A method for treating cancer by intratumoral administration of a combination of tumor cell lysis and immunotherapy components

Abstract

The present disclosure provides a method for treating cancer, which includes, inter alia, a) an intratumoral cell lysis step mediated by cryolysis, and b) an intratumoral administration step of a combination of immunotherapeutic agents including: 1) i) a TLR9 agonist CpG oligodeoxynucleotide, ii) an agonistic anti-CD40 monoclonal antibody, iii) an agonistic anti-OX40 monoclonal antibody, and iv) an anti-CTLA4 monoclonal antibody, or 2) i) a TLR9 agonist CpG oligodeoxynucleotide, ii) an agonistic anti-CD40 monoclonal antibody, iii) an anti-PD1 monoclonal antibody, and iv) an anti-CTLA4 monoclonal antibody.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 347,690, filed on June 1, 2022, which is hereby incorporated by reference in its entirety for all purposes.

Background Art

[0002] Current clinical approaches to cancer immunotherapy rely on the systemic administration of high doses of one or two biological immunomodulatory agents. These agents aim to restore the already established state of tumor - induced immunosuppression while stimulating the adaptive immune response against tumor - specific antigens (TSAs) and tumor - associated antigens (TAAs).

[0003] Cancer treatment has advanced significantly in the past decade through the use of antagonistic monoclonal antibodies (mAbs) that block immune checkpoint inhibitor molecules such as PD - 1, PD - L1, and CTLA - 4. However, despite the significant benefits obtained by the use of these mAbs, a substantial proportion of patients still do not respond to treatment or become resistant to it. Furthermore, the high - dose systemic use of these checkpoint inhibitors, especially when used in combination with other immunotherapy agents, results in a high frequency of severe adverse events, which limits the number and dosage of agents that can be combined to address the multifactorial nature of tumor immune resistance (Vilgelm, Johnson et al. 2016). There is a need in the art for new approaches to cancer immunotherapy.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Current cancer immunotherapy approaches are effective only in a limited subset of patients, and their use often results in only partial responses for a limited period of time. Additionally, current cancer immunotherapy approaches are associated with treatment-acquired resistance and a high rate of immune-related adverse events, which limit treatment compliance and applicability. The present disclosure describes methods and compositions for treating cancer that overcome many of the limitations of current cancer immunotherapy approaches.

Means for Solving the Problems

[0005] In some embodiments, the immunotherapy methods of the present disclosure are not antigen-specific and do not require prior antigen identification. The methods of the present disclosure, in many embodiments, include combinations of different pharmacological agents that mediate different and synergistic anti-tumor immune activation effects, thereby enhancing efficacy and reducing the likelihood of immune-acquired resistance compared to treatments that rely on a single therapeutic agent. Additionally, the use of the compositions of the present disclosure via the intratumoral administration route mediates a local area treatment that improves the accessibility of the drug and the biodistribution of the drug to immune-related tumor-associated immune cells and related secondary and tertiary lymphoid tissues. In addition, as described herein, the intratumoral administration method allows for a significant reduction in dosage, which reduces the probability of toxicity and adverse events. This allows for an increase in the number of therapeutic agents in the drug combinations of the present disclosure and an expansion of the breadth of pharmacological effects that enhance the anti-tumor effect. The expansion of the therapeutic window by the use of low dosages and the increase in the number of therapeutic agents with synergistic mechanisms of action improve the effectiveness of treatment while reducing the frequency and severity of adverse events. Thus, the present disclosure provides, among other things, methods for treating cancer that overcome the limitations of current cancer immunotherapy options.

[0006] The present disclosure further provides combinations and compositions of immunotherapeutic agents that mediate, at least, (1) potent activation of the innate arm of the immune response that mediates a local anti-tumor effect, (2) recruitment of antigen-presenting cells, activation and maturation of APCs to activate the adaptive arm of the anti-tumor immune response, (3) increased activation, proliferation, and survival of T cells, (4) reduction of the immunosuppressive effect of regulatory T cells, and / or (5) restoration and prevention of T cell exhaustion by factors that dominate the suppressive tumor microenvironment.

[0007] The present disclosure also provides dosages of the various immunotherapeutic agents present in the compositions and formulations described herein. Indeed, the present disclosure further provides compositions for treating cancer, particularly for overcoming the limitations of current cancer immunotherapy options.

[0008] For example, in some embodiments, the present disclosure provides a method for treating cancer by including a first step aimed at initiating in situ immunization by using an intratumoral cryoablation device to cause lysis of tumor cells and immunogenic cell death, and a second step including intratumoral administration of a combination of immunotherapeutic agents that mediate different pharmacological effects. In some embodiments, the combination of immunotherapeutic agents mediates a synergistic pharmacological effect when administered to a subject.

[0009] Furthermore, in some embodiments, the present disclosure provides methods and compositions comprising an immunotherapeutic combination of active ingredients administered by intratumoral administration. In some embodiments, the composition, or combination formulation, comprises a TLR9 agonist, an agonistic anti-CD40 monoclonal antibody, an agonistic anti-OX40 monoclonal antibody, and an anti-CTLA4 monoclonal antibody. In some embodiments, the composition, or combination formulation, consists of a TLR9 agonist, an agonistic anti-CD40 monoclonal antibody, an agonistic anti-OX40 monoclonal antibody, and an anti-CTLA4 monoclonal antibody. In some embodiments, the composition, or combination formulation, comprises a TLR9 agonist, an agonistic anti-CD40 monoclonal antibody, an anti-PD1 monoclonal antibody, and an anti-CTLA4 monoclonal antibody. In some embodiments, the composition, or combination formulation, consists of a TLR9 agonist, an agonistic anti-CD40 monoclonal antibody, an anti-PD1 monoclonal antibody, and an anti-CTLA4 monoclonal antibody.

[0010] Furthermore, in some embodiments, the present disclosure provides dosage ranges for each active ingredient for different combinations of the pharmaceutical formulations disclosed herein.

[0011] The present disclosure provides a method of treating cancer in a subject, the method comprising: a) administering a treatment that causes tumor cell lysis to the tumor of the subject; and b) administering to the subject a therapeutically effective amount of a combination formulation, the combination formulation comprising: a. a TLR9 agonist; b. an agonistic anti-CD40 monoclonal antibody; c. an anti-CTLA4 monoclonal antibody; and d. an agonistic anti-OX40 monoclonal antibody or an anti-PD1 monoclonal antibody.

[0012] In some embodiments, the combination formulation comprises: a. a TLR9 agonist; b. an agonistic anti-CD40 monoclonal antibody; c. an agonistic anti-OX40 monoclonal antibody; and d. an anti-CTLA4 monoclonal antibody.

[0013] In some embodiments, the combination formulation consists of: a. a TLR9 agonist, b. an agonistic anti-CD40 monoclonal antibody, c. an agonistic anti-OX40 monoclonal antibody, and d. an anti-CTLA4 monoclonal antibody.

[0014] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0015] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0016] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 2; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0017] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0018] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35.

[0019] In some embodiments, the combination formulation comprises the TLR9 agonist set forth in SEQ ID NO: 1; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 31; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0020] In some embodiments, the combination formulation comprises the TLR9 agonist set forth in SEQ ID NO: 2; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 31; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0021] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 31; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0022] In some embodiments, the combination formulation comprises: a. a TLR9 agonist, b. an agonistic anti-CD40 monoclonal antibody, c. an anti-PD1 monoclonal antibody, and d. an anti-CTLA4 monoclonal antibody.

[0023] In some embodiments, the combination formulation consists of: a. a TLR9 agonist, b. an agonistic anti-CD40 monoclonal antibody, c. an anti-PD1 monoclonal antibody, and d. an anti-CTLA4 monoclonal antibody.

[0024] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0025] In some embodiments, the combination formulation comprises the TLR9 agonist set forth in SEQ ID NO: 1; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0026] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 2; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0027] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0028] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35.

[0029] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 33; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0030] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 2; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 33; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0031] In some embodiments, the combination formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 33; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0032] In some embodiments, the TLR9 agonist, the agonistic anti-CD40 monoclonal antibody, the agonistic anti-OX40 monoclonal antibody, and the anti-CTLA4 monoclonal antibody are co-administered.

[0033] In some embodiments, the TLR9 agonist, the agonistic anti-CD40 monoclonal antibody, the agonistic anti-OX40 monoclonal antibody, and the anti-CTLA4 monoclonal antibody are administered sequentially.

[0034] In some embodiments, the TLR9 agonist is administered sequentially in a separate composition from a composition comprising the agonistic anti-CD40 monoclonal antibody, the agonistic anti-OX40 monoclonal antibody, and the anti-CTLA4 monoclonal antibody.

[0035] In some embodiments, the TLR9 agonist, the agonistic anti-CD40 monoclonal antibody, the anti-PD1 monoclonal antibody, and the anti-CTLA4 monoclonal antibody are co-administered.

[0036] In some embodiments, the TLR9 agonist, the agonistic anti-CD40 monoclonal antibody, the anti-PD1 monoclonal antibody, and the anti-CTLA4 monoclonal antibody are administered sequentially.

[0037] In some embodiments, the TLR9 agonist is administered sequentially in a separate composition from a composition comprising an agonistic anti-CD40 monoclonal antibody, an anti-PD1 monoclonal antibody, and an anti-CTLA4 monoclonal antibody.

[0038] In some embodiments, the treatment administered to the tumor is cryoablation therapy that mediates tumor cell lysis by cryoablation.

[0039] In some embodiments, the administration of cryoablation therapy includes at least 1 cycle, at least 2 cycles, or at least 3 cycles of freezing and thawing. In some embodiments, the administration of cryoablation therapy includes 1 cycle of freezing and thawing. In some embodiments, the administration of cryoablation therapy includes 2 cycles of freezing and thawing. In some embodiments, the administration of cryoablation therapy includes 3 cycles of freezing and thawing.

[0040] In some embodiments, the administration of cryoablation therapy includes 1 cycle or less, 2 cycles or less, or 3 cycles or less of freezing and thawing. In some embodiments, the administration of cryoablation therapy includes 1 cycle or less of freezing and thawing. In some embodiments, the administration of cryoablation therapy includes 2 cycles or less of freezing and thawing. In some embodiments, cryoablation therapy includes 3 cycles or less of freezing and thawing.

[0041] In some embodiments, cryoablation therapy is mediated by an intratumoral cryoablation device that contacts the tumor cells during the freeze-thaw cycle and includes a cryoprobe for freezing. In some embodiments, the coolant circulates within the cryoprobe. In some embodiments, the coolant is selected from the group consisting of argon, nitrous oxide, carbon dioxide, and liquid nitrogen.

[0042] In some embodiments, the intratumoral cryoablation device is set to a duty cycle of about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, and is operated to generate a cryoprobe temperature of about -40°C or lower, about -45°C or lower, about -50°C or lower, about -55°C or lower, about -60°C or lower, about -65°C or lower, about -70°C or lower, about -75°C or lower, about -80°C or lower, about -85°C or lower, about -90°C or lower, about -100°C or lower, about -110°C or lower, or about -120°C or lower in less than about 1 minute during the freeze-thaw cycle, and then a passive thawing process is performed. In some embodiments, the intratumoral cryoablation device is set to a duty cycle of about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, and is operated to generate a cryoprobe temperature of about -40°C or lower, about -45°C or lower, about -50°C or lower, about -55°C or lower, about -60°C or lower, about -65°C or lower, about -70°C or lower, about -75°C or lower, about -80°C or lower, about -85°C or lower, about -90°C or lower, about -100°C or lower, about -110°C or lower, or about -120°C or lower in less than about 30 seconds during the freeze-thaw cycle, and then a passive thawing process is performed. In some embodiments, the intratumoral cryoablation device is set to a duty cycle of about 70% to about 100%, and is operated to generate a cryoprobe temperature of about -40°C to about -60°C in less than about 1 minute during the freeze-thaw cycle, and then a passive thawing process is performed. In some embodiments, the intratumoral cryoablation device is set to a duty cycle of about 70% to about 100%, and is operated to generate a cryoprobe temperature of about -40°C to about -60°C in less than about 30 seconds during the freeze-thaw cycle, and then a passive thawing process is performed. In some embodiments, the intratumoral cryoablation device is set to a 100% duty cycle, and is operated to generate a cryoprobe temperature of -40°C or lower in less than about 1 minute during the freeze-thaw cycle, and then a passive thawing process is performed. In some embodiments, the temperature of the generated cryoprobe is maintained for about 1 minute before passive thawing. In some embodiments, the temperature of the generated cryoprobe is maintained for about 2 minutes before passive thawing. In some embodiments, the temperature of the generated cryoprobe is maintained for about 3 minutes before passive thawing.In some embodiments, the temperature of the generated cryoprobe continues to be cooled to a temperature of about -70°C or lower, about -80°C or lower, about -90°C or lower, about -100°C or lower, about -110°C or lower, about -120°C or lower, about -130°C or lower, about -140°C or lower, or about -150°C or lower for a total of about 5 minutes, about 4.5 minutes, about 4 minutes, about 3.5 minutes, about 3 minutes, about 2.5 minutes, about 2 minutes, about 1.75 minutes, about 1.5 minutes, about 1.25 minutes, about 1 minute, about 50 seconds, about 40 seconds, about 30 seconds, about 20 seconds, or about 10 seconds.

[0043] In some embodiments, the cryoprobe is cooled to a temperature that creates an ice ball having a diameter of about 8 mm to about 30 mm, about 10 mm to about 30 mm, about 15 mm to about 30 mm, about 20 mm to about 30 mm, or about 25 mm to about 30 mm, about 8 mm to about 25 mm, about 8 mm to about 20 mm, about 8 mm to about 15 mm, about 8 mm to about 10 mm, about 10 mm to about 18 mm, or about 12 mm to about 16 mm within the tumor. In some embodiments, the cryoprobe is cooled to a temperature that creates an ice ball having a diameter of about 12 mm to about 16 mm within the tumor. In some particularly preferred embodiments, the cryoprobe is cooled to a temperature that creates an ice ball having a diameter of about 14 mm within the tumor.

[0044] In some embodiments, most of the ice ball has a temperature of about -40°C. In some embodiments, most of the ice ball has a temperature of about -50°C. In some embodiments, most of the ice ball has a temperature of about -60°C. In some embodiments, most of the ice ball has a temperature of about -40°C or lower. In some embodiments, most of the ice ball has a temperature of about -50°C or lower. In some embodiments, most of the ice ball has a temperature of about -60°C or lower. In some embodiments, most of the ice ball has a temperature of about -40°C to about -60°C.

[0045] In some embodiments, the ice ball includes a region having a diameter of about 1 cm (10 mm) and a temperature of about -40°C inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 1 cm and a temperature of about -50°C inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 1 cm and a temperature of about -60°C inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 1 cm and a temperature of about -40°C or less inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 1 cm and a temperature of about -50°C or less inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 1 cm and a temperature of about -60°C or less inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 0.5 cm to about 1.5 cm and a temperature of about -40°C inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 0.5 cm to about 1.5 cm and a temperature of about -50°C inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 0.5 cm to about 1.5 cm and a temperature of about -60°C inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 0.5 cm to about 1.5 cm and a temperature of about -40°C or less inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 0.5 cm to about 1.5 cm and a temperature of about -50°C or less inside thereof. In some embodiments, the ice ball includes a region having a diameter of about 0.5 cm to about 1.5 cm and a temperature of about -60°C or less inside thereof.

[0046] In some embodiments, cryoablation does not excise the entire tumor but rather causes partial lysis of tumor cells.

[0047] In some embodiments, cryoablation causes necrotic cell death in a zone of about 14 mm in diameter of the tumor tissue. In some embodiments, cryoablation causes necrotic cell death in a zone of about 8 mm to about 30 mm, about 10 mm to about 30 mm, about 15 mm to about 30 mm, about 20 mm to about 30 mm, about 25 mm to about 30 mm, about 8 mm to about 25 mm, about 8 mm to about 20 mm, about 8 mm to about 15 mm, about 8 mm to about 10 mm, about 10 mm to about 18 mm, or about 12 mm to about 16 mm of the tumor tissue.

[0048] In some embodiments, the TLR9 agonist is a class B or C CpG ODN. In some embodiments, the TLR9 agonist is a class B CpG ODN. In some embodiments, the TLR9 agonist is a class C CpG ODN.

[0049] In some embodiments, the TLR9 agonist comprises a nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 1. In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 2. In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 27.

[0050] In some embodiments, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody is of the human IgG2k isotype.

[0051] In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0052] In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0053] In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28, and the light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28, and the light chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 28, and the light chain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 28, and the light chain comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 28, and the light chain comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 28, and the light chain comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 29.In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 28, and the light chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 28, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 29.

[0054] In some embodiments, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody is of the human IgG1k isotype.

[0055] In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30.

[0056] In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 31.

[0057] In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 31.In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 30, and the light chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 30, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 31.

[0058] In some embodiments, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody is of the human IgG4k isotype.

[0059] In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0060] In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 33.

[0061] In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32, and the light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 32, and the light chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 32, and the light chain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 32, and the light chain comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 32, and the light chain comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 32, and the light chain comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 32, and the light chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 33.In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 32, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0062] In some embodiments, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0063] In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34.

[0064] In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0065] In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34, and the light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 34, and the light chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 34, and the light chain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 34, and the light chain comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 34, and the light chain comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 34, and the light chain comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 35.In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 34, and the light chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 34, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0066] In some embodiments, the treatment that causes tumor cell lysis, and the combination formulation, are administered into the same tumor. In some embodiments, the treatment that causes tumor cell lysis, and the combination formulation, are administered into different tumors.

[0067] In some embodiments, the treatment is repeated about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, about once every ten weeks, about once every eleven weeks, about once every twelve weeks, about once every thirteen weeks, about once every fourteen weeks, about once every fifteen weeks, or about once every sixteen weeks. In some embodiments, the treatment is repeated about every 3 - 16 weeks, about every 6 - 16 weeks, about every 8 - 16 weeks, about every 10 - 16 weeks, about every 12 - 16 weeks, about every 14 - 16 weeks, about every 3 - 14 weeks, about every 3 - 12 weeks, about every 3 - 10 weeks, about every 3 - 8 weeks, about every 3 - 6 weeks, or about every 3 - 4 weeks. In some embodiments, the treatment is repeated about every 4 - 8 weeks, about every 5 - 8 weeks, about every 6 - 8 weeks, about every 7 - 8 weeks, about every 4 - 7 weeks, about every 4 - 6 weeks, or about every 4 - 5 weeks. In some embodiments, the treatment is repeated about every 4 - 8 weeks.

[0068] In some embodiments, the treatment is repeated for 1 to 12 cycles, 1 to 10 cycles, 1 to 8 cycles, 1 to 6 cycles, 1 to 4 cycles, 1 to 2 cycles, 3 to 12 cycles, 5 to 12 cycles, 7 to 12 cycles, 9 to 12 cycles, or 11 to 12 cycles. In some embodiments, the treatment is repeated for 3 to 6 cycles, 3 to 5 cycles, 3 to 4 cycles, 4 to 6 cycles, or 5 to 6 cycles. In some embodiments, the treatment is repeated for 3 to 6 cycles.

[0069] In some embodiments, the TLR9 agonist is administered at a dose in the range of about 0.5 mg to about 10 mg, about 1 mg to about 10 mg, about 2 mg to about 10 mg, about 4 mg to about 10 mg, about 8 mg to about 10 mg, about 0.5 mg to about 8 mg, about 0.5 mg to about 6 mg, about 0.5 mg to about 4 mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 1 mg, about 1 mg to about 4 mg, about 2 mg to about 4 mg, or about 3 mg to about 4 mg.

[0070] In some embodiments, the anti-CD40 agonistic monoclonal antibody is administered at a dose in the range of about 0.5 mg to about 10 mg, about 1 mg to about 10 mg, about 2 mg to about 10 mg, about 4 mg to about 10 mg, about 8 mg to about 10 mg, about 0.5 mg to about 8 mg, about 0.5 mg to about 6 mg, about 0.5 mg to about 4 mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 1 mg, about 1 mg to about 8 mg, or about 4 mg to 8 mg.

[0071] In some embodiments, the anti-OX40 agonistic monoclonal antibody is administered at a dose in the range of about 0.5 mg to about 10 mg, about 1 mg to about 10 mg, about 2 mg to about 10 mg, about 4 mg to about 10 mg, about 8 mg to about 10 mg, about 0.5 mg to about 8 mg, about 0.5 mg to about 6 mg, about 0.5 mg to about 4 mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 1 mg, about 1 mg to about 8 mg, or about 4 mg to 8 mg.

[0072] In some embodiments, the anti-PD1 monoclonal antibody is administered at a dosage in the range of about 1 mg to about 100 mg, about 2 mg to about 100 mg, about 4 mg to about 100 mg, about 8 mg to about 100 mg, about 10 mg to about 100 mg, about 20 mg to about 100 mg, about 30 mg to about 100 mg, about 40 mg to about 100 mg, about 50 mg to about 100 mg, about 70 mg to about 100 mg, about 90 mg to about 100 mg, about 1 mg to about 90 mg, about 1 mg to about 70 mg, about 1 mg to about 50 mg, about 1 mg to about 40 mg, about 1 mg to about 30 mg, about 1 mg to about 20 mg, about 1 mg to about 10 mg, about 3 mg to about 10 mg, 3 mg to about 30 mg, about 3 mg to about 100 mg, or about 10 mg to about 30 mg.

[0073] In some embodiments, the anti-CTLA4 monoclonal antibody is administered at a dosage in the range of about 1 mg to about 50 mg, about 2 mg to about 50 mg, about 4 mg to about 50 mg, about 8 mg to about 50 mg, about 10 mg to about 50 mg, about 20 mg to about 50 mg, about 30 mg to about 50 mg, about 40 mg to about 50 mg, about 1 mg to about 40 mg, about 1 mg to about 30 mg, about 1 mg to about 20 mg, about 1 mg to about 10 mg, about 1 mg to about 5 mg, about 1 mg to about 2 mg, about 5 mg to about 15 mg, about 5 mg to about 40 mg, or about 15 mg to about 40 mg.

[0074] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-OX40 agonist antibody, and anti-CTLA4 antibody are each administered at a dosage of about 1 mg, about 1 mg, about 1 mg, and about 1 mg, respectively.

[0075] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-OX40 agonist antibody, and anti-CTLA4 antibody are each administered at a dosage of about 2 mg, about 5 mg, about 5 mg, and about 5 mg, respectively.

[0076] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-OX40 agonist antibody, and anti-CTLA4 antibody are each administered at a dosage of about 3 mg, about 7.5 mg, about 7.5 mg, and about 15 mg, respectively.

[0077] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-OX40 agonist antibody, and anti-CTLA4 antibody are each administered at a dose of about 4 mg, about 10 mg, about 10 mg, and about 40 mg, respectively.

[0078] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody, and anti-CTLA4 antibody are each administered at a dose of about 1 mg, about 1 mg, about 3 mg, and about 1 mg, respectively.

[0079] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody, and anti-CTLA4 antibody are each administered at a dose of about 2 mg, about 5 mg, about 10 mg, and about 5 mg, respectively.

[0080] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody, and anti-CTLA4 antibody are each administered at a dose of about 3 mg, about 7.5 mg, about 30 mg, and about 15 mg, respectively.

[0081] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody, and anti-CTLA4 antibody are each administered at a dose of about 4 mg, about 10 mg, about 80 mg, and about 40 mg, respectively.

[0082] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody, and anti-CTLA4 antibody are each administered at a dose of about 4 mg, about 10 mg, about 100 mg, and about 40 mg, respectively.

[0083] In some embodiments, the combination formulation is administered in a total volume in the range of about 1 mL to about 30 mL, about 1 mL to about 25 mL, about 1 mL to about 20 mL, about 1 mL to about 15 mL, about 1 mL to about 10 mL, about 1 mL to about 5 mL, about 1 mL to about 2 mL, about 2 mL to about 30 mL, about 5 mL to about 30 mL, about 10 mL to about 30 mL, about 15 mL to about 30 mL, about 20 mL to about 30 mL, about 25 mL to about 30 mL, about 5 mL to about 25 mL, about 10 mL to about 20 mL, about 10 mL to about 15 mL, or about 15 mL to about 20 mL. In some embodiments, the combination formulation is administered in a total volume in the range of about 1 mL to about 30 mL. In some embodiments, the total volume is about 15 mL.

[0084] In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from adenocarcinoma, astrocytoma, bladder cancer, osteosarcoma, breast cancer, cervical cancer, chordoma, colorectal cancer, endometrial cancer, esophageal cancer, glioblastoma, glioma, kidney cancer, liver cancer, medulloblastoma, melanoma, meningioma, mesothelioma, metastatic pituitary cancer, prostate cancer, neuroblastoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer, ovarian cancer, pancreatic cancer, renal cell cancer, retinoblastoma, sarcoma, small cell lung cancer, squamous cell carcinoma (including head and neck cancer), gastric cancer, testicular cancer, thyroid cancer, and Wilms tumor. In some embodiments, the cancer is metastatic prostate cancer. In some embodiments, the cancer is metastatic breast cancer. In some embodiments, the cancer is non-small cell lung cancer.

[0085] The present disclosure provides a pharmaceutical composition comprising a. a TLR9 agonist, b. an agonistic anti-CD40 monoclonal antibody, c. an anti-CTLA4 monoclonal antibody, d. an agonistic anti-OX40 monoclonal antibody, and e. a pharmaceutically acceptable excipient. The present disclosure further provides a pharmaceutical composition consisting of a. a TLR9 agonist, b. an agonistic anti-CD40 monoclonal antibody, c. an anti-CTLA4 monoclonal antibody, d. an agonistic anti-OX40 monoclonal antibody, and e. a pharmaceutically acceptable excipient.

[0086] In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0087] In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29. In some embodiments, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31. In some embodiments, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35.

[0088] In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0089] In some embodiments, the respective dosages of the TLR9 agonist, the anti-CD40 agonist antibody, the anti-OX40 agonist antibody, and the anti-CTLA4 antibody are selected from: a. approximately 1 mg, approximately 1 mg, approximately 1 mg, and approximately 1 mg, respectively; b. approximately 2 mg, approximately 5 mg, approximately 5 mg, and approximately 5 mg, respectively; c. approximately 3 mg, approximately 7.5 mg, approximately 7.5 mg, and approximately 15 mg, respectively; and d. approximately 4 mg, approximately 10 mg, approximately 10 mg, and approximately 40 mg, respectively.

[0090] The present disclosure provides a pharmaceutical composition comprising: a. a TLR9 agonist; b. an agonistic anti-CD40 monoclonal antibody; c. an anti-CTLA4 monoclonal antibody; d. an agonistic anti-PD1 monoclonal antibody; and e. a pharmaceutically acceptable excipient. The present disclosure further provides a pharmaceutical composition consisting of: a. a TLR9 agonist; b. an agonistic anti-CD40 monoclonal antibody; c. an anti-CTLA4 monoclonal antibody; d. an agonistic anti-PD1 monoclonal antibody; and e. a pharmaceutically acceptable excipient.

[0091] In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0092] In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29. In some embodiments, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In some embodiments, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35.

[0093] In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 28, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 32, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 34, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0094] In some embodiments, the respective dosages of the TLR9 agonist, the anti-CD40 agonist antibody, the anti-PD1 antibody, and the anti-CTLA4 antibody are selected from: a. approximately 1 mg, approximately 1 mg, approximately 3 mg, and approximately 1 mg, respectively; b. approximately 2 mg, approximately 5 mg, approximately 10 mg, and approximately 5 mg, respectively; c. approximately 3 mg, approximately 7.5 mg, approximately 30 mg, and approximately 15 mg, respectively; and d. approximately 4 mg, approximately 10 mg, approximately 100 mg, and approximately 40 mg, respectively.

[0095] In some embodiments, the respective dosages of the TLR9 agonist, the anti-CD40 agonist antibody, the anti-PD1 antibody, and the anti-CTLA4 antibody are selected from: a. approximately 1 mg, approximately 1 mg, approximately 3 mg, and approximately 1 mg, respectively; b. approximately 2 mg, approximately 5 mg, approximately 10 mg, and approximately 5 mg, respectively; c. approximately 3 mg, approximately 7.5 mg, approximately 30 mg, and approximately 15 mg, respectively; d. approximately 4 mg, approximately 10 mg, approximately 80 mg, and approximately 40 mg, respectively; and e. approximately 4 mg, approximately 10 mg, approximately 100 mg, and approximately 40 mg, respectively.

[0096] The present disclosure provides, inter alia, a method of treating cancer in a subject, the method comprising: a) administering cryoablation therapy that causes partial tumor cell lysis in the subject's tumor; and b) administering a therapeutically effective amount of a combination formulation to the same tumor of step a), the combination formulation comprising: a. a TLR9 agonist; b. an agonistic anti-CD40 monoclonal antibody; c. an anti-CTLA4 monoclonal antibody; and d. an agonistic anti-OX40 monoclonal antibody, wherein the cryoablation therapy is administered prior to the administration of the combination formulation.

[0097] The present disclosure provides, inter alia, a method of treating cancer in a subject, the method comprising: a) administering cryoablation therapy that causes partial tumor cell lysis in the subject's tumor; and b) administering a therapeutically effective amount of a combination formulation to the same tumor of step a), the combination formulation comprising: a. a TLR9 agonist; b. an agonistic anti-CD40 monoclonal antibody; c. an anti-CTLA4 monoclonal antibody; and d. an anti-PD1 monoclonal antibody, wherein the cryoablation therapy is administered prior to the administration of the combination formulation.

[0098] In some embodiments, the combination formulation is any pharmaceutical composition described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0099]

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Mode for Carrying Out the Invention

[0100] Definitions Administration: As used herein, the term "administration" refers to the administration of treatment (e.g., a composition / formulation, or other treatment modalities such as cryotherapy) to a subject or system. Administration to an animal subject (e.g., a human) can be by any suitable route. For example, in some embodiments, administration can be intratumoral. In some embodiments, administration is by injection or perfusion (e.g., using the methods described herein). In some embodiments, administration is intratumoral administration for the purpose of local regional therapy where it is expected that the injected formulation will not completely fit within the tumor tissue and will actually enter the lymphatic system structures connecting to the tumor and the peritumoral tissue through lymphatics.

[0101] Agent: As used herein, the term "agent" can refer to a compound or entity of any chemical classification, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations thereof. In some embodiments, the agent is one or more entities that are artifacts in that they are designed, engineered, and / or produced by the action of human hands and / or are not found in nature. In some embodiments, the agent can be utilized in isolated form or in pure form. In some embodiments, the agent can be utilized in crude form. Some specific embodiments of agents that can be utilized in accordance with the present disclosure include antibodies, antibody fragments, nucleic acids. In some specific embodiments, the agent of the present disclosure is a CpG oligodeoxynucleotide (CpG ODN). In some specific embodiments, the agent of the present disclosure is a monoclonal antibody. In some aspects of the present disclosure, the agent is a "therapeutic agent" or "pharmacological agent" that elicits a desired pharmacological effect when administered to an organism (e.g., a subject). In some embodiments, the above-mentioned agent that elicits a desired pharmacological effect when administered to an organism can be said to be "active" or "pharmacologically active". In some embodiments, an agent is considered a therapeutic agent if it shows a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be a population of model organisms. In some embodiments, the appropriate population can be defined by various criteria such as a particular age group, gender, genetic background, existing clinical condition, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, reduce, inhibit, prevent, delay the onset thereof, reduce the severity thereof, and / or reduce the incidence thereof of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, the agent is a component in a combination formulation or composition. In some embodiments, a "therapeutic agent" is an agent that has been approved or needs to be approved by a government agency before being marketed for administration to humans. In some embodiments, a "therapeutic agent" is an agent that requires a medical prescription for administration to humans.

[0102] Antibody: As used herein, the term "antibody" as used herein may include the whole antibody and any antigen-binding fragment (i.e., "antigen-binding portion"), or single chains thereof. "Antibody" refers, in some embodiments, to a glycoprotein comprising at least two heavy chains and two light chains interconnected by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region is composed of three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. In some embodiments, the light chain constant region is composed of one domain CL. The VH region and the VL region can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four framework regions (FRs), which are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy chain and / or the light chain contain the binding domains that interact with the antigen. In some embodiments, the constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Unless otherwise indicated, the antibody can be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. The IgG isotype is divided into subclasses in certain species: in humans, IgG1, IgG2, IgG3, and IgG4; in mice, IgG1, IgG2a, IgG2b, and IgG3. Immunoglobulins, such as human IgG1, exist in several allotypes that differ from each other by up to a few amino acids. Unless otherwise indicated, "antibody" may include, by way of example, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, fully synthetic antibodies, as well as single-chain antibodies.

[0103] Combination therapy: As used herein, the term "combination therapy" can refer to a situation where a subject is simultaneously exposed to two or more treatment regimens or treatments (e.g., including two or more therapeutic agents). In some embodiments, two or more agents can be administered simultaneously (i.e., co-administered). In some embodiments, such agents can be administered sequentially (in a particular order or in any order indicated by the context). In some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, combination therapy is administered in the form of a combination formulation or composition (e.g., the combination formulations and compositions described herein). In some embodiments, the plurality of agents of the composition or formulation are mixed immediately prior to administration.

[0104] Cryoablation: As used herein, the term "cryoablation" can refer to a process that uses local ultra-low temperatures to destroy target tissue, such as abnormal or diseased tissue, e.g., a tumor. Cryoablation is performed using a hollow needle (e.g., a cryoprobe) through which a cooled heat-conductive fluid circulates. During cryoablation, the cryoprobe is placed adjacent to or within the target tissue (e.g., intratumoral placement) in such a way that the freezing process destroys the abnormal or diseased tissue, such as a tumor, completely or to a substantially complete extent. Once the cryoprobe is positioned at a given location, an attached cryogenic freezing unit extracts heat ( "cools") from the tip of the cryoprobe and, as it extends, from the surrounding tissue of the target zone. Cryoablation occurs in frozen tissue by at least three mechanisms: 1) the formation of ice crystals within cells, which thereby disrupts the membranes and inhibits cell metabolism among other processes, 2) the coagulation of blood, which thereby inhibits blood flow to the tissue, resulting in ischemia and cell death, and 3) the induction of apoptosis, the so-called programmed cell death cascade. Cryoablation is applicable to the treatment of most solid tumors. One of ordinary skill in the art will understand which tumors are amenable to treatment by cryogenic therapies such as cryoablation or cryolysis (e.g., the cryolysis therapy described herein). In many embodiments of the present disclosure, the administration of cryogenic therapy is performed in such a way as to avoid cryoablation. In some embodiments of the present disclosure, cryogenic therapy is performed in such a way as to promote cryolysis (i.e., partial dissolution of tumor tissue), as opposed to cryoablation.

[0105] Cryoablation: As used herein, the term "cryoablation" can refer to a process that uses local ultra-low temperatures to partially destroy target tissue, such as abnormal or diseased tissue, such as a tumor. Thus, cryoablation performed by the methods described herein is similar to cryoablation, but cryoablation is not aimed at excising the entire tumor. Thus, during administration of a treatment that causes cell lysis by cryoablation, the cryoprobe is placed adjacent to or within the target tissue (e.g., intratumoral placement) in such a way that the freezing process partially destroys abnormal or diseased tissue such as a tumor. In many embodiments of the present disclosure, the goal of cryoablation is not to achieve complete resection of tumor tissue (i.e., cryoablation), but rather to induce a partial necrotic region within the tumor and release a sufficient amount of TSA / TAA and DAMP to initiate an immune response. Thus, cryoablation may differ from cryoablation in the method of administration, including, but not limited to, the minimum temperature achieved in the treatment zone of the target tissue, the cooling rate of the target tissue treatment zone (which is related, inter alia, to the cooling rate of a cryotherapy device such as a cryoprobe), the diameter of the ice ball formed in the target tissue treatment zone, the diameter of the isotherm within the ice ball that reaches the critical temperature for necrosis and apoptosis, the duty cycle of the cryotherapy device, the thawing rate, and the duration of the freeze-thaw cycle (Cooperberg, M. and Kim D.K. (2020); Littrup, P.J., et al. (2009); Shah, T.T., et al. (2016)).

[0106] Cryoprobe: As used herein, the term "cryoprobe" may refer to a hollow needle through which a cooled thermally conductive fluid circulates. The thermally conductive fluid can be any acceptable coolant used in the art, such as argon, nitrous oxide, carbon dioxide, or liquid nitrogen. The cryoprobe can be of any diameter commonly used in the field of cryoablation therapy. In some embodiments, the cryoprobe has a diameter of from about 1.5 mm to about 3.4 mm. In some embodiments, the cryoprobe has a diameter of about 1.5 mm. In some embodiments, the cryoprobe has a diameter of about 3.4 mm. In some embodiments, the diameter of the cryoprobe is selected to allow for a rapid cooling rate. In some embodiments, to achieve a rapid cooling rate, it is necessary for the cooling probe to reach a temperature of about -40 °C or less, about -50 °C or less, or about -60 °C or less in less than 1 minute. In some embodiments, the cryoprobe surface can drop to -130 °C to -150 °C and, in cases where rapid cooling of the target tissue is required, down to -190 °C, although it is generally not recognized that such low temperatures are necessary for cell death (Littrup, P.J., et al. (2009); Shah, T.T., et al. (2016)). In some embodiments, the diameter of the cryoprobe is selected to allow for efficient cryo-lysis of the target tissue, e.g., tumor cells, i.e., the selected cryoprobe effectively induces a partial necrotic region within the tumor and releases a sufficient amount of TSA and DAMP to initiate an immune response.

[0107] Cryotherapy: As used herein, the terms "cryotherapy" or "cryosurgery" can refer to the controlled local use of freezing temperatures in medical therapy. Cryotherapy can be used to treat various tissue lesions. For example, in cancer treatment, cryosurgery involves applying extremely low temperatures to completely (i.e., cryoablation) or partially (i.e., cryolysis or partial cryolysis) destroy abnormal or diseased tissue (e.g., tumor tissue). In some embodiments, when a cryotherapy treatment is administered, damage occurs to the treated tissue due to intracellular ice formation. The extent of damage to the target tissue depends on many factors including, but not limited to, the minimum temperature achieved in the treatment zone of the target tissue, the cooling rate of the target tissue treatment zone (which is related, among other things, to the cooling rate of a cryotherapy device such as a cryoprobe), the diameter of the ice ball formed in the target tissue treatment zone, the diameter of the isotherm within the ice ball that reaches the critical temperature for necrosis and / or apoptosis, the thawing rate, the duty cycle of the cryotherapy device, and the duration of the freeze-thaw cycle (Cooperberg, M. and Kim D.K. (2020); Littrup, P.J., et al. (2009); Shah, T.T., et al. (2016)).

[0108] Duty cycle: As used herein, the term "duty cycle" can refer to the percentage of time during one cycle that a signal, system, or device is active. A cycle is the time it takes for a signal, system, or device to complete one cycle between on and off cycles or two states. The duty cycle is generally expressed as a percentage or ratio. In some embodiments of the present disclosure, the duty cycle represents the percentage of time that the cryogen is flowing or circulating within the cryoprobe. In some embodiments, a duty cycle of about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% is used. In some embodiments, a duty cycle of about 100% is used. In some embodiments of the present disclosure, the duty cycle is selected to provide a rapid freezing rate of tumor cells such that the tumor cell zone reaches about -40°C or less, about -50°C or less, or about -60°C or less in less than 2 minutes, less than 1.75 minutes, less than 1.5 minutes, less than 1.25 minutes, less than 1 minute, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 20 seconds, or less than 10 seconds. In some embodiments of the present disclosure, the duty cycle is selected to provide a rapid freezing rate of tumor cells such that the tumor cell zone reaches about -40°C or less, about -50°C or less, or about -60°C or less in less than 1 minute.

[0109] Subject: As used herein, the terms "subject" or "patient" refer to any animal, e.g., a mammal, to which the provided methods and / or compositions are administered or can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc. The subject is preferably a human. In some embodiments, the subject has or is susceptible to one or more disorders or conditions. In some embodiments, the subject exhibits one or more symptoms of a disorder or condition. In some embodiments, the subject is diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is a proliferative disease such as cancer or includes it. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the subject has received or has previously received a particular treatment for diagnosing and / or treating a disease, disorder, or condition.

[0110] Substantially: As used herein, the term "substantially" refers to a qualitative state that exhibits the characteristic or property of interest to a complete or nearly complete degree or extent. Those skilled in the biological arts will appreciate that it is rare for biological and chemical phenomena to reach completion and / or proceed to completion or to reach or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena, including those related to therapeutic treatment.

[0111] Treatment: As used herein, the term "treatment" (or "treating" or "to treat") refers to the optional administration of a composition or formulation (e.g., those described herein) or a provided method that partially or completely alleviates, ameliorates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition (e.g., cancer). Such treatment may be of a subject that does not exhibit signs of the associated disease, disorder, and / or condition and / or a subject that exhibits only initial signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of a subject that exhibits one or more established signs of one or more of the associated diseases, disorders, and / or conditions. In some embodiments, treatment may be of a subject diagnosed with having the associated disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder, and / or condition. In some embodiments, it refers to the improvement, prevention, or reversal of a disease or disorder, or at least one recognizable symptom thereof. For example, it is a treatment for cancer, particularly solid tumor cancer. Treatment may be administered in cycles or treatment cycles. In some embodiments, a treatment cycle involves the administration of a treatment regimen (e.g., a method described in the present disclosure) to a subject and a subsequent drug-free period for the subject to recover. In some embodiments, the drug-free period after a subject has received treatment is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, or about 16 weeks. In some embodiments, treatment is repeated for 1 to 12 cycles, 1 to 10 cycles, 1 to 8 cycles, 1 to 6 cycles, 1 to 4 cycles, 1 to 2 cycles, 3 to 12 cycles, 5 to 12 cycles, 7 to 12 cycles, 9 to 12 cycles, or 11 to 12 cycles. In some embodiments, treatment is repeated for 3 to 6 cycles, 3 to 5 cycles, 3 to 4 cycles, 4 to 6 cycles, or 5 to 6 cycles.In some embodiments, the treatment is repeated for 3 to 6 cycles.

[0112] Therapeutically effective amount: As used herein, a "therapeutically effective amount" of a drug or composition / formulation of the present disclosure (e.g., an antibody, an oligonucleotide, etc.) means an amount of the drug or composition / formulation that is effective to treat a condition or its signs or symptoms or to prevent its recurrence. A complete therapeutic effect is not necessarily produced by a single administration and may occur only after administration of a series of doses. Thus, a therapeutically effective amount can be administered in one or multiple administrations of a unit dosage form.

[0113] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" when applied to a carrier, diluent, or excipient used to formulate the compositions disclosed herein means that the carrier, diluent, or excipient must be compatible with the other components of the composition and not harmful to its recipient.

[0114] Detailed Description The present disclosure recognizes that, considering the complex and multifactorial nature of the mechanisms mediating tumor immune evasion, it is becoming increasingly clear that in cancer immunotherapy, it is necessary to combine and administer multiple therapeutic agents that elicit multiple immunopharmacological effects (Melero, Berman et al. 2015, Vilgelm, Johnson et al. 2016).

[0115] The present disclosure further understands that a comprehensive anti-tumor immunotherapy approach would ideally: a) induce immunogenic tumor cell death, characterized by necrosis and the release of TAAs / TSAs and danger-associated molecular patterns (DAMPs); b) mediate the priming of an adaptive immune response against such antigens by recruiting and activating innate immune cells (e.g., NK, macrophages, etc.) and antigen-presenting cells (APCs) that take up, process, and present peptides derived from TSAs / TAAs; c) avoid counter-regulatory mechanisms that limit T cell priming while stimulating the activation and proliferation of effector T cells; and d) block existing and inducible immunosuppressive mechanisms by reversing mechanisms that mediate T cell exhaustion, inhibiting the function of regulatory T cells (Tregs) or depleting Tregs, and preventing the recruitment of myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment (Chen and Mellman 2013).

[0116] The present disclosure recognizes that there are multiple therapeutic approaches that promote immunogenic tumor cell death and the release of TAAs / TSAs (Green, Ferguson et al. 2009, Dudek, Garg et al. 2013, Zhou, Wang et al. 2019). These include radiotherapy, chemotherapy, oncolytic viruses, toxins, lytic peptides, lytic compounds, cryoablation, electroporation, radiofrequency ablation, antibody-drug conjugates, and the like.

[0117] Furthermore, different approaches for promoting the recruitment, activation, and immunogenic maturation of APCs to the tumor bed have been investigated, and these involve the use of cytokines (e.g., GM-CSF, FLT-3 ligand), chemokines (e.g., CCL21, CXCL10, etc.), TLR ligands (e.g., CpG ODN), STING agonists, and agonistic anti-CD40 (αCD40) ligand. Additionally, vaccination strategies based on the administration of peptides / adjuvants, DNA vaccines, or RNA vaccines encoding single or individualized TSAs aim to provide tumor antigens in a context favorable for antigen uptake and presentation by professional APCs, with the goal of stimulating a strong adaptive immune response. Similarly, several strategies for promoting the activation and proliferation of NK cells and effector CD4 helper T cells and CD8 cytotoxic T cells have also been studied. These studies include the use of interleukins (e.g., IL-2, IL-12, IL-15, etc.) and agonistic ligands of the TNFR family such as OX40, 4-1BB, GITR, ICOS, CD27, etc. (Choi, Shi et al. 2020).

[0118] The present disclosure further recognizes that one of the most effective immunotherapy approaches in a clinical setting is to block the Treg-mediated immunosuppressive mechanism that affects the functions of helper T cells, effector T cells, and dendritic cells (DC). The major checkpoint inhibitor expressed by Treg is CTLA-4, and its blockade by antagonistic anti-CTLA4 (αCTLA) mAb has led to a quantum leap in cancer immunotherapy (Wei, Duffy et al. 2018). Furthermore, effector T cells express a number of immune checkpoints that mediate exhaustion and anergy, such as PD-1, TIM-3, TIGIT, LAG-3, VISTA, and BTLA, and their blockade by antagonistic mAbs is being vigorously investigated (Melero, Berman et al. 2015). The most advanced of these approaches is the blockade of the PD-1 checkpoint (or its ligand PD-L1 expressed by tumor cells or immunosuppressive DC), which reactivates exhausted T cells and currently constitutes the central backbone of almost all immunotherapy approaches. However, the combination of CTLA-4 and PD-1 / PD-L1 blockade relies on existing anti-tumor immunity and tumor-infiltrating T cells and has not fully addressed the mechanisms of enhanced tumor lysis, activation of dendritic cells, promotion of antigen presentation, activation of naive T cells, and direct stimulation of effector T cell proliferation. For these reasons, anti-tumor responses using this combination are frequently observed only in certain "hot" immunogenic cancers (e.g., melanoma, lung cancer) and less so in "cold" non-immunogenic cancers (e.g., breast cancer, prostate cancer, pancreatic cancer, etc.) (Das, Verma et al. 2015, Winograd, Byrne et al. 2015, Vilgelm, Johnson et al. 2016, Chae, Arya et al. 2018, Chowdhury, Chamoto et al. 2018).

[0119] Therefore, there is strong rationale for combining therapeutic approaches that target different stages and functions of the anti-tumor immune response, synchronizing the release of tumor antigens with the recruitment and activation of APCs, promoting the activation and proliferation of effector T cells, and simultaneously suppressing existing and inducible counter-regulatory immunosuppressive mechanisms mediated by Tregs, MDSCs, and the tumor itself. However, despite the apparent potential of combination therapy approaches, there are several barriers to the development of immunotherapies involving the use of combinations of investigational drugs, and as a result, some of the expected combinations have not produced the expected results, such as an increase in anti-tumor response. One such barrier is due to the fact that drugs are often developed individually or in combination with only one or two other drugs. When the activity of individual or a small number of drugs is suboptimal, it is often compensated for by relying on high doses, which maximizes the potential for their serious adverse effects (AEs). Another barrier is due to the fact that most investigational drugs are administered systemically, which requires more than the dose actually needed at the site of action (e.g., the tumor, tumor draining lymph nodes (TDLNs), and tertiary lymphoid-like structures surrounding the tumor), resulting in increased systemic exposure and a higher risk of off-target toxicity. Furthermore, the additional efficacy benefits of using specific drug combinations are often offset by additional serious AEs such as cytokine storm, ulcerative colitis, hepatitis, thyroiditis, hypophysitis, and other immune-related AEs, often necessitating the discontinuation of these treatments (Xu, Xu et al. 2020, Biniakewitz, Kasler et al. 2021).

[0120] As a potential solution to the systemic administration of multiple immunotherapeutic agents at high doses, a portion of these agents can be administered by intratumoral or peritumoral injection at lower doses, which has shown good efficacy and reduced AEs in preclinical models (Marabelle, Kohrt et al. 2014, Aznar, Tinari et al. 2017). However, these approaches still rely on the intratumoral administration of only one or two agents and the systemic administration of other agents at high doses.

[0121] Therefore, current immunotherapy approaches that rely on the use of one or two agents only partially address some of these immunological mechanisms and thus have not yet provided a comprehensive solution to the problem of tumor-mediated immune evasion.

[0122] The methods and compositions of the present disclosure provide solutions to these problems, maximizing the anti-tumor effect through coordinated and simultaneous targeting of multiple immunological mechanisms, and optimizing the in vivo distribution of active pharmacological components while minimizing the potential for severe adverse events. In some embodiments, the methods and compositions of the present disclosure combine the induction of immunogenic tumor cell death by physical and / or pharmacological methods, which mediates the mobilization and activation of APCs and promotes the activation and proliferation of effector T cells, with a tumor-infiltrating multi-drug combination immunotherapy strategy that simultaneously suppresses existing and inducible counter-regulatory immunosuppressive mechanisms mediated by Tregs and cells of the tumor microenvironment, thereby stimulating autologous vaccination against endogenous TSA / TAA. In some embodiments, the methods of the present disclosure aim to generate a systemic immune response against a tumor by local immune stimulation by using the tumor itself as its own vaccine, thereby generating a polyclonal adaptive immune response mediated by T cells and / or B cells against existing TSA / TAA. An advantage of the methods and compositions of the present disclosure over conventional cancer vaccines is that, in some embodiments, they use off-the-shelf immunostimulatory products and do not require pretreatment, identification of molecular targets of tumor antigens, identification and selection of HLA types of patients, and as a result, represent a more universal treatment strategy compared to individualized cancer vaccine approaches that typically target specific tumor antigens that must be shared between the vaccine and the tumor.

[0123] The methods and compositions provided herein mediate multiple pharmacological effects aimed at initiating a coordinated and multifaceted anti-tumor immune response.

[0124] Administration of tumor cell lysis therapy In some embodiments, the methods of the present disclosure include a first step aimed at initiating in situ immunization by causing cell lysis and immunogenic cell death of tumor cells using an intratumoral cryoablation device, and a second step including intratumoral administration of a combination of immunotherapeutic agents that mediate different pharmacological effects (Figure 6).

[0125] In some embodiments, the method of the present disclosure includes a first step of causing cytolysis and immunogenic cell death of tumor cells (e.g., by cryoablation within the tumor), which mediates necrotic cell death, and thereafter, TSA / TAA and DAMPs are released into the tumor microenvironment. One goal of the cryoablation step performed in some embodiments of the present disclosure is not to achieve complete resection of the tumor tissue (i.e., cryoablation), but rather to induce a partial necrotic region within the tumor (as a result of partial cryoablation) and release a sufficient amount of TSA and DAMPs to initiate an immune response (den Brok, Sutmuller et al. 2004, Sidana 2014). In some embodiments of the present disclosure, partial cryoablation resulting from 1 to 2 freeze-thaw cycles is preferred to preserve the vascular and lymphatic structures that facilitate the movement of antigens, cytokines, therapeutic agents, and immune cells between the TDLN and the tumor microenvironment. In some embodiments, the number of freeze-thaw cycles is selected to minimize damage to the vascular and lymphatic structures and / or immunosuppressive mechanisms. In some embodiments, 1 freeze-thaw cycle is preferred. In some embodiments, 2 freeze-thaw cycles are preferred. In some embodiments, 1 or fewer freeze-thaw cycles are preferred. In some embodiments, 2 or fewer freeze-thaw cycles are preferred. In some embodiments, 3 or fewer freeze-thaw cycles are preferred. In some embodiments, the step of causing cytolysis and immunogenic cell death of tumor cells is the first step in the method described herein. In some embodiments, the step of causing cytolysis and immunogenic cell death of tumor cells is the first step in the method described herein, and thereafter, a step of administering a combination formulation comprising the immunotherapeutic agent described herein is performed.

[0126] This disclosure recognizes that cryotherapy (e.g., cryoablation and cryosurgery) can directly induce necrosis by damaging cell membranes and organelles through the formation of ice crystals, and can indirectly induce necrosis through ischemia due to osmotic stress and microvascular thrombosis (Fraser and Gill 1967, Whittaker 1984, Karlsson, Cravalho et al. 1993, Hoffmann and Bischof 2002, Sabel 2009). Its mechanism of action is due to the mechanical forces and osmotic changes induced by the crystallization of water, and the ischemic effects of microvascular injury contributing to the coagulative necrosis of tumors (Gage and Baust 1998). Upon thawing, necrotic tumor cells within the ice ball (e.g., the ice ball formed during cryosurgery) release intact tumor antigens and DAMPs, such as endogenous TLR ligands like cytokines, intracellular ATP, nuclear proteins, and HMGB1 protein (Sidana 2014). DAMPs stimulate the innate immune response and attract granulocytes, macrophages, and NK cells. DCs take up tumor antigens, migrate to the TDLN, and initiate an adaptive immune response (structurally similar to the mechanism of in situ immunization) (den Brok, Sutmuller et al. 2006). Subsequently, immature DCs loaded with antigens are activated, migrate to the TDLN, where they process and present tumor antigens in the context of MHC molecules, and prime their activation by presenting antigens to T cells, promoting the proliferation of Th1 cell cytotoxic CD8+ T cells. Additionally, soluble antigens can reach the TDLN, where they can be loaded and cross-presented by DCs. Therefore, controlled tumor lysis by cryosurgery of solid tumors is mechanistically similar to in situ immunization where a heterogeneous population of tumor cells releases many unique TAAs / TSAs along with danger signals that can prime and initiate an immune response against such antigens (Bastianpillai, Petrides et al. 2015, Yakkala, Chiang et al. 2019).In multiple animal experiments, induction of a systemic anti-tumor immune response by tumor cryoablation has been confirmed (Blackwood and Cooper 1972, Neel and DeSanto 1973, Bagley, Faraci et al. 1974, Faraci, Bagley et al. 1975, Faraci, Bagley et al. 1975, Muller, Micksche et al. 1985, Joosten, Muijen et al. 2001, Urano, Tanaka et al. 2003, Sabel and Nehs 2005, Sabel, Nehs et al. 2005, den Brok, Sutmuller et al. 2006, den Brok, Sutmuller et al. 2006, Sabel, Arora et al. 2006) (see (Sabel 2009) for a review). In these studies, cryotherapy (e.g., cryoablation) alone was able to increase the amount of tumor antigen and mature DCs loaded in the TDLN (den Brok, Sutmuller et al. 2006), increase the levels of Th1 cytokines (IL2 and IFNγ), enhance the tumor rejection rate, and increase protection against tumor rechallenge.

[0127] In this field, it is understood that there is a threshold with respect to the antigenic stimuli necessary to assist anti-tumor immunity, and this requires the removal of a majority of the tumor mass (i.e., cryoablation) (Blackwood and Cooper 1972). In fact, prostate cancer patients with primary tumors treated by cryoablation showed a reduction in tumor mass, and this immune response was enhanced after repeated cryotherapy treatments leading to cryodestruction of prostate tissue (Soanes, Ablin et al. 1970, Soanes, Gonder et al. 1970, Ablin 1972). Also, patients with metastatic prostate cancer showed relief of bone pain after cryosurgery and an associated increase in IgM antibodies (Gursel, Roberts et al. 1973). Furthermore, cryoablation of high-risk prostate cancer showed an increase in serum levels of TNFα and IFNγ at 4 weeks after tumor resection, an increase in tumor-specific CTLs, and an increase in the Th1:Th2 cytokine ratio (Soanes, Ablin et al. 1970).

[0128] Of note, cryotherapy does not always lead to immune stimulation and may sometimes result in immunosuppression and tumor growth promotion (Hanawa 1993, Shibata, Yamashita et al. 1998). While many studies have shown a certain degree of anti-tumor immune response after cryosurgery, many other studies have shown no response at all. There are multiple reasons for these contradictions. First, although it is not desirable to be constrained by theory, the freezing rate is thought to play a role in determining the immune-stimulatory and immunosuppressive effects of cryotherapy. A fast freezing rate leads to the formation of ice crystals, which is associated with necrotic immunogenic cell death, an increase in IFNγ expression, and a decrease in the number of Tregs (Sabel, Su et al. 2010). In contrast, when the freezing rate is slow (observed at the periphery of the ice ball), cells die by osmotic shock and subsequent apoptosis occurs, which is less immunogenic and correlates with an immunosuppressive increase in Treg cells, low IFNγ levels, and a Th2-type T cell response (e.g., TGF-β and IL-10) (Sabel 2009, Sabel, Su et al. 2010). Therefore, the ratio of necrosis to apoptosis is understood to play an important role in determining the stimulatory or suppressive nature of the immune response to cryotherapy (Sabel 2009, Ferguson, Choi et al. 2011). In addition, when a large amount of tumor is frozen, the large number of immune complexes generated may cause "high zone tolerance", a phenomenon in which antigen overload may lead to immunosuppression (Sabel 2009).

[0129] The partial cryoablation of tumor tissue, which is performed in many embodiments of the present disclosure, fundamentally changes the tumor microenvironment within the treatment zone. In particular, most tumor cells lyse by rupture of the cell membrane (necrotic cell death), thereby releasing the cell contents of the tumor cells (including antigens, damage-associated molecular patterns (DAMPs), and other cellular debris) into the extracellular space, while minimizing damage to the lymphatic drainage of the tissue and denaturation of the released tumor proteins. The partial cryoablation performed in many embodiments of the present disclosure results in a different outcome than complete tumor cryoablation. For example, unlike more commonly performed tumor cryoablation, which requires complete cell death of both the entire tumor and the margin of surrounding healthy tissue, in the partial cryoablation method of the present disclosure, only most of the necrotic tumor cells in the partial tumor treatment zone lyse, and there is no margin of healthy cells in the target treatment zone. Such cryoablation methods will fail for several reasons, including that only a portion of the tumor is lysed, there is no margin of healthy surrounding tissue, and a large number of tumor cells remain viable in the partial cryoablation treatment zone.

[0130] The present disclosure understands that partial tumor lysis resulting in necrotic cell death by rupture of the cell membrane can potentially be achieved by many modalities including radiation, high frequency, high intensity focused ultrasound (HIFU), microwaves, oncolytic viruses, oncolytic chemicals, oncolytic peptides, and cryoablation. In many embodiments, cryoablation is preferred over other modalities due to its efficiency, reproducibility, induction of a superior immune response compared to heat-based modalities, quality of the released proteins (minimal denaturation), overall safety profile, and lack of off-target effects. In some embodiments of the present disclosure, cryoablation is mediated by an intratumoral cryoablation device, and then at least one cycle of freezing and thawing is performed using standard approved cryoablation devices and probes. In some preferred embodiments, cryoablation is administered as the first step in the methods described herein.

[0131] In some embodiments of the present disclosure, to mediate cryoablation, a cryoprobe is inserted into the tumor (e.g., within the tumor). Once at a predetermined location within the tumor, the cryoprobe is cooled to a temperature that creates an ice ball having a diameter of about 10 mm to about 14 mm within the tumor. In some embodiments, the ice ball within the tumor has a diameter of about 8 mm to about 14 mm. In some embodiments, the ice ball within the tumor has a diameter of about 8 mm to about 12 mm. In some embodiments, the ice ball within the tumor has a diameter of about 8 mm to about 10 mm. In some embodiments, the ice ball within the tumor has a diameter of about 12 mm to about 16 mm. In some embodiments, the ice ball within the tumor has a diameter of about 13 mm to about 15 mm. In some embodiments, the ice ball within the tumor has a diameter of about 14 mm to about 18 mm. In some embodiments, the ice ball within the tumor has a diameter of about 14 mm to about 30 mm. In some embodiments, the ice ball within the tumor has a diameter of about 16 mm to about 30 mm. In some embodiments, the ice ball within the tumor has a diameter of about 18 mm to about 30 mm. In some embodiments, the ice ball within the tumor has a diameter of about 20 mm to about 30 mm. In some embodiments, the ice ball within the tumor has a diameter of about 25 mm to about 30 mm. In some embodiments, the ice ball within the tumor has a diameter of about 14 mm to about 25 mm. In some embodiments, the ice ball within the tumor has a diameter of about 14 mm to about 20 mm. In some embodiments, the ice ball within the tumor has a diameter of about 14 mm to about 15 mm. In some embodiments, the ice ball within the tumor has a diameter of about 13 mm to about 14 mm. In some embodiments, the ice ball within the tumor has a diameter of about 8 mm. In some embodiments, the ice ball within the tumor has a diameter of about 9 mm. In some embodiments, the ice ball within the tumor has a diameter of about 10 mm. In some embodiments, the ice ball within the tumor has a diameter of about 11 mm. In some embodiments, the ice ball within the tumor has a diameter of about 12 mm. In some embodiments, the ice ball within the tumor has a diameter of about 13 mm.In some embodiments, the ice balls within the tumor have a diameter of about 14 mm. In some embodiments, the ice balls within the tumor have a diameter of about 15 mm. In some embodiments, the ice balls within the tumor have a diameter of about 16 mm. In some embodiments, the ice balls within the tumor have a diameter of about 17 mm. In some embodiments, the ice balls within the tumor have a diameter of about 18 mm. In some embodiments, the ice balls within the tumor have a diameter of about 19 mm. In some embodiments, the ice balls within the tumor have a diameter of about 20 mm. In some embodiments, the ice balls within the tumor have a diameter of about 21 mm. In some embodiments, the ice balls within the tumor have a diameter of about 22 mm. In some embodiments, the ice balls within the tumor have a diameter of about 23 mm. In some embodiments, the ice balls within the tumor have a diameter of about 24 mm. In some embodiments, the ice balls within the tumor have a diameter of about 25 mm. In some embodiments, the ice balls within the tumor have a diameter of about 30 mm.

[0132] In some embodiments, the ice ball formed by the methods described herein has a temperature gradient within the ice ball, and the temperature of the ice near the cryoprobe surface is approximately the same as the cryoprobe surface. Further, as the distance from the cryoprobe increases, the temperature within the ice ball changes and approaches 0°C near the ice ball / tissue interface (also referred to as the leading edge). Due to this temperature change, in some embodiments, the temperature generated by the cryoprobe is below -40°C to -60°C. In some embodiments, the temperature generated by the cryoprobe is less than -40°C. In some embodiments, the temperature generated by the cryoprobe is less than -60°C. In some embodiments, the temperature generated by the cryoprobe is less than -80°C. In some embodiments, the temperature generated by the cryoprobe is less than -100°C. In some embodiments, the temperature generated by the cryoprobe is less than -120°C. In some embodiments, the temperature generated by the cryoprobe is less than -140°C. In some embodiments, cooling of the cryoprobe creates an ice ball in which most of the ice has a temperature of approximately -40°C. In some embodiments, lethal ice (e.g., ice that causes cell death by necrosis, apoptosis, or osmotic shock) in the ice ball (at approximately -40°C or below) is present at approximately 5 mm from the leading edge (the ice at the ice ball / tissue interface where the temperature is approximately 0°C). In some embodiments, the lethal ice in the ice ball is present at approximately 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm from the leading edge. After freezing, the ice ball of the frozen tumor tissue formed by the cryoprobe can be thawed passively (without a heating cycle of the cryoprobe). This thawing is automatically determined by timing and can be further confirmed by any suitable imaging method known in the art (e.g., thermocouple, ultrasound).

[0133] In some embodiments of the present disclosure, the size of the partial tumor cryoablation treatment zone is the same regardless of the size of the tumor being treated and is defined by the size of the ice ball generated. In some preferred embodiments, the size of the diameter of the ice ball ranges from about 8 mm to about 30 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 10 mm to about 30 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 12 mm to about 30 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 14 mm to about 30 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 16 mm to about 30 mm. In some preferred embodiments, the size of the diameter of the ice ball ranges from about 18 mm to about 30 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 20 mm to about 30 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 25 mm to about 30 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 8 mm to about 25 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 8 mm to about 20 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 8 mm to about 18 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 8 mm to about 16 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 8 mm to about 14 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 8 mm to about 12 mm. In some preferred embodiments, the size of the diameter of the ice ball ranges from about 12 mm to about 16 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 10 mm to about 14 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 11 mm to about 15 mm. In some embodiments, the size of the diameter of the ice ball ranges from about 13 mm to about 15 mm.

[0134] In some preferred embodiments, the diameter of the ice ball is about 14 mm, and as a result, the spherical volume of the partially dissolved tissue is about 1.44 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 1.2 ml to about 1.6 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 0.3 ml to about 14 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 0.3 ml to about 10 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 0.3 ml to about 8 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 0.3 ml to about 4 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 0.3 ml to about 2 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 0.3 ml to about 1.4 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 0.3 ml to about 1 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 1 ml to about 14 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 1.4 ml to about 14 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 2 ml to about 14 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 4 ml to about 14 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 8 ml to about 14 ml. In some embodiments, the ice ball has a diameter that results in a spherical volume of the partially dissolved tissue of from about 10 ml to about 14 ml.

[0135] In some embodiments, during the cryoablation cycle, the ice ball cools the temperature of the cryoprobe to about -40°C to about -60°C, or to a temperature lower than that, in about 1 minute or less, and continues to cool the temperature to about -70°C or lower, about -80°C or lower, about -90°C or lower, about -100°C or lower, about -110°C or lower, about -120°C or lower, about -130°C or lower, about -140°C or lower, or about -150°C or lower for a total of about 5 minutes, about 4.5 minutes, about 4 minutes, about 3.5 minutes, about 3 minutes, about 2.5 minutes, about 2 minutes, about 1.75 minutes, about 1.5 minutes, about 1.25 minutes, about 1 minute, about 50 seconds, about 40 seconds, about 30 seconds, about 20 seconds, or about 10 seconds. In some embodiments, the ice ball cools the temperature of the cryoprobe to about -40°C to about -60°C, or to a temperature lower than that, in about 1 minute or less, and continues to cool the temperature to about -70°C or lower, about -80°C or lower, about -90°C or lower, about -100°C or lower, about -110°C or lower, about -120°C or lower, about -130°C or lower, about -140°C or lower, or about -150°C or lower for a total of about 5 minutes. In some embodiments, the ice ball cools the temperature of the cryoprobe to about -40°C to about -60°C, or to a temperature lower than that, in about 1 minute or less, and continues to cool the temperature to about -120°C to about -150°C or lower for a total of about 5 minutes. In some embodiments, the ice ball cools the temperature of the cryoprobe to about -40°C to about -60°C, or to a temperature lower than that, in about 1 minute or less, and continues to cool the temperature to about -70°C or lower, about -80°C or lower, about -90°C or lower, about -100°C or lower, about -110°C or lower, about -120°C or lower, about -130°C or lower, about -140°C or lower, or about -150°C or lower for a total of about 2 minutes. In some embodiments, the ice ball cools the temperature of the cryoprobe to about -40°C to about -60°C, or to a temperature lower than that, in about 1 minute or less, and continues to cool the temperature to about -120°C to about -150°C or lower for a total of about 2 minutes.

[0136] In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in about 1 minute. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in about 50 seconds. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in about 40 seconds. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in about 30 seconds. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in about 20 seconds. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in about 10 seconds. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in less than about 1 minute. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in less than about 50 seconds. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in less than about 40 seconds. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in less than about 30 seconds. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in less than about 20 seconds. In some embodiments, the temperature generated by the cryoprobe reaches from about -40°C to about -60°C or less in less than about 10 seconds.

[0137] In some embodiments, the temperature generated by the cryoprobe reaches a temperature of about -60°C to about -70°C, about -40°C to about -70°C, about -50°C to about -70°C, about -60°C to about -80°C, about -70°C to about -90°C, about -80°C to about -100°C, about -90°C to about -110°C, about -100°C to about -120°C, about -110°C to about -130°C, about -120°C to about -150°C, or lower in about 5 minutes, about 4.5 minutes, about 4 minutes, about 3.5 minutes, about 3 minutes, about 2.5 minutes, about 2 minutes, about 1.75 minutes, about 1.5 minutes, about 1.25 minutes, about 1 minute, about 50 seconds, about 40 seconds, about 30 seconds, about 20 seconds, or about 10 seconds. In some embodiments, the temperature generated by the cryoprobe reaches a temperature of about -60°C to about -70°C, about -40°C to about -70°C, about -50°C to about -70°C, about -60°C to about -80°C, about -70°C to about -90°C, about -80°C to about -100°C, about -90°C to about -110°C, about -100°C to about -120°C, about -110°C to about -130°C, about -120°C to about -150°C, or lower in about 5 minutes or less, about 4.5 minutes or less, about 4 minutes or less, about 3.5 minutes or less, about 3 minutes or less, about 2.5 minutes or less, about 2 minutes or less, about 1.75 minutes or less, about 1.5 minutes or less, about 1.25 minutes or less, about 1 minute or less, about 50 seconds or less, about 40 seconds or less, about 30 seconds or less, about 20 seconds or less, or about 10 seconds or less.

[0138] In some embodiments, the ice ball generation cycle time is predetermined, and after the ice ball reaches its predetermined maximum size of a diameter of about 8 mm to about 30 mm, about 10 mm to about 30 mm, about 12 mm to about 30 mm, about 14 mm to about 30 mm, about 16 mm to about 30 mm, about 18 mm to about 30 mm, about 20 mm to about 30 mm, about 25 mm to about 30 mm, about 8 mm to about 25 mm, about 8 mm to about 20 mm, about 8 mm to about 18 mm, about 8 mm to about 16 mm, about 8 mm to about 14 mm, about 8 mm to about 12 mm, about 8 mm to about 10 mm, about 12 mm to about 16 mm, about 13 mm to about 15 mm, or about 14 mm, it automatically ends in a few seconds. In some embodiments, the ice ball generation cycle time is predetermined, and after the ice ball reaches its predetermined maximum size of a diameter of about 8 mm to about 30 mm, about 12 mm to about 16 mm or about 14 mm, it automatically ends in a few seconds.

[0139] In some embodiments, cooling of the cryoprobe creates an ice ball in which most of the ice has a temperature of about -40°C or lower, about -45°C or lower, about -50°C or lower, about -55°C or lower, about -60°C or lower, about -65°C, about -70°C, about -80°C or lower, about -90°C or lower, about -100°C or lower, about -110°C or lower, about -120°C or lower, about -130°C or lower, about -140°C or lower, or about -150°C or lower. In some embodiments, cooling of the cryoprobe creates an ice ball in which most of the ice has a temperature of about -40°C or lower. In some embodiments, cooling of the cryoprobe creates an ice ball in which most of the ice has a temperature of about -60°C or lower.

[0140] In some embodiments, the ice ball includes a region having a temperature of about -40°C or lower, about -50°C or lower, about -60°C or lower, about -70°C or lower, about -80°C or lower, about -90°C or lower, about -100°C or lower, about -110°C or lower, about -120°C or lower, about -130°C or lower, about -140°C or lower, or about -150°C or lower and a diameter of about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, or about 15 mm within it. In some embodiments, the ice ball includes a diameter of about 10 mm having a temperature of about -40°C, about -50°C, about -60°C or lower, about -70°C or lower, about -80°C or lower, about -90°C or lower, about -100°C or lower, about -110°C or lower, about -120°C or lower, about -130°C or lower, about -140°C or lower, or about -150°C or lower within it. In some embodiments, the ice ball includes a diameter of about 10 mm having a temperature of about -40°C or lower within it.

[0141] In some embodiments, after the ice ball reaches its predetermined maximum size, a thawing process (e.g., passive thawing) is performed. In some embodiments, the passive thawing process lasts for about 10 minutes, about 9.5 minutes, about 9 minutes, about 8.5 minutes, about 8 minutes, about 7.5 minutes, about 7 minutes, about 6.5 minutes, about 6 minutes, about 5.5 minutes, about 5 minutes, about 4.5 minutes, about 4 minutes, about 3.5 minutes, about 3 minutes, about 2.75 minutes, about 2.5 minutes, about 2.25 minutes, about 2 minutes, about 1.75 minutes, about 1.5 minutes, about 1.25 minutes, or about 1 minute. In some embodiments, the passive thawing process lasts for less than about 10 minutes, less than about 9.5 minutes, less than about 9 minutes, less than about 8.5 minutes, less than about 8 minutes, less than about 7.5 minutes, less than about 7 minutes, less than about 6.5 minutes, less than about 6 minutes, less than about 5.5 minutes, less than about 5 minutes, less than about 4.5 minutes, less than about 4 minutes, less than about 3.5 minutes, less than about 3 minutes, less than about 2.75 minutes, less than about 2.5 minutes, less than about 2.25 minutes, less than about 2 minutes, less than about 1.75 minutes, less than about 1.5 minutes, less than about 1.25 minutes, or less than about 1 minute. In some embodiments, the passive thawing process lasts for about 5 minutes. In some embodiments, the passive thawing process lasts for about 2 minutes. In some embodiments, the passive thawing process continues until the ice ball disappears (e.g., as determined by a suitable visualization method).

[0142] In some embodiments of the present disclosure, a second cycle of cryolysis is repeated after passive thawing of the frozen tissue and is confirmed by any suitable imaging method known in the art. In some embodiments, a third cycle of cryolysis is repeated after passive thawing of the frozen tissue and is confirmed by any suitable imaging method known in the art.

[0143] The cryoablation cycles described herein are intended to produce a tissue effect in the tumor treatment zone in which most tumor cells lyse (i.e., their cell membranes rupture), thereby releasing cell contents into the extracellular space. The freeze / thaw cycles described herein are intended not to impair the lymphatic drainage of tumor tissue within and around the treatment zone. Frozen tumor tissue within an ice ball (e.g., an ice ball formed by a cryoprobe) can be thawed completely passively (without a heating cycle of the cryoprobe). This thawing is automatically determined by timing and can be confirmed by any suitable imaging method known in the art.

[0144] In some embodiments, the cryoablation therapy administered in accordance with the present disclosure causes necrotic cell death in a zone having a diameter of about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, or about 15 mm of tumor tissue. In some embodiments, the cryoablation therapy causes necrotic cell death in a zone having a diameter of about 12 mm to about 16 mm of tumor tissue. In some embodiments, the cryoablation therapy causes necrotic cell death in a zone having a diameter of about 13 mm to about 15 mm of tumor tissue. In some embodiments, the cryoablation therapy causes necrotic cell death in a zone having a diameter of about 14 mm of tumor tissue.

[0145] In some embodiments of the present disclosure, the selected tumor site is prepared for a percutaneous approach under precise image guidance using a coaxial cryoprobe / injection needle system (SCINS). The SCINS is inserted by standard percutaneous techniques under precise image guidance, and the tip of the SCINS is accurately positioned such that the volume of the resulting cryoablation treatment zone fits entirely within the selected tumor. The length of the cryoablation zone using the SCINS can be visualized under imaging as the length of the cryoprobe exposed from the tip of the coaxial injection needle.

[0146] In some embodiments, the cryoablation device is set to a duty cycle that enables generation of a cryoprobe temperature of about -40°C, about -50°C, about -60°C, or lower in about 5 minutes, about 4.5 minutes, about 4 minutes, about 3.5 minutes, about 3 minutes, about 2.75 minutes, about 2.5 minutes, about 2.25 minutes, about 2 minutes, about 1.75 minutes, about 1.5 minutes, about 1.25 minutes, about 1 minute, about 50 seconds, about 40 seconds, about 30 seconds, about 20 seconds, or about 10 seconds. In some embodiments, the cryoablation device is set to a duty cycle that enables generation of a cryoprobe temperature of about -40°C, about -50°C, about -60°C, or lower in about 5 minutes or less, about 4.5 minutes or less, about 4 minutes or less, about 3.5 minutes or less, about 3 minutes or less, about 2.75 minutes, about 2.5 minutes or less, about 2.25 minutes or less, about 2 minutes or less, about 1.75 minutes or less, about 1.5 minutes or less, about 1.25 minutes or less, or about 1 minute or less. In some embodiments, the cryoablation device is set to a duty cycle that enables generation of a cryoprobe temperature of about -40°C or lower in less than about 1 minute. In some embodiments, the cryoablation device is set to a duty cycle that enables generation of a cryoprobe temperature of about -60°C or lower in less than about 1 minute. In some embodiments, the cryoablation device is set to a duty cycle of about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 80%, or about 70%. In some embodiments, the cryoablation device is set to a duty cycle of at least about 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 80%, or at least about 70%. In some embodiments, the cryoablation device is set to a 100% duty cycle.

[0147] Administration of combination formulations The methods and compositions of the present disclosure are based on intratumoral administration of either a TLR9 agonist, an agonistic anti-CD40 monoclonal antibody, an anti-CTLA4 monoclonal antibody, and either an agonistic anti-OX40 monoclonal antibody or an anti-PD1 monoclonal antibody. These combinations of immunostimulatory reagents are intended to promote and augment an immunogenic antitumor response following tumor cell lysis mediated by cryolysis, and to ensure that the results of cryolysis are pro-inflammatory and avoid situations that could be immunosuppressive.

[0148] TLR9 is an intracellular pattern recognition receptor present in the endosomal compartments of murine plasmacytoid dendritic cells, macrophages, natural killer cells, and other APCs (Karapetyan, Luke et al. 2020). In humans, TLR9 is mainly expressed in B cells and pDCs (Krieg 2007). The physiological ligand of TLR9 is a bacterial-derived dsDNA fragment with unmethylated cytidine phosphate guanosine (CpG) dinucleotides. TLR9 agonists are artificial oligonucleotides with unmethylated CpG motifs. They can induce signaling cascades that lead to transcriptional programs resulting in inflammatory processes, enhanced killing of cancer cells, and generation of adaptive immune responses. According to compelling data, intratumoral administration of TLR9 agonists has been shown to improve the activation of APCs, particularly pDCs in TDLNs, and result in the release of pro-inflammatory Th1 cytokines such as TNFα, IFNγ, IL2, IL-6, IL-12, and type I IFN genes (Suek, Campesato et al. 2019, Karapetyan, Luke et al. 2020). IFNα acts directly on tumors, such as inhibiting angiogenesis, anti-proliferative effects, and increasing MHC-I expression by tumor cells, which enhances target visibility and promotes the killing of tumor cells by T cells (Suek, Campesato et al. 2019). In addition, IFNα enhances the killing ability of NKs and promotes the maturation of conventional DCs (Hervas-Stubbs, Perez-Gracia et al. 2011). Furthermore, TLR9 agonists increase the expression of co-stimulatory molecules CD80 and CD86, increase IP10 (CXCL10), a chemoattractant for monocytes, macrophages, DCs, T cells, NK cells, and increase the expression of the lymph node homing signal CCR7, resulting in an ultimately increased priming of T cells and tumor rejection, and in some cases, a potent abscopal effect on distant untreated tumors can be achieved (Suek, Campesato et al. 2019, Karapetyan, Luke et al. 2020) (Figure 1).

[0149] CD40 is a membrane molecule that is a member of the TNFR family and is expressed on antigen-presenting cells (APCs) such as dendritic cells (DCs), B cells, monocytes, and macrophages. The important physiological functions of the CD40 / CD40L pathway are mainly mediated by ligation of CD40 on APCs, particularly dendritic cells (DCs), by CD40L expressed by CD4 helper T cells (Schonbeck and Libby 2001, Vonderheide and Glennie 2013, Vonderheide 2020). Although CD40 is not a direct T cell agonist, its ability to increase antigen presentation indirectly enhances T cell activation. CD40 is expressed on APCs such as DCs, B cells, and macrophages, and when ligated by CD40L expressed by helper T cells or agonistic mAbs, it activates APCs by improving the antigen-presenting ability of APCs through upregulation of HLA class II and costimulatory molecules CD80 / 86, and also improves the T cell priming ability of APCs through upregulation of OX40-L, 4-1BB-L, GITR-L, while downregulating immunosuppressive molecules such as PD-L1. CD40 agonism also increases the release of various Th1 cytokines (IL-1β, IL-6, IL-8, IL-12, TNFα, and IFNγ) by DCs, which enhances the cytotoxic response (Schonbeck and Libby 2001, Vonderheide and Glennie 2013, Vonderheide 2020). In particular, αCD40 agonistic mAbs induce IL-12 secretion by APCs, which i) reduces the immunosuppressive property of Tregs by downregulating the expression of CTLA4 and PD1 on Tregs, and ii) downregulates PD1 on effector CD8+ T cells and restores the T cell response to αPD1 mAbs (Ngiow, Young et al. 2016). CD40 also affects the expression of pro-apoptotic genes and anti-apoptotic genes (Bcl-XL) in various types of cells (Choi, Shi et al. 2020). In B cells, αCD40 agonistic mAbs can upregulate HLA-II, CD40L, Th1 cytokine production, and induce the secretion of antigen-specific IgM / IgG.Agonistic αCD40 mAb can also induce the secretion of cytokines important for macrophage cross-priming and cytotoxic function, thereby activating macrophages to directly kill tumor cells in a T cell-independent manner. αCD40 antibodies of the IgG2 isotype have been shown to have potent CD40 agonist activity, mimic signals from CD40L-expressing helper CD4+ T cells to activate APCs, and license APCs to prime CD8+ T cells (Vonderheide and Glennie 2013, Vonderheide 2020, Yu, Chan et al. 2020). Activation of CD40 requires oligomerization of the CD40 receptor to induce an agonist effect. Oligomerization of the receptor can be enhanced in trans by interaction of the Fc domain of the αCD40 agonistic mAb with Fc receptors on adjacent cells (White, Chan et al. 2011) (Figure 2).

[0150] CTLA4 is an inhibitory molecule that is constitutively expressed in Tregs and is inducible upon TCR engagement and activation in effector CD4+ and CD8+ T cells (Wei, Duffy et al. 2018). The expression level of CTLA4 in the immune synapse correlates with the strength of TCR affinity for the MHC I / peptide complex (Buchbinder and Desai 2016). CTLA4 binds to the CD80 and CD86 costimulatory molecules on APCs with higher affinity than the costimulatory ligand CD28 expressed on T cells. Thus, CTLA4 negates the positive activation signal mediated by CD28 after binding to CD80 / 86. The competition between CD28 and CTLA4 has the function of controlling peripheral immune tolerance to highly reactive T cell clones by proliferating only T cell clones with moderate TCR / MHC-I / peptide affinity and suppressing the proliferation of T cell clones with very strong TCR / MHC-I / peptide affinity (Buchbinder and Desai 2016). In addition to this cell-intrinsic inhibitory mechanism in which CTLA4 and CD28 derived from the same T cell compete with each other for binding to CD80 / 86, CTLA4 also has a cell-extrinsic inhibitory mechanism, in which CTLA4 expressed by Tregs blocks CD80 / 86 on the surface of APCs and sequesters CD80 / 86 by trans-endocytosis (Qureshi, Zheng et al. 2011), thereby reducing the availability of CD80 / 86 and interfering with the interaction with CD28 on T cells activated by such APCs (Buchbinder and Desai 2016, Wei, Duffy et al. 2018). Based on the above mechanisms, blockade of CTLA4 by αCTLA4 mAb is expected to increase immune activation by interfering with both these cell-intrinsic and cell-extrinsic mechanisms. First, by blocking CTLA4 on activated T cells and Tregs, it is thought that activation of effector T cells becomes possible by increasing the costimulatory interaction between CD80 / 86 and CD28, regardless of TCR affinity and specificity.This results in the activation and proliferation of a greater number of effector T cell clones, which can contribute to anti-tumor activity by enhancing T cell priming and by expanding the TCR repertoire and spectrum of tumor antigens to which CD8+ T cells respond (Cha, Klinger et al. 2014, Kvistborg, Philips et al. 2014, Robert, Harview et al. 2014, Robert, Tsoi et al. 2014). Second, CTLA4 blockade results in the expansion of tumor neoantigen-specific CD8+ T cells within the tumor microenvironment (Fehlings, Simoni et al. 2017) and in the expansion of a specific subset of exhausted Th1 CD4+ T cells (Wei, Levine et al. 2017). Third, there is evidence that αCTLA4 mAb contributes to anti-tumor immunity through a second mechanism involving ADCC-mediated depletion of Tregs, which can not only enhance T cell priming in the TDLN but also reset the cold TME by removing tumor-infiltrating Tregs (Wei, Duffy et al. 2018). Thus, αCTLA4 mAb mediates its function by a dual mechanism involving Fc-dependent ADCC-mediated depletion of Tregs and blockade of the co-stimulatory signal of APCs provided by CD80 / 86 on CD28+ T cells, thereby promoting CD28-mediated activation of T cells (Tang, Du et al. 2018, Wei, Duffy et al. 2018). With regard to the site of action where CTLA4 blockade exerts its pharmacological effect, CTLA4 blockade is thought to most likely affect the early stage of T cell activation in draining regional lymph nodes, where CTLA4+ Tregs remove CD80 / CD86 from the surface of APCs by transcytosis and compete with CD28 for binding to CD80 / 86 (Qureshi, Zheng et al. 2011). Furthermore, CTLA4 blockade may exert its effect at the tumor site by the accumulation of exhausted CTLA4+ T cells and Tregs within the tumor microenvironment (Curran, Montalvo et al. 2010).Blocking CTLA4 with an antagonistic mAb has been shown to increase the CD8:Treg ratio in the tumor microenvironment (Figure 3).

[0151] OX40 is a member of the TNFR superfamily and is expressed by effector CD4+ and CD8+ T cells, Tregs, NKTs, and NKs within 1 to 4 days after TCR engagement. Ligation of OX40 by OX40L expressed by APCs or agonistic anti-OX40 mAbs promotes distinct signals that enhance Teff cell survival and memory T cell generation (Croft 2010). Immunopharmacological effects include promotion of cytokine production, proliferation, and upregulation of CD40L, which can help stimulate DCs via the CD40 / CD40L axis (Colombo 2017). Thus, agonistic anti-OX40 mAbs such as YH002 mimic the function of activated APCs on effector T cell activation. Clustering and trimerization of the OX40 receptor molecule mediated by many agonistic αOX40 mAbs require the Fc function of the antibody and accessory cells expressing appropriate Fc receptor molecules, such as NKs, DCs, macrophages, or B cells (Zhang, Armstrong et al. 2017, Kuang, Jing et al. 2020). In addition to having an activating function on Teff cells, agonistic αOX40 mAbs can inhibit Treg generation and Treg suppressive activity by directly inhibiting FoxP3 expression (Zhang, Xiao et al. 2018), which leads to reduced expression of the checkpoint inhibitory molecule CTLA4 and better co-stimulation of Teff cells via the CD28 / CD80 axis. Furthermore, inhibition of FoxP3 in Tregs leads to decreased IL10 secretion by Tregs, which promotes good signaling between CD40 on DCs and CD40L on memory T cells (Burocchi, Pittoni et al. 2011).Furthermore, agonistic αOX40 mAbs of the IgG1 isotype can also mediate depletion of Tregs by ADCC against intratumoral Tregs that express particularly high levels of OX40, and can promote reprogramming of Tregs into TH17 T cells (Piconese, Valzasina et al. 2008, Croft 2010, Aspeslagh, Postel-Vinay et al. 2016, Colombo 2017, Deng, Zhao et al. 2019, Alves Costa Silva, Facchinetti et al. 2020, Choi, Shi et al. 2020) (Figure 4).

[0152] PD-1 is expressed on activated T cells after TCR-mediated antigen stimulation, as well as on B cells, NK cells, and monocytes. The function of PD1 is to control the activation of T cells in peripheral tissues and prevent tissue damage caused by ongoing inflammation. When exposed to antigens for a long time or at high levels, such as in advanced cancers, T cell exhaustion mediated by PD1 occurs. During T cell priming, PD-L1 is induced by IFNγ in APCs as a counter-regulatory mechanism after T cell activation (Keir, Butte et al. 2008). Ligation of PD-1 and PD-L1 under conditions of TCR activation results in phosphorylation of PD1 by the Lck tyrosine kinase, followed by recruitment of the SHP2 phosphatase to the intracellular domain of PD1, which dephosphorylates and inactivates both TCR and CD28, resulting in blockade of the activation signaling downstream of the stimulatory and co-stimulatory signals that occur during T cell priming (Buchbinder and Desai 2016, Wei, Duffy et al. 2018). Similarly, in the effector phase of tumors, PD-L1 is induced by IFNγ and expressed by tumor cells. Ligation of PD-L1 in PD1+ Teff cells that recognize tumor antigens presented by MHC-I via the TCR recruits SHP2, which inactivates TCR signaling, reduces cytokine production, induces T cell apoptosis or exhaustion and anergy of PD1+ TILs, and as a result, inhibits the tumor infiltration of Teff cells (Keir, Butte et al. 2008, Shrimali, Janik et al. 2015). Blockade of PD1 by antagonistic mAbs functions by restoring the immune function of exhausted T cells in the periphery (Shrimali, Janik et al. 2015), leading to direct inhibition of T cell apoptosis that leads to restoration of T cell effector function while providing resistance to the inducible expression of PD-L1 in tumors, enhancement of APC-mediated T cell activation, downregulation of the Treg-mediated inhibitory effect, downregulation of the immunosuppressive cytokines IL-10 and TGFβ, and upregulation of IL-2 and IFN-γ, and can enhance tumor killing by various immune mechanisms (Wei, Duffy et al. 2018).Clinically, a model is supported that the blockade of the PD1 signaling axis is most effective in tumors in which an endogenous T cell response has already been induced but is suppressed through PD1 engagement by the PD1 ligands PD-L1 and PD-L2. However, the response to αPD1 mAb in PD-L1(−) tumors indicates that the presence of an existing immune response is not necessarily an absolute requirement for tumor rejection after αPD1 treatment (Figure 5).

[0153] Preclinical results have shown broad responses in different tumor models when αPD1 mAb is combined with agonistic αOX40 mAb (Guo, Wang et al. 2014, Vilgelm, Johnson et al. 2016, Messenheimer, Jensen et al. 2017, Shrimali, Ahmad et al. 2017). These responses range from synergistic to antagonistic, and the timing of administration of these two mAbs seems to play an important role in the type of response. For at least these reasons, the methods and compositions of the present disclosure do not combine the simultaneous administration of an agonistic anti-OX40 antibody and an antagonistic αPD1 mAb.

[0154] In some embodiments, the method of the present disclosure comprises the step of intratumoral administration of SV-101, a combination formulation of an immunostimulatory therapeutic agent comprising a TLR9 agonist, an αCD40 agonist, an αOX40 agonist, and an αCTLA4 checkpoint blockade inhibitor. In some embodiments, the method of the present disclosure comprises the step of intratumoral administration of an SV-101 combination formulation consisting of a TLR9 agonist, an αCD40 agonist, an αOX40 agonist, and an αCTLA4 checkpoint blockade inhibitor (FIG. 7). In some embodiments, the method of the present disclosure comprises the step of administering an SV-101 formulation, and the SV-101 administration step is the second step in the method described herein. In some embodiments, the step of administering SV-101 is the second step following the first step of lysing the tumor by cryoablation in a method (e.g., the method described herein). In many embodiments of the present disclosure, such a combination of agents promotes the priming of a new immune response, strongly aids in the activation and maturation of APCs, while also aiding in the priming of naive T cells and promoting their stimulation, proliferation, survival, and the formation of memory T cells. This combination of active ingredients targets primarily "cold" tumors that do not exhibit severe T cell infiltration or strong expression of PD-L1. In many embodiments of the present disclosure, administration of SV-101 may exhibit some T cell infiltration and have the characteristics of a "hot" tumor, but primes a new round of immune response in tumors that have become unresponsive to treatment with an αPD1 checkpoint inhibitor.

[0155] One of the first changes following intratumoral cryoablation and intratumoral injection of SV-101 described herein is the recruitment and activation of different classes of APCs such as macrophages, B cells, and DCs. The combination of TLR9 agonist and αCD40 agonist in the TME can activate these cells in a unique synergistic manner that cannot be achieved by the use of either agent alone. First, a T cell-independent synergistic effect on the direct antitumor activity of macrophages was observed (Figure 9). Treatment with the αCD40 agonist mAb can increase the expression of TLR9, which in turn increases the response to the TLR9 agonist. Macrophages stimulated in this way show high levels of expression of TNFα, IFNγ, and IL12p70, which helps to bias the macrophage phenotype towards a pro-inflammatory M1 phenotype. Importantly, the secretion of IL12 is only observed when both αCD40 and TLR9 agonists are used in combination. Furthermore, this synergistic effect results in an increase in NO production and direct T cell-independent antitumor activity. Additionally, the secreted Th1 cytokines cause M1 polarization, help to present TA by increasing the expression of MHC molecules, and improve T cell activation and proliferation.

[0156] Tumors, tertiary lymphoid-like structures (TLS), and B cells present in the TDLN are also stimulated by the dual combination of αCD40 mAb and TLR9 agonist (Figure 10). B cells express CD40 and TLR9 receptors and can bind to TA via their membrane IgM. Agonism by αCD40 mAb requires the help of FcγRIIB-expressing cells, such as macrophages or another B cell, to assist in cross-linking of the CD40 trimer. Agonism of the CD40 receptor results in increased expression of the TLR9 receptor, which increases sensitivity to CpG ODN. This combined stimulation results in increased expression of TNFα, IFNγ, IL6, IL10, and IL12. There is also an increase in the expression of CD40L by B cells, which can assist and enhance the agonist effect of αCD40 mAb and assist in the activation of pDC. B cells thus stimulated can increase the secretion of TA-specific IgM and IgG. Furthermore, B cells can present processed TSA peptides to helper T cells via the MHC-II receptor of B cells. Interestingly, B cells can induce the proliferation and Th1 differentiation of allogeneic naive CD4+ T cells via an IL-12-dependent mechanism only when exposed to both CpG ODN and CD40L. IL12p70 is an important Th1 cytokine that can result in changes in helper T cells, such as increased expression of stimulatory molecules such as CD40-L and OX40, decreased expression of immunosuppressive molecules associated with T cell stimulation such as CTLA4 and PD1, and increased Th1 cytokines such as TNFα, IFNγ, and IL-2.

[0157] Similar effects of αCD40 and TLR9 agonists are observed in pDC and cDC (Figure 10). The combinatorial activity of these two agents has been shown to increase Th1 cytokines IFNα, IL12p70, TNFα, IL1β, IL6, which leads to the activation and stimulation of the chemokine IP10 (or CXCL10), a chemokine that promotes Th1 T cell responses, as well as the recruitment of immature pDC and activated T cells. More importantly, significantly higher amounts of IFNα and bioactive IL12p70 production are observed only when both the αCD40 agonist and the TLR9 agonist are present, and the downstream Th1 response is IL12-dependent. Furthermore, in pDC, the following occur: 1) an increase in CD40, which is expected to enhance pDC sensitivity to the αCD40 agonist and assist in stimulation by T helper cells expressing CD40L; 2) an increase in OX40L expression, which helps DC stimulate and activate T cells expressing OX40, potentially leading to improved survival and the establishment of memory T cells; 3) an increase in the expression of CCR7, a ligand for the CXCL10 chemokine, which promotes the mobilization and homing of activated DC to the LN; 4) an increase in the expression of co-stimulatory molecules CD80 and CD86, which provide co-stimulatory signals during T cell priming in the TDLN; 5) an increase in MHC-II expression, which helps stimulate CD4+ helper T cells; 6) an increase in MHC-I expression and cross-presentation responses, which help stimulate cytotoxic CD8+ T cells; 7) an increase in the expression of co-stimulatory molecules 4-1BB-L, GITR-L; and 8) downregulation of the immunosuppressive molecule PD-L1, as well as other related phenotypic and functional changes.

[0158] Naive CD4+ and CD8+ T cell priming and activation occur in the TDLN and further expand in the TLS of the TME (Figure 11). CD4+ T cells with an appropriate TCR can recognize the processed TA peptide in the context of MHC-II (Signal 1). Similarly, CD8+ T cells with an appropriate TCR recognize the processed TA peptide in the context of MHC-I as a result of cross-presentation of exogenously endocytosed TA (Signal 1). Both CD4+ and CD8+ T cells receive a co-stimulatory signal (Signal 2) through the interaction of CD28 on the T cell with co-stimulatory receptors CD80 and CD86 on the DC. IFNα secreted by activated DCs provides additional stimulatory signals (Signal 3) to T cells and the DCs themselves. As T cell activation progresses, counter-regulatory mechanisms are induced to prevent the activation and survival of T cells with TCR / MHC / Ag interactions that are too weak or too strong. One of these inducible signals expressed on Teff cells is CTLA4, which is induced when TCR / MHC / Ag interactions are too strong or prolonged. CTLA-4 has the function of overriding CD28 and thereby blocking the co-stimulatory signal provided by CD80 / 86. This restricts the polyclonality of TCR variants and T cell clones that are selected, proliferate, and survive. Additionally, CTLA-4 is constitutively expressed at high levels by Tregs, and CTLA-4 can also suppress the co-stimulatory signal of CD80 / 86 by directly blocking the interaction with CD28 and by trans-endocytosis of membrane patches containing CD80 / 86 from the DC. As T cell stimulation increases, the secretion of Th1 cytokines such as TNFα, IL2, and IFNγ increases, and T cell proliferation improves (↑CD3 / Ki67). Thus, blocking CTLA4 with an αCTLA mAb will result in an increase and progression of the co-stimulatory signal provided by CD28, ultimately enabling the survival and proliferation of T cell clones with strong TCR / MHC / Ag avidity and increasing the diversity of TCR variants.Furthermore, when the αCTLA4 mAb used to block CTLA4 is of the IgG1 isotype (such as YH001), ADCC-mediated elimination of Tregs expressing high levels of CTLA4 may occur, which can be an additional mechanism to assist in the priming of naive Teff cells.

[0159] Another important mechanistic effect of the activators of SV-101, such as αCD40 agonist and TLR9 agonist, is the secretion of IL12, which has the effect of increasing the expression of OX40 and CD40-L on T cells. When the expression of OX40 increases, CD4 T cells are more susceptible to the agonistic effect of αOX40 mAb, which brings about an additional effect of upregulation of CD40-L in Th cells, thereby promoting the activation of cDC via CD40.

[0160] Also, OX40 is highly expressed in Tregs, and αOX40 mAb agonism can suppress the function of FoxP3, thereby resulting in the downregulation of CTLA4 and IL10, as well as the promotion of Treg reprogramming into Th17 cells. Furthermore, αOX40 of the IgG1 isotype can mediate the death of Tregs via ADCC, and this activity has been shown to have a synergistic effect with the ADCC activity of the IgG1 isotype αCTLA4 mAb. The overall effect provided by the four components present in SV-101 is to increase T cell priming while reducing counter-regulatory mechanisms, thereby helping to increase CD4 Th cell stimulation, proliferation, survival, and memory differentiation, and increasing the polyclonality of the TCR.

[0161] When activated, CD8+ Teff cells proliferate in the TDLN and migrate to the TME (which is further promoted by the expression of CXCL10 by DCs activated by both the TLR9 agonist and the αCD40 agonist), initiating the effector phase of the anti-tumor immune response (Figure 13).

[0162] In the TME, the function of CD8+ Teff cells is to recognize the same TA peptide in the context of MHC-I and kill tumor cells by releasing GrzB, thereby restarting another cycle of TA and DAMP from tumor cells and maintaining the T cell stimulation cycle. An important immunosuppressive mechanism to be overcome in the TME is mediated by Tregs. Tregs mediate immunosuppression of the effector phase of Teff cells through several mechanisms, including: 1) degradation of extracellular ATP, a DAMP signal, to adenosine by the combined action of the membrane enzymes CD39 and CD72; 2) sequestering immunostimulatory cytokines such as IL-2 by overexpressing the high-affinity IL-2R CD25, thereby suppressing the helper effect of CD4 Th cells; 3) secretion of immunosuppressive cytokines such as TGFβ, IL10, and IL35; and 4) induction of Teff lysis by secretion of GrzB and perforin. The presence of the αOX40 and αCTLA4 components in SV-101 is also important in the effector phase of the immune response since these molecules target the function and number of Tregs. Tregs express both CTLA4 and OX40 at high levels, and the presence of IgG1 αCTLA and αOX40 can mediate ADCC of Tregs in the TME and reduce the immunosuppressive effect.

[0163] In summary, the αCD40 agonistic mAb and TLR9 agonist present in SV-101 mediate potent activation and maturation of APCs (primarily immature dendritic cells, but also including macrophages and B cells) in the TME, promoting the uptake and cross-presentation of tumor antigens, as well as upregulating pro-inflammatory molecules (MHC-I, MHC-II, CD80 / CD86) and cytokines (TNFα, INFα / β, IL-12) that stimulate helper T cells and effector cytotoxic T cells during antigen presentation, while downregulating immunosuppressive molecules against T cells such as PD-L1. Activation of dendritic cells promotes their migration to tumor-draining regional lymph nodes, where they activate antigen-specific naive T cells and memory T cells (both CD4 and CD8), promoting a cytotoxic Th1 anti-tumor immune response. Activation of effector T cells by APCs is promoted by the presence of αCTLA4 mAb, which has a dual function of promoting direct co-stimulation through CD80 / 86-CD28 interaction on activated T cells expressing inhibitory CTLA4 and indirect co-stimulation by limiting the sequestration of CD80 / 86 by Tregs expressing CTLA4. Further positive stimulation of effector T cells is obtained by the agonistic function of αOX40 mAb, which can trigger signaling that promotes survival and differentiation into memory T cells while inhibiting the differentiation of Tregs by inhibiting FoxP3 signaling. Additionally, the αCTLA4 mAb and αOX40 mAb in SV-101 inhibit the function of Tregs and promote Treg depletion by ADCC, thereby inhibiting the existing immunosuppressive mechanisms mediated by Tregs. A decrease in Tregs in the TME also prevents the mobilization of MDSCs, thereby preventing the re-establishment of an inhibitory TME after immunogenic cell death. Furthermore, IL-12 induced by αCD40 mAb and TLR9 ligand (e.g., agonist) can reduce the expression of PD-1 on exhausted T cells, which can help the infiltration of T cells into tumors by preventing the exclusion of lymphocytes from the TME by PD-L1 expressed by tumors.

[0164] In some embodiments, the method of the present disclosure includes the step of intratumoral administration of SV-102, which is a combined preparation of immunostimulatory therapeutic agents including a TLR9 agonist, an αCD40 agonist, and αPD1 and αCTLA4 checkpoint blockade inhibitors. In some embodiments, the method of the present disclosure includes the step of intratumoral administration of an SV-102 combination preparation consisting of a TLR9 agonist, an αCD40 agonist, an αPD1 checkpoint blockade inhibitor, and an αCTLA4 checkpoint blockade inhibitor. In some embodiments, the method of the present disclosure includes the step of administering an SV-102 preparation, and the SV-102 administration step is the second step in the method described herein. In some embodiments, the step of administering SV-101 is the second step following the first step of lysing the tumor by cryoablation in a method (e.g., the method described herein).

[0165] In many embodiments of the present disclosure, such a combination of agents promotes the priming of a new immune response, helps to strongly activate and mature APCs while assisting in the priming of naive T cells, and promotes the reactivation of existing exhausted Teff cells while counteracting the immunosuppressive effects of Tregs. This combination of active ingredients is targeted at treating mainly hot tumors showing severe T cell infiltration, and "cold" tumors that have not been treated previously and are no longer responsive to αPD1 checkpoint inhibitors, or tumors showing strong expression of PD-L1.

[0166] The mechanism of action of SV-102 shares similar immunopharmacological effects with SV-101 with respect to the activation of macrophages (Figure 9), B cells (Figure 10), and dendritic cells (Figure 11). However, there are specific differences in the mechanism of action between SV-102 and SV-101 in the activation / priming of T cell responses and in the effector phase in the tumor microenvironment.

[0167] Naive CD4+ and CD8+ T cell priming and activation occur in the TDLN and further expand in the TLS of the TME (Figure 12). CD4+ T cells with an appropriate TCR can recognize processed TA peptides in the context of MHC-II (Signal 1). Similarly, CD8+ T cells with an appropriate TCR recognize processed TA peptides in the context of MHC-I as a result of cross-presentation of exogenously endocytosed TA (Signal 1). Both CD4+ and CD8+ T cells receive co-stimulatory signals (Signal 2) through the interaction of CD28 on T cells with co-stimulatory receptors CD80 and CD86 on DCs. IFNα secreted by activated DCs provides additional stimulatory signals (Signal 3) to T cells and DCs themselves. As T cell activation progresses, counter-regulatory mechanisms are induced to prevent the activation and survival of T cells with TCR / MHC / Ag interactions that are either too weak or too strong. One of these inducible signals expressed on Teff cells is CTLA4, which is induced when TCR / MHC / Ag interactions are too strong or prolonged. CTLA-4 has the function of overriding CD28 and thereby blocking the co-stimulatory signals provided by CD80 / 86. This restricts the polyclonality of TCR variants and T cell clones that are selected, proliferate, and survive. In addition, CTLA-4 is constitutively expressed at high levels by Tregs, and CTLA-4 can also suppress the co-stimulatory signals of CD80 / 86 by directly blocking the interaction with CD28 and by trans-endocytosis of membrane patches containing CD80 / 86 from DCs. As T cell stimulation increases, the secretion of Th1 cytokines such as TNFα, IL2, and IFNγ increases, and T cell proliferation improves (↑CD3 / Ki67). Therefore, blocking CTLA4 with an αCTLA mAb will result in an increase and progression of the co-stimulatory signals provided by CD28, ultimately enabling the survival and proliferation of T cell clones with strong TCR / MHC / Ag avidity and increasing the diversity of TCR variants.Furthermore, when the αCTLA4 mAb used to block CTLA4 is of the IgG1 isotype, ADCC-mediated elimination of Tregs expressing high levels of CTLA4 may occur, which can be an additional mechanism to assist in the priming of naive Teff cells.

[0168] Another counter-regulatory mechanism induced by persistent TCR activation is the expression of PD1, which can be activated by PD-L1-expressing cells (immature DCs, MDSCs, M2 macrophages) in the TDLN and is usually upregulated in response to increased levels of IFNγ. The PD-1 / PD-L1 interaction recruits the SHP-2 phosphatase, which can inhibit T cell activation by dephosphorylating the intracellular domains of the Lck / TCR complex and CD28. Thus, blocking the PD-1 / PDL1 interaction with αPD1 can be another mechanism that can help maintain T cell priming in the TDLN.

[0169] Another important mechanistic effect of αCD40 agonist and TLR9 agonist agents is the secretion of IL12, which reduces the expression of PD-1, thereby making the effect of the αPD1 mAb more susceptible, and has the effect of increasing the expression of OX40 and CD40-L on T cells. When these effects are combined with the increased levels of the cognate ligands OX40-L and CD40 expressed by DCs activated in this way, an increase in CD4 Th cell stimulation, survival, and memory differentiation can be expected.

[0170] The overall effect provided by the four components present in SV-102 is to increase T cell priming while reducing counter-regulatory mechanisms, thereby helping to increase CD4 Th cell stimulation, proliferation, survival, and memory differentiation, and increasing TCR polyclonality.

[0171] When activated, CD8+ Teff cells proliferate in the TDLN and migrate to the TME, which is further promoted by the expression of CXCL10 by DCs activated by both TLR9 agonists and αCD40 agonists, initiating the effector phase of the anti-tumor immune response (Figure 14).

[0172] In the TME, the function of CD8+ Teff cells is to recognize the same TA peptide in the context of MHC-I and kill tumor cells by releasing GrzB, thereby restarting another cycle of TA and DAMP from tumor cells and maintaining the T cell stimulation cycle.

[0173] As a result of continuous TCR-mediated T cell stimulation, the expression of PD1 and the release of IFNγ increase, which leads to the induction of the expression of PD-L1 on tumor cells. The PD-1 / PD-L1 interaction results in the phosphorylation of PD1 by Lck, mobilizing the SHP2 phosphatase, which in turn results in the dephosphorylation of the TCR, leading to exhausted PD1hi Teff, which is often resistant to PD1 checkpoint inhibitors. Exhausted PD1hi T cells also express CD40 in the TME. These exhausted PD1hi T cells can be reactivated by several mechanisms mediated by the agents present in SV-102. First, an increase in the level of IL12p70 in the TME (which is achieved by the combined action of αCD40 agonists and TLR9 agonists) helps to reduce the expression level from PD1hi to PD1lo, thereby making the cells more sensitive to the αPD1 mAb blocker. Also, the agonism of CD40 by the αCD40 agonistic mAb can reactivate the metabolism of exhausted T cells by activating mTORC1.

[0174] Another important immunosuppressive mechanism to be overcome in the TME is the Treg-mediated immunosuppressive mechanism (Figure 15). Tregs mediate immunosuppression of the effector phase of Teff cells through several mechanisms, including: 1) degradation of extracellular ATP, a DAMP signal, to adenosine by the combined action of the membrane-bound enzymes CD39 and CD72; 2) sequestering immunostimulatory cytokines such as IL-2 by overexpressing the high-affinity IL-2R CD25, thereby suppressing the helper effect of CD4 Th cells; 3) secretion of immunosuppressive cytokines such as TGFβ, IL10, and IL35; and 4) induction of Teff lysis by secretion of GrzB and perforin.

[0175] Tregs express high levels of CTLA4, and the presence of IgG1 αCTLA can mediate ADCC of Tregs in the TME and suppress the immunosuppressive effect. Therefore, the presence of αCTLA4 in SV-102 is also important in the effector phase of the immune response.

[0176] In summary, the αCD40 agonistic mAb and TLR9 agonist present in SV-102 mediate the strong activation and maturation of APCs (primarily immature dendritic cells, but also including macrophages and B cells) in the TME, promoting the uptake and cross-presentation of tumor antigens, as well as upregulating pro-inflammatory molecules (MHC-I, MHC-II, CD80 / CD86) and cytokines (TNFα, INFα / β, IL-12) that stimulate helper T cells and effector cytotoxic T cells during antigen presentation, while downregulating immunosuppressive molecules against T cells such as PD-L1. The activation of dendritic cells promotes their migration to tumor-draining regional lymph nodes, where they activate antigen-specific naive T cells and memory T cells (both CD4 and CD8), promoting a cytotoxic Th1 anti-tumor immune response. The activation of effector T cells by APCs is promoted by the presence of αCTLA4 mAb, which has a dual function of promoting direct co-stimulation through the CD80 / 86-CD28 interaction on activated T cells expressing inhibitory CTLA4 and indirect co-stimulation by limiting the sequestration of CD80 / 86 by Tregs expressing CTLA4. Further positive stimulation of effector T cells is obtained by αPD1 blocking mAb, which can reduce the inhibitory signals provided by PD-L1+DCs. Additionally, the αCTLA4 mAb in SV-102 inhibits the function of Tregs and promotes the depletion of Tregs by ADCC, thereby inhibiting the existing immunosuppressive mechanisms mediated by Tregs. The reduction of Tregs in the TME also prevents the mobilization of MDSCs, thereby preventing the re-establishment of an inhibitory TME after immunogenic cell death. Furthermore, IL-12 induced by αCD40 mAb and TLR9 ligands (e.g., agonists) can reduce the expression of PD-1 on exhausted T cells, which can prevent the elimination of lymphocytes from the TME by PD-L1 expressed by tumors and make the lymphocytes responsive to αPD1 mAb treatment, thereby assisting in the tumor infiltration of T cells.The presence of αPD1 mAb in SV-102 can rescue TILs from exhaustion and extend their functional cytotoxic phenotype by preventing TILs from becoming exhausted.

[0177] In addition to the pharmacological activities of the individual active ingredients or agents injected intratumorally in the combination formulations SV-101 and SV-102, the biomechanics of intratumor injection and regional injection play an important role in controlling the biodistribution of the drug and mediating the biological activity and antitumor efficacy of the methods of the present disclosure. As a well-known general principle of hydrodynamics, liquids flow (volume / time) from regions of high pressure to regions of low pressure through the path of least resistance (change in pressure / flow) and highest compliance (change in volume / change in pressure). During injection, tissues exhibit resistance and compliance along various scales of elasticity, and as the paths of various resistances and compliances become saturated with fluid and the resistance (back pressure) increases, the path of least resistance changes. Almost all normal and healthy tissues maintain fluid balance within the tissue mainly using the lymphatic drainage system. Lymphatic vessels are typically the main drainage routes for interstitial fluid and extravascular leakage fluid. When lymphatic drainage is blocked, edema (excess fluid in the tissue) occurs. Also, metastatic tumors often have an overabundance of lymphatic systems compared to healthy tissues or early tumors, and it is well understood that they utilize the lymphatic system extensively to transport small and large molecules in and out of the tumor region and communicate with other tissues such as TDLN. Furthermore, tumors are associated with tertiary lymphoid-like structures (TLS) either within the tumor, around the tumor, or in adjacent tissues, and these TLS communicate with secondary lymphoid structures such as the tumor and TDLN via lymphatic vessels. The drainage system of lymphoid tissue represents an important biomechanically significant aspect of the methods of the present disclosure in that it presents the path of least resistance for tumor tissue to resolve the excess (edema) of interstitial fluid and extravascular leakage fluid caused by drug injection into the treatment zone. The relatively low-speed and low-pressure injection rate (compared to subcutaneous and intramuscular injections) allows the drug to first mix with relatively low resistance (via the shortest path of least resistance) immediately surrounding the lysed cell debris in the treatment zone containing tumor antigens, DAMPS, and other cell contents. As the lysed treatment zone becomes saturated, the injected fluid spreads through the path of least resistance towards the surrounding intact tumor tissue and is also drained first through the available lymphatic vessel drainage route and to a much lesser extent through the vascular route.This continues until the compliance limit of the tissue (the ability of the tissue to expand to accept additional fluid) is reached, at which point the continuous infusion pressure reaches equilibrium and the flow rate out of the tumor, mainly through the draining lymphatics and blood vessels, becomes approximately equal to the inflow rate injected into the tumor. This point varies based on the tissue compliance within and around the tumor (some tissues are more compliant than others), but it is expected that the equilibrium point will be reached when approximately 5 mL of the drug (e.g., the formulations described herein) has been injected, or earlier (based on the fact that the tissue compliance limits associated with intramuscular injection range from 1.5 mL to 5 mL). At the equilibrium point, the continuous infusion flow rate / pressure saturates and expands the target tumor tissue to its compliance limit, and any diffusible substances, including the injected drug and dissolved tumor cell contents, tumor antigens, DAMPs, etc., mix and can be washed away into the surrounding intratumoral tissue, peritumoral tissue, and lymphatic drainage ducts. Thus, most of this excess fluid mixture of the injected drug and some of the mobile substances is expected to be drained from the treatment zone and reach their cellular targets within the tumor microenvironment, TLS, and TDLN through the draining lymphatics and enter the inflow region lymph nodes. When the injection is complete, the injection pressure head ends, and the expanded and elastic intratumoral and peritumoral tissues also drain the excess drug and debris fluid mixture through the least resistant pathway, which is expected to be mainly the lymphatic drainage system, and return to the pre-injection pressure at rest. The remaining mixture of extravasated drug, damaged tissue, intact mobile cells, and cell debris (tumor antigens, DAMPs, cell fragments, etc.) is expected to be highly immunogenic and stimulate the recruitment and activation of APCs, the migration of APCs to the TDLN, antigen presentation, and the activation of T cells, promoting cytokines that facilitate the infiltration of T cells into the TME. Additionally, it is expected that the drug injected into the TME will promote the reactivation of exhausted TILs and shift the balance towards a cytotoxic anti-tumor immune response by blocking the immunosuppressive function of Tregs.

[0178] For at least the reasons described above, in some embodiments, the intratumoral injection procedure described herein, unlike other intratumoral therapies, is not expected to have the injected liquid completely contained within the tumor tissue, but rather is considered an intratumoral injection for the purpose of a local regional therapy that is actually expected to enter lymphatic structures leading to the tumor and peritumoral tissue through lymphatic vessels. In fact, a characteristic parameter in which the method of the present disclosure differs from most intratumoral therapies is that the injection volume of the drug combination exceeds the volume of the cryoablation zone and the volume that can theoretically be contained or retained within the tumor, and the exudation of this liquid composition outside the tumor margin is achieved by a large volume of injection and an increase in hydrodynamic pressure.

[0179] Accordingly, the method of the present disclosure is thought to produce local regional effects in at least two physical locations (intratumoral / peritumoral and lymphoid organs) that synchronize and bring into proximity a drug (e.g., the formulations described herein), an antigen, and immune cells, thereby increasing the likelihood of interaction and ultimately eliciting local and systemic anti-tumor responses.

[0180] In some embodiments of the present disclosure, the active ingredient or mixture of agents in SV-101 or SV-102 is intratumorally injected using the following procedure. After a partial tumor cryoablation treatment zone has been established within the selected tumor, the next step is to advance a coaxial cryoprobe / injection needle system (SCINS) under precise image guidance and position the tip of the outer coaxial injection needle approximately at the center of the cryoablation treatment zone. Once this is completed, the cryoprobe is withdrawn from the SCINS, leaving the tip of the outer coaxial injection needle remaining approximately at the center of the cryoablation treatment zone within the tumor, and preparation for drug injection is made.

[0181] In some embodiments of the present disclosure, the combination formulation SV-101 or the combination formulation SV-102 is prepared for injection in a volume of about 1 mL to about 30 mL. In some embodiments, the combination formulation is prepared in a volume of about 1 mL to about 5 mL, about 1 mL to about 10 mL, about 1 mL to about 15 mL, about 1 mL to about 20 mL, about 1 mL to about 25 mL, about 5 mL to about 30 mL, about 10 mL to about 30 mL, about 20 mL to about 30 mL, about 5 mL to about 10 mL, about 10 mL to about 15 mL, about 15 mL to about 20 mL, or about 20 mL to about 30 mL. In a preferred embodiment, the combination formulation is prepared in a volume of about 15 mL.

[0182] In some embodiments of the present disclosure, a syringe containing a drug (e.g., the formulation described herein) is attached to a pump, and a connection tube with a low priming volume (e.g., a MEDLINE 60-inch extension set with a microbore tube) is connected to the syringe via a one-way stopcock. The syringe pump is advanced to prime the system with the drug until the drug can be seen coming out of the connection tube. Next, the primed connection tube is attached to the SCINS injection needle. The priming volume of the connection tube is about 0.6 ml. The infusion syringe pump is set to a flow rate of about 1 - 5 mL / min, and the recommended value is 3 mL / min. The overpressure alarm is not set. Next, the pump is started, and a total volume of about 15 ml is injected in 5 minutes. Care is taken to ensure that the tip of the injection needle remains in the correct position and that the drug does not flow back along the needle cannula. When the drug infusion pump completes its infusion cycle and 15 ml has been delivered from the syringe into the connection tube, the one-way stopcock is closed. At this point, there is a residual volume of about 0.6 ml of the drug in the connection tube. This volume of the drug needs to be advanced by removing the one-way stopcock from the infusion syringe and injecting 0.6 mL of sterile water into the one-way stopcock.

[0183] In some embodiments, the infusion syringe pump is set to flow at a rate of about 1 mL / min, about 2 mL / min, about 3 mL / min, about 4 mL / min, or about 5 mL / min. In some embodiments, the infusion syringe pump is set to flow at a rate of about 1 mL / min. In some embodiments, the infusion syringe pump is set to flow at a rate of about 2 mL / min. In some embodiments, the infusion syringe pump is set to flow at a rate of about 3 mL / min. In some embodiments, the infusion syringe pump is set to flow at a rate of about 4 mL / min. In some embodiments, the infusion syringe pump is set to flow at a rate of about 5 mL / min.

[0184] In some embodiments of the present disclosure, the TLR9 agonist is a class B or C CpG oligodeoxynucleotide (ODN). In some embodiments of the present disclosure, the TLR9 agonist is a class B CpG oligodeoxynucleotide (ODN). In some embodiments of the present disclosure, the TLR9 agonist is a class C CpG oligodeoxynucleotide (ODN).

[0185] In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 1. In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 2. In some embodiments, the TLR9 agonist is a nucleic acid set forth in SEQ ID NO: 27.

[0186] In some embodiments, the TLR9 agonist is administered in accordance with the present disclosure at a dosage in the range of about 0.5 mg to about 10 mg, about 1 mg to about 10 mg, about 2 mg to about 10 mg, about 4 mg to about 10 mg, about 8 mg to about 10 mg, about 0.5 mg to about 8 mg, about 0.5 mg to about 6 mg, about 0.5 mg to about 4 mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 1 mg, about 1 mg to about 4 mg, about 2 mg to about 4 mg, or about 3 mg to about 4 mg. In some embodiments, the TLR9 agonist is administered at a dosage of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 7.5 mg, about 10 mg, about 12 mg, or about 15 mg. In some embodiments, the TLR9 agonist is administered at a dosage of about 1 mg. In some embodiments, the TLR9 agonist is administered at a dosage of about 2 mg. In some embodiments, the TLR9 agonist is administered at a dosage of about 3 mg. In some embodiments, the TLR9 agonist is administered at a dosage of about 4 mg.

[0187] In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody is one of the human IgG2k isotypes. In some embodiments, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0188] In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 28. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0189] In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a light chain, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0190] In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 29.In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 28, and the light chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 29. In some embodiments of the present disclosure, the agonistic anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 28, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 29.

[0191] In some embodiments, the anti-CD40 agonistic monoclonal antibody is administered in accordance with the present disclosure at a dose in the range of about 0.5 mg to about 10 mg, about 1 mg to about 10 mg, about 2 mg to about 10 mg, about 4 mg to about 10 mg, about 8 mg to about 10 mg, about 0.5 mg to about 8 mg, about 0.5 mg to about 6 mg, about 0.5 mg to about 4 mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 1 mg, about 1 mg to about 8 mg, or about 4 mg to 8 mg. In some embodiments, the anti-CD40 agonistic monoclonal antibody is administered at a dose of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 7.5 mg, about 10 mg, about 12 mg, about 15 mg, or about 20 mg. In some embodiments, the anti-CD40 agonistic monoclonal antibody is administered at a dose of about 1 mg. In some embodiments, the anti-CD40 agonistic monoclonal antibody is administered at a dose of about 5 mg. In some embodiments, the anti-CD40 agonistic monoclonal antibody is administered at a dose of about 7.5 mg. In some embodiments, the anti-CD40 agonistic monoclonal antibody is administered at a dose of about 10 mg.

[0192] In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody is one of the human IgG1k isotypes. In some embodiments, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0193] In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 30. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30.

[0194] In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti - OX40 monoclonal antibody comprises a light chain, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 31.

[0195] In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 31.In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 30, and the light chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 31. In some embodiments of the present disclosure, the agonistic anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 30, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 31.

[0196] In some embodiments, the anti-OX40 agonistic monoclonal antibody is administered according to the present disclosure at a dose in the range of about 0.5 mg to about 10 mg, about 1 mg to about 10 mg, about 2 mg to about 10 mg, about 4 mg to about 10 mg, about 8 mg to about 10 mg, about 0.5 mg to about 8 mg, about 0.5 mg to about 6 mg, about 0.5 mg to about 4 mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 1 mg, about 1 mg to about 8 mg, or about 4 mg to 8 mg. In some embodiments, the anti-OX40 agonistic monoclonal antibody is administered at a dose of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 7.5 mg, about 10 mg, about 12 mg, about 15 mg, or about 20 mg. In some embodiments, the anti-OX40 agonistic monoclonal antibody is administered at a dose of about 1 mg. In some embodiments, the anti-OX40 agonistic monoclonal antibody is administered at a dose of about 5 mg. In some embodiments, the anti-OX40 agonistic monoclonal antibody is administered at a dose of about 7.5 mg. In some embodiments, the anti-OX40 agonistic monoclonal antibody is administered at a dose of about 10 mg.

[0197] In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody is of the human IgG4k isotype. In some embodiments, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0198] In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 32. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0199] In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a light chain, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 33.

[0200] In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 33. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 33.In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 33.

[0201] In some embodiments, the anti-PD1 monoclonal antibody is administered in accordance with the present disclosure at a dose in the range of about 1 mg to about 100 mg, about 2 mg to about 100 mg, about 4 mg to about 100 mg, about 8 mg to about 100 mg, about 10 mg to about 100 mg, about 20 mg to about 100 mg, about 30 mg to about 100 mg, about 40 mg to about 100 mg, about 50 mg to about 100 mg, about 70 mg to about 100 mg, about 90 mg to about 100 mg, about 1 mg to about 90 mg, about 1 mg to about 70 mg, about 1 mg to about 50 mg, about 1 mg to about 40 mg, about 1 mg to about 30 mg, about 1 mg to about 20 mg, about 1 mg to about 10 mg, about 3 mg to about 10 mg, 3 mg to about 30 mg, about 3 mg to about 100 mg, or about 10 mg to about 30 mg. In some embodiments, the anti-PD1 monoclonal antibody is administered at a dose of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 7.5 mg, about 10 mg, about 10 mg, about 12 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, or about 150 mg. In some embodiments, the anti-PD1 monoclonal antibody is administered at a dose of about 3 mg. In some embodiments, the anti-PD1 monoclonal antibody is administered at a dose of about 10 mg. In some embodiments, the anti-PD1 monoclonal antibody is administered at a dose of about 30 mg. In some embodiments, the anti-PD1 monoclonal antibody is administered at a dose of about 80 mg. In some embodiments, the anti-PD1 monoclonal antibody is administered at a dose of about 100 mg.

[0202] In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody is of the human IgG1κ isotype. In some embodiments, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprising a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0203] In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 34. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain, and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34.

[0204] In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a light chain, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0205] In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 35.In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 34, and the light chain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 35. In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 34, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0206] In some embodiments, the anti-CTLA4 monoclonal antibody is administered according to the present disclosure at a dose within the range of about 1 mg to about 50 mg, about 2 mg to about 50 mg, about 4 mg to about 50 mg, about 8 mg to about 50 mg, about 10 mg to about 50 mg, about 20 mg to about 50 mg, about 30 mg to about 50 mg, about 40 mg to about 50 mg, about 1 mg to about 40 mg, about 1 mg to about 30 mg, about 1 mg to about 20 mg, about 1 mg to about 10 mg, about 1 mg to about 5 mg, about 1 mg to about 2 mg, about 5 mg to about 15 mg, about 5 mg to about 40 mg, or about 15 mg to about 40 mg. In some embodiments, the anti-CTLA4 monoclonal antibody is administered at a dose of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 7.5 mg, about 10 mg, about 10 mg, about 12 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg. In some embodiments, the anti-CTLA4 monoclonal antibody is administered at a dose of about 1 mg. In some embodiments, the anti-CTLA4 monoclonal antibody is administered at a dose of about 5 mg. In some embodiments, the anti-CTLA4 monoclonal antibody is administered at a dose of about 15 mg. In some embodiments, the anti-CTLA4 monoclonal antibody is administered at a dose of about 40 mg.

[0207] In some preferred embodiments of the present disclosure, intratumoral cryoablation of solid tumors and intratumoral injection of SV-101 or SV-102 are repeated every 3 to 8 weeks in the same lesion or different metastatic lesions.

[0208] In some embodiments, intratumoral cryoablation of solid tumors and intratumoral injection of the combination formulations provided by the present disclosure are repeated about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, about once every ten weeks, about once every eleven weeks, about once every twelve weeks, about once every thirteen weeks, about once every fourteen weeks, about once every fifteen weeks, or about once every sixteen weeks. In some embodiments, the treatment is repeated about every 3 - 16 weeks, about every 6 - 16 weeks, about every 8 - 16 weeks, about every 10 - 16 weeks, about every 12 - 16 weeks, about every 14 - 16 weeks, about every 3 - 14 weeks, about every 3 - 12 weeks, about every 3 - 10 weeks, about every 3 - 8 weeks, about every 3 - 6 weeks, or about every 3 - 4 weeks. In some embodiments, the treatment is repeated about every 4 - 8 weeks, about every 5 - 8 weeks, about every 6 - 8 weeks, about every 7 - 8 weeks, about every 4 - 7 weeks, about every 4 - 6 weeks, or about every 4 - 5 weeks. In some embodiments, the treatment is repeated every 4 - 8 weeks.

[0209] In some embodiments of the present disclosure, the SV-101 formulation contains: i) a TLR9 agonist at a dosage level of about 0.5 mg to about 10 mg per administration; ii) an anti-CD40 monoclonal antibody at a dosage level of about 0.5 mg to about 10 mg per administration; iii) an anti-OX40 monoclonal antibody at a dosage level of about 0.5 mg to about 10 mg per administration; and iv) an anti-CTLA4 monoclonal antibody in the range of about 1 mg to about 50 mg per administration.

[0210] In some embodiments, the SV-101 formulation contains a TLR9 agonist, an anti-CD40 agonist antibody, an anti-OX40 agonist antibody, and an anti-CTLA4 antibody at dosage levels of about 1 mg, about 1 mg, about 1 mg, and about 1 mg, respectively.

[0211] In some embodiments, the SV-101 formulation contains a TLR9 agonist, an anti-CD40 agonist antibody, an anti-OX40 agonist antibody, and an anti-CTLA4 antibody at dosage levels of about 2 mg, about 5 mg, about 5 mg, and about 5 mg, respectively.

[0212] In some embodiments, the SV-101 formulation contains a TLR9 agonist, an anti-CD40 agonist antibody, an anti-OX40 agonist antibody, and an anti-CTLA4 antibody at dosage levels of about 3 mg, about 7.5 mg, about 7.5 mg, and about 15 mg, respectively.

[0213] In some embodiments, the SV-101 formulation contains a TLR9 agonist, an anti-CD40 agonist antibody, an anti-OX40 agonist antibody, and an anti-CTLA4 antibody at dosage levels of about 4 mg, about 10 mg, about 10 mg, and about 40 mg, respectively.

[0214] In some embodiments, the SV-101 formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0215] In some embodiments, the SV-101 formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0216] In some embodiments, the SV-101 formulation comprises a TLR9 agonist set forth in SEQ ID NO: 2; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0217] In some embodiments, the SV-101 formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0218] In some embodiments, the SV-101 formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35.

[0219] In some embodiments, the SV-101 formulation comprises a TLR9 agonist set forth in SEQ ID NO: 1; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 31; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0220] In some embodiments, the SV-101 formulation comprises a TLR9 agonist set forth in SEQ ID NO: 2; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 31; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0221] In some embodiments, the SV-101 formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an agonistic anti-OX40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 31; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0222] In some embodiments of the present disclosure, the SV-102 formulation contains: i) a TLR9 agonist at a dosage level of about 0.5 mg to about 10 mg per administration; ii) an anti-CD40 monoclonal antibody at a dosage level of about 0.5 mg to about 10 mg per administration; iii) an anti-PD1 monoclonal antibody at a dosage level of about 1 mg to about 100 mg per administration; and iv) an anti-CTLA4 monoclonal antibody in the range of about 1 mg to about 50 mg per administration.

[0223] In some embodiments, the SV-102 formulation contains a TLR9 agonist, an anti-CD40 agonist antibody, an anti-PD1 antibody, and an anti-CTLA4 antibody at dosage levels of about 1 mg, about 1 mg, about 3 mg, and about 1 mg, respectively.

[0224] In some embodiments, the SV-102 formulation contains a TLR9 agonist, an anti-CD40 agonist antibody, an anti-PD1 antibody, and an anti-CTLA4 antibody at dosage levels of about 2 mg, about 5 mg, about 10 mg, and about 5 mg, respectively.

[0225] In some embodiments, the SV-102 formulation contains a TLR9 agonist, an anti-CD40 agonist antibody, an anti-PD1 antibody, and an anti-CTLA4 antibody at dose levels of about 3 mg, about 7.5 mg, about 30 mg, and about 15 mg, respectively.

[0226] In some embodiments, the SV-102 formulation contains a TLR9 agonist, an anti-CD40 agonist antibody, an anti-PD1 antibody, and an anti-CTLA4 antibody at dose levels of about 4 mg, about 10 mg, about 80 mg, and about 40 mg, respectively.

[0227] In some embodiments, the SV-102 formulation contains a TLR9 agonist, an anti-CD40 agonist antibody, an anti-PD1 antibody, and an anti-CTLA4 antibody at dose levels of about 4 mg, about 10 mg, about 100 mg, and about 40 mg, respectively.

[0228] In some embodiments, the SV-102 formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0229] In some embodiments, the SV-102 formulation comprises the TLR9 agonist set forth in SEQ ID NO: 1; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0230] In some embodiments, the SV-102 formulation comprises the TLR9 agonist set forth in SEQ ID NO: 2; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0231] In some embodiments, the SV-102 formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0232] In some embodiments, the SV-102 formulation comprises a TLR9 agonist as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 29; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35.

[0233] In some embodiments, the SV-102 formulation comprises the TLR9 agonist set forth in SEQ ID NO: 1; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 33; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0234] In some embodiments, the SV-102 formulation comprises the TLR9 agonist set forth in SEQ ID NO: 2; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 33; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0235] In some embodiments, the SV-102 formulation comprises the TLR9 agonist set forth in SEQ ID NO: 27; an agonistic anti-CD40 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 29; an anti-PD1 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 33; and an anti-CTLA4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0236] In some embodiments of the present disclosure, the individual components of SV-101 and SV-102 are sequentially injected in different treatment cycles into the same tumor lesion being treated.

[0237] In some embodiments, the individual components of SV-101 and SV-102 are premixed together and simultaneously injected into the tumor lesion being treated.

[0238] In one embodiment of the present disclosure, the combination formulation is administered in a fixed volume within the range of 1 to 30 mL, most preferably 15 mL. In some embodiments, the combination formulation is about 1 to 30 mL, about 2 to 30 mL, about 5 to 30 mL, about 10 to 30 mL, about 12 to 30 mL, about 14 to 30 mL, about 16 to 30 mL, about 18 to 30 mL, about 20 to 30 mL, about 22 to 30 mL, about 24 to 30 mL, about 26 to 30 mL, about 28 to 30 mL, about 1 to 28 mL, about 1 to 26 mL, about 1 to 24 mL, about 1 to 22 mL, about 1 to 20 mL, about 1 to 18 mL, about 1 to 16 mL, about 1 to 14 mL, about 1 to 12 mL, about 1 to 10 mL, about 1 to 5 mL, about 1 to 2 mL, about 5 to 25 mL, about 10 to 20 mL, about 12 to 16 mL, or about 13 to 14 mL. In some embodiments, the combination formulation is administered in a fixed volume of about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 25 mL, or about 30 mL. In some embodiments, the combination formulation is administered in a fixed volume of about 15 mL.

[0239] In one embodiment of the present disclosure, the cancer is a solid tumor cancer selected from adenocarcinoma, astrocytoma, bladder cancer, osteosarcoma, breast cancer, cervical cancer, chordoma, colorectal cancer, endometrial cancer, esophageal cancer, glioblastoma, glioma, kidney cancer, liver cancer, medulloblastoma, melanoma, meningioma, mesothelioma, metastatic pituitary cancer, prostate cancer, neuroblastoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer, ovarian cancer, pancreatic cancer, renal cell carcinoma, retinoblastoma, sarcoma, small cell lung cancer, squamous cell carcinoma (including head and neck cancer), gastric cancer, testicular cancer, thyroid cancer, and Wilms tumor.

[0240] In one embodiment of the present disclosure, a method for treating cancer involves: a) a tumor cell lysis step; and b) an intratumoral administration step of a combined preparation of four immunotherapeutic active ingredients including a TLR9 agonist, an agonistic anti-CD40 monoclonal antibody, an anti-CTLA4 monoclonal antibody, and an agonistic anti-OX40 monoclonal antibody or an anti-PD1 monoclonal antibody to the same lesion. This is repeated every 3 to 16 weeks, preferably every 4 to 8 weeks, for a total of 1 to 12 cycles of treatment, preferably 6 cycles if there are treatable lesions.

[0241] In some embodiments, the treatment methods provided herein are repeated for 1 to 12 cycles, 1 to 10 cycles, 1 to 8 cycles, 1 to 6 cycles, 1 to 4 cycles, 1 to 2 cycles, 3 to 12 cycles, 5 to 12 cycles, 7 to 12 cycles, 9 to 12 cycles, or 11 to 12 cycles. In some embodiments, the treatment is repeated for 3 to 6 cycles, 3 to 5 cycles, 3 to 4 cycles, 4 to 6 cycles, or 5 to 6 cycles. In some embodiments, the treatment is repeated for 3 to 6 cycles. In some embodiments, the treatment is repeated for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles. In some embodiments, the treatment is repeated for 2 cycles. In some embodiments, the treatment is repeated for 3 cycles. In some embodiments, the treatment is repeated for 4 cycles. In some embodiments, the treatment is repeated for 5 cycles. In some embodiments, the treatment is repeated for 6 cycles.

[0242] Throughout this specification, when a composition is described as having, including, or comprising a particular component, or when a process and method are described as having, including, or comprising a particular step, it is contemplated that there are also compositions of the present disclosure that consist essentially of, or consist of, the recited components, and processes and methods according to the present disclosure that consist essentially of, or consist of, the recited process steps.

[0243] The use of the terms "include", "includes", "including", "have", "has", "having", "contain", "contains", or "containing", including their grammatical equivalents, is generally open-ended and non-limiting, unless specifically stated otherwise or understood from the context, and is not to be construed as excluding additional unrecited elements or steps. For example, it should not be understood to exclude additional unrecited elements or steps.

[0244] When the term "about" or "approximately" is used before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically described otherwise. As used herein, the term "about" or "approximately" refers to a variation of ±10% from the nominal value, unless otherwise indicated or inferred.

[0245] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, references to "an antibody" include a plurality of antibodies, and in some embodiments, references to "an antibody" include a plurality of antibodies (e.g., antibodies of different types (e.g., class / isotype, light chain type), antibody fragments, or engineered antibodies, etc.).

[0246] The use of alternatives (e.g., "or") is to be understood to mean either one, both, or any combination of the alternatives.

Examples

[0247] The following examples are provided for illustrative purposes only and are in no way intended to limit the scope of the present disclosure.

[0248] Example 1: Preparation of SV-101 - Low Dose In this example, the preparation of the SV-101 combination formulation will be described. In a sterile 20 mL vial, the following components are mixed and diluted to a volume of 15 mL in sterile physiological saline. In one SV-101 combination formulation, the CTLA4 antibody is the YH001 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 21-26 and / or the VH / VL sequences described in SEQ ID NOs: 34-35), the OX40 antibody is the YH002 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 9-14 and / or the VH / VL sequences described in SEQ ID NOs: 30-31), the CD40 antibody is the YH003 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 3-8 and / or the VH / VL sequences described in SEQ ID NOs: 28-29), and the CpG ODN is the one of SEQ ID NO: 1. In another SV-101 combination formulation, the CpG ODN is the one of SEQ ID NO: 2 or SEQ ID NO: 27. Those skilled in the art will understand that derivatives or variants of the SV-101 components can be used.

[0249]

Table 1

[0250] Example 2: Preparation of SV-101 - Intermediate Dose In this example, the preparation of the SV-101 combination formulation will be described. In a sterile 20 mL vial, the following components are mixed and diluted to a volume of 15 mL in sterile physiological saline. In one SV-101 combination formulation, the CTLA4 antibody is the YH001 antibody (e.g., defined by the CDR sequences set forth in SEQ ID NOs: 21-26 and / or the VH / VL sequences set forth in SEQ ID NOs: 34-35), the OX40 antibody is the YH002 antibody (e.g., defined by the CDR sequences set forth in SEQ ID NOs: 9-14 and / or the VH / VL sequences set forth in SEQ ID NOs: 30-31), the CD40 antibody is the YH003 antibody (e.g., defined by the CDR sequences set forth in SEQ ID NOs: 3-8 and / or the VH / VL sequences set forth in SEQ ID NOs: 28-29), and the CpG ODN is the one of SEQ ID NO: 1. In another SV-101 combination formulation, the CpG ODN is the one of SEQ ID NO: 2 or SEQ ID NO: 27. Those skilled in the art will understand that derivatives or variants of the SV-101 components can be used.

[0251]

Table 2

[0252] Example 3: Preparation of SV-101 - High Dose In this example, the preparation of the SV-101 combination formulation will be described. In a sterile 20 mL vial, the following components are mixed and diluted to a volume of 15 mL in sterile physiological saline. In one SV-101 combination formulation, the CTLA4 antibody is the YH001 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 21-26 and / or the VH / VL sequences described in SEQ ID NOs: 34-35), the OX40 antibody is the YH002 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 9-14 and / or the VH / VL sequences described in SEQ ID NOs: 30-31), the CD40 antibody is the YH003 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 3-8 and / or the VH / VL sequences described in SEQ ID NOs: 28-29), and the CpG ODN is the one of SEQ ID NO: 1. In another SV-101 combination formulation, the CpG ODN is the one of SEQ ID NO: 2 or SEQ ID NO: 27. Those skilled in the art will understand that derivatives or variants of the SV-101 components can be used.

[0253]

Table 3

[0254] Example 4: Preparation of SV-101 - Maximum Dose Level In this example, the preparation of the SV-101 combination formulation will be described. In a sterile 20 mL vial, the following components are mixed and diluted to a volume of 15 mL in sterile physiological saline. In one SV-101 combination formulation, the CTLA4 antibody is the YH001 antibody (e.g., defined by the CDR sequences set forth in SEQ ID NOs: 21-26 and / or the VH / VL sequences set forth in SEQ ID NOs: 34-35), the OX40 antibody is the YH002 antibody (e.g., defined by the CDR sequences set forth in SEQ ID NOs: 9-14 and / or the VH / VL sequences set forth in SEQ ID NOs: 30-31), the CD40 antibody is the YH003 antibody (e.g., defined by the CDR sequences set forth in SEQ ID NOs: 3-8 and / or the VH / VL sequences set forth in SEQ ID NOs: 28-29), and the CpG ODN is the one of SEQ ID NO: 1. In another SV-101 combination formulation, the CpG ODN is the one of SEQ ID NO: 2 or SEQ ID NO: 27. Those skilled in the art will understand that derivatives or variants of the SV-101 components can be used.

[0255]

Table 4

[0256] Example 5: Preparation of SV-102 - Low Dose In this example, the preparation of the SV-102 combination formulation will be described. In a sterile 20 mL vial, the following components are mixed and diluted to a volume of 15 mL in sterile physiological saline. In one SV-102 combination formulation, the CTLA4 antibody is the YH001 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 21-26 and / or the VH / VL sequences described in SEQ ID NOs: 34-35), the PD-1 antibody is the 609A antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 15-20 and / or the VH / VL sequences described in SEQ ID NOs: 32-33), the CD40 antibody is the YH003 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 3-8 and / or the VH / VL sequences described in SEQ ID NOs: 28-29), and the CpG ODN is the one of SEQ ID NO: 1. In another SV-102 combination formulation, the CpG ODN is the one of SEQ ID NO: 2 or SEQ ID NO: 27. Those skilled in the art will understand that derivatives or variants of the SV-102 components can be used.

[0257]

Table 5

[0258] Example 6: Preparation of SV-102 - Intermediate Dose In this example, the preparation of the SV-102 combination formulation will be described. In a sterile 20 mL vial, the following components are mixed and diluted to a volume of 15 mL in sterile physiological saline. In one SV-102 combination formulation, the CTLA4 antibody is the YH001 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 21-26 and / or the VH / VL sequences described in SEQ ID NOs: 34-35), the PD-1 antibody is the 609A antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 15-20 and / or the VH / VL sequences described in SEQ ID NOs: 32-33), the CD40 antibody is the YH003 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 3-8 and / or the VH / VL sequences described in SEQ ID NOs: 28-29), and the CpG ODN is the one of SEQ ID NO: 1. In another SV-102 combination formulation, the CpG ODN is the one of SEQ ID NO: 2 or SEQ ID NO: 27. Those skilled in the art will understand that derivatives or variants of the SV-102 components can be used.

[0259]

Table 6

[0260] Example 7: Preparation of SV-102 - High Dose In this example, the preparation of the SV-102 combination formulation will be described. In a sterile 20 mL vial, the following components are mixed and diluted to a volume of 15 mL in sterile physiological saline. In one SV-102 combination formulation, the CTLA4 antibody is the YH001 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 21-26 and / or the VH / VL sequences described in SEQ ID NOs: 34-35), the PD-1 antibody is the 609A antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 15-20 and / or the VH / VL sequences described in SEQ ID NOs: 32-33), the CD40 antibody is the YH003 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 3-8 and / or the VH / VL sequences described in SEQ ID NOs: 28-29), and the CpG ODN is the one of SEQ ID NO: 1. In another SV-102 combination formulation, the CpG ODN is the one of SEQ ID NO: 2 or SEQ ID NO: 27. Those skilled in the art will understand that derivatives or variants of the SV-102 components can be used.

[0261]

Table 7

[0262] Example 8: Preparation of SV-102 - Maximum Dose Level In this example, the preparation of the SV-102 combination formulation will be described. In a sterile 20 mL vial, the following components (from Table A or Table B) are mixed and diluted to a volume of 15 mL in sterile physiological saline. In one SV-102 combination formulation, the CTLA4 antibody is the YH001 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 21-26 and / or the VH / VL sequences described in SEQ ID NOs: 34-35), the PD-1 antibody is the 609A antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 15-20 and / or the VH / VL sequences described in SEQ ID NOs: 32-33), the CD40 antibody is the YH003 antibody (e.g., defined by the CDR sequences described in SEQ ID NOs: 3-8 and / or the VH / VL sequences described in SEQ ID NOs: 28-29), and the CpG ODN is the one of SEQ ID NO: 1. In another SV-102 combination formulation, the CpG ODN is the one of SEQ ID NO: 2 or SEQ ID NO: 27. Those skilled in the art will understand that derivatives or variants of the SV-102 components can be used.

[0263]

Table 8

[0264]

Table 9

[0265] Example 9: Treatment Plan and Patient Preparation In this example, exemplary patient screening and treatment criteria are described. As a preliminary step, detailed imaging examinations including, but not limited to, CT, PET / CT, and MRI are performed and reviewed for planning purposes to identify tumors that may be treatment targets. From the target tumor candidates, primary or metastatic tumors are selected for treatment, which are assumed to have an antigenic repertoire that at least partially represents the metastatic lesions. The selected tumor may be present in soft tissue, lymph nodes, or bone and must be readily accessible by a percutaneous approach under precise image guidance. The selected tumor is also of a volume and shape large enough (e.g., at least 2 cm or more in two measurement axes) to reliably contain the entire cryoablation partial tumor treatment zone within the tumor. The patient undergoes either general anesthesia or conscious sedation anesthesia and is fully monitored.

[0266] Example 10: Cryoablation Procedure This example describes an exemplary cryoablation procedure. The selected tumor site (e.g., the one selected in Example 9) is prepared for a percutaneous approach under precise image guidance using a coaxial cryoprobe / injection needle system (SCINS). The SCINS is inserted by standard percutaneous techniques under precise image guidance, and the tip of the SCINS is accurately positioned such that the volume of the resulting cryoablation treatment zone fits completely within the selected tumor. The length of the cryoablation zone using the SCINS can be visualized under imaging as the length of the cryoprobe exposed from the tip of the coaxial injection needle.

[0267] The cryoprobe is inserted into the tumor through the injection needle. After the position is confirmed, the injection needle is withdrawn, exposing 2.5 cm to 3 cm from the distal end of the cryoprobe. The cryoablation treatment is started by pressing a button on the cryo console. Thereby, the cryogenic fluid flowing through the probe begins to flow, and the cooling process starts. The cryoprobe is cooled so that the probe temperature reaches -40°C to -60°C in less than 1 minute. The cooling continues for a total of 2 minutes (during which the temperature of the cryoprobe could reach a maximum of about -120°C to about -150°C or lower). An ice ball with a diameter of about 14 mm is created. This ice ball can be confirmed by typical imaging methods (such as ultrasound (US), CT, PET / CT, etc.) implemented in the art. After 2 minutes, the flow of the cryogenic gas automatically stops (however, depending on the size of the lesion being treated, cooling may be performed for up to about 5 minutes or more). The ice ball is thawed passively (without heating with electricity or gas). The thawing and disappearance of the ice ball can be confirmed by typical imaging methods implemented in the art as discussed herein. When the ice ball has completely disappeared, the injection needle is advanced below the cryoprobe and inserted into the cryoablation treatment zone.

[0268] Example 11: Intratumoral injection and infusion of SV-101 or SV-102 In this example, an exemplary intratumoral administration of a combination formulation for immunotherapy is described. After a partial tumor cryoablation treatment zone is established in the selected tumor (e.g., as in Example 9 and / or Example 10), the injection needle (which is part of a coaxial cryoprobe / injection needle system (SCINS)) is advanced under precise image guidance, and the tip of the outer coaxial injection needle is placed approximately at the center of the cryoablation treatment zone.

[0269] When this is completed, the cryoprobe is withdrawn from the SCINS, leaving the tip of the outer coaxial injection needle still located approximately at the center of the cryoablation treatment zone within the tumor, and preparations for drug injection are made.

[0270] Prepare the pharmaceutical preparation SV-101 or SV-102 for injection at a final volume of 15 mL and place it in a single syringe suitable for use with a syringe infusion pump. Attach the syringe containing the drug to the pump and connect a low priming volume connecting tube (e.g., a MEDLINE 60-inch extension set with a microbore tube) to the syringe via a one-way stopcock. Advance the syringe pump to prime the system with the drug until it is seen that the drug emerges from the connecting tube. Next, attach the primed connecting tube to the SCINS injection needle. Set the infusion syringe pump to a flow rate of 3 mL / min. The overpressure alarm is not set. Next, start the pump and inject the total volume of 15 ml in 5 minutes.

[0271] Pay attention to ensure that the tip of the injection needle remains in the correct position and that the drug does not flow back along the needle tube. When the drug infusion pump completes its infusion cycle and 15 ml has been delivered from the syringe into the connecting tube, close the one-way stopcock. At this point, there is a residual volume of 0.6 ml of drug in the connecting tube. This volume of drug needs to be advanced by removing the one-way stopcock from the infusion syringe and injecting 0.6 mL of sterile saline into the one-way stopcock.

[0272] Example 12: Treatment of mCRPC patient #1 with SV-102 A male patient with metastatic prostate cancer confirmed by biopsy and bone scan had previously received six sessions of chemotherapy and hormonal blocking agents (enzalutamide, abiraterone, docetaxel), as well as radiation therapy for bone metastases. The patient did not show a response and the disease was progressing. On PET / CT, a prostate tumor and multiple SUV-positive osteosclerotic lesions were shown prior to treatment. Using the method provided by the present disclosure (see, for example, Examples 9-11), a first treatment of the prostate lesion was performed, including a first cycle of cryoablation and injection of SV-102 at a high dose level (see Example 7 and below). Two months after the first treatment, the MRI scan showed no evidence of active disease in the prostate (Figure 18).

[0273] The components of the SV-102 combination formulation included an anti-CTLA4 antibody (YH001; CDR sequences described in SEQ ID NOs: 21-26 and VH / VL sequences described in SEQ ID NOs: 34-35), an anti-PD-1 an...

Claims

1. A combination formulation for use in a method of treating cancer in a subject, The aforementioned combination formulation, a. TLR9 agonist, b. Agonist anti-CD40 monoclonal antibody, c. Anti-CTLA4 monoclonal antibody, and d. Containing an agonist anti-OX40 monoclonal antibody or an anti-PD1 monoclonal antibody, The method comprises the step of administering the combination formulation to the tumor in the subject before administering the combination formulation, wherein the combination formulation is subjected to a treatment that induces partial intratumor cytolysis.

2. The combination formulation according to claim 1, wherein the TLR9 agonist, the agonist-type anti-CD40 monoclonal antibody, the agonist-type anti-OX40 monoclonal antibody, and the anti-CTLA4 monoclonal antibody are co-administered or administered sequentially, or the TLR9 agonist is administered sequentially in a separate composition with a composition comprising the agonist-type anti-CD40 monoclonal antibody, the agonist-type anti-OX40 monoclonal antibody, and the anti-CTLA4 monoclonal antibody.

3. The combination formulation according to claim 1, wherein the TLR9 agonist, the agonist-type anti-CD40 monoclonal antibody, the anti-PD1 monoclonal antibody, and the anti-CTLA4 monoclonal antibody are co-administered or administered sequentially, or the TLR9 agonist is administered sequentially in a separate composition with a composition comprising the agonist-type anti-CD40 monoclonal antibody, the anti-PD1 monoclonal antibody, and the anti-CTLA4 monoclonal antibody.

4. The combination formulation according to any one of claims 1 to 3, wherein the treatment applied to the tumor is a cryolysis treatment that mediates partial intratumor cell lysis by cryolysis.

5. The combination formulation according to claim 4, wherein the cryo-thaw treatment is mediated by an intratumor cryo-thaw device comprising a cryoprobe for contacting and freezing tumor cells during the cryo-thaw cycle.

6. The combination formulation according to claim 5, wherein the cryoprobe is cooled to a temperature that creates an ice ball within the tumor.

7. The combination formulation according to claim 6, wherein the ice ball includes a region within the ice ball having a temperature of approximately -40°C or lower.

8. The combination formulation according to claim 4, wherein the cryolysis treatment causes partial intratumoral cell lysis rather than excision of the entire tumor.

9. The combination formulation according to claim 4, wherein the cryo-thaw treatment induces necrotic cell death in the tumor tissue zone.

10. The TLR9 agonist is a Class B or C CpG ODN, and / or The TLR9 agonist contains, or consists of, the nucleic acid sequence described in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27, and / or The agonist anti-CD40 monoclonal antibody comprises a heavy chain and a light chain. The heavy chain comprises a heavy chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 3, a heavy chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 4, and a heavy chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

5. The light chain comprises a light chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 6, a light chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 7, and / or a light chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO: 8, and / or The agonist anti-OX40 monoclonal antibody comprises a heavy chain and a light chain. The heavy chain comprises a heavy chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 9, a heavy chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 10, and a heavy chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

11. The light chain comprises a light chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 12, a light chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 13, and a light chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO: 14, and / or The anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, The heavy chain comprises a heavy chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 15, a heavy chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 16, and a heavy chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

17. The light chain comprises a light chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 18, a light chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO: 20, and / or The anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain. The heavy chain comprises a heavy chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 21, a heavy chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 22, and a heavy chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

23. The combination formulation according to any one of claims 1 to 3, wherein the light chain comprises a light chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 24, a light chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 25, and a light chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

26.

11. The combination formulation according to any one of claims 1 to 3, wherein the treatment that causes tumor cell lysis and the combination formulation are administered to the same tumor or to different tumors.

12. a. TLR9 agonist, b. Agonist anti-CD40 monoclonal antibody, c. Anti-CTLA4 monoclonal antibody, d. Agonist anti-OX40 monoclonal antibody, and e. A pharmaceutical composition comprising a pharmaceutically acceptable excipient, or comprising a. to e.

13. a. The TLR9 agonist is a nucleic acid described in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

27. b. The agonist anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, The heavy chain comprises a heavy chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 3, a heavy chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 4, and a heavy chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

5. The light chain comprises a light chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 6, a light chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 7, and a light chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

8. c. The agonist anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, The heavy chain comprises a heavy chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 9, a heavy chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 10, and a heavy chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

11. The light chain comprises a light chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 12, a light chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 13, and a light chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

14. d. The anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, The heavy chain comprises a heavy chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 21, a heavy chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 22, and a heavy chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

23. The pharmaceutical composition according to claim 12, wherein the light chain comprises a light chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 24, a light chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 25, and a light chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

26.

14. a. TLR9 agonist, b. Agonist anti-CD40 monoclonal antibody, c. Anti-CTLA4 monoclonal antibody, d. Agonist anti-PD1 monoclonal antibody, and e. A pharmaceutical composition comprising a pharmaceutically acceptable excipient, or comprising a. to e.

15. a. The TLR9 agonist is a nucleic acid described in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

27. b. The agonist anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, The heavy chain comprises a heavy chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 3, a heavy chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 4, and a heavy chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

5. The light chain comprises a light chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 6, a light chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 7, and a light chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

8. c. The anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, The heavy chain comprises a heavy chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 15, a heavy chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 16, and a heavy chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

17. The light chain comprises a light chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 18, a light chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

20. d. The anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, The heavy chain comprises a heavy chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 21, a heavy chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 22, and a heavy chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

23. The pharmaceutical composition according to claim 14, wherein the light chain comprises a light chain variable region CDR1 containing the amino acid sequence described in SEQ ID NO: 24, a light chain variable region CDR2 containing the amino acid sequence described in SEQ ID NO: 25, and a light chain variable region CDR3 containing the amino acid sequence described in SEQ ID NO:

26.

16. A composition comprising an immunotherapy agent for use in a method for tumor lysis treatment of cancer in a subject, wherein the use is a. Intratumoral cryolysis mediating partial tumor cell lysis within a tumor, wherein the composition is administered intratumorally to the tumor, and the immunotherapy agent promotes and enhances the immunogenic antitumor response following the cryolysis-mediated partial tumor cell lysis. Here, the immunotherapy agent is i. Potent activation of the innate immune sector of the immune response to mediate local antitumor effects; ii. Activation of the adaptive immune sector of the antitumor immune response through the recruitment, activation, and maturation of antigen-presenting cells (APCs); iii. Increased activation, proliferation, and survival rate of T cells; iv. Reduction of the immunosuppressive effect of regulatory T cells; and / or v. A composition that at least mediates the recovery and prevention of T cell exhaustion caused by factors that govern the suppressive tumor microenvironment.

17. A system for treating cancer, An intratumoral cryolysis device configured to induce partial necrotic cell death in a zone of tumor tissue; and A system comprising an intratumor injection device configured to deliver the immunotherapy agent according to claim 16 to tumor tissue at a site of partial necrotic cell death.

18. A combination formulation for use in tumor lysis treatment for cancer, It contains, in a colocalized manner, at least three components necessary to induce a systemic T cell response in mammals, and the at least three components are i. Release of tumor antigens by intratumoral cryo-thawing sufficient to induce necrotic tumor cell death in a sufficient number of tumor cells, up to the majority of cells in the tumor, at the cryo-thawing site in the tumor, thereby maintaining drainage lymphatic vessels in the most optimal condition possible at the cryo-thawing site; ii. A composition of at least four immunomodulators sufficient to induce the systemic T cell response, wherein the composition is provided intratumorally at the cryothreshing site in the tumor, and is provided in a dose, volume, and rate calculated to minimize adverse events when systemically administered at a dose, volume, and rate sufficient to be effective at the cryothreshing site and to produce an antitumor effect; and iii. A method comprising synchronized release of the antigen and immune cells activated by administration of the at least tetravalent composition delivered via the lymphatic vessels.

19. A system for treating cancer via partial tumor cell lysis and intratumoral administration of a combination of immunotherapy components, An intratumor cell lysis device configured to induce partial cell lysis in a zone of tumor tissue, wherein only a subset of the tumor cells are lysed by the cell lysis; A system comprising: an intratumor injection device configured to deliver an immunotherapy agent to tumor tissue within a zone of the tumor tissue.

20. The intratumor cell lysis device and the intratumor injection device are coaxial, and the immunotherapy agent is a. TLR9 agonist, b. Agonist anti-CD40 monoclonal antibody, c. Anti-CTLA4 monoclonal antibody, and d. The system according to claim 19, comprising one or more agonist anti-OX40 monoclonal antibodies or anti-PD1 monoclonal antibodies.

21. The system according to claim 20, wherein the intratumor injection device is configured to deliver the immunotherapy agent at a predetermined flow rate in the range of about 1 mL / min to about 5 mL / min and in a fixed volume in the range of about 1 mL to about 30 mL.

22. The system according to claim 19, wherein the intratumor cell lysis device is a cryoprobe having a distal end designed to contact at least a portion of the tumor tissue within a zone of the tumor tissue.

23. The system according to claim 22, further comprising a console for communicating with the cryoprobe, wherein the console is configured to cool the distal end of the cryoprobe for a period of at least one freeze-thaw cycle of about 1 minute to about 5 minutes and a duty cycle of about 70% to about 100%.

24. The system according to claim 23, wherein contact of the distal end of the cryoprobe with at least a portion of the tumor tissue creates an ice ball within the tumor, the temperature within the ice ball being about -40°C to -150°C.

25. The system according to claim 24, wherein the intratumor injection device is inserted into the zone of tumor tissue after the ice ball has completely disappeared, and the intratumor cell lysis device and the intratumor injection device are arranged coaxially with each other in a coaxial cryoprobe injection needle system (SCINS) system, the SCINS system includes an injection needle that houses the intratumor cell lysis device.

26. The system according to claim 25, wherein the injection needle is at least partially retractable so as to expose the distal end of the intratumor cell lysis device over a range of about 2.5 cm to about 3 cm, and the intratumor injection device includes a syringe connected to a syringe pump.

27. ​​A method for using a therapeutically effective combination formulation in the manufacture of a pharmaceutical product for use in a method of treating cancer in a subject, The combination formulation comprises at least a quadrivalent composition of an immunomodulator sufficient to induce a systemic T cell response, the systemic T cell response being activated by synchronized release of a tumor antigen and administration of at least the quadrivalent composition. The aforementioned use is, a) A step of administering a treatment to the tumor in the subject to induce partial tumor cell lysis, and b) A method of use comprising the step of administering a therapeutically effective amount of the combination formulation to the subject at the site of partial tumor cell lysis.

28. The at least tetravalent composition of the immunotherapy agent, i. At least one compound capable of potently activating the innate immune sector of the immune response to mediate a local antitumor effect; ii. At least one compound that mobilizes antigen-presenting cells (APCs) and, by activation and maturation of APCs, results in activation of the adaptive immune sector of the antitumor immune response; iii. At least one compound that results in increased activation, proliferation, and viability of T cells; and iv. The method of use according to claim 27, comprising at least one compound that reduces the immunosuppressive effect of regulatory T cells; and / or at least one compound that restores and prevents T cell exhaustion caused by factors that govern the suppressive tumor microenvironment.

29. i. A potent activator of the innate immune sector of the immune response for mediating a local antitumor effect; ii. Activators of the adaptive immune sector of the antitumor immune response through the recruitment of antigen-presenting cells (APCs), and the activation and maturation of APCs; iii. Activating substances for increasing the proliferation and survival rate of T cells; and iv. Substances that inhibit the immunosuppressive effect of regulatory T cells; or substances that restore and prevent T cell exhaustion caused by factors that control the suppressive tumor microenvironment. A pharmaceutical composition consisting of a substantial amount.

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