Methods of treating cancer via combination of tumor cell lysis and intratumoral administration of immunotherapeutic components

Abstract

The present disclosure provides, inter alia, a method of cancer treatment comprising the steps of: a) intratumoral cell lysis mediated by cryolysis; and b) administering in the tumor 1) a combination of immunotherapeutic agents comprising i) a TLR9 agonist CpG oligodeoxydinucleotide, ii) an agonistic anti-CD40 monoclonal antibody, iii) an agonistic anti-OX40 monoclonal antibody, and iv) an anti-CTLA4 monoclonal antibody; or 2) a combination of immunotherapeutic agents comprising i) a TLR9 agonist CpG oligodeoxydinucleotide, ii) an agonistic anti-CD40 monoclonal antibody, iii) an anti-PD1 monoclonal antibody, and iv) an anti-CTLA4 monoclonal antibody.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 347,690, filed on June 1, 2022, which is incorporated herein by reference in its entirety for all purposes. Background Art

[0003] Current clinical approaches to cancer immunotherapy rely on systemic administration of high doses of one or two biological immunomodulatory agents. These agents are designed to reverse the predetermined conditions of tumor-induced immunosuppression while stimulating adaptive immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs).

[0004] Over the past decade, significant advances have been made in cancer treatment 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 considerable benefits obtained using these mAbs, a significant proportion of patients still do not respond to treatment or become refractory to therapy. In addition, the use of high systemic doses of these checkpoint inhibitors is accompanied by an extremely frequent occurrence of serious adverse events, especially when used in combination with other immunotherapeutic agents, which limits the number and dose of agents that can be used in combination to address the multifactorial nature of tumor immune resistance (Vilgelm, Johnson et al., 2016). New cancer immunotherapy approaches are needed in the art. Summary of the invention

[0005] Current cancer immunotherapy approaches only provide benefit to a limited subset of patients, and their use typically results in only partial responses of limited duration. In addition, current cancer immunotherapy approaches are associated with treatment-acquired resistance and a high percentage of immune adverse events, which limits their compliance and applicability. The present disclosure describes methods and compositions for treating cancer that overcome many of the limitations of current cancer immunotherapy approaches.

[0006] In some embodiments, the immunotherapy methods disclosed herein are not antigen-specific and do not require prior antigen identification. In many embodiments, the methods disclosed herein include a combination of different pharmacological agents that mediate different and synergistic anti-tumor immune activation effects, thereby improving efficacy and reducing the possibility of immune-acquired resistance compared to therapies that rely on a single therapeutic agent. In addition, the use of intratumoral routes to administer the compositions disclosed herein also mediate local regional treatments, thereby increasing drug accessibility and the biodistribution of drugs in immune-related tumor-associated immune cells and related secondary and tertiary lymphoid tissues. In addition, the intratumoral administration methods described herein allow for a significant reduction in doses, thereby reducing the possibility of toxicity and adverse events. This can increase the number of therapeutic agents in the drug combination disclosed herein, thereby increasing the breadth of pharmacological effects to improve anti-tumor efficacy. The increase in the therapeutic window achieved by using a lower dose and the increase in the number of therapeutic agents with a synergistic mechanism will improve the therapeutic efficacy while reducing the frequency and severity of adverse events. Therefore, the present disclosure particularly provides a cancer treatment method that overcomes the limitations of current cancer immunotherapy options.

[0007] The present disclosure further provides combinations and compositions of immunotherapeutic agents that mediate at least (1) robust activation of innate immune responses, thereby mediating local anti-tumor effects; (2) activation of adaptive anti-tumor immune responses achieved through antigen presenting cell recruitment, APC activation and maturation; (3) increased activation, proliferation and survival of T cells; (4) reduction of the immunosuppressive effects of regulatory T cells; and / or (5) reversal and prevention of T cell exhaustion achieved by factors that dominate the suppressive tumor microenvironment.

[0008] The present disclosure also provides dosages of various immunotherapeutic agents present in the compositions and formulations described herein. Indeed, the present disclosure provides, inter alia, compositions for treating cancer that overcome the limitations of current cancer immunotherapy options.

[0009] For example, in some embodiments, the present disclosure provides a method for treating cancer, wherein the method comprises: a first step, which is intended to trigger in situ immunity by using an intratumoral cryolysis device to trigger lysis and immunogenic cell death of tumor cells; a second step, which comprises intratumoral administration of a combination of immunotherapeutic agents, the immunotherapeutic agents mediating different pharmacological effects. In some embodiments, the provided immunotherapeutic combination mediates a synergistic pharmacological effect when administered to a subject.

[0010] In addition, in some embodiments, the present disclosure provides methods and compositions, which include immunotherapy combinations of active ingredients administered by intratumoral administration. In some embodiments, the composition or combined preparation comprises a TLR9 agonist, an anti-CD40 agonist monoclonal antibody, an anti-OX40 agonist monoclonal antibody, and an anti-CTLA4 monoclonal antibody. In some embodiments, the composition or combined preparation consists of a TLR9 agonist, an anti-CD40 agonist monoclonal antibody, an anti-OX40 agonist monoclonal antibody, and an anti-CTLA4 monoclonal antibody. In some embodiments, the composition or combined preparation comprises a TLR9 agonist, an anti-CD40 agonist monoclonal antibody, an anti-PD1 monoclonal antibody, and an anti-CTLA4 monoclonal antibody. In some embodiments, the composition or combined preparation consists of a TLR9 agonist, an anti-CD40 agonist monoclonal antibody, an anti-PD1 monoclonal antibody, and an anti-CTLA4 monoclonal antibody.

[0011] Furthermore, in some embodiments, the present disclosure provides dosage ranges for each active ingredient in the various pharmaceutical formulation combinations disclosed herein.

[0012] The present disclosure provides a method for treating cancer in a subject, the method comprising the steps of: a) administering a therapy that causes tumor cell lysis to the subject's tumor; and b) administering a therapeutically effective amount of a combined preparation to the subject, wherein the combined preparation comprises: 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.

[0013] In some embodiments, the combination preparation 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.

[0014] In some embodiments, the combination preparation 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.

[0015] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 27; an agonist 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 shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8; an agonist 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 shown in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11. NO:11, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:12, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0016] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:9, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:10, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:11, and wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:12, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:13, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:14. NO:12, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0017] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:9, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:10, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:11, and wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:12, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:13, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:14. NO:12, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0018] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:9, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:10, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:11, and wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:12, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:13, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:14. NO:12, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0019] In some embodiments, the combination preparation comprises: a TLR9 agonist as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonist 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 agonist 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: NO:30 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, 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: NO:35 has an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0020] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:28, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:30, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:34, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35.

[0021] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:28, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:30, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:34, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35.

[0022] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:28, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:30, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:34, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35.

[0023] In some embodiments, the combination preparation 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.

[0024] In some embodiments, the combined preparation 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.

[0025] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 27; an agonist 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 shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9. NO:17, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:18, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:19, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0026] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18. NO:18, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:19, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0027] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown in SEQ ID NO:2; an agonist 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 shown in SEQ ID NO:3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:17, and wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:18 NO:18, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:19, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0028] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown in SEQ ID NO:27; an agonist 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 shown in SEQ ID NO:3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:17, and wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:18 NO:18, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:19, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0029] In some embodiments, the combination preparation comprises: a TLR9 agonist as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonist 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: NO:32 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, 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: NO:35 has an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0030] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:28, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:32, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:34, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35.

[0031] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:28, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:32, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:34, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35.

[0032] In some embodiments, the combination preparation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:28, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:32, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:34, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35.

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

[0034] 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.

[0035] In some embodiments, the TLR9 agonist is administered as a separate composition sequentially with the composition comprising an agonistic anti-CD40 monoclonal antibody, an agonistic anti-OX40 monoclonal antibody, and an anti-CTLA4 monoclonal antibody.

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

[0037] 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.

[0038] In some embodiments, the TLR9 agonist is administered as a separate composition sequentially with the composition comprising an agonistic anti-CD40 monoclonal antibody, an anti-PD1 monoclonal antibody, and an anti-CTLA4 monoclonal antibody.

[0039] In some embodiments, the therapy administered to the tumor is cryolysis therapy by cryolysis-mediated lysis of tumor cells.

[0040] In some embodiments, the administration of cryolysis therapy comprises at least one, at least two, or at least three freeze-thaw cycles. In some embodiments, the administration of cryolysis therapy comprises one freeze-thaw cycle. In some embodiments, the administration of cryolysis therapy comprises two freeze-thaw cycles. In some embodiments, the administration of cryolysis therapy comprises three freeze-thaw cycles.

[0041] In some embodiments, the administration of cryolysis therapy includes no more than one, no more than two, or no more than three freeze-thaw cycles. In some embodiments, the administration of cryolysis therapy includes only one freeze-thaw cycle. In some embodiments, the administration of cryolysis therapy includes only two freeze-thaw cycles. In some embodiments, the administration of cryolysis therapy includes only three freeze-thaw cycles.

[0042] In some embodiments, cryolysis therapy is mediated by an intratumoral cryolysis device comprising a cryoprobe for contacting and freezing tumor cells during freeze-thaw cycles. In some embodiments, a cryogen circulates within the cryoprobe. In some embodiments, the cryogen is selected from the group consisting of argon, nitrous oxide, carbon dioxide, and liquid nitrogen.

[0043] In some embodiments, the intratumoral cryolysis device is set to a duty cycle of about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% and operated during a freeze-thaw cycle to produce a cryoprobe temperature of about -40°C or colder, about -45°C or colder, about -50°C or colder, about -55°C or colder, about -60°C or colder, about -65°C or colder, about -70°C or colder, about -75°C or colder, about -80°C or colder, about -85°C or colder, about -90°C or colder, about -100°C or colder, about -110°C or colder, or about -120°C or colder in less than about one minute, followed by a passive thawing step. In some embodiments, the intratumoral cryolysis device is set to a duty cycle of about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% and operated to produce a cryoprobe temperature of about -40°C or colder, about -45°C or colder, about -50°C or colder, about -55°C or colder, about -60°C or colder, about -65°C or colder, about -70°C or colder, about -75°C or colder, about -80°C or colder, about -85°C or colder, about -90°C or colder, about -100°C or colder, about -110°C or colder, or about -120°C or colder in less than about 30 seconds during a freeze-thaw cycle, followed by a passive thawing step. In some embodiments, the intratumoral cryolysis device is set to a duty cycle of about 70% to about 100% and is operated during the freeze-thaw cycle to produce a cryoprobe temperature of about -40°C to about -60°C in less than about one minute, followed by a passive thawing step. In some embodiments, the intratumoral cryolysis device is set to a duty cycle of about 70% to about 100% and is operated during the freeze-thaw cycle to produce a cryoprobe temperature of about -40°C to about -60°C in less than about 30 seconds, followed by a passive thawing step. In some embodiments, the intratumoral cryolysis device is set to a duty cycle of 100% and is operated during the freeze-thaw cycle to produce a cryoprobe temperature of about -40°C to colder in less than about one minute, followed by a passive thawing step. In some embodiments, the cryoprobe temperature produced is maintained for about one minute before passive thawing. In some embodiments, the cryoprobe temperature produced is maintained for about two minutes before passive thawing. In some embodiments, the cryoprobe temperature produced is maintained for about three minutes before passive thawing.In some embodiments, the resulting cryoprobe temperature continues to cool to a temperature of about -70°C or colder, about -80°C or colder, about -90°C or colder, about -100°C or colder, about -110°C or colder, about -120°C or colder, about -130°C or colder, about -140°C or colder, or about -150°C or colder for a total time 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.

[0044] In some embodiments, the cryoprobe is cooled to a temperature to create an ice ball within the tumor 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. In some embodiments, the cryoprobe is cooled to a temperature to create an ice ball within the tumor having a diameter of about 12 mm to about 16 mm. In some embodiments, the cryoprobe is cooled to a temperature to create an ice ball within the tumor having a diameter of about 14 mm.

[0045] In some embodiments, the temperature of most of the ice balls is about -40°C. In some embodiments, the temperature of most of the ice balls is about -50°C. In some embodiments, the temperature of most of the ice balls is about -60°C. In some embodiments, the temperature of most of the ice balls is about -40°C or colder. In some embodiments, the temperature of most of the ice balls is about -50°C or colder. In some embodiments, the temperature of most of the ice balls is about -60°C or colder. In some embodiments, the temperature of most of the ice balls is about -40°C to about -60°C.

[0046] In some embodiments, the ice ball contains an area of ​​about 1 cm (10 mm) diameter, and the temperature within the area is about -40°C. In some embodiments, the ice ball contains an area of ​​about 1 cm diameter, and the temperature within the area is about -50°C. In some embodiments, the ice ball contains an area of ​​about 1 cm diameter, and the temperature within the area is about -60°C. In some embodiments, the ice ball contains an area of ​​about 1 cm diameter, and the temperature within the area is about -40°C or colder. In some embodiments, the ice ball contains an area of ​​about 1 cm diameter, and the temperature within the area is about -50°C or colder. In some embodiments, the ice ball contains an area of ​​about 1 cm diameter, and the temperature within the area is about -60°C or colder. In some embodiments, the ice ball contains an area of ​​about 0.5 cm to about 1.5 cm diameter, and the temperature within the area is about -40°C. In some embodiments, the ice ball contains an area of ​​about 0.5 cm to about 1.5 cm diameter, and the temperature within the area is about -50°C. In some embodiments, the ice ball comprises an area of ​​about 0.5 cm to about 1.5 cm diameter, and the temperature within the area is about -60°C. In some embodiments, the ice ball comprises an area of ​​about 0.5 cm to about 1.5 cm diameter, and the temperature within the area is about -40°C or colder. In some embodiments, the ice ball comprises an area of ​​about 0.5 cm to about 1.5 cm diameter, and the temperature within the area is about -50°C or colder. In some embodiments, the ice ball comprises an area of ​​about 0.5 cm to about 1.5 cm diameter, and the temperature within the area is about -60°C or colder.

[0047] In some embodiments, cryolysis therapy does not ablate the entire tumor, but causes lysis of some tumor cells.

[0048] In some embodiments, cryolipolysis therapy causes necrotic cell death in a region of tumor tissue, wherein the diameter of the region is about 14 mm. In some embodiments, cryolipolysis therapy causes necrotic cell death in a region of tumor tissue, wherein the region is 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.

[0049] In some embodiments, the TLR9 agonist is a CpG ODN of class B or class C. In some embodiments, the TLR9 agonist is a CpG ODN of class B. In some embodiments, the TLR9 agonist is a CpG ODN of class C.

[0050] In some embodiments, the TLR9 agonist comprises a nucleic acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 1. In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 2. In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 27.

[0051] In some embodiments, the agonist 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 shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8. In some embodiments of the present disclosure, the agonist anti-CD40 monoclonal antibody is of human IgG2k isotype.

[0052] In some embodiments of the present disclosure, an agonist anti-CD40 monoclonal antibody comprises a heavy 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. In some embodiments of the present disclosure, an agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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, an agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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, an agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28.

[0053] In some embodiments of the present disclosure, the agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 29.

[0054] In some embodiments of the present disclosure, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist anti-CD40 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: 28, 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, an agonist anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 29.

[0055] In some embodiments, the agonist anti-OX40 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 shown in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 14. In some embodiments of the present disclosure, the agonist anti-OX40 monoclonal antibody is of human IgG1k isotype.

[0056] In some embodiments of the present disclosure, an agonist anti-OX40 monoclonal antibody comprises a heavy 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. In some embodiments of the present disclosure, an agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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, an agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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, an agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 30.

[0057] In some embodiments of the present disclosure, an agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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, an agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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, an agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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, an agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 31.

[0058] In some embodiments of the present disclosure, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist anti-OX40 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: 30, 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, an agonist anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 30, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 31.

[0059] In some embodiments, the anti-PD1 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 shown in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 20. In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody is of human IgG4k isotype.

[0060] In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a heavy 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. In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein 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, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein 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, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein 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, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein 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, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 32. In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 32. In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 32.

[0061] In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a light chain, wherein 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, an anti-PD1 monoclonal antibody comprises a light chain, wherein 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, an anti-PD1 monoclonal antibody comprises a light chain, wherein 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, an anti-PD1 monoclonal antibody comprises a light chain, wherein 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, an anti-PD1 monoclonal antibody comprises a light chain, wherein 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, wherein 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, wherein 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, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 33.

[0062] In some embodiments of the present disclosure, an 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, an 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, an 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 with SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 33. In some embodiments of the present disclosure, an 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 with SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 96% sequence identity with SEQ ID NO: 33. In some embodiments of the present disclosure, an 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 with SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 33. In some embodiments of the present disclosure, an 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 with SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 33. In some embodiments of the present disclosure, an 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 with SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 33. In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 32, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 33.

[0063] In some embodiments, the anti-CTLA4 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0064] In some embodiments of the present disclosure, an anti-CTLA4 monoclonal antibody comprises a heavy 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. In some embodiments of the present disclosure, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 34.

[0065] In some embodiments of the present disclosure, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 35.

[0066] In some embodiments of the present disclosure, an 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, an 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, an 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, an 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, an 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, an 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, an anti-CTLA4 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: 34, 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 35.

[0067] In some embodiments, the therapy that causes tumor cell lysis and the combination formulation are administered to the same tumor. In some embodiments, the therapy that causes tumor cell lysis and the combination formulation are administered to different tumors.

[0068] In some embodiments, the treatment is repeated about every week, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, about every 9 weeks, about every 10 weeks, about every 11 weeks, about every 12 weeks, about every 13 weeks, about every 14 weeks, about every 15 weeks, or about every 16 weeks. In some embodiments, the treatment is repeated about every 3 to 16 weeks, about every 6 to 16 weeks, about every 8 to 16 weeks, about every 10 to 16 weeks, about every 12 to 16 weeks, about every 14 to 16 weeks, about every 3 to 14 weeks, about every 3 to 12 weeks, about every 3 to 10 weeks, about every 3 to 8 weeks, about every 3 to 6 weeks, or about every 3 to 4 weeks. In some embodiments, the treatment is repeated about every 4 to 8 weeks, about every 5 to 8 weeks, about every 6 to 8 weeks, about every 7 to 8 weeks, about every 4 to 7 weeks, about every 4 to 6 weeks, or about every 4 to 5 weeks. In some embodiments, the treatment is repeated about every 4 to 8 weeks.

[0069] 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.

[0070] In some embodiments, the TLR9 agonist is administered at a dose ranging from 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.

[0071] In some embodiments, the anti-CD40 agonist is administered at a dose ranging from 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 about 8 mg.

[0072] In some embodiments, the anti-OX40 agonist is administered at a dose ranging from 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 about 8 mg.

[0073] In some embodiments, the anti-PD1 monoclonal antibody is administered at a dose within 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.

[0074] In some embodiments, the anti-CTLA4 monoclonal antibody is administered at a dose ranging from 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.

[0075] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-OX40 agonist antibody, and anti-CTLA4 antibody are administered at doses of about 1 mg, about 1 mg, about 1 mg, and about 1 mg, respectively.

[0076] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-OX40 agonist antibody, and anti-CTLA4 antibody are administered at doses of about 2 mg, about 5 mg, about 5 mg, and about 5 mg, respectively.

[0077] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-OX40 agonist antibody, and anti-CTLA4 antibody are administered at doses of about 3 mg, about 7.5 mg, about 7.5 mg, and about 15 mg, respectively.

[0078] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-OX40 agonist antibody, and anti-CTLA4 antibody are administered at doses of about 4 mg, about 10 mg, about 10 mg, and about 40 mg, respectively.

[0079] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody, and anti-CTLA4 antibody are administered at doses of about 1 mg, about 1 mg, about 3 mg, and about 1 mg, respectively.

[0080] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody, and anti-CTLA4 antibody are administered at doses of about 2 mg, about 5 mg, about 10 mg, and about 5 mg, respectively.

[0081] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody, and anti-CTLA4 antibody are administered at doses of about 3 mg, about 7.5 mg, about 30 mg, and about 15 mg, respectively.

[0082] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody, and anti-CTLA4 antibody are administered at doses of about 4 mg, about 10 mg, about 80 mg, and about 40 mg, respectively.

[0083] In some embodiments, the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody, and anti-CTLA4 antibody are administered at doses of about 4 mg, about 10 mg, about 100 mg, and about 40 mg, respectively.

[0084] In some embodiments, the combined formulation is applied in a total volume within 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 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 combined formulation is applied in a total volume within the range of about 1 mL to about 30 mL. In some embodiments, the total volume is about 15 mL.

[0085] In some embodiments, cancer is a solid tumor cancer. In some embodiments, 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 carcinoma, retinoblastoma, sarcoma, small cell lung cancer, squamous cell carcinoma (including head and neck cancer), gastric cancer, testicular cancer, thyroid cancer and Wilms tumor (Wilms tumor). In some embodiments, cancer is metastatic prostate cancer. In some embodiments, cancer is metastatic breast cancer. In some embodiments, cancer is non-small cell lung cancer.

[0086] The present disclosure provides a pharmaceutical composition, which comprises: a. TLR9 agonist; b. agonistic anti-CD40 monoclonal antibody; c. anti-CTLA4 monoclonal antibody; d. agonistic anti-OX40 monoclonal antibody; and e. pharmaceutically acceptable excipients. The present disclosure also provides a pharmaceutical composition, which consists of the following: a. TLR9 agonist; b. agonistic anti-CD40 monoclonal antibody; c. anti-CTLA4 monoclonal antibody; d. agonistic anti-OX40 monoclonal antibody; and e. pharmaceutically acceptable excipients.

[0087] In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the agonist 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 shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the agonist anti-OX40 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 shown in SEQ ID NO:9, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:10, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:11, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:12, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:14. In some embodiments, the anti-CTLA4 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0088] In some embodiments, the TLR9 agonist is a nucleic acid as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the agonist 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 agonist 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, an 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.

[0089] In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, an agonist anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 28, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 29. In some embodiments, an agonist anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 30, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 31. In some embodiments, an anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 34, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 35.

[0090] In some embodiments, the dose of each of the TLR9 agonist, anti-CD40 agonist antibody, anti-OX40 agonist antibody, and anti-CTLA4 antibody is selected from: a. about 1 mg, about 1 mg, about 1 mg, and about 1 mg, respectively; b. about 2 mg, about 5 mg, about 5 mg, and about 5 mg, respectively; c. about 3 mg, about 7.5 mg, about 7.5 mg, and about 15 mg, respectively; and d. about 4 mg, about 10 mg, about 10 mg, and about 40 mg, respectively.

[0091] The present disclosure provides a pharmaceutical composition, which comprises: a. TLR9 agonist; b. agonistic anti-CD40 monoclonal antibody; c. anti-CTLA4 monoclonal antibody; d. agonistic anti-PD1 monoclonal antibody; and e. pharmaceutically acceptable excipients. The present disclosure also provides a pharmaceutical composition, which consists of the following: a. TLR9 agonist; b. agonistic anti-CD40 monoclonal antibody; c. anti-CTLA4 monoclonal antibody; d. agonistic anti-PD1 monoclonal antibody; and e. pharmaceutically acceptable excipients.

[0092] In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the agonist 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 shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the anti-PD1 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 shown in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the anti-CTLA4 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0093] In some embodiments, the TLR9 agonist is a nucleic acid as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the agonist 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, an 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.

[0094] In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the agonist anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 28, and the light chain comprises the amino acid sequence as shown in 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 the amino acid sequence as shown in SEQ ID NO: 32, and the light chain comprises the amino acid sequence as shown in 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 the amino acid sequence as shown in SEQ ID NO: 34, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 35.

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

[0096] In some embodiments, the dose of each of the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody and anti-CTLA4 antibody is selected from: a. about 1 mg, about 1 mg, about 3 mg and about 1 mg, respectively; b. about 2 mg, about 5 mg, about 10 mg and about 5 mg, respectively; c. about 3 mg, about 7.5 mg, about 30 mg and about 15 mg, respectively; d. about 4 mg, about 10 mg, about 80 mg and about 40 mg, respectively; and e. about 4 mg, about 10 mg, about 100 mg and about 40 mg, respectively.

[0097] The present disclosure provides, inter alia, a method for treating cancer in a subject, the method comprising the steps of: a) administering cryolysis therapy to a tumor of the subject to cause partial tumor cell lysis; and b) administering a therapeutically effective amount of a combination formulation to the same tumor in step a), wherein the combination formulation comprises: 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 cryolysis therapy is administered prior to administering the combination formulation.

[0098] The present disclosure provides, inter alia, a method for treating cancer in a subject, the method comprising the steps of: a) administering cryolysis therapy to a tumor of the subject to cause partial tumor cell lysis; and b) administering a therapeutically effective amount of a combination formulation to the same tumor in step a), wherein the combination formulation comprises: 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 cryolysis therapy is administered prior to administering the combination formulation.

[0099] In some embodiments, the combined preparation is any pharmaceutical composition described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 Shown is a representation of the mechanism of action of CpG oligodeoxynucleotides as TLR9 agonists on human B cells and plasmacytoid dendritic cells.

[0102] Figure 2 A representation of the mechanism of action of agonistic anti-CD40 monoclonal antibodies on B cells, dendritic cells, and macrophages is shown.

[0103] Figure 3 Representation showing the mechanism of action of a blocking anti-CTLA4 monoclonal antibody

[0104] Figure 4 Representation of the mechanism of action of agonistic anti-OX40 monoclonal antibodies

[0105] Figure 5 Representation showing the mechanism of action of blocking anti-PD1 monoclonal antibodies

[0106] Figure 6 A representation of the three immune states of the tumor is shown along with a schematic diagram of partial cryolysis within the tumor and the formation of ice balls smaller than the tumor.

[0107] Figure 7 A schematic diagram of intratumoral injection and infusion of the therapeutic component of SV-101 into an area previously treated by partial cryolysis is shown.

[0108] Figure 8 A schematic diagram of intratumoral injection and infusion of the therapeutic component of SV-102 into an area previously treated by partial cryolysis is shown.

[0109] Fig. 9 A representation of the proposed mechanism of action for the synergistic activation of macrophages mediated by simultaneous treatment with anti-CD40 agonistic mAb and TLR9 agonist is shown.

[0110] Fig.10A representation of the proposed mechanism of action for the synergistic activation of B cells mediated by simultaneous treatment with anti-CD40 agonistic mAb and TLR9 agonist is shown.

[0111] Fig.11 Shown is a representation of the proposed mechanism of action for the synergistic activation of dendritic cells (eg, pDCs and cDCs) mediated by simultaneous treatment with anti-CD40 agonistic mAb and TLR9 agonist.

[0112] Fig.12 A representation of the proposed mechanism of action for synergistic activation of CD4+ and CD8+ T cells by dendritic cells and suppression of Treg function in TDLN, TME, and TLS mediated by combination therapy with anti-CTLA4 mAb, anti-CD40 agonistic mAb, and anti-OX40 agonistic mAb is shown.

[0113] Fig.13 Representation of the proposed mechanism of action for synergistic activation of CD4+ and CD8+ T cells by dendritic cells and suppression of Treg function in TDLN, TME, and TLS mediated by combination therapy with anti-CTLA4 mAb, anti-CD40 agonistic mAb, and anti-PD1 mAb is shown.

[0114] Fig.14 A representation of the proposed mechanism of action for the synergistic inhibition of Treg function mediated by the combination of anti-OX40 mAb and anti-CTLA4 mAb in the TME is shown.

[0115] Fig.15 A representation of the proposed mechanism of action for the synergistic reactivation of Teff cells in the TME by the combined action of anti-CD40 agonistic mAb and anti-PD1 mAb is shown.

[0116] Fig.16 A representation of the proposed mechanism of action for the synergistic inhibition of Treg function and reactivation of exhausted Teff cells in the TME mediated by the combination of anti-PD1 mAb and anti-CTLA4 mAb is shown.

[0117] Fig.17 A representation of the isotherms around the cryoprobe during the freeze-thaw process is shown. The temperatures within the freeze-thaw region represented are -40°C to -60°C.

[0118] FIG18 shows a multiparametric MRI with a diffusion-weighted sequence showing no evidence of residual tumor in the prostate (Panels AC).

[0119] Fig.19Coronal images showing lung windows from CT scans performed on treatment days 1, 28, and 56. The right pleural effusion has resolved to an almost undetectable level, and the combined diameter of the lesions within the lung parenchyma has decreased by 40%, indicating a partial response. DETAILED DESCRIPTION

[0120] definition

[0121] Administration: As used herein, the term "administering" refers to administering a therapy (e.g., a composition / formulation or other form of treatment, such as cryotherapy) to a subject or system. Administration to an animal subject (e.g., a human) can be by any appropriate route. For example, in some embodiments, administration can be intratumoral administration. In some embodiments, administration is performed by infusion or perfusion (e.g., using the methods described herein). In some embodiments, administration is intratumoral administration, intended for local regional treatment, in which the injected formulation is not expected to be completely contained within the tumor tissue, but is actually expected to enter the lymphatic structures connected to the tumor and the peritumoral tissue via the lymphatic vessels.

[0122] Agent: The term "agent" as used herein may refer to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations thereof. In some embodiments, an agent is or comprises one or more artificial entities, i.e., it is artificially designed, engineered, and / or produced, and / or does not exist in nature. In some embodiments, the agent used may be in an isolated or purified form; in some embodiments, the agent used may be in a crude form. Some specific embodiments of agents that can be utilized according to 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 causes a desired pharmacological effect when administered to an organism (e.g., a subject). In some embodiments, the agent that causes a desired pharmacological effect when administered to an organism may be considered "active" or "pharmacologically active". In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect in an appropriate population. In some embodiments, the appropriate colony can be a model organism colony. In some embodiments, the appropriate colony can be defined by various criteria, such as a specific age group, sex, genetic background, pre-existing clinical conditions, etc. In some embodiments, the therapeutic agent is a substance that can be used to alleviate, improve, alleviate, inhibit, prevent one or more symptoms or features of a disease, disorder and / or illness, delay its onset, alleviate its severity and / or reduce its incidence. In some embodiments, the agent is a component in a combined preparation or composition. In some embodiments, a "therapeutic agent" is an agent that has been obtained or needs to be approved by a government agency to be listed for human administration. In some embodiments, a "therapeutic agent" is an agent that requires a doctor's prescription to be administered to a person.

[0123] Antibody: The term "antibody" as used herein may include whole antibodies and any antigen binding fragments thereof (i.e., "antigen binding portion") or single chains. In some embodiments, "antibody" refers to a glycoprotein comprising at least two heavy chains and two light chains connected by disulfide bonds, or an antigen binding fragment thereof. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. In some naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region comprises three domains, namely CH1, CH2 and CH3. In some naturally occurring antibodies, each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. In some embodiments, the light chain constant region comprises one domain, namely CL. The VH region and the VL region can be further subdivided into hypervariable regions interspersed with more conserved regions, called complementarity determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of the heavy chain and / or light chain contains a binding domain that interacts with the antigen. In some embodiments, the constant region of the antibody can 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 from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into several subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans; and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, such as human IgG1, have several allotypes that differ from each other by only a few amino acids at most. Unless otherwise indicated, "antibody" may include, for example, monoclonal antibodies and polyclonal antibodies; chimeric antibodies and humanized antibodies; human antibodies and non-human antibodies; fully synthetic antibodies; and single-chain antibodies.

[0124] Combination therapy: As used herein, the term "combination therapy" may refer to a situation in which a subject is simultaneously exposed to two or more treatment regimens or therapies (e.g., using two or more therapeutic agents). In some embodiments, two or more agents may be administered simultaneously (i.e., co-administered); in some embodiments, the agents may be administered sequentially (in a specific order or in any order, as indicated by the context); in some embodiments, the agents are administered in overlapping dosing regimens. In some embodiments, combination therapy is administered in the form of a combined preparation or composition (e.g., the combined preparations and compositions described herein). In some embodiments, the multiple agents in the composition or preparation are mixed just prior to administration.

[0125] Cryoablation: The term "cryoablation" as used herein may refer to a method of using localized extreme cold to destroy target tissue (e.g., abnormal or diseased tissue, such as a tumor). Cryoablation is performed using a hollow needle (e.g., a cryoprobe) through which a cooled heat-conducting fluid is circulated. During cryoablation, the cryoprobe is placed near or within the target tissue (e.g., positioned within a tumor), thereby allowing the freezing process to completely or substantially completely destroy the abnormal or diseased tissue, such as a tumor. Once the cryoprobe is in place, an attached cryogenic freezing device removes heat ("cooling") from the tip of the cryoprobe and extends through the surrounding tissue to the target area. Cryoablation occurs in frozen tissue by at least three mechanisms: 1. Formation of ice crystals within cells, thereby disrupting cell membranes and interrupting processes such as cell metabolism; 2. Coagulation of blood, thereby interrupting blood flow to the tissue, thereby causing ischemia and cell death; and 3. Induction of apoptosis, the so-called programmed cell death cascade. Cryoablation is suitable for treating most solid tumors. A skilled medical practitioner will understand which tumors are suitable for treatment by cryotherapy such as cryoablation or cryolysis (e.g., cryolysis therapy described herein). In many embodiments of the present disclosure, cryotherapy is administered in a manner that avoids cryoablation. In some embodiments of the present disclosure, cryotherapy is performed in a manner that promotes cryolysis (i.e., partial lysis of tumor tissue), as opposed to cryoablation.

[0126] Cryolysis: The term "cryolysis" as used herein may refer to a method of using local extreme cold to partially destroy a target tissue (e.g., abnormal or diseased tissue, such as a tumor). Therefore, the cryolysis performed in the methods described herein is similar to cryoablation, but the purpose of cryolysis is not to ablate the entire tumor. Therefore, during the administration of a therapy that causes cell lysis by cryolysis, a cryoprobe is placed near or inside the target tissue (e.g., positioned within a tumor), so that the freezing process will partially destroy the abnormal or diseased tissue, such as a tumor. In many embodiments of the present disclosure, the goal of cryolysis is not to achieve complete ablation of tumor tissue (i.e., cryoablation), but to induce partial necrotic areas within the tumor to release sufficient amounts of TSA / TAA and DAMPs to initiate an immune response. Therefore, cryolysis and cryoablation may differ in the method of administration, including but not limited to the lowest temperature reached in the target tissue treatment area, the cooling rate of the target tissue treatment area (which is related to the cooling rate of cryotherapy devices such as cryoprobes, etc.), the diameter of the ice ball formed in the target tissue treatment area, the diameter of the isotherm inside the ice ball that reaches the critical temperature of 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 DK (2020); Littrup, PJ, et al. (2009); Shah, TT et al., (2016)).

[0127] Cryoprobe: The term "cryoprobe" as used herein may refer to a hollow needle through which a cooled heat transfer fluid is circulated. The heat transfer fluid may be any acceptable cryogen for use on site, such as argon, nitrous oxide, carbon dioxide, or liquid nitrogen. The cryoprobe may have any diameter commonly used in the field of intratumoral cryotherapy. In some embodiments, the diameter of the cryoprobe is about 1.5 mm to about 3.4 mm. In some embodiments, the diameter of the cryoprobe is about 1.5 mm. In some embodiments, the diameter of the cryoprobe is about 3.4 mm. In some embodiments, the selected cryoprobe diameter enables a faster cooling rate. In some embodiments, achieving a rapid cooling rate requires the cooling probe to reach a temperature of about -40°C or colder, about -50°C or colder, or about -60°C or colder in less than a minute. In some embodiments, the cryoprobe surface can reach -130°C to -150°C, and as low as -190°C, depending on the need for rapid cooling of the target tissue, however, it is generally believed that such low temperatures are not necessary for cell death (Littrup, PJ et al. (2009); Shah, TT et al. (2016)). In some embodiments, the selected cryoprobe diameter can effectively freeze and dissolve the target tissue, such as tumor cells, that is, the selected cryoprobe effectively triggers the partial necrotic area within the tumor to release sufficient amounts of TSA and DAMP to initiate an immune response.

[0128] Cryotherapy: The term "cryotherapy" or "cryosurgery" as used herein may refer to the controlled local use of freezing temperatures in medical therapy. Cryotherapy can be used to treat a variety of tissue lesions. For example, in cancer treatment, cryosurgery is the application of 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, the application of cryotherapy treatment causes damage to the treated tissue due to the formation of intracellular ice. The extent of target tissue damage depends on a variety of factors, including but not limited to the lowest temperature reached in the target tissue treatment area, the cooling rate of the target tissue treatment area (which is related to the cooling rate of cryotherapy devices such as cryoprobes, etc.), the diameter of the ice ball formed in the target tissue treatment area, the diameter of the isotherm in the ice ball reaching the critical temperature of 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 DK (2020); Littrup, PJ et al. (2009); Shah, TT et al., (2016)).

[0129] Duty cycle: The term "duty cycle" as used herein may refer to the portion of a cycle during which a signal, system, or device is in an active state. A cycle is the time required for a signal, system, or device to complete an on and off cycle or a cycle between two states. Duty cycles are typically expressed as a percentage or ratio. In some embodiments of the present disclosure, the duty cycle describes the percentage of time that a cryogen flows or circulates within a 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 selected duty cycle provides a rapid freezing rate of tumor cells, wherein the tumor cell region reaches about -40°C or lower, about -50°C or lower, or about -60°C or lower 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 selected duty cycle provides a rapid freezing rate of tumor cells, wherein the tumor cell region reaches about -40°C or colder, about -50°C or colder, or about -60°C or colder in less than a minute.

[0130] Subject: The term "subject" or "patient" as used herein refers to any animal, including mammals, to which the provided methods and / or compositions are or can be administered, for example, to achieve experimental, diagnostic, preventive and / or therapeutic purposes. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like. The subject is preferably a human. In some embodiments, the subject suffers from 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 has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes a proliferative disease, such as cancer. In some embodiments, cancer is a solid tumor cancer. In some embodiments, the subject is receiving or has received a therapy to diagnose and / or treat a disease, disorder or condition.

[0131] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena are rarely, if ever, completed and / or carried out to perfection or to achieve or avoid an absolute outcome. Thus, the term "substantially" is used herein to capture the potential imperfections inherent in many biological and chemical phenomena, including those relevant to therapy.

[0132] Treatment: As used herein, the term "treatment" (as well as "treat" or "treating") refers to any administration of a composition or formulation (e.g., a composition or formulation described herein) or a provided method that partially or completely alleviates, ameliorates, inhibits, delays the onset of, lessens 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. Such treatment may be directed to subjects who do not show signs of the relevant disease, disorder, and / or condition, and / or subjects who only show early signs of the disease, disorder, and / or condition. Alternatively or in addition, such treatment may be directed to subjects who show one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition. In some embodiments, refers to the improvement, prevention, or reversal of a disease or condition, or at least one discernible symptom. For example, cancer is treated, particularly solid tumor cancer. Treatment can be carried out in cycles or treatment cycles. In some embodiments, the treatment cycle involves administering a treatment regimen (e.g., the method proposed in the present disclosure) to the subject, followed by a period of rest to allow the subject to recover. In some embodiments, the rest period after the subject receives 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, repeated treatment is 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.

[0133] Therapeutically effective amount: As used herein, a "therapeutically effective amount" of an agent or composition / formulation (e.g., an antibody, oligonucleotide, etc.) of the present disclosure refers to an amount of an agent or composition / formulation that is effective for treating or preventing a disease or recurrence of its signs or symptoms. A complete therapeutic effect may not occur after administration of a single dose, but may only occur after administration of a series of doses. Therefore, a therapeutically effective amount can be administered by one or more administrations of a unit dosage form.

[0134] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" as 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 ingredients of the composition and not deleterious to the recipient thereof.

[0135] The present disclosure recognizes that due to the complexity and multifactorial nature of the mechanisms mediating tumor immune escape, it is becoming increasingly clear that cancer immunotherapy requires the implementation of a combination of many therapeutic agents to elicit multiple immunopharmacological effects (Melero, Berman et al., 2015; Vilgelm, Johnson et al., 2016).

[0136] The present disclosure also recognizes that a comprehensive anti-tumor immunotherapy approach ideally requires: a) inducing immunogenic tumor cell death, characterized by necrotic death and release of TAAs / TSAs and danger-associated molecular patterns (DAMPs); b) recruiting and activating innate immune cells (e.g., NK, macrophages, etc.) and antigen-presenting cells (APCs), which take up, process and present TSA / TAA-derived peptides to mediate the initiation of adaptive immune responses against such antigens; c) stimulating the activation and proliferation of effector T cells while avoiding counter-regulatory mechanisms that limit T cell initiation; and d) blocking existing and inducible immunosuppressive mechanisms by reversing mechanisms that mediate T cell exhaustion, blocking the function of regulatory T cells (Tregs) or depleting Tregs, and preventing the recruitment of myeloid suppressor cells (MDSCs) to the tumor microenvironment (Chen and Mellman 2013).

[0137] The present disclosure recognizes that there are a variety of treatments that will promote immunogenic tumor cell death and TAA / TSA release (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, etc.

[0138] In addition, different approaches are being investigated to recruit, activate, and promote the immunogenic maturation of APCs in the tumor bed, and these approaches involve the use of cytokines (e.g., GM-CSF, FLT-3 ligands), chemokines (e.g., CCL21, CXCL10, etc.), TLR ligands (e.g., CpG ODN), STING agonists, and agonistic anti-CD40 (αCD40) ligands. In addition, vaccination strategies based on the administration of peptides / adjuvants, DNA or RNA vaccines encoding single or personalized TSAs aim to provide tumor antigens in an environment that is conducive to the uptake and presentation of antigens by professional APCs to stimulate a strong adaptive immune response. Similarly, there are several research strategies that can promote the activation and proliferation of NK cells as well as effector CD4 helper T cells and CD8 cytotoxic T cells. 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).

[0139] The present disclosure also recognizes that one of the most effective immunotherapy methods in clinical settings is to block the immunosuppressive mechanism mediated by Treg, thereby affecting the function of helper T cells and effector T cells and dendritic cells (DC). The main checkpoint inhibitor expressed by Treg is CTLA-4, and blocking the inhibitor with antagonistic anti-CTLA4 (αCTLA) mAb represents a major breakthrough in cancer immunotherapy (Wei, Duffy et al., 2018). In addition, effector T cells express a variety of immune checkpoints that mediate exhaustion and incompetence, such as PD-1, TIM-3, TIGIT, LAG-3, VISTA and BTLA, and the use of antagonistic mAbs to block these immune checkpoints is being actively studied (Melero, Berman et al., 2015). The most advanced method in these methods is to block the PD-1 checkpoint (or its ligand PD-L1, expressed by tumor cells or immunosuppressive dendritic cells), thereby regenerating exhausted T cells, and this method has now become the core pillar of almost all immunotherapy methods. However, the combined use of CTLA-4 and PD-1 / PD-L1 blockade relies on pre-existing anti-tumor immunity and tumor-infiltrating T cells, and still cannot fully address the mechanisms of enhanced tumor lysis, dendritic cell activation, promotion of antigen presentation, activation of naive T cells, and direct stimulation of effector T cell proliferation. For these reasons, the anti-tumor response achieved using this combination is only more commonly observed in certain "hot" immunogenic cancers (e.g., melanoma, lung cancer), and less frequently 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).

[0140] Therefore, there is a strong rationale for combining therapeutic approaches targeting different stages and functions of the antitumor immune response, synchronizing tumor antigen release with APC recruitment and activation, promoting the activation and proliferation of effector T cells, and inhibiting existing and inducible counter-regulatory immunosuppressive mechanisms mediated by Tregs, MDSCs, and the tumor itself. However, despite the obvious therapeutic potential of combinatorial therapeutic approaches, there are still some obstacles in the development of immunotherapies involving the use of combinations of investigational agents, and as a result, several expected combinations have not produced the expected results, such as enhanced antitumor responses. One reason for this obstacle is that each agent is usually developed alone or in combination with only one or two other agents. The suboptimal activity of individual or a few agents is usually compensated by using high doses, which maximizes their potential serious adverse events (AEs). Another obstacle is that most investigational agents are administered systemically, which requires higher doses than are actually required at the site of action (e.g., tumors, tumor-draining lymph nodes (TDLN), and peritumoral tertiary lymphoid structures), thereby increasing systemic exposure and the risk of off-target toxicity. Furthermore, any additional efficacy benefits from using specific combinations of agents are often offset by additional serious adverse events, such as cytokine storm, ulcerative colitis, hepatitis, thyroiditis, hypophysitis, and other immune-related adverse events, which often force the discontinuation of these therapies (Xu, Xu et al., 2020, Biniakewitz, Kasler et al., 2021).

[0141] A potential solution to the high systemic doses of multiple immunotherapeutic agents is to administer lower doses of some of these immunotherapeutic agents by intratumoral or peritumoral injection, which has shown good efficacy and reduced adverse events in preclinical models (Marabelle, Kohrt et al., 2014; Aznar, Tinari et al., 2017). However, these approaches still rely on administering only one or two intratumoral agents, while the others are administered systemically at high doses.

[0142] Therefore, current immunotherapy approaches that rely on the use of one or two agents only partially address certain immune mechanisms and thus still fail to provide a comprehensive solution to the problem of tumor-mediated immune escape.

[0143] The methods and compositions of the present disclosure provide solutions to these problems by simultaneously and simultaneously targeting multiple immune mechanisms to maximize anti-tumor efficacy, optimize the biodistribution of active pharmacological ingredients, and minimize the likelihood of serious adverse events. In some embodiments, the methods and compositions of the present disclosure stimulate autologous vaccination against unique TSA / TAA by implementing a strategy that combines the induction of immunogenic tumor cell death using physical and / or pharmacological methods with a multi-drug immunotherapy strategy within the tumor that mediates the promotion of APC recruitment and activation, activation and expansion of effector T cells, while inhibiting existing and inducible counter-regulatory immunosuppressive mechanisms mediated by Tregs and cells in the tumor microenvironment. In some embodiments, the methods of the present disclosure are intended to generate a systemic immune response against the tumor following local immune stimulation by using the tumor as its own vaccine, thereby generating a polyclonal adaptive immune response mediated by T cells and / or B cells against existing TSA / TAA. The advantages of the methods and compositions of the present disclosure over conventional cancer vaccines are that, in some embodiments, they use readily available immunostimulatory products and do not require pretreatment, molecular target identification of tumor antigens, or identification and selection of HLA-typed patients, thereby providing a more versatile treatment strategy than personalized cancer vaccine approaches, which typically target specific tumor antigens that must be shared between the vaccine and the tumor.

[0144] The methods and compositions provided herein mediate multiple pharmacological actions designed to elicit a synchronized and multifaceted anti-tumor immune response.

[0145] Administration of tumor cell lytic therapy

[0146] In some embodiments, the methods of the present disclosure include: a first step aimed at eliciting in situ immunity by triggering cell lysis and immunogenic cell death of tumor cells using an intratumoral cryolysis device; a second step comprising intratumoral administration of a combination of immunotherapeutic agents that mediate different pharmacological effects ( Figure 6 ).

[0147] In some embodiments, the method of the present disclosure includes a first step, that is, triggering cell lysis and immunogenic cell death of tumor cells (e.g., achieved by intratumoral cryolysis), thereby mediating necrotic cell death, and subsequently releasing TSA / TAA and DAMP into the tumor microenvironment. As performed in some embodiments of the present disclosure, one goal of the cryolysis step is not to achieve complete ablation of tumor tissue (i.e., cryoablation), but to induce a partial necrotic area (caused by partial cryoablation) in the tumor to release a sufficient amount of TSA and DAMP to initiate an immune response (den Brok, Sutmuller et al., 2004; Sidana 2014). In some embodiments of the present disclosure, it is preferred to cause partial cryolysis by 1-2 freeze-thaw cycles, thereby preserving blood vessels and lymphatic structures to facilitate the migration of antigens, cytokines, therapeutic agents and immune cells between TDLN and the tumor microenvironment. In some embodiments, the number of freeze-thaw cycles selected will minimize damage to blood vessels and lymphatic structures and / or immunosuppressive mechanisms. In some embodiments, one freeze-thaw cycle is preferred. In some embodiments, two freeze-thaw cycles are preferred. In some embodiments, only one freeze-thaw cycle is preferred. In some embodiments, no more than two freeze-thaw cycles are preferred. In some embodiments, no more than three freeze-thaw cycles are preferred. In some embodiments, the step of triggering cell lysis and immunogenic cell death of tumor cells is the first step of the methods described herein. In some embodiments, the step of triggering cell lysis and immunogenic cell death of tumor cells is the first step of the methods described herein, followed by the step of administering a combination formulation comprising an immunotherapeutic agent described herein.

[0148] The present disclosure recognizes that cryotherapy (e.g., cryoablation and cryothawing) can directly induce necrosis by forming ice crystals that damage cell membranes and organelles, as well as indirectly induce necrosis through osmotic stress and local ischemia caused by microvascular thrombosis (Fraser and Gill 1967; Whittaker 1984; Karlsson, Cravalho et al., 1993; Hoffmann and Bischof 2002; Sabel 2009). Its mechanism of action is attributed to mechanical and osmotic changes induced by water crystallization and local ischemic effects of microvascular damage causing coagulative necrosis of tumors (Gage and Baust 1998). After thawing, necrotic tumor cells within the ice ball (e.g., formed during cryothawing) will release intact tumor antigens and DAMPs, such as cytokines, intracellular ATP, nuclear proteins, and endogenous TLR ligands, such as HMGB1 protein (Sidana 2014). DAMPS stimulate innate immune responses and attract granulocytes, macrophages, and NK cells. DC absorbs tumor antigens, migrates to TDLN and initiates adaptive immune response (mechanistically similar to in situ immunity) (den Brok, Sutmuller et al., 2006). Then, immature dendritic cells (DC) loaded with antigens are activated and migrate to TDLN, where they process and display tumor antigens in the context of MHC molecules, and present antigens to T cells, thereby triggering their activation and promoting the expansion of Th1 cytotoxic CD8+T cells. In addition, soluble antigens arrive at TDLN, where these antigens can be loaded and cross-presented by DC. Therefore, controlling tumor lysis by freezing and thawing solid tumors is mechanistically similar to in situ immunity, in which many unique TAA / TSA from heterogeneous tumor cell populations are released together with danger signals, which can trigger and initiate immune responses against these antigens (Bastianpillai, Petrides et al., 2015; Yakkala, Chiang et al., 2019).Several animal studies have demonstrated that tumor cryothawing can induce systemic anti-tumor immune responses (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) (for details, please see (Sabel 2009)). These studies have shown that cryotherapy alone (i.e., cryothawing) can increase tumor antigen load and the amount of mature dendritic cells in TDLN (den Brok, Sutmuller et al., 2006), increase levels of Th1 cytokines (IL2 and IFNγ), improve tumor rejection rates, and increase protection against tumor rechallenge.

[0149] It is understood in the art that there appears to be a threshold for the antigenic stimulus required to support anti-tumor immunity, which requires the removal of a large portion of the mass (i.e., cryoablation) (Blackwood and Cooper 1972). In fact, prostate cancer patients treated with cryoablation of primary tumors showed a reduction in metastatic burden and an enhanced immune response after repeated cryotherapy treatments, resulting in cryodestruction of prostate tissue (Soanes, Ablin et al., 1970; Soanes, Gonder et al., 1970; Ablin 1972). In addition, patients with metastatic prostate cancer experienced relief of bone pain symptoms after cryosurgery, accompanied by an increase in IgM antibodies (Gursel, Roberts et al., 1973). In addition, cryoablated high-risk prostate cancer showed increased levels of TNFα and IFNγ in serum four weeks after tumor ablation, as well as increased tumor-specific CTLs and an increased Th1:Th2 cytokine ratio (Soanes, Ablin et al., 1970).

[0150] It is worth noting that cryotherapy does not always lead to immunostimulation, and sometimes it can even cause immunosuppression and tumor growth promotion (Hanawa 1993; Shibata, Yamashita et al., 1998). Although many studies have shown a certain degree of anti-tumor immune response after cryosurgery, many studies have not shown any response. There may be many reasons for these differences. First, without wishing to be bound by theory, the freezing rate is believed to play a role in determining the immunostimulatory versus immunosuppressive effects of cryotherapy. High freezing rates lead to the formation of ice crystals, which cause necrotic immunogenic cell death, accompanied by increased IFNγ expression and decreased Treg numbers (Sabel, Su et al., 2010). In contrast, the slower freezing rate observed at the periphery of the ice ball causes cell death due to osmotic shock, followed by apoptosis, reduced immunogenicity, and is associated with increased immunosuppression of Treg cells, reduced IFNγ levels, and Th2-type T cell responses (such as TGF-β and IL-10) (Sabel 2009; Sabel, Su et al., 2010). Therefore, the ratio of necrosis to apoptosis is thought to play a key role in determining whether the immune response to cryotherapy is stimulatory or suppressive (Sabel 2009; Ferguson, Choi et al., 2011). In addition, when large amounts of tumor are frozen, the large amount of immune complexes produced may cause "high zone tolerance", a phenomenon in which antigen overload leads to immunosuppression (Sabel 2009).

[0151] The partial cryolysis of tumor tissue performed in many embodiments of the present disclosure fundamentally changes the tumor microenvironment within the treatment area, and in particular achieves the lysis of most tumor cells and rupture of cell membranes (necrotic cell death), thereby releasing their cellular contents (including antigens and damage-associated molecular patterns (DAMPS) and other cell debris) into the extracellular space, while also minimizing damage to tissue lymphatic drainage and denaturing released tumor proteins. The partial cryolysis performed in many embodiments of the present disclosure produces different results from complete tumor cryoablation. For example, unlike the more commonly practiced tumor cryoablation, which requires complete cell death of the entire tumor and the surrounding healthy tissue margin, the partial cryolysis method of the present disclosure only causes the death of most necrotic tumor cells in the dissolved partial tumor treatment area, and there is no healthy cell margin at all in the target treatment area. This cryolysis method can cause cryoablation to fail for several reasons, including that only a portion of the tumor is dissolved, there is no healthy tissue margin around, and a large number of tumor cells are still alive in the partial cryolysis treatment area.

[0152] The present disclosure recognizes that partial tumor lysis via ruptured cell membranes causing necrotic cell death can potentially be achieved via a variety of methods, including radiation, radiofrequency, high-intensity focused ultrasound (HIFU), microwaves, oncolytic viruses, oncolytic chemicals, oncolytic peptides, and cryolysis. In many embodiments, cryolysis is more favored than other methods because it is highly efficient, repeatable, can induce an immune response better than heat-based methods, has good quality (minimum denaturation) of released proteins, has high overall safety, and has no off-target effects. In some embodiments of the present disclosure, cryolysis is mediated by an intratumoral cryolysis device, and then at least one freezing and thawing cycle is performed using a standard and approved cryolysis device and probe. In some preferred embodiments, cryolysis is administered as the first step of the method described herein.

[0153] In some embodiments of the present disclosure, in order to mediate cryolysis, a cryoprobe is inserted into a tumor (e.g., within a tumor). Once inserted into the appropriate position in the tumor, the cryoprobe will cool to a certain temperature, producing an ice ball with a diameter of about 10mm to about 14mm in the tumor. In some embodiments, the diameter of the ice ball in the tumor is about 8mm to about 14mm. In some embodiments, the diameter of the ice ball in the tumor is about 8mm to about 12mm. In some embodiments, the diameter of the ice ball in the tumor is about 8mm to about 10mm. In some embodiments, the diameter of the ice ball in the tumor is about 12mm to about 16mm. In some embodiments, the diameter of the ice ball in the tumor is about 13mm to about 15mm. In some embodiments, the diameter of the ice ball in the tumor is about 14mm to about 18mm. In some embodiments, the diameter of the ice ball in the tumor is about 14mm to about 30mm. In some embodiments, the diameter of the ice ball in the tumor is about 16mm to about 30mm. In some embodiments, the diameter of the ice ball in the tumor is about 18mm to about 30mm. In some embodiments, the diameter of the ice ball within the tumor is about 20mm to about 30mm. In some embodiments, the diameter of the ice ball within the tumor is about 25mm to about 30mm. In some embodiments, the diameter of the ice ball within the tumor is about 14mm to about 25mm. In some embodiments, the diameter of the ice ball within the tumor is about 14mm to about 20mm. In some embodiments, the diameter of the ice ball within the tumor is about 14mm to about 15mm. In some embodiments, the diameter of the ice ball within the tumor is about 13mm to about 14mm. In some embodiments, the diameter of the ice ball within the tumor is about 8mm. In some embodiments, the diameter of the ice ball within the tumor is about 9mm. In some embodiments, the diameter of the ice ball within the tumor is about 10mm. In some embodiments, the diameter of the ice ball within the tumor is about 11mm. In some embodiments, the diameter of the ice ball within the tumor is about 12mm. In some embodiments, the diameter of the ice ball within the tumor is about 13mm. In some embodiments, the diameter of the ice ball within the tumor is about 14mm. In some embodiments, the diameter of the ice ball within the tumor is about 15mm. In some embodiments, the diameter of the ice ball within the tumor is about 16 mm. In some embodiments, the diameter of the ice ball within the tumor is about 17 mm. In some embodiments, the diameter of the ice ball within the tumor is about 18 mm. In some embodiments, the diameter of the ice ball within the tumor is about 19 mm. In some embodiments, the diameter of the ice ball within the tumor is about 20 mm. In some embodiments, the diameter of the ice ball within the tumor is about 21 mm. In some embodiments, the diameter of the ice ball within the tumor is about 22 mm. In some embodiments, the diameter of the ice ball within the tumor is about 23 mm. In some embodiments, the diameter of the ice ball within the tumor is about 24 mm.In some embodiments, the diameter of the ice ball within the tumor is about 25 mm. In some embodiments, the diameter of the ice ball within the tumor is about 30 mm.

[0154] In some embodiments, the ice ball formed according to the methods described herein has a temperature gradient within the ice ball, wherein the temperature of the ice near the surface of the cryoprobe is approximately the same as the temperature of the surface of the cryoprobe. In addition, as the distance from the cryoprobe increases, the temperature within the ice ball changes, and near the ice ball / tissue interface (also referred to as the leading edge) is close to 0°C. Due to this temperature change, in some embodiments, the temperature generated at the cryoprobe is lower than -40°C to -60°C. In some embodiments, the temperature generated at the cryoprobe is lower than -40°C. In some embodiments, the temperature generated at the cryoprobe is lower than -60°C. In some embodiments, the temperature generated at the cryoprobe is lower than -80°C. In some embodiments, the temperature generated at the cryoprobe is lower than -100°C. In some embodiments, the temperature generated at the cryoprobe is lower than -120°C. In some embodiments, the temperature generated at the cryoprobe is lower than -140°C. In some embodiments, the cooling of the cryoprobe produces an ice ball in which the temperature of most of the ice is about -40°C. In some embodiments, the lethal ice (e.g., ice that causes death by necrosis, apoptosis, or osmotic shock) in the ice ball (about -40°C or lower) is about 5 mm from the leading edge (ice at the ice ball / tissue interface, which is about 0°C). In some embodiments, the lethal ice in the ice ball is about 1 mm, about 2 mm, about 3 mm, about 4 mm, or about 5 mm from the leading edge. After freezing, the frozen tumor tissue ice ball formed by the cryoprobe is allowed to thaw completely passively (without thermal cycling of the cryoprobe), which is automatically determined by timing and can be further verified by any suitable imaging method known in the art (e.g., thermocouple, ultrasound).

[0155] In some embodiments of the present disclosure, the size of the partial tumor cryolysis treatment area is the same regardless of the size of the treated tumor and is defined by the size of the ice ball produced. In some preferred embodiments, the size range of the ice ball diameter is about 8mm to about 30mm. In some embodiments, the size range of the ice ball diameter is about 10mm to about 30mm. In some embodiments, the size range of the ice ball diameter is about 12mm to about 30mm. In some embodiments, the size range of the ice ball diameter is about 14mm to about 30mm. In some embodiments, the size range of the ice ball diameter is about 16mm to about 30mm. In some embodiments, the size range of the ice ball diameter is about 18mm to about 30mm. In some embodiments, the size range of the ice ball diameter is about 20mm to about 30mm. In some embodiments, the size range of the ice ball diameter is about 25mm to about 30mm. In some embodiments, the size range of the ice ball diameter is about 8mm to about 25mm. In some embodiments, the size range of the ice ball diameter is about 8mm to about 20mm. In some embodiments, the size range of the ice ball diameter is about 8mm to about 18mm. In some embodiments, the size range of the ice ball diameter is about 8mm to about 16mm. In some embodiments, the size range of the ice ball diameter is about 8mm to about 14mm. In some embodiments, the size range of the ice ball diameter is about 8mm to about 12mm. In some preferred embodiments, the size range of the ice ball diameter is about 12mm to about 16mm. In some embodiments, the size range of the ice ball diameter is about 10mm to about 14mm. In some embodiments, the size range of the ice ball diameter is about 11mm to about 15mm. In some embodiments, the size range of the ice ball diameter is about 13mm to about 15mm.

[0156] In some preferred embodiments, the diameter of the ice ball is about 14mm, thereby making the spherical volume of the partially dissolved tissue about 1.44ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 1.2ml to about 1.6ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 0.3ml to about 14ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 0.3ml to about 10ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 0.3ml to about 8ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 0.3ml to about 4ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 0.3ml to about 2ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 0.3ml to about 1.4ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 0.3ml to about 1ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 1 ml to about 14 ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 1.4 ml to about 14 ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 2 ml to about 14 ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 4 ml to about 14 ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 8 ml to about 14 ml. In some embodiments, the diameter of the ice ball is such that the spherical volume of the partially dissolved tissue is about 10 ml to about 14 ml.

[0157] In some embodiments, in a freeze-thaw cycle, an ice ball is generated by cooling the temperature at the cryoprobe to about -40°C to about -60°C, or colder, in about 1 minute or less, and continuing to cool the temperature to a temperature of -70°C or colder, about -80°C or colder, about -90°C or colder, about -100°C or colder, about -110°C or colder, about -120°C or colder, about -130°C or colder, about -140°C or colder, or about -150°C or colder for a total time 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 is generated by cooling the temperature at the cryoprobe to about -40°C to about -60°C, or colder, in about 1 minute or less, and continuing to cool the temperature to about -70°C or colder, about -80°C or colder, about -90°C or colder, about -100°C or colder, about -110°C or colder, about -120°C or colder, about -130°C or colder, about -140°C or colder, or about -150°C or colder for a total time of about 5 minutes. In some embodiments, the ice ball is generated by cooling the temperature at the cryoprobe to about -40°C to about -60°C, or colder, in about 1 minute or less, and continuing to cool the temperature to about -120°C to about -150°C, or colder, for a total time of about 5 minutes. In some embodiments, the ice ball is generated by cooling the temperature at the cryoprobe to about -40°C to about -60°C, or colder, in about 1 minute or less, and continuing to cool the temperature to about -70°C or colder, about -80°C or colder, about -90°C or colder, about -100°C or colder, about -110°C or colder, about -120°C or colder, about -130°C or colder, about -140°C or colder, or about -150°C or colder for a total time of about 2 minutes. In some embodiments, the ice ball is generated by cooling the temperature at the cryoprobe to about -40°C to about -60°C, or colder, in about 1 minute or less, and continuing to cool the temperature to about -120°C to about -150°C, or colder, for a total time of about 2 minutes.

[0158] In some embodiments, the temperature generated at the cryoprobe reaches about -40°C to about -60°C or colder in about 1 minute. In some embodiments, the temperature generated at the cryoprobe reaches about -40°C to about -60°C or colder in about 50 seconds. In some embodiments, the temperature generated at the cryoprobe reaches about -40°C to about -60°C or colder in about 40 seconds. In some embodiments, the temperature generated at the cryoprobe reaches about -40°C to about -60°C or colder in about 30 seconds. In some embodiments, the temperature generated at the cryoprobe reaches about -40°C to about -60°C or colder in about 20 seconds. In some embodiments, the temperature generated at the cryoprobe reaches about -40°C to about -60°C or colder in about 10 seconds. In some embodiments, the temperature generated at the cryoprobe reaches about -40°C to about -60°C or colder in about 1 minute or less. In some embodiments, the temperature generated at the cryoprobe reaches about -40°C to about -60°C or colder in about 50 seconds or less. In some embodiments, the temperature produced at the cryoprobe reaches about -40°C to about -60°C or colder in about 40 seconds or less. In some embodiments, the temperature produced at the cryoprobe reaches about -40°C to about -60°C or colder in about 30 seconds or less. In some embodiments, the temperature produced at the cryoprobe reaches about -40°C to about -60°C or colder in about 20 seconds or less. In some embodiments, the temperature produced at the cryoprobe reaches about -40°C to about -60°C or colder in about 10 seconds or less.

[0159] In some embodiments, the temperature produced at the cryoprobe reaches 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 colder 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 produced at the cryoprobe is 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 The temperature may be 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 colder in about 30 seconds or less, about 20 seconds or less, or about 10 seconds or less.

[0160] In some embodiments, the cycle time for ice ball generation is predetermined and ends automatically a few seconds after the ice ball reaches its predetermined maximum size 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 in diameter. In some embodiments, the cycle time for ice ball generation is predetermined and ends automatically a few seconds after the ice ball reaches its predetermined maximum size of about 8 mm to about 30 mm, about 12 mm to about 16 mm, or about 14 mm in diameter.

[0161] In some embodiments, cooling of the cryoprobe produces an ice ball wherein the temperature of the majority of the ice is about -40°C or colder, about -45°C or colder, about -50°C or colder, about -55°C or colder, about -60°C or colder, about -65°C, about -70°C, about -80°C or colder, about -90°C or colder, about -100°C or colder, about -110°C or colder, about -120°C or colder, about -130°C or colder, about -140°C or colder, or about -150°C or colder. In some embodiments, cooling of the cryoprobe produces an ice ball wherein the temperature of the majority of the ice is about -40°C or colder. In some embodiments, cooling of the cryoprobe produces an ice ball wherein the temperature of the majority of the ice is about -60°C or colder.

[0162] In some embodiments, the ice ball comprises an area 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 diameter, wherein the temperature within the area is about -40°C or colder, about -50°C or colder, about -60°C or colder, about -70°C or colder, about -80°C or colder, about -90°C or colder, about -100°C or colder, about -110°C or colder, about -120°C or colder, about -130°C or colder, about -140°C or colder, or about -150°C or colder. In some embodiments, the puck comprises an area of ​​about 10 mm diameter within which the temperature is about -40°C, about -50°C, about -60°C or colder, about -70°C or colder, about -80°C or colder, about -90°C or colder, about -100°C or colder, about -110°C or colder, about -120°C or colder, about -130°C or colder, about -140°C or colder, or about -150°C or colder. In some embodiments, the puck comprises an area of ​​about 10 mm diameter within which the temperature is about -40°C or colder.

[0163] In some embodiments, a thawing step (e.g., passive thawing) is performed after the puck has reached its predetermined maximum size. In some embodiments, the passive thawing step 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 step lasts 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 step lasts about 5 minutes. In some embodiments, the passive thawing step lasts about 2 minutes. In some embodiments, the passive thawing step lasts until the ice ball dissipates (e.g., as judged by a suitable visualization method).

[0164] In some embodiments of the present disclosure, a second freeze-thaw cycle is repeated after the frozen tissue is passively thawed and verified by any suitable imaging method known in the art. In some embodiments, a second freeze-thaw cycle is repeated after the frozen tissue is passively thawed and verified by any suitable imaging method known in the art.

[0165] The cryo-thaw cycles described herein are intended to produce a tissue effect in the tumor treatment area, whereby a large portion of the tumor cells are lysed (i.e., their cell membranes are ruptured), thereby releasing their cellular contents into the extracellular space. The freeze / thaw cycles described herein are intended not to impair lymphatic drainage in the tumor tissue in and around the treatment area. Tumor tissue frozen in an ice ball (e.g., formed by a cryoprobe) is completely thawed passively (without thermal cycling of the cryoprobe), which is automatically determined by timing and can be verified by any suitable imaging method known in the art.

[0166] In some embodiments, cryolipolysis therapy administered in accordance with the present disclosure causes necrotic cell death in a tumor tissue region, wherein the diameter of the region is 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. In some embodiments, cryolipolysis therapy causes necrotic cell death in a tumor tissue region, wherein the diameter of the region is about 12 mm to about 16 mm. In some embodiments, cryolipolysis therapy causes necrotic cell death in a tumor tissue region, wherein the diameter of the region is about 13 mm to about 15 mm. In some embodiments, cryolipolysis therapy causes necrotic cell death in a tumor tissue region, wherein the diameter of the region is about 14 mm.

[0167] In some embodiments of the present disclosure, a selected tumor site is prepared for image-guided precision percutaneous access using a coaxial cryoprobe / infusion needle system (SCINS). The SCINS is inserted via standard image-guided precision percutaneous access techniques, and the tip of the SCINS is precisely positioned so that the volume of the resulting cryolysis treatment zone is completely within the selected tumor. The length of the cryolysis zone with the SCINS can be visualized under imaging as the length of the cryoprobe exposed from the tip of the coaxial infusion needle.

[0168] In some embodiments, the freeze-thaw device is set to a certain duty cycle to allow a cryoprobe temperature of about -40°C, about -50°C, about -60°C or colder to be generated 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 freeze-thaw device is set to a duty cycle to allow a cryoprobe temperature of about -40°C, about -50°C, about -60°C or colder to be generated 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 or less, 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 freeze-thaw device is set to a duty cycle to allow a cryoprobe temperature of about -40°C or colder to be generated in less than about 1 minute. In some embodiments, the freeze-thaw device is set to a duty cycle to allow a cryoprobe temperature of about -60°C or colder to be generated in less than about 1 minute. In some embodiments, the duty cycle of the freeze lysis device is set to 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 duty cycle of the freeze lysis device is set to 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 duty cycle of the freeze lysis device is set to about 100%.

[0169] Administration of combined preparations

[0170] The methods and compositions disclosed herein are based on the intratumoral administration of TLR9 agonists, anti-CD40 agonistic monoclonal antibodies, anti-CTLA4 monoclonal antibodies, and agonistic anti-OX40 monoclonal antibodies or anti-PD1 monoclonal antibodies. The combination of these immunostimulatory agents is designed to promote and facilitate an immunogenic anti-tumor response after cryolysis-mediated tumor cell lysis, and to ensure that the result of cryolysis is pro-inflammatory, thereby avoiding situations that may have an immunosuppressive effect.

[0171] TLR9 is an intracellular pattern recognition receptor present in the endosomal compartments of mouse 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 double-stranded DNA fragment with unmethylated cytidine phosphate guanosine (CpG) dinucleotides. TLR9 agonists are artificial oligonucleotides with unmethylated CpG motifs. They can induce signal transduction cascades, cause transcriptional programs, thereby initiating inflammatory processes, enhancing cancer cell killing, and generating adaptive immune responses. Convincing data show that intratumoral administration of TLR9 agonists improves APC activation, especially pDC in TDLN, causing the release of proinflammatory 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 has direct effects on tumors, including inhibition of angiogenesis, antiproliferative effects, and increased MHC-I expression of tumor cells, thereby enhancing target visibility and promoting T cell killing of tumor cells (Suek, Campesato et al., 2019). In addition, IFNα also enhances NK killing ability and the maturation of conventional DC (Hervas-Stubbs, Perez-Gracia et al., 2011). In addition, TLR9 agonists can increase the expression of co-stimulatory molecules CD80 and CD86, increase IP10 (CXCL10) as a chemoattractant for monocytes, macrophages, DCs, T cells, and NK cells, and increase the expression of lymph node homing signal CCR7, ultimately leading to increased T cell stimulation and tumor rejection, and in some cases, causing powerful abscopal effects in distant untreated tumors (Suek, Campesato et al., 2019; Karapetyan, Luke et al., 2020)( Figure 1 ).

[0172] CD40 is a membrane molecule and a member of the TNFR family, expressed on APCs such as DCs, B cells, monocytes, and macrophages. The key physiological function of the CD40 / CD40L pathway is mediated by the ligation of CD40L, which is primarily expressed by CD4 helper T cells, with CD40 on APCs, especially dendritic cells (DCs) (Schonbeck and Libby 2001; Vonderheide and Glennie 2013; Vonderheide 2020). CD40 is not a direct T cell agonist, but its ability to increase antigen presentation indirectly enhances T cell activation. CD40 is expressed in APCs such as DCs, B cells, and macrophages, and its ligation with CD40L expressed by helper T cells or agonistic mAbs will activate APCs by upregulating class II HLA and co-stimulatory molecules CD80 / 86 to improve their antigen presentation ability, and by upregulating OX40-L, 4-1BB-L, GITR-L, while downregulating immunosuppressive molecules such as PD-L1 to improve APCs’ T cell-stimulating ability. CD40 agonism also increases the release of various Th1 cytokines (IL-1β, IL-6, IL-8, IL-12, TNFα, and IFNg) by DCs, thereby enhancing cytotoxic responses (Schonbeck and Libby 2001; Vonderheide and Glennie 2013; Vonderheide 2020). Specifically, αCD40 agonistic mAbs cause APCs to secrete IL-12, thereby i) downregulating the expression of CTLA4 and PD1 on Tregs, reducing their immunosuppressive effects; and ii) downregulating PD1 on effector CD8+ T cells, thereby restoring T cell responses to αPD1 mAbs (Ngiow, Young et al., 2016). CD40 also affects the expression of pro-apoptotic and anti-apoptotic genes (Bcl-XL) on different cell types (Choi, Shi et al., 2020). In B cells, αCD40 agonistic mAbs can upregulate HLA-II, CD40L, Th1 cytokine production and cause antigen-specific IgM / IgG secretion. Agonistic αCD40 mAbs can also induce the secretion of cytokines that are critical for macrophage cross-priming and cytotoxic function, thereby activating macrophages to directly kill tumor cells in a T cell-independent manner. Studies have shown that αCD40 antibodies of the IgG2 isotype have potent CD40 agonistic activity and can activate APCs by mimicking signals from helper CD4+ T cells expressing CD40L, allowing them to stimulate CD8+ T cells (Vonderheide and Glennie 2013; Vonderheide 2020; Yu, Chan et al., 2020).CD40 activation requires CD40 receptor oligomerization to induce agonism. The Fc domain of αCD40 agonistic mAb interacts with Fc receptors of neighboring cells and can enhance receptor oligomerization in a trans manner (White, Chan et al., 2011). Figure 2 ).

[0173] CTLA4 is an inhibitory molecule that is constitutively expressed in Tregs and inducibly expressed in effector CD4+ and CD8+ T cells upon TCR engagement and activation (Wei, Duffy et al., 2018). The expression level of CTLA4 in the immune synapse correlates with the strength of TCR affinity for the MHCI / peptide complex (Buchbinder and Desai 2016). CTLA4 binds to the CD80 and CD86 co-stimulatory molecules on APCs with higher binding affinity than to the co-stimulatory ligand CD28 expressed on T cells. Therefore, CTLA4 will outcompete CD28-mediated positive activation signals in the negative direction after binding to CD80 / 86. Competition between CD28 and CTLA4 has the function of controlling peripheral tolerance to highly reactive T cell clones by allowing only clones with moderate TCR / MHC-I / peptide affinity to expand, while reducing the proliferation of clones with extremely strong TCR / MHC-I / peptide affinity (Buchbinder and Desai 2016). In addition to this cell-intrinsic inhibitory mechanism, where CTLA4 and CD28 from the same T cell compete with each other for binding to CD80 / 86, CTLA4 also has a cell-extrinsic inhibitory mechanism, where CTLA4 expressed by Treg blocks CD80 / 86 on the surface of APCs and sequesters CD80 / 86 by transendocytosis (Qureshi, Zheng et al., 2011), thereby reducing their availability and preventing them from interacting with CD28 on T cells activated by such APCs (Buchbinder and Desai 2016; Wei, Duffy et al., 2018). Based on the above mechanisms, blocking CTLA4 using αCTLA4 mAb is expected to enhance immune activation by interfering with both cell-intrinsic and cell-extrinsic mechanisms. First, blocking CTLA4 on activated T cells and Tregs should activate effector T cells by increasing the co-stimulatory interaction between CD80 / 86 and CD28, regardless of their TCR affinity and specificity. This will lead to the activation and proliferation of a large number of effector T cell clones, thereby enhancing antitumor activity by enhancing T cell stimulation and broadening the TCR repertoire and tumor antigen spectrum that reacts with CD8+ T cells (Cha, Klinger et al., 2014; Kviskangtborg, Philips et al., 2014; Robert, Harview et al., 2014; Robert, Tsoi et al., 2014). Second, CTLA4 blockade will lead to the expansion of tumor neoantigen-specific CD8+ T cells within the tumor microenvironment (Fehlings, Simoni et al., 2017) and the expansion of specific subsets of exhausted Th1 CD4+ T cells (Wei, Levine et al., 2017).Again, there is evidence that αCTLA4 mAb can promote antitumor immunity via another mechanism involving ADCC-mediated Treg depletion, which can not only enhance T cell stimulation in TDLN, but also reconstitute the cold TME by deleting tumor-infiltrating Tregs (Wei, Duffy et al., 2018). Therefore, αCTLA4 mAb mediates its function through a dual mechanism, which includes depleting Tregs through Fc-dependent ADCC and inhibiting CD28+ T cells by blocking the co-stimulatory signals provided by CD80 / 86 in APCs, thereby promoting CD28-mediated T cell activation (Tang, Du et al., 2018; Wei, Duffy et al., 2018). Regarding the site of action where CTLA4 blockade exerts its pharmacological effects, it is thought that blocking CTLA4 is likely to affect the early stages of T cell activation in the draining lymph nodes, when CTLA4+Tregs remove CD80 / CD86 from the surface of APCs by transendocytosis and compete with CD28 for binding to CD80 / 86 (Qureshi, Zheng et al., 2011). In addition, CTLA4 blockade may also work at the tumor site, as exhausted CTLA4+T cells and Tregs can accumulate in the tumor microenvironment (Curran, Montalvo et al., 2010). Studies have shown that blocking CTLA4 with antagonistic mAbs will increase the ratio of CD8:Tregs in the tumor microenvironment (. Figure 3 ).

[0174] OX40 is a member of the TNFR superfamily and is expressed by effector CD4+ and CD8+ T cells, Treg, NKT, and NK within 1-4 days after TCR engagement. OX40L expressed by APC or agonistic anti-OX40 mAb ligation to OX40 promotes different signals, thereby enhancing the survival of Teff cells and the generation of memory T cells (Croft 2010). Immunopharmacological effects include promoting cytokine production, proliferation, and upregulation of CD40L, which helps stimulate DC via the CD40 / CD40L axis (Colombo 2017). Therefore, agonistic anti-OX40 mAbs such as YH002 simulate the effect of activated APC on effector T cell activation. Many agonistic αOX40 mAb-mediated aggregation and trimerization of OX40 receptor molecules require the Fc function of the antibody and auxiliary cells expressing appropriate Fc receptor molecules, such as NK cells, DC cells, macrophages, or B cells (Zhang, Armstrong et al., 2017; Kuang, Jing et al., 2020). In addition to its activation function for Teff cells, the agonistic αOX40 mAb can also inhibit the generation of Treg and the inhibitory activity of Treg by directly inhibiting FoxP3 expression (Zhang, Xiao et al., 2018), thereby causing a decrease in the expression of the checkpoint inhibitory molecule CTLA4, thereby allowing better co-stimulation of Teff cells via the CD28 / CD80 axis. In addition, inhibition of FoxP3 on Treg causes a decrease in IL10 secretion by Treg, which is conducive to better signal transduction between CD40 in DC and CD40L on memory T cells (Burocchi, Pittoni et al., 2011). In addition, agonistic αOX40 mAbs of the IgG1 isotype can also mediate the depletion of Tregs, especially intratumoral Tregs that express high levels of OX40, through ADCC, and can help reprogram 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 ).

[0175] PD-1 is expressed on activated T cells after TCR-mediated antigen stimulation, and is also expressed on B cells, NK cells, and monocytes. The function of PD1 is to control T cell activation in peripheral tissues to prevent tissue damage caused by ongoing inflammation. Chronic or high-level antigen exposure (e.g., in advanced cancer) can lead to PD1-mediated T cell exhaustion. During T cell stimulation, IFNγ induces PD-L1 in APCs as a counter-regulatory mechanism after T cell activation (Keir, Butte et al., 2008). In the case of TCR activation, PD-1 ligation with PD-L1 will cause phosphorylation of PD1 by Lck tyrosine kinase, followed by recruitment of SHP2 phosphatase to the intracellular domain of PD1, which in turn dephosphorylates and inactivates TCR and CD28, thereby blocking stimulatory and co-stimulatory downstream activation signaling that occurs during T cell stimulation (Buchbinder and Desai 2016; Wei, Duffy et al., 2018). Similarly, during tumor effects, IFNγ is induced and tumor cells express PD-L1. PD-L1 connects to PD1+Teff cells, recognizes tumor antigens presented by MHC-I via TCR, recruits SHP2, thereby inactivating TCR signaling, reducing cytokine production, and inducing T cell apoptosis or exhaustion and PD1+TIL inactivation, thereby preventing Teff cells from infiltrating tumors (Keir, Butte et al., 2008; Shrimali, Janik et al., 2015). Blockade of PD1 using antagonist mAbs works by restoring immune function to peripheral exhausted T cells (Shrimali, Janik et al., 2015) and can enhance tumor killing through various immune mechanisms, including direct inhibition of T cell apoptosis to restore T cell effector function, enhancement of APC-mediated T cell activation, downregulation of Treg-mediated suppression, downregulation of the immunosuppressive cytokines IL-10 and TGFβ, and upregulation of IL-2 and IFN-γ, while counteracting the inducible expression of PD-L1 in tumors (Wei, Duffy et al., 2018). Clinical evidence supports a model in which blockade of the PD1 signaling axis is most effective against tumors that have already elicited endogenous T cell responses but have been suppressed by PD1 engagement of its ligands PD-L1 and PD-L2. However, the response of PD-L1(-) tumors to αPD1 mAbs suggests that the presence of a pre-existing immune response is not always an absolute requirement for tumor rejection after αPD1 treatment ( Figure 5 ).

[0176] Preclinical results show that when αPD1 mAb is combined with agonistic αOX40 mAb, there is a broad spectrum of responses in different tumor models (Guo, Wang et al., 2014; Vilgelm, Johnson et al., 2016; Messenheimer, Jensen et al., 2017; Shrimali, Ahmad et al., 2017). These responses range from synergy to antagonism, with the timing of administration of the two mAbs appearing 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 simultaneous administration of agonistic anti-OX40 antibodies with antagonistic αPD1 mAbs.

[0177] In some embodiments, the methods of the present disclosure include the step of intratumoral administration of SV-101, which is a combination preparation of an immune-activating therapeutic agent comprising a TLR9 agonist, an αCD40 agonist, an αOX40 agonist, and an αCTLA4 checkpoint blockade inhibitor. In some embodiments, the methods of the present disclosure include the step of intratumoral administration of SV-101, which is a combination preparation consisting of a TLR9 agonist, an αCD40 agonist, an αOX40 agonist, and an αCTLA4 checkpoint blockade inhibitor ( Figure 7 ). In some embodiments, the method of the present disclosure includes the step of administering an SV-101 preparation, wherein the step of administering SV-101 is the second step in the method described herein. In some embodiments, the step of administering SV-101 is the second step in the method (e.g., the method described herein), which is followed by the first step of dissolving the tumor by cryolysis. In many embodiments of the present disclosure, this combination of agents is conducive to stimulating a new immune response, effectively helping the activation and maturation of APCs, while helping to stimulate naive T cells and promote their stimulation, proliferation, survival, and formation of memory T cells. This combination of active ingredients is mainly targeted at "cold" tumors, which do not show severe T cell infiltration or do not show strong PD-L1 expression. In many embodiments of the present disclosure, the administration of SV-101 stimulates a new round of immune response in tumors that show a certain degree of T cell infiltration, which may have the characteristics of "hot" tumors but have stopped responding to αPD1 checkpoint inhibitor treatment.

[0178] As described here, one of the first changes that occurs after intratumoral cryolysis and intratumoral infusion of SV-101 is the recruitment and activation of different classes of APCs, such as macrophages, B cells, and DCs. The combination of a TLR9 agonist and an αCD40 agonist in the TME can activate these cells in a unique, synergistic manner that is not achieved with either agent alone. First, a T cell-independent synergistic effect on the direct tumoricidal activity of macrophages was observed ( Fig. 9). Treatment with an αCD40 agonistic mAb increased TLR9 expression and subsequently increased responses to TLR9 agonists. Macrophages stimulated in this manner showed high levels of expression of TNFα, IFNγ, and IL12p70, which helped skew their phenotype toward a proinflammatory M1 phenotype. Importantly, IL12 secretion was only observed when αCD40 and TLR9 agonists were used in combination. Moreover, this synergistic effect also caused increased NO production and direct T cell-independent tumoricidal activity. Additionally, secreted Th1 cytokines caused M1 polarization and increased expression of MHC molecules, which facilitated TA presentation and increased T cell activation and proliferation.

[0179] B cells present in tumors, tertiary lymphoid structures (TLS), and TDLN were also stimulated by a dual combination of αCD40 mAb and TLR9 agonist ( Fig.10 ). B cells express CD40 and TLR9 receptors and can bind to TA via their membrane IgM. The agonism of αCD40 mAb requires the help of FcγRIIB expressing cells (such as macrophages or another B cell) to help CD40 trimer cross-linking. The agonism of CD40 receptor increases the expression of TLR9 receptor, thereby increasing sensitivity to CpG ODN. Combined stimulation increases the expression of TNFα, IFNγ, IL6, IL10 and IL12. In addition, the expression of CD40L in B cells increases, which can help and enhance the agonism of αCD40 mAb and contribute to the activation of pDC. B cells stimulated in this way can increase the secretion of TA-specific IgM and IgG. In addition, B cells can present the processed TSA peptide to helper T cells via their MHC-II receptors. Interestingly, B cells can induce the proliferation and Th1 differentiation of allogeneic naive CD4+T cells via an IL12-dependent mechanism only when exposed to CpG ODN and CD40L. IL12p70 is a key Th1 cytokine that can cause changes in helper T cells, such as increased expression of stimulatory molecules such as CD40-L and OX40, decreased immunosuppressive molecules associated with T cell stimulation such as CTLA4 and PD1, and increased Th1 cytokines such as TNFα, IFNγ, and IL-2.

[0180] Similar effects of αCD40 and TLR9 agonists were observed in pDCs and cDCs ( Fig.10). The study showed that the combined activity of these two agents would increase the Th1 cytokines IFNα, IL12p70, TNFα, IL1β, IL6, thereby causing the activation and stimulation of Th1 T cell responses and IP10 (or CXCL10), a chemokine that promotes the recruitment of immature pDCs and activated T cells. More importantly, significantly higher amounts of IFNα and bioactive IL12p70 were observed only in the presence of both αCD40 agonists and TLR9 agonists, and the downstream Th1 response was dependent on IL12. In addition, other relevant phenotypic and functional changes occurred on pDCs, including: 1) increased CD40, which is expected to increase their sensitivity to αCD40 agonists and facilitate the stimulation of CD40L-expressing T helper cells; 2) increased OX40L expression, which can help DCs stimulate and activate OX40-expressing T cells, thereby improving survival and establishing memory T cells; 3) increased expression of the CXCL10 chemokine ligand CCR7, which favors the recruitment and homing of activated DCs to LNs; 4) increased expression of the costimulatory molecules CD80 and CD86, which provide costimulatory signals during T cell stimulation in TDLNs; 5) increased MHC-II expression, which helps stimulate CD4+ helper T cells; 6) increased MHC-I expression and cross-presentation reactions, which help stimulate cytotoxic CD8+ T cells; 7) increased expression of the costimulatory molecules 4-1BB-L, GITR-L; and 8) downregulation of the immunosuppressive molecule PD-L1.

[0181] Priming and activation of naive CD4+ and CD8+ T cells occurs in TDLN and, to a certain extent, in TLS in the TME ( Fig.11). CD4+T cells with appropriate TCR can recognize processed TA peptides in the context of MHC-II (signal 1). Similarly, CD8+T cells with correct TCR recognize processed TA peptides in the context of MHC-I due to the cross-presentation of externally endocytosed TA (signal 1). Both CD4+ and CD8+T cells obtain costimulatory signals (signal 2) through the interaction of CD28 in T cells with costimulatory receptors CD80 and CD86 on DC. IFNα secreted by activated DCs provides additional stimulation signals for T cells and DCs themselves (signal 3). With the activation of T cells, counter-regulatory mechanisms are induced, which prevent the activation and survival of T cells whose TCR / MHC / Ag interactions are too weak or too strong. One of these inducible signals expressed on Teff cells is CTLA4, which will be induced when the TCR / MHC / Ag interaction is too strong or lasts too long. CTLA-4 has the function of outperforming CD28, thereby blocking the costimulatory signal provided by CD80 / 86. This limits the polyclonality of TCR variants and T cell clones that are selected, proliferated, and survived. In addition, CTLA-4 is constitutively expressed at high levels by Tregs, and it can also inhibit the co-stimulatory signal of CD80 / 86 by directly blocking the interaction with CD28 and by transendocytosis of DC membrane patches containing CD80 / 86. Increased T cell stimulation will cause increased secretion of Th1 cytokines such as TNFα, IL2, and IFNγ, as well as increased T cell proliferation (↑CD3 / Ki67). Therefore, blocking CTLA4 with αCTLA mAb will increase the co-stimulatory signal provided by CD28, ultimately allowing T cell clones with stronger TCR / MHC / Ag affinity to survive and proliferate, thereby increasing TCR variant diversity. In addition, if the αCTLA4 mAb used to block CTLA4 is an IgG1 isotype (e.g., YH001), ADCC-mediated loss of Tregs expressing high levels of CTLA4 may occur, which will be another mechanism to help stimulate naive Teff cells.

[0182] Another important mechanistic effect of the active agents in SV-101 (such as αCD40 agonists and TLR9 agonists) is the secretion of IL12, which has the effect of increasing the expression of OX40 and CD40-L on T cells. Increased expression of OX40 will make CD4 T cells susceptible to agonism by αOX40 mAb, which has the additional effect of upregulating CD40-L on Th cells, thus promoting cDC activation via CD40.

[0183] Furthermore, OX40 is expressed at high levels on Tregs, and agonism by αOX40 mAb inhibits FoxP3 function, leading to downregulation of CTLA4 and IL10, and promoting Treg reprogramming into Th17 cells. In addition, studies have shown that αOX40, which is of the IgG1 isotype, can mediate Treg death via ADCC, and this activity can synergize with the ADCC activity of αCTLA4 mAb, which is of the IgG1 isotype. The overall effect provided by the four components present in SV-101 is to increase T cell excitation while attenuating counter-regulatory mechanisms, thereby helping to increase CD4 Th cell stimulation, proliferation, survival, and memory differentiation, and increase TCR polyclonality.

[0184] After activation, CD8+ Teff cells in TDLN proliferate and migrate to the TME (DCs activated by TLR9 agonists and αCD40 agonists expressing CXCL10 further promote this process) to stimulate the effector phase of anti-tumor immune response ( Fig.13 ).

[0185] 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, which will restart another TA and DAMP cycle in tumor cells to maintain the T cell stimulation cycle. An important immunosuppressive mechanism that needs to be overcome in the TME is that mediated by Tregs. Tregs mediate immunosuppression in the effector phase of Teff cells through several mechanisms, including: 1) degradation of extracellular ATP (a DAMP signal) to adenosine through the combined action of membrane enzymes CD39 and CD72; 2) sequestration of immunostimulatory cytokines such as IL-2 by overexpression of the high-affinity IL-2R CD25, thereby inhibiting the helper effect of CD4 Th cells; 3) secretion of immunosuppressive cytokines such as TGFβ, IL10 and IL35; and 4) direct cleavage of Teff by secretion of GrzB and perforin. The presence of αOX40 and αCTLA4 components in SV-101 is also important in the effector phase of the immune response because these molecules target the function and number of Tregs. Tregs express high levels of CTLA4 and OX40, and the presence of IgG1αCTLA and αOX40 can mediate ADCC of Tregs in the TME and reduce immunosuppression.

[0186] In summary, the αCD40 agonistic mAb and TLR9 agonist present in SV-101 mediate robust activation and maturation of APCs (primarily immature dendritic cells, as well as macrophages and B cells) in the TME, inducing them to upregulate proinflammatory molecules (MHC-I, MHC-II, CD80 / CD86) that promote tumor antigen uptake and cross-presentation, as well as cytokines that stimulate helper and effector cytotoxic T cells during antigen presentation (TNFα, INFα / β, IL-12), while downregulating immunosuppressive molecules against T cells, such as PD-L1. Activation of dendritic cells will prompt their migration to tumor-draining lymph nodes, where they activate antigen-specific naive and memory T cells (CD4 and CD8), thereby promoting cytotoxic Th1 antitumor immune responses. The presence of αCTLA4 mAb favors the activation of effector T cells by APCs, which has a dual function, namely, direct co-stimulation of activated T cells expressing inhibitory CTLA4 through CD80 / 86-CD28 interactions; and indirect co-stimulation by limiting the sequestration of CD80 / 86 by Tregs expressing CTLA4. The agonistic effect of αOX40 mAb will obtain additional positive stimulation of effector T cells, which can trigger signaling that favors survival and differentiation into memory T cells, while inhibiting the differentiation of Tregs by inhibiting FoxP3 signaling. In addition, αCTLA4 mAb and αOX40 mAb in SV-101 inhibit existing immunosuppressive mechanisms mediated by Tregs by inhibiting their function and promoting Treg exhaustion through ADCC. Reducing Tregs in the TME will also prevent the recruitment of MDSCs, thereby preventing the re-establishment of the suppressive TME after immunogenic cell death. Furthermore, IL-12 induced by αCD40 mAb and TLR9 ligands (e.g., agonists) can reduce PD-1 expression on exhausted T cells, which could aid T cell tumor infiltration by preventing lymphocytes from being excluded from the TME by tumor-expressed PD-L1.

[0187] In some embodiments, the method of the present disclosure includes the step of administering SV-102 intratumorally, SV-102 is a combination preparation of an immune activation therapeutic agent comprising a TLR9 agonist, an αCD40 agonist, an αPD1, and an αCTLA4 checkpoint blocking inhibitor. In some embodiments, the method of the present disclosure includes the step of administering SV-102 intratumorally, SV-102 is a combination preparation consisting of a TLR9 agonist, an αCD40 agonist, an αPD1 checkpoint blocking inhibitor, and an αCTLA4 checkpoint blocking inhibitor. In some embodiments, the method of the present disclosure includes the step of administering an SV-102 preparation, wherein the step of administering SV-102 is the second step in the method described herein. In some embodiments, the step of administering SV-101 is the second step in a method (e.g., a method described herein), which is followed by the first step of dissolving the tumor by cryolysis.

[0188] In many embodiments of the present disclosure, this combination of agents is intended to promote the initiation of new immune responses, strongly aiding the activation and maturation of APCs, while aiding the initiation of naive T cells, counteracting the immunosuppressive effects of Tregs, while also promoting the reactivation of existing exhausted Teff cells. This combination of active ingredients is intended to treat predominantly hot tumors that show severe T cell infiltration, as well as "cold" tumors that have not been previously treated and have ceased to respond to αPD1 checkpoint inhibitors, or tumors that show strong expression of PD-L1.

[0189] Regarding activation of macrophages ( Fig. 9 ), B cells ( Fig.10 ) and dendritic cells ( Fig.11 ), the mechanism of action of SV-102 shares similar immunopharmacological effects with SV-101. However, SV-102 and SV-101 have unique mechanistic differences in the activation / stimulation of T cell responses and in the effector phase in the tumor microenvironment.

[0190] Priming and activation of naive CD4+ and CD8+ T cells occurs in TDLN and, to a certain extent, in TLS in the TME ( Fig.12). CD4+T cells with appropriate TCR can recognize processed TA peptides in the context of MHC-II (signal 1). Similarly, CD8+T cells with correct TCR recognize processed TA peptides in the context of MHC-I due to the cross-presentation of externally endocytosed TA (signal 1). Both CD4+ and CD8+T cells obtain costimulatory signals (signal 2) through the interaction of CD28 in T cells with costimulatory receptors CD80 and CD86 on DC. IFNα secreted by activated DCs provides additional stimulation signals for T cells and DCs themselves (signal 3). With the activation of T cells, counter-regulatory mechanisms are induced, which prevent the activation and survival of T cells whose TCR / MHC / Ag interactions are too weak or too strong. One of these inducible signals expressed on Teff cells is CTLA4, which will be induced when the TCR / MHC / Ag interaction is too strong or lasts too long. CTLA-4 has the function of outperforming CD28, thereby blocking the costimulatory signal provided by CD80 / 86. This limits the polyclonality of TCR variants and T cell clones that are selected, proliferated, and survived. In addition, CTLA-4 is constitutively expressed at high levels by Tregs, and it can also inhibit the co-stimulatory signal of CD80 / 86 by directly blocking the interaction with CD28 and by transendocytosis of DC membrane patches containing CD80 / 86. Increased T cell stimulation will cause increased secretion of Th1 cytokines such as TNFα, IL2, and IFNγ, as well as increased T cell proliferation (↑CD3 / Ki67). Therefore, blocking CTLA4 with αCTLA mAbs will increase the co-stimulatory signal provided by CD28, ultimately allowing T cell clones with stronger TCR / MHC / Ag avidity to survive and proliferate, thereby increasing TCR variant diversity. In addition, if the αCTLA4 mAb used to block CTLA4 is an IgG1 isotype, ADCC-mediated loss of Tregs expressing high levels of CTLA4 may occur, which will be another mechanism to help stimulate naive Teff cells.

[0191] Another counter-regulatory mechanism induced by sustained TCR activation is the expression of PD1, which can be activated by cells in TDLN that express PD-L1 (immature DCs, MDSCs, M2 macrophages), which is usually upregulated by elevated IFNγ levels. The PD-1 / PD-L1 interaction recruits the SHP-2 phosphatase, which can inhibit T cell activation by dephosphorylating the Lck / TCR complex and the intracellular domain of CD28. Therefore, blocking the interaction of PD-1 / PDL1 with αPD1 is another mechanism that can help maintain T cell activation in TDLN.

[0192] Another important mechanistic action of αCD40 agonists and TLR9 agonists is the secretion of IL12, which has the effect of reducing PD-1 expression, thereby making it more susceptible to the action of αPD1 mAbs, and increasing the expression of OX40 and CD40-L on T cells. When these actions are combined with the increased levels of cognate ligands OX40-L and CD40 expressed by DC activated in this way, it can be expected that CD4 Th cell stimulation and survival and memory differentiation will increase.

[0193] The overall effect provided by the four components present in SV-102 is to increase T cell stimulation while attenuating counter-regulatory mechanisms, thereby helping to increase CD4 Th cell stimulation, proliferation, survival and memory differentiation, and increase TCR polyclonality.

[0194] After activation, CD8+ Teff cells in TDLN proliferate and migrate to the TME (DCs activated by TLR9 agonists and αCD40 agonists expressing CXCL10 will further promote this process) to initiate the effector phase of anti-tumor immune response ( Fig.14 ).

[0195] In the TME, the function of CD8+ Teff cells is to recognize the same TA peptides in the context of MHC-I and kill tumor cells by releasing GrzB, which will reinitiate another TA and DAMP cycle in tumor cells to maintain the T cell stimulation cycle.

[0196] The result of sustained T cell stimulation by the TCR is increased PD1 expression and release of IFNγ, thereby inducing PD-L1 expression on tumor cells. The PD-1 / PD-L1 interaction will cause phosphorylation of PD1 by Lck, which will recruit the SHP2 phosphatase, causing TCR dephosphorylation and generating exhausted PD1hi Teff, which is usually refractory to PD1 checkpoint inhibitors. Exhausted PD1hi T cells also express CD40 in the TME. Such exhausted PD1hi T cells can be reactivated by several mechanisms mediated by the active agents present in SV-102. First, the increased levels of IL12p70 in the TME (achieved by the combined action of αCD40 agonists and TLR9 agonists) help to reduce the expression levels of PD1hi to PD1lo, which makes the cells more susceptible to αPD1 mAb blockade. In addition, the agonism of CD40 by αCD40 agonist mAbs can reactivate the metabolism of exhausted T cells by activating mTORC1.

[0197] Another important immunosuppressive mechanism to be overcome in TME is Treg-mediated immunosuppression ( Fig.15Tregs mediate immunosuppression during the Teff cell effector phase through several mechanisms, including: 1) degradation of extracellular ATP (a DAMP signal) to adenosine through the combined action of membrane enzymes CD39 and CD72; 2) sequestration of immunostimulatory cytokines such as IL-2 by overexpression of the high-affinity IL-2RCD25, thereby inhibiting the helper effect of CD4 Th cells; 3) secretion of immunosuppressive cytokines such as TGFβ, IL10, and IL35; and 4) direct cleavage of Teffs by secretion of GrzB and perforin.

[0198] The presence of αCTLA4 in SV-102 is also important during the effector phase of the immune response, as Tregs express CTLA4 at high levels, and the presence of IgG1αCTLA can mediate ADCC of Tregs in the TME and reduce immunosuppression.

[0199] In summary, the αCD40 agonistic mAb and TLR9 agonist present in SV-102 mediate robust activation and maturation of APCs (mainly immature dendritic cells, as well as macrophages and B cells) in the TME, inducing them to upregulate proinflammatory molecules (MHC-I, MHC-II, CD80 / CD86) that promote tumor antigen uptake and cross-presentation, as well as cytokines that stimulate helper and effector cytotoxic T cells during antigen presentation (TNFα, INFα / β, IL-12), while downregulating immunosuppressive molecules against T cells, such as PD-L1. Activation of dendritic cells will prompt their migration to tumor-draining lymph nodes, where they activate antigen-specific naive and memory T cells (CD4 and CD8), thereby promoting cytotoxic Th1 antitumor immune responses. The presence of αCTLA4 mAb favors the activation of effector T cells by APCs, which has a dual function, namely, direct co-stimulation of activated T cells expressing inhibitory CTLA4 through CD80 / 86-CD28 interactions; and indirect co-stimulation by limiting the sequestration of CD80 / 86 by Tregs expressing CTLA4. Additional positive stimulation of effector T cells will be obtained using αPD1 blocking mAb, which can reduce the inhibitory signal provided by PD-L1+ DC. In addition, the αCTLA4 mAb in SV-102 inhibits existing immunosuppressive mechanisms mediated by Tregs by inhibiting their function and promoting their exhaustion through ADCC. Reducing Tregs in the TME also prevents the recruitment of MDSCs, thereby preventing the re-establishment of the suppressive TME after immunogenic cell death. In addition, αCD40 mAb and IL-12 induced by TLR9 ligands (e.g., agonists) can reduce the expression of PD-1 on exhausted T cells, which can help T cell tumor infiltration by preventing lymphocytes from being excluded from the TME by tumor-expressed PD-L1 and making them responsive to αPD1 mAb therapy. The presence of αPD1 mAb in SV-102 will rescue TILs from exhaustion and can prolong their functional cytotoxic state by preventing their exhaustion.

[0200] In addition to the pharmacological activity of each active ingredient or agent injected intratumorally in the form of the combined formulations SV-101 and SV-102, the biomechanics of intratumoral injection and local regional infusion play an important role in controlling the biodistribution of the drug and mediating the biological activity and anti-tumor efficacy of the disclosed method. It is well known that the general principle of fluid dynamics is that liquid will flow from an area of ​​higher pressure to an area of ​​lower pressure (volume / time) via a path with the least resistance (change in pressure / flow) and the highest compliance (volume change / pressure change). During the infusion process, the tissue will show resistance and compliance along different degrees of elasticity, and as various resistance and compliance pathways are saturated with liquid and show increasing resistance (back pressure), the path with the least resistance will change. Almost all normal, healthy tissues use the lymphatic drainage system primarily to maintain fluid balance in the tissue. Lymphatic drainage vessels are usually the main drainage pathways for interstitial fluid and exudate. If lymphatic drainage is affected, edema (fluid overload in the tissue) will occur. It should also be fully understood that metastatic tumors are usually lymphatic hyperfunction when compared to healthy tissue or early tumors, and they extensively use the lymphatic system to transport small and large molecules into and out of the tumor area to communicate with other tissues such as TDLN. In addition, tumors are associated with tertiary lymphatic structures (TLS) in the tumor, surrounding the tumor, or nearby tissues, and these TLS are connected to the tumor and secondary lymphatic structures (such as TDLN) via lymphatic vessels. The lymphatic tissue drainage system represents an important aspect of the biomechanics of the disclosed method because it provides an important path of least resistance for tumor tissue to solve the interstitial fluid and exudate overload (edema) caused by drug infusion into the treatment area. The relatively slow and low-pressure (compared to subcutaneous or intramuscular injection) infusion rate allows the drug to first (via the closest path of least resistance) mix with the surrounding lysed cell fragments with relatively low resistance in the treatment area, and the lysed cell fragments include tumor antigens, DAMPS and other cell contents. After saturation of the lysed treatment zone is achieved, the infusion fluid will expand into the surrounding intact tumor tissue via the path of least resistance and also outflow via the first available lymphatic drainage pathway and, to a lesser extent, into the vascular pathway until the compliance limit of the tissue (the ability of the tissue to expand to accept additional fluid) is reached, at which point the continued infusion pressure will reach equilibrium, where the amount of fluid flowing out of the tumor, primarily via the draining lymphatics and blood vessels, is approximately equal to the inflow rate injected into the tumor. This point will vary depending on the compliance of the tissues within and around the tumor (some tissues are more compliant than others), but the equilibrium point is expected to be reached at or before the time of infusion of about 5 mL of drug (e.g., the formulations described herein) (based on the limits of tissue compliance associated with intramuscular infusion, which range from 1.5 mL to 5 mL).At the equilibrium point, the flow / pressure of the continuous infusion will saturate and expand the target tumor tissue to its compliance limit, and the infused drug and any diffusible entities including dissolved tumor cell contents, tumor antigens, DAMPS, etc. will likely mix with and be flushed into the surrounding intra-tumoral tissue, peritumoral tissue, and lymphatic drainage vessels. Therefore, it is expected that most of this excess liquid mixture of infused drug and some mobile entities will be drained into the treatment area and reach the cell targets within the tumor microenvironment, TLS and TDLN via the draining lymphatic vessels and enter the draining lymph nodes. When the infusion is completed, the pressure head of the infusion fluid is terminated, and the expanded and elastic intra-tumoral and peritumoral tissues will also drain to the mixture of excess drug and debris fluid via the path of least resistance (expected to be mainly the lymphatic drainage system) and return to their pre-injection resting pressure. It is expected that the mixture of remaining extravasated drugs, damaged tissue, intact mobile cells and cell debris (tumor antigens, DAMPS, cell debris, etc.) will be highly immunogenic and is expected to stimulate APC recruitment and activation, APC migration to TDLN, antigen presentation and T cell activation, and promote cytokines that favor T cell infiltration into the TME. In addition, it is expected that drugs injected into the TME will be beneficial for reactivating exhausted TILs and blocking the immunosuppressive function of Tregs, thereby shifting the balance toward cytotoxic antitumor immune responses.

[0201] For at least the reasons described above, in some embodiments, the intratumoral injection procedures described herein are different from other intratumoral therapies and should be considered intratumoral injections for locoregional treatment, in which the injected liquid is not expected to be completely contained within the tumor tissue, but is actually expected to enter lymphatic structures connected to the tumor and peritumoral tissue via lymphatic vessels. In fact, a distinguishing parameter of the disclosed method compared to most intratumoral therapies is that the injection volume of the drug combination exceeds the volume of the cryolysis zone and the volume that can theoretically be contained or retained within the tumor, and that extravasation of this liquid composition beyond the tumor margin will be achieved by large injection and elevated hydrodynamic pressure.

[0202] Thus, the methods of the present disclosure are expected to produce a local-regional effect in at least two body locations (intra-tumor / peritumoral and lymphoid organs) where drugs (e.g., the formulations described herein), antigens, and immune cells are brought to synchronous locations and close proximity, thereby increasing the likelihood that they interact and ultimately elicit local and systemic anti-tumor responses.

[0203] In some embodiments of the present disclosure, the active ingredients or mixtures of agents in SV-101 or SV-102 are injected into the tumor using the following procedure. After a partial tumor cryolysis treatment zone is established in the selected tumor, the next step is to advance the coaxial cryoprobe / infusion needle system (SCINS) under precise imaging guidance until the coaxial infusion needle tip on the outside appears in the approximate center of the cryolysis treatment zone. After this step is completed, the cryoprobe is withdrawn from the SCINS, so that the coaxial infusion needle tip on the outside is approximately located in the center of the cryolysis treatment zone in the tumor and is ready for drug infusion.

[0204] In some embodiments of the present disclosure, the combined preparation SV-101 or the combined preparation SV-102 is prepared as an infusion with a volume of about 1 mL to about 30 mL. In some embodiments, the volume of the prepared combined preparation is 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 volume of the prepared combined preparation is about 15 mL.

[0205] In some embodiments of the present disclosure, a syringe containing a drug (e.g., a formulation described herein) is mounted on a pump, and a low-infusion connecting tube (e.g., a MEDLINE 60" Extension Set with microbore tubing) is connected to the pump. Set)) is connected to the syringe via a one-way stopcock, and the system is infused with drug by pushing the syringe pump until the drug is seen flowing out of the connecting tube. Then, the prepared connecting tube is connected to the SCINS infusion needle. The infusion volume of the connecting tube is about 0.6ml. The flow rate of the syringe pump for infusion is set to about 1-5mL / min, and the preferred value is 3mL / min. Do not set the overpressure alarm. Then, start the pump and inject a total volume of about 15ml within 5 minutes. Be careful to ensure that the infusion needle tip is still in the correct position and that no drug flows back along the needle tract. When the drug infusion pump completes its infusion cycle and delivers 15ml of drug from the syringe to the connecting tube, close the one-way stopcock. At this point, the volume of drug remaining in the connecting tube is about 0.6ml. This volume of drug should now be pushed by removing the one-way stopcock from the infusion syringe and injecting 0.6ml of sterile water into the one-way stopcock.

[0206] In some embodiments, the infusion syringe pump is configured 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 configured to flow at a rate of about 1 mL / min. In some embodiments, the infusion syringe pump is configured to flow at a rate of about 2 mL / min. In some embodiments, the infusion syringe pump is configured to flow at a rate of about 3 mL / min. In some embodiments, the infusion syringe pump is configured to flow at a rate of about 4 mL / min. In some embodiments, the infusion syringe pump is configured to flow at a rate of about 5 mL / min.

[0207] 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).

[0208] In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27. In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 1. In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 2. In some embodiments, the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 27.

[0209] In some embodiments, according to the present disclosure, TLR9 agonists are administered at a dosage within the following range: 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, TLR9 agonists are 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, TLR9 agonists are administered at a dosage of about 1 mg. In some embodiments, TLR9 agonists are administered at a dosage of about 2 mg. In some embodiments, TLR9 agonists are administered at a dosage of about 3 mg. In some embodiments, the TLR9 agonist is administered at a dose of about 4 mg.

[0210] In some embodiments of the present disclosure, the agonist anti-CD40 monoclonal antibody is a human IgG2k isotype. In some embodiments, the agonist 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 shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8.

[0211] In some embodiments of the present disclosure, an agonist anti-CD40 monoclonal antibody comprises a heavy 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. In some embodiments of the present disclosure, an agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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, an agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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, an agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28.

[0212] In some embodiments of the present disclosure, the agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-CD40 monoclonal antibody comprises a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 29.

[0213] In some embodiments of the present disclosure, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist anti-CD40 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: 28, 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, an agonist anti-CD40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 29.

[0214] In some embodiments, according to the present disclosure, the anti-CD40 agonist monoclonal antibody is administered at a dose in the following range: 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 about 8 mg. In some embodiments, the anti-CD40 agonist 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 agonist monoclonal antibody is administered at a dose of about 1 mg. In some embodiments, the anti-CD40 agonist monoclonal antibody is administered at a dose of about 5 mg. In some embodiments, the anti-CD40 agonist monoclonal antibody is administered at a dose of about 7.5 mg. In some embodiments, the anti-CD40 agonist monoclonal antibody is administered at a dose of about 10 mg.

[0215] In some embodiments of the present disclosure, the agonist anti-OX40 monoclonal antibody is a human IgG1k isotype. In some embodiments, the agonist anti-OX40 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 shown in SEQ ID NO:9, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:10, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:11, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:12, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:14.

[0216] In some embodiments of the present disclosure, an agonist anti-OX40 monoclonal antibody comprises a heavy 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. In some embodiments of the present disclosure, an agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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, an agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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, an agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 30.

[0217] In some embodiments of the present disclosure, an agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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, an agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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, an agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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, an agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a light chain, wherein 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 agonist anti-OX40 monoclonal antibody comprises a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 31.

[0218] In some embodiments of the present disclosure, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist 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, an agonist anti-OX40 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: 30, 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, an agonist anti-OX40 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 30, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 31.

[0219] In some embodiments, according to the present disclosure, the anti-OX40 agonist monoclonal antibody is administered at a dose in the following range: 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 about 8 mg. In some embodiments, the anti-OX40 agonist 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 agonist monoclonal antibody is administered at a dose of about 1 mg. In some embodiments, the anti-OX40 agonist monoclonal antibody is administered at a dose of about 5 mg. In some embodiments, the anti-OX40 agonist monoclonal antibody is administered at a dose of about 7.5 mg. In some embodiments, the anti-OX40 agonist monoclonal antibody is administered at a dose of about 10 mg.

[0220] In some embodiments of the present disclosure, the anti-PD1 monoclonal antibody is a human IgG4k isotype. In some embodiments, the anti-PD1 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 shown in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 20.

[0221] In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a heavy 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. In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein 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, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein 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, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein 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, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein 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, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 32. In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 32. In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 32.

[0222] In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a light chain, wherein 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, an anti-PD1 monoclonal antibody comprises a light chain, wherein 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, an anti-PD1 monoclonal antibody comprises a light chain, wherein 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, an anti-PD1 monoclonal antibody comprises a light chain, wherein 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, an anti-PD1 monoclonal antibody comprises a light chain, wherein 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, wherein 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, wherein 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, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 33.

[0223] In some embodiments of the present disclosure, an 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, an 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, an 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 with SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 33. In some embodiments of the present disclosure, an 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 with SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 96% sequence identity with SEQ ID NO: 33. In some embodiments of the present disclosure, an 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 with SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 33. In some embodiments of the present disclosure, an 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 with SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 33. In some embodiments of the present disclosure, an 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 with SEQ ID NO: 32, and the light chain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 33. In some embodiments of the present disclosure, an anti-PD1 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 32, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 33.

[0224] In some embodiments, according to the present disclosure, an anti-PD1 monoclonal antibody is administered at a dose within 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.

[0225] In some embodiments of the present disclosure, the anti-CTLA4 monoclonal antibody is a human IgG1k isotype. In some embodiments, the anti-CTLA4 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 shown in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 26.

[0226] In some embodiments of the present disclosure, an anti-CTLA4 monoclonal antibody comprises a heavy 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. In some embodiments of the present disclosure, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 34.

[0227] In some embodiments of the present disclosure, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein 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, an anti-CTLA4 monoclonal antibody comprises a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 35.

[0228] In some embodiments of the present disclosure, an 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, an 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, an 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, an 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, an 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, an 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, an anti-CTLA4 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: 34, 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, an anti-CTLA4 monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 35.

[0229] In some embodiments, according to the present disclosure, an anti-CTLA4 monoclonal antibody is administered at a dose ranging from 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.

[0230] In some preferred embodiments of the present disclosure, intratumoral cryolysis of solid tumors and intratumoral infusion of SV-101 or SV-102 are repeated every 3 to 8 weeks in the same lesion or in different metastatic lesions.

[0231] In some embodiments, intratumoral cryolysis of solid tumors and intratumoral infusion of the combination formulation provided by the present disclosure are repeated about every week, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, about every 9 weeks, about every 10 weeks, about every 11 weeks, about every 12 weeks, about every 13 weeks, about every 14 weeks, about every 15 weeks, or about every 16 weeks. In some embodiments, the treatment is repeated about every 3 to 16 weeks, about every 6 to 16 weeks, about every 8 to 16 weeks, about every 10 to 16 weeks, about every 12 to 16 weeks, about every 14 to 16 weeks, about every 3 to 14 weeks, about every 3 to 12 weeks, about every 3 to 10 weeks, about every 3 to 8 weeks, about every 3 to 6 weeks, or about every 3 to 4 weeks. In some embodiments, the treatment is repeated about every 4 to 8 weeks, about every 5 to 8 weeks, about every 6 to 8 weeks, about every 7 to 8 weeks, about every 4 to 7 weeks, about every 4 to 6 weeks, or about every 4 to 5 weeks. In some embodiments, the treatment is repeated every 4 to 8 weeks.

[0232] 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 dose, ii) an anti-CD40 monoclonal antibody at a dosage level of about 0.5 mg to about 10 mg per dose, iii) an anti-OX40 monoclonal antibody at a dosage level of about 0.5 mg to about 10 mg per dose, and iv) an anti-CTLA4 monoclonal antibody in the range of about 1 mg to about 50 mg per dose.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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 agonist 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 wherein 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 agonist 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 light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:11. NO:11, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:12, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0238] 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 wherein 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 wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:12, NO:12, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0239] In some embodiments, the SV-101 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 wherein 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 wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:12, NO:12, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0240] 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 wherein 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 wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:12 NO:12, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0241] 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 agonist 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 agonist 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: NO:30 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, 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: NO:35 has an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0242] 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.

[0243] 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.

[0244] In some embodiments, the SV-101 formulation comprises a 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 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.

[0245] 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 dose, ii) an anti-CD40 monoclonal antibody at a dosage level of about 0.5 mg to about 10 mg per dose, iii) an anti-PD1 monoclonal antibody at a dosage level of about 1 mg to about 100 mg per dose, and iv) an anti-CTLA4 monoclonal antibody in the range of about 1 mg to about 50 mg per dose.

[0246] 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.

[0247] 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.

[0248] 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 3 mg, about 7.5 mg, about 30 mg, and about 15 mg, respectively.

[0249] 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 4 mg, about 10 mg, about 80 mg, and about 40 mg, respectively.

[0250] 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 4 mg, about 10 mg, about 100 mg, and about 40 mg, respectively.

[0251] In some embodiments, the SV-102 formulation comprises: a TLR9 agonist as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 27; an agonist 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 shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9. NO:17, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:18, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:19, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0252] In some embodiments, the SV-102 formulation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18. NO:18, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:19, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0253] In some embodiments, the SV-102 formulation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:17, and wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:18 NO:18, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:19, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0254] In some embodiments, the SV-102 formulation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:5, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:17, and wherein the light chain comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:17 NO:18, a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:19, and a light chain variable region CDR3 comprising the amino acid sequence shown 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 shown in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:23, and wherein the light chain comprises a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0255] 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 agonist 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: NO:32 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, 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: NO:35 has an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0256] In some embodiments, the SV-102 formulation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO: 28, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO: 32, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO: 34, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 35.

[0257] In some embodiments, the SV-102 formulation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:28, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:32, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:34, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35.

[0258] In some embodiments, the SV-102 formulation comprises: a TLR9 agonist as shown 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 shown in SEQ ID NO:28, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:32, and the light chain comprises the amino acid sequence shown 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 shown in SEQ ID NO:34, and the light chain comprises the amino acid sequence shown in SEQ ID NO:35.

[0259] In some embodiments of the present disclosure, the individual components of SV-101 and SV-102 are injected sequentially into the same tumor lesion being treated in different treatment cycles.

[0260] In some embodiments, the individual components of SV-101 and SV-102 are premixed together and injected simultaneously into the tumor lesion to be treated.

[0261] In one embodiment of the present disclosure, the combined formulation is administered in a fixed volume ranging from 1 to 30 mL, most preferably 15 mL. In some embodiments, the combination formulation is administered in a fixed volume ranging from 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.

[0262] In one embodiment of the present disclosure, the cancer is a solid tumor cancer selected from the group consisting of 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.

[0263] In one embodiment of the present disclosure, the method for treating cancer involves the following steps: a) tumor cell lysis and b) intratumoral administration of a combined preparation of four immunotherapeutic active ingredients comprising 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 into the same lesion, repeated every 3 to 16 weeks, and preferably at a frequency of 4-8 weeks, for a total number of treatments of 1 to 12 treatment cycles, wherein if the lesion is available for treatment, 6 cycles are preferred.

[0264] 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, 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, treatment is repeated for 3 to 6 cycles. In some embodiments, treatment is repeated for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles. In some embodiments, treatment is repeated for 2 cycles. In some embodiments, treatment is repeated for 3 cycles. In some embodiments, treatment is repeated for 4 cycles. In some embodiments, treatment is repeated for 5 cycles. In some embodiments, treatment is repeated for 6 cycles.

[0265] Throughout the specification, when compositions are described as having, including, or comprising specific components, or when processes and methods are described as having, including, or comprising specific steps, it is contemplated that, in addition, the compositions of the present disclosure consist essentially of or consist of the recited components, and the processes and methods according to the present disclosure consist essentially of or consist of the recited processing steps.

[0266] Unless otherwise clearly stated or understood from the context, use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing" (including grammatical equivalents thereof) should generally be understood to be open-ended and non-limiting, e.g., not excluding other elements or steps not listed.

[0267] When the term "about" is used before a quantitative value, unless expressly stated otherwise, the disclosure also includes the specific quantitative value itself. Unless otherwise indicated or inferred, as used herein, the term "about" or "approximately" refers to a ±10% variation relative to the nominal value.

[0268] Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "an antibody" includes plural antibodies, and in some embodiments reference to "an antibody" includes plural antibodies (e.g., antibodies of different types (e.g., class / isotype, light chain type), antibody fragments, or engineered antibodies, etc.), and so forth.

[0269] The use of alternatives (eg, "or") should be understood to mean one, both, or any combination of the alternatives.

[0270] Example

[0271] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way.

[0272] Example 1: Preparation of SV-101 - Low Dose

[0273] This example describes the preparation of SV-101 combination formulations. In a 20 mL sterile vial, the following components are mixed and diluted to a volume of 15 mL in sterile saline solution. In a SV-101 combination formulation, the CTLA4 antibody is a YH001 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 21-26 and / or the VH / VL sequences shown in SEQ ID NOs: 34-35), the OX40 antibody is a YH002 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 9-14 and / or the VH / VL sequences shown in SEQ ID NOs: 30-31), the CD40 antibody is a YH003 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 3-8 and / or the VH / VL sequences shown in SEQ ID NOs: 28-29), and the CpG ODN has SEQ ID NO: 1. In another SV-101 combination formulation, the CpG ODN has SEQ ID NO: 2 or SEQ ID NO: 27. It will be appreciated by those skilled in the art that derivatives or variants of the SV-101 components may be used.

[0274]

[0275] Example 2: Preparation of SV-101—Medium Dose

[0276] This example describes the preparation of SV-101 combination formulations. In a 20 mL sterile vial, the following components are mixed and diluted to a volume of 15 mL in sterile saline solution. In a SV-101 combination formulation, the CTLA4 antibody is a YH001 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 21-26 and / or the VH / VL sequences shown in SEQ ID NOs: 34-35), the OX40 antibody is a YH002 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 9-14 and / or the VH / VL sequences shown in SEQ ID NOs: 30-31), the CD40 antibody is a YH003 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 3-8 and / or the VH / VL sequences shown in SEQ ID NOs: 28-29), and the CpG ODN has SEQ ID NO: 1. In another SV-101 combination formulation, the CpG ODN has SEQ ID NO: 2 or SEQ ID NO: 27. It will be appreciated by those skilled in the art that derivatives or variants of the SV-101 components may be used.

[0277]

[0278] Example 3: Preparation of SV-101—High Dose

[0279] This example describes the preparation of SV-101 combination formulations. In a 20 mL sterile vial, the following components are mixed and diluted to a volume of 15 mL in sterile saline solution. In a SV-101 combination formulation, the CTLA4 antibody is a YH001 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 21-26 and / or the VH / VL sequences shown in SEQ ID NOs: 34-35), the OX40 antibody is a YH002 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 9-14 and / or the VH / VL sequences shown in SEQ ID NOs: 30-31), the CD40 antibody is a YH003 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 3-8 and / or the VH / VL sequences shown in SEQ ID NOs: 28-29), and the CpG ODN has SEQ ID NO: 1. In another SV-101 combination formulation, the CpG ODN has SEQ ID NO: 2 or SEQ ID NO: 27. It will be appreciated by those skilled in the art that derivatives or variants of the SV-101 components may be used.

[0280]

[0281] Example 4: Preparation of SV-101 - Highest Dose Level

[0282] This example describes the preparation of SV-101 combination formulations. In a 20 mL sterile vial, the following components are mixed and diluted to a volume of 15 mL in sterile saline solution. In a SV-101 combination formulation, the CTLA4 antibody is a YH001 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 21-26 and / or the VH / VL sequences shown in SEQ ID NOs: 34-35), the OX40 antibody is a YH002 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 9-14 and / or the VH / VL sequences shown in SEQ ID NOs: 30-31), the CD40 antibody is a YH003 antibody (e.g., defined by the CDR sequences shown in SEQ ID NOs: 3-8 and / or the VH / VL sequences shown in SEQ ID NOs: 28-29), and the CpG ODN has SEQ ID NO: 1. In another SV-101 combination formulation, the CpG ODN has SEQ ID NO: 2 or SEQ ID NO: 27. It will be appreciated by those skilled in the art that derivatives or variants of the SV-101 components may be used.

[0283]

[0284] Example 5: Preparation of SV-102 - Low Dose

[0285] This example describes the preparation of SV-102 combination formulations. In a 20mL sterile vial, the following components are mixed and diluted to a volume of 15mL in a sterile saline solution. In a SV-102 combination formulation, the CTLA4 antibody is a YH001 antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 21-26 and / or the VH / VL sequence shown in SEQ ID NO: 34-35), the PD-1 antibody is a 609A antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 15-20 and / or the VH / VL sequence shown in SEQ ID NO: 32-33), the CD40 antibody is a YH003 antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 3-8 and / or the VH / VL sequence shown in SEQ ID NO: 28-29), and the CpG ODN has SEQ ID NO: 1. In another SV-102 combination formulation, the CpG ODN has SEQ ID NO: 2 or SEQ ID NO: 27. It will be appreciated by those skilled in the art that derivatives or variants of the SV-102 components may be used.

[0286]

[0287] Example 6: Preparation of SV-102—Medium Dose

[0288] This example describes the preparation of SV-102 combination formulations. In a 20mL sterile vial, the following components are mixed and diluted to a volume of 15mL in a sterile saline solution. In a SV-102 combination formulation, the CTLA4 antibody is a YH001 antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 21-26 and / or the VH / VL sequence shown in SEQ ID NO: 34-35), the PD-1 antibody is a 609A antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 15-20 and / or the VH / VL sequence shown in SEQ ID NO: 32-33), the CD40 antibody is a YH003 antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 3-8 and / or the VH / VL sequence shown in SEQ ID NO: 28-29), and the CpG ODN has SEQ ID NO: 1. In another SV-102 combination formulation, the CpG ODN has SEQ ID NO: 2 or SEQ ID NO: 27. It will be appreciated by those skilled in the art that derivatives or variants of the SV-102 components may be used.

[0289]

[0290] Example 7: Preparation of SV-102—High Dose

[0291] This example describes the preparation of SV-102 combination formulations. In a 20mL sterile vial, the following components are mixed and diluted to a volume of 15mL in a sterile saline solution. In a SV-102 combination formulation, the CTLA4 antibody is a YH001 antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 21-26 and / or the VH / VL sequence shown in SEQ ID NO: 34-35), the PD-1 antibody is a 609A antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 15-20 and / or the VH / VL sequence shown in SEQ ID NO: 32-33), the CD40 antibody is a YH003 antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 3-8 and / or the VH / VL sequence shown in SEQ ID NO: 28-29), and the CpG ODN has SEQ ID NO: 1. In another SV-102 combination formulation, the CpG ODN has SEQ ID NO: 2 or SEQ ID NO: 27. It will be appreciated by those skilled in the art that derivatives or variants of the SV-102 components may be used.

[0292]

[0293] Example 8: Preparation of SV-102 - Highest Dose Level

[0294] This example describes the preparation of SV-102 combination formulations. In a 20 mL sterile vial, the following components (see Table A or Table B) are mixed and diluted to a volume of 15 mL in a sterile saline solution. In a SV-102 combination formulation, the CTLA4 antibody is a YH001 antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 21-26 and / or the VH / VL sequence shown in SEQ ID NO: 34-35), the PD-1 antibody is a 609A antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 15-20 and / or the VH / VL sequence shown in SEQ ID NO: 32-33), the CD40 antibody is a YH003 antibody (e.g., defined by the CDR sequence shown in SEQ ID NO: 3-8 and / or the VH / VL sequence shown in SEQ ID NO: 28-29), and the CpG ODN has SEQ ID NO: 1. In another SV-102 combination formulation, the CpG ODN has SEQ ID NO: 2 or SEQ ID NO: 27. It will be appreciated by those skilled in the art that derivatives or variants of the SV-102 components may be used.

[0295] Table A

[0296]

[0297] Table B

[0298]

[0299] Example 9: Treatment Planning and Patient Preparation

[0300] This embodiment describes exemplary patient screening and treatment criteria. As a preliminary step, a detailed imaging study will be performed, including but not limited to CT, PET / CT and MRI, and a review will be conducted to develop a plan to identify potential target tumors to be treated. Primary or metastatic tumors are selected from target tumor candidates for treatment, assuming that their antigen library represents metastatic lesions at least in part. The selected tumor can be located in soft tissue, lymph nodes or bones, and should be easily accessible via a percutaneous method guided by precise imaging. The volume and shape of the selected tumor are also large enough (e.g., the size on at least two measuring axes is equal to or greater than 2 cm) so that the entire cryolysis partial tumor treatment area can be confidently contained in the tumor. Under comprehensive monitoring, the patient is subjected to general anesthesia or conscious sedation anesthesia.

[0301] Example 10: Freeze-thaw procedure

[0302] This embodiment describes an exemplary cryolysis procedure. A selected tumor site (e.g., the one selected in Example 9) is prepared for image-guided precision percutaneous access using a coaxial cryoprobe / infusion needle system (SCINS). The SCINS is inserted via a standard image-guided precision percutaneous access technique, and the tip of the SCINS is precisely positioned so that the volume of the resulting cryolysis treatment zone is completely within the selected tumor. The length of the cryolysis zone with the SCINS can be visualized under imaging as the length of the cryoprobe exposed from the tip of the coaxial infusion needle.

[0303] The cryoprobe is inserted into the tumor through the infusion needle. After confirming the position, the infusion needle is pulled back to expose 2.5 cm–3 cm distal to the cryoprobe. Cryolysis treatment is initiated by pressing a button on the cryo-console. This will cause the cryogenic fluid to begin flowing through the probe and begin the cooling process. The cryoprobe is cooled so that the probe temperature reaches -40°C to -60°C in less than one minute. Cooling lasts for a total of two minutes (during which the temperature of the cryoprobe can be brought to about -120°C to about -150°C, or colder). An ice ball with a diameter of about 14 mm is formed. This ice ball can be seen by typical imaging methods commonly used in the art (e.g., ultrasound (US), CT, PET / CT, etc.). After 2 minutes, the cryogenic gas flow is automatically stopped (however, depending on the size of the lesion being treated, cooling can be performed for up to about 5 minutes or more). The ice ball is passively thawed (no electrical or gas heating is required). As discussed herein, the thawing and dissipation of the ice ball can be seen using typical imaging methods practiced in the art. When the ice ball has completely dissipated, the infusion needle is advanced down the cryoprobe and into the cryolysis treatment area.

[0304] Example 11: Intratumoral injection and infusion of SV-101 or SV-102

[0305] This example describes an exemplary intratumoral administration of an immunotherapeutic combination formulation. After a partial tumor cryolysis treatment zone has been established within a selected tumor (e.g., as described in Example 9 and / or Example 10), an infusion needle (which is part of a coaxial cryoprobe / infusion needle system (SCINS)) is advanced under precise image guidance until the outer coaxial infusion needle tip is located in the approximate center of the cryolysis treatment zone.

[0306] Once this is accomplished, the cryoprobe is withdrawn from the SCINS, leaving the external coaxial infusion needle tip approximately centered within the cryolysis treatment zone in the tumor and ready for drug infusion.

[0307] Prepare the drug formulation SV-101 or SV-102 to a final volume of 15 mL of infusion solution and place it in a syringe suitable for a syringe infusion pump. Mount the syringe containing the drug on the pump, connect the low-infusion volume connecting tube (such as MEDLINE 60" extension set with microbore tubing) to the syringe via a one-way stopcock, and prime the system with the drug by pushing the syringe pump until the drug is seen flowing out of the connecting tube. Then, connect the prepared connecting tube to the SCINS infusion needle. Set the flow rate of the syringe pump for infusion to 3 mL / min. Do not set the overpressure alarm. Then, start the pump and inject a total volume of 15 ml within 5 minutes.

[0308] Take care to ensure that the infusion needle tip remains in the correct position and that no drug flows back down the needle tract. When the drug infusion pump has completed its infusion cycle and delivered 15 mL of drug from the syringe to the connecting tube, close the one-way stopcock. At this point, the volume of drug remaining in the connecting tube is 0.6 mL. This volume of drug should now be pushed by removing the one-way stopcock from the infusion syringe and injecting 0.6 mL of sterile saline into the one-way stopcock.

[0309] Example 12: Treatment of mCRPC Patient #1 with SV-102

[0310] A male patient diagnosed with metastatic prostate cancer by biopsy and bone scan had received 6 courses of chemotherapy and hormone blockers (enzalutamide, abiraterone, docetaxel), as well as radiotherapy for bone metastases. The patient did not respond and showed progression of the disease. Before treatment, PET / CT showed a prostate mass and multiple SUV-positive sclerotic bone lesions. The prostate lesions were first treated using the method provided by the present disclosure (see, e.g., Examples 9-11), including a first round of cryolysis and injection of high-dose levels of SV-102 (see Example 7 and below). Two months after the first treatment, an MRI scan showed no signs of active disease in the prostate (Figure 18).

[0311] The components of the SV-102 combination preparation include anti-CTLA4 antibody (YH001; its CDR sequence is as shown in SEQ ID NO: 21-26 and VH / VL sequence is as shown in SEQ ID NO: 34-35), anti-PD-1 antibody (609A; its CDR sequence is as shown in SEQ ID NO: 15-20 and VH / VL sequence is as shown in SEQ ID NO: 32-33), anti-CD40 antibody (YH003; its CDR sequence is as shown in SEQ ID NO: 3-8 and VH / VL sequence is as shown in SEQ ID NO: 28-29) and CpG ODN as shown in SEQ ID NO: 1. The dosage of each component is as shown in Example 7.

[0312] Example 13: Treatment of mCRPC Patient #2 with SV-102

[0313] A male patient diagnosed with metastatic prostate cancer by MRI and prostate biopsy was treated with irreversible electroporation of prostate lesions. After 2.5 years, his PSA increased to 12.8ng / mL, and PET / CT showed a 4×3cm mass with extracapsular invasion. The patient had received 10 courses of radiation therapy for bone metastasis, and six months of hormone therapy (Leuprorelin). The patient did not show a response and PET / CT examination showed more bone metastases, and before treatment, a prostate mass and multiple SUV-positive sclerotic bone lesions were shown. The method provided by the present disclosure was used to treat prostate lesions for the first time (see, for example, Examples 9-11), including the first round of cryolysis and injection of high-dose levels of SV-102 (see Example 7 and below). In the first follow-up examination after 5 weeks, the PSA level dropped to 8.56ng / mL, indicating that the treatment was responsive.

[0314] The components of the SV-102 combination preparation include anti-CTLA4 antibody (YH001; its CDR sequence is as shown in SEQ ID NO: 21-26 and VH / VL sequence is as shown in SEQ ID NO: 34-35), anti-PD-1 antibody (609A; its CDR sequence is as shown in SEQ ID NO: 15-20 and VH / VL sequence is as shown in SEQ ID NO: 32-33), anti-CD40 antibody (YH003; its CDR sequence is as shown in SEQ ID NO: 3-8 and VH / VL sequence is as shown in SEQ ID NO: 28-29) and CpG ODN as shown in SEQ ID NO: 1. The dosage of each component is as shown in Example 7.

[0315] Example 14: Treatment of mCRPC Patient #3 with SV-102

[0316] A male patient diagnosed with metastatic prostate cancer by prostate biopsy (Gleason score of 8 and 9) and MRI (5×3 prostate mass with extracapsular invasion) had a PSA of 2.97 ng / mL and had not received previous treatment. The prostate lesions were first treated using the methods provided by the present disclosure (see, e.g., Examples 9-11), including a first round of cryothawing and injection of high-dose levels of SV-102 (see Example 7 et seq.). At the first follow-up examination 5 weeks later, the PSA level dropped to 0.19 ng / mL, indicating a response to treatment.

[0317] The components of the SV-102 combination preparation include anti-CTLA4 antibody (YH001; its CDR sequence is as shown in SEQ ID NO: 21-26 and VH / VL sequence is as shown in SEQ ID NO: 34-35), anti-PD-1 antibody (609A; its CDR sequence is as shown in SEQ ID NO: 15-20 and VH / VL sequence is as shown in SEQ ID NO: 32-33), anti-CD40 antibody (YH003; its CDR sequence is as shown in SEQ ID NO: 3-8 and VH / VL sequence is as shown in SEQ ID NO: 28-29) and CpG ODN as shown in SEQ ID NO: 1. The dosage of each component is as shown in Example 7.

[0318] Example 15: Treatment of metastatic breast cancer patient #4 with SV-101

[0319] A female patient has stage 4 breast cancer that has metastasized to the lungs, pelvis, chest, and lymph nodes (invasive ductal carcinoma, intermediate nuclear grade, ER+, PR+, HER2 / NEU-, Ki-67+). The patient had extensive lung metastases and a large amount of pleural effusion. The patient was treated using the methods...

Claims

1. A method for treating cancer in a subject in need thereof, the method comprising: a) administering to the subject's tumor a therapy that causes tumor cell lysis; as well as b) administering to said subject a therapeutically effective amount of the combined preparation, Wherein the combined preparation comprises: a. TLR9 agonist; b. Agonistic anti-CD40 monoclonal antibody; c. anti-CTLA4 monoclonal antibody; and d. Agonistic anti-OX40 monoclonal antibody or anti-PD1 monoclonal antibody.

2. The method of claim 1, wherein the combined preparation comprises: a. the TLR9 agonist; b. the agonistic anti-CD40 monoclonal antibody; c. the agonistic anti-OX40 monoclonal antibody; and d. The anti-CTLA4 monoclonal antibody.

3. The method of claim 1 or claim 2, wherein the combined preparation consists of: a. the TLR9 agonist; b. the agonistic anti-CD40 monoclonal antibody; c. the agonistic anti-OX40 monoclonal antibody; and d. The anti-CTLA4 monoclonal antibody.

4. The method of claim 1, wherein the combined preparation comprises: a. the TLR9 agonist; b. the agonistic anti-CD40 monoclonal antibody; c. the anti-PD1 monoclonal antibody; and d. The anti-CTLA4 monoclonal antibody.

5. The method of claim 1 or claim 4, wherein the combined preparation consists of: a. the TLR9 agonist; b. the agonistic anti-CD40 monoclonal antibody; c. the anti-PD1 monoclonal antibody; and d. The anti-CTLA4 monoclonal antibody.

6. The method of any one of claims 1-3, wherein the TLR9 agonist, the agonistic anti-CD40 monoclonal antibody, the agonistic anti-OX40 monoclonal antibody, and the anti-CTLA4 monoclonal antibody are co-administered.

7. The method of any one of claims 1-3, wherein the TLR9 agonist, the agonistic anti-CD40 monoclonal antibody, the agonistic anti-OX40 monoclonal antibody, and the anti-CTLA4 monoclonal antibody are administered sequentially.

8. The method of any one of claims 1-3, wherein the TLR9 agonist is administered as a separate composition sequentially with the composition comprising the agonistic anti-CD40 monoclonal antibody, the agonistic anti-OX40 monoclonal antibody, and the anti-CTLA4 monoclonal antibody.

9. The method of any one of claims 1, 4 or 5, wherein the TLR9 agonist, the agonistic anti-CD40 monoclonal antibody, the anti-PD1 monoclonal antibody and the anti-CTLA4 monoclonal antibody are co-administered.

10. The method of any one of claims 1, 4 or 5, wherein the TLR9 agonist, the agonistic anti-CD40 monoclonal antibody, the anti-PD1 monoclonal antibody and the anti-CTLA4 monoclonal antibody are administered sequentially.

11. The method of any one of claims 1, 4 or 5, wherein the TLR9 agonist is administered sequentially as a separate composition with the composition comprising the agonistic anti-CD40 monoclonal antibody, the anti-PD1 monoclonal antibody and the anti-CTLA4 monoclonal antibody.

12. The method of any one of claims 1-11, wherein the therapy administered to the tumor is cryolysis therapy by cryolysis-mediated tumor cell lysis.

13. The method of claim 12, wherein the administration of cryolysis therapy comprises at least one, at least two, or at least three freeze-thaw cycles.

14. The method of claim 12 or claim 13, wherein the administration of cryolysis therapy comprises one freeze-thaw cycle.

15. The method of claim 12 or claim 13, wherein the administration of cryolysis therapy comprises two freeze-thaw cycles.

16. The method of claim 12 or claim 13, wherein the administration of cryolysis therapy comprises no more than one, no more than two, or no more than three freeze-thaw cycles.

17. The method of any one of claims 12-16, wherein the cryolysis therapy is mediated by an intratumoral cryolysis device comprising a cryoprobe for contacting and freezing tumor cells during freeze-thaw cycles.

18. The method of claim 17, wherein a cryogen is circulated within the cryoprobe.

19. The method of claim 18, wherein the cryogen is selected from the group consisting of argon, nitrous oxide, carbon dioxide, and liquid nitrogen.

20. The method of any of claims 17-19, wherein the intratumoral cryolysis device is set to a 100% duty cycle and operated during a freeze-thaw cycle to produce a cryoprobe temperature of -40°C or colder in less than about one minute, followed by a passive thawing step.

21. The method of claim 20, wherein the cryoprobe temperature produced is: a. Maintain for approximately two minutes, followed by passive thawing; or b. Continue cooling to a temperature of about -70°C or colder, about -80°C or colder, about -90°C or colder, about -100°C or colder, about -110°C or colder, about -120°C or colder, about -130°C or colder, about -140°C or colder, or about -150°C or colder for a total time 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.

22. The method of any one of claims 17-21, wherein the cryoprobe is cooled to a temperature that produces an ice ball within the tumor having a diameter of about 14 mm.

23. The method of claim 22, wherein the temperature of a majority of the ice ball is about -40°C or colder.

24. The method of claim 22 or claim 23, wherein the ice ball comprises a region of about 1 cm diameter, the temperature within the region being about -40°C or colder.

25. The method of any one of claims 12-24, wherein the cryolysis therapy does not ablate the entire tumor, but rather causes lysis of a portion of the tumor cells.

26. The method of any one of claims 12-25, wherein the cryolysis therapy causes necrotic cell death in a region of tumor tissue, wherein the diameter of the region is about 14 mm.

27. The method of any one of claims 1-26, wherein the TLR9 agonist is a class B or C CpG ODN.

28. The method of any one of claims 1-27, wherein the TLR9 agonist is a class B CpG ODN.

29. The method of any one of claims 1-27, wherein the TLR9 agonist is a C-class CpG ODN.

30. The method of any one of claims 1-29, wherein the TLR9 agonist comprises a nucleic acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

27.

31. The method of any one of claims 1-30, wherein the TLR9 agonist is a nucleic acid as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

27.

32. The method of any one of claims 1-31, wherein the TLR9 agonist is a nucleic acid as shown in SEQ ID NO:

1.

33. The method of any one of claims 1-31, wherein the TLR9 agonist is a nucleic acid as shown in SEQ ID NO:

2.

34. The method of any one of claims 1-31, wherein the TLR9 agonist is a nucleic acid as shown in SEQ ID NO:

27.

35. The method of any one of claims 1-34, wherein the agonist 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 shown in SEQ ID NO:3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:5, and The light chain comprises: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

8.

36. The method of any one of claims 1-35, wherein the agonist anti-OX40 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 shown in SEQ ID NO:9, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:10, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:11, and The light chain comprises: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:12, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

14.

37. The method of any one of claims 1-35, wherein the anti-PD1 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 shown in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and The light chain comprises: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

20.

38. The method of any one of claims 1-37, wherein the anti-CTLA4 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 shown in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 23, and The light chain comprises: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

26.

39. The method of any one of claims 1-38, wherein the therapy that causes tumor cell lysis and the combined preparation are administered to the same tumor.

40. The method of any one of claims 1-38, wherein the therapy that causes tumor cell lysis and the combination formulation are administered to different tumors.

41. The method of any one of claims 1-40, wherein the treatment is repeated about every week, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, about every 9 weeks, about every 10 weeks, about every 11 weeks, about every 12 weeks, about every 13 weeks, about every 14 weeks, about every 15 weeks, or about every 16 weeks.

42. The method of any one of claims 1-41, wherein the treatment is repeated about every 3 to 16 weeks, about every 6 to 16 weeks, about every 8 to 16 weeks, about every 10 to 16 weeks, about every 12 to 16 weeks, about every 14 to 16 weeks, about every 3 to 14 weeks, about every 3 to 12 weeks, about every 3 to 10 weeks, about every 3 to 8 weeks, about every 3 to 6 weeks, or about every 3 to 4 weeks.

43. The method of any one of claims 1-42, wherein the treatment is repeated approximately every 4 to 8 weeks, approximately every 5 to 8 weeks, approximately every 6 to 8 weeks, approximately every 7 to 8 weeks, approximately every 4 to 7 weeks, approximately every 4 to 6 weeks, or approximately every 4 to 5 weeks.

44. The method of claim 43, wherein the treatment is repeated approximately every 4 to 8 weeks.

45. The method of any one of claims 1-44, wherein 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.

46. ​​The method of any one of claims 1-45, wherein 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.

47. The method of claim 46, wherein the treatment is repeated for 3 to 6 cycles.

48. The method of any one of claims 1-47, wherein the TLR9 agonist is administered at a dose ranging from 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.

49. The method of any one of claims 1-48, wherein the anti-CD40 agonist is administered at a dose ranging from 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 about 8 mg.

50. The method of any one of claims 1-49, wherein the anti-OX40 agonist is administered at a dose ranging from 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 about 8 mg.

51. The method of any one of claims 1-49, wherein the anti-PDl monoclonal antibody is administered at a dose ranging from 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.

52. The method of any one of claims 1-51, wherein the anti-CTLA4 monoclonal antibody is administered at a dose ranging from 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.

53. The method of any one of claims 1-52, wherein the TLR9 agonist, the anti-CD40 agonist antibody, the anti-OX40 agonist antibody, and the anti-CTLA4 antibody are administered at doses of about 1 mg, about 1 mg, about 1 mg, and about 1 mg, respectively.

54. The method of any one of claims 1-52, wherein the TLR9 agonist, the anti-CD40 agonist antibody, the anti-OX40 agonist antibody, and the anti-CTLA4 antibody are administered at doses of about 2 mg, about 5 mg, about 5 mg, and about 5 mg, respectively.

55. The method of any one of claims 1-52, wherein the TLR9 agonist, the anti-CD40 agonist antibody, the anti-OX40 agonist antibody, and the anti-CTLA4 antibody are administered at doses of about 3 mg, about 7.5 mg, about 7.5 mg, and about 15 mg, respectively.

56. The method of any one of claims 1-52, wherein the TLR9 agonist, the anti-CD40 agonist antibody, the anti-OX40 agonist antibody, and the anti-CTLA4 antibody are administered at doses of about 4 mg, about 10 mg, about 10 mg, and about 40 mg, respectively.

57. The method of any one of claims 1-52, wherein the TLR9 agonist, the anti-CD40 agonist antibody, the anti-PD1 antibody, and the anti-CTLA4 antibody are administered at doses of about 1 mg, about 1 mg, about 3 mg, and about 1 mg, respectively.

58. The method of any one of claims 1-52, wherein the TLR9 agonist, the anti-CD40 agonist antibody, the anti-PD1 antibody, and the anti-CTLA4 antibody are administered at doses of about 2 mg, about 5 mg, about 10 mg, and about 5 mg, respectively.

59. The method of any one of claims 1-52, wherein the TLR9 agonist, the anti-CD40 agonist antibody, the anti-PD1 antibody, and the anti-CTLA4 antibody are administered at doses of about 3 mg, about 7.5 mg, about 30 mg, and about 15 mg, respectively.

60. The method of any one of claims 1-52, wherein the TLR9 agonist, the anti-CD40 agonist antibody, the anti-PD1 antibody, and the anti-CTLA4 antibody are administered at doses of about 4 mg, about 10 mg, about 100 mg, and about 40 mg, respectively; or about 4 mg, about 10 mg, about 80 mg, and about 40 mg.

61. The method of any one of claims 1-60, wherein the combined formulation is administered in a total volume ranging from about 1 mL to about 30 mL.

62. The method of claim 61, wherein the total volume is about 15 mL.

63. The method of any one of claims 1-62, wherein the cancer is a solid tumor cancer.

64. The method of claim 63, wherein 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 carcinoma, retinoblastoma, sarcoma, small cell lung cancer, squamous cell carcinoma (including head and neck cancer), gastric cancer, testicular cancer, thyroid cancer, and Wilms' tumor.

65. The method of claim 64, wherein the cancer is metastatic prostate cancer.

66. The method of claim 64, wherein the cancer is metastatic breast cancer.

67. The method of claim 64, wherein the cancer is non-small cell lung cancer.

68. A pharmaceutical composition comprising: a. TLR9 agonist; b. Agonistic anti-CD40 monoclonal antibody; c. Anti-CTLA4 monoclonal antibody; d. Agonistic anti-OX40 monoclonal antibodies; and e. Pharmaceutically acceptable excipients.

69. A pharmaceutical composition, the pharmaceutical composition consisting of: a. TLR9 agonist; b. Agonistic anti-CD40 monoclonal antibody; c. Anti-CTLA4 monoclonal antibody; d. Agonistic anti-OX40 monoclonal antibodies; and e. Pharmaceutically acceptable excipients.

70. The pharmaceutical composition of claim 68 or claim 69, wherein: a. The TLR9 agonist is a nucleic acid as shown 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, wherein the heavy chain comprises: a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:8; c. the agonist anti-OX40 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 shown in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, and wherein the light chain comprises: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 14; and d. the anti-CTLA4 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 shown in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 23, and The light chain comprises: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

26.

71. The pharmaceutical composition of any one of claims 68-70, wherein the dose of each of the TLR9 agonist, the anti-CD40 agonist antibody, the anti-OX40 agonist antibody, and the anti-CTLA4 antibody is selected from: a. about 1 mg, about 1 mg, about 1 mg, and about 1 mg, respectively; b. about 2 mg, about 5 mg, about 5 mg and about 5 mg, respectively; c. about 3 mg, about 7.5 mg, about 7.5 mg and about 15 mg, respectively; and d. about 4 mg, about 10 mg, about 10 mg and about 40 mg, respectively.

72. A pharmaceutical composition comprising: a. TLR9 agonist; b. Agonistic anti-CD40 monoclonal antibody; c. Anti-CTLA4 monoclonal antibody; d. agonistic anti-PD1 monoclonal antibodies; and e. Pharmaceutically acceptable excipients.

73. A pharmaceutical composition, the pharmaceutical composition consisting of: a. TLR9 agonist; b. Agonistic anti-CD40 monoclonal antibody; c. Anti-CTLA4 monoclonal antibody; d. agonistic anti-PD1 monoclonal antibodies; and e. Pharmaceutically acceptable excipients.

74. A pharmaceutical composition according to claim 72 or claim 73, wherein: a. The TLR9 agonist is a nucleic acid as shown 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, wherein the heavy chain comprises: a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 4, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, and wherein the light chain comprises: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:8; c. the anti-PD1 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 shown in SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 16, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 17, and wherein the light chain comprises: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 20; and d. the anti-CTLA4 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 shown in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 23, and The light chain comprises: a light chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO:24, a light chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

26.

75. A pharmaceutical composition as described in any one of claims 72-74, wherein the dosage of each of the TLR9 agonist, anti-CD40 agonist antibody, anti-PD1 antibody and anti-CTLA4 antibody is selected from: a. about 1 mg, about 1 mg, about 3 mg and about 1 mg respectively; b. about 2 mg, about 5 mg, about 10 mg and about 5 mg, respectively; c. about 3 mg, about 7.5 mg, about 30 mg and about 15 mg, respectively; d. about 4 mg, about 10 mg, about 80 mg and about 40 mg, respectively; and e. about 4 mg, about 10 mg, about 100 mg and about 40 mg, respectively.

76. A method of treating cancer in a subject, the method comprising the steps of: a) administering cryolysis therapy to the subject's tumor to cause partial tumor cell lysis; and b) administering a therapeutically effective amount of the combined preparation to the same tumor in step a), Wherein the combined preparation comprises: a. TLR9 agonist; b. Agonistic anti-CD40 monoclonal antibody; c. anti-CTLA4 monoclonal antibody; and d. Agonistic anti-OX40 monoclonal antibody; wherein the cryolysis therapy is administered prior to administering the combined preparation.

77. A method of treating cancer in a subject, the method comprising the steps of: a) administering cryolysis therapy to the subject's tumor to cause partial tumor cell lysis; and b) administering a therapeutically effective amount of the combined preparation to the same tumor in step a), Wherein the combined preparation comprises: a. TLR9 agonist; b. Agonistic anti-CD40 monoclonal antibody; c. anti-CTLA4 monoclonal antibody; and d. Anti-PD1 monoclonal antibody; wherein the cryolysis therapy is administered prior to administering the combined preparation.

78. The method of claim 76, wherein the combined preparation is a pharmaceutical composition of any one of claims 68-71.

79. The method of claim 77, wherein the combined preparation is a pharmaceutical composition of any one of claims 72-75.

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