ameliorating the severity and / or slowing the progression of a disease in a subject in need thereof, comprising: administering, to the patient, a CD4 lymphocyte-depleting agent; administering at least one additional agent selected from the group consisting of an immune checkpoint inhibitor, an adoptive immunotherapeutic agent, an immune adjuvant and an immune modulating agent; and treating, preventing, ameliorating the severity and / or slowing the progression of the disease in the subject by administering, to the subject, therapeutically effective amounts of the CD4 lymphocyte-depleting agent and the additional agent.SELECTED DRAWING: Figure 11

Description

[Technical Field]

[0001] 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 combination with any one or more of immune checkpoint inhibitors, adoptive immunotherapeutics, immunoadjuvants, or immunomodulatory agents. [Background technology]

[0002] All documents cited herein are incorporated by reference in their entirety to the same extent as if each individual document or patent application was specifically and individually indicated to be incorporated by reference. The following description contains information that may be useful in understanding the present invention. None of the information presented herein is to be construed as prior art or related to the presently claimed invention, or any document specifically or implicitly referenced thereto is to be construed as prior art.

[0003] The immune system can prevent cancer. Effective immune stimulation produces long-term memory lymphocytes that can rapidly respond to repeated antigen challenges. CD4-expressing lymphocytes include both helper T cells and regulatory T cells. Helper T cells are essential for mounting adaptive immune responses. However, regulatory T cells (Tregs) inhibit the function of cytotoxic T cells and generally function to limit immune responses. Therefore, CD4 depletion has been evaluated as a method to eliminate Treg activity. Although Treg cells represent only a small fraction of CD4 lymphocytes, CD4 depletion remains an effective approach to eliminate Treg activity and, importantly, has the potential for rapid translation to clinical use. Humanized CD4-depleting antibodies are being evaluated as a method to inhibit the immune system in clinical trials for autoimmune diseases. However, this paper describes the use of CD4 lymphocyte-depleting antibodies to stimulate the immune system. Provided herein are treatments for cancer and infectious diseases in a subject using CD4+ lymphocyte depletion in combination with any one or more of immune checkpoint inhibitors, adoptive immunotherapeutics, immunoadjuvants, and immunomodulatory agents, or combinations thereof. Summary of the Invention

[0004] In various embodiments of the present invention, there is provided a method of treating, preventing, reducing the severity, and / or delaying the progression of a disease in a subject, comprising 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, and / or delaying the progression of the disease in the subject.

[0005] In various embodiments of the present invention, methods are provided for treating, preventing, reducing the severity, and / or delaying the progression of a disease 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 checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulator; and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent and a therapeutically effective amount of the at least one immune checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulator to the subject, thereby treating, preventing, reducing the severity, and / or delaying the progression of the disease in the subject. In some embodiments, the methods further include administering a therapeutically effective amount of an mTOR inhibitor.

[0006] In various embodiments of the present invention, there is provided a method of treating, preventing, reducing the severity, and / or delaying the progression of a disease in a subject, comprising providing a CD4 lymphocyte depleting agent and an adoptive immunotherapy agent (e.g., 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 immunotherapy agent, thereby treating, preventing, reducing the severity, and / or delaying the progression of the disease in the subject.

[0007] In various embodiments of the present invention, there is provided a method of treating, preventing, reducing the severity of, and / or delaying the progression of a disease in a subject, the method comprising providing a CD4 lymphocyte depleting agent and a checkpoint inhibitor (e.g., 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 delaying the progression of the disease in the subject.

[0008] In various embodiments of the present invention, there are provided methods for treating, preventing, reducing the severity, and / or delaying the progression of a disease in a subject, comprising providing a CD4 lymphocyte depleting agent, a dendritic cell (DC) vaccine, and an mTOR inhibitor, and administering effective amounts of the CD4 lymphocyte depleting agent, dendritic cell vaccine, and mTOR inhibitor, respectively, thereby treating, preventing, reducing the severity, and / or delaying the progression of the disease in the subject.

[0009] In various embodiments of the present invention, a pharmaceutical composition is provided that includes a CD4 lymphocyte depleting agent. In further embodiments, a pharmaceutical composition is provided that includes any one of an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulatory agent.

[0010] In various embodiments of the present invention, a kit for treating, preventing, reducing the severity of, and / or delaying the progression of a disease in a subject is provided, the kit comprising a CD4 lymphocyte depleting agent and instructions for using the composition to treat, prevent, reduce the severity of, and / or delay the progression of the disease in the subject.

[0011] In various embodiments of the present invention, a kit for treating, preventing, reducing the severity of, and / or delaying the progression of a disease in a subject is provided. The kit includes a CD4 lymphocyte depleting agent and at least one of an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulatory agent. The kit further includes instructions for using the CD4 lymphocyte depleting agent and the at least one of the immune checkpoint inhibitor, the adoptive immunotherapy agent, the immunoadjuvant, and the immunomodulatory agent to treat, prevent, reduce the severity of, and / or delay the progression of the disease in the subject.

[0012] In various embodiments, the disease is cancer. In typical embodiments, the cancer can be any of kidney cancer, melanoma, prostate cancer, breast cancer, glioblastoma, lung cancer, colon cancer, or bladder cancer. In some embodiments, the disease is an infectious disease.

[0013] Various methods, compositions, and kits of the present invention find utility in the treatment of cancer or infectious diseases. In exemplary embodiments, the methods, compositions, and kits of the present invention find utility in the treatment of specific subsets of malignant cell proliferative disorders or diseases, including, but not limited to, carcinomas and melanomas. In exemplary embodiments, carcinomas include renal cell carcinoma. [The present invention 1001] 1. A method of treating, preventing, reducing the severity of, and / or delaying the progression of a disease in a subject in need thereof, comprising: giving CD4 lymphocyte depleting agents; providing at least one additional agent selected from the group consisting of an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulatory agent; and treating, preventing, reducing the severity of, and / or slowing the progression of, said disease in said subject by administering to said subject therapeutically effective amounts of said CD4 lymphocyte depleting agent and said additional agent. The method comprising: [The present invention 1002] 1. A method of treating, preventing, reducing the severity of, and / or slowing the progression of a disease in a subject, comprising: providing a composition comprising a CD4 lymphocyte depleting agent; and The method comprises administering to the subject a therapeutically effective amount of the composition, thereby treating, preventing, reducing the severity of, and / or delaying the progression of the disease in the subject. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein the disease is an infectious disease or cancer. [The present invention 1004] 1004. The method of claim 1003, wherein said cancer is kidney cancer, melanoma, prostate cancer, breast cancer, glioblastoma, lung cancer, colon cancer, or bladder cancer. [The present invention 1005] The method of any one of claims 1002 to 1001, wherein the subject is a human. [The present invention 1006] The method of claim 1002, wherein the subject has been diagnosed with cancer or an infectious disease. [The present invention 1007] 1002 or 1001, wherein said CD4 lymphocyte depleting agent is selected from the group consisting of a small molecule, a peptide, an antibody or fragment thereof, and a nucleic acid molecule. [The present invention 1008] 1002 or 1001, wherein said CD4 lymphocyte depleting agent is a monoclonal antibody or a 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. [The present invention 1009] 1002 or 1001, wherein said CD4 lymphocyte depleting agent is a humanized anti-CD4 antibody. [The present invention 1010] 1002 or 1001, wherein said CD4 lymphocyte depleting agent is zanolimumab. [The present invention 1011] The CD4 lymphocyte depleting agent is administered in a dose of 100 to 200 mg / day, 200 to 300 mg / day, 300 to 400 mg / day, 400 to 500 mg / day, 500 to 600 mg / day, 600 to 700 mg / day, 700 to 800 mg / day, 800 to 900 mg / day, 900 to 1000 mg / day, 1000 to 1100 mg / day, 1100 to 1200 mg / day, 1200 to 1400 mg / day, 1400 to 1600 mg / day, 1600 to 2000 mg / day, 1800 to 2200 mg / day, 1900 to 2300 mg / day, 2000 to 2500 mg / day, 2100 to 2400 mg / day, 2200 to 2600 mg / day, 2300 to 2500 mg / day, 2400 to 2600 mg / day, 2500 to 3000 mg / day, 2600 to 3200 mg / day, 2700 to 3600 mg / day, 2800 to 3800 mg / day, 2900 to 3900 mg / day, 3000 to 4000 mg / day, 3100 to 3200 mg / day, 3300 to 3400 mg / day, 3400 to 3600 mg / day, 3500 to 3700 mg / day, 3600 to 3800 mg / day, 3700 to 3900 mg / day, 3800 to 4000 mg / day, 39 0 mg / day, 1200-1300 mg / day, 1300-1400 mg / day, 1400-1500 mg / day, 1500-1600 mg / day, 1600-1700 mg / day, 1700-1800 mg / day, 1800-1900 mg / day, or 1900-2000 mg / day. [The present invention 1012] 1003. The method of claim 1001 or 1002, wherein the adoptive immunotherapy agent is a dendritic cell vaccine. [The present invention 1013] 1001 or 1002, wherein said immune checkpoint inhibitor is an anti-PD1 antibody. [The present invention 1014] 1003. The method of claim 1001 or 1002, wherein the composition further comprises an mTOR inhibitor. [The present invention 1015] 1002. The method of any one of claims 1002 to 1001, wherein said composition is administered intravenously, intramuscularly, subcutaneously, intraperitoneally, orally, or via inhalation. [The present invention 1016] 1001. The method of claim 1001, wherein said CD4 lymphocyte depleting agent and said additional agent are administered simultaneously. [The present invention 1017] 1001. The method of claim 1001, wherein said CD4 lymphocyte depleting agent is administered before, during, or after administration of said additional agent. [The present invention 1018] The additional drug is 0.1 to 0.5 mg / day, 0.5 to 1.0 mg / day, 1.0 to 1.5 mg / day, 1.5 to 2.0 mg / day, 2.0 to 2.5 mg / day, 2.5 to 5 mg / day, 5 to 10 mg / day, 10 to 15 mg / day, 15 to 20 mg / day, 20 to 25 mg / day, 25 to 30 mg / day, 30 to 35 mg / day, 35 to 40 mg / day, 40-45 mg / day, 45-50 mg / day, 50-55 mg / day, 55-60 mg / day, 60-65 mg / day, 65-70 mg / day, 70-75 mg / day, 75-80 mg / day, 80-85 mg / day, 85-90 mg / day, 90-95 mg / day, or 95-100 mg / day. [The present invention 1019] 1001. The method of claim 1001, wherein said additional agent is an immune checkpoint inhibitor. [The present invention 1020] 1019. The method of the present invention, wherein 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 IDO1, an antibody against IDO2, an antibody against TIM-3, an antibody against LAG-3, an antibody against OX40R, and an antibody against PS, or a combination thereof. [The present invention 1021] 1001. The method of claim 1001, wherein said additional agent is an adoptive immunotherapy agent. [The present invention 1022] The method of the present invention 1021, wherein the adoptive immunotherapy agent is selected from the group consisting of dendritic cell vaccines, peptide vaccines, chimeric T cell antigen-based therapies, immunocytokines, heat shock protein-based vaccines, tumor lysate-based vaccines, viral vectors containing tumor antigens, viral vaccines, bacterial vaccines, and fungal vaccines, or combinations thereof. [The present invention 1023] 1001. The method of claim 1001, wherein said additional agent is an immune adjuvant. [The present invention 1024] 1023. The method of claim 1023, wherein said immune adjuvant is selected from the group consisting of aluminum salts, virosomes, and oil-based adjuvants, or combinations thereof. [The present invention 1025] 1001. The method of claim 10, wherein said additional agent is an immunomodulatory agent. [The present invention 1026] 1025. The method of claim 1025, wherein said immunomodulatory agent is selected from the group consisting of an mTOR inhibitor, a STAT inhibitor, a TGFβ receptor inhibitor, and a tyrosine kinase inhibitor, or a combination thereof. [The present invention 1027] 1014. The method of claim 10, wherein said mTOR inhibitor is selected from the group consisting of a small molecule, a peptide, an antibody or fragment thereof, a nucleic acid molecule, and a macrolide compound. [The present invention 1028] The method of claim 1027, wherein the mTOR inhibitor is selected from the group consisting of (i) temsirolimus (CCI-779) or a pharmaceutical equivalent, analog, derivative, or salt thereof, (ii) everolimus (RAD-001) or a pharmaceutical equivalent, analog, derivative, or salt thereof, and (iii) sirolimus (rapamycin) or a pharmaceutical equivalent, analog, derivative, or salt thereof. [The present invention 1029] A composition comprising a CD4 lymphocyte depleting agent. [The present invention 1030] A composition comprising a CD4 lymphocyte depleting agent and at least one additional agent selected from the group consisting of an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulatory agent. [The present invention 1031] The composition of any one of claims 1029 to 1030, further comprising a pharmaceutically acceptable carrier. [The present invention 1032] 1. A kit for treating, preventing, reducing the severity of, and / or delaying the progression of a disease in a subject, comprising: a composition comprising a CD4 lymphocyte depleting agent; and Instructions for using said composition to treat, prevent, reduce the severity of, and / or slow the progression of said disease in said subject. The kit comprises: [The present invention 1033] 1. A kit for treating, preventing, reducing the severity of, and / or delaying the progression of a disease in a subject, comprising: CD4 lymphocyte depleting agents; at least one additional agent selected from the group consisting of an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulatory agent; and Instructions for using said composition to treat, prevent, reduce the severity of, and / or slow the progression of said disease in said subject. The kit comprises:

[0014] Exemplary embodiments will now be described with reference to the drawings, in which: The embodiments and drawings disclosed herein are intended to be considered illustrative, not restrictive. [Brief explanation of the drawings]

[0015] [Figure 1A]As with various embodiments of the present invention, we demonstrate both immunostimulatory and inhibitory effects of mTOR inhibition, with the net effect being enhanced antitumor immunity. (a) Experimental scheme for a melanoma tumor prevention model: Mice (n=5 / group) received a tumor lysate-pulsed DC vaccine on days -30 and -23 and received daily intraperitoneal injections of temsirolimus from days -23 to -13. On day 0, B16 tumor cells were subcutaneously injected into the flank. B16 tumor growth (left) and survival (right) curves are shown. Results are representative of duplicate experiments. (b) Experimental scheme for characterizing lymphocytes after DC vaccine and temsirolimus treatment: Thy1.1 Pmel-1 lymphocytes were adoptively transferred into Thy1.2 B6 mice, which received a tumor lysate-pulsed DC vaccine on day -6 and daily temsirolimus for 5 days. Splenocytes were harvested on day 0, stained for CD8, Thy1.1, Tbet, Eomes, and CD4 / FoxP3, and analyzed by flow cytometry. Representative results (left) and summary data (right) are shown. Results are representative of duplicate experiments. (c) Lymphocytes were characterized in a 48-hour in vitro mixed culture with pmel-1 lymphocytes and tumor lysate-pulsed, CpG-activated, temsirolimus-treated DCs. Lymphocytes were stained for CD8, Thy1.1, Tbet, Eomes, and CD4 / FoxP3 and analyzed by flow cytometry. Representative results (left) and summary data (right) are shown. Results are representative of duplicate experiments. *p<0.05, **p<0.01, ***p<0.005. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2-1]This figure demonstrates that CD4 depletion enhanced the antitumor effect of mTor inhibition, in accordance with various embodiments of the present invention. (a) RENCA-CA9 tumor cells were injected into Balb / C mice (n=5 / group) on day 0. CD4 lymphocytes were depleted with αCD4 antibody on days 6 and 10. Mice were treated with temsirolimus daily from days 14 to 34. Tumor growth was monitored. Results are representative of triplicate experiments. (b) In the same experiment, lymphocytes were harvested on day 45, restimulated with CA9 peptide, and stained for CD8 and IFNγ. (c) After CD4 depletion, spleens, lymph nodes, and blood were harvested on days 0, 1, and 10. Lymphocytes were stained for CD4, CD8, and FoxP3 and analyzed by flow cytometry. (c-e) Percentages of CD4 cells in spleens, lymph nodes, and blood on days 0, 1, and 10 after CD4 depletion are shown. (d) Percentage of CD4 or CD8 positive splenocytes after CD4 depletion and percentage of FoxP3 positive CD4 cells are shown. (e) Absolute numbers of CD4, CD8, and CD4 / FoxP3 positive splenocytes from the same experiment. *p<0.05, **p<0.01, ***p<0.005. [Figure 2-2] See description of Figure 2-1. [Figure 3A]These results demonstrate that, in accordance with various embodiments of the present invention, the combination of CD4 depletion and temsirolimus generated anti-tumor immunity dependent on memory CD8 cells. (a) Tumor-bearing mice (n=8) were treated daily with temsirolimus and then rechallenged with RENCA-CA9 35 days after the initial tumor injection. CD8 cells were depleted on day 36 by injection of αCD8 antibody. (b) Tumor-bearing mice (n=8) were treated with temsirolimus and CD4 depletion and then rechallenged with RENCA-CA9 35 days after the initial tumor injection. CD8 cells were depleted on day 36 by injection of αCD8 antibody. (c-e) Lymphocytes were harvested from mice treated with temsirolimus and CD4 depletion. The lymphocytes were cultured in vitro with CA9 peptide and IL2 (10 μg / ml) for 3 days and then adoptively transferred into naive B6 mice. 24 hours later, the mice were intravenously injected with 2 x 10 RENCA tumor cells for a challenge test. 30 days after intravenous tumor challenge, lungs were harvested. Lung weight (d) and the number of lung tumor deposits (e) were measured. IL2, interleukin-2: *p<0.05, **p<0.01, ***p<0.005. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 4A]This figure demonstrates that the combination of CD4 depletion and temsirolimus treatment enhanced the function of CD8 memory cells, in accordance with various embodiments of the present invention. (a) Experimental scheme: Lymphocytes from Thy1.1 Pmel-1 mice were adoptively transferred into Thy1.2 B6 mice, which were stimulated with tumor lysate-pulsed DC vaccine and treated with αCD4 antibody on days 7 and 10, and with temsirolimus daily from days 10 to 24. Results are representative of triplicate experiments. (b) Splenocytes (n=3 / group) were harvested 1 day before the rechallenge with tumor lysate-pulsed DC vaccine, stained with antibodies, and analyzed by flow cytometry. The percentage of CD8 cells positive for the indicated markers is shown. (c) Splenocytes (n=3 / group) were harvested 4 days after the rechallenge with tumor lysate-pulsed DC vaccine, stained with antibodies, and analyzed by flow cytometry. The percentage of CD8 cells positive for the indicated markers is shown. *p<0.05, **p<0.01, ***p<0.005. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5A] According to various embodiments of the present invention, the combination of FoxP3+ Treg depletion and temsirolimus enhances CTL function in vivo. (a) Experimental scheme: DEREG mice received tumor lysate-pulsed DC vaccine and were treated intraperitoneally with diphtheria toxin on days 6 and 10, and with temsirolimus daily from days 10 to 20. In vivo CTL analysis was performed on day 35. (b) CD4+FoxP3+ cells were assessed by flow cytometry using peripheral lymphocytes collected before and after mouse treatment with diphtheria toxin. (c) The in vivo CTL results were analyzed by flow cytometry using splenocytes collected 14 hours after injection of target cells. CTL, cytotoxic T lymphocyte; *p<0.05, **p<0.01, ***p<0.005. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6A]These results demonstrate that adoptive transfer of FoxP3+ Tregs reduced in vivo CTL function in mice treated with a combination of CD4 depletion and temsirolimus, in accordance with various embodiments of the present invention. (a) Experimental scheme: B6 mice were administered a tumor lysate-pulsed DC vaccine and treated intraperitoneally with αCD4 antibody on days 6 and 10, and with temsirolimus daily from days 14 to 24. Tregs selected from lymphocytes from GFP-FoxP3 mice administered a tumor lysate-pulsed DC vaccine were adoptively transferred on day 20, and in vivo CTL analysis was performed on day 35. (b) The in vivo CTL results were analyzed by flow cytometry using splenocytes harvested 14 hours after target cell injection (n=3 / group). *p<0.05, **p<0.01, ***p<0.005. [Figure 6B] See legend to Figure 6A. [Figure 7-1] In accordance with various embodiments of the present invention, the recovered Treg population after CD4-depleting treatment demonstrates reduced immunosuppressive properties. (a) 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) Splenocytes recovered from CD4-depletion were used to enrich for CD4+ cells, which were then sorted by CD25 status. Representative flow cytometry data for CD4+CD25+ and CD4+CD25- status for the resulting groups are shown. (c) CD4+CD25- cells were cocultured with DCs pulsed with B16 tumor lysate. The CD4+CD25- cells were analyzed by flow cytometry for IFNγ or IL-4 expression. (d) CD4+CD25+ cells were cocultured 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 CFSE dilution analysis. *p<0.05, **p<0.01, ***p<0.005. [Figure 7-2] See description of Figure 7-1. [Figure 8]In accordance with various embodiments of the present invention, tumors have been shown to increase Treg expression. The percentage of CD4+ cells expressing FoxP3 was examined in lymph nodes, spleens, bone marrow, and peripheral blood lymphocytes (PBLs) from tumor-bearing mice (approximately 1 cm in diameter) and tumor-free control mice. Flow cytometry results are shown. [Figure 9] Figure 1 shows that αCD4 antibodies eliminate effector CD4 subtypes, in accordance with various embodiments of the present invention. (a) Splenocytes were collected before administration of αCD4 antibodies (day 0) and 1 or 10 days after administration and analyzed by flow cytometry. The total number of splenocytes staining for CD4 and IL-2, IL-4, or IL-17 is shown. (b) The percentage of CD4 cells staining for IL-2, IL-4, or IL-17 is shown. [Figure 10] These results demonstrate that CD4 depletion prior to immune stimulation prevents the formation of CD8 memory cells, consistent with various embodiments of the present invention. The experimental scheme is similar to that shown in Figure 4. An additional group (Group A) in which αCD4 antibody was administered on days -1 and +1 was included. (a) Percentage of CD8 cells expressing Thy1.1 from splenocytes harvested 5 days after re-administration of memory cells is shown. (b) Percentage of CD8 cells expressing Eomes is shown. *p<0.05, **p<0.01, ***p<0.005. [Figure 11] As per various embodiments of the present invention, depletion of Tregs or CD4 has been shown to enhance anti-tumor effects. [Figure 12] In accordance with various embodiments of the present invention, depletion of CD4 has been shown to enhance the anti-tumor effects of PD-1 blockade. [Figure 13] In accordance with various embodiments of the present invention, CD4 depletion enhances tumor-specific cellular immunity. A. Experimental diagram. B. CD4 depletion stimulated tumor-specific interferon-gamma secretion from CD8 cells. Thus, CD4 depletion led to the education of CD8 cells capable of being activated in response to tumors. C. These same CD8 cells were capable of killing RENCA tumor cells. D. B16 tumor growth curve. [Figure 14]

[0023] Figure 1 shows that the combination of anti-CD4 antibody, temsirolimus, and dendritic cell vaccine, according to various embodiments of the present invention, exhibited enhanced anti-tumor activity. A. Experimental diagram. B. B16 tumor growth curve with standard error of the mean (SEM). **p<0.001. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description of the Invention All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22 nd ed.,Pharmaceutical Press (September 15,2012);Hornyak et al.,Introduction to Nanoscience and Nanotechnology,CRC Press (2008);Singleton and Sainsbury,Dictionary of Microbiology and Molecular Biology 3 rd ed.,revised ed.,J. Wiley & Sons(New York,NY 2006);Smith, March's Advanced Organic Chemistry Reactions,Mechanisms and Structure 7 th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3 rded., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide those of skill in the art with a general guide to many of the terms used in this application. For reference to methods of producing antibodies, see Greenfield, Antibodies A Laboratory Manual 2 nd ed., Cold Spring Harbor Press (Cold Spring Harbor, NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, US Patent No. 5,585,089 (1996 December); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 March 24, 332(6162):323-7.

[0017] For pediatric references, see Schwartz et al., The 5-Minute Pediatric Consult 4 th ed., Lippincott Williams & Wilkins,(June 16, 2005);Robertson et al.,The Harriet Lane Handbook:A Manual for Pediatric House Officers 17 thed., Mosby (June 24, 2005); and Hay et al., Current Diagnosis and Treatment in Pediatrics (Current Pediatrics Diagnosis & Treatment) 18 th ed., McGraw-Hill Medical (September 25, 2006).

[0018] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Other features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For convenience, certain terms employed herein in the specification, examples, and appended claims are collected here.

[0019] Unless otherwise specified or implicit from context, the following terms and phrases have the meanings indicated below. Unless expressly specified otherwise or apparent from context, the following terms and phrases do not exclude the meaning acquired in the art to which the term or phrase pertains. Since the scope of the present invention is limited only by the claims, these definitions are provided to help describe particular embodiments and are not intended to limit the claimed invention. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0020] Herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective component(s) that are useful to the embodiments and that embrace the inclusion of unspecified elements, whether or not they are useful. Those skilled in the art will generally understand that the terms used herein are generally intended as "open" terms (e.g., the term "including" shall be interpreted as "including, but not limited to," the term "having" shall be interpreted as "having at least," the term "includes" shall be interpreted as "including, but not limited to," etc.).

[0021] Unless otherwise specified, the terms "a," "an," and "the" and similar references used in the description of specific embodiments in this application (particularly in the claims) may be construed to cover both the singular and the plural. The description of ranges herein is intended merely to serve as a shorthand method of individually referring to each separate value within that range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually set forth herein. Methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or illustrative language (e.g., "such as") in connection with specific embodiments herein is intended merely to facilitate understanding of the application and does not pose a limitation on the scope of the application as otherwise claimed. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Accordingly, the abbreviation "eg" is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0022] Herein, 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, the purpose of which is to prevent, ameliorate, alleviate, ameliorate, suppress, reduce, slow, or halt the progression or severity of a symptom or disease. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a disease. Treatment is typically "effective" if one or more symptoms or clinical indicators are reduced. Alternatively, treatment is "effective" if the progression of the pathology is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or indicators, but also the halting or at least slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Furthermore, "treatment" can mean pursuing or achieving a beneficial result or reducing the likelihood of an individual developing a disease, even if the treatment ultimately fails. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

[0023] A "beneficial outcome" or "desired outcome" can include, but is not limited to, reducing or alleviating the severity of a condition, preventing a condition from worsening, curing a condition, preventing the onset of a condition, reducing the likelihood that a patient will develop a condition, reducing mortality and morbidity, and increasing a patient's lifespan or life expectancy. By way of non-limiting example, a "beneficial outcome" or "desired outcome" can be the alleviation of one or more symptoms, a reduction in the extent of a defect, stabilization (i.e., not worsening) of the progression of the cancer, a delay or slowing of metastasis or invasiveness, and an improvement or alleviation of symptoms associated with cancer.

[0024] As used herein, the term "administering" refers to placing an agent disclosed herein into a subject by a method or route that results in at least partial localization of the agent at a desired site.

[0025] As used herein, "cancer" or "tumor" refers to uncontrolled cell proliferation that interferes with the normal function of the body's 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 within this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. As used herein, the term "epithelial malignancy" refers to cancer arising from epithelial cells. As used herein, the term "invasive" refers to the ability to invade and destroy surrounding tissues. Melanoma is an invasive form of skin tumor. Examples of cancer include, but are not limited to, B-cell lymphoma (Hodgkin's lymphoma and / or non-Hodgkin's lymphoma), brain tumor, breast cancer, colon cancer, lung cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, thyroid cancer, kidney 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.

[0026] Here, "disease" and "condition" can include cancer, tumor, or infectious disease. In typical embodiments, the disease includes, but is not limited to, any type of malignant cell proliferative disorder or disease. In typical embodiments, the disease includes any one or more of kidney cancer, melanoma, prostate cancer, breast cancer, glioblastoma, lung cancer, colon cancer, or bladder cancer.

[0027] As used herein, "immune cells" refers to 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.

[0028] As used herein, "immune response" refers to immunity including, but not limited to, innate immunity, humoral immunity, cellular immunity, immune response, inflammatory response, acquired (adaptive) immunity, autoimmunity, and / or hyperimmunity.

[0029] As used herein, the term "sample" or "biological sample" refers to 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, bodily fluid samples; serum; plasma; urine; saliva; tumor samples; tumor biopsy specimens and / or tissue samples; and the like. The term also encompasses mixtures of the above-mentioned samples. The term "sample" also encompasses untreated or pre-treated (or pre-processed) biological samples. In some embodiments, a sample can comprise one or more cells from a subject. In some embodiments, a sample can be a tumor cell sample, and can, for example, comprise cancer cells, cells derived from a tumor, and / or a tumor biopsy specimen.

[0030] The term "functional," when used in conjunction with "derivative," "variant," or "fragment," refers to a polypeptide that has substantially the same biological activity as the entity or molecule of which it is a derivative, variant, or fragment.

[0031] As used herein, "subject" refers to a human or an animal. Typically, the animal is a vertebrate, such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, water buffalo, feline species, e.g., domestic cats, and canine species, e.g., dogs, foxes, and wolves. As used herein, the terms "patient," "individual," and "subject" are used interchangeably. In embodiments, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Furthermore, the methods described herein can be used to treat livestock and / or pet animals.

[0032] The subject may have previously been diagnosed or identified as suffering from or having a condition (e.g., cancer or infectious disease) that requires monitoring or one or more complications associated with such a condition, and may optionally have already received treatment for the condition or one or more complications associated with the disease / disorder. Alternatively, the subject may not have previously been diagnosed with the condition or one or more complications associated with the disease / disorder. For example, the subject may be one who exhibits one or more risk factors for the condition or one or more complications associated with the condition, or may be a subject who does not exhibit risk factors. A "subject in need" of treatment for a particular condition may be a subject who has the disease / disorder, has been diagnosed with the disease, or is at risk of developing the disease.

[0033] The term "statistically significant" or "significantly" refers to statistical evidence of a difference. This is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is in fact true. The decision is often made using a p-value.

[0034] Here, "CD4 lymphocytes" refers to lymphocytes that express CD4, i.e., lymphocytes that are CD4+. CD4 lymphocytes may be T cells that express CD4.

[0035] The terms "T cells" and "T lymphocytes" are interchangeable and are used interchangeably herein. Examples include, but are not limited to, naive T cells, central memory T cells, effector memory T cells, or combinations thereof.

[0036] Here, the term "therapeutic agent" refers to, for example, an agent used to treat, suppress, prevent, alleviate the effects of, reduce the severity of, reduce the likelihood of onset of, delay the progression of, and / or cure a disease. Diseases targeted by the therapeutic agent include, but are not limited to, infectious diseases, epithelial malignancies, non-epithelial malignancies, lymphomas, leukemias, germ cell tumors, blastomas, antigens expressed in various immune cells, and antigens expressed in cells associated with various blood disorders, and / or inflammatory diseases.

[0037] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is to be understood that this invention is not limited to the particular means, protocols, and reagents described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0038] Provided herein are methods, compositions, and kits for treating cancer and / or infectious diseases using CD4 lymphocyte depleting agents, alone or in combination with various other therapies and drugs that target the immune system. Non-limiting examples of therapies and drugs that can be used in combination with CD4 lymphocyte depleting agents include, but are not limited to, immune checkpoint inhibitors, adoptive immunotherapeutics, immunoadjuvants, and immunomodulatory agents.

[0039] Treatment method In various embodiments, the present invention provides methods for treating, preventing, reducing the severity, and / or delaying the progression of a disease in a subject. The methods include 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, and / or delaying the progression of the disease in the subject. In some embodiments, the disease is cancer or an infectious disease. In some embodiments, the subject has been diagnosed with cancer. In some embodiments, the subject has been diagnosed with an infectious disease. In various embodiments, the subject has suffered from the disease long enough to prime the immune system prior to administration of the composition comprising the CD4 lymphocyte depleting agent. In various embodiments, the subject has been ill for 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 a combination thereof.

[0040] In various embodiments, the present invention provides methods for treating, preventing, reducing the severity, and / or delaying the progression of a disease 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 checkpoint inhibitor, an adoptive immunotherapy, an immunoadjuvant, and an immunomodulator; and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent and a therapeutically effective amount of at least one of the immune checkpoint inhibitor, the adoptive immunotherapy, the immunoadjuvant, and the immunomodulator to the subject, thereby treating, preventing, reducing the severity, and / or delaying the progression of the disease in the subject. In some embodiments, the method further includes administering an mTOR inhibitor to the subject. In various embodiments, the CD4 lymphocyte depleting agent and the additional agent can be administered sequentially or simultaneously. In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease.

[0041] In various embodiments, the present invention provides methods for treating, preventing, reducing the severity, and / or delaying the progression of a disease in a subject. The methods include providing a CD4 lymphocyte depleting agent and an adoptive immunotherapy agent, and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent and a therapeutically effective amount of the adoptive immunotherapy agent to the subject, thereby treating, suppressing, preventing, reducing the severity, and / or delaying the progression of the disease in the subject. In embodiments, the CD4 lymphocyte depleting agent is an anti-CD4 antibody, fragment thereof, or variant thereof, and the adoptive immunotherapy agent is a dendritic cell vaccine. In various embodiments, the disease is cancer or an infectious disease. In some embodiments, the anti-CD4 antibody and the 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 method can further include administering an effective amount of an mTOR inhibitor sequentially or simultaneously with the CD4 lymphocyte depleting agent and the adoptive immunotherapy agent.

[0042] In various embodiments, the present invention provides methods for treating, preventing, reducing the severity, and / or delaying the progression of a disease 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, thereby treating, suppressing, preventing, reducing the severity, and / or delaying the progression of the disease in the subject. In embodiments, the CD4 lymphocyte depleting agent is an anti-CD4 antibody, fragment thereof, or variant thereof, and the checkpoint inhibitor is an anti-PD-1 antibody, fragment thereof, or variant thereof. In various embodiments, the disease is cancer or an infectious disease. In some embodiments, the anti-CD4 antibody and the 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 include administering an mTOR inhibitor. In some embodiments, the method may further comprise administering an effective amount of an mTOR inhibitor sequentially or simultaneously with the CD4 lymphocyte depleting agent and the checkpoint inhibitor.

[0043] In various embodiments, the present invention provides methods for treating, preventing, reducing the severity, and / or delaying the progression of a disease in a subject. The methods include providing a CD4 lymphocyte depleting agent, an adoptive immunotherapy agent, and an mTOR inhibitor, and administering a therapeutically effective amount of the CD4 lymphocyte depleting agent, the adoptive immunotherapy agent, and the mTOR inhibitor to the subject, thereby treating, suppressing, preventing, reducing the severity, and / or delaying the progression of the disease in the subject. In embodiments, the CD4 lymphocyte depleting agent is an anti-CD4 antibody, a fragment thereof, or a variant thereof. In embodiments, the adoptive immunotherapy agent is a dendritic cell vaccine. In embodiments, the mTOR inhibitor is temsirolimus. In various embodiments, the disease is cancer or an infectious disease. In some embodiments, the anti-CD4 antibody, the dendritic cell vaccine, and the mTOR inhibitor (e.g., temsirolimus) are administered simultaneously. In some embodiments, the anti-CD4 antibody, the dendritic cell vaccine, and the mTOR inhibitor (eg, temsirolimus) are administered sequentially.

[0044] In various embodiments, the methods described herein can be used to prevent cancer metastasis or prevent cancer recurrence in a subject in need thereof.

[0045] In various embodiments of the invention, the CD4 lymphocyte depleting agent can be any one or more of a small molecule, a peptide, an antibody or fragment thereof, and a nucleic acid molecule. In embodiments, the antibody specifically binds CD4 to CD4-expressing T cells, such as regulatory T cells (Treg cells).

[0046] The antibody (e.g., an anti-CD4 antibody or an anti-PD-1 antibody) may be any one or more of a monoclonal antibody or fragment thereof, a polyclonal antibody or fragment thereof, a chimeric antibody, a humanized antibody, a human antibody or fragment thereof, or a single-chain antibody. These antibodies may be of any origin, such as rat, mouse, guinea pig, dog, cat, rabbit, pig, cow, horse, goat, donkey, or human. The antibody fragment may be any one or more of a Fab, F(ab')2, or Fv fragment, or a fusion protein thereof.

[0047] In various embodiments, the CD4 lymphocyte depleting agent is a humanized anti-CD4 antibody or fragment thereof. In various embodiments, the CD4 lymphocyte depleting agent is zanolimumab, keliximab, and OKT4.

[0048] In some embodiments of the present invention, the CD4 lymphocyte depleting agent and the additional agent are administered simultaneously. 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 the administration of the additional agent. In further embodiments, the CD4 lymphocyte depleting agent and the additional agent are administered with or without food. According to the present 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 may be any one or more of an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immune adjuvant, and an immunomodulatory agent.

[0049] In various embodiments, the additional agent is an immune checkpoint inhibitor. Examples of 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-L1 antibodies, such as MPDL3280A (RG7446), MEDI4736, and BMS-936559, anti-PD-L2 antibodies, B7-DC-Fc fusion proteins, such as AM P-224, anti-CTLA-4 antibodies, e.g., tremelimumab (CP-675,206) and ipilimumab (MDX-010), antibodies against the B7 / CD28 receptor superfamily, anti-indoleamine (2,3)-dioxygenase (IDO) antibodies, anti-IDO1 antibodies, anti-IDO2 antibodies, tryptophan, tryptophan mimetics, 1-methyltryptophan (1-MT), indoximod (D- 1-methyltryptophan (D-1-MT)), L-1-methyltryptophan (L-1-MT), TX-2274, hydroxyamidine inhibitors, e.g., INCB024360, anti-TIM-3 antibodies, anti-LAG-3 antibodies, e.g., BMS-986016, recombinant soluble LAG-3Ig fusion proteins that activate MHC class II-driven dendritic cell activation, e.g., IMP321, anti-KIR2DL1 / 2 / 3 or anti-KIR) antibodies, e.g., lirilumab (IPH2102), urelumab (BMS-663513), anti-phosphatidylserine (anti-PS) antibodies, e.g., bavituximab, anti-idiotypic mouse monoclonal antibodies against human monoclonal antibodies to N-glycolyl-GM3 ganglioside, e.g., racotumomab (formerly known as 1E10), anti-OX40R antibodies, e.g., IgG These include CD134 mAb, anti-B7-H3 antibodies such as MGA271, as well as small interfering (si) RNA-based cancer vaccines designed to treat cancer by suppressing immune checkpoint genes.Further information can be found in Creelan BC (Update on immune checkpoint inhibitors in lung cancer, Cancer Control. 2014 Jan;21(1):80-9) and Jane de Lartigue (Another Immune Checkpoint Emerges as Anticancer Target, Published online by onclive.com, Tuesday, September 24, 2013), which are incorporated by reference in their entirety as if fully set forth. In some embodiments, 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 IDO1, an antibody against IDO2, an antibody against TIM-3, an antibody against LAG-3, an antibody against OX40R, and an antibody against PS, or a combination thereof.

[0050] In various embodiments, the additional agent is an adoptive immunotherapy agent. In some embodiments, the adoptive immunotherapy agent is selected from the group consisting of a dendritic cell vaccine, a peptide vaccine, a chimeric T cell antigen-based therapy, a T cell-based therapy, an immunocytokine, a heat shock protein-based vaccine, a tumor lysate-based vaccine, a viral vector containing a tumor antigen, a viral vaccine, a bacterial vaccine, and a fungal vaccine, or a combination thereof. Further 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 if fully set forth.

[0051] In various embodiments, the additional agent is an immune adjuvant. Examples of 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™, complete Freund's adjuvant, incomplete Freund's adjuvant, CpG DNA vaccine adjuvant, cholera toxin, cholera toxin B subunit, liposomes, saponin vaccine adjuvant, DDA adjuvant, squalene-based adjuvant, Etx B subunit adjuvant, IL-12 vaccine adjuvant, LTK63 vaccine mutant adjuvant, TiterMax Gold adjuvant, Ribi vaccine adjuvant, and Montanide ISA. 720 adjuvant, Corynebacterium-derived P40 vaccine adjuvant, MPL™ adjuvant, AS04, AS02, lipopolysaccharide vaccine adjuvant, muramyl dipeptide adjuvant, CRL1005, killed Corynebacterium parvum vaccine adjuvant, Montanide ISA 51, Bordetella pertussis component vaccine adjuvant, cationic liposomal vaccine adjuvant, adamantylamide dipeptide vaccine adjuvant, Alacel A, VSA-3 adjuvant, aluminum vaccine adjuvant, Polygen vaccine adjuvant, Adjumer™, algal glucan, Bay R1005, Theramide®, stearyl tyrosine, Specol, Algammulin, Avridine®, calcium phosphate gel, CTA1-DD gene fusion protein, DOC / Alum Complex, gamma inulin, Gerbu adjuvant, GM-CSF, GMDP, recombinant hIFN-gamma / interferon-g, interleukin-1β, interleukin-2, interleukin-7, Sclavo peptide, Rehydragel LV, RehydragelHPA, Loxoribine, MF59, MTP-PE liposome, Murametide, Murapalmitin, D-Murapalmitin, NAGO, Nonionic surfactant vesicle, PMMA, Cochleate protein, QS-21, SPT (antigen formulation), Nanoemulsion vaccine adjuvant, AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR192G vaccine adjuvant, E. coli heat-labile toxin, LT, Amorphous aluminum hydroxyphosphate sulfate adjuvant, Calcium phosphate vaccine adjuvant, Montanide Incomplete Seppic adjuvant, imiquimod, resiquimod, AF03, flagellin, poly(I:C), ISCOMATRIX®, Abisco-100 vaccine adjuvant, albumin-heparin microparticle vaccine adjuvant, AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, immunoliposomes containing antibodies against costimulatory molecules, SAF-1, Sendai proteoliposomes, Sendai-containing lipid matrix, threonylmuramyl dipeptide (TMDP), Ty particle vaccine adjuvant, bupivacaine vaccine adjuvant, DL-PGL (polyester Examples of vaccine adjuvants include poly(DL-lactide-co-glycolide) vaccine adjuvants, IL-15 vaccine adjuvants, LTK72 vaccine adjuvants, MPL-SE vaccine adjuvants, mCT-E112K, a non-toxic mutant of cholera toxin, E112K, Matrix-S, and water-soluble triterpene glucoside compounds. Further information can be found in Sayers et al. (VAXJO: A WEB-BASED VACCINE ADJUVANT DATABASE AND ITS APPLICATION FOR ANALYSIS OF VACCINE ADJUVANTS AND THEIR USES IN VACCINE DEVELOPMENT, 2012; 2012:831486; Epub 2012 Mar 13), which is incorporated by reference in its entirety as if fully set forth herein. In some embodiments, the immune adjuvant is selected from the group consisting of aluminum salts, virosomes, and oil-based adjuvants, or a combination thereof.

[0052] In various embodiments, the additional agent is an immunomodulatory agent, hi some embodiments, the immunomodulatory agent is selected from the group consisting of an mTOR inhibitor, a STAT inhibitor, a TGFβ receptor inhibitor, and a tyrosine kinase inhibitor, or a combination thereof.

[0053] In various embodiments, the method may further comprise administering an mTOR inhibitor to the subject. According to the present invention, the mTOR inhibitor may be any one or more of a small molecule, a peptide, an antibody or fragment thereof, a nucleic acid molecule, and / or a macrolide compound. In embodiments, the antibody specifically binds to mTOR and inhibits mTOR. The antibody may be any one or more of a monoclonal antibody or fragment thereof, a polyclonal antibody or fragment thereof, a chimeric antibody, a humanized antibody, a human antibody or fragment thereof, or a single-chain antibody. These antibodies may be derived from any origin, for example, rat, mouse, guinea pig, dog, cat, rabbit, pig, cow, horse, goat, donkey, or human. The antibody fragment may be any one or more of a Fab, F(ab')2, Fv fragment, or a fusion protein thereof.

[0054] In one embodiment of the invention, the mTOR inhibitor is a macrolide compound. Examples of 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 salt thereof, everolimus (RAD-001) or a pharmaceutical equivalent, analog, derivative, or salt thereof, and / or sirolimus (rapamycin) or a pharmaceutical equivalent, analog, derivative, or salt thereof.

[0055] In various embodiments, the additional agent is a STAT inhibitor. Examples of STAT inhibitors include, but are not limited to, SOCS (suppressors of cytokine signaling cascade), PIAS (suppressor of activated stat), including PIAS1, PIAS2, PIAS3, PIAS4, PIASxa, PIASxb, and PIASy, nifuroxazide (5-nitro-2-furaldehyde-p-hydroxybenzoylhydrazone), N-[2-(1,3,4-oxadiazolyl)]-4-quinolinecarbohydrate, and cisplatin. oxamide, non-peptide small molecule inhibitor, Stattic, STA-21, LLL-3, LLL12, XZH-5, S31-201, SF-1066, SF-1087, 17o, cryptotanshinone, FLL32, C188-9, LY5, BP-1108, BP-1075, galiellalactone, JQ1, STX-0119, FLLL11, FLLL12, FLLL32, FLLL62, hormone-derived nicotinic acid hydrazide, IS3 295, oligonucleotides targeting the STAT pathway, antisense oligonucleotides (ASOs) targeting the STAT pathway, AZD9150 (ISIS-STAT3Rx or ISIS481464, synthetic ASOs against STAT3, STAT3 decoy oligonucleotides (ODNs), STAT3-siRNA, STAT3-G-quartets, STAT5-ODNs, STAT5-siRNA, OPB-31121, peptide and peptidomimetic inhibitors, XpYL, Ac-pYLPQTV-NH3, ISS610, S31-M2001, and CJ-1383. Further information can be found in Furqan et al. (STAT inhibitors for cancer therapy, J Hematol Oncol. 2013 Dec 5;6:90), which is incorporated herein by reference in its entirety as if fully described.

[0056] In various embodiments, the additional agent is a JAK inhibitor. Examples of JAK inhibitors include, but are not limited to, tyrphostin AG490, CP-690550, ruxolitinib (INCB018424), TG101348 (SAR 30253), lestaurtinib (CEP701), CYT387, pacritinib (SB1518), AZD1480, XL019, and LY2784544. Further 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 fully incorporated herein by reference in its entirety.

[0057] In various embodiments, the additional agent is a TGF-beta receptor inhibitor. Examples of TGF-beta receptor inhibitors include, but are not limited to, dominant-negative TGF-beta type II receptor (TβRII), ligand traps, soluble TβRII extracellular domain, soluble betaglycan extracellular domain, recombinant Fc-fusion proteins containing the soluble extracellular domain of either TβRII (TβRII-Fc) or type III receptor / betaglycan, soluble human α2 macroglobulin plasma protein, fully humanized pan-TGF-beta monoclonal neutralizing antibodies, including lerdelimumab (CAT-152), meterimumab (CAT-192), and GC-1008 (fresolimumab), and 1D11; their complementary antibodies. These include antisense oligonucleotides (ASOs) AP12009 (travedersen) and AP-11014, which are designed to hybridize to RNA sequences and promote mRNA degradation; receptor kinase inhibitors SB505124, SB-431542, LY550410, LY580276, LY215729, LY364937, LY2109761, Ki26894, SD-093, and SD-208; peptide aptamers, such as the Trx-SARA aptamer; vectors encoding small hairpin RNAs that suppress TGF-β receptor type II gene expression by RNA interference, and shRNA lentiviral or adeno-associated vectors. Further information can be found in Connolly et al. (Complexities of TGF-β targeted cancer therapy, Int J Biol Sci. 2012;8(7):964-78; Epub 2012 Jul 12) and Kaminska et al. (TGF beta signaling and its role in tumor pathogenesis; Acta Biochim Pol. 2005;52(2):329-37. Epub 2005 Jun 25), which are incorporated herein by reference in their entireties as fully set forth.

[0058] In various embodiments, the additional agent is a tyrosine kinase inhibitor. Examples of tyrosine kinase inhibitors include, but are not limited to, sunitinib, erlotinib, vandetanib, cediranib, brivanib, foretinib, and dovitinib. Further information can be found in Huynh H. (Molecularly targeted therapy in hepatocellular carcinoma, Biochem Pharmacol. 2010 Sep 1; 80(5):550-60. Epub 2010 Apr 4), which is fully incorporated herein by reference in its entirety.

[0059] In some embodiments, the methods described herein further comprise administering a chemotherapeutic agent to a subject receiving a composition comprising a 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 checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulatory agent. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs KW-2189 and CB1 -TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembicine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, e.g., calmus cin, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicin, dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and dexo xorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, chelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, oxate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenergics such as aminoglutethimide, mitotane, trilostane; folic acid supplements , for example, folic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diazicon; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, for example maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanol; nitraelin; pentostatin; fenameth; pirarubicin; losoxantrone;Podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oregon); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, veracrine A, roridin A, and anguidin); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, New Jersey), ABRAXANE® cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumburg, Ill.), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including regimens of irinotecan with 5-FU and leucovorin); the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, oxaliplatin regimen (FOLFOX); lapatinib (Tykerb);Examples of inhibitors that reduce cell proliferation include PKC-alpha inhibitors, Raf inhibitors, H-Ras inhibitors, EGFR inhibitors (e.g., erlotinib (Tarceva®)), and VEGF-A inhibitors, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above. In addition, the treatment method may further include the use of radiation therapy.

[0060] Examples of cancer include, but are not limited to, carcinomas, blastomas, and non-carcinomas. More specific examples of such cancers include, but are not limited to, basal cell carcinoma, cholangiocarcinoma; bladder cancer; osteosarcoma; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intraepithelial neoplasia; kidney cancer; laryngeal cancer; liver cancer. cancer); lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer); lymphoma, Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; non-epithelial malignancies; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine and endometrial cancer; cancer of the urinary system; vulvar cancer; and other epithelial and non-epithelial malignancies.

[0061] According to the present invention, the disease may be a malignant tumor cell proliferative disorder or disease. Further according to the present invention, the disease is renal cell carcinoma or melanoma. Diseases targeted by the therapeutic agent include epithelial malignancies, non-epithelial malignancies, germ cell tumors, and / or blastomas.

[0062] In various embodiments, the infection is caused by an infectious bacterium. Examples of infectious bacteria include: Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria species (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes, and others. pyogenes (Streptococcus group A), Streptococcus agalactiae (Streptococcus group B), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium species.), Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelii Other infectious microorganisms (e.g., protozoa) include: Plasmodium falciparum and Toxoplasma gondii. The compositions and methods described herein contemplate use in treating infections caused by these bacterial agents. Other infectious microorganisms (e.g., protozoa) include: Plasmodium falciparum and Toxoplasma gondii. The compositions and methods described herein contemplate use in treating infections caused by these agents.

[0063] In various embodiments, the viral antigen can be any antigen present in an infectious virus that induces an immune response in a subject. Examples of infectious viruses include: Retroviridae (e.g., HIV); Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., types that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., hantavirus, bunyavirus, phlebovirus, and nairovirus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orthomyxoviridae). Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (mostly adenovirus); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (variola virus, vaccinia virus, poxvirus) and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., agents of spongiform encephalopathies, agents of delta hepatitis (thought to be an incomplete satellite of hepatitis B virus), pathogens of non-A, non-B hepatitis (Class 1 = internally transmitted; Class 2 = parenterally transmitted (i.e., hepatitis C); Norwalk and related viruses, and astrovirus). The compositions and methods described herein contemplate use in treating infections with these viral agents.

[0064] 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. Accordingly, examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. The compositions and methods described herein are contemplated for use in treating infections with these fungal organisms.

[0065] In various embodiments, the subject is a human. In various embodiments, the subject is a mammal, including, but not limited to, a human, a monkey, an ape, a dog, a cat, a cow, a horse, a goat, a pig, a rabbit, a mouse, and a rat.

[0066] In various embodiments, the compositions are administered intravenously, intramuscularly, subcutaneously, intraperitoneally, orally, or via inhalation. According to the present invention, various routes may be used for administration of the compositions of the claimed methods, including, but not limited to, aerosol, intranasal, oral, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsules, and / or injection.

[0067] In various embodiments, the CD4 lymphocyte depleting agent is administered at 100-200 mg / day, 200-300 mg / day, 300-400 mg / day, 400-500 mg / day, 500-600 mg / day, 600-700 mg / day, 700-800 mg / day, 800-900 mg / day, 900-1000 mg / day, 1000-1100 mg / day, 1100-1200 mg / day, 1200-1300 mg / day, 1300-1400 mg / day, 1400-1500 mg / day, 1500-1600 mg / day, 1600-1700 mg / day, 1700-1800 mg / day, 1800-1900 mg / day, or 1900-2000 mg / day. In various embodiments, the additional agent is administered at a dose of 0.1-0.5 mg / day, 0.5-1.0 mg / day, 1.0-1.5 mg / day, 1.5-2.0 mg / day, 2.0-2.5 mg / day, 2.5-5 mg / day, 5-10 mg / day, 10-15 mg / day, 15-20 mg / day, 20-25 mg / day, 25-30 mg / day, 30-35 mg / day g / day, 35–40 mg / day, 40–45 mg / day, 45–50 mg / day, 50–55 mg / day, 55–60 mg / day, 60–65 mg / day, 65–70 mg / day, 70–75 mg / day, 75–80 mg / day, 80–85 mg / day, 85–90 mg / day, 90–95 mg / day, or 95–100 mg / day.

[0068] Pharmaceutical Composition In various embodiments, the present invention provides a composition comprising a CD4 lymphocyte depleting agent. In various embodiments, the present invention provides a composition comprising at least one of an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulatory agent. Examples of immune checkpoint inhibitors, adoptive immunotherapy agents, immunoadjuvants, and immunomodulatory agents are described herein. In various embodiments, the composition comprising the CD4 lymphocyte depleting agent and the composition comprising the additional agent are two separate compositions.

[0069] In accordance with the present invention, the CD4 lymphocyte depleting agents and / or the additional agents useful for treating disease in mammals are often prepared substantially free of naturally occurring immunoglobulins or other biomolecules. Preferred CD4 lymphocyte depleting agents and / or the additional agents also exhibit minimal toxicity when administered to a mammal.

[0070] The pharmaceutical compositions of the present invention may contain any pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" generally refers to an excipient that is safe, non-toxic, and useful in the manufacture of desirable pharmaceutical compositions, including excipients acceptable for human pharmaceutical use as well as veterinary applications. Such excipients may be solid, liquid, semisolid, or, in the case of aerosol compositions, gaseous. Examples of excipients include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, wetting agents, emulsifiers, colorants, release agents, coating agents, sweeteners, flavoring agents, perfumes, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, setting agents, suspending agents, surfactants, humectants, carriers, stabilizers, and combinations thereof.

[0071] In various embodiments, the pharmaceutical compositions of the present invention can be formulated for delivery via any route of administration. "Route of administration" can refer to any route of administration known in the art, including, but not limited to, aerosol, nasal, oral, transmucosal, transdermal, parenteral, enteral, topical, or local routes. "Parenteral" refers to a route of administration that typically involves injection, including intraorbital, infusion, intraarterial, intravesical, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intrathecal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. The composition via the parenteral route can be in the form of a solution or suspension for injection or infusion, or a lyophilized powder. The composition via the parenteral route can be in the form of a solution or suspension for injection or infusion. The pharmaceutical composition for enteral route can be in the form of tablets, gel capsules, sugar-coated tablets, syrup, suspension, solution, powder, granules, emulsion, controlled-release microspheres, nanospheres, lipid vesicles or polymer vesicles.Usually, the composition is administered by injection.These administration methods are well known to those skilled in the art.

[0072] The pharmaceutical composition of the present invention can contain any pharmaceutically acceptable carrier. Here, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle involved in the delivery or transport of the target compound from one body tissue, organ, or part to another body tissue, organ, or part. For example, the carrier can 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 the sense that it must be compatible with the other components of the formulation. Each component of the carrier must also be suitable for use in contact with any tissue or organ with which it may come into contact, meaning that it must not pose a risk of toxicity, irritation, allergic reaction, immunogenicity, or other complications that unduly outweigh its therapeutic benefits.

[0073] The pharmaceutical compositions of the present invention may be encapsulated, tableted, or formulated into emulsions or syrups for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to improve or stabilize the composition or to facilitate its manufacture. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohol, 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.

[0074] The pharmaceutical preparation is prepared according to conventional pharmaceutical technology, including milling, mixing, granulating, and optionally compressing for tablet form; or milling, mixing, and filling for hard gelatin capsule form.When a liquid carrier is used, the preparation will be in the form of syrup, elixir, emulsion, or aqueous or non-aqueous suspension.Such liquid formulations can be administered directly or filled into soft gelatin capsules.

[0075] The pharmaceutical compositions of the present invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is the amount of the composition that will achieve the most effective results in terms of therapeutic efficacy in a given subject. This amount will vary depending on various 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, type and stage of disease, general physical condition, response to a given dose, and type of drug therapy), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. Those skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for example, by observing the subject's response to administration of the compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0076] Formulants may be added to the composition prior to administration to a patient. Liquid formulations may be preferred. For example, these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, bulking agents, or combinations thereof.

[0077] Carbohydrate formulants include sugars or sugar alcohols, such as monosaccharides, disaccharides, or polysaccharides, or water-soluble glucans. Examples of sugars or glucans include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha- and beta-cyclodextrin, soluble starch, hydroxyethyl starch, and carboxymethylcellulose, or mixtures thereof. "Sugar alcohol" is defined as a C4 to C8 hydrocarbon having an OH group, including galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. These sugars or sugar alcohols may be used alone or in combination. There is no specific limit to the amount of sugar or sugar alcohol used, as long as it is soluble in the aqueous formulation. In one embodiment, the concentration of the sugar or sugar alcohol is between 1.0 and 7.0 w / v%, more preferably between 2.0 and 6.0 w / v%.

[0078] Amino acid formulants include the levorotatory (L) forms of carnitine, arginine, and betaine, although other amino acids may be added.

[0079] In some embodiments, the polymer used as the formulant includes polyvinylpyrrolidone (PVP) having an average molecular weight of 2,000 to 3,000 or polyethylene glycol (PEG) having an average molecular weight of 3,000 to 5,000.

[0080] It is preferable to use a buffer in the composition to minimize changes in the pH of the solution before lyophilization or after reconstitution. Almost any physiological buffer can be used, including, but not limited to, citrate, phosphate, succinate, and glutamate buffers, or mixtures thereof. In some embodiments, the concentration is 0.01 to 0.3 molar. Surfactants that can be added to the formulation are described in EP Nos. 270,799 and 268,110.

[0081] Another drug delivery system for increasing circulation half-life is liposomes. Methods for preparing liposome delivery systems are described 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 well known in the art and are described, for example, in Poznansky et al., DRUG DELIVERY SYSTEMS (RL Juliano, ed., Oxford, NY 1980), pp. 253-315; M.L. Poznansky, Pharm Revs (1984) 36:277.

[0082] After preparation of the liquid pharmaceutical composition, it may be freeze-dried to prevent degradation and maintain sterility. Methods for freeze-drying liquid compositions are well known to those skilled in the art. Just before use, the composition may be reconstituted with a sterile diluent (e.g., Ringer's solution, distilled water, or sterile saline) that may contain additional components. Once reconstituted, the composition is administered to a subject using methods well known to those skilled in the art.

[0083] The compositions of the present invention can be sterilized using conventional, well-known sterilization techniques. The resulting solution can be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized preparation can be mixed with a sterilized solution before administration. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting agents, buffers, isotonicity adjusting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and stabilizers (e.g., 1-20% maltose, etc.).

[0084] Kits of the Invention In various embodiments, the present invention provides kits for treating, preventing, reducing the severity of, and / or delaying the progression of a disease in a subject, the kit comprising a composition comprising a CD4 lymphocyte depleting agent and instructions for using the composition to treat, prevent, reduce the severity of, and / or delay the progression of the disease in the subject.

[0085] In various embodiments, the present invention provides kits for treating, preventing, reducing the severity of, and / or delaying the progression of a disease in a subject, the kit comprising a composition comprising a CD4 lymphocyte depleting agent and at least one additional agent selected from the group consisting of an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulatory agent; and instructions for using the composition to treat, prevent, reduce the severity of, and / or delay the progression of the disease in the subject.

[0086] The kit is a combination of materials or components, including at least one of the compositions of the invention. Thus, in some embodiments, the kit includes a composition comprising a drug delivery molecule conjugated to a therapeutic agent, as described above.

[0087] The exact nature of the components comprising the kit of the present invention will depend on its intended use. In one embodiment, the kit is specifically configured for the treatment of mammalian subjects. In another embodiment, the kit is specifically configured for the treatment of human subjects. In a further embodiment, the kit is specifically configured for veterinary applications, including but not limited to the treatment of subjects such as farm animals, livestock, and laboratory animals.

[0088] The kit may include instructions for use. "Instructions for use" typically include specific language describing techniques to be employed in using the components of the kit to affect a desired result. Optionally, the kit also includes other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, dispensing or measuring devices, dressings, or other useful tools readily recognized by those skilled in the art.

[0089] The materials or components organized in the kit can be stored and provided to the practitioner in any convenient and appropriate manner that preserves their operability and usefulness. For example, the components can be dissolved, dehydrated, or lyophilized; they can be provided at room temperature, refrigerated, or frozen. The components are typically contained in suitable packaging material(s). Here, the term "packaging material" refers to one or more physical structures used to store the contents of the kit, such as the inventive compositions. The packaging material is preferably configured, in a well-known manner, to provide a sterile, contaminant-free environment. Here, the term "package" refers to any suitable solid matrix or material, such as glass, plastic, paper, foil, etc., capable of maintaining the individual kit components. Thus, for example, the package can be a glass vial used to contain an appropriate amount of a composition comprising a CD4 lymphocyte depleting agent or a composition comprising a CD4 lymphocyte depleting agent and at least one additional agent selected from the group consisting of an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, and an immunomodulatory agent. The packaging material typically has an exterior label indicating the contents and / or purpose of the kit and / or its components.

[0090] Dosage of the present invention In some embodiments, the effective amount of the CD4 lymphocyte depleting agent in the claimed methods, compositions, and / or kits is about 100-200 mg / day, 200-300 mg / day, 300-400 mg / day, 400-500 mg / day, 500-600 mg / day, 600-700 mg / day, 700-800 mg / day, 800-900 mg / day, 900- The dose may be in the range of 1000 mg / day, 1000-1100 mg / day, 1100-1200 mg / day, 1200-1300 mg / day, 1300-1400 mg / day, 1400-1500 mg / day, 1500-1600 mg / day, 1600-1700 mg / day, 1700-1800 mg / day, 1800-1900 mg / day, or 1900-2000 mg / day. In one embodiment of the present invention, the CD4 lymphocyte depleting agent is a humanized anti-CD4 antibody (e.g., zanolimumab).

[0091] In other embodiments, the effective amount of the CD4 lymphocyte depleting agent in the claimed methods, compositions, and / or kits is about 100-200 mg / week, 200-300 mg / week, 300-400 mg / week, 400-500 mg / week, 500-600 mg / week, 600-700 mg / week, 700-800 mg / week, 800-900 mg / week, 900-1000 mg / week, 1000-1200 mg / week, 1300-1400 mg / week, 1400-1500 mg / week, 1500-1600 mg / week, 1600-1700 mg / week, 1700-1800 mg / week, 1800-2000 mg / week, 1900-2100 mg / week, 2100-2200 mg / week, 2200-2300 mg / week, 2300-2400 mg / week, 2400-2500 mg / week, 2500-2600 mg / week, 2600-2700 mg / week, 2700-2800 mg / week, 2800-3000 mg / week, 3000-3100 mg / week, 3100-3200 mg / week, 3200-3300 mg / week, 3300-3400 mg / week, 3400-3500 mg / week, 3500-3600 mg / week, 3600-3700 mg / week, 3700-3800 mg / week, 3800-3900 mg / week, 3900-4000 mg / week, 4000-4 The dose may range from 1000 mg / week, 1000-1100 mg / week, 1100-1200 mg / week, 1200-1300 mg / week, 1300-1400 mg / week, 1400-1500 mg / week, 1500-1600 mg / week, 1600-1700 mg / week, 1700-1800 mg / week, 1800-1900 mg / week, or 1900-2000 mg / week. In one embodiment of the present invention, the CD4 lymphocyte depleting agent is a humanized anti-CD4 antibody (e.g., zanolimumab). Zanolimumab may be administered at a dose of 980 mg per week.

[0092] In some embodiments, the effective amount of the additional agent in the claimed methods, compositions, and / or kits is about 0.1-0.5 mg / day, 0.5-1.0 mg / day, 1.0-1.5 mg / day, 1.5-2 mg / day, 2.0-2.5 mg / day, 2.5-5 mg / day, 5-10 mg / day, 10-15 mg / day, 15-20 mg / day, 20-25 mg / day, 25-30 mg / day, 30-40 mg / day, 40-45 mg / day, 50-55 mg / day, 60-65 mg / day, 70-75 mg / day, 80-85 mg / day, 90-95 mg / day, 100-125 mg / day, 110-145 mg / day, 120-165 mg / day, 130-175 mg / day, 140-155 mg / day, 150-185 mg / day, 160-185 mg / day, 170-195 mg / day, 180-215 mg / day, 190-215 mg / day, 200-225 mg / day, 210-235 mg / day, 220-245 mg / day, 230-250 mg / day, 240-250 mg / day, 250-265 mg / day, 260-275 mg / day, 270-285 mg / day, 280-295 mg / day, 290-300 mg / day, 300-325 mg / day, 300-345 mg / day, 310-350 mg / day, 320-365 mg / day, 330-345 mg / day The dose can range from 35 mg / day, 35-40 mg / day, 40-45 mg / day, 45-50 mg / day, 50-55 mg / day, 55-60 mg / day, 60-65 mg / day, 65-70 mg / day, 70-75 mg / day, 75-80 mg / day, 80-85 mg / day, 85-90 mg / day, 90-95 mg / day, 95-100 mg / day, 0.75-10 mg / day, or 2-10 mg / day. In various embodiments, the additional agent is an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immune adjuvant, or an immunomodulatory agent.

[0093] In some embodiments, the effective amount of the additional agent in the claimed methods, compositions, and / or kits can be in the range of about 1-5 mg / week, 5-10 mg / week, 10-15 mg / week, 15-20 mg / week, 20-25 mg / week, 25-30 mg / week, 30-35 mg / week, 35-40 mg / week, 40-45 mg / week, 45-50 mg / week, 50-55 mg / week, 55-60 mg / week, 60-65 mg / week, 65-70 mg / week, 70-75 mg / day, 75-80 mg / mg, 80-85 mg / mg, 85-90 mg / week, 90-95 mg / week, or 95-100 mg / week. In various embodiments, the additional agent is an immune checkpoint inhibitor, an adoptive immunotherapy agent, an immunoadjuvant, or an immunomodulatory agent.

[0094] In some embodiments, the at least one additional agent comprises temsirolimus administered at a dose of 25 mg over 30-60 minutes weekly, everolimus administered at a dose of 0.75-10 mg per day, and / or rapamycin administered at a dose of 2-10 mg per day.

[0095] In embodiments of the claimed methods of the present invention, the CD4 lymphocyte depleting agent and the additional agent may be administered simultaneously in the dosages described above using an appropriate administration method, e.g., an administration method recommended by the respective manufacturers of the CD4 lymphocyte depleting agent and the additional agent.

[0096] Alternatively, the CD4 lymphocyte depleting agent and the additional agent are administered sequentially at the doses described above. For example, the additional agent may be administered, for example, daily at the dose described above, and the CD4 lymphocyte depleting agent (e.g., a humanized anti-CD4 antibody) may be administered, for example, daily, weekly, twice weekly, every two weeks, and / or monthly at the dose described above. Alternatively, the additional agent may be administered, for example, daily, weekly, twice weekly, every two weeks, and / or monthly at the dose described above, and the CD4 lymphocyte depleting agent (e.g., a humanized anti-CD4 antibody) may be administered, for example, daily at the dose described above. Furthermore, the additional agent and the CD4 lymphocyte depleting agent (e.g., a humanized anti-CD4 antibody) may each be administered daily, weekly, twice weekly, every two weeks, and / or monthly, where the additional agent is administered at the dose described above on a day different from the day on which the CD4 lymphocyte depleting agent is administered at the dose described above.

[0097] The dosage of the cancer vaccine depends on the vaccine used. The effective amount of the cancer vaccine may be determined by a skilled artisan (e.g., a physician) or may be administered per manufacturer's recommendations. In one embodiment, the first administration of the cancer vaccine is administered on day 0, and the second administration on day 7. The booster agent may be administered at the doses described above on days 2 to 32. Additionally, an anti-CD4 depleting agent may be administered every 2 to 3 weeks, starting on day 10, at the doses described above. For example, when a heat shock protein-based vaccine is used, a heat shock protein (e.g., hsp110 or grp170) is complexed with a tumor antigen (e.g., gp100) and then administered. In an embodiment, for a melanoma vaccine, a complex of hsp110 and gp100 may be administered intradermally at 2.5 mg / kg.

[0098] Typical dosages of an effective amount of the CD4 lymphocyte depleting agent or the additional agent may be in the range recommended by the manufacturer when known therapeutic compounds are used, or as indicated to one skilled in the art by in vitro responses of cells or in vivo responses in animal models.

[0099] For example, the FDA-approved dose of temsirolimus is 25 mg administered intravenously over 30-60 minutes once weekly; everolimus is administered orally at 0.75 mg to 10 mg per day; rapamycin is administered orally at about 2-10 mg per day; and zanolimumab is administered intravenously at about 980 mg per week. The same or similar dosages may be used in accordance with various embodiments of the invention, or different dosages may be used in conjunction with different embodiments of the invention. The actual dosage may depend on the physician's judgment, the patient's condition, and the effectiveness of the therapy, based, for example, on the in vitro reactivity of relevant cell culture or tissue culture samples or responses observed in appropriate animal models. [Example]

[0100] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0101] Example 1: Experimental Method Mice and tumor cells Six- to eight-week-old female C57BL / 6J, BALB / c, and Pmel-1 mice were obtained from the 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 (Depleted Regulatory T Cells) transgenic mice were generated and described by Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, et al. J Exp Med. 2007;204:57-63. All animal experiments were conducted in accordance with federal and state standards, including the Federal Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals.

[0102] B16 cells (a murine melanoma cell line) were transduced with human gp100 (human melanoma antigen) (B16-gp100). RENCA is a murine renal cell carcinoma (RCC) cell line. These 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 L-glutamine, 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0103] Mice were treated with 2x10 tumors in the flank. 5 B16 tumors were generated in C57BL / 6 mice, and RENCA tumors were generated in Balb / C mice by subcutaneous injection of the cells. The diameter of the tumor was measured twice a week using a caliper, and the tumor volume was calculated (the shortest diameter). 2 × longest diameter / 2). In the lung metastasis model, tumor cells were injected intravenously via the tail vein. Lung metastases were counted using a dissecting microscope.

[0104] Antibodies and reagents The following monoclonal antibodies (mAbs), with or without fluorescent conjugates, were obtained from Biolegend (San Diego): anti-CD4 (GK1.5 and RM4-5), anti-CD8 (53-6.7), anti-CD16 / CD32 (9.3), anti-CD90.1 (OX-7), anti-CD11c (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-γ (XMG1.2), anti-IL-2 (JES6.5H4), anti-IL-4 (11B11), and IL-17A (eBio1787). CellTrace 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kit was obtained from Molecular Probes (Eugene, OR). Temsirolimus was obtained from LC Laboratory (Woburn, MA).

[0105] T cell enrichment and Treg selection Mouse spleens and lymph nodes were harvested and processed into single-cell suspensions. CD8 and CD4 T cells were negatively enriched using a mouse CD8 or CD4 recovery column kit (Cedarlane Labs, Burlington, NC). The purity of CD8 and CD4 cells after negative selection exceeded 85%. FoxP3-GFP cells or antibody-stained CD4+CD25+ cells were sorted using a MoFlo Cell Sorter (Fort Collins, CO).

[0106] DC production and T cell stimulation The production of DCs has been described (Wang Y, Wang XY, Subjeck JR, Shrikant PA, Kim HL. Br J Cancer. 2011;104:643-52). Briefly, mouse bone marrow was harvested from the femur and tibia and then plated in a 12-well plate at 1x10 cells per well. 6 The cells were plated at a cell density of 1000 x g with 10% FBS and 10 ng / ml mouse GM-CSF. The cells were fed every two days and harvested after 7–9 days. 75–90% of the cells were CD11c positive. To prepare DC vaccines for mouse treatment, DCs were pulsed with tumor cell lysate and activated with 10 μg / ml CpG. The DCs were injected subcutaneously into mice. For in vitro activation of Pmel-1 cells, DCs were pulsed with 10 ng / ml mouse gp100 peptide (amino acids 25–33, produced by the H2-Db class I molecule; Alpha Diagnostic International, San Antonio, TX) and activated with 10 μg / ml CpG for 2 hours. The DCs were washed with PBS and cocultured with CFSE-labeled Pmel-1 cells. Pmel-1 cell proliferation was analyzed by FACscan.

[0107] Adoptive transfer, CD4 cell depletion, and mTOR inhibition Pmel-1 lymphocytes were isolated from the lymph nodes and spleens of naive Pmel-1 mice. CD8 lymphocytes were enriched by negative selection using Cedarlane purification columns. At least 85% of the resulting cells were CD8+. 5x10 5The cells were transferred into B57BL / 6 mice. The day after adoptive transfer, mice received a tumor lysate-pulsed DC vaccine. To deplete CD4 cells, αCD4 was administered approximately 7 and 9 days later; mice were intraperitoneally injected with 250 μg of CD4 mAb (clone GK1.5). To deplete CD8 cells, mice were intraperitoneally injected with 250 μg of CD8 mAb (clone 2.43). To deplete FoxP3 cells, DEREG mice were injected with 5 μg of DT. Target cell depletion was confirmed using flow cytometry. For mTor inhibitor treatment, 15 μg of temsirolimus was injected intraperitoneally daily for 2 weeks. Memory and Treg cells were analyzed using flow cytometry.

[0108] In vivo CTL analysis In vivo CTL analysis has been described (Wang Y, Wang XY, Subjeck JR, Shrikant PA, Kim HL. Br J Cancer. 2011;104:643-52). Briefly, single-cell suspensions of splenocytes (1x10 7 2x10 cells / ml) were obtained from naive mice and pulsed with or without 10 μM peptide in DMEM containing 10% FBS for 30 min at 37°C. Each cell population was then inoculated with different concentrations of CFSE (0.5 or 12.5 μM) at 2x10 7 Cells were labeled at 1000 cells / ml in PBS / 0.1% BSA. CFSE labeling was stopped by adding an equal volume of FBS for 1 minute and washed three times with RPMI complete medium. Five hundred 10 cells were collected from each of the peptide-pulsed and unpulsed populations. 6 The cells were mixed and injected intravenously into immunized and non-immunized mice. 16 hours after transfer, the mice were sacrificed and splenocytes were harvested. Single-cell suspensions of splenocytes were prepared and analyzed by flow cytometry. The percentage of specific lysis of fluorescent donor splenocytes was calculated as follows: [(number of unpulsed targets × A − number of pulsed targets) / number of unpulsed targets × A] × 100, where A = [number of pulsed targets / number of unpulsed targets] in non-immunized recipient mice.

[0109] statistics Differences in tumor growth were assessed using repeated measures ANOVA. Statistical differences in the number of lung metastases, in vivo CTL killing rates, and mean percentages from flow cytometry were assessed by two-tailed Student's T-test. All statistical analyses were performed using Stata 8.0 (StataCorp, College Station, TX). A P value of <0.05 was considered significant.

[0110] Example 2: mTOR inhibition enhances antitumor immunity In animal models, pharmacological mTOR inhibition can promote the formation of immunological memory, which helps clear infection and reduce tumor growth. This was a surprising finding, given that mTOR inhibitors are used to suppress the immune system in patients undergoing solid organ transplants. Temsirolimus is a rapamycin analog and one of the first mTOR inhibitors approved by the U.S. Food and Drug Administration (FDA) for cancer treatment. In our preclinical model, mTOR inhibition with temsirolimus enhanced the antitumor immunity of DCs pulsed with tumor lysate (referred to here as DC vaccine) (Figure 1a). Temsirolimus can directly inhibit the growth of a portion of tumors, so a tumor prevention study was conducted to evaluate the immunological effects of temsirolimus. Administration of the DC vaccine and temsirolimus 13 days before tumor challenge likely did not have a direct antitumor effect, and immune stimulation may be responsible for all tumor growth reduction. Administration of the DC vaccine alone reduced B16 tumor cell growth in mice, but most mice eventually died from tumor growth. In contrast, the combination of DC vaccine and temsirolimus resulted in 100% survival and completely suppressed the growth of B16 tumor cells.

[0111] To evaluate the immunological effects of temsirolimus on specific CD8 lymphocytes, Thy1.1 Pmel-1 lymphocytes were adoptively transferred into Thy1.2 B6 mice (Figure 1b). Pmel-1 transgenic mice possess a rearranged T cell receptor that recognizes the gp100 epitope (amino acids 25–33) produced by H2-Db MHC class I molecules. Lymphocytes were collected from B6 mice after treatment with DC vaccine and temsirolimus. Temsirolimus, when administered with DC vaccine, had both immunostimulatory and immunosuppressive effects. Temsirolimus slightly reduced the proportion of CD8 cells that were Pmel-1 lymphocytes (p 0.08), but Pmel-1 lymphocytes increased the expression of Eomes, an early marker of memory cell formation. Potential immunosuppressive effects included a decrease in Tbet expression and an increase in Tregs in Pmel-1 lymphocytes. These findings were highly similar to in vitro mixed lymphocyte culture studies (Fig. 1c), in which temsirolimus significantly reduced the proliferation of Pmel-1 lymphocytes induced by DC vaccines.

[0112] Example 3: CD4 depletion enhances the antitumor effect of mTor inhibition Temsirolimus induced a net antitumor immune response despite the increase in Tregs. Furthermore, the presence of tumors themselves increased Tregs (Figure 8). Therefore, we hypothesized that antitumor immunity induced by mTOR inhibition could be further enhanced by targeting Tregs. Currently, there are no clinical strategies for selectively depleting Tregs, but it is possible to deplete all CD4 lymphocytes. However, CD4 effector cells are necessary for immune activation. Therefore, in our mouse model, CD4 lymphocytes were depleted using αCD4 antibody (αCD4) starting 6 days after immune stimulation with the injected tumor (Figure 2). Figure 1 shows the results in a mouse model of melanoma. We tested this approach in a second model of gastric carcinoma, another classically immune-sensitive tumor. In the tumor-treated model, palpable RENCA tumors were observed in Balb / c mice. Temsirolimus has been shown to directly inhibit the growth of RENCA tumor cells in vitro (Wang Y, Wang XY, Subjeck JR, Shrikant PA, Kim HL. Br J Cancer. 2011;104:643-52) and was also effective in reducing tumor growth in our mouse model (Figure 2a). While αCD4 alone was ineffective, adding αCD4 to temsirolimus treatment further reduced tumor growth. Interestingly, the combination of αCD4 and temsirolimus reduced tumor growth even when a cancer vaccine was not used and the injected tumor was the only source of specific immune stimulation.

[0113] In the same experiment, lymphocytes were harvested on day 45 to assess tumor-specific IFN-γ responses (Figure 2b). Untreated tumor-bearing mice had no IFN-γ responses. Treatment with either αCD4 or temsirolimus induced some IFN-γ responses, but treatment with the combination induced the maximal IFN-γ response. To characterize the CD4 lymphocyte depletion in response to αCD4, naive mice were treated with a single dose of αCD4. Nearly all CD4 cells were depleted from the peripheral blood, spleen, and lymph nodes by the next day (Figure 2c), whereas CD8 cells were spared (Figure 2d). Importantly, FoxP3+CD4+ cells were depleted and remained low even 10 days after αCD4 administration (Figure 2e). A single dose of αCD4 reduced all CD4 subset populations (Figure 9).

[0114] Example 4: Combination of CD4 depletion and temsirolimus created anti-tumor immunity dependent on memory CD8 cells Because temsirolimus may have a direct antitumor effect, it was important to confirm that the combination of αCD4 and temsirolimus generated 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 secondary RENCA tumors (Figure 3a). Mice injected with αCD8 antibody (αCD8) to eliminate CD8 effector cells exhibited increased growth of the secondary RENCA tumors, indicating that temsirolimus alone acts, at least in part, by stimulating an immune response. The combination of temsirolimus and αCD4 completely prevented the growth of the secondary RENCA tumors, whereas αCD8 eliminated the antitumor effect against the secondary tumors, demonstrating the importance of cell-mediated immunity for tumor control (Figure 3b).

[0115] To further confirm the role of the immune system and test our proposed treatment of more invasive tumor models, we evaluated whether antitumor immunity could be transferred to prevent the growth of metastatic lung deposits. We used a combination of αCD4 and temsirolimus to treat established, subcutaneous RENCA tumors (Figure 3c). Lymphocytes from these mice were adoptively transferred into naive mice, which were then challenged intravenously with RENCA cells. The combined treatment significantly reduced the establishment and growth of lung deposits (Figure 3c), as quantified by comparison of lung weights (Figure 3d) and lung deposit numbers (Figure 3e). Thus, memory cells were successfully transferred into naive mice and helped control tumor growth. Depletion of CD8 cells demonstrated that antitumor activity was dependent on CD8 cells. Further confirmation of immune stimulation was provided by transferring CD8 lymphocytes from treated mice to naive mice. In a highly invasive cancer model, the transferred lymphocytes were effective in controlling the growth of metastatic deposits.

[0116] Example 5: Combining CD4 depletion and temsirolimus treatment enhanced CD8 memory cell function An important mechanism by which temsirolimus inhibits tumor growth is by enhancing the quality of specific CD8 memory. Therefore, we characterized the quality of CD8 memory cells to assess whether αCD4 further enhances specific CD8 memory formed in the presence of mTOR inhibition. Because the experimental period in tumor-bearing mice was limited due to rapid tumor growth in the control group, we used a model in which a DC vaccine stimulated the immune response, rather than the tumor itself. Using a DC vaccine allows us to assess long-term memory, including recall responses. Thy1.1 Pmel-1 lymphocytes were adoptively transferred into B6 mice, which were subsequently administered the B16-DC vaccine and treated with αCD4 and temsirolimus (Figure 4a).

[0117] To evaluate memory cells, splenocytes were collected before (Figure 4b) or after (Figure 4c) the rechallenge of mice with the DC vaccine on day 46. There was no significant difference in the proportion of Pmel-1 lymphocytes between the experimental groups immediately before the rechallenge (Figure 4b). However, CD8 lymphocytes from mice treated with both αCD4 and temsirolimus showed significantly higher expression of the memory markers Eomes and BCL2. The CD8 lymphocytes from this group also showed significantly higher expression of CD62L, a marker of highly effective central memory cells. Consistent with high-quality memory cells, after rechallenge with the DC vaccine, Pmel-1 cells in the combination treatment group showed the greatest expansion, and CD8 cells showed the highest expression of Tbet and IL2 (Figure 4c). Interestingly, even after the rechallenge, the expanded CD8 cells from the combination treatment group showed the highest expression of the early memory marker Eomes. The combination therapy produced CD8 lymphocytes with the strongest memory phenotype that could be rapidly expanded in response to repeated antigen challenge. In our model, Tregs were present during immune priming because CD4 depletion began at least 6 days after immune stimulation.

[0118] Others have reported that Tregs are required for CD8 memory formation during immune priming. Therefore, in our treatment model, Tregs were removed at least 6 days after the initial immune stimulation. However, we confirmed the importance of having CD4 cells during immune priming. When CD4 removal was performed before immune priming, CD8 memory formation was insufficient, as indicated by the low expansion of tumor-specific CD8 and low Eomes expression after memory cell stimulation (Figure 10).

[0119] Example 6: Deletion or replacement of Foxp3 Treg cells alters in vivo CTL function Our original hypothesis was that depletion of Tregs successfully induced by temsirolimus would enhance antitumor immunity. We chose CD4 depletion as the method of Treg depletion because CD4 depletion is feasible in patients. However, we wanted to test whether the effects of CD4 depletion could be directly attributed to Treg depletion. Therefore, we used DEREG (depleted regulatory T cell) transduced mice, which carry DTR-eGFP under the control of the Foxp3 promoter and allow specific Treg depletion by administration of diphtheria toxin (DT) (Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, et al. J Exp Med. 2007;204:57-63). In an experiment similar to that shown in Figure 2, DT was administered on days 6 and 10 instead of αCD4 (Figure 5a). The immune system was stimulated with DC vaccine, and specific immune memory was evaluated by in vivo CTL analysis on day 35 (Fig. 5c). DT administration eliminated almost all CD4+FoxP3+ lymphocytes (Fig. 5b). Specific killing was significantly increased in mice treated with DT and temsirolimus compared with the control group (Fig. 5c, far right panel). Thus, Treg depletion exhibited an immune effect similar to that of CD4 depletion.

[0120] To fully establish Treg depletion as the underlying mechanism of immune stimulation after CD4 depletion, Tregs were restored after CD4 depletion (Figure 6a). Mice treated with αCD4 and temsirolimus developed the best specific immune memory, as assessed by in vivo CTL (Figure 6b). However, when Tregs from DC vaccine-treated mice were adoptively transferred, specific killing was reduced to that of control mice that received only DC vaccine (Figure 6b, far right panel). These experiments confirm that it is Treg depletion that enhances specific immune memory formation in conjunction with αCD4.

[0121] Example 7: Treg populations that are ultimately recovered after CD4 depletion are less immunosuppressive After treatment with DC vaccine, αCD4, and temsirolimus, the Treg population eventually recovered (Figure 7a). The difference in absolute Treg numbers in the spleen between the experimental groups was not statistically significant. However, the treatment may have had long-term effects on Treg function. Therefore, we evaluated the immunosuppressive function of recovered Tregs. CD4 lymphocytes were classified by CD25 status (Figure 7b). The majority of CD4+CD25+ cells were FoxP3 positive and therefore considered Tregs, while the majority of CD4+CD25- cells were FoxP3 negative and therefore considered CD4 effector cells. In functional studies, control CD4+CD25+ cells suppressed the proliferation of CD8 lymphocytes. However, CD4+CD25+ cells recovered after CD4 depletion were less immunosuppressive, likely because they were less likely to represent tumor-specific Tregs (Figure 7d). Interestingly, after CD4 depletion, the recovered CD4 effector cells were also less effective, as indicated by lower IFNγ and IL-4 secretion (Fig. 7c), suggesting that both CD4 effector cells and Tregs were less likely to be tumor-specific.

[0122] Immunotherapeutic approaches have proven effective in the treatment of solid tumors. The FDA approved sipuleucel-T, becoming the first commercially available cancer vaccine for the treatment of solid tumors. The monoclonal antibody ipilimumab, which targets CTLA4, was recently approved for the treatment of melanoma. Immune checkpoint inhibitors targeting CTLA4 and PD-1 are being actively investigated in numerous clinical trials for a variety of malignancies. While there has always been some indication that immune-based treatments may be curative, even for some patients with metastatic disease, recent advances in immunotherapy have reaffirmed that durable complete remissions are possible. Thus, immunotherapy represents one of the most promising approaches to cancer treatment.

[0123] Example 8: Combining CD4 lymphocyte depleting agents with adoptive immunotherapy drugs enhances antitumor effects B16 tumor cells were injected into DREG mice, which are B6 mice engineered to express the diphtheria toxin receptor under the FoxP3 promoter. Mice received a DC vaccine pulsed with tumor lysate on day 2. Tregs were specifically depleted with diphtheria toxin, and CD4 cells were depleted with αCD4 antibody on days 6 and 10. Tumor growth was monitored. * p=0.05, ** p=0.0009. As shown in Figure 11, the combination of CD4 lymphocyte depletion and DC vaccination enhances the anti-tumor effect compared to DC vaccination alone.

[0124] Example 9: Combining CD4 lymphocyte depleting agents with immune checkpoint inhibitors enhances antitumor effects B16 tumor cells were injected into B6 mice on day 0. CD4 cells were depleted with αCD4 antibody on days 6 and 10. Mice were treated with αPD-1 antibody on days 10 and 24. Tumor growth was monitored. * p=0.02. As shown in Figure 12, the combination of CD4 lymphocyte depletion and anti-PD-1 antibody enhances the anti-tumor effect compared to anti-PD-1 antibody alone.

[0125] Example 10: Anti-CD4 antibodies exhibit anti-tumor activity RENCA-CA9 tumor cells were injected into Balb / C mice (n = 5 / group) on day 0. CD4 lymphocytes were depleted with αCD4 antibody on days 6 and 10 (Figure 13A). Lymphocytes were harvested on day 45, restimulated with CA9 peptide, and stained for CD8 and IFNγ. Results are representative of duplicate experiments (Figure 13B). For in vitro CTL analysis, splenocytes were harvested on day 45 and cultured with IL2, RENCA lysate, and CA9 peptide. Target cells were obtained by CFSE-labeling RENCA cells. Effector and target cells were cocultured at a 50:1 ratio and analyzed by FACS for the percentage of 7-AAD-positive, Annexin V-negative, CFSE+ cells (Figure 13C). Histograms show the mean + SEM. *p<0.05. B16 tumor growth curves demonstrate that anti-CD4 antibodies have an anti-tumor effect (Figure 13D). As shown in Figure 13, CD4 depletion produces tumor-specific CD8 cells that are capable of activation and tumor cell killing in the presence of tumor antigens.

[0126] Example 11: Combination of CD4 lymphocyte depleting agents, adoptive immunotherapeutic drugs, and mTOR inhibitors enhances antitumor efficacy B16 tumor cells were injected subcutaneously into the flank of B6 mice (n=5 / group) on day 0. Mice received tumor lysate-pulsed DC vaccine on day 3 and underwent CD4 lymphocyte depletion (using anti-CD4 antibody) on days 6 and 9. Temsirolimus was injected intraperitoneally daily from day 9 to day 20. B16 tumor cell curves are shown with SEM. ** p<0.001. As shown in Figure 14, the combination of CD4 lymphocyte depleting agent, dendritic cell vaccine, and mTOR inhibitor (temsirolimus) effectively controlled tumor growth.

[0127] The various methods and techniques described above provide many ways of implementing the present application. It should be understood, of course, that not all of the described objectives or advantages can be achieved in accordance with any particular embodiment described herein. Thus, for example, one skilled in the art will recognize that a method can be performed in a manner that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other objectives or advantages taught or suggested herein. Various options are listed herein. It should be understood that some preferred embodiments specifically include one, another, or several features; others specifically exclude one, another, or several features; and still others mitigate a particular feature by including one, another, or several advantageous features.

[0128] Moreover, those skilled in the art will recognize the applicability of various features from different embodiments. Similarly, the various elements, features, and steps described above, as well as other known equivalents of each such element, feature, or step, may be employed by those skilled in the art in various combinations to perform methods consistent with the principles described herein. Among the various elements, features, and steps, some are specifically included and others are specifically excluded in various embodiments.

[0129] While the present application has been disclosed in particular embodiments and examples, those skilled in the art will appreciate that the embodiments of the present application extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses, modifications, and equivalents thereof.

[0130] Preferred embodiments of the present application are described herein, including the best mode known to the inventors for carrying out the application. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. Those skilled in the art may employ such variations, if desired, and it is understood that the application may be practiced otherwise than as specifically described herein. Accordingly, many embodiments of the present application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is covered by this application unless otherwise indicated herein or clearly contradicted by context.

[0131] All patents, patent applications, published patent applications, and other materials, e.g., literature, books, specifications, publications, documents, articles, and / or the like, referenced herein are hereby incorporated by this reference in their entirety, except for any accompanying prosecution history, any that contradicts or conflicts with this document, or any that may have a limiting effect on the broadest scope of the claims herein or later related to this document. By way of example, in the event of a conflict or inconsistency between the explanation, definition, and / or terminology used in connection with any of the incorporated materials and that used in connection with this document, the explanation, definition, and / or terminology used in this document shall control.

[0132] It should be understood that the embodiments of the present application disclosed herein are illustrative of the principles of the embodiments of the present application. Other modifications that may be adopted may be within the scope of the present application. Thus, by way of non-limiting example, alternatives to the embodiments of the present application may be utilized in accordance with the teachings herein. Thus, the embodiments of the present application are not limited to that precisely as shown and described.

[0133] Various embodiments of the present invention have been described in the foregoing detailed description. While these descriptions directly describe the embodiments, it is understood that those skilled in the art will envision modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of this description are intended to be encompassed therein as well. Unless specifically noted, it is the inventors' intention that the words and phrases in the specification and claims be given the ordinary and accustomed meaning to one skilled in the applicable art(s).

[0134] While the foregoing description of various embodiments of the present invention known to applicant at the time of filing this application has been presented, it has been done for purposes of illustration and description. This description is not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in light of the above teachings. The described embodiments serve to explain the principles and practical applications of the present invention and to enable others skilled in the art to utilize the invention in its various embodiments and with various modifications as may be suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0135] While particular embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that, based on the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects, and it is therefore intended in the appended claims to cover within their scope all such changes and modifications as fall within the true spirit and scope of this invention.

Claims

1. 1. A pharmaceutical composition for use in combination with an immune checkpoint inhibitor to treat, prevent, reduce the severity of, and / or delay the progression of cancer in a subject in need thereof, comprising a therapeutically effective amount of an anti-CD4 antibody, administering an anti-CD4 antibody to deplete CD4+ regulatory T cells (Tregs) after the cancer has primed the subject's immune system to stimulate an immune response; the immune checkpoint inhibitor is selected from the group consisting of an antibody against PD-1, an antibody against PD-L1, and a combination thereof; the cancer is melanoma, breast cancer, colon cancer, kidney cancer, or a combination thereof; The medicine.

2. The pharmaceutical described in claim 1, wherein the cancer is renal cancer and administration of the anti-CD4 antibody induces an interferon-gamma (IFN-γ) response in the subject.

3. The pharmaceutical of claim 1 , wherein the subject is a human.

4. The pharmaceutical composition according to claim 1, wherein the anti-CD4 antibody is a monoclonal antibody or a 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.

5. The pharmaceutical composition of claim 1 , wherein the anti-CD4 antibody is a humanized anti-CD4 antibody.

6. The pharmaceutical composition of claim 1, wherein the anti-CD4 antibody is zanolimumab.

7. 2. The pharmaceutical composition of claim 1, wherein the anti-CD4 antibody is administered at a dose of 100 to 200 mg / day, 200 to 300 mg / day, 300 to 400 mg / day, 400 to 500 mg / day, 500 to 600 mg / day, 600 to 700 mg / day, 700 to 800 mg / day, 800 to 900 mg / day, 900 to 1000 mg / day, 1000 to 1100 mg / day, 1100 to 1200 mg / day, 1200 to 1300 mg / day, 1300 to 1400 mg / day, 1400 to 1500 mg / day, 1500 to 1600 mg / day, 1600 to 1700 mg / day, 1700 to 1800 mg / day, 1800 to 1900 mg / day, or 1900 to 2000 mg / day.

8. The pharmaceutical composition of claim 1, wherein the anti-CD4 antibody and the immune checkpoint inhibitor are administered intravenously, intramuscularly, subcutaneously, intraperitoneally, orally, or via inhalation.

9. The pharmaceutical composition of claim 1, wherein the anti-CD4 antibody and the immune checkpoint inhibitor are administered simultaneously.

10. The pharmaceutical composition of claim 1, wherein the anti-CD4 antibody is administered before or after administration of the immune checkpoint inhibitor.

11. The immune checkpoint inhibitor is 0.1 to 0.5 mg / day, 0.5 to 1.0 mg / day, 1.0 to 1.5 mg / day, 1.5 to 2.0 mg / day, 2.0 to 2.5 mg / day, 2.5 to 5 mg / day, 5 to 10 mg / day, 10 to 15 mg / day, 15 to 20 mg / day, 20 to 25 mg / day, 25 to 30 mg / day, 30 to 35 mg / day, 35 to 40 mg / day, 40 to 45 mg / day, 45 to 50 mg / day, 45 to 50 mg / day, 45 to 60 mg / day, 45 to 65 mg / day, 45 to 70 mg / day, 45 to 75 mg / day, 45 to 80 mg / day, 45 to 85 mg / The medicament of claim 1, which is administered at a dose of up to 40 mg / day, 40-45 mg / day, 45-50 mg / day, 50-55 mg / day, 55-60 mg / day, 60-65 mg / day, 65-70 mg / day, 70-75 mg / day, 75-80 mg / day, 80-85 mg / day, 85-90 mg / day, 90-95 mg / day, or 95-100 mg / day.

12. The pharmaceutical of claim 1 for use in further combination with a therapeutically effective amount of an adoptive immunotherapy agent.

13. The pharmaceutical of claim 12, wherein the adoptive immunotherapy agent is selected from the group consisting of dendritic cell vaccines, peptide vaccines, chimeric T cell antigen-based therapies, immunocytokines, heat shock protein-based vaccines, tumor lysate-based vaccines, viral vectors containing tumor antigens, and combinations thereof.

14. The pharmaceutical according to claim 12, wherein the adoptive immunotherapy agent is a dendritic cell vaccine.

15. The pharmaceutical according to claim 1, for use in further combination with an effective amount of an immune adjuvant.

16. The pharmaceutical according to claim 15, wherein the immunoadjuvant is selected from the group consisting of aluminum salts, virosomes, oil-based adjuvants, and combinations thereof.

17. The pharmaceutical of claim 1 for use in further combination with a therapeutically effective amount of an immunomodulator.

18. The pharmaceutical composition of claim 17, wherein the immunomodulatory agent is an mTOR inhibitor.

19. 19. The pharmaceutical agent of claim 18, wherein the mTOR inhibitor is selected from the group consisting of (i) temsirolimus (CCI-779), (ii) everolimus (RAD-001), and (iii) sirolimus (rapamycin).

20. 1. A kit for treating, preventing the progression of, reducing the severity of, and / or delaying the progression of cancer in a subject, comprising: a composition comprising an anti-CD4 antibody; A composition comprising an immune checkpoint inhibitor; and Instructions for using said composition to treat, prevent the progression of, reduce the severity of, and / or slow the progression of said cancer in said subject. Including, administering an anti-CD4 antibody to deplete CD4+ regulatory T cells (Tregs) after the cancer has primed the subject's immune system to stimulate an immune response; the immune checkpoint inhibitor is selected from the group consisting of an antibody against PD-1, an antibody against PD-L1, and a combination thereof; the cancer is melanoma, breast cancer, colon cancer, kidney cancer, or a combination thereof; The kit.

21. Compositions containing anti-IFN-γ antibodies further comprising An anti-IFN-γ antibody is used to detect the level of IFN-γ after administration of an anti-CD4 antibody; 21. The kit of claim 20.

22. The kit of claim 20, wherein the cancer is renal cancer and administration of an anti-CD4 antibody induces an interferon-γ (IFN-γ) response in the subject.

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