WO2018203875A1 - Antitumor effect of cryptotanshinone - Google Patents

Abstract

Methods are disclosed for treating a tumor in a subject. The methods include administering to the subject a therapeutically effective amount of a) cryptotanshinone (CT) or a salt or a derivative thereof, and b) a Programmed Death (PD-1) antagonist, a Cytotoxic T- lymphocyte-Associated Protein 4 (CTLA-4) antagonist, a B- and T-lymphocyte Attenuator (BTLA) antagonist, T-cell Immunoglobulin and Mucin-domain containing-3 (TIM-3) antagonist, a Lymphocyte- Activation Gene 3 (LAG3) antagonist, or a combination thereof, thereby treating the tumor in the subject. In some non-limiting examples the tumor is a hepatic cancer, such as a hepatic carcinoma, or a lung cancer, such as a small cell carcinoma of the lung or a non-small cell carcinoma of the lung.

Description

ANTITUMOR EFFECT OF CRYPTOTANSHINONE

FIELD OF THE DISCLOSURE

This relates to the field of cancer, specifically to methods of treating a cancer, such as hepatic cancer or lung cancer.

BACKGROUND

Lung cancer is the leading cause of cancer related mortality world-wide including the United States, with a 5-year overall survival rate of only 15% for all stages of patients. The majority (-75%) of lung cancer patients are diagnosed at an advanced stage of the disease.

Nonspecific cytotoxic chemotherapy is associated with severe side effects, while surgery is not effective for late-stage disease. Targeted therapy against epidermal growth factor (e.g. erlotinib, afatinib, etc) or anaplastic lymphoma kinase (crizotinib and ceritinib) is helpful only for a small subgroup of patients with relevant targetable genomic alterations. Recently, checkpoint inhibitor antibodies against PD-1 (Nivolumab and Pembrolizumab) or PD-L1 (Duvalumab, Atezolizumab, and Avelumab) have been tried for the treatment of lung cancer patients. The overall response rate is approximately 20% to 25%, and 75% of lung cancer patients failed to respond. Therefore, development of additional effective therapies for lung cancers is still needed.

Liver cancer is the fifth most prevalent neoplasm in the world and the third most common cause of cancer-related mortality. According to the American Cancer Society, hepatocellular carcinoma (HCC) accounts for about 75 percent of liver cancer cases. There are often no symptoms of liver cancer until the later stages. Surgery is the standard treatment for liver cancer as this type of cancer does not respond well to most chemotherapy drugs. Therefore, development of additional effective therapies for liver cancers is still needed.

SUMMARY OF THE DISCLOSURE

Methods are disclosed for treating a tumor in a subject. The methods include administering to the subject a therapeutically effective amount of a) cryptotanshinone (CT) or a salt or a derivative thereof, and b) a Programmed cell Death protein 1 (PD-1) antagonist (including antagonists of PD- 1, PD-L1, and PD-L2), a Cytotoxic T-lymphocyte- Associated Protein 4 (CTLA-4) antagonist, a B- and T-lymphocyte Attenuator (BTLA) antagonist, a T-cell Immunoglobulin and Mucin-domain containing-3 (TEV1-3) antagonist, a Lymphocyte- Activation Gene 3 (LAG3) antagonist, or a combination thereof, thereby treating the tumor in the subject. In some non- limiting examples the tumor is a hepatic cancer, such as a hepatic carcinoma, or a lung cancer, such as a small cell carcinoma of the lung or a non-small cell carcinoma of the lung. The subject can be human.

In some embodiments the PD-1 antagonist, the CTLA-4, the TIM-3 antagonist, the LAG3 antagonist, and/or the BTLA antagonist can be an inhibitory RNA, a dominant negative protein, or an antibody that specifically binds to PD-1, PD-L1, PD-L2, TIM-3, LAG3, BLTA, or CLTA-4, or an antigen binding fragment thereof. For example, the antibody can be a monoclonal antibody, or an antigen binding fragment thereof, which specifically binds to human PD-1, PD-L1, or PD-L2 and blocks the binding of human PD-L1 or PD-L2 to human PD-1. In some embodiments, the antibody is one of nivolumab, pembrolizumab, pidilizumab, atezolizumab, avelumab, durvalumab, or ipilimumab.

The CT (or a salt or a derivative thereof) and the PD-1 antagonist, the CTLA-4 antagonist, the TIM-3 antagonist, the LAG3 antagonist, the BTLA antagonist, or a combination thereof, can be administered to the subject by any suitable means. In some embodiments, the CT is administered by oral, intratumor, intramuscular or intravenous administration. In some embodiments, the PD-1 antagonist, the CTLA-4 antagonist, the TIM-3 antagonist, the LAG3 antagonist, or the BTLA antagonist, is administered by intratumor intramuscular or intravenous administration.

In additional embodiments, the disclosed methods can comprise surgically resecting the tumor from the subject in addition to administration of the therapeutically effective amount of CT (or a salt or a derivative thereof) and the PD-1 antagonist, the CTLA-4 antagonist, the TIM-3 antagonist, the LAG3 antagonist, the BTLA antagonist, or the combination thereof.

In several embodiments, the disclosed methods of treating the tumor in the subject comprise decreasing tumor volume, decreasing the number or size of metastases, or lessening a symptom of the tumor. In some embodiments, the disclosed methods of treating the tumor in the subject further inhibit reoccurrence of the tumor in the subject.

The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES FIGs. 1A-1B. CT exhibited the dual capacities of inhibiting the proliferation of human lung cancer cells and inducing the maturation of human DCs. FIG. 1A, A549 human lung cancer cells seeded in a 96- well plate at 4 xlO³ cells/well were treated in triplicate with indicated concentrations of CT for 48 hours in a CO2 incubator (37°C humidified air containing 5% CO2) and pulsed with 0.5 μCi/well of ³H-TdR for the last 4 hours. After cell harvest and β- scintillation counting, the % proliferation was calculated as % proliferation = (CPM with compound - CPM blank) ÷ (CPM without compound - CPM blank) x 100. IC50 was the calculated concentration at which 50% of the proliferation was inhibited. FIG. IB, Human DCs were cultured in a CO2 incubator for 48 hours in the absence (sham) or presence of LPS at 100 ng/ml or CT at 10 μg/ml before they were immunostained and analyzed by flow cytometry. Shown are the overlay histograms illustrating the expression of surface ostimulatory (CD80, and CD86) and MHC (HLA- ABC and HLA-DR) of sham (solid line) and treated (grey area) DCs.

FIGs. 2A-2B. CT was not cytotoxic for human erythrocytes and macrophages. FIG. 2A, Human erythrocytes were suspended in PBS (2%) containing CT at 0-20 g/ml or water for positive control (upper panel). After incubation for 30 minutes at room temperature, the tubes were centrifuged at 500xg for 5 minutes (lower panel). FIG. 2B, Human macrophages seeded in a 24- well plate were cultured with indicated concentrations of CT in a CO2 incubator for 48 hours before the plate was stained with Toluidine blue. After photo recording (upper panel), 0.5 ml of 1% SDS were added into each well to solubilize the dye. The absorbance of the wells at 620 nm was measured using a spectrometer and graphed as the average of triplicate wells.

FIGs. 3A- 3C. CT inhibited the proliferation of LLC through G2/M arrest. FIG. 3A, LLC cancer cells were treated in triplicate in a 96-well plate for 48 hours in a CO2 incubator with CT at concentrations indicated before their proliferation was assessed by ³H-TdR incorporation. The % proliferation was calculated as (CPM with compound - CPM blank) ÷ (CPM without compound - CPM blank) x 100. FIG. 3B, LLC cells seeded in a 12-well plate at

3xl0⁵ cells/ml/well were treated with various concentrations of CT or 1% NaN3 (as a positive control) for 24 hours in a CO2 incubator. Subsequently, the cells were harvested and stained with an apoptosis detection kit. Only the dot plot (PI vs annexin V) of cells treated with 10 μg/ml of CT and 1% NaN3 are shown. FIG. 3C, Synchronized LLC treated with various concentrations of CT for 48 hours in a C02 incubator were stained with PI and subsequently analyzed for cell cycle. The data were graphed using FlowJo.

FIGs. 4A-4B. CT altered intracellular signaling compatible with G2/M arrest in LLC. FIG. 4A, LLC cells serum-starved for 24 hours were treated with indicated concentrations of CT and solubilized in lxSDS sample buffer at 10⁷ cells/ml. The samples were separated on a 4-12% gradient NuPAGE™ gel, transferred on a piece of Immobilon™ membrane, blocked, and reacted with anti-phospho-p53, anti-cyclin Bl, or anti-Cdc2. The membrane used for probing phosphor-p53 was stripped, and re-probed with anti-p53. After the images were taken, the membranes were stripped and re-probed with anti-GAPDH. FIG. 4B, a chart illustrating CT-induced signaling pathways responsible for CT-induced G2/M arrest in LLC cells.

FIGs. 5A-5B. CT induced maturation of mouse DCs. FIG. 5A, Mouse bone marrow- derived DCs were incubated in a CO2 incubator for 48 hours in the absence or presence of CT or LPS at the concentrations specified before they were immunostained for detection of surface marker (CD80, CD83, CD86, and I-A/E) expression by flow cytometry. Shown are the overlay histograms of sham (blue line) and treated (red line) DCs. FIG. 5B, Mouse bone marrow-derived DCs were cultured in the absence (sham) or presence of various concentrations of CT for 24 or 48 hours before the supernatants were harvested for the measurement of indicated cytokines. Shown is the average (mean ± SD) of three independent experiments. *p<0.05 and **p<0.001.

FIG. 6. CT regulation of NF-κΒ and MAPK activation. Mouse bone marrow-derived DCs were treated with 10 g/ml of CT for indicated time periods were solubilized in lxSDS sample buffer at 10⁷ cells/ml. The samples were separated on a 4-12% gradient NuPAGE™ gel, transferred on a piece of Immobilon™ membrane, blocked, and reacted with anti-ΙκΒα, anti- phospho-Erks, anti-phospho-p38, or anti-phospho-JNK. After the images were taken, the membranes were stripped and re-probed with anti-GAPDH, anti-Erks, anti-p38, and anti-JNK, respectively.

FIGs. 7A-7B. Comparison of CT induced maturation of wild-type (WT), TLR4^{_ "}, and MyD88^v- mouse DCs. FIG. 7A, WT (C57BL/6), TLR4^_/", and MyD88^_/" mouse DCs were incubated in a CO2 incubator for 48 hours with CT or LPS at the concentrations specified before they were immunostained for the detection of surface marker (CD80, CD83, CD86, and I-A/E) expression by flow cytometry. Shown are the overlay histograms of sham (blue line) and treated (red line) DCs. FIG. 7B, Mouse bone marrow-derived DCs were cultured in the absence (sham) or presence of various concentrations of CT for 24 or 48 hours before the supernatants were harvested for the measurement of indicated cytokines. Shown is the average (mean ± SD) of three independent experiments. *p<0.05 and **p<0.001.

FIGs. 8A-8D. Therapeutic effect of CT on mouse LLC. FIG. 8A, C57BL/6 (female, 8 week-old, n=5) were subcutaneously inoculated with 5xl0⁶/mouse of LLC into the right flank on day 1. From day 7, LLC-bearing mice were treated every other day with i.t. injection of PBS (control) or CT at the doses as indicated for two weeks. Tumor growth (mean ± SD) was monitored and graphed (*p<0.05). FIGs. 8B-8C, LLC-bearing mice were prepared as in A and treated, starting on day 7, with i.t. injection of CT (100 μg/mouse) every other day or CT combined with i.t.

injection of anti-PD-Ll antibody (10 μg/mouse, Bio X Cell, clone 10F.9G2) twice weekly for two weeks. Tumor growth (FIG. 8B) and mouse survival (FIG. 8C) were monitored and graphed. FIG. 8D, The mice cured of LLC by treatment with CT+anti-PD-Ll in C were s.c. inoculated with 5xl0⁶/mouse of LLC in the right flank and EG7 thymoma in the contralateral flank. The growth of tumors on both flanks was monitored and graphed. All the mice grew EG7 tumors without LLC tumors, with the photo image confirmation of one euthanized mouse at the end of the experiment (insert).

FIGs. 9A-9B. The in vivo therapeutic effect of CT on smaller Hepal-6 tumor.

C57BL/6 mice (female, 8 week-old, n = 5) were inoculated s.c. with 2xl0⁶/mouse of Hepal-6 cells in the right flank on day 1 and the formation of tumors were monitored. When tumors reached approximately 0.5 cm in diameter (day 6), tumor-bearing mice were treated by intratumoral (i.t.) injection of 0.1 ml PBS or PBS containing different doses of CT (100 or 500 μg/injection tumor) twice weekly for two weeks, and subsequently once every other day for one week. Tumor growth (FIG. 9A, mean + SD) and survival (FIG. 9B) were monitored and plotted. Shown are the results of one experiment representative of three.

FIGs. 10A-10E. The therapeutic effect of CT alone or in combination with anti-PD-Ll on mice harboring large Hepal-6 tumors. FIG. 10A, Treatment schedule. FIGs. lOB-lOC, C57BL/6 mice (female, 8 week-old, n = 5) were inoculated s.c. with 2xl0⁶/mouse of Hepal-6 cells in the right flank on day 1 and the formation of tumors were monitored. When tumors reached approximately 1 cm in diameter (day 8), the mice were treated with i.t. CT (100 μg/injection tumor) every other day started on day 8 and/or four i.t. injections of control antibody (10

μg/injection/tumor, Bio X Cell, clone LTF-2) or anti-PD-Ll (10 μg/injection/tumor, Bio X Cell, clone 10F.9G2) on day 8, 12, 16, and 20. Tumor growth (FIG. 10B, mean + SD) and survival (FIG. IOC) were monitored and plotted. Shown are the results of one experiment representative of three. FIGs. 10D-10E, The Hepal-6 tumor- free mice cured by the treatment with the combination of CT+ anti-PD-Ll in FIGs. lOB-lOC were inoculated s.c. with 2xl0⁶/mouse of Hepal-6 cells in the right flank and 2xl0⁵/mouse of EG7 cells in the left flank. The growth (FIG. 10D) and appearance (FIG. 10E) of the resultant solid tumors were monitored for up to five weeks. The formation of EG7 tumor but not Hepal-6 tumors was documented by photographing of the naked flank regions of two randomly chosen euthanized mice.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

Vertebrates are endowed with an adaptive immune system that generates specific immune responses against malignant tumor cells such as the production of IFNyand tumor- specific cytotoxic CD8 T cells. At advanced stages of cancers including lung cancer and liver cancer, the tumor tissues become highly immunosuppressive due to the infiltration by immunoinhibitory cells such as regulatory T cells and myeloid-derived suppressor cells or generation of inhibitory factors such as PD-1/PD-L1, lymphocyte activation gene-3, IL-10, transforming growth factor β (TGF ), and vascular endothelial cell growth factor. The immunosuppressive tumor microenvironment yields at least two dire immunologic consequences. One is to nullify the cancer cell-killing capacity of preexisting CTLs, which can be countered by checkpoint inhibitor antibodies in a subset of lung cancer patients. The other is to incapacitate antigen-presenting dendritic cells (DCs) in the tumor tissue by preventing DC maturation and presentation of tumor-associated antigens to naive T cells in the secondary lymphoid organs, hampering the generation of additional tumor-specific CTLs.

Among factors that contribute to the development of immunosuppressive microenvironment in lung cancers, increases in cancer cell number and tumor size as seen in advanced tumors play an essential role by producing many immunosuppressive factors such as TGF , which results in the promotion of IL-10 production and generation of regulatory T cells and myeloid-derived suppressor cells (Thomas et al., Ann Oncol 2015;26:2213-2220; Fukuyama et al., Cancer Sci 2007;98: 1048- 1054; Hasegawa et al., Cancer 2001 ;91:964-971; Liu et al., J Immunol 2007;178:2883-2892). Therefore, reducing tumor burden would facilitate curtailing the immunosuppressive

microenvironment in lung cancer. Another means of overcoming the immunosuppressive microenvironment in lung cancer would be to directly activate antigen-presenting DCs in lung cancer tissues so that DCs regain the antigen-presenting capacity and produce proinflammatory cytokines (such as TNFoc and IL-12) capable of countering immunosuppressive cytokines. Thus, any agent capable of simultaneously inhibiting the proliferation of lung cancer cells and inducing the maturation of DCs would be highly desirable.

Traditional Chinese Medicines (TCMs) are widely employed in China and some southeastern Asian countries for the treatment of cancers. More than a dozen of TCMs and purified TCM compounds that are used for the treatment of cancer patients. This led to the identification of CT, a compound isolated from the TCM Danshen/S /vz^' miltiorrhiza Bunge (Chen et al.,

"Molecular evidence of cryptotanshinone for treatment and prevention of human cancer,"

Anticancer Agents Med Chem 2013;13:979-987, which is incorproated by reference herein), as an inhibitor of the proliferation of a human lung cancer cell line A549 and concomitant inducer of the maturation of human DCs. CT is biochemically well characterized (Takiura K. "Study on components of Danshen: the structure of cryptotanshinone." J Pharm Soc JPN 1941;61:482-490, which is incorproated by reference herein), can be chemically synthesized (Inouye et al., "Total Syntheses of Tanshinone-I, Tanshinone-Π and Crytotanshinone." Bull Chem Soc 1969;42:3318- 3323, which is incorporated by reference herein), and is available commercially with more than 98% purity.

As shown in the examples, CT inhibits the proliferation of lung cancer cells and induces DC maturation via distinct signaling pathways, and has a curative effect on Lewis lung carcinoma in immunocompetent mice. Furthermore, CT synergizes with a checkpoint inhibitor (an anti-PD-Ll antibody), to eliminate lung cancers and liver cancers, and prevent their recurrence, in an animal model system.

Accordingly methods of treating a tumor (such as a lung or liver tumor) in a subject by administering a therapeutically effective amount of (a) CT and (b) a PD-1 antagonist (including agents that inhibit the activity and/or expression of PD-1, PD-L1, and/or PD-L2) , a TEV1-3 antagonist, a LAG3 antagonist, a CTLA-4 antagonist, or a combination thereof, are provided. As discussed in more detail herein, the synergistic action of the CT and the PD- 1 antagonist, a CTLA-4 antagonist, the BTLA antagonist, the LAG3 antagonist, the TEV1-3 antagonist or a combination thereof provide for surprisingly effective reduction in tumor burden and inhibition of tumor recurrence relative to either agent alone.

Terms

Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Antibody: A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen, such as a PD-1, PD-L1, or CTLA-4 polypeptide, or a fragment thereof. Immunoglobulin molecules are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.

Antibodies include intact immunoglobulins and the variants and portions of antibodies well known in the art, such as single-domain antibodies (e.g. VH domain antibodies), Fab fragments, Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3^rd Ed., W. H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs has been defined according to Kabat et al. (see, Kabat et al. , Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991) and ImMunoGeneTics database (IMGT) (see, Lefranc, Nucleic Acids Res 29:207-9, 2001 ; and imgt.cines.fr/IMGT_vquest/vquest?_ livret=0&Option=humanIg). The Kabat database is maintained online (ncbi.nlm.nih.gov/igblast/). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 (or H-CDR3) is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDRl (or L-CDR1) is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds PD-1, PD-Ll, or PD-L2, for example, will have a specific VH region and the VL region sequence, and thus specific CDR sequences. Antibodies with different specificities (i.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A "monoclonal antibody" is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and/or heavy chain genes of a single antibody have been transfected.

Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

A "chimeric antibody" has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds a PD-1, PD-L1, or CTLA-4 polypeptide.

A "human" antibody (also called a "fully human" antibody) is an antibody that includes human framework regions and all of the CDRs from a human immunoglobulin. In one example, the framework and the CDRs are from the same originating human heavy and/or light chain amino acid sequence. However, frameworks from one human antibody can be engineered to include CDRs from a different human antibody. A "humanized" immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a "donor," and the human immunoglobulin providing the framework is termed an

"acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e. , at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Patent No. 5,585,089).

B- and T-lymphocyte attenuator (BTLA): A protein also known as CD272. BTLA expression is induced during activation of T cells, and BTLA remains expressed on Thl cells. BTLA interacts with a B7 homolog, B7H4, and plays a role in T-cell inhibition via interaction with tumor necrosis family receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). BTLA-HVEM complexes negatively regulate T-cell immune responses. A specific, non-limiting BTLA amino acid sequence, and an mRNA sequence encoding BTLA, is provided in GENBANK® Accession No. NM_001085357, September 1, 2016, incorporated herein by reference. BTLA antagonists include agents that reduce the expression or activity of BTLA or inhibits the T-cell inhibition function of BTLA, for example, by specifically binding to BTLA and inhibiting binding of BTLA to tumor necrosis factor receptors. Exemplary compounds include antibodies (such as an anti-BTLA antibody), RNAi molecules, antisense molecules, and dominant negative proteins.

Binding affinity: Affinity of an antibody for an antigen. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et al. , Mol. Immunol. , 16: 101-106, 1979. In another embodiment, binding affinity is measured by an antigen/antibody dissociation rate. In another embodiment, a high binding affinity is measured by a competition radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. An antibody that "specifically binds" an antigen (such as PD-1, PD-L1, or PD-L2) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.

Chemotherapeutic agents: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth such as psoriasis. In one embodiment, a chemotherapeutic agent is an agent of use in treating liver cancer, such as HCC, or another tumor. In one embodiment, a chemotherapeutic agent is a radioactive compound. One of skill in the art can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al , Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^nd ed., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to

Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, HJ. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of an antibody that binds PD-1, PD-Ll, or CTLA-4 polypeptide used in combination with a radioactive or chemical compound, such as CT or a pharmaceutically acceptable salt or derivative thereof.

Conservative variants: "Conservative" amino acid substitutions are those substitutions that do not substantially affect or decrease the affinity of a protein, such as an antibody. For example, a human antibody that specifically binds PD-1, PD-Ll, BTLA, TIM-3, LAG3 or CTLA-4 can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind the PD-1, PD-Ll, BTLA, TIM-3, LAG3 or

CTLA-4 polypeptide. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibody specifically binds the PD-1, PD-Ll, BTLA, TIM-3, LAG3 or CTLA-4 polypeptide. Non-conservative substitutions are those that reduce an activity or binding to a PD-1, PD-Ll, BTLA, TIM-3, LAG3 or CTLA-4 polypeptide.

Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Contacting: Placement in direct physical association; includes both in solid and liquid form.

Cytotoxic T-lymphocyte-Associated Protein 4 (CTLA-4): A protein also known as CD152. CTLA-4 is a member of the immunoglobulin superfamily. CTLA-4 is a protein receptor that functions as an immune checkpoint, and thus downregulates immune responses. CTLA-4 is constitutively expressed in regulatory T cells (Tregs) and is upregulated in conventional T cells after activation. CLTA4 binds CD80 or CD86 on the surface of antigen-presenting cells, and is an inhibitor of T cells. Specific non-limiting examples of a CTLA-4 protein and an mRNA encoding CTLA-4 are disclosed, for example, in GENBANK® Accession No. NM_001037631, October 7, 2016, incorporated herein by reference. CTLA-4 antagonists include agents that reduce the expression or activity of CTLA-4 or inhibits the T-cell inhibition function of CTLA-4, for example, by specifically binding to CTLA-4 and inhibiting binding of CTLA-4 to CD80 or CD86 on the surface of antigen-presenting cells. Exemplary compounds include antibodies (such as an anti- CTLA-4 antibody), RNAi molecules, antisense molecules, and dominant negative proteins.

Cytotoxicity: The toxicity of a molecule, such as an antibody, to the cells intended to be targeted, as opposed to the cells of the rest of an organism. In one embodiment, in contrast, the term "toxicity" refers to toxicity of an antibody to cells other than those that are the cells intended to be targeted by the antibody, and the term "animal toxicity" refers to toxicity of the antibody to an animal by toxicity of the antibody to cells other than those intended to be targeted by the antibody.

Diagnostic: Identifying the presence or nature of a pathologic condition, such as, but not limited to, liver cancer, ovarian cancer, melanoma or lung cancer. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives). The "specificity" of a diagnostic assay is one minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. "Prognostic" is the probability of development (e.g., severity) of a pathologic condition, such as liver cancer or metastasis.

Effector molecule: The portion of a chimeric molecule that is intended to have a desired effect on a cell to which the chimeric molecule is targeted. Effector molecule is also known as an effector moiety (EM), therapeutic agent, or diagnostic agent, or similar terms.

Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides. Alternatively, the molecule linked to a targeting moiety, such as an anti- PD-1, PD-L1 , or PD-L2 antibody, may be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (such as an antisense nucleic acid), or another therapeutic moiety that can be shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known to those of skill in the art (see, for example, U.S. Patent No. 4,957,735; and Connor et al, Pharm. Ther. 28:341-365, 1985).

Diagnostic agents or moieties include radioisotopes and other detectable labels. Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as S, ⁿC, ¹³N, ¹⁵0, ¹⁸F, ¹⁹F, ^99mTc, ¹³¹1, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, "Tc, ^{i n}In and ¹²⁵I, fluorophores,

chemiluminescent agents, and enzymes.

Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide, such as PD-1, PD-L1, or PD-L2.

Hepatic Cancer: A primary cancer of the liver. This includes hepatocellular carcinoma, fibrolamellar carcinoma, angiosarcoma, and hepatoblastoma.

Hepatocellular carcinoma (HCC): A primary malignancy of the liver typically occurring in patients with inflammatory livers resulting from viral hepatitis, liver toxins or hepatic cirrhosis (often caused by alcoholism). HCC is also called malignant hepatoma.

Immune Checkpoint Inhibitor: A type of agent that blocks biological pathways in specific types of immune system cells, such as, but no limited to, T cells, and some cancer cells. These inhibitors inhibit T cells from killing cancer cells. When a checkpoint inhibitor is blocked, an "inhibition" on the immune system is reduced and T cells become activated against cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD- 1/PD-L1 and CTLA- 4/B7-1/B7-2.

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, an immune response is a T cell response, such as a CD4⁺ response or a CD8⁺ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.

Isolated: An "isolated" biological component, such as a nucleic acid, protein (including antibodies) or organelle, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component naturally occurs, i.e. , other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

Lymphocyte-activation gene 3 (LAG3): A protein which in humans is encoded by the LAG3 gene, also called CD223. LAG-3 is a cell surface molecule with diverse biologic effects on T cell function, and is an immune checkpoint receptor. LAG3 negatively regulates cellular proliferation, activation, and homeostasis of T cells, and has been reported to play a role in Treg suppressive function. An exemplary amino acid and mRNA encoding human LAG3 is provided in GENBANK® Accession No. NM_002286.5, April 9, 2017, incorporated herein by reference.

Lung cancer: The main type of lung cancer is carcinoma of the lung, which includes small cell lung carcinoma and non-small cell lung carcinoma. Non-small cell lung carcinoma (NSCLC) is sometimes treated with surgery, while small cell lung carcinoma (SCLC) usually responds to chemotherapy and radiation. The most common cause of lung cancer is long-term exposure to tobacco smoke.

The non-small cell lung carcinomas are grouped together because their prognosis and management are similar. There are three main sub-types: squamous cell lung carcinoma, adenocarcinoma, and large cell lung carcinoma. Squamous cell lung carcinoma usually starts near a central bronchus. Cavitation and necrosis within the center of the cancer is a common finding. Well-differentiated squamous cell lung cancers often grow more slowly than other cancer types. Adenocarcinoma accounts for 29.4% of lung cancers. It usually originates in peripheral lung tissue. Most cases of adenocarcinoma are associated with smoking; however, among people who have never smoked, adenocarcinoma is the most common form of lung cancer. A subtype of adenocarcinoma, the bronchioloalveolar carcinoma, is more common in females.

Small cell lung cancers (SCLC, also called "oat cell carcinoma") is less common. It tends to arise in the larger airways (primary and secondary bronchi) and grows rapidly, becoming quite large. The "oat" cell contains dense neurosecretory granules (vesicles containing neuroendocrine hormones), which give this an endocrine/paraneoplastic syndrome association. While initially more sensitive to chemotherapy, it ultimately carries a worse prognosis and is often metastatic at presentation. Small cell lung cancers are divided into limited stage and extensive stage disease. This type of lung cancer also is strongly associated with smoking.

Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

Neoplasia, malignancy, cancer or tumor: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." Examples of hematological tumors include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic,

promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

In several examples, a tumor is a liver cancer, such HCC or hepatoblastoma, melanoma, a squamous cell carcinoma, such as squamous cell carcinoma of the lung, a clear cell carcinoma, such as clear cell carcinoma of the ovary, thyroid cancer, Wilms' tumor, neuroblastoma, or a testicular germ cell tumor.

Parenteral: Administered outside of the intestine, e.g. , not via the alimentary tract.

Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, intraarticularly, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for

pharmaceutical delivery of the antibodies herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Preventing, treating or ameliorating a disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.

Programmed cell Death protein (PD)-l: PD-1 molecules are members of the

immunoglobulin gene superfamily. The human PD- 1 has an extracellular region containing an immunoglobulin superfamily domain, a transmembrane domain, and an intracellular region including an immunoreceptor tyrosine-based inhibitory motif (ΠΊΜ) ((Ishida et al., EMBO J. 11:3887, 1992; Shinohara et al, Genomics 23:704, 1994; U.S. Patent No. 5,698,520,incorporated herein by reference). These features also define a larger family of molecules, called the immunoinhibitory receptors, which also includes gp49B, PIR-B, and the killer inhibitory receptors (KIRs) (Vivier and Daeron (1997) Immunol. Today 18:286). Without being bound by theory, it is believed that the tyrosyl phosphorylated ITIM motif of these receptors interacts with the SI 12- domain containing phosphatase, which leads to inhibitory signals. A subset of these immunoinhibitory receptors bind to major histocompatibility complex (MHC) molecules, such as the KIRs, and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) binds to B7-1 and B7-2. In humans, PD-1 is a 50-55 kDa type I transmembrane receptor that was originally identified in a T cell line undergoing activation-induced apoptosis. PD-1 is expressed on T cells, B cells, and macrophages. The ligands for PD-1 are the B7 family members PD-ligand 1 (PD-L1, also known as B7-H1) and PD-L2 (also known as B7-DC).

In vivo, PD-1 is expressed on activated T cells, B cells, and monocytes. Experimental data implicates the interactions of PD- 1 with its ligands in down regulation of central and peripheral immune responses. In particular, proliferation in wild-type T cells but not in PD-1 -deficient T cells is inhibited in the presence of PD-L1. Additionally, PD-1 -deficient mice exhibit an autoimmune phenotype. An exemplary amino acid sequence of human PD-1 is set forth in Ishida et al, EMBO J. 11 :3887, 1992; Shinohara et al. Genomics 23:704,1994; U.S. Pat. No. 5,698,520):

Engagement of PD-1 (for example by crosslinking or by aggregation), leads to the transmission of an inhibitory signal in an immune cell, resulting in a reduction of immune responses concomitant with an increase in immune cell anergy. PD-1 binds two ligands, PD-Ll and PD-L2, both of which are human PD-1 ligand polypeptides, that are members of the B7 family of polypeptides.

PD-1 antagonists include agents that reduce the expression or activity of a PD ligand 1 (PD- Ll) or a PD ligand 2 (PD-L2) or reduces the interactions between PD-1 and PD-Ll, or PD-L2.

Exemplary compounds include antibodies (such as an anti-PD-1 antibody, an anti-PD-Ll antibody, and an anti-PD-L2 antibody), RNAi molecules (such as anti-PD-1 RNAi molecules, anti-PD-Ll RNAi, and an anti-PD-L2 RNAi), antisense molecules (such as an anti-PD-1 antisense RNA, an anti-PD-Ll antisense RNA, and an anti-PD-L2 antisense RNA), dominant negative proteins (such as a dominant negative PD-1 protein, a dominant negative PD-Ll protein, and a dominant negative PD-L2 protein), see, for example, PCT Publication No. 2008/083174, incorporated herein by reference.

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.

Squamous cell carcinoma: A type of cancer that originates in squamous cells, thin, flat cells that form the surface of the skin, eyes, various internal organs, and the lining of hollow organs and ducts of some glands. Squamous cell carcinoma is also referred to as epidermoid carcinoma. One type of squamous cell carcinoma is squamous cell carcinoma of the lung.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.

T-cell immunoglobulin and mucin-domain containing-3 (TIM-3): A protein that in humans is encoded by the HAVCR2 gene. TEV13 is an immune checkpoint that is a Thl-specific cell surface protein that regulates macrophage activation and enhances the severity of experimental autoimmune encephalomyelitis in mice. The Tim-3 pathway can interact with the PD- 1 pathway in the exhausted CD8+ T cells in cancer. An exemplary mRNA and protein sequence for human TIM-3 is provided in GENBANK® Accession No. NM_032782.4, April 30, 2017, incorporated herein by reference.

Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor. When

administered to a subject, a dosage will generally be used that will achieve target tissue

concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Hence "comprising A or B" means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are

incorporated by reference in their entirety. All GENBANK® Accession numbers are herein incorporated by reference as they appear in the database on April 28, 2017. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Cryptotanshinone ( CT)

CT is material belonging to tanshinone class and its structural formula is as follows:

CT is available commercially (e.g., from Sigma- Aldrich, No. C5624). Further, method manufacturing, for example, as described in U.S. Pat. Pub. Nos. 20030031690 and 2013018275, which are incorporated herein by reference. In some embodiments. CT can be obtained by a process of chemical synthesis (see, e.g., Inouye et al., "Total Syntheses of Tanshinone-I,

Tanshinone-II and Crytotanshinone." Bull Chem Soc 1969;42:3318-3323, which is incorporated by reference herein) or by a process of extraction from plants, such as Salvia miltiorrhiza Bunge (see, e.g., Chen et al., "Molecular evidence of cryptotanshinone for treatment and prevention of human cancer," Anticancer Agents Med Chem 2013;13:979-987, which is incorproated by reference herein). For example, CT can be extracted from roots of the perennial herbal plant Salvia militiorrhira. In one embodiment, the CT can be included in a composition at a concentration of 1 to 50 μΜ.

In some embodiments, a pharmaceutically acceptable salt of CT or a derivative thereof is provided. The terms "pharmaceutically acceptable salt" refers to salts prepared by conventional means that include salts, e.g. , of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. "Pharmaceutically acceptable salts" of the presently disclosed compounds also include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, Ν,Ν'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. "Pharmaceutically acceptable salts" are also inclusive of the free acid, base, and zwitterionic forms. Descriptions of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). When compounds disclosed herein include an acidic function such as a carboxy group, then suitable pharmaceutically acceptable cation pairs for the carboxy group are well known to those skilled in the art and include alkaline, alkaline earth, ammonium, quaternary ammonium cations and the like. Such salts are known to those of skill in the art. For additional examples of "pharmacologically acceptable salts," see Berge et al., /. Pharm. Sci. 66: 1 (1977).

In several embodiments, salts of the compounds are those wherein the counter-ion is pharmaceutically acceptable. However, salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

The pharmaceutically acceptable acid and base addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid and base addition salt forms which the compounds are able to form. The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric, methanesulfonic,

ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. Conversely said salt forms can be converted by treatment with an appropriate base into the free base form.

The compounds containing an acidic proton may also be converted into their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like. PD-1, CTLA-4, TIMS, LAG3 and BTLA Antagonists

Check-point inhibitors, such as PD-1 antagonists, CTLA-4 antagonists, LAG3 antagonists, TIM-3 antagonists and/or BTLA antagonists are of use in the method disclosed herein, for example in combination with CT. The antagonist can be a chemical or biological compound. The antagonist can be an antibody, including but not limited to a chimeric, humanized, or human antibody.

Suitable antagonists also include antigen binding fragments of these antibodies (see above for a description of antigen binding fragments). The antagonist can be, for example, an inhibitor nucleic acid molecule or a small molecule, such as a molecule less than 900 daltons or less than 800 daltons. A PD- 1 antagonist can be any chemical compound or biological molecule that blocks binding of PD-Ll or PD-L2 expressed on a cell to human PD-1 expressed on an immune cell (T cell, B cell or NKT cell). Alternative names or synonyms for PD-1 and its ligands include:

PDCDl, PDl, CD279 and SLEB2 for PD-1 ; PDCDILI, PD-Ll, B7H1, B7-4, CD274 and B7-H for PD-Ll; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. Exemplary human PD-1 amino acid sequences can be found in NCBI Accession No.: NP_005009. Exemplary human PD-Ll and PD-L2 amino acid sequences can be found in NCBI Accession No.: NP_054862 and

NP 079515, respectively, April 28, 2017, incorporated by reference. In vivo, PD-1 is expressed on activated T cells, B cells, and monocytes. In humans, PD-1 is a 50-55 kDa type I

transmembrane receptor that was originally identified in a T cell line undergoing activation-induced apoptosis. PD-1 is expressed on T cells, B cells, and macrophages. The ligands for PD-1 are the B7 family members PD-ligand 1 (PD-Ll, also known as B7-H1) and PD-L2 (also known as B7- DC). A PD-Ll or PD-L2 inhibitor can be used in the methods disclosed herein.

Experimental data implicates the interactions of PD- 1 with its ligands in downregulation of central and peripheral immune responses. In particular, proliferation in wild-type T cells but not in PD-1 -deficient T cells is inhibited in the presence of PD-Ll. (See e.g., Ishida et al., EMBO J. 11 :3887, 1992; Shinohara et al. Genomics 23:704,1994; U.S. Pat. No. 5,698,520, incorporated herein by reference).

Additional PD-1 amino acid sequences are disclosed in U.S. Patent No. 6,808,710 and U.S. Patent Application Publication Nos. 2004/0137577, 2003/0232323, 2003/0166531, 2003/0064380, 2003/0044768, 2003/0039653, 2002/0164600, 2002/0160000, 2002/0110836, 2002/0107363, and 2002/0106730, which are incorporated herein by reference.

PD- 1 is a member of the CD28/CTLA-4 family of molecules based on its ability to bind to PD-Ll. In vivo, like CTLA-4, PD-1 is rapidly induced on the surface of T-cells in response to anti- CD3 (Agata et al. Int. Immunol. 8:765, 1996). T cell exhaustion is concomitant with an induction in PD-1 expression, see PCT Publication No. 2008/083174, incorporated herein by reference. T- cell cytotoxicity can be increased by contacting a T-cell with an agent that reduces the expression or activity of PD-1. An agent that reduces the expression or activity of PD-1 can be used to increase an immune response, such as to a tumor. Without being bound by theory, reduction of PD- 1 expression or activity results in an increase in cytotoxic T cell activity, increasing the specific immune response.

PD-1 family members bind to one or more receptors, such as PD-Ll and PD-L2 on antigen presenting cells. An exemplary amino acid sequence for PD-Ll is provided as GENBANK® Accession No. AAG18508, which is incorporated by reference herein as available October 4, 2000. An exemplary PD-L2 precursor amino acid sequence is provided as GENBANK® Accession No. AAK15370, which is incorporated by reference herein as available April 8, 2002. An exemplary variant PD-L2 precursor amino acid sequence is provided as GENBANK® Accession No.

Q9BQ51, which is incorporated by reference herein as available December 12, 2006.

Antagonists of use in the methods disclosed herein include agents that reduce the expression or activity of a PD ligand 1 (PD-Ll) or a PD ligand 2 (PD-L2) or reduces the interaction between PD-1 and PD-Ll or the interaction between PD-1 and PD-L2; these are PD-antagonists. Exemplary compounds include antibodies (such as an anti-PD-1 antibody, an anti-PD-Ll antibody, and an anti- PD-L2 antibody), RNAi molecules (such as anti-PD-1 RNAi molecules, anti-PD-Ll RNAi, and an anti-PD-L2 RNAi), antisense molecules (such as an anti-PD-1 antisense RNA, an anti-PD-Ll antisense RNA, and an anti-PD-L2 antisense RNA), dominant negative proteins (such as a dominant negative PD-1 protein, a dominant negative PD-Ll protein, and a dominant negative PD- L2 protein), and small molecule inhibitors. Any of these PD-1 antagonists are of use in the methods disclosed herein.

Other antibodies are of use in the methods disclosed herein (such as an anti-CTLA-4 antibody, and anti-LAG3 antibody, an-anti- TIM-3 antibody or an anti-BTLA antibody), RNAi molecules (such as anti-CTLA-4 RNAi molecules, anti-LAG3 RNAi, anti-TIM-3 RNAi and an anti-BTLA RNAi), antisense molecules (such as an anti-CTLA-4 antisense RNA, anti-LAG3 antisense RNA, anti-TIM-3 antisense RNA and an anti-BTLA antisense RNA). Dominant negative proteins also of use are a dominant negative CTLA-4 protein, a dominant negative LAG3 protein, a dominant negative LAG-3 protein and a dominant negative BTLA protein). Any of these antagonists are of use in the methods disclosed herein.

An antagonist is an agent having the ability to reduce the expression or the activity of the target in a cell. In some embodiments, PD-1, PD-Ll, PD-L2, LAG3, TIM-3, CTLA-4 or BTLA expression or activity is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to such expression or activity in a control. Exemplary reductions in activity are at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or a complete absence of detectable activity. In one example, the control is a cell that has not been treated with the PD-1 antagonist. In another example, the control is a standard value, or a cell contacted with an agent, such as a carrier, known not to affect activity. Expression or activity can be determined by any standard method in the art. In one non- limiting example, a PD-1 antagonist inhibits or reduces binding of PD-1 to PD-Ll, PD-L2, or both. In one non-limiting example, a PD-L1 antagonist reduces the binding of PD-L1 or PD-1.

A. Antibodies

In some embodiments, the antagonist is an antibody. Exemplary amino acid sequence of antibodies that bind PD-1 are disclosed, for example, in U.S. Patent Publication No. 2006/0210567, which is incorporated herein by reference. Antibodies that bind PD-1 are also disclosed in U.S. Patent Publication No. 2006/0034826, which is also incorporated herein by reference. Antibodies that bind PD-1 are also disclosed in U.S. Patent No. 7,488,802, U.S. Patent No. 7,521,051, U.S. Patent No. 8,008,449, U.S. Patent No. 8,354,509, U.S. Patent No. 8,168,757, and U.S. PCT Publication No. WO2004/004771, PCT Publication No. WO2004/072286, PCT Publication No. WO2004/056875, and US Published Patent Application No. 2011/0271358. The antibody can be KEYTRUDA® (pembrolizumab). The antibody can be an anti-PD-1 antibody such as Nivolumab (ONO-4538/BMS-936558) or OPDrVO® from Ono Pharmaceuticals. PD-L1 binding antagonists include YW243.55.S70, MPDL3280A, MDX-1105 and MEDI 4736, see U.S. Published Patent Application No. 2017/0044256. Examples of monoclonal antibodies that specifically bind to human PD-L1, and are useful in the disclosed methods and compositions are disclosed in PCT Publication No. WO2013/019906, PCT Publication No. WO2010/077634 Al and U.S. Patent No. 8,383,796. The checkpoint inhibitor antibodies against PD-1 (e.g., Nivolumab, pidilizumab, and Pembrolizumab) or PD-L1 (e.g., Durvalumab, Atezolizumab, and Avelumab) are of use in any of the methods disclosed herein. Antibodies that bind PD-1, PD-L2 and PD-1 are also disclosed in Patent No. 8,552, 154. In several examples, the antibody specifically binds CTLA-4, BTLA, PD-1, PD-L1, or PD-L2 with an affinity constant of at least 10⁷ M^"1, such as at least 10⁸ M^"1 at least 5 X 10⁸ M^"1 or at least 10⁹ M^"1. Any of these antibodies, and antigen binding fragments, are of use in the methods disclosed herein.

Exemplary antibodies that specifically bind CTLA-4 are disclosed in PCT Publication No.

WO 2001/014424, PCT Publication No. WO 2004/035607, U.S. Publication No. 2005/0201994, European Patent No. EP1141028, and European Patent No. EP 1212422 Bl. Additional CTLA-4 antibodies are disclosed in U.S. Patent No. 5,811,097, U.S. Patent No 5,855,887, U.S. Patent No 6,051,227, U.S. Patent No 6,984,720, U.S. Patent No. 6,682,736, U.S. Patent No. 6,207,156, U.S. Patent No. 5,977,318, U.S. Patent No. 6,682,736, U.S. Patent No. 7,109,003, U.S. Patent No.

7,132,281, U.S. Patent No. 7,452,535, and U.S. Patent No. 7,605,238; PCT Publication No. WO 01/14424, PCT Publication No. WO 00/37504, PCT Publication No. WO 98/42752, U.S. Published Patent Application No. 2000/037504, U.S. Published Application No. 2002/0039581, and U.S. Published Application No. 2002/086014. Antibodies that specifically bind CTLA-4 are also disclosed in Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho et al., J. Clin. Oncol., 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998). In some embodiments the CTLA-4 antagonist is Ipilmumab (also known as MDX-010 and MDX-101 and YERVOY®), see PCT Publication No. WO 2001/014424, incorporated herein by reference. These antibodies, and antigen binding fragments, are of use in the methods disclosed herein.

In further embodiments, a BTLA antagonist is utilized in the methods disclosed herein. Antibodies that specifically bind BTLA are disclosed, for example, in U.S. Published Patent Application No. 2016/0222114, U.S. Published Patent Application No. 2015/0147344, and U.S. published Patent Application No. 2012/0288500, all incorporated herein by reference. Biological agents that modulate BTLA activity, specifically using Herpesvirus entry mediator (HVEM) cis complexes are disclosed in U.S. Published Patent Application No. 2014/0220051 and U.S.

Published Patent Application No. 2010/0104559, both incorporated herein by reference. In yet other embodiments, the antibody specifically binds TIM-3, such as TSR-022. In further embodiments, the antibody specifically binds LAG3, such as BMS-986016, GSK2831781, or the antibodies disclosed in PCT Publication No. WO2015042246 Al, incorporated herein by reference. See also Clinical trial number NCT01968109 for "Safety Study of Anti-LAG-3 With and Without Anti-PD-1 in the Treatment of Solid Tumors" available on the internet at clinicaltrials.gov and incorporated by reference herein. These antibodies, and antigen binding fragments, are of use in the methods disclosed herein.

The antibodies of use in the disclosed methods include monoclonal antibodies, humanized antibodies, deimmunized antibodies (such as to reduce a human-anti-mouse response), chimeric antibodies, and immunoglobulin (Ig) fusion proteins. Antigen binding fragments of these antibodies are also of use in the methods disclosed herein. Polyclonal antibodies can be prepared by one of skill in the art, such as by immunizing a suitable subject (such as a veterinary subject) with an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized antigen. In one example, an antibody that specifically bind CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 (or combinations thereof) can be isolated from the mammal (such as from serum) and further purified by techniques known to one of skill in the art. For example, antibodies can be purified using protein A chromatography to isolate IgG antibodies. Antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques (see Kohler and Milstein Nature 256:495 49, 1995; Brown et al., J. Immunol. 127:539 46, 1981; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96, 1985; Gefter, M. L. et al. (1977) Somatic Cell Genet. 3:231 36; Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In Biological Analyses. Plenum Publishing Corp., New York, N.Y. (1980); Kozbor et al. Immunol. Today 4:72, 1983; Lerner, E. A. (1981) Yale J. Biol. Med. 54:387 402; Yeh et al., Proc. Natl. Acad. Sci. 76:2927 31, 1976). In one example, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with PD-1, PD-Ll, PD-L2, TIM-3, LAG3, BTLA or CTLA-4 and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that specifically binds to the polypeptide of interest.

In one embodiment, to produce a hybridoma, an immortal cell line (such as a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with a CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 peptide with an immortalized mouse cell line. In one example, a mouse myeloma cell line is utilized that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, including, for example, P3- NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines, which are available from the American Type Culture Collection (ATCC), Rockville, Md. HAT-sensitive mouse myeloma cells can be fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused (and unproductively fused) myeloma cells. Hybridoma cells producing a monoclonal antibody of interest can be detected, for example, by screening the hybridoma culture supernatants for the production antibodies that bind a PD-1, PD-Ll, TIM-3, LAG3, BTLA, CTLA-4 or PD-L2 molecule, such as by using an immunological assay (such as an enzyme-linked immunosorbant assay(ELISA) or radioimmunoassay (RIA).

As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody that specifically binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (such as an antibody phage display library) with CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 to isolate immunoglobulin library members that specifically bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (such as, but not limited to, Pharmacia and Stratagene). Examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No.

5,223,409; PCT Publication No. WO 90/02809; PCT Publication No. WO 91/17271; PCT

Publication No. WO 92/18619; PCT Publication WO 92/20791; PCT Publication No. WO

92/15679; PCT Publication No. WO 92/01047; PCT Publication WO 93/01288; PCT Publication No. WO 92/09690; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978 7982, 1991; Hoogenboom et al., Nucleic Acids Res. 19:4133 4137, 1991.

In one example the sequence of the specificity determining regions of each CDR is determined. Residues are outside the SDR (non-ligand contacting sites) are substituted. For example, in any of the CDR sequences as in the table above, at most one, two or three amino acids can be substituted. The production of chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, is well known in the art. For example, humanized antibodies can be routinely produced. The antibody or antibody fragment can be a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor monoclonal antibody that binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2, and immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks. Humanized monoclonal antibodies can be produced by transferring donor complementarity determining regions (CDRs) from heavy and light variable chains of the donor mouse immunoglobulin (such a CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 specific antibody) into a human variable domain, and then substituting human residues in the framework regions when required to retain affinity. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of the constant regions of the donor antibody. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al, Nature 321:522, 1986; Riechmann et al, Nature 332:323, 1988; Verhoeyen et al, Science 239:1534, 1988; Carter et al, Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer et al, J. Immunol.150:2844, 1993. The antibody may be of any isotype, but in several embodiments the antibody is an IgG, including but not limited to, IgGi, IgG2, IgG3 and IgG₄.In some embodiments, the humanized immunoglobulin specifically binds to the antigen of interest (e.g., CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2) with an affinity constant of at least 10⁷ M ¹, such as at least 10⁸ M ¹ at least 5 X 10⁸ M ¹ or at least 10⁹ M^"1.

In one embodiment, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 99% or at least about 95%, identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Human framework regions, and mutations that can be made in humanized antibody framework regions, are known in the art (see, for example, in U.S. Patent No. 5,585,089, which is incorporated herein by reference).

Antibodies, such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which include a heavy chain and light chain variable region and are capable of binding specific epitope determinants. These antibody fragments retain some ability to selectively bind with their antigen or receptor. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

(5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of making these antigen binding fragments are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). In several examples, the variable region includes the variable region of the light chain and the variable region of the heavy chain expressed as individual polypeptides. Fv antibodies are typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain. To produce these antibodies, the VH and the VL can be expressed from two individual nucleic acid constructs in a host cell. If the VH and the VL are expressed non- contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.

In an additional example, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are known in the art (see Whitlow et al, Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al, Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al,

Bio/Technology 11:1271, 1993; and Sandhu, supra).

Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No.

4,036,945 and U.S. Patent No. 4,331,647, and references contained therein; Nisonhoff et al, Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al, Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

One of skill will realize that conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the VH and the VL regions to increase yield. Thus, one of skill in the art can readily review the amino acid sequence of an antibody of interest, locate one or more of the amino acids in the brief table above, identify a conservative substitution, and produce the conservative variant using well-known molecular techniques.

Effector molecules, such as therapeutic, diagnostic, or detection moieties can be linked to an antibody that specifically binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH₂) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

Nucleic acid sequences encoding the antibodies can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151, 1979; the

diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22: 1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20): 1859- 1862, 1981, for example, using an automated synthesizer as described in, for example, Needham- VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

Exemplary nucleic acids encoding sequences encoding an antibody that specifically binds CTLA-4, BTLA, TEV1-3, LAG3, PD-1, PD-L1, or PD-L2 can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life

Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

In one example, an antibody of use is prepared by inserting the cDNA which encodes a variable region from an antibody that specifically binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD- Ll, or PD-L2 into a vector which comprises the cDNA encoding an effector molecule (EM). The insertion is made so that the variable region and the EM are read in frame so that one continuous polypeptide is produced. Thus, the encoded polypeptide contains a functional Fv region and a functional EM region. In one embodiment, cDNA encoding a detectable marker (such as an enzyme) is ligated to a scFv so that the marker is located at the carboxyl terminus of the scFv. In another example, a detectable marker is located at the amino terminus of the scFv. In a further example, cDNA encoding a detectable marker is ligated to a heavy chain variable region of an antibody that specifically binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2, so that the marker is located at the carboxyl terminus of the heavy chain variable region. The heavy chain- variable region can subsequently be ligated to a light chain variable region of the antibody that specifically binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 using disulfide bonds. In a yet another example, cDNA encoding a marker is ligated to a light chain variable region of an antibody that binds CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2, so that the marker is located at the carboxyl terminus of the light chain variable region. The light chain-variable region can subsequently be ligated to a heavy chain variable region of the antibody that specifically binds CTLA-4, BTLA, TEV1-3, LAG3, PD-1, PD-L1, or PD-L2 using disulfide bonds.

Once the nucleic acids encoding the antibody or functional fragment thereof are isolated and cloned, the protein can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. One or more DNA sequences encoding the antibody or functional fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.

Polynucleotide sequences encoding the antibody or functional fragment thereof can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

The polynucleotide sequences encoding the antibody or functional fragment thereof can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCh method using procedures well known in the art. Alternatively, MgC or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation. When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding the antibody of functional fragment thereof and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). One of skill in the art can readily use expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide can be carried out by conventional means including preparative chromatography and immunological separations. Once expressed, the recombinant antibodies can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, Protein Purification, Springer- Verlag, N.Y., 1982). Substantially pure compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.

Methods for expression of single chain antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are well-known and are applicable to the antibodies disclosed herein. See, Buchner et al., Anal.

Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991 ; Huse et al, Science

246: 1275, 1989 and Ward et al, Nature 341 :544, 1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent refolding. During the solubilization step, as is well known in the art, a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, incorporated by reference herein, and especially as described by Buchner et al., supra. Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L- arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution. An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. It is desirable to add excess oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution after the redox-shuffling is completed.

In addition to recombinant methods, the antibodies and functional fragments thereof that are disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. , Pierce Chem. Co., Rockford, 111., 1984. Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicycylohexylcarbodimide) are well known in the art.

B. Inhibitory Nucleic Acids

Inhibitory nucleic acids that decrease the expression and/or activity of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 can also be used in the methods disclosed herein. One embodiment is a small inhibitory RNA (siRNA) for interference or inhibition of expression of a target gene. Nucleic acid sequences encoding PD-1, PD-L1 and PD-L2 are disclosed in

GENBANK® Accession Nos. NM_005018, AF344424, NP_079515, and NP_054862, all incorporated by reference as available on April 28, 2017.

Generally, siRNAs are generated by the cleavage of relatively long double-stranded RNA molecules by Dicer or DCL enzymes (Zamore, Science, 296: 1265-1269, 2002; Bernstein et al., Nature, 409:363-366, 2001). In animals and plants, siRNAs are assembled into RISC and guide the sequence specific ribonucleolytic activity of RISC, thereby resulting in the cleavage of mRNAs or other RNA target molecules in the cytoplasm. In the nucleus, siRNAs also guide heterochromatin- associated histone and DNA methylation, resulting in transcriptional silencing of individual genes or large chromatin domains. PD-1 siRNAs are commercially available, such as from Santa Cruz Biotechnology, Inc.

The present disclosure provides RNA suitable for interference or inhibition of expression of a target gene, which RNA includes double stranded RNA of about 15 to about 40 nucleotides containing a 0 to 5-nucleotide 3' and/or 5' overhang on each strand. The sequence of the RNA is substantially identical to a portion of an mRNA or transcript of a target gene, such as CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2, for which interference or inhibition of expression is desired. For purposes of this disclosure, a sequence of the RNA "substantially identical" to a specific portion of the mRNA or transcript of the target gene for which interference or inhibition of expression is desired differs by no more than about 30 percent, and in some embodiments no more than about 10 percent, from the specific portion of the mRNA or transcript of the target gene. In particular embodiments, the sequence of the RNA is exactly identical to a specific portion of the mRNA or transcript of the target gene.

Thus, siRNAs disclosed herein include double- stranded RNA of about 15 to about 40 nucleotides in length and a 3' or 5' overhang having a length of 0 to 5-nucleotides on each strand, wherein the sequence of the double stranded RNA is substantially identical to (see above) a portion of a mRNA or transcript of a nucleic acid encoding CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2. In particular examples, the double stranded RNA contains about 19 to about 25 nucleotides, for instance 20, 21, or 22 nucleotides substantially identical to a nucleic acid encoding CTLA-4, BTLA, TEVI-3, LAG3, PD-1, PD-Ll, or PD-L2. In additional examples, the double stranded RNA contains about 19 to about 25 nucleotides 100% identical to a nucleic acid encoding CTLA-4, BTLA, TEVI-3, LAG3, PD-1, PD-Ll, or PD-L2. It should be not that in this context "about" refers to integer amounts only. In one example, "about" 20 nucleotides refers to a nucleotide of 19 to 21 nucleotides in length.

Regarding the overhang on the double-stranded RNA, the length of the overhang is independent between the two strands, in that the length of one overhang is not dependent on the length of the overhang on other strand. In specific examples, the length of the 3' or 5' overhang is 0-nucleotide on at least one strand, and in some cases it is 0-nucleotide on both strands (thus, a blunt dsRNA). In other examples, the length of the 3' or 5' overhang is 1 -nucleotide to 5- nucleotides on at least one strand. More particularly, in some examples the length of the 3 ' or 5 ' overhang is 2-nucleotides on at least one strand, or 2-nucleotides on both strands. In particular examples, the dsRNA molecule has 3' overhangs of 2-nucleotides on both strands.

Thus, in one particular provided RNA embodiment, the double- stranded RNA contains 20, 21, or 22 nucleotides, and the length of the 3' overhang is 2-nucleotides on both strands. In embodiments of the RNAs provided herein, the double-stranded RNA contains about 40-60% adenine+uracil (AU) and about 60-40% guanine+cytosine (GC). More particularly, in specific examples the double- stranded RNA contains about 50% AU and about 50% GC.

Also described herein are RNAs that further include at least one modified ribonucleotide, for instance in the sense strand of the double- stranded RNA. In particular examples, the modified ribonucleotide is in the 3' overhang of at least one strand, or more particularly in the 3' overhang of the sense strand. It is particularly contemplated that examples of modified ribonucleotides include ribonucleotides that include a detectable label (for instance, a fluorophore, such as rhodamine or FITC), a thiophosphate nucleotide analog, a deoxynucleotide (considered modified because the base molecule is ribonucleic acid), a 2'-fluorouracil, a 2'-aminouracil, a 2'-aminocytidine, a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, an inosine, or a 2'0-Me-nucleotide analog.

Antisense and ribozyme molecules for CTLA-4, BTLA, TEVI-3, LAG3, PD-1, PD-L1, or PD-L2 are also of use in the method disclosed herein. Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double- stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate an mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target cell producing CTLA-4, BTLA, TEVI-3, LAG3, PD-1, PD-L1, or PD-L2. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (see, for example, Marcus-Sakura, Anal. Biochem. 172:289, 1988).

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, such as phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridin- e, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, amongst others.

Use of an oligonucleotide to stall transcription is known as the triplex strategy since the bloomer winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al. , Antisense Res. and Dev. 1(3):227, 1991 ; Helene, C, Anticancer Drug Design 6(6):569), 1991. This type of inhibitory oligonucleotide is also of use in the methods disclosed herein.

Ribozymes, which are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases, are also of use. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, /. Amer. Med. Assn. 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type, (Hasselhoff, Nature 334:585, 1988) and "hammerhead"-type. Tetrahymena-type, ribozymes recognize sequences which are four bases in length, while "hammerhead"-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-base recognition sequences are preferable to shorter recognition sequences.

Various delivery systems are known and can be used to administer the siRNAs and other inhibitory nucleic acid molecules as therapeutics. Such systems include, for example,

encapsulation in liposomes, microparticles, microcapsules, nanoparticles, recombinant cells capable of expressing the therapeutic molecule(s) (see, e.g. , Wu et al. , J. Biol. Chem. 262, 4429, 1987), construction of a therapeutic nucleic acid as part of a retroviral or other vector, and the like.

C. Small Molecule Inhibitors

CTLA-4, BTLA, TEV1-3, LAG3, PD-1, PD-L1, or PD-L2 antagonists include molecules that are identified from large libraries of both natural product or synthetic (or semi- synthetic) extracts or chemical libraries according to methods known in the art. The screening methods that detect decreases in CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 activity (such as detecting cell death for PD-1, PD-Ll and PD-L2) are useful for identifying compounds from a variety of sources for activity. The initial screens may be performed using a diverse library of compounds, a variety of other compounds and compound libraries. Thus, molecules that bind CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 molecules that inhibit the expression of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 and molecules that inhibit the activity of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 can be identified. These small molecules can be identified from combinatorial libraries, natural product libraries, or other small molecule libraries. In addition, CTLA-4, BTLA, TEVI-3, LAG3, PD-1, PD-Ll, and PD-L2 antagonists can be identified as compounds from commercial sources, as well as commercially available analogs of identified inhibitors. In some embodiments, the small molecule is less than 900 daltons, or less than 800 daltons.

The precise source of test extracts or compounds is not critical to the identification of antagonists. Accordingly, antagonists can be identified from virtually any number of chemical extracts or compounds. Examples of such extracts or compounds that can be antagonists include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from

Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). CTLA-4, BTLA, and PD- 1 antagonists can be identified from synthetic compound libraries that are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, and PD-L2 antagonists can be identified from a rare chemical library, such as the library that is available from Aldrich

(Milwaukee, Wis.). CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, and PD-L2 antagonists can be identified in libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.

Useful compounds may be found within numerous chemical classes, though typically they are organic compounds, including small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, such as less than about 750 or less than about 350 daltons can be utilized in the methods disclosed herein. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. In several embodiments, compounds of use has a Kd for CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 of less than InM, less than lOnm, less than 1 μΜ, less than 10μΜ, or less than lmM.

D. Peptide variants as antagonists

An immunoadhesin that specifically binds to human CTLA-4, human BTLA, human TIM-3, human LAG3, human PD-1, human PD-Ll, or human PD-L2 can also be utilized. An

immunoadhesin is a fusion protein containing the extracellular or a binding portion of a protein fused to a constant region such as an Fc region of an immunoglobulin molecule. Examples of immunoadhesion molecules that specifically bind to PD-1 are disclosed in PCT Publication Nos. WO2010/027827 and WO2011/066342, both incorporated by reference. These immunoadhesion molecules include AMP- 224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein.

Additional PD-1 antagonists that are fusion proteins are disclosed, for example, in U.S. Published Patent Application No. 2014/0227262, incorporated herein by reference.

In one embodiment, a LAG3 antagonist of use in the disclosed methods is IMP321, a soluble LAG3 which has been used to activate dendritic cells. In another embodiment, aTEVI-3 antagonists if use in the disclosed methods is CA-327 (Curis).

In one embodiment, variants of a CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 protein which function as an antagonist can be identified by screening combinatorial libraries of mutants, such as point mutants or truncation mutants, of a CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 protein to identify proteins with antagonist activity. In one example, the antagonist is a soluble protein.

Thus, a library of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-Ll, or PD-L2 variants can be generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A library of CTLA-4, BTLA, TEVI-3, LAG3, PD-1, PD-Ll, or PD-L2 variants can be produced by, for example, by enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential PD-1 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (such as for phage display) containing the set of PD-1 sequences. There are a variety of methods which can be used to produce libraries of potential CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential CTLA-4, BTLA, TEV1-3, LAG3, PD-1, PD-L1, or PD-L2 antagonist sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang, et al., Tetrahedron 39:3, 1983; Itakura et al. Annu. Rev. Biochem. 53:323, 1984; Itakura et al. Science 198: 1056, 1984).

In addition, libraries of fragments of a CTLA-4, BTLA, TEV1-3, LAG3, PD-1, PD-L1, or PD-

L2 protein coding sequence can be used to generate a population of fragments for screening and subsequent selection of variants of a specified antagonist. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM) can be used in combination with the screening assays to identify CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 antagonists (Arkin and Youvan, Proc. Natl. Acad. Sci. USA 89:7811 7815, 1992; Delagrave et al., Protein Eng. 6(3):327 331, 1993). In one embodiment, cell based assays can be exploited to analyze a library of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 variants. For example, a library of expression vectors can be transfected into a cell line which ordinarily synthesizes and secretes CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2. The transfected cells are then cultured such that CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 and a particular CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 (respectively) variant are secreted. The effect of expression of the mutant on activity in cells or in supematants can be detected, such as by any of a functional assay. Plasmid DNA can then be recovered from the cells wherein endogenous activity is inhibited, and the individual clones further characterized.

Peptidomimetics can also be used as CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-

L2 antagonists. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds and are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (for example, polypeptide that has a PD-1 biological activity), but has one or more peptide linkages optionally replaced by a— CH2NH-, ~CH₂S~,— CH2-CH2-, -CH.=.CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO— linkages. These peptide linkages can be replaced by methods known in the art (see, for example, Morley, Trends Pharm. Sci. pp. 463 468, 1980; Hudson et al. Int. J. Pept. Prot. Res. 14: 177 185, 1979; Spatola, Life Sci. 38: 1243 1249, 1986; Holladay, et al. Tetrahedron Lett. 24:4401 4404, 1983). Peptide mimetics can be procured economical, be stable, and can have increased have-life or absorption. Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (such as by an amide group), to non- interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions that do not form direct contacts with the macromolecules(s) to which the peptidomimetic binds to produce the therapeutic effect. Derivitization of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the

peptidomimetic.

A dominant negative protein or a nucleic acid encoding a dominant negative protein that interferes with the biological activity of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 can also be used in the methods disclosed herein. A dominant negative protein is any amino acid molecule having a sequence that has at least 50%, 70%, 80%, 90%, 95%, or even 99% sequence identity to at least 10, 20, 35, 50, 100, or more than 150 amino acids of the wild type protein to which the dominant negative protein corresponds. For example, a dominant-negative PD-L1 has mutation such that it binds PD- 1 more tightly than native (wild-type) PD- 1 but does not activate any cellular signaling through PD- 1.

The dominant negative protein may be administered as an expression vector. The expression vector may be a non-viral vector or a viral vector (e.g., retrovirus, recombinant adeno-associated virus, or a recombinant adenoviral vector). Alternatively, the dominant negative protein may be directly administered as a recombinant protein systemically or to the infected area using, for example, microinjection techniques.

Polypeptide antagonists can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding the amino acid sequence, frequently as part of a larger polypeptide (a fusion protein, such as with ras or an enzyme). Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art (see Maniatis el al. Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Kaiser et al., Science 243: 187, 1989; Merrifield, Science 232:342, 1986; Kent, Annu. Rev. Biochem. 57:957, 1988).

Peptides can be produced, such as by direct chemical synthesis, and used as antagonists. Peptides can be produced as modified peptides, with nonpeptide moieties attached by covalent linkage to the N-terminus and/or C-terminus. In certain preferred embodiments, either the carboxy- terminus or the amino-terminus, or both, are chemically modified. The most common

modifications of the terminal amino and carboxyl groups are acetylation and amidation, respectively. Amino-terminal modifications such as acylation (for example, acetylation) or alkylation (for example, methylation) and carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization, can be incorporated into various embodiments. Certain amino-terminal and/or carboxy-terminal modifications and/or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as: enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others.

The CTLA-4 antagonist can be a dominant negative protein or an immunoadhesins, see for example U.S. Published Patent Application No. 2016/0264643, incorporated herein by reference. Additional anti-CTLA-4 antagonists include any inhibitor, including but not limited to a small molecule, that can inhibit the ability of CTLA-4 to bind to its cognate ligand, disrupt the ability of B7 to CTLA-4, disrupt the ability of CD80 to bind to CTLA-4, disrupt the ability of CD86 to bind to CTLA-4. Pharmaceutical Compositions and Methods of Use

Methods are disclosed herein for producing an immune response to a tumor in a subject. Methods are also provided for treating a tumor in a subject. In some embodiments, the methods include treating an existing tumor in a subject. In additional embodiments, methods are disclosed herein for preventing conversion of a benign to a malignant lesion, or preventing metastasis in a subject. In some examples, the methods reduce a symptom of the tumor in the subject. In additional examples, the tumor is a solid tumor. In some embodiments, the disclosed methods can increase the survival of the subject. In further embodiments, the disclosed methods can delay or prevent reoccurrence of the tumor in the subject. Any of the antagonists disclosed above are of use in these methods.

Generally, the methods include selecting a subject having a tumor, such as a benign or malignant tumor, and administering to the subject a therapeutically effective amount of (1) CT or a pharmaceutical salt or derivative thereof and (2) a checkpoint inhibitor antagonist, such as a PD-1 antagonist (which includes antagonists that specifically inhibit PD-1 itself, PD-Ll or PD-L2), a BTLA antagonist, a TIM-3 antagonist, a LAG3 antagonist, or a CTLA-4 antagonist. The PD-1 antagonist, BTLA antagonist, TIM-3 antagonist, LAG3 antagonist or CTLA-4 antagonist, can, in some non-limiting examples, be an antibody (or antigen binding fragment thereof) that specifically binds PD-1, PD-Ll, PD-Ll, PD-L2, TEVI-3, LAG3, BTLA, or CTLA-4. The methods are of use for treating the tumor, preventing metastasis, preventing the conversion of a benign to a malignant tumor and/or preventing or inhibiting reoccurrence of the tumor. The administration can be local or systemic.

In some embodiments, an advantage of the methods provided herein is that the synergistic combination of CT with checkpoint inhibitors such as a PD- 1 antagonist (including antagonists that specifically inhibit PD-1 itself, PD-Ll or PD-L2), BTLA antagonist, TEVI-3 antagonist, LAG3 antagonist and/or a CTLA-4 antagonist, allows for reduced dosage of active agents for cancer therapy, while also reducing any corresponding undesired side-effects (such as cytotoxicity) of the therapy. In further embodiments, another advantage of the methods provided herein is that that the synergistic combination of CT with checkpoint inhibitors such as a PD-1 antagonist (including antagonists that specifically inhibit PD-1 itself, PD-Ll or PD-L2), BTLA antagonist, TIM-3 antagonist, LAG3 antagonist and/or a CTLA-4 antagonist, allows for reduce tumor reoccurrence in a subject. In additional embodiments, a further advantage of the method provided herein is that the synergistic combination of CT with checkpoint inhibitors such as a PD-1 antagonist (including antagonists that specifically inhibit PD-1 itself, PD-L1 or PD-L2), BTLA antagonist, TIM-3 antagonist, LAG3 antagonist and/or a CTLA-4 antagonist, allows for increased survival.

Additional agents can also be administered to the subject of interest, such as, but not limited to, chemotherapeutic agents. Additional treatments can also be administered to the subject, such as, but not limited to, surgical resection of the tumor.

The subject can be selected for treatment. For example, a diagnostic assay (such as an IHC assay) can be performed on the tumor (or a sample on the tumor) to identify the subject as one likely to respond to the disclosed method of treatment. In some embodiments, the subject is selected for treatment with a therapeutically effective amount of CT and a PD- 1 antagonist if the tumor tests positive for PD-L1 or PD-L2 expression by an immunohistochemical (IHC) assay. Exemplary assays for detecting a tumor that tests positive for PD-L1 expression are provided in Topalian et al. 2012. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med. 366:2443-2454; Wolchok et al. 2013. Nivolumab plus ipilimumab in advanced melanoma. N. Engl. J. Med. 369:122-133; Herbst et al. 2014. Predictive correlates of response to the anti-PD-Ll antibody MPDL3280A in cancer patients. Nature. 515:563-567; Garon et al. 2015. Pembrolizumab for the treatment of non-small-cell lung cancer. N. Engl. J. Med. 372:2018-2028; and Reck et al. Pembrolizumab versus chemotherapy for PD-Ll-positive non-small-cell lung cancer. N. Engl. J. Med. 375:1823-1833, each of which is incorporated by reference herein.

The tumor can be benign or malignant. The tumor can be any tumor of interest, including, but not limited to, hepatic cancer and lung cancer. The lung cancer can be small cell or non-small cell carcinoma of the lung. The hepatic cancer can be a hepatic carcinoma. Additional examples are skin tumors, breast tumors, brain tumors, cervical carcinomas, testicular carcinomas, head and neck tumors, gastrointestinal tract tumors, genitourinary system tumors, gynecological system tumors, breast, endocrine system tumors, skin tumors, a sarcoma of the soft tissue and bone, a mesothelioma, a melanoma, a neoplasm of the central nervous system, or a leukemia. In some embodiments, the tumor is a head and neck tumor, such as tumors of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands and paragangliomas. In other embodiments, the tumor is a lung tumor, such as a non-small cell lung cancer or a small cell lung cancer. In further embodiments, the tumor can be a tumor of the gastrointestinal tract, such as cancer of the esophagus, stomach, pancreas, liver, biliary tree, small intestine, colon, rectum and anal region. In yet other embodiments, the tumor can be a tumor of the genitourinary system, such as cancer of the kidney, urethra, bladder, prostate, urethra, penis and testis. In some embodiments, the tumor is a gynecologic tumor, such as cancer of the cervix, vagina, vulva, uterine body, gestational trophoblastic diseases, ovarian, fallopian tube, peritoneal, or breast. In other embodiments, the tumor is an endocrine system tumor, such as a thyroid tumor, parathyroid tumor, adrenal cortex tumor, pancreatic endocrine tumor, carcinoid tumor and carcinoid syndrome. The tumor can be a sarcoma of the soft tissue and bone, a mesothelioma, a cancer of the skin, a melanoma, comprising cutaneous melanomas and intraocular melanomas, a neoplasm of the central nervous system, a cancer of the childhood, comprising retinoblastoma, Wilm's tumor, neurofibromatoses, neuroblastoma, Ewing's sarcoma family of tumors, rhabdomyosarcoma. The tumor can be a lymphoma, comprising non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, primary central nervous system lymphoma, and Hodgkin's disease. The tumor can be a leukemia, such as acute leukemia, chronic myelogenous leukemia and lymphocytic leukemia. The tumor can be plasma cell neoplasms, a cancer of unknown primary site, a peritoneal carcinomastosis, a Kaposi's sarcoma, AIDS-associated lymphomas, AIDS-associated primary central nervous system lymphoma, AIDS-associated Hodgkin's disease and AIDS-associated anogenital cancers, a metastatic cancer to the liver, metastatic cancer to the bone, malignant pleural and pericardial effusions and malignant ascites. In specific non-liming examples, the tumor is lung cancer or hepatic cancer.

Treatment of the tumor is generally initiated after the diagnosis of the tumor, or after the initiation of a precursor condition (such as dysplasia or development of a benign tumor). Treatment can be initiated at the early stages of cancer, for instance, can be initiated before a subject manifests symptoms of a condition, such as during a stage I diagnosis or at the time dysplasia is diagnosed. However, treatment can be initiated during any stage of the disease, such as but not limited to stage I, stage II, stage ΠΙ and stage IV cancers. In some examples, treatment is administered to these subjects with a benign tumor that can convert into a malignant or even metastatic tumor.

The presence of a tumor can be determined by methods known in the art, and typically include cytological and morphological evaluation. The tumor can be an established tumor. The cells can be in vivo or ex vivo, including cells obtained from a biopsy.

Treatment initiated after the development of a condition, such as malignant cancer, may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms, or reducing metastasis, tumor volume or number of tumors. In some example, the tumor becomes undetectable following treatment. Treatment can also include increasing the immune response to the tumor, such as by increasing the humoral response, or cytokines, NK cells, activated CTLs, such as CD8+ T cells, or MDSCs.

In one aspect of the disclosure, the formation of tumors, such as metastasis, is delayed, prevented or decreased. In another aspect, the size of the primary tumor is decreased. In a further aspect, a symptom of the tumor is decreased. In yet another aspect, tumor volume is decreased. In yet another aspect reoccurrence of the tumor is delayed or prevented, such as for 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2, 22, 23, or 24 months, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

In some embodiments immune response can be measured, tumor volume can be measured, the number of metastatic lesions can be measured, and/or a symptom of a tumor can be measured. A therapeutically effective dose can increase the immune response, decrease tumor volume, decrease the number and/or size of metastases, and/or decrease one or more symptoms of the tumor.

Treatment prior to the development of the condition, such as treatment upon detecting dysplasia or an early (benign) precursor condition, is referred to herein as treatment of a subject that is "at risk" of developing the condition. In some embodiments, administration of a composition can be performed during or after the occurrence of the conditions described herein. The compositions can be administered to a subject at risk of developing the tumor

Pharmaceutical compositions can include (1) CT or a salt or derivative thereof and (2) a PD- 1 (including a PD-1, PD-L1 and PD-L2 specific antagonist), BTLA, TEVI-3, LAG3, or CTLA-4 antagonist. These compositions can also include an additional agent, such as an additional chemotherapeutic agent. These compositions are of use for threating a tumor. These compositions can be formulated in a variety of ways for administration to a subject to induce an immune response to a tumor, or to delay, prevent, reduce the risk of developing, or treat, any tumor of interest. The compositions described herein can also be formulated for application such that they prevent metastasis of an initial lesion. In some embodiments, the compositions are formulated for local administration, such as intratumoral administration or for systemic administration, such as intravenous administration. Pharmaceutical compositions are thus provided for both local use and for systemic use, formulated for use in human or veterinary medicine.

While the disclosed methods and compositions will typically be used to treat human subjects they may also be used to treat similar or identical diseases in other vertebrates, such as other primates, dogs, cats, horses, and cows. A suitable administration format may best be determined by a medical practitioner for each subject individually. Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S, 1988. The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen.

CT, salts and derivatives thereof and/or a PD-1, PD-L1, PD-L2, BTLA, TIM-3, LAG3, or

CTLA-4 antagonist can be administered by any route, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intraperitoneal, intrasternal, or intraarticular injection or infusion, or by sublingual, oral, topical, intranasal, or transmucosal administration, or by pulmonary inhalation. In some embodiments, the CT, salts and derivatives thereof and/or the PD-1, PD-L1, PD-L2, BTLA, TIM-3, LAG3, or CTLA-4 antagonist are administered to a tissue wherein the tumor is located, or directly into the tumor (intratumoral). When a parenteral composition is provided, e.g. for injection or infusion, active agents are generally suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate- acetic acid buffers. A form of repository or "depot" slow release preparation may be used so that

therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.

In certain embodiments, the PD-1, PD-L1 or PD-L2 antagonist (such as, but not limited to, an antibody or antigen binding fragment that specifically binds to PD-1, PD-L1, or PD-L2) can be administered at a dose in the range of from about 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosing schedule of administration including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician. As part of the combination therapy, the CT can be administered to the subject before, after, or concurrent to the PD-1 antagonist, as long as the administration schedule provides for a sufficient physiological concentrations of the agents to provide a therapeutic benefit. In some embodiments, the CT is administered at a dose in the range of 0.001-100 mg/kg, 0.001-10 mg/kg, 0.001-1 mg/kg, 0.001-0.1 mg/kg, 0.05-10 mg/kg, 0.05-1 mg/kg, 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosing schedule of administration including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician. In certain embodiments, the CTLA-4 antagonist (such as an antibody or antigen binding fragment that specifically binds to CTLA-4) can be administered at a dose in the range of from about 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosing schedule of administration including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician. As part of the combination therapy, the CT can be administered to the subject before, after, or concurrent to the CTLA-4 antagonist, as long as the administration schedule provides for a sufficient physiological concentrations of the agents to provide a therapeutic benefit. In some embodiments, the CT is administered at a dose in the range of 0.001-100 mg/kg, 0.001-10 mg/kg, 0.001-1 mg/kg, 0.001- 0.01 mg/kg, 0.05-10 mg/kg, 0.05-10 mg/kg, 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosing schedule of administration including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician.

In certain embodiments, the BTLA antagonist (such as an antibody or antigen binding fragment that specifically binds to BTLA) can be administered at a dose in the range of from about 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosing schedule of administration including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician. As part of the combination therapy, the CT can be administered to the subject before, after, or concurrent to the BTLA antagonist, as long as the administration schedule provides for a sufficient physiological concentrations of the agents to provide a therapeutic benefit. In some embodiments, the CT is administered at a dose in the range of 0.001-100 mg/kg, 0.001-10 mg/kg, 0.001-1 mg/kg, 0.001- 0.01 mg/kg, 0.05-10 mg/kg, 0.05-10 mg/kg, 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosing schedule of administration including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician.

In additional embodiments, the LAG3 or TIM-3 antagonist (such as an antibody or antigen binding fragment that specifically binds to LAG3 or TIM-3) can be administered at a dose in the range of from about 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosing schedule of administration including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician. As part of the combination therapy, the CT can be administered to the subject before, after, or concurrent to the LAG3 or TIM-3 antagonist, as long as the administration schedule provides for a sufficient physiological concentrations of the agents to provide a therapeutic benefit. In some embodiments, the CT is administered at a dose in the range of 0.001-100 mg/kg, 0.001-10 mg/kg, 0.001-1 mg/kg, 0.001-0.01 mg/kg, 0.05-10 mg/kg, 0.05-10 mg/kg, 0.01-10 mg/kg, 0.01-5 mg/kg, 0.01-1 mg/kg, 0.01-0.1 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-3 mg/kg, 0.5-1.0 mg/kg, 0.05-0.5 mg/kg, according to a dosing schedule of administration including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician.

Suitable examples of sustained-release compositions include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (such as, for example, an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt). Sustained-release formulations may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray, depending on the location of the tumor.

The pharmaceutically acceptable carriers and excipients useful in the disclosed methods are conventional. For instance, parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients that can be included are, for instance, proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

Kits are also provided. CT, salts and derivatives thereof and/or the PD-1, PD-L1, PD-L2,

LAG3, TEVI-3, BTLA, or CTLA-4 antagonist can be formulated in unit dosage form, suitable for individual administration of precise dosages. The amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated. Multiple treatments are envisioned, such as over defined intervals of time, such as daily, bi-weekly, weekly, bi-monthly or monthly, such that chronic administration is achieved.

Additional agents can be administered, such as a cytokine, a chemokine, or a

chemotherapeutic agent. These can be included in the disclosed pharmaceutical compositions. A cytokine can be administered, such as interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), or interferon, such as interferon (IFN) β. In one example, for the prevention and treatment of cancer, surgical treatment can be administered to the subject. In one example, this administration is sequential. In other examples, this administration is simultaneous.

Examples of chemotherapeutic agents are alkylating agents, antimetabolites, natural products, or hormones and their antagonists. Examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or

chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine). Examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine. Examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase). Examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum Π also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide). Examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as

hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,

Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea,

Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP- 16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples of immunomodulators that can be used include AS- 101 (Wyeth- Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech). In some embodiments, the subject is administered sorafenib.

Embodiments

Clause 1: A method of treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of: a) cryptotanshinone (CT) or a salt or derivative thereof; and b) a Programmed Death (PD-1) antagonist, a Cytotoxic T-lymphocyte- Associated Protein 4 (CTLA-4) antagonist, a B- and T-lymphocyte Attenuator (BTLA) antagonist, T-cell Immunoglobulin and Mucin-domain containing-3 (TIM-3) antagonist, a Lymphocyte- Activation Gene 3 (LAG3) antagonist, or a combination thereof; thereby treating the tumor in the subject.

Clause 2: The method of clause 1, wherein the tumor is a hepatic cancer or a lung cancer. Clause 3: The method of clause 2, wherein the lung cancer is a small cell carcinoma of the lung or a non-small cell carcinoma of the lung, or wherein the hepatic cancer is a hepatocellular carcinoma.

Clause 4: The method of any one of the prior clauses, wherein the subject is human.

Clause 5: The method of any one of the prior clauses, wherein the PD-1 antagonist, the CTLA-4 antagonist, the TIM-3 antagonist, the LAG3 antagonist and/or the BTLA antagonist is an inhibitory RNA, a dominant negative protein, or an antibody that specifically binds to PD-1, PD-Ll, PD-L2, BLTA, TEVI-3, LAG3 or CLTA-4, or an antigen binding fragment thereof.

Clause 6: The method of clause 5, wherein the monoclonal antibody is a human monoclonal antibody or a humanized monoclonal antibody.

Clause 7: The method of any one of the prior clauses, comprising administering to the subject the PD-1 antagonist.

Clause 8: The method of clause 7, wherein the tumor tests positive for PD-Ll expression by an immunohistochemical (IHC) assay.

Clause 9: The method of clause 7 or clause 8, wherein the PD-1 antagonist is a monoclonal antibody, or an antigen binding fragment thereof, which specifically binds to human PD-1, human PD-Ll, or human PD-L2, and blocks the binding of human PD-Ll to human PD-1 or human PD-L2 to human PD-1.

Clause 10: The method of any one of clauses 7- 9, wherein the PD-1 antagonist is one of nivolumab, pembrolizumab, pidilizumab, atezolizumab, avelumab, or durvalumab.

Clause 11: The method of any one of clauses 1-6, comprising administering to the subject the CTLA-4 antagonist.

Clause 12: The method of clause 11, wherein the CTLA-4 antagonist is ipilimumab.

Clause 13: The method of any one of clauses 1-6, comprising administering to the subject the CTLA-4 antagonist.

Clause 14: The method of any one of the prior clauses, wherein the CT is administered by oral, intratumor, intramuscular, or intravenous administration; and/or wherein the PD- 1 antagonist, the CTLA-4 antagonist, the TIM-3 antagonist, the LAG3 antagonist, the BTLA antagonist, or the combination thereof, is administered by intratumor, intramuscular, or intravenous administration.

Clause 15: The method of any one of the prior clauses, wherein treating the tumor comprises decreasing tumor volume, decreasing the number or size of metastases, or lessening a symptom of the tumor.

Clause 16: The method of any one of the prior clauses, further comprising surgically resecting the tumor.

Clause 17: The method of any one of the prior clauses, further comprising administering to the subject a therapeutically effective amount of an additional chemotherapeutic agent.

Clause 18: The method of any one of the prior clauses, wherein the method inhibits reoccurrence of the tumor in the subject.

Clause: 19: Use of a therapeutically effective amount of a combination therapy of a) CT and b) a PD-1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3 antagonist, a LAG3 antagonist or a combination thereof, to treat a tumor in the subject.

Clause 20: The use of clause 19, wherein the PD-1 antagonist is an antibody that specifically binds PD-Ll or an antigen binding fragment thereof.

EXAMPLES

Lung cancer is the leading cause of cancer-related mortality, with very limited effective therapeutics. Screening of a variety of TCMs for capacity to inhibit the proliferation of human lung cancer A549 cells and to induce the maturation of human DCs led to the identification of CT, a compound purified from the TCM Salvia miltiorrhiza Bunge. CT inhibited the proliferation of mouse Lewis lung carcinoma (LLC) cells by upregulating p53 and downregulating cyclin Bl and Cdc2 and consequently inducing G2/M cell-cycle arrest. CT promoted phenotypic maturation of DCs with upregulation of costimulatory and MHC molecules, and stimulated DC to produce TNFa, IL-Ιβ and IL-12p70, but not IL-10. CT-induced DC maturation depended on MyD88 and also involved the activation of NF-κΒ, p38, and JNK. CT was effective in the treatment of established LLC tumors and, when used in combination with low-dose anti-PD-Ll, can cure LLC -bearing mice with the induction of subsequent anti-LLC specific immunity. The data provided herein indicate that CT has unique activities that may provide a new promising therapeutic for the treatment of human lung cancers.

Example 1

Materials and methods

TCMs, cell lines and mice: The TCM FuFang Kushen injection was obtained from Shanxi Zhendong Pharmaceutical Co. Ltd (Shanxi, China). Compounds purified from various TCMs including CT were obtained from the National Institutes for Food and Drug Control (Beijing,

China). CT was dissolved at a stock concentration of 15-20 mg/ml in dimethylsulfoxide and diluted into physiologic solutions or medium for experiments.

All cell lines used were originally obtained from the American Type Culture Collection (Manassas, VA). Lewis lung carcinoma (LLC) cell line was passaged and maintained in DMEM medium (Meditech, Manassas, VA) supplemented with 10% FBS (Hyclone, Logan, UT), 2 mM L- glutamine, 25 mM HEPES, 100 U/ml penicillin, and 100 g/ml streptomycin. A549, a human lung carcinoma cell line and EG7, a cell line derived from EL4 thymoma, was passaged and maintained in RPMI 1640 medium (Meditech) supplemented with 10% FBS, 2 mM glutamine, 25 mM HEPES, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μΜ 2-mercaptoethanol.

C57BL/6 mice were obtained from Charles River (Frederick, MD). TLR4^_/" and MyD88^_/" mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were kept under specific pathogen-free conditions with water and food given ad libitum. All experiments with mice were performed in compliance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals and were approved by the National Cancer Institute at Frederick Animal Care and Use Committee.

Proliferation assay: A549 or LLC cells were seeded into a 96-well flat-bottomed tissue culture plate at 4 xl0³/well in appropriate medium and cultured in a CO2 incubator (37°C humidified air containing 5% CO2) overnight. CT or other compounds was added at final concentrations as specified and incubated for 48 hours. Tritiated thymidine (³H-TdR, New England Nuclear, North Billerica, MA) was added at 0.5 μΟΛνεΙΙ for the last 4 h of culture. At the end of culture, the cells were collected on a membrane using a 96-well automatic harvester (INOTECHAG IH-280, Dottikon, Switzerland) and ³H-TdR incorporation (CPM) was measured using an automatic MicroBeta counter (Wallac). The change in the percentage (%) of cell proliferation was calculated as: % Proliferation = (CPM with compound - CPM blank) ÷ (CPM without compound - CPM blank) x 100. The concentration at which 50% of the proliferation was inhibited (IC50) was calculated using GraphPad Prism.

DC generation and treatment: Human DCs were generated by culturing purified monocytes at 5 x 10⁵cells/ml in RPMI 1640 medium (Meditech) supplemented with 10% FBS (Hyclone, Logan, UT), 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 g/ml streptomycin, and 50 μΜ of 2-ME in the presence of 50 ng/ml of hGM-CSF (PeproTech, Rocky Hill, NJ) and 50 ng/ml of hIL-4 (PeproTech) in a CO2 incubator for 5 days as previously reported (19). Mouse DCs were generated by culturing mouse hematopoietic progenitors isolated from the femurs and tibias of C57BL/6, TLR4^_/", or MyD88^_/" mice in complete RPMI 1640 containing 20 ng/ml of mGM-CSF (PeproTech) for 5-7 days as previously reported (20). To measure phenotypic maturation of human or mouse DCs, they were incubated at 5 x 10⁵ cells/ml in a CO2 incubator in the presence or absence of various compounds at concentrations specified for 48 hours before immunostaining. To measure cytokine production by DCs, human or mouse DCs were cultured at 5xl0⁵ cells/ml in a CO2 incubator without or with CT at concentrations as specified for 24 hours before collection of culture supematants for cytokine measurement. For investigating CT-induced signaling, mouse DCs were treated with CT at 10 g/ml in serum- free RPMI 1640 in a CO2 incubator for various time as indicated. Subsequently, DCs were solubilized in lxSDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8 at 25°C, 2% w/v SDS, 10% glycerol, 50 mM dithiothretol, 0.01% bromophenol blue) at 10⁷ cells/ml, boiled for 5 min, and stored at -20°C until use.

Immunostaining and flow cytometry: Immunostaining of DCs was done using a routine protocol. In brief, DCs suspended in FACS buffer (PBS containing 0.5% BSA and 0.05% NaN₃) were blocked with 2% normal human AB or mouse serum on ice for 20 minutes and stained with various combinations of fluorophore-conjugated antibodies against human or mouse DC surface markers on ice for 30 minutes in the dark. The antibodies used to stain human DCs were FITC- anti-human HLA-ABC (clone W6/32, eBioscience, San Diego, CA), PE-anti-human HLA-DR (clone G46-6, BD/PharMingen, San Diego, CA), PerCP-Cy5.5-anti-human CD86 (clone 2331, BD), BV421 -anti-human CDl lc (clone Bul5, BioLegend, San Diego, CA), and APC-anti-human CD80 (clone 2D10, BioLegend). Mouse DCs were immunostained with FITC-anti-mouse CD86 (clone GL1, TONBO Biosciences, San Diego, CA), PE-anti-mouse CD80 (clone 16-10A1, TONBO), Pacific Blue-anti-mouse CD83 (clone Michel- 19, BD), APC-anti-mouse I-A/E (clone M5/114.15.2, eBioscience). Data of the stained samples were acquired using a LSR Π flow cytometer (BD) and analyzed using the software FlowJo.

Measurement of cytotoxicity to erythrocytes and macrophages: To measure potential cytotoxic effect of CT on erythrocytes, human erythrocytes suspended in PBS at 2% (vol./vol.) were aliquoted into 9 Falcon 4-ml tubes at 1 ml/tube. The tubes were labeled and centrifuged at 500xg for 5 minutes to pellet the erythrocytes. After removal of the supernatant, 1 ml of PBS in the absence or presence of CT at various concentrations were added into corresponding tubes to resuspend the erythrocytes. For positive hemolysis control, 1 ml of H2O instead of PBS was added into the 9^th tube. All the tubes were incubated at room temperature for 30 min, and then centrifuged at 500xg for 5 min. The photo images before and after centrifugation were recorded.

For the detection of potential cytotoxic effect of CT on human macrophages, purified human monocytes were cultured in a CO2 incubator in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μΜ 2- mercaptoethanol and 50 ng/ml rhM-CSF (PeproTech) in a 48-well tissue culture plate at 2 x 10⁵/well for 7 days with 50% medium replacement on day 3 and day 5. Subsequently, CT was added into triplicate wells at concentrations as specified and incubated for another 48 hours. At the end of the incubation, the plate was stained with 1% Toluidine blue (Sigma, St. Louis, MO) dissolved in 1% sodium tetraborate (Sigma) for 45 minutes at room temperature, followed by washing with distilled water 3 times. After air-drying, the plate was photo-imaged and the dye was solubilized by adding 0.5 ml of 1% SDS and the absorbance at 620 nm was measured using a spectrometer.

Detection ofapoptosis: LLC cancer cells seeded into a 12-well plate at 3xl0⁵ cells/ml/well were cultured in a CO2 incubator overnight to allow the cells to adhere. Subsequently, 1 ml of medium containing various concentrations of CT or 2% NaZ3 (for positive control) was added into each wells and the plate was incubated for another 24 hours. The cells were harvested by digestion with 0.25% trypsin-2.21 mM EDTA, washed three times, and stained with an apoptosis detection kit (BMS500FI/300, eBioscience) consisting of FITC-conjugated annexin V and propidium iodide (PI) following the vendor's recommendation. The stained samples were assayed using a LSR Π (BD) flow cytometer and analyzed using FlowJo. Cell cycle analysis: LLC cells with 70-80% confluency were washed and serum-starved in a CO2 incubator in DMEM medium containing 0.2% FBS for 48 hours for synchronization. The synchronized LLC cells were plated into a 6-well plate at 5xl0⁵/well in DMEM medium (10% FBS) containing various concentrations of CT and cultured in a C02 incubator for 24 hours. The resultant cells were transferred into corresponding Falcon tubes, washed twice with PBS, and fixed in 70% ethanol for 30 minutes at 4°C. After fixation, the cells were washed twice with PBS and treated with 50 μΐ/tube of 100 g/ml of ribonuclease for 30 minutes at room temperature. Finally, 200 μΐ of PI at 50 g/ml was added into each tube and the cells were analyzed using a LSR Π flow cytometer.

Treatment of LLC for signaling analysis: LLC cells were serum-starved in a CO2 incubator in DMEM medium containing 0.1% FBS overnight before they were treated with various concentrations of CT for 24 hours. The treated cells were solubilized in lxSDS-PAGE sample buffer at 10⁷ cells/ml, boiled for 5 min, and stored at -20°C until use.

SDS-PAGE and Western blot: Samples (10-20 μΐ/lane) and SEEBLUE® Plus2 Prestained Standard (Invitrogen, Carlsbad, CA) were separated on a 4-12% NUPAGE™ Bis-Tris Gel

(Invitrogen) by running at a constant voltage of 100 volts using lx NUPAGE™ MES or MOPS electrode buffer. After transfer onto a piece of IMMOBILON™ membrane (Millipore, Bedford, MA), the membranes were rinsed with washing buffer (TBST, tris-buffered saline containing 0.05% Tween 20), blocked with 5% nonfat dry milk (in TBST) at room temperature for 1 h, and incubated with appropriately diluted (1 :500-2000) 1^st antibodies overnight at in a cold room. All of 1^st antibodies were rabbit IgG, including anti-Ι-κΒα (Cell Signaling #9242, Beverly, MA), anti- GAPDH (Cell Signaling #2118), anti-phospho-p44/42 (Cell Signaling #9101), anti-p44/42 (Cell Signaling #9102), anti-phosphorylated p38 (Cell Signaling #9211), anti-p38 (Cell Signaling #9212), anti-phosphorylated JNK (Cell Signaling #9251), anti-JNK (Cell Signaling #9252), anti- phosphorylated p53 (Cell Signaling #9284), anti-p53 (Santa Cruz, Biotech, sc-6243, Dallas, TX), anti-Cdc2 (Cell Signaling #77055), and anti-cyclin Bl (Cell Signaling #4138). After washing 3 times with TBST, the membranes were reacted with 1:2000 diluted HRP-conjugated goat anti- rabbit IgG (Cell Signaling, #70741) for 1 h at room temperature, washed, and soaked in

SUPERSIGNAL^® West Dura Extended Duration Substrate (Thermo Fisher Scientific Inc., Hanover Park, IL). The images were collected using the G BOX Chemi systems (Syngene, Frederick, MD).

Cytokine quantitation: TNFoc, IL-Ιβ, IL-10, and IL-12p70 in the culture supernatants were quantitated by human and mouse Customary Cytokine Arrays following the manufacturer' s protocol (MesoScale Diagonostics, Rockville, MD). LLC mouse model and treatment: C57BL/6 mice (female, 8-10 week-old, n = 5) were implanted subcutaneously with 0.2 ml sterile PBS containing 5xl0⁶/mouse of LLC into the right flank. The appearance and growth of tumors were monitored twice a week. The greatest longitudinal diameter (length) and the greatest transverse diameter (width) of a palpable tumor were measured to the nearest 0.1 mm using a caliper. Tumor volume (mm³) was calculated by the formula Tumor volume = (length x width² )/2. LLC -bearing mice were treated every other day, starting on day 7, with intratumoral (i.t.) injection of CT at various doses for two weeks. In some experiments, LLC-bearing mice were also treated with i.t. injection of control or anti-PD-Ll twice weekly for two weeks. In accordance with the institutional guideline, mice with big tumors (volume>2000 mm³) undergoing necrosis were considered morbid and euthanized. The control (clone LTF-2) or anti-PD-Ll (clone 10F.9G2) antibodies were obtained from Bio X Cell and used at 10 μg/injection/tumor.

Statistical analysis: Unless otherwise specified, all experiments were performed at least three times, and the results of one representative experiment or the mean of multiple experiments are shown. The difference between groups in terms of cytokine production was determined by

Student' s t test. Differences in the in vivo tumor growth were determined by Repeated Measures of ANOVA, whereas differences between control group and experimentally treated groups were evaluated by one-way ANOVA after arcsine square-root transformation. The percent of survival data were compared and analyzed by Logrank test, using GraphPad Prism.

Example 2

CT inhibited A549 proliferation and stimulated the maturation of human DCs

To identify compounds capable of simultaneously inhibiting the proliferation of lung cancer cells and enhancing host antitumor immune responses, we investigated a variety of TCMs and TCM-derived compounds over a wide range (0.1 ~ 10 mg/ml) of concentrations. As shown in FIG. 1, CT dose-dependently inhibited the proliferation of A549 cells in vitro, with a 50% inhibitory concentration of 0.228 g/ml (FIG. 1A). In addition, CT at 10 g/ml upregulated the expression of surface CD80, CD86, HLA-ABC, and HLA-DR on human monocyte-derived DCs when treated for 48 hours, demonstrating that CT could also inducing the phenotypic maturation of human DCs (FIG. IB).

Incubation of human erythrocytes in PBS containing CT at 0-20 g/ml for 30 minutes at room temperature did not cause hemolysis. All the erythrocytes sedimented to the bottom of the tubes and the supernatant remained clear (FIG. 2). In addition, human monocyte-derived macrophages (ΙιΜφ) were treated with CT at 0-20 g/ml for 48 hours in a 37°C CO2 incubator and subsequently stained. There was no obvious difference between sham-treated (CT at 0 g/ml) and CT treated groups (FIG. 2B, upper panel). After solubilizing the dye by adding 1 ml of 1% SDS into each wells, the ABS620 of the wells was measured and graphed (FIG. 2B, lower panel). Almost identical ABS620 reading for all the groups confirmed that CT was not cytotoxic for ΙιΜφ.

Therefore, CT at the concentrations capable of inhibiting A549 proliferation and inducing DC maturation was not cytotoxic for normal cells.

Example 3

The anti- proliferative effect of CT on Lewis lung carcinoma (LLC) cells was based on G2/M cell cycle arrest

The proliferation of LLC cells, a C57BL/6 mouse-derived NSCLC cell line, was dose- dependently inhibited by CT, with an IC50 at 2.8 g/ml (FIG. 3A). CT has been previously reported to inhibit the proliferation of a number of tumor cell lines in vitro through distinct mechanisms such as blocking STAT3 in prostate and colon cancer cells (21, 22), inducing apoptosis in lung cancer cells (23), and causing cell cycle arrest in leukemic cells (24). To investigate how CT inhibited the proliferation of LLC, LLC cells were cultured with various concentrations of CT or 1% NaN3 (as a positive control) for 24 hours and subsequently stained with FITC-conjugated annexin V and propidium iodide (PI) for the detection of potential apoptosis by flow cytometry (FIG. 3B). LLC treated with 1 % NaN3 as positive control showed apoptotic death as evidenced by a dramatic increase in the percentage of annexin V-positive cells (FIG. 3B, Q3 quadrant of the right plot). In contrast, CT over a wide concentration range did not induce apoptosis. Even at 10 g/ml that completely inhibited the proliferation of LLC (FIG. 3 A), CT did not induce apoptosis of LLC cells, since most of the treated were annexin V-negative and Pi-negative (FIG. B, left plot).

To determine whether CT treatment could affect cell cycle progression, LLC cells synchronized by serum starvation were cultured in the presence of 0-2 μg/ml of CT for 24 hours. At the end of the culture period, the cells were stained with PI and analyzed by flow cytometry to quantitate the relative fractions of cells in every phase of the cell cycle (FIG. 3C). CT dose dependently increased the percentage of cells in G2/M phase with concomitant reduction in the percentage of cells in G0/G1 phase, demonstrating that CT treatment resulted in G2/M arrest of LLC (FIG. 3C).

To further investigate how CT caused G2/M cell cycle arrest, LLC cells were treated with various concentrations of CT for 24 hours and the signaling molecules that regulate cell cycle progression were measured by Western blot (FIG. 4A). After detecting the target molecules, the same membrane was stripped and re-probed with anti-GAPDH to confirm loading of similar amount of proteins into each lane (FIG. 4A). CT in a dose-dependent manner elevated the levels of phosphorylated p53, which was mirrored by a reduction of unphosphorylated p53, indicating CT caused p53 activation (FIG. 4A). In accordance with activation of p53, treatment with CT decreased the levels of both cyclin Bl and Cdc2. Therefore, the data demonstrate a signaling scenario in which CT induced the activation of p53 and consequently the downregulation of both Cdc2 and cyclin Bl, which, in turn, prevents cell cycle progression through the mitotic phase in LLC, resulting in G2/M arrest (FIG. 4B). CT treatment did not alter the level of p21, in agreement with the fact that CT did not cause G0/G1 arrest in LLC (FIG. 3C).

Example 4

CT induced maturation of mouse DCs in a MyD88-dependent manner

In order to determine the effect of CT on the maturation of mouse DCs, mouse bone marrow-derived DCs were incubated at 37°C for 48 hours in humidified air containing 5% C02 in the presence of CT or LPS (as a positive control), and subsequently analyzed for the expression of costimulatory molecules and I-A/E by flow cytometry. Overlay histograms showed that CT at 5 μg/ml upregulated the expression of CD80, CD83, CD86, and I-A/E, indicating that CT could induce phenotypic maturation of mouse DCs (FIG. 5 A). Noticeably, CT at 5 g/ml was even more effective than LPS at 100 ng/ml in inducing phenotypic maturation of mouse DCs as shown by a more robust upregulation of CD80, CD83, CD86, and I-A/E (FIG. 5A).

Another characteristic of DC maturation is the enhancement of production of

proinflammatory cytokines (25). The supematants of mouse bone marrow-derived DCs treated with different concentrations of CT were assayed and CT was found to stimulate DCs to produce TNFoc, IL-Ιβ, and IL-12p70 in a dose-dependent manner when measured at either 24 hours or 48 hours after the start of the treatment (FIG. 5B). Since CT did not induce IL-10 production by mouse DCs (FIG. 5B), CT-matured DCs are likely to preferentially induce Thl-polarized immune responses, which would favor the induction of antitumor immune responses.

To identify the CT-triggered signaling events, mouse bone marrow-derived DCs were treated with CT at 10 g/ml as indicated and the activation of NF-κΒ and mitogen-activated protein kinases (MAPKs) determined (FIG. 6). CT decreased the levels of Ι-κΒα protein, which became obvious within 10 minutes (FIG. 6A, upper panel). Re-probing the same membrane with anti- GAPDH revealed that similar amounts of proteins were loaded onto each lane, indicating the reduction of Ι-κΒα was not due to uneven loading (FIG. 6A, lower panel). Since degradation of I- κΒα frees the p50/p65 dimer of NF-κΒ and enables its nuclear translocation, the data indicate that CT promoted NF-κΒ activation. Determination of the effect of CT on the three major MAPKs revealed that CT in a time-dependent manner lowered the levels of phosphorylated Erks (FIG. 6B, upper panel) without affecting the levels of unphosphorylated Erks (FIG. 6B, lower panel), suggesting that CT downregulated the activation of Erks in DCs. In contrast, CT induced phosphorylation of p38 and JNK with different kinetics which peaked at 90 and 10 minutes, respectively (FIGs. 6C & 6D). Therefore, CT activated p38 and JNK, but decreased the activation of Erks in mouse DCs.

To gain further insight into the mechanistic basis of CT-induced DC maturation, we studied whether TLR4 or MyD88 contributed to CT-induced DC maturation. To this end, WT, TLR4^_/", and MyD88^_/" DCs were treated in parallel with CT at 5 g/ml or LPS (100 ng/ml) as a positive control for 48 hours and subsequently measured for the expression of surface markers by flow cytometry. As shown in FIG. 7A, CT induced similar levels of upregulation of CD80, CD83, CD86, and I-A/E in WT and TLR4^_/" DCs, indicating that the DC-maturing effect of CT was not dependent on TLR4. This data also indicated that there was no endotoxin contamination in the CT preparation used in the present study. A comparison of CT-treated WT with MyD88^_/" DCs revealed that CT only upregulated CD80 and CD83 in MyDSS^"7" DCs, while CT-induced upregulation of CD86 and I-A/E were markedly reduced in MyD88^' DCs (FIG. 7A). As anticipated, LPS-induced upregulation of CD80, CD83, CD86, and I-A/E in WT was completely deficient in TLR4^_/" DCs and markedly reduced in MyD88^' DCs (FIG. 7A). The production of proinflammatory cytokines (TNF-oc, IL-Ιβ, or IL-12p70) was absent in LPS -stimulated TLR4^' DCs, but showed no reduction in CT-treated TLR4^_/" DCs (FIG. 7B, upper panel). In contrast, My SS ^A DCs showed inhibition of both CT- and LPS-induced TNF-oc, IL-Ιβ, and IL-12p70 production (FIG. 7B, lower panel). These results demonstrated that CT activated DC maturation in a MyD88-dependent manner.

Example 5

Therapeutic antitumor effect of CT on LLC tumors

To test the potential therapeutic effect of CT, C57BL/6 mice harboring established subcutaneous LLC tumors on the flank were treated with intratumoral (i.t.) injection of CT at different doses every other day for two weeks and the growth of tumors was monitored (FIG. 8A). CT significantly inhibited the growth of LLC tumors at 100 μg/mouse (FIG. 8A).

However, CT alone did not eliminate the LLC tumors. Lung tumors and antigen-presenting cells in the tumor microenvironment express PD-L1 which by interacting with PD-1 on T cells can inactivate the effector functions of both CD4⁺ Thl T cells and CD8⁺ CTLs, leading to evasion of antitumor immune responses (26, 27). To block this potential inhibitory pathway, we investigated whether a combination of CT and anti-PD-Ll antibody would exert a more robust therapeutic effect on LLC tumors. Combination of i.t. CT together with i.t. injection of anti-PD-Ll at 10 μg/mouse twice weekly for two weeks initially arrested LLC growth, and subsequently caused LLC tumors to shrink and to be eliminated (FIG. 8B). However, anti-PD-Ll at 10 μg/mouse twice weekly alone did not eliminate LLC tumors. All LLC-bearing mice treated with i.t. PBS died by day 40, while those treated with CT survived until day 60, a significant improvement over PBS-treated group (FIG. 8C). Strikingly, none of the LLC -bearing mice treated with the combination of CT and anti- PD-Ll died and they all became tumor-free (FIG. 8C).

To determine whether mice cured from LLC by the combination of CT and anti-PD-Ll acquired specific immune protection against LLC, the tumor-free mice were subcutaneously inoculated with LLC and EG7 mouse thymoma cells on the contralateral flanks and the formation of solid tumors on both flanks was monitored. All the mice formed solid EG7 tumors, whereas none of the mice formed LLC tumors, as illustrated by one of the mice photographed after euthanization on day 20 (FIG. 8D). These data demonstrated that the mice cured from LLC in response to treatment with CT and anti-PD-Ll acquired LLC-specific immunity.

Thus, the unique capacity of CT to induce the maturation of both human and mouse DCs was determined, as evidenced by DC upregulation of costimulatory and MHC molecules on the surface as well as enhanced production of TNFoc, IL-Ιβ, and IL-12 (FIGs. 1, 5 & 7) in response to CT (FIGs. 1, 5 & 7). The purified CT preparation used in this study contained no contaminating endotoxin since it stimulated the maturation of both WT and TLR4 knockout DCs. Importantly, CT at concentrations capable of promoting DC production of TNFoc, IL-Ιβ, and IL-12 did not induce DC production of IL-10 (FIG. 5). This is a remarkable feature for CT in the context of cancer treatment for two reasons. First, DCs matured by CT are likely to be preferentially polarized for the induction of Thl -type immune responses that are important for antitumor immune defense.

Although the induction of Thl -type immune responses is promoted by DC-derived IL-12, it is inhibited by DC-derived IL-10 through the induction of Tregs (28, 29). Secondly, the failure of IL- 10 production by DCs in tumor tissues in response to CT treatment would also serve to reduce the level of immunosuppression in the tumor microenvironment because IL-10 is a potent

immunosuppressive cytokine (6, 7).

CT treatment resulted in the downregulation of Ι-κΒα and upregulation of phosphorylated p38 and JNK in DCs (FIG. 6). Reduction of Ι-κΒα level allows p50/p65 complex to translocate from the cytosol to the nucleus, bind to promoters with NF-KB-binding sites for promoting the production of cytokines such as TNFoc, IL-Ιβ, and IL-12 (25, 30). Activation of p38 and JNK in DCs is important for the upregulation of the expression of surface costimulatory and MHC molecules as well as production of IL-12 (25, 28). Therefore, both NF-κΒ and MAPK signaling pathways are involved in CT-induced DC maturation. The signaling pathway upstream of NF-KB and MAPK involves MyD88, because CT-induced DC upregulation of CD86 and I-A/E as well as induction of TNFcc, IL-Ιβ, and IL-12 were greatly compromised in MyD88^_/" DCs (FIG. 7).

However, CT-induced upregulation of 80 and CD83 was not completely inhibited by MyD88 knockout (FIG. 7), suggesting that additional signaling mechanism(s) may also be involved.

The anti-proliferative effect on both human lung cancer cell line A549 (FIG. 1) and mouse lung cancer cell line LLC (FIG. 3) was not based on an overall cytotoxic effect because CT at concentrations as high as 20 g/ml did not lyse human erythrocytes and was not cytotoxic for human macrophages (FIG. 2). Inhibition of LLC proliferation based on the induction of cell cycle arrest at the G2/M phase (FIG. 3). Measurement of cell cycle regulators revealed that treatment of LLC cells with CT dramatically activated p53 (FIG. 4). p53 is an important tumor suppressor that regulates both Gl and G2/M cell-cycle checkpoints of mammalian cells (31, 32). p53 induces Gl arrest by promoting the transcription and expression of p2l^WAF1/CIP1, a cyclin dependent kinase (Cdk) inhibitor (31, 33). Binding of p21^WAF1/CIP1 to a number of cyclin/Cdk complexes inhibits their kinase activities, resulting in hypophosphorylation of Rb, sequestration of E2F, and failure to activate E2F-responsive genes leading to Gl arrest (32, 34). Consistent with the fact that CT did not induce Gl arrest in LLC cells (FIG. 3), CT treatment of LLC cells did not alter the cellular levels of p21^WAF1/CIP1 or the phosphorylation of Rb. p53 causes G2/M arrest by transcriptionally repressing cyclin B 1 and inhibiting Cdc2 because cyclin B 1/Cdc2 complex is required for cell cycle entry into mitosis (31, 32, 35). In full agreement with CT-induced G2/M arrest (FIG. 3), CT-induced activation of p53 was also accompanied by reduction of cyclin Bl and Cdc2 (FIG. 4). Thus, the signaling pathway responsible for CT-induced G2/M arrest in LLC cells involves activation of p53 that downregulates cyclin Bl and Cdc2, leading to G2/M cell cycle arrest (FIG. 4). It was previous reported that treatment of human lung cancer cell line A549 with CT also resulted in

downregulation of cyclin Bl and G2/M cell cycle arrest (23). Therefore, it is likely that induction of G2/M cell cycle arrest is a common pathway for CT-induced inhibition of proliferation of both human and mouse lung cancer cells. In addition to inducing G2/M arrest, CT treatment also caused apoptotic cell death of human lung cancer A549 cells (see also 23). Very recently, CT has also been reported to inhibit the proliferation of A549 human lung cancer cells through a signaling pathway involving induction of reactive oxygen species, activation of JNK, downregulation of mTOR, and ultimately formation of pro-death autophagy (36). Irrespective of the underlying mechanistic basis, administration of CT into nude mice harboring human lung cancers inhibits the growth of xenograft tumors (23, 36), indicating that CT exhibits direct anti-proliferative effect against lung cancer cells in vivo.

It was hypothesized that CT would exhibit therapeutic anti-lung cancer effect in

immunocompetent mice by augmenting the generation of antitumor immunity. Indeed, CT exhibited remarkable therapeutic effect on established LLC tumors when used alone or in combination anti-PD-Ll antibody in mice (FIG. 8). Mice bearing established s.c. LLC tumors were cured by a combination of CT and low doses of anti-PD-Ll (FIG. 8). The data showing that the resultant tumor-free mice were resistant to re-challenge with LLC, but not B16 melanoma, demonstrate that treatment with CT plus low doses of anti-PD-Ll promoted the generation of LLC- specific antitumor immune responses and immunological memory. CT in combination with anti- PD-Ll provides a robust treatment for human lung cancers.

CT has previously been reported to inhibit the proliferation of diverse types of tumor cells in vitro, such as prostate cancer (21, 37, 38), human rhabdomyosarcoma (39), human leukemia (24), breast cancer (40), pancreatic cancer (41), and colon cancer (22). CT treatment of prostate cancer stem cells could inhibit their proliferation and tumorigenesis by downregulating the expression of sternness genes including Nanog, SOX2, Oct4, and CXCR4 (42). CT can promote antitumor immunity to other types of cancers in addition to lung cancer, see FIG. 9 and 10 for the therapeutic effect of CT or CT in combination with anti-PD-Ll on liver tumor.

In conclusion, a TCM-derived compound, CT, was determined to have unique dual capabilities of inhibiting the proliferation of lung cancer cells and inducing DC maturation. This inhibition of lung cancer cell proliferation by CT is mediated by G2/M cell cycle arrest through promotion of p53 activation, whereas CT induces DC maturation via a MyD 88 -dependent pathway involving the activation of NF-κΒ, p38, and JNK. CT is effective for the treatment of established LLC and liver cancers alone or even more effectively in combination with anti-PD-Ll in immunocompetent mice (see FIG. 8 and FIG. 10). Thus, the combination therapy can be used for the treatment of a variety of tumors. References

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In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

It is claimed:

1. A method of treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of:

a) cryptotanshinone (CT) or a salt or derivative thereof; and

b) a Programmed Death (PD-1) antagonist;

thereby treating the tumor in the subject.

2. The method of claim 1, wherein the tumor is a hepatic cancer or a lung cancer.

3. The method of claim 2, wherein the lung cancer is a small cell carcinoma of the lung or a non-small cell carcinoma of the lung.

4. The method of any one of the prior claims, wherein the subject is human.

5. The method of any one of the prior claims, wherein the PD-1 antagonist is an inhibitory RNA, an antisense RNA, a dominant negative protein, or an antibody that specifically binds to PD-1, PD-Ll, PD-L2, or an antigen binding fragment thereof.

6. The method of claim 5, wherein the monoclonal antibody is a human monoclonal antibody or a humanized monoclonal antibody.

7. The method of any one of the prior claims, wherein the method increases survival of the subject.

8. The method of any one of the prior claims, wherein the tumor is positive for PD-Ll expression by an immunohistochemical (IHC) assay.

9. The method of any one of the prior claims, wherein the PD-1 antagonist is a monoclonal antibody, or an antigen binding fragment thereof, which specifically binds to human PD-1, human PD-Ll, or human PD-L2, and blocks the binding of human PD-Ll to human PD-1 and/or human PD-L2 to human PD- 1.

10. The method of any one of the prior claims, wherein the PD-1 antagonist is one of nivolumab, pembrolizumab, pidilizumab, atezolizumab, avelumab, or durvalumab.

11. The method of any one of claims 1-9, wherein the PD-1 antagonist is a) a monoclonal antibody that specifically binds PD-Ll, or an antigen binding fragment thereof; or b) a monoclonal antibody that specifically binds PD-1, or an antigen binding fragment thereof.

12. The method of any one of the prior claims, wherein the hepatic cancer is hepatocellular carcinoma.

13. The method of any one of the prior claims, wherein the CT is administered by oral, intratumor, intramuscular, or intravenous administration

14. The method of any one of the prior claims, wherein the PD-1 antagonist is administered by intratumor, intramuscular, or intravenous administration.

15. The method of any one of the prior claims, wherein treating the tumor comprises decreasing tumor volume, decreasing the number or size of metastases, or lessening a symptom of the tumor.

16. The method of any one of the prior claims, further comprising surgically resecting the tumor.

17. The method of any one of the prior claims, further comprising administering to the subject a therapeutically effective amount of an additional chemotherapeutic agent.

18. The method of any one of the prior claims, wherein the method inhibits reoccurrence of the tumor in the subject.

19. Use of a therapeutically effective amount of a combination therapy of a) CT and b) a PD-1 antagonist to treat a tumor in the subject.

20. The use of claim 19, wherein the PD-1 antagonist is an antibody that specifically binds PD-Ll or an antigen binding fragment thereof.