CN117693350A

CN117693350A - Combination of checkpoint inhibitors with oncolytic viruses for the treatment of cancer

Info

Publication number: CN117693350A
Application number: CN202280050519.3A
Authority: CN
Inventors: 赛义达·达迪-穆罕默塔伊; 马库斯·莫尔斯; 加文·瑟斯顿
Original assignee: Veriard Co ltd; Regeneron Pharmaceuticals Inc
Current assignee: Veriard Co ltd; Regeneron Pharmaceuticals Inc
Priority date: 2021-07-19
Filing date: 2022-07-18
Publication date: 2024-03-12

Abstract

The present disclosure relates to novel triple combination therapies of oncolytic viruses, PD-1 pathway inhibitors and CTLA4 inhibitors for treating tumors or inhibiting tumor growth in patients suffering from cancer.

Description

Combination of checkpoint inhibitors with oncolytic viruses for the treatment of cancer

Technical Field

The present disclosure relates generally to combination therapies for cancer treatment with an oncolytic virus and a checkpoint inhibitor, such as a programmed death 1 (pd-1) pathway inhibitor and a cytotoxic T lymphocyte antigen-4 (cytotoc T-lymphocyte antigen-4, ctla 4) inhibitor.

Background

Until recently, cancer immunotherapy has focused on methods of enhancing anti-tumor immune responses by adoptive transfer of activated effector cells, immunization against the relevant antigen, or provision of non-specific immunostimulants (e.g., cytokines). However, over the last decade, a great deal of effort to develop specific immune checkpoint pathway inhibitors has begun to provide new immunotherapeutic approaches for the treatment of cancer, including the development of anti-PD-1 antibodies and anti-CTLA 4 antibodies.

PD-1 (also known as CD 279) plays an important role in autoimmunity, immunity against infection and anti-tumor immunity. Blocking of PD-1 with antagonists (including monoclonal antibodies) has been studied in the treatment of cancer and chronic viral infections. Blockade of PD-1 is also an effective and well-tolerated method to stimulate immune responses, and therapeutic advantages have been achieved for a variety of human cancers, including melanoma, renal cell carcinoma (renal cell cancer, RCC) and non-small cell lung carcinoma (non-small cell lung cancer, NSCLC). (Sheridan 2012, nat. Biotechnol.,30:729-730; poston et al, 2015,J Clin Oncol,33:1974-1982).

CTLA4 (also known as CD 152) is a type I transmembrane T cell inhibitory checkpoint receptor expressed on normal and regulatory T cells. CTLA4 down-regulates T cell activation by outcompeting the binding of the stimulatory receptor CD28 to its cognate natural ligands B7-1 (CD 80) and B7-2 (CD 86).

Initial T cell activation is achieved by stimulating a T Cell Receptor (TCR) that recognizes specific peptides presented by major histocompatibility complex class I or class II (major histocompatibility complex class I or II, MHCI or MHCII) proteins on antigen presenting cells (Goldrate et al 1999, nature 402:255-262). The activated TCR complex in turn initiates a cascade of signaling events driven by promoters that regulate the expression of a variety of transcription factors, such as activator-protein 1, ap-1, nuclear factor of activated T cells (Nuclear Factor of Activated T-cell, NFAT), or nuclear factor kappa-light chain enhancer of activated B cells (nuclear factor kappa-light-chain-enhancer of activated B cell, NF-kappa-B). The T cell response is then further modulated by participation of co-stimulatory or co-inhibitory receptors constitutively or inducibly expressed on the T cells, such as CD28, CTLA4, PD-1, lymphocyte Activation Gene 3 (LAG-3) or other molecules (Sharpe et al 2002, nat. Rev. Immunol. 2:116-126).

Oncolytic viruses are also promising for the treatment of cancer. These viruses infect, replicate specifically in and kill malignant cells, while normal tissues are unaffected. A variety of oncolytic viruses have reached a later stage for clinical evaluation of treatment of a variety of tumors. However, immunosuppression of tumors and premature clearance of viruses often result in only weak tumor-specific immune responses, limiting the potential of these viruses as cancer therapeutics.

Thus, there is a strong need for more effective therapies for cancer treatments, including combination therapies comprising an oncolytic virus and a checkpoint inhibitor (e.g., a PD-1 pathway inhibitor and a CTLA4 inhibitor) as disclosed herein.

Summary of The Invention

In one aspect, the disclosed technology relates to a method of treating a tumor or inhibiting tumor growth, comprising: (a) selecting a patient with cancer; and (b) administering to the patient in need thereof: a combination of (i) a therapeutically effective amount of an oncolytic virus and (ii) a therapeutically effective amount of an inhibitor of the programmed death 1 (PD-1) pathway and (iii) a therapeutically effective amount of an inhibitor of the cytotoxic T lymphocyte antigen-4 (CTLA 4). In some embodiments, the oncolytic virus comprises an oncolytic vesicular virus. In some embodiments, the oncolytic vesicular virus comprises an oncolytic vesicular stomatitis virus (vesicular stomatitis virus, VSV). In some embodiments, the VSV comprises a recombinant VSV. In some embodiments, the recombinant VSV comprises one or more mutations, e.g., an M51R substitution. In some embodiments, the recombinant VSV expresses a cytokine. In some embodiments, the recombinant VSV comprises a nucleic acid sequence encoding an immunostimulatory molecule (e.g., a cytokine).

In some embodiments, the cytokine comprises interferon-beta (IFNb), such as human or mouse IFNb or variants thereof. In some embodiments, the nucleic acid sequence encoding the IFNb is located between M and G viral genes.

In some embodiments, the recombinant VSV further expresses sodium/iodine symporter (NIS). In some embodiments, the recombinant VSV further comprises a nucleic acid sequence encoding a sodium/iodine symporter (NIS) or variant thereof. In some embodiments, the nucleic acid sequence encoding NIS is located between G and L viral genes. In some embodiments, the oncolytic virus is Voyager V1. In some embodiments, the oncolytic virus, the PD-1 pathway inhibitor, and the CTLA4 inhibitor are administered to the patient simultaneously. In some embodiments, one or more doses of the oncolytic virus are administered in sequence in combination with one or more doses of the PD-1 pathway inhibitor and one or more doses of the CTLA4 inhibitor.

In some embodiments, the oncolytic virus is administered to the patient before or after the PD-1 pathway inhibitor and/or CTLA4 inhibitor. In some embodiments, the PD-1 pathway inhibitor is administered to the patient before or after the oncolytic virus and/or CTLA4 inhibitor. In some embodiments, the CTLA4 inhibitor is administered to the patient before or after the oncolytic virus and/or PD-1 pathway inhibitor. In some embodiments, at least one of an oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor is administered to the patient once daily, once every two days, once every three days, once every four days, once every five days, once weekly, once every two weeks, or once every three weeks. In some embodiments, the dose of oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor is administered to the patient 1 day to 12 weeks after the immediately preceding dose (immediately preceding dose) of oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor, respectively.

In some embodiments, one or more doses of CTLA4 inhibitor comprise a single dose of the CTLA4 inhibitor, and wherein administration of a single dose of the CTLA4 inhibitor results in an anti-tumor efficacy comparable to that of a combination therapy comprising two or more doses of the CTLA4 inhibitor.

In some embodiments, the anti-tumor efficacy is characterized by the following decreases: tumor volume mean or average tumor volume, percent survival, number of tumor-free patients in each treatment group, or a combination thereof. In some embodiments, the oncolytic virus is administered to the patient in one or more of the following doses: 10 ⁴ To 10 ¹⁴ TCID ₅₀ (50% Tissue Culture Infectious Dose,50% tissue culture infection dose), 10% ⁴ To 10 ¹² TCID ₅₀ 、10 ⁶ To 10 ¹² TCID ₅₀ 、10 ⁸ To 10 ¹⁴ TCID ₅₀ 、10 ⁸ To 10 ¹² TCID ₅₀ Or 10 ¹⁰ To 10 ¹² TCID ₅₀ . In some embodiments, the PD-1 pathway inhibitor is administered to the patient in one or more doses of about 0.1mg/kg to about 20mg/kg of patient body weight. In some embodiments, the PD-1 pathway inhibitor is administered to a patient in one or more doses of about 1mg to about 1000 mg. In some embodiments, the CTLA4 inhibitor is administered to the patient at one or more doses of about 0.1mg/kg to about 15mg/kg of patient body weight. In some embodiments, the The CTLA4 inhibitor is administered to the patient in a single dose of about 0.1mg/kg to about 15mg/kg of patient body weight. In some embodiments, the CTLA4 inhibitor is administered to a patient in one or more doses of about 1mg to about 600 mg. In some embodiments, the oncolytic virus is administered intratumorally or intravenously to a patient. In some embodiments, the PD-1 pathway inhibitor and the CTLA4 inhibitor are administered to a patient intravenously, subcutaneously, or intraperitoneally.

In some embodiments, the cancer is selected from adrenal tumor, bile duct cancer, bladder cancer, brain cancer, breast cancer, epithelial cancer, cancer of central or peripheral nervous system tissue, cervical cancer, colon cancer, cancer of the endocrine or neuroendocrine or hematopoietic system, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, li-Fraumeni tumor (Li-Fraumeni tumor), liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple neuroendocrine type I and type II tumors, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma tumor, ovarian cancer, pancreatic cancer, islet cell cancer, parathyroid cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, renal cancer, respiratory cancer, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, tracheal cancer, genitourinary cancer, and uterine cancer. In some embodiments, the cancer is resistant to treatment with at least one anti-PD-1 agent or treatment.

In some embodiments, the PD-1 pathway inhibitor comprises an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, or an anti-PD-L2 antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of cimetidine Li Shan antibody, nivolumab, pembrolizumab (pembrolizumab), picolizumab (pidilizumab), MEDI0608, BI 754091, PF-06801591, stadazumab (spartalizumab), carilizumab (camrelizumab), JNJ-63723283, and MCLA-134.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (heavy chain complementarity determining region, HCDR) comprising the heavy chain variable region (heavy chain variable region, HCVR) of the amino acid sequence of SEQ ID No. 1 and a light chain complementarity determining region (light chain complementarity determining region, LCDR) comprising the light chain variable region (light chain variable region, LCVR) of the amino acid sequence of SEQ ID No. 2. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) (HCDR 1, HCDR2 and HCDR 3) comprising the corresponding amino acid sequences of SEQ ID NOs 3, 4 and 5; and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) comprising the corresponding amino acid sequences of SEQ ID NOS: 6, 7 and 8.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) comprising the amino acid sequence of SEQ ID No. 1; and a Light Chain Variable Region (LCVR) comprising the amino acid sequence of SEQ ID NO. 2. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy and light chain sequence pair of SEQ ID NOs 9 and 10.

In some embodiments, the anti-PD-L1 antibody is selected from REGN3504, aviumab (avelumab), atezolizumab (atezolizumab), duvalumab (durvalumab), MDX-1105, LY3300054, FAZ053, STI-1014, CX-072, KN035, and CK-301. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) comprising the amino acid sequence of SEQ ID NO. 11; and a Light Chain Variable Region (LCVR) comprising the amino acid sequence of SEQ ID NO. 12. In some embodiments, the anti-PD-L1 antibody comprises REGN3504.

In some embodiments, the CTLA4 inhibitor comprises an anti-CTLA 4 antibody or antigen-binding fragment thereof. In some embodiments, the anti-CTLA 4 antibody is selected from ipilimumab (ipilimumab), tremelimumab (tremelimumab), and REGN4659. In some embodiments, the anti-CTLA 4 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (HCDR) comprising a Heavy Chain Variable Region (HCVR) of the amino acid sequence of SEQ ID No. 13 and a light chain complementarity determining region (LCDR) comprising a Light Chain Variable Region (LCVR) of the amino acid sequence of SEQ ID No. 14.

In some embodiments, the anti-CTLA 4 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR) (HCDR 1, HCDR2 and HCDR 3) comprising the corresponding amino acid sequences of SEQ ID NOs 15, 16 and 17; and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) comprising the corresponding amino acid sequences of SEQ ID NOS: 18, 19 and 20. In some embodiments, the anti-CTLA 4 antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) comprising the amino acid sequence of SEQ ID No. 13; and a Light Chain Variable Region (LCVR) comprising the amino acid sequence of SEQ ID NO. 14. In some embodiments, the anti-CTLA 4 antibodies or antigen-binding fragments thereof comprise pairs of heavy and light chain sequences of SEQ ID NOs 21 and 22.

In some embodiments, the treatment produces a therapeutic effect selected from one or more of the following: delay in tumor growth, reduced tumor cell number, tumor regression, increased survival, partial response, and complete response. In some embodiments, the tumor growth is inhibited by at least 50% compared to an untreated patient. In some embodiments, the tumor growth is inhibited by at least 50% as compared to a patient administered the oncolytic virus, the PD-1 pathway inhibitor, or the CTLA4 inhibitor as monotherapy. In some embodiments, the tumor growth is inhibited by at least 50% as compared to a patient administered any two of the oncolytic virus, the PD-1 pathway inhibitor, and the CTLA4 inhibitor.

In some embodiments, the method further comprises administering an additional therapeutic agent or treatment to the patient. In some embodiments, the additional therapeutic agent or treatment is selected from the group consisting of: radiation, surgery, chemotherapeutics, cancer vaccines, B7-H3 inhibitors, B7-H4 inhibitors, lymphocyte activation gene 3 (lymphocyte activation gene, LAG 3) inhibitors, inhibitors containing T-cell immunoglobulin and mucin domain-3 (T cell immunoglobulin and mucin-domain containing-3, TIM 3), galectin 9 (galectin 9, GAL 9) inhibitors, inhibitors of T-cell activated V-domain containing immunoglobulin (Ig) inhibitors (V-domain immunoglobulin-containing suppressor of T-cell activation, VISTA), killer cell immunoglobulin-Like Receptor (Killer-Cell Immunoglobulin-Like Receptor, KIR) inhibitors, B and T-lymphocyte attenuators (B and T lymphocyte attenuator, BTLA) inhibitors, T-cell immune Receptor (TIGIT) inhibitors with Ig and ITIM domains, CD47 inhibitors, indoleamine-2, 3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (vascular endothelial growth factor, VEGF) antagonists, angiopoietin-2 (ang2) inhibitors, transforming growth factor beta (transforming growth factor beta, tgfp) inhibitors, epidermal growth factor Receptor (epidermal growth factor Receptor, EGFR) inhibitors, antibodies to tumor-specific antigens, bcg (Bacillus Calmette-Guerin vaccinee), granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony-stimulating factor, GM-CSF), cytotoxins, interleukin 6Receptor (interleukin 6 Receptor), IL-6R) inhibitors, interleukin 4receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-12, IL-21, IL-15, antibody-drug conjugates, anti-inflammatory drugs, and combinations thereof.

In another aspect, the disclosed technology relates to a combination of an oncolytic virus, a PD-1 pathway inhibitor, and a CTLA4 inhibitor for use in a method of treating a tumor or inhibiting tumor growth, the method comprising: (a) selecting a patient with cancer; and (b) administering to a patient in need thereof: a combination of (i) a therapeutically effective amount of an oncolytic virus with (ii) a therapeutically effective amount of a PD-1 pathway inhibitor and (iii) a therapeutically effective amount of a CTLA inhibitor.

In another aspect, the disclosed technology relates to a kit comprising a combination of an oncolytic virus, a PD-1 pathway inhibitor, and a CTLA4 inhibitor, and written instructions for using a therapeutically effective amount of a combination of the oncolytic virus, the PD-1 pathway inhibitor, and the CTLA4 inhibitor to treat a tumor or inhibit tumor growth in a patient.

The foregoing summary is not intended to limit each aspect of the disclosure, and additional aspects are described in other sections, such as in the detailed description below. The entire document is intended to be associated as a unified disclosure and it should be understood that all combinations of features described herein are contemplated even if they do not exist together in the same sentence, or paragraph, or portion of the document. Other features and advantages of the present invention will become apparent from the following detailed description of the invention. It should be understood, however, that the detailed description and the specific examples, while indicating some specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Brief Description of Drawings

Fig. 1 is a diagram showing the following: is carried by 150mm ³ The antitumor efficacy of treatment with a combination of anti-PD-1, anti-CTLA 4 and intratumoral delivery of oncolytic virus VSV-M51R-Fluc in mice with average MC38 tumors is as described in example 1. The mean tumor volume (mm) in each treatment group at time points after implantation of multiple tumors is shown ³ +/-SEM), where treatment days are indicated by arrows, as described in example 1.

Fig. 2 is a diagram showing the following: individual tumor volumes at day 11 after treatment initiation (at day 26 after tumor implantation) for each treatment group described in example 1.

FIG. 3 is a graph showing the Kaplan-Meier survival curves for each of the treatment groups described in example 1.

Fig. 4A, 4B, 4C, 4D, and 4E are atlases showing the following: the anti-tumor efficacy of triple combination anti-PD-1, anti-CTLA 4 and intratumorally delivered oncolytic virus VSV-M51R-GFP can be achieved with only one dose of anti-CTLA 4 mIgG2a antibody, as described in example 2. Figure 4A shows the average tumor volume in PBS-treated groups at time points after multiple tumor implants. Figure 4B shows the average tumor volumes in PBS, anti-PD-1 antibody and anti-CTLA 4 antibody (4 doses) treated groups at time points after multiple tumor implants. Figure 4C shows the average tumor volumes in VSV, anti-PD-1 antibody and anti-CTLA 4 antibody (1 dose) treated groups at time points after multiple tumor implants. Figure 4D shows the average tumor volumes in VSV, anti-PD-1 antibody and anti-CTLA 4 antibody (2 doses) treated groups at time points after multiple tumor implants. Figure 4E shows the average tumor volumes in VSV IT, anti-PD-1 antibody and anti-CTLA 4 antibody (4 doses) treated groups at time points after multiple tumor implants. Days of treatment are indicated by arrows. TF: no tumor.

Fig. 5 is a graph showing Kaplan-Meier survival curves for each treatment group described in example 2.

Fig. 6 is a diagram showing the following: the anti-tumor efficacy of the triple combination anti-PD-1, anti-CTLA 4 and oncolytic virus VSV-M51R-GFP can be achieved with intratumoral or intravenous delivery of the virus, as described in example 3. The mean tumor volume (mm) in each treatment group at time points after implantation of multiple tumors is shown ³ +/-SEM). The treatment was applied as described in table 5 and example 3.

Fig. 7 is a graph showing Kaplan-Meier survival curves for each treatment group described in example 3.

Fig. 8 is a diagram showing the following: is carried by 150mm ³ The antitumor efficacy of treatment with a combination of anti-PD-1, anti-CTLA 4 and intravenous delivery of oncolytic virus VSV-mIFNb-NIS in mice with MC38 tumors was averaged as described in example 4. The mean tumor volume (mm) in each treatment group at time points after implantation of multiple tumors is shown ³ +/-SEM). The treatment was applied as described in table 7 and example 4.

Fig. 9 is a diagram showing the following: individual tumor volumes at day 10 after treatment initiation for each treatment group described in example 4. Statistical significance (< 0.01, <0.0001, < p) was determined by one-way ANOVA and Dunnett multiple comparison post hoc test.

Fig. 10 is a graph showing Kaplan-Meier survival curves for each treatment group described in example 4.

Fig. 11 is a diagram showing the following: average tumor volume (mm) in each treatment group described in example 5 at multiple post-tumor implantation time points ³ +/-SEM), where treatment days are indicated by arrows.

Fig. 12 is a diagram showing the following: individual tumor volumes at day 29 after treatment initiation for each treatment group described in example 5.

Fig. 13 is a graph showing Kaplan-Meier survival curves for each treatment group described in example 5.

Fig. 14 is a diagram showing the following: average tumor volume (mm) in each treatment group described in example 6 at multiple post-tumor implantation time points ³ +/-SEM), where treatment days are indicated by arrows.

Fig. 15 is a diagram showing the following: individual tumor volumes at day 22 after tumor implantation (10 days after treatment initiation) for each treatment group described in example 6.

FIG. 16 is a graph showing the Kaplan-Meier survival curves for each of the treatment groups described in example 6.

Fig. 17 is a diagram showing the following: average tumor volume (mm) in each treatment group described in example 7 at multiple post-tumor implantation time points ³ +/-SEM), where treatment days are indicated by arrows.

Fig. 18 is a diagram showing the following: average tumor volume (mm) in each treatment group described in example 8 at multiple post-tumor implantation time points ³ +/-SEM), where treatment days are indicated by arrows.

Fig. 19 is a diagram showing the following: average tumor volume (mm) in each treatment group described in example 9 at multiple post-tumor implantation time points ³ +/-SEM)。

Fig. 20 is a diagram showing the following: average plaque forming units of IFNg released from CD8 TIL harvested from tumors and again overnight exposed to the indicated tumor antigen or VSV-NP on day 12 and on day 17 in each of the treatment groups described in example 10 after receiving VSV on day 12 and two doses of anti-PD-1 and a-CTLA4 on day 12 and 14 (spot forming unit, SFU). DMSO and PMA/ionomycin were used as negative and positive controls, respectively, at time points after implantation of multiple tumors, with treatment days indicated by arrows.

Detailed Description

The present disclosure is based, at least in part, on the unexpected discovery that: the novel triple combination therapy of an oncolytic virus, a programmed death 1 (PD-1) pathway inhibitor and a cytotoxic T lymphocyte antigen-4 (CTLA 4) inhibitor exhibits synergistic activity in inhibiting tumor growth compared to any monotherapy or double combination therapy of an oncolytic virus, a PD-1 pathway inhibitor and a CTLA4 inhibitor. As demonstrated herein, the disclosed triple combination therapies comprising administering one dose of CTLA4 inhibitor achieve anti-tumor efficacy comparable to combination therapies comprising 2, 3, 4 or more doses of CTLA4 inhibitor. In addition, intravenous administration of oncolytic viruses is at least as effective as intratumoral administration of the virus. Thus, triple combination therapies as disclosed herein represent unexpectedly effective treatments for cancer treatment with reduced risk of treatment-related toxicity.

Accordingly, in one aspect, the present disclosure provides a method of treating a tumor or inhibiting tumor growth comprising: (a) selecting a patient with cancer; and (b) administering to a patient in need thereof: a combination of (i) a therapeutically effective amount of an oncolytic virus with (ii) a therapeutically effective amount of a PD-1 pathway inhibitor (e.g., an anti-PD-1, anti-PD-L1, or anti-PD-L2 antibody, or antigen-binding fragment thereof) and (iii) a therapeutically effective amount of a CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof).

The term "patient" as used herein may be used interchangeably with the term "subject". The expression "subject in need thereof" means a human or non-human mammal that exhibits one or more symptoms or indications of cancer and/or has been diagnosed with cancer. In some embodiments, a human subject may be diagnosed with a primary or metastatic tumor and/or with one or more symptoms or indications including, but not limited to: lymph node enlargement, abdominal swelling, chest pain/pressure, weight loss due to unknown reasons, fever, night sweat, sustained fatigue, anorexia, spleen enlargement, itching. The expression includes patients who have been subjected to one or more cycles of chemotherapy with toxic side effects. In some embodiments, the expression "a subject in need thereof" includes patients suffering from cancer who have been treated but who subsequently relapse or metastasis. For example, such patients are treated with the methods of the present disclosure: the patient may have received treatment with one or more anti-cancer agents, resulting in tumor regression, however, subsequently relapsed cancer that is resistant to the one or more anti-cancer agents (e.g., a chemotherapy-resistant cancer).

The term "treatment" and variations thereof as used herein means reducing or lessening the severity of at least one symptom or indication, eliminating the cause of symptoms on a temporary or permanent basis, delaying or inhibiting tumor growth, reducing tumor cell burden or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis and/or disappearance, preventing tumor recurrence, preventing or inhibiting metastasis, inhibiting metastatic tumor growth, eliminating the need for radiation or surgery, and/or increasing the duration of survival of a subject.

In many embodiments, the terms "tumor," "lesion," "neoplastic lesion," "cancer," and "malignancy" are used interchangeably and refer to one or more cancerous growths. In some embodiments, the cancer is selected from adrenal tumor, bile duct cancer, bladder cancer, brain cancer, breast cancer, epithelial cancer, cancer of central or peripheral nervous system tissue, cervical cancer, colon cancer, cancer of the endocrine or neuroendocrine or hematopoietic system, esophageal cancer, fibromas, gastrointestinal cancer, glioma, head and neck cancer, li-frernia tumor, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple neuroendocrine type I and type II tumors, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma tumor, ovarian cancer, pancreatic cancer, islet cell cancer, parathyroid cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, renal cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tracheal cancer, genitourinary cancer, and uterine cancer.

According to some embodiments, the present disclosure includes methods for treating tumors, delaying or inhibiting tumor growth. In some embodiments, the disclosure includes methods of promoting tumor regression. In some embodiments, the disclosure includes methods of reducing tumor cell burden or reducing tumor burden. In some embodiments, the disclosure includes methods of preventing tumor recurrence.

According to some embodiments, the methods of the present disclosure comprise administering to a subject in need thereof an oncolytic virus, a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof), or a CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof).

In some embodiments, the method comprises administering one or more doses of an oncolytic virus to a subject before, after, or simultaneously with administering one or more doses of a PD-1 pathway inhibitor and/or one or more doses of a CTLA4 inhibitor to the subject. In some embodiments, one or more doses of the PD-1 pathway inhibitor may be administered in combination with one or more doses of the CTLA4 inhibitor.

The term "in combination with … …" as used herein also includes sequential or simultaneous administration of an oncolytic virus, a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof), and a CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof). For example, when administered "prior to" the CTLA4 inhibitor, one or more doses of the PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof) can be administered more than about 12 weeks, about 11 weeks, about 10 weeks, about 9 weeks, about 8 weeks, about 7 weeks, about 6 weeks, about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, about 150 hours, about 100 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes prior to administration of the one or more doses of the CTLA inhibitor.

When administered "after" a CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof), a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof) can be administered about 12 weeks, about 11 weeks, about 10 weeks, about 9 weeks, about 8 weeks, about 7 weeks, about 6 weeks, about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, about 150 hours, about 100 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes after administration of the CTLA4 inhibitor.

By "simultaneous" administration with a CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof) is meant that the PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof) is administered to a subject in a separate dosage form within less than 10 minutes of administration of the CTLA4 inhibitor (prior to, subsequent to, or simultaneous to), or as a single combined dosage formulation comprising both the PD-1 pathway inhibitor and the CTLA4 inhibitor.

In some embodiments, the disclosed methods may further comprise administering an anti-tumor therapy. Antitumor therapies include, but are not limited to, conventional antitumor therapies, such as chemotherapy, radiation, surgery, or as described elsewhere herein.

In some embodiments, the treatment produces a therapeutic effect selected from one or more of the following: delay in tumor growth, reduced tumor cell number, tumor regression, increased survival, partial response, and complete response. In some embodiments, tumor growth in the patient is delayed by at least 10 days as compared to tumor growth in untreated patients. In some embodiments, tumor growth is inhibited by at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%) as compared to an untreated patient. In some embodiments, tumor growth is inhibited by at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%) as compared to a patient administered an oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor as monotherapy. In some embodiments, tumor growth is inhibited by at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%) as compared to a patient administered both the oncolytic virus, the PD-1 pathway inhibitor, and the CTLA4 inhibitor.

Oncolytic viruses

Oncolytic viruses are cancer treatments that use engineered or naturally evolved cancer-philic viruses to stimulate tumor cell death in the treated patient. In general, when oncolytic viruses are administered that are replicating, the infected tumor cells have the potential to produce progeny viruses that allow the spread of destructive infection to neighboring tumor cells. The potential for viral replication is determined by the ability of the cell to sense and respond to viral infection. In addition, oncolytic viruses carry pathogen-associated molecular patterns (PAMPs), which can act as adjuvants to stimulate myeloid cells (macrophages and dendritic cells) to enhance T cell stimulation.

In some embodiments, the oncolytic virus is replication competent oncolytic rhabdovirus (rhabdovirus). Such oncolytic rhabdoviruses include, but are not limited to, wild-type or genetically modified Arajas virus, chandiprara virus (Chandiura virus), kecal virus (Cocal virus), isfahan virus (Isfahan virus), maraba virus (Maraba virus), margarit virus (Piry virus), vesicular stomatitis Alangry virus (Vesicular stomatitis Alagoas virus), vesicular Stomatitis Virus (VSV), bean 157575virus (Bean 157575 virus), botek virus (Boteke virus), cal Cha Ji virus (Calcha virus), american eel virus (Eel virus American), gee Lei Luoji virus (Gray Lodge virus), zhu Luona virus (Jurona virus), keamas virus (Kamata virus), ketta virus (Kwatta virus), joya virus (Joya virus), pegya virus (Vesicular stomatitis Alagoas virus), hila virus (Ducheon virus (Ubban virus), hillla virus (In virus) (55), inpad virus (Inward virus) (Tara virus), and Inward virus (Tara virus), bayiya virus (Bahia Grande virus), moslarines virus (Muir Springs virus), reed Ranch virus, hart Park virus, fei Landu virus (Flanders virus), kamex virus (Kamese virus), mosqueiro virus, mo Suli virus (Mossuril virus), barur virus, fukuoka virus, crohn valley virus (Kern Canyon virus), enkebison virus (Nkolbisson virus), li Dante virus (Le Dantec virus), keuraliba virus, connecticut virus (Connecticut virus), new Minoto virus (New Minto virus), cyperus virus (Sawglass virus), chakka virus, sana Ma Dulei Lavirus (Sena Madureira virus), timbo virus (Timbo virus), armharwal virus (Almpiwar virus), arhaka virus (Aruac virus), bugoran virus (Bangoran virus), binbo virus (Bimbo virus), bi Wensi Arm virus (Bivens m virus), blue crab virus (Blue crab virus), sha Lewei Er virus (Charleville virus), carson plagion virus (Coastal Plains virus), dakurK 7292virus (DakurK 7292 virus) Entamoeba virus (Entamoeba virus), garba virus (Garba virus), gossas virus (Gossas virus), haplocalyx Du Bingdu (Humpty Doo virus), joinkaka virus (Joinjaka virus), kennaman virus (Kannamangalam virus), colon virus (Kolongo virus), kurthan Ping Ye virus (Koolpinyah virus), kotonkoku virus (Kotonkon virus), landeya virus (Landjia virus), hantolba virus (Manitoba virus), marcota virus (Marco virus), nasoule virus (Nasouls virus), navarrovirus (Navarrov), engahnjia virus (Ngaingan virus), oak-Vale viruses, obond Hiedge viruses (Obodhiams), dacron viruses (Oita viruses), au An Ge viruses (Ouango viruses), parry Crek viruses (Parry Crek viruses), reed-Paus virus (Rio Grande ci chlid vims), st.Joba viruses (Sandjimms), sigma viruses (Sigma viruses), st.Bull viruses (Sripur viruses), st.Witermate viruses (Sweetwater Branch vims), tibrogargan viruses (Tibrogargan viruses), sib Mars viruses (Xibura viruses), yata viruses, rhode Island, ardelad viruses (Adelaide River vims), bei Lima viruses (Berrimah viruses), kimberley viruses (Kimberley viruses) or transient fever viruses (Bovine ephemeral fever vims).

As described above, vesicular Stomatitis Virus (VSV) is a member of the Rhabdoviridae family (Rhabdoviridae family). The VSV genome is a single molecule of negative sense RNA that encodes 5 major polypeptides: nucleocapsid (N) polypeptides, phosphoprotein (P) polypeptides, matrix (M) polypeptides, glycoprotein (G) polypeptides and viral polymerase (L) polypeptides.

In some embodiments, the oncolytic virus is a wild-type or recombinant VSV. In some embodiments, the recombinant VSV comprises one or more mutations, such as an M51R substitution (also referred to herein as VSV-M51R).

In some embodiments, oncolytic viruses may be engineered to express one or more cytokines, such as interferon- β (IFNb). In some embodiments, the IFNb (e.g., interferon beta-1 a) can be human or mouse IFNb or a variant thereof. In some embodiments, the IFNb comprises an amino acid sequence having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID NO:23 or SEQ ID NO:24, or comprises the amino acid sequence of SEQ ID NO:23 or SEQ ID NO: 24. In some embodiments, the nucleic acid sequence encoding IFNb is located between M and G viral genes. Such a position allows the virus to express such amounts of IFNb polypeptide: the amount is effective to activate an antiviral immune response in non-cancerous tissue and thereby mitigate potential viral toxicity without interfering with efficient viral replication in cancerous cells.

In some embodiments, the recombinant VSV further expresses a sodium/iodine symporter (NIS) or variant thereof. In some embodiments, NIS comprises an amino acid sequence having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID NO. 25, or comprises the amino acid sequence of SEQ ID NO. 25. In some embodiments, the nucleic acid sequence encoding NIS is located between G and L viral genes, which allows for proper expression levels of NIS polypeptides.

In certain embodiments, the oncolytic virus is a recombinant VSV known in the art, such as Voyager V1 described, for example, in US 9428736, which US 9428736 is incorporated herein by reference in its entirety.

PD-1 pathway inhibitors

The methods disclosed herein comprise administering a therapeutically effective amount of a PD-1 pathway inhibitor. As used herein, a "PD-1 pathway inhibitor" refers to any molecule capable of inhibiting, blocking, eliminating, or interfering with the activity or expression of PD-1. In some embodiments, the PD-1 pathway inhibitor may be an antibody, a small molecule compound, a nucleic acid, a polypeptide, or a functional fragment or variant thereof. Some non-limiting examples of suitable PD-1 pathway inhibitors include anti-PD-1 antibodies and antigen-binding fragments thereof, anti-PD-L1 antibodies and antigen-binding fragments thereof, and anti-PD-L2 antibodies and antigen-binding fragments thereof.

Further non-limiting examples of suitable PD-1 pathway inhibitors include RNAi molecules such as anti-PD-1 RNAi molecules, anti-PD-L1 RNAi and anti-PD-L2 RNAi molecules, antisense molecules such as anti-PD-1 antisense RNA, anti-PD-L1 antisense RNA and anti-PD-L2 antisense RNA, and dominant negative proteins such as dominant negative PD-1 protein, dominant negative PD-L1 protein and dominant negative PD-L2 protein. Some examples of the foregoing PD-1 pathway inhibitors are described, for example, in US 9308236, US10011656 and US20170290808, wherein the identification of portions of PD-1 pathway inhibitors is incorporated herein by reference.

The term "antibody" as used herein is intended to refer to immunoglobulin molecules (i.e., "whole antibody molecules") composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, and multimers (e.g., igms) or antigen-binding fragments thereof. Each heavy chain comprises a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (made up of domains CH1, CH2 and CH 3). Each light chain comprises a light chain variable region ("LCVR" or "VL") and a light chain constant region (light chain constant region, CL). The VH and VL regions can be further subdivided into regions of hypervariability (termed complementarity determining regions (complementarity determining region, CDRs)) interspersed with regions that are more conserved (termed Framework Regions (FR)). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FR of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequence, or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on parallel analysis (side-by-side analysis) of two or more CDRs. The term "antibody" as used herein also includes antigen binding fragments of whole antibody molecules.

The terms "antigen-binding fragment" of an antibody, an "antigen-binding portion" of an antibody, and the like as used herein include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The antigen binding fragment of an antibody may be derived, e.g., from an intact antibody molecule, using any suitable standard technique involving manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody, e.g., proteolytic digestion or recombinant genetic engineering techniques. Such DNA is known and/or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated by chemical means or by using molecular biological techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, produce cysteine residues, modify, add or delete amino acids, and the like.

Some non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) a F (ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) a dAb fragment; and (vii) a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated Complementarity Determining Region (CDR), such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies (minibodies), nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), miniblock immunodrugs (small modular immunopharmaceutical, SMIP), and shark variable IgNAR domains are also encompassed by the expression "antigen-binding fragments" as used herein.

The antigen binding fragment of an antibody typically comprises at least one variable domain. The variable domain may have any size or amino acid composition and typically comprises at least one CDR adjacent to or in frame with one or more framework sequences. In the presence of V _L Domain associated V _H In the antigen binding fragment of the domain, V _H And V _L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimerAnd comprises V _H -V _H 、V _H -V _L Or V _L -V _L A dimer. Alternatively, the antigen-binding fragment of the antibody may comprise monomer V _H Or V _L A domain.

In some embodiments, the antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be present within an antigen binding fragment of an antibody of the present disclosure include:

(i)V _H -C _H 1；(ii)V _H -C _H 2；(iii)V _H -C _H 3；(iv)V _H -C _H 1-C _H 2；(v)V _H -C _H 1-C _H 2-C _H 3；(Vi)V _H -C _H 2-C _H 3；(vii)V _H -C _L ；(viii)V _L -C _H 1；(ix)V _L -C _H 2；(x)V _L -C _H 3；(xi)V _L -C _H 1-C _H 2；(xii)V _L -C _H 1-C _H 2-C _H 3；(xiii)V _L -C _H 2-C _H 3, a step of; and (xiv) V _L -C _L 。

In any configuration of variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly linked to each other, or may be linked by whole or part of a hinge or linker region. The hinge region can be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Furthermore, antigen binding fragments of antibodies of the present disclosure may comprise one or more monomers V with each other and/or with one or more monomers V _H Or V _L The domains are non-covalently associated (e.g., via disulfide bonds) with homodimers or heterodimers (or other multimers) of any of the variable domain and constant domain configurations listed above.

The antibodies used in the methods disclosed herein may be human antibodies. The term "human antibody" as used herein refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Nonetheless, the human antibodies of the present disclosure may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in CDRs, and particularly in CDR 3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

The antibodies used in the methods disclosed herein may be recombinant human antibodies. The term "recombinant human antibody" as used herein includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies isolated from recombinant, combinatorial human antibody libraries (described further below), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., taylor et al (1992) nucleic acids res.20:6287-6295), or antibodies prepared, expressed, produced or isolated by any other means that involves splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and thus, the V of the recombinant antibodies _H And V _L The amino acid sequence of the region is such that: although derived from human germline V _H And V _L Sequences and related thereto, but may not naturally occur in human antibody germline libraries in vivo.

anti-PD-1 antibodies and antigen binding fragments thereof

In some embodiments, the PD-1 pathway inhibitors used in the methods disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind to PD-1 (e.g., anti-PD-1 antibodies). The term "specificBy "sexually binds" or the like is meant that the antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds" PD-1 as used in the context of the present disclosure includes an antibody that is K below _D Binding to PD-1 or a portion thereof: less than about 500nM, less than about 300nM, less than about 200nM, less than about 100nM, less than about 90nM, less than about 80nM, less than about 70nM, less than about 60nM, less than about 50nM, less than about 40nM, less than about 30nM, less than about 20nM, less than about 10nM, less than about 5nM, less than about 4nM, less than about 3nM, less than about 2nM, less than about 1nM, or less than about 0.5nM, as measured in a surface plasmon resonance assay. However, isolated antibodies that specifically bind to human PD-1 may have cross-reactivity with other antigens (e.g., PD-1 molecules from other (non-human) species).

According to certain exemplary embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR), a Light Chain Variable Region (LCVR), and/or a Complementarity Determining Region (CDR) comprising the amino acid sequence of any of the anti-PD-1 antibodies shown in US 9987500, which are incorporated herein by reference in their entirety.

In certain exemplary embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof useful in the context of the present disclosure comprises a heavy chain complementarity determining region (HCDR) comprising a Heavy Chain Variable Region (HCVR) of the amino acid sequence of SEQ ID NO. 1, and a light chain complementarity determining region (LCDR) comprising a Light Chain Variable Region (LCVR) of the amino acid sequence of SEQ ID NO. 2.

According to some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR 1, HCDR2, and HCDR 3) and three LCDRs (LCDR 1, LCDR2, and LCDR 3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO. 4; HCDR3 comprises the amino acid sequence of SEQ ID NO. 5; LCDR1 comprises the amino acid sequence of SEQ ID NO. 6; LCDR2 comprises the amino acid sequence of SEQ ID NO. 7; and LCDR3 comprises the amino acid sequence of SEQ ID NO. 8.

In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a HCVR comprising SEQ ID NO. 1 and a LCVR comprising SEQ ID NO. 2. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 9. In some embodiments, the anti-PD-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO. 10.

An exemplary antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 1 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 2 is known as a siegesbeck Li Shan antibody (also known as REGN2810;) Is a fully human anti-PD-1 antibody.

According to certain exemplary embodiments, the methods of the present disclosure include the use of a cimrpu Li Shan antibody or a biological equivalent thereof. As used herein, the term "biological equivalent" with respect to a PD-1 pathway inhibitor refers to an anti-PD-1 antibody or PD-1 binding protein or fragment thereof that is a pharmaceutical equivalent or a pharmaceutical substitute that does not exhibit a significant difference in its absorbance and/or extent of absorption from a reference antibody (e.g., a cimetidine Li Shan antibody) when administered in the same molar dose (single or multiple doses) under similar experimental conditions. In the context of the present disclosure, the term "biological equivalent" includes antigen binding proteins that bind to PD-1 and that differ from cimetidine Li Shan in terms of safety, purity and/or potency in no clinical sense.

According to some embodiments of the disclosure, the anti-human PD-1 or antigen-binding fragment thereof comprises a HCVR having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID No. 1.

According to some embodiments of the present disclosure, the anti-human PD-1 or antigen-binding fragment thereof comprises an LCVR having (e.g., 90%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID No. 2. Sequence identity may be measured by methods known in the art (e.g., GAP, BESTFIT, and BLAST).

According to some embodiments of the present disclosure, the anti-human PD-1 or antigen-binding fragment thereof comprises a HCVR comprising an amino acid sequence of SEQ ID NO:1 with NO more than 10 amino acid substitutions. According to some embodiments of the present disclosure, the anti-human PD-1 or antigen-binding fragment thereof comprises a LCVR comprising an amino acid sequence of SEQ ID NO:2 with NO more than 10 amino acid substitutions.

Variants of any HCVR, LCVR and/or CDR amino acid sequences disclosed herein having one or more conservative amino acid substitutions are also within the scope of the present disclosure. For example, the present disclosure includes anti-PD-L1 antibodies having HCVR, LCVR and/or CDR amino acid sequences with conservative amino acid substitutions, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., relative to any HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

Other anti-PD-1 antibodies or antigen-binding fragments thereof that may be used in the context of the methods of the present disclosure include, for example, antibodies referred to in the art and known as follows: nivolumab, pembrolizumab, MEDI0608, dermatitidab, BI 754091, swadazumab (also known as PDR 001), carlizumab (also known as SHR-1210), JNJ-63723283, MCLA-134, or any anti-PD-1 antibody shown in US patent nos. 6808710, 7488802, 8008449, 8168757, 8354509, 8609089, 8686119, 8779105, 8900587 and 9987500 and in patent publications WO 2006/121168, WO 2009/114335. All of the above publications are incorporated herein by reference for part of the identification of anti-PD-1 antibodies.

anti-PD-1 antibodies used in the context of the methods of the present disclosure may have pH-dependent binding properties. For example, the anti-PD-1 antibodies used in the methods of the present disclosure may exhibit reduced binding to PD-1 at acidic pH as compared to neutral pH. Alternatively, the anti-PD-1 antibodies of the present disclosure may exhibit enhanced binding to their antigen at acidic pH as compared to neutral pH. The expression "acidic pH" includes pH values of less than about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

In some cases, "reduced binding to PD-1 at acidic pH compared to neutral pH" is based on K of an antibody that binds to PD-1 at acidic pH _D Value and K of antibodies binding to PD-1 at neutral pH _D The ratio of values (and vice versa). For example, if the antibody or antigen binding fragment thereof exhibits an acidic/neutral K of about 3.0 or greater _D For purposes of this disclosure, an antibody or antigen-binding fragment thereof may be considered to exhibit "reduced binding to PD-1 at acidic pH compared to neutral pH". In certain exemplary embodiments, the acid/neutral K of the antibodies or antigen binding fragments of the disclosure _D The ratio may be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening a population of antibodies that have reduced (or enhanced) binding to a particular antigen at an acidic pH as compared to a neutral pH. In addition, modification of the antigen binding domain at the amino acid level can result in antibodies with pH-dependent characteristics. For example, by replacing one or more amino acids of an antigen binding domain (e.g., within a CDR) with histidine residues, antibodies can be obtained that have reduced antigen binding at acidic pH relative to neutral pH. As used herein, the expression "acidic pH" means a pH of 6.0 or less. anti-PD-L1 antibodies and antigen binding fragments thereof

In some embodiments, the PD-1 pathway inhibitors used in the methods disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind to PD-L1 (e.g., anti-PD-L1 antibodies). For example, an antibody that "specifically binds" PD-L1 as used in the context of the present disclosure includes at about 1 x 10 ^-8 M or less K _D (e.g., smaller K _D Representing tighter binding) antibodies that bind to PD-L1 or a portion thereof. By "high affinity" anti-PD-L1 antibody is meant an antibody having a binding affinity for PD-L1 (denoted K _D ) Is described as (I), a K _D At least 10 ^-8 M (e.g. 10 ^-9 M、10 ^-10 M、10 ^-11 M or 10 ^-12 M), e.g. by surface plasmon resonance (e.g. BIACORE ^TM Or solution affinity ELISA). However, isolated antibodies that specifically bind to human PD-L1 may have cross-reactivity with other antigens (e.g., PD-L1 molecules from other (non-human) species).

According to certain exemplary embodiments, an anti-PD-Ll antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR), a Light Chain Variable Region (LCVR), and/or a Complementarity Determining Region (CDR) comprising the amino acid sequence of any of the anti-PD-L1 antibodies shown in US 9938345, which are incorporated herein by reference in their entirety.

In certain exemplary embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof that can be used in the context of the present disclosure comprises a heavy chain complementarity determining region (HCDR) comprising the Heavy Chain Variable Region (HCVR) of SEQ ID NO. 11 and a light chain complementarity determining region (LCDR) comprising the Light Chain Variable Region (LCVR) of SEQ ID NO. 12. An exemplary anti-PD-L1 antibody comprising the HCVR of SEQ ID NO. 11 and the LCVR of SEQ ID NO. 12 is REGN3504.

According to some embodiments of the disclosure, an anti-human PD-L1 antibody or antigen-binding fragment thereof comprises a HCVR having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID No. 11. According to some embodiments of the disclosure, an anti-human PD-L1 antibody or antigen-binding fragment thereof comprises a LCVR having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID No. 12.

According to some embodiments of the present disclosure, an anti-human PD-L1 antibody or antigen-binding fragment thereof comprises a HCVR comprising an amino acid sequence of SEQ ID No. 11 with NO more than 10 amino acid substitutions. According to some embodiments of the present disclosure, an anti-human PD-L1 antibody or antigen-binding fragment thereof comprises a LCVR containing an amino acid sequence of SEQ ID NO. 12 with NO more than 10 amino acid substitutions.

Variants of any HCVR, LCVR and/or CDR amino acid sequences disclosed herein having one or more conservative amino acid substitutions are also within the scope of the present disclosure. For example, the present disclosure includes the use of anti-PD-L1 antibodies having HCVR, LCVR and/or CDR amino acid sequences with conservative amino acid substitutions, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., relative to any HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

Other anti-PD-Ll antibodies that may be used in the context of the methods of the present disclosure include, for example, antibodies known in the art as follows and known as follows: MDX-1105, ab bead mab (TECENTRIQ) ^TM ) Devacizumab (IMFINZI) ^TM ) Avermectin (BAVENCIO) ^TM ) LY3300054, FAZ053, STI-1014, CX-072, KN035 (Zhang et al, cell Discovery,3,170004 (3 months of 2017)), CK-301 (Gorelik et al, american Association for Cancer Research Annual Meeting (AACR), 2016-04 abstract 4606), or any other anti-PD-L1 antibody shown in U.S. Pat. Nos. 7943743, 8217149, 9402899, 9624298, and 9938345 and patent publications WO 2007/005874, WO 2010/077634, WO 2013/181452, WO 2013/181634, WO 2016/14971, WO 2017/034916, or EP 3177649. All of the above publications are incorporated herein by reference for part of the identification of anti-PD-L1 antibodies.

anti-PD-L2 antibodies and antigen binding fragments thereof

In some embodiments, the PD-1 pathway inhibitors used in the methods disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind to PD-L2 (e.g., anti-PD-L2 antibodies). For example, an antibody that "specifically binds" PD-L2 as used in the context of the present disclosure includes antibodies that bind to PD-L2 in an amount of about 1 x 10 ^-8 Antibodies that bind to PD-L2 or a portion thereof with a KD of M or less (e.g., a smaller KD indicates tighter binding). "high affinity" anti-PD-L2 antibodies refer to those mAbs having a binding affinity for PD-L2 (denoted KD) of at least 10 ^-8 M (e.g. 10 ^-9 M、10 ^-10 M、10 ^-11 M or 10 ^-12 M), e.g. by surface plasmon resonance (e.g. BIACORETM or solution affinityELISA) was measured. However, isolated antibodies that specifically bind to human PD-L2 may have cross-reactivity with other antigens (e.g., PD-L2 molecules from other (non-human) species).

anti-PD-L2 antibodies that may be used in the context of the methods of the present disclosure include anti-PD-L2 antibodies shown, for example, in US patent nos. 8552154 and 10647771. All of the above publications are incorporated herein by reference for part of the identification of anti-PD-L2 antibodies.

CTLA4 inhibitors

The methods disclosed herein comprise administering a therapeutically effective amount of a CTLA4 inhibitor. As used herein, a "CTLA4 inhibitor" refers to any molecule capable of inhibiting, blocking, eliminating, or interfering with the activity or expression of CTLA 4. In some embodiments, the CTLA4 inhibitor can be an antibody, a small molecule compound, a nucleic acid, a polypeptide, or a functional fragment or variant thereof. Some non-limiting examples of suitable CTLA4 inhibitors include anti-CTLA 4 antibodies and antigen-binding fragments thereof. Other non-limiting examples of suitable CTLA4 inhibitors include RNAi molecules (e.g., anti-CTLA 4RNAi molecules) and dominant negative proteins (e.g., dominant negative CTLA4 proteins).

anti-CTLA 4 antibodies and antigen-binding fragments thereof

In some embodiments, the CTLA4 inhibitors used in the methods disclosed herein are antibodies or antigen-binding fragments thereof that specifically bind CTLA4 (e.g., anti-CTLA 4 antibodies). The term "specifically binds" or the like means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, antibodies that "specifically bind" CTLA4 as used in the context of the present disclosure include antibodies that bind to the following K _D Binding CTLA4 antibody or portion thereof: less than about 500nM, less than about 300nM, less than about 200nM, less than about 100nM, less than about 90nM, less than about 80nM, less than about 70nM, less than about 60nM, less than about 50nM, less than about 40nM, less than about 30nM, less than about 20nM, less than about 10nM, less than about 5nM, less than about 4nM, less than about3nM, less than about 2nM, less than about 1nM, or less than about 0.5nM, as measured in the surface plasmon resonance assay. However, isolated antibodies that specifically bind to human CTLA4 can be cross-reactive with other antigens (e.g., CTLA4 molecules from other (non-human) species).

In certain exemplary embodiments, an anti-CTLA 4 antibody or antigen-binding fragment thereof that can be used in the context of the present disclosure comprises a heavy chain complementarity determining region (HCDR) comprising a Heavy Chain Variable Region (HCVR) of the amino acid sequence of SEQ ID No. 13 and a light chain complementarity determining region (LCDR) comprising a Light Chain Variable Region (LCVR) of the amino acid sequence of SEQ ID No. 14.

According to some embodiments, the anti-CTLA 4 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR 1, HCDR2, and HCDR 3) and three LCDRs (LCDR 1, LCDR2, and LCDR 3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 15; HCDR2 comprises the amino acid sequence of SEQ ID NO. 16; HCDR3 comprises the amino acid sequence of SEQ ID NO. 17; LCDR1 comprises the amino acid sequence of SEQ ID NO. 18; LCDR2 comprises the amino acid sequence of SEQ ID NO. 19; and LCDR3 comprises the amino acid sequence of SEQ ID NO. 20.

In other embodiments, the anti-CTLA 4 antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO. 13 and a LCVR comprising the amino acid sequence of SEQ ID NO. 14. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 21. In some embodiments, the anti-CTLA 4 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO. 22.

An exemplary antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 14 is a fully human anti-CTLA 4 antibody known as REGN 4659.

According to certain exemplary embodiments, the methods of the present disclosure include the use of REGN4659 or a biological equivalent thereof. As used herein, the term "biological equivalent" with respect to CTLA4 inhibitor refers to an anti-CTLA 4 antibody or CTLA4 binding protein or fragment thereof that is a drug equivalent or drug substitute that does not exhibit a significant difference in absorbance and/or extent of absorbance from a reference antibody (e.g., REGN 4659) when administered in the same molar dose (single dose or multiple doses) under similar experimental conditions. In the context of the present disclosure, the term "biological equivalent" includes antigen binding proteins that bind CTLA4 and that have no clinically significant differences in safety, purity, and/or potency from REGN 4659.

According to some embodiments of the disclosure, the anti-human CTLA4 or antigen-binding fragment thereof comprises an HCVR having at least 90% (e.g., 90%, 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID No. 13.

According to some embodiments of the present disclosure, the anti-human CTLA4 or antigen-binding fragment thereof comprises LCVR having (e.g., 90%, 95%, 96%, 97%, 98%, 99%) sequence identity to the amino acid sequence of SEQ ID No. 14.

According to some embodiments of the present disclosure, the anti-human CTLA4 or antigen-binding fragment thereof comprises HCVR comprising an amino acid sequence of SEQ ID No. 13 with NO more than 10 amino acid substitutions. According to some embodiments of the present disclosure, the anti-human CTLA4 or antigen-binding fragment thereof comprises LCVR comprising an amino acid sequence of SEQ ID No. 14 with NO more than 10 amino acid substitutions.

Other anti-CTLA 4 antibodies or antigen-binding fragments thereof that may be used in the context of the methods of the present disclosure include, for example, antibodies known and known in the art as ipilimumab, tremelimumab, or any of the anti-CTLA 4 antibodies shown in US patent nos. 7527969, 8779098, 7666424, 7737258, 7740845, 8148154, 8414892, 8501471, and 9062110, and in patent publications US 2013/0078234, US2010/0143245, WO 2017062615A2, WO 2004/001381, and WO 2012/147713. All of the above publications are incorporated herein by reference for part of identifying anti-CTLA 4 antibodies.

Pharmaceutical composition and administration

The present disclosure includes methods comprising administering an oncolytic virus, a PD-1 pathway inhibitor, and/or a CTLA4 inhibitor to a subject, wherein the antibodies are contained in a (single) pharmaceutical composition, alone or in combination. The pharmaceutical compositions of the present disclosure may be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. A number of suitable formulations can be found in the prescription set known to all pharmaceutical chemists: remington's Pharmaceutical Sciences, mack Publishing Company, easton, pa. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (e.g., LIPOFECTIN), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al PDA (1998) J Pharm Sci Technol 52:52:238-311.

A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., wu et al (1987) j. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intratumoral, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (mucocutaneous lining) (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other bioactive agents.

Pharmaceutical compositions comprising an oncolytic virus, a PD-1 pathway inhibitor, or a CTLA4 inhibitor can be delivered intratumorally, subcutaneously, or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable to delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices typically use a replaceable cartridge containing a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device may then be reused. In disposable pen delivery devices, there is no replaceable cartridge. In contrast, disposable pen delivery devices are pre-filled with a pharmaceutical composition contained in a reservoir within the device. Once the reservoir is free of the pharmaceutical composition, the entire device is discarded.

In some cases, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material may be used; see, e.g., medical Applications of Controlled Release, langer and Wise (eds.), 1974, crc Pres., boca Raton, fla. In another embodiment, the controlled release system may be placed in proximity to the target of the composition, thus requiring only a portion of the systemic dose (see, e.g., goodson,1984, vol. Medical Applications of Controlled Release, supra, vol. 2, pages 115 to 138). Other controlled release systems are discussed in the review by Langer,1990, science 249:1527-1533.

Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, intratumoral and intramuscular injection, instillation, and the like. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared, for example, by dissolving, suspending or emulsifying the above-described antibodies or salts thereof in a sterile aqueous or oily medium conventionally used for injection. As the aqueous medium for injection, there are, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with suitable solubilizing agents such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [ e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adducts of hydrogenated castor oil) ] and the like. As the oily medium, for example, sesame oil, soybean oil, or the like is used, which may be used in combination with a solubilizing agent (for example, benzyl benzoate, benzyl alcohol, or the like). The injection thus prepared is preferably filled in a suitable ampoule.

Advantageously, the pharmaceutical compositions described above for oral or parenteral use are prepared in unit dosage forms suitable for the dosage of the active ingredient. Such dosage forms in unit dosage form include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like.

The present disclosure also provides a kit comprising a combination of an oncolytic virus, a PD-1 pathway inhibitor, and a CTLA4 inhibitor with written instructions for using a therapeutically effective amount of a combination of an oncolytic virus, a PD-1 pathway inhibitor, and a CTLA4 inhibitor to treat a tumor or inhibit tumor growth in a patient.

Administration protocol

The methods of the disclosure can include administering to a subject an oncolytic virus, a PD-1 pathway inhibitor (e.g., an anti-PD-1, anti-PD-L1, or anti-PD-L2 antibody, or antigen-binding fragment thereof), or a CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof) at the following dosing frequency: about four times per week, twice per week, once per two weeks, once per three weeks, once per four weeks, once per five weeks, once per six weeks, once per eight weeks, once per twelve weeks, or less frequently, so long as a therapeutic response is achieved. The methods of the present disclosure may further comprise administering a single dose of each of the oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor.

In some embodiments, the patient is administered at least one of an oncolytic virus, a PD-1 pathway inhibitor, or a CTLA4 inhibitor once a day, once every two days, once every three days, once every four days, once every five days, once a week, once every two weeks, or once every three weeks.

In some embodiments, the oncolytic virus, PD-1 pathway inhibitor, and CTLA4 inhibitor are administered to the patient simultaneously.

In some embodiments, the methods can include sequentially administering two or more of an oncolytic virus, a PD-1 pathway inhibitor, and a CTLA4 inhibitor to a subject. In some embodiments, the oncolytic virus is administered to the patient before or after the PD-1 pathway inhibitor and CTLA4 inhibitor. In some embodiments, the PD-1 pathway inhibitor is administered to the patient before or after the oncolytic virus and CTLA4 inhibitor. In some embodiments, the CTLA4 inhibitor is administered to the patient before or after the oncolytic virus and PD-1 pathway inhibitor.

As used herein, "sequentially administering" means that each dose of oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor is administered to a subject at different time points (e.g., on different days) at predetermined intervals (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering a single initial dose of an oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor to a patient, followed by one or more second doses of an oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor, and optionally followed by one or more third doses of an oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor. In some embodiments, the method further comprises sequentially administering a single initial dose of an oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor to the patient, followed by one or more second doses of an oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor, and optionally followed by one or more third doses of an oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor.

The terms "initial dose", "second dose" and "third dose" refer to the temporal sequence of administration. Thus, an "initial dose" is the dose administered at the beginning of a treatment regimen (also referred to as the "baseline dose"); a "second dose" is a dose administered after the initial dose; and "third dose" is the dose administered after the second dose. The initial, second, and third doses may all comprise the same amount of oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor. However, in some embodiments, during the course of treatment, the amounts contained in the initial, second, and/or third doses are different from each other (e.g., adjusted up or down as the case may be). In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as a "loading dose" followed by subsequent doses (e.g., a "maintenance dose") that are administered based on a lower frequency.

In one exemplary embodiment of the present disclosure, each second and/or third dose is 1/2 to 14 weeks (e.g., 1/2, 1 ^1/2 、2、2 ^1/2 、3、3 ^1/2 、4、4 ^1/2 、5、5 ^1/2 、6、6 ^1/2 、7、7 ^1/2 、8、8 ^1/2 、9、9 ¹ ^/2 、10、10 ^1/2 、11、11 ^1/2 、12、12 ^1/2 、13、13 ^1/2 、14、14 ^1/2 Or more weeks). The phrase "immediately preceding dose" as used herein means a dose of an oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor administered to a subject in the order of multiple administrations immediately prior to the administration of the next dose in order without intervening doses.

In some embodiments, the methods can include administering to the patient any number of second and/or third doses of an oncolytic virus, a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof), or a CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof). For example, in some embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Also, in some embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to the patient.

In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered at the same frequency as the other third doses. For example, each third dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the second and/or third doses are administered to the patient may vary over the course of the treatment regimen. The physician may also adjust the frequency of administration during the course of treatment according to the needs of the individual patient after the clinical examination.

In some embodiments, one or more doses of an oncolytic virus, PD-1 pathway inhibitor, or CTLA4 inhibitor are administered at the beginning of the treatment regimen as an "induction dose" based on a higher frequency (twice weekly, once weekly, or once 2 weeks), followed by a subsequent dose ("consolidation dose" or "maintenance dose") based on a lower frequency (e.g., once 4 to 12 weeks) of administration.

Dosage of

The amount of an oncolytic virus, PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof), or CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof) administered to a subject according to the methods disclosed herein is generally a therapeutically effective amount. As used herein, the term "therapeutically effective amount" means an amount of an oncolytic virus, PD-1 pathway inhibitor, and/or CTLA4 inhibitor that results in one or more of the following: (a) Reducing the severity or duration of symptoms or indications of cancer (e.g., neoplastic lesions); (b) Inhibit tumor growth, or increase tumor necrosis, tumor shrinkage, and/or tumor disappearance; (c) delay tumor growth and development; (d) inhibiting tumor metastasis; (e) preventing recurrence of tumor growth; (f) increasing survival of a subject suffering from cancer; and/or (g) the use or need for conventional anti-cancer therapy is reduced (e.g., the need for surgery is eliminated or the use of chemotherapeutic or cytotoxic agents is reduced or eliminated) compared to an untreated subject, a subject treated with a monotherapy, or a subject treated with any two of the three therapeutic agents disclosed herein (PD-1 pathway inhibitor, CTLA4 inhibitor, and oncolytic virus).

In some embodiments, the combined oncolytic viruses may be according to one or more of 10, 100, 10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ 、10 ¹⁰ 、10 ¹¹ 、10 ¹² 、10 ¹³ 、10 ¹⁴ Or more viral particles (vp)A unit dose of plaque-forming units (pfu) is administered. In some embodiments, the oncolytic virus is an oncolytic rhabdovirus (e.g., wild-type or genetically modified VSV), and is according to one or more 10 ⁶ To 10 ¹⁴ pfu、10 ⁶ To 10 ¹² pfu、10 ⁸ To 10 ¹⁴ pfu、10 ⁸ To 10 ¹² Or 10 ¹⁰ To 10 ¹² pfu, or any range of doses therebetween, is administered to a human suffering from cancer.

In some embodiments, the combined oncolytic viruses may be according to one or more of 10, 100, 10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ 、10 ¹⁰ 、10 ¹¹ 、10 ¹² 、10 ¹³ 、10 ¹⁴ Or more than 50% Tissue Culture Infection Dose (TCID) ₅₀ ) Is administered in unit doses of (a). In some embodiments, the oncolytic virus is an oncolytic rhabdovirus (e.g., wild-type or genetically modified VSV), and is according to one or more 10 ⁴ To 10 ¹⁴ TCID ₅₀ 、10 ⁴ To 10 ¹⁴ TCID ₅₀ 、10 ⁴ To 10 ¹² TCID ₅₀ 、10 ⁸ To 10 ¹⁴ TCID ₅₀ 、10 ⁸ To 10 ¹² Or 10 ¹⁰ To 10 ¹² TCID ₅₀ Or any range of dosages therebetween, to a person suffering from cancer.

In some embodiments, a therapeutically effective amount of a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof, such as a cimetidine Li Shan antibody or biological equivalent thereof) may be about 0.05mg to about 1500mg, about 1mg to about 800mg, about 5mg to about 600mg, about 10mg to about 550mg, about 50mg to about 400mg, about 75mg to about 350mg, or about 100mg to about 300mg of the antibody. For example, in various embodiments, the amount of the PD-1 pathway inhibitor is about 0.05mg, about 0.1mg, about 1.0mg, about 1.5mg, about 2.0mg, about 5mg, about 10mg, about 15mg, about 20mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg, about 480mg, about 490mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 600mg, about 610mg, about 620mg, about 630mg, about 640mg, about 650mg, about 660mg, about 670mg, about about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg, about 480mg, about about 490mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 600mg, about 610mg, about 620mg, about 630mg, about 640mg, about 650mg, about 660mg, about 670mg, about.

The amount of PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof) included in a single dose can be expressed in terms of milligrams of antibody per kilogram of subject body weight (i.e., mg/kg). In some embodiments, the PD-1 pathway inhibitors used in the methods disclosed herein may be administered to a subject at a dose of about 0.0001 to about 100mg/kg of body weight of the subject. In some embodiments, the anti-PD-1 antibody may be administered at a dose of about 0.1mg/kg to about 20mg/kg of patient body weight. In some embodiments, the methods of the present disclosure comprise administering a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof) at a dose of about 1mg/kg to 3mg/kg, 1mg/kg to 5mg/kg, 1mg/kg to 10mg/kg, 1mg/kg, 3mg/kg, 5mg/kg, or 10mg/kg of patient body weight.

In some embodiments, each dose comprises 0.1 to 10mg/kg (e.g., 0.3mg/kg, 1mg/kg, 3mg/kg, or 10 mg/kg) of the subject's body weight. In certain further embodiments, each dose comprises 5 to 1500mg of a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof), e.g., 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 40mg, 45mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 1050mg, 1100mg, 1150mg, 1200mg, 1550mg, 1300mg, 1350mg, 1400mg, 1450mg, or 1500mg of the PD-1 pathway inhibitor.

In some embodiments, a therapeutically effective amount of a CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof, or biological equivalent thereof) can be about 0.05mg to about 1000mg, about 1mg to about 800mg, about 5mg to about 600mg, about 10mg to about 550mg, about 50mg to about 400mg, about 75mg to about 350mg, or about 100mg to about 300mg of antibody. For example, in various embodiments, the CTLA4 inhibitor is present in an amount of about 0.05mg, about 0.1mg, about 1.0mg, about 1.5mg, about 2.0mg, about 5mg, about 10mg, about 15mg, about 20mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about about 470mg, about 480mg, about 490mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 600mg, about 610mg, about 620mg, about 630mg, about 640mg, about 650mg, about 660mg, about 670mg, about 680mg, about 690mg, about 700mg, about 710mg, about 720mg, about 730mg, about 740mg, about 750mg, about 760mg, about 770mg, about 780mg, about 790mg, about 800mg, about 810mg, about 820mg, about 830mg, about 840mg, about 850mg, about 860mg, about 870mg, about 880mg, about 890mg, about 900mg, about 910mg, about 920mg, about 930mg, about 940mg, about 950mg, about 960mg, about 970mg, about 980mg, about 990mg or about 1000mg.

The amount of CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof) contained in a single dose can be expressed in terms of milligrams of antibody per kilogram of subject body weight (i.e., mg/kg). In some embodiments, the anti-CTLA 4 antibody can be administered at a dose of about 0.1mg/kg to about 20mg/kg of patient body weight. In some embodiments, the methods of the disclosure comprise administering a CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof) at a dose of about 1mg/kg to 3mg/kg, 1mg/kg to 5mg/kg, 1mg/kg to 10mg/kg, 1mg/kg, 3mg/kg, 5mg/kg, 10mg/kg, or 15mg/kg of patient body weight.

In some embodiments, each dose comprises 0.1 to 10mg/kg (e.g., 0.3mg/kg, 1mg/kg, 3mg/kg, or 10 mg/kg) of the subject's body weight. In certain further embodiments, each dose comprises 5 to 1000mg of CTLA4 inhibitor (e.g., an anti-CTLA 4 antibody or antigen-binding fragment thereof), e.g., 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 40mg, 45mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, or 1000mg of CTLA4 inhibitor.

In some embodiments, the methods of the present disclosure further comprise administering an additional therapeutic agent or treatment to the subject. Additional therapeutic agents or treatments may be administered for increasing anti-tumor efficacy, for reducing the toxic effects of one or more treatments, and/or for reducing the dose of one or more treatments. In various embodiments, the additional therapeutic agent or treatment may include one or more of the following: radiation, surgery, cancer vaccines, imiquimod (imiquimod), antiviral agents (e.g., cidofovir (cidofovir)), photodynamic therapy, lymphocyte activating gene 3 (LAG 3) inhibitors (e.g., anti-LAG 3 antibodies, glucocorticoid-induced tumor necrosis factor receptor (glucosporicoid-induced tumor necrosis factor receptor, GITR) agonists (e.g., anti-GITR antibodies), inhibitors containing T-cell immunoglobulins and mucin-3 (TIM 3), B and T-lymphocyte attenuator (BTLA) inhibitors, T-cell immunoreceptor (TIGIT) inhibitors having Ig and ITIM domains, CD38 inhibitors, CD47 inhibitors, indoleamine-2, 3-dioxygenase (IDO) inhibitors, CD28 activators, vascular Endothelial Growth Factor (VEGF) antagonists (e.g., "VEGF-Trap" (e.g., aflibercept), or antigen binding fragments thereof (e.g., bevacizumab) or mucin-3 (timiber)), or antigen-binding inhibitors (e.g., bevacizumab) or vascular growth factor receptor (tgfjoba), vascular growth factor (tga) or receptor-37-35, vascular growth factor (tgla), tumor-3-associated antigen (granny) inhibitors, such as vascular growth factor (tgla), vascular growth factor (tgla) inhibitors, vascular growth factor (tgla) 2, 37-receptor (tgla) inhibitors, vascular Endothelial Growth Factor (VEGF) or antigen-binding fragments thereof (e.g., bevacizumab-35) Carcinoembryonic antigen (carcinoembryonic antigen, CEA), vimentin, tumor-M2-PK, prostate Specific Antigen (PSA), mucin-1, MART-1, and CA 19-9), vaccine (e.g., BCG), granulocyte-macrophage colony stimulating factor (GM-CSF), second oncolytic virus, cytotoxin, chemotherapeutic agent (e.g., pemetrexed (pemetrexed), dacarbazine (dacarbazine), temozolomide (temozolomide), cyclophosphamide (cyclophosphamide), docetaxel (docetaxel), doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine (gemcitabine), methotrexate, mitoxantrone (mitoxantrone), oxaliplatin (oxaptin), paclitaxel, topotecan (topotecan), irinotecan (irinotecan), vinorelbine (vinorelbine) and vincristine (vincristine)), an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, cytokines (e.g., IL-2, IL-7, IL-12, IL-21 and IL-15), antibody drug conjugates, anti-inflammatory drugs (e.g., corticosteroids, non-steroidal anti-inflammatory drugs (non-sterioi anti-inflammatory drug, anti-tumor drugs, HPV resections), laser therapy, HPV, cell therapy, and combinations thereof.

In some embodiments, the method further comprises administering an additional therapeutic agent, such as an anticancer drug. As used herein, "anticancer drug" means any agent useful in the treatment of cancer, including, but not limited to, cytotoxins and agents, such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotics, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O, P' - (DDD)), biologicals (e.g., antibodies and interferons), and radiopharmaceuticals. As used herein, "cytotoxin or cytotoxic agent" also refers to a chemotherapeutic agent and refers to any agent that is detrimental to cells. Examples include TAXOL, temozolomide, cytochalasin B (cytochalasin B), poncirin D (gramicidin D), ethidium bromide, emetine, cisplatin, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax-dione, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.

Additional definitions

To facilitate an understanding of specific embodiments of compositions and methods according to the present disclosure, some express definitions are provided to facilitate an express disclosure of aspects of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used herein, the term "pharmaceutical agent" refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule (e.g., a nucleic acid, antibody, protein, or portion thereof (e.g., peptide)), or an extract made from biological material such as bacterial, plant, fungal, or animal (particularly mammalian) cells or tissues. The activity of such agents may make them suitable as "therapeutic agents" which are biologically, physiologically or pharmacologically active substance(s) that act locally or systemically in a subject. In the context of the present disclosure, the term "therapeutic agent" refers to any PD-1 pathway inhibitor, CTLA4 inhibitor, or oncolytic virus disclosed herein.

As used herein, the terms "therapeutic agent," "therapeutically effective agent," or "therapeutic agent" are used interchangeably and refer to a molecule or compound that imparts some beneficial effect upon administration to a subject. Beneficial effects include enabling diagnostic determinations; improvement of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally combat diseases, symptoms, disorders or pathological conditions.

As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not destroy the biological activity or characteristics of the composition and is relatively non-toxic, i.e., the material may be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, which participate in carrying or transporting the compounds of the present disclosure within or to a subject so that they can perform their intended function. Typically, such compounds are carried or transported from one organ or body part to another organ or body part. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Some examples of materials that may be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; non-thermal raw water; isotonic saline; ringer's solution; ethanol; phosphate buffer solution; a diluent; granulating agent; a lubricant; an adhesive; a disintegrant; a wetting agent; an emulsifying agent; a colorant; a release agent; a coating agent; a sweetener; a flavoring agent; a fragrance; a preservative; an antioxidant; a plasticizer; a gelling agent; a thickener; a hardening agent; a setting agent; a suspending agent; a surfactant; a humectant; a carrier; a stabilizer; and other non-toxic compatible substances for pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, absorption delaying agents, and the like that are compatible with the activity of one or more components of the present disclosure and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.

Dosage is generally expressed as being weight dependent. Thus, expressed as [ g, mg or other units ]]The dose of/kg (or g, mg, etc.) is generally referred to as [ g, mg, or other units ]]"/kg (or g, mg, etc.) of body weight", even though the term "body weight" is not explicitly mentioned. Treatment may include a variety of "unit doses". A unit dose is defined as containing a predetermined amount of a therapeutic composition. The unit dose need not be administered as a single injection, but may comprise continuous infusion over a set period of time. For oncolytic viruses, unit doses can be described in terms of plaque forming units (pfu) or viral particles for viral constructs. The unit dosage range is 10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ 、10 ¹⁰ 、10 ¹¹ 、10 ¹² 、10 ¹³ pfu or vp and higher. Alternatively, depending on the type of virus and the achievable titer, 1 to 100, 10 to 50, 100 to 1000, or up to about 1X 10 ⁴ 、1×10 ⁵ 、1×10 ⁶ 、1×10 ⁷ 、1×10 ⁸ 、1×10 ⁹ 、1×10 ¹⁰ 、1×10 ¹¹ 、1×10 ¹² 、1×10 ¹³ 、1×10 ¹⁴ Or 1X 10 ¹⁵ Or more infectious viral particles (vp) to the patient or patient cells. Alternatively, the oncolytic virus unit dose is defined by TCID ₅₀ And (3) representing. "TCID ₅₀ "refers to" tissue culture infectious dose "and is defined as the viral dilution required to infect 50% of a given batch of inoculated cell culture. Various methods known to those skilled in the art may be used to calculate the TCID ₅₀ The method includes the Spearman-Karber method used throughout this specification. For a description of the Spearman-Karber method, see b.w. mahy &H.0.Kangro, virology Methods Manual 25-46 (1996). In some embodiments, the unit dosage range is 10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ 、10 ¹⁰ 、10 ¹¹ 、10 ¹² 、10 ¹³ TCID ₅₀ And higher or any range therebetween.

As used herein, the term "disease" is intended to be generally synonymous and interchangeably used with the terms "disorder" and "condition" (as in a medical condition), as they both reflect an abnormal condition (e.g., cancer) of one of the human or animal body or its parts that impair normal function, which is generally manifested by distinguishing between signs and symptoms, and resulting in a reduction in the duration of life or quality of life of the human or animal.

As used herein, the term "in vitro" refers to events that occur in an artificial environment (e.g., in a tube or reaction vessel, in a cell culture, etc.), rather than within a multicellular organism.

As used herein, the term "in vivo" refers to events that occur in multicellular organisms (e.g., in non-human animals).

Unless the context clearly indicates otherwise, nouns without quantitative word modifications as used herein mean one and more.

The terms "comprising," "including," "containing," or "having," and variations thereof, as used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter, unless otherwise specified.

As used herein, the phrases "in one embodiment," "in embodiments," "in some embodiments," and the like are reused. Such phrases are not necessarily referring to the same embodiment, but they may refer to the same embodiment unless the context indicates otherwise.

As used herein, the term "and/or"/"means any item, any combination of items, or all items associated with the term.

As used herein, the word "substantially" does not exclude "complete", e.g., a composition that is "substantially free" of Y may be completely free of Y. The word "substantially" may be omitted from the definition of the present disclosure, if necessary.

As used herein, the term "each" when used in relation to a collection of items is intended to identify a single item in the collection, but does not necessarily refer to each item in the collection. An exception may occur if an explicit disclosure or context clearly dictates otherwise.

As used herein, the term "about" or "approximately" when applied to one or more destination values refers to values similar to the reference value. In some embodiments, unless otherwise indicated or otherwise apparent from the context, the term "about" or "approximately" refers to a range of values that fall within either direction (greater than or less than) of the reference value(s) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less (except where such number would exceed 100% of the possible values). Unless otherwise indicated herein, the term "about" is intended to include values approaching the stated range, e.g., weight percentages, which are equivalent in terms of the function of the individual ingredients, compositions or embodiments.

As disclosed herein, a plurality of value ranges are provided. It is to be understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is also specifically disclosed. Every smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within the stated range is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where neither, nor both limits are included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. With respect to any method provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur in sequence, the steps may occur in any order unless otherwise indicated. Where a method includes a combination of steps, each and every combination or sub-combination of steps is encompassed within the scope of the present disclosure unless otherwise indicated herein.

Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent not inconsistent with this disclosure. The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present disclosure. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such publication by virtue of prior disclosure. Furthermore, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present disclosure, and are not intended to limit the scope of what the inventors regard as their invention. Likewise, the present disclosure is not limited to any particular preferred embodiment described herein. Indeed, modifications and variations of the present embodiments may be apparent to those of ordinary skill in the art upon reading the present specification, and may be made without departing from the spirit and scope thereof. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees celsius, room temperature is about 25 ℃, and pressure is at or near atmospheric pressure.

Example 1: is carried by 150mm ³ Antitumor efficacy in mice with average MC38 tumors treated with a combination of anti-PD-1, anti-CTLA 4 and intratumoral delivery of oncolytic virus VSV-M51R-Fluc

This example describes the anti-tumor efficacy of using the oncolytic virus Vesicular Stomatitis Virus (VSV) in combination with the anti-PD-1 and anti-CTLA 4 triplets in wild-type mice implanted with MC38 tumors. The VSV used in this example (and examples 2 to 3 that follow) is a genetically attenuated virus designated VSV-M51R-Fluc, because it encodes a mutation in the M protein (M51R) (M protein inhibits host cell protein production, but M51R mutation maintains host cell protein production) and encodes firefly luciferase inserted between the G and L viral genes. The anti-PD-1 antibody used in this example (and subsequent examples 2 to 4) was an anti-mouse PD-1 rat IgG2a antibody (clone 29f1.a12 from Bioxcell), and the anti-CTLA 4 antibody used in this example (and subsequent examples 2 to 4) was an anti-mouse CTLA4-mIgG2a antibody (clone 9D 9).

Background was small on day 0 for C57BL/6 strain from Jackson laboratoriesMouse subcutaneous implantation of MC38 cells (3X 10 suspended in 100. Mu.l DMEM) ⁵ Individual cells/mice). Tumors were measured using calipers and combined with the formula (L ² Tumor volume was calculated by x W)/2, where L is the smallest dimension. When the average tumor size reached 150mm on day 15 ³ At this time, mice were randomly aliquoted into 7 treatment groups. On days 15, 19, 22 and 26, mice were intratumorally injected with 50 μl of 5×10 resuspended in 50 μl of PBS ⁵ TCID ₅₀ The dose of VSV-M51R-Fluc virus or PBS was used as control and/or 250. Mu.g isotype control antibody (mIgG 2a and/or rat IgG2 a) and/or 250. Mu.g anti-CTLA 4 antibody and/or anti-PD-1 antibody was intraperitoneally injected. Experimental dosing and treatment regimens for the various groups are shown in table 1.

Table 1: experimental dosing and treatment regimen for groups of mice

Tumor volumes were monitored twice weekly by caliper measurements until the end of the study on day 60.

The mean tumor volume over time for each group indicated that monotherapy with VSV or anti-PD-1 antibodies showed partial tumor growth inhibition compared to the PBS-treated and isotype-control-treated groups (fig. 1). The individual tumor volumes at day 26 after the start of treatment (fig. 2) were used for statistical analysis, as this was the last time point for survival of all animals in all groups in the study. Statistical significance (< 0.01, <0.0001, < p) was determined by one-way ANOVA and Dunnett multiple comparison post hoc test. The monotherapy efficacy of anti-PD-1 antibodies or VSV did not reach statistical significance. Combination treatment of VSV with anti-CTLA 4 antibody or with anti-PD-1 antibody resulted in more effective tumor growth inhibition compared to monotherapy with anti-PD-1 antibody or control, with statistically significantly smaller tumors in the combination treatment group than in the anti-PD-1 antibody treatment group on day 26 (fig. 3). The combined treatment with anti-CTLA 4 and anti-PD-1 antibodies resulted in a statistically significant reduction in tumor growth compared to all other single and dual combinations, without any tumor-free mice by day 26. Notably, triple combination treatment of VSV with anti-CTLA 4 and anti-PD-1 antibodies was more effective than all other groups, with all mice cleared their tumor by day 29, and this tumor clearance continued until the end of the study by day 60 (fig. 1, 2, 3). Table 2 summarizes the average tumor volume, percent survival, and number of tumor-free mice in each treatment group.

Table 2: average tumor volume, percent survival, and number of tumor-free mice in each treatment group

As shown in table 2, mice treated with the triple combination of VSV with anti-PD-1 and anti-CTLA 4 antibodies were very effective in controlling and clearing large tumors during the course of the study, with six out of six mice no tumors by day 29. On study day 26, mice treated with anti-PD-1 or anti-CTLA 4 antibodies (with or without combination with VSV) showed moderately reduced tumor volumes compared to controls. In contrast, in this study, treatment with the anti-PD 1 and anti-CTLA 4 antibodies in a double combination showed significant efficacy in reducing tumor volume compared to the control, with one of the five mice achieving tumor clearance; but it is not as effective as when VSV is also delivered in combination with anti-PD 1 and anti-CTLA 4 antibodies. By day 29 of the study, all mice were eliminated (elimate) in five of six of the control PBS group, anti-PD-1 group, VSV treated group, double combined VSV and anti-PD-1 treated group, double combined VSV and three of six of the anti-CTLA 4 group, and all mice remained alive in the anti-PD 1 and anti-CTLA 4 antibody double combined and triple combined VSV and anti-PD-1 and anti-CTLA 4 antibody groups. At the end of the study, only the triple combination group remained tumor-free and survived, along with only one mouse out of five in the anti-PD 1 and anti-CTLA 4 antibody double combination group. As a result of the triple combination treatment, no signs of weight loss were observed.

In summary, treatment with VSV in combination with anti-mCTLA 4 and anti-PD-1 antibodies resulted in reduced tumor growth and longer survival compared to monotherapy or dual therapy with antibody and/or VSV.

Example 2: the anti-tumor efficacy of the triple combination of anti-PD-1, anti-CTLA 4 and intratumorally delivered oncolytic virus VSV-M51R-GFP can be achieved with only one dose of anti-CTLA 4mIgG2a antibody

This example describes the number of doses of anti-CTLA 4 antibody necessary to achieve anti-tumor efficacy in wild-type mice implanted with MC38 tumors using the oncolytic virus Vesicular Stomatitis Virus (VSV) in triple combination with anti-PD-1 and anti-CTLA 4. The VSV used in this study was a genetically attenuated virus designated VSV-M51R-GFP, because it encodes a mutation in the M protein (M51R) (M protein inhibits host cell protein production, but M51R mutation maintains host cell protein production) and encodes GFP inserted between the G and L viral genes. Subcutaneous implantation of MC38 cells (3×10) on day 0 in background mice of the C57BL/6 strain from Jackson laboratories ⁵ Individual cells/mice). Tumors were measured using calipers and combined with the formula (L ² Tumor volume was calculated by x W)/2, where L is the smallest dimension. When the average tumor size reached 150mm on day 15 ³ At this time, mice were randomly aliquoted into 5 treatment groups. On days 15, 18, 22 and 25, mice were intratumorally injected with 50 μl of 5×10 resuspended in PBS ⁸ TCID ₅₀ Doses of VSV-M51R-GFP virus or PBS served as control, and/or intraperitoneal injections of 250 μg of isotype control antibody and/or anti-PD-1 antibody, and/or 250 μg of anti-CTLA 4 antibody were performed at different dose amounts, 4 doses (on days 15, 18, 22 and 25) or 1 dose (on day 15) or 2 doses (on days 15 and 18) (table 3).

Table 3: experimental dosing and treatment regimen for groups of mice

Table 4 summarizes the average tumor volume, percent survival, and number of tumor-free mice in each treatment group. The anti-tumor efficacy of VSV in combination with the triple combination of anti-PD-1 antibody and anti-CTLA 4 antibody in the form of mIgG2a was very similar in the group receiving one dose or two doses or four doses of anti-CTLA 4 antibody (fig. 4A, 4B, 4C, 4D, 4E), with six of the eight mice (75%) being tumor-free in one and two dose groups compared to five of the seven mice (71%) in the 4 dose group by day 45. By day 60, one and two dose groups had 62.5% of their mice surviving and tumor-free compared to 71.4% of the 4 dose groups (fig. 5). These results demonstrate that the potent anti-tumor efficacy of the triple combination of VSV with anti-PD-1 and anti-CTLA 4 antibodies can be reproduced with only one dose of anti-CTLA 4 administered concurrently with the virus and the first dose of anti-PD-1 antibody.

Table 4: average tumor volume, percent survival, and number of tumor-free mice in each treatment group

Example 3: the anti-tumor efficacy of the triple combination of anti-PD-1, anti-CTLA 4 and oncolytic virus VSV-M51R-GFP can be achieved with intratumoral or intravenous delivery of the virus

This example describes a delivery route whereby virus can be delivered in wild-type mice implanted with MC38 tumors to achieve anti-tumor efficacy using the oncolytic virus Vesicular Stomatitis Virus (VSV) in triple combination with anti-PD-1 and anti-CTLA 4. The VSV used in this example is a genetically attenuated virus designated VSV-M51R-GFP, because it encodes a mutation in the M protein (M51R) (M protein inhibits host cell protein production, but M51R mutation maintains host cell protein production) and encodes GFP inserted between the G and L viral genes. Subcutaneous implantation of MC38 cells (3×10) on day 0 in background mice of the C57BL/6 strain from Jackson laboratories ⁵ Individual cells/mice). Tumors were measured using calipers and combined with the formula (L ² Tumor volume was calculated by x W)/2, where L is the smallest dimension. When the average tumor size reached 150mm on day 15 ³ At this time, mice were randomly aliquoted into 4 treatment groups. In the first place15. Mice were intratumorally injected with 50 μl of 5×10 resuspended in PBS for 18, 22 and 25 days ⁸ TCID ₅₀ Doses of VSV-M51R-GFP virus, or 200. Mu.l of intravenous injection 1X 10 resuspended in PBS ⁹ TCID ₅₀ The dose of VSV-M51R-GFP virus, or PBS was used as a control, and/or 250. Mu.g of isotype control antibody and/or anti-PD-1 antibody and/or 250. Mu.g of anti-CTLA 4 antibody was intraperitoneally injected (Table 5). Tumor volumes were monitored until the study ended on day 60.

Table 5: experimental dosing and treatment regimen for groups of mice

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Table 6 summarizes the average tumor volume, percent survival, and number of tumor-free mice in each treatment group in this experiment.

The anti-tumor efficacy of VSV in combination with the triplets of anti-PD-1 and anti-CTLA 4 antibodies was very robust in groups receiving VSV as an intratumoral single dose or as an intravenous single dose, where intravenous delivery tended to be more effective than intratumoral (fig. 6), where eight of the eight mice in the intravenous administration group (100%) were tumor-free by day 45 compared to five of the seven mice in the intratumoral group (71%). By day 60, 100% of the intravenous group survived and tumor-free compared to 71.4% of the intratumoral group (fig. 7). Intravenous delivery of VSV robustly enhanced anti-PD-1 and anti-CTLA 4 combination checkpoint treatment and demonstrated that triple combination efficacy can be achieved with intratumoral or intravenous delivered viruses.

Table 6: average tumor volume, percent survival, and number of tumor-free mice in each treatment group

Example 4: is carried by 150mm ³ Antitumor efficacy in mice with average MC38 tumors treated with a combination of anti-PD-1, anti-CTLA 4 and intravenous delivered oncolytic virus VSV-mIFNb-NIS

This example describes the anti-tumor efficacy in wild-type mice implanted with MC38 tumors using the intravenously delivered oncolytic virus Vesicular Stomatitis Virus (VSV) in triple combination with anti-PD-1 and anti-CTLA 4 antibodies. The VSV used in this study was the genetically attenuated virus VSV-mIFNb-NIS (or mVV 1) encoding mouse interferon-beta (IFNb) inserted between M and G viral genes and encoding sodium/iodine symporter (NIS) inserted between G and L viral genes.

Subcutaneous implantation of MC38 cells (3×10) on day 0 in background mice of the C57BL/6 strain from Jackson laboratories ⁵ Individual cells/mice). Tumors were measured using calipers and combined with the formula (L ² Tumor volume was calculated by x W)/2, where L is the smallest dimension. When the average tumor size reached 150mm on day 15 ³ At this time, mice were randomly aliquoted into eight treatment groups. On days 15, 18, 21 and 24, mice received 200 μl of intravenous injection of 1×10 resuspended in PBS ⁹ TCID ₅₀ Doses of mVV or PBS served as controls, and/or 250 μg isotype control antibodies (mIgG 2a and/or rat IgG2 a) and/or 250 μg anti-CTLA 4 antibodies and/or anti-PD-1 antibodies were intraperitoneally injected (table 7). Tumor volumes were monitored twice weekly by caliper measurements until the end of the study on day 60.

Table 7: experimental dosing and treatment regimen for groups of mice

Table 8 summarizes the average tumor volume, percent survival, and number of tumor-free mice in each treatment group. The mean tumor volume over time for each group indicated that monotherapy with mVV1 or anti-PD-1 or anti-CTLA 4 antibodies showed less tumor growth inhibition than the PBS-treated and isotype control-treated groups (fig. 8). The single tumor volume at day 24 after the start of treatment (fig. 9) was used for statistical analysis, as this was the last time point for survival of all animals in all groups in the study. Statistical significance (< 0.01, <0.0001, < p) was determined by one-way ANOVA and Dunnett multiple comparison post hoc test. Monotherapy with anti-PD-1 or anti-CTLA 4 antibodies or mVV1 did not reach statistical significance, nor did mVV1 in combination with anti-PD-1 antibodies. The combination of mVV1 with anti-CTLA 4 antibody treatment resulted in more effective tumor growth inhibition compared to monotherapy with anti-PD-1 or anti-CTLA 4 antibody or mVV1 or control (fig. 10). The combination of anti-CTLA 4 with anti-PD-1 antibodies resulted in reduced tumor growth, but did not result in a statistically significant reduction at day 24 as compared to all other single and dual combinations. Notably, the triple combination of intravenously delivered mVV1 with anti-CTLA 4 and anti-PD-1 antibody treatment was very effective compared to all other groups, with two of the seven mice remaining tumor-free by day 60 study end (fig. 8, 9, 10). No signs of weight loss were observed as a result of the triple combination treatment. In summary, treatment with the combination of mVV1 with anti-mCTLA 4 and anti-PD-1 antibodies delivered intravenously resulted in reduced tumor growth and increased survival compared to monotherapy or dual therapy with antibody and/or mVV 1.

Table 8: average tumor volume, percent survival, and number of tumor-free mice in each treatment group

Example 5: the anti-tumor efficacy of the triple combination of anti-PD-1, anti-CTLA 4 and intratumorally delivered oncolytic virus VSV-M51R-GFP can be achieved with lower doses of anti-CTLA 4 mIgG2a antibodies

This example describes reduced doses of anti-CTLA 4 antibody that still achieve anti-tumor efficacy in wild-type mice implanted with MC38 tumors using the oncolytic virus Vesicular Stomatitis Virus (VSV) in triple combination with anti-PD-1 and anti-CTLA 4. The VSV used in this study was a genetically attenuated virus designated VSV-M51R-GFP, because it encodes a mutation in the M protein (M51R) (M protein inhibits host cell protein production, but M51R mutation maintains protein production) and encodes GFP inserted between the G and L viral genes. The anti-PD-1 antibody used in this study was clone 29F1.A12 rat IgG2a from Bioxcell, usedThe anti-CTLA 4 antibody was clone 9D9 in the form of mIgG2a purchased from invitrogen. MC38 cells (3×105 cells/mouse) were subcutaneously implanted on day 0 in background mice from Jackson labs, strain C57 BL/6. The tumors were measured using calipers and tumor volumes were calculated using formula (l2×w)/2, where L is the smallest dimension. When the average tumor size reached 150mm on day 15 ³ At this time, mice were randomly aliquoted into four treatment groups. On days 15, 18, 22 and 25, mice were intratumorally injected with 50. Mu.l of a 5X 108TCID50 dose of VSV-M51R-GFP virus resuspended in PBS or PBS as control, and/or 250. Mu.g of isotype control antibody and/or anti-mouse PD-1 rat IgG2a antibody (29F 1. A12), and/or 250. Mu.g or 50. Mu.g of anti-mouse CTLA4-mIgG2a (clone 9D 9), 4 doses (on days 15, 18, 22 and 25). Tumor volumes were monitored twice weekly by caliper measurements until the end of the study on day 60.

The anti-tumor efficacy of VSV in combination with the triplets of anti-PD-1 and anti-CTLA 4 antibodies was observed in the group receiving the lower dose of anti-CTLA 4 antibody (fig. 11, 12), wherein four of the eight mice (50%) were tumor-free by 45 days as compared to five of the seven mice (71%) in the higher dose group. By day 60, the low dose group had 50% of its mice surviving and tumor-free compared to the 4 dose group of 71.4% (fig. 13). These results indicate that the amount of anti-CTLA 4 administered in a triple combination of VSV with anti-PD-1 and anti-CTLA 4 antibodies can be reduced to still achieve anti-tumor efficacy.

Table 9: average tumor volume, percent survival, and number of tumor-free mice in each treatment group from in vivo tumors

As shown in table 9, mice treated with the triple combination of VSV i.t. (intratumoral) with anti-PD-1 and anti-CTLA 4 antibodies were very effective in controlling tumor growth, with five of the seven mice being tumor-free by day 45. When combined with VSV and anti-PD-1 antibodies, reducing the anti-CTLA 4 dose to one fifth (from 250 μg to 50 μg) showed unexpectedly significant anti-tumor efficacy.

Example 6: is carried by 150mm ³ Antitumor efficacy in mice with average MC38 tumors treated with anti-PD-1, one dose of anti-CTLA 4 in combination with intravenous delivery of oncolytic virus VSV-mIFNb-NIS

This example describes the anti-tumor efficacy of using the oncolytic virus Vesicular Stomatitis Virus (VSV) encoding IFNb and NIS in combination with a triple anti-PD-1 and a single dose of anti-CTLA 4 in wild-type mice implanted with MC38 tumors. The VSV used in this study was a genetically attenuated virus designated VSV-mIFNb-NIS (or mVV 1) because it encodes mouse interferon beta (IFNb) inserted between M and G viral genes, and encodes sodium/iodine symporter (NIS) inserted between G and L viral genes. The anti-PD-1 antibody used in this study was clone 29f1.a12 rat IgG2a from Bioxcell, and the anti-CTLA 4 antibody used was clone 9D9 in the form of mIgG2a from invitrogen.

Subcutaneous implantation of MC38 cells (3×10) on day 0 in background mice of the C57BL/6 strain from Jackson laboratories ⁵ Individual cells/mice). Tumors were measured using calipers and combined with the formula (L ² Tumor volume was calculated by x W)/2, where L is the smallest dimension. When the average tumor size reached 150mm on day 12 ³ At this time, mice were randomly aliquoted into treatment groups. On days 12, 15, 19 and 22, mice received 200 μl of intravenous injection of 1×10 resuspended in PBS ⁹ The TCID50 dose of mVV1 or PBS served as control, and/or 250 μg isotype control antibody (mIgG 2a and/or rat IgG2 a) and/or anti-mouse PD-1 rat IgG2a antibody (29 f1. A12), and/or 250 μg anti-mouse CTLA4-mIg a antibody at either day 12 or 15 in a single dose or at four doses at days 12, 15, 19 and 22 (clone 9D 9). Tumor volumes were monitored twice weekly by caliper measurements until the end of the study on day 60. The tumor volume average over time for each group indicated that one dose of anti-CTLA 4 antibody showed similar tumor growth inhibition when administered in combination with mVV1 and anti-PD-1, as compared to treatment with four doses of anti-CTLA 4 (fig. 14). The individual tumor volumes at day 22 after the start of treatment (fig. 15) were used for statistical analysis, as this is the present The last time point at which all animals survived in all groups in the study. Statistical significance (p) was determined by one-way ANOVA and Dunnett multiple comparison post-hoc test<0.01，****p<0.0001). The combination of mVV1 with one dose of anti-CTLA 4 antibody treatment resulted in equivalent efficacy in tumor growth inhibition compared to four doses of anti-CTLA 4 antibody (fig. 14, 15). Notably, triple combination treatment of intravenously delivered mVV1 with one dose of anti-CTLA 4 and anti-PD-1 antibodies also resulted in increased survival, similar to the group receiving four doses of CTLA4 (fig. 16). In summary, combined treatment of intravenously delivered mVV1 with anti-PD-1 and one dose of anti-mcta 4 antibody resulted in efficacy similar to administration of four doses of anti-mcta 4 antibody.

Table 10: experimental dosing and treatment regimen for groups of mice

Table 11: average tumor volume, percent survival, and number of tumor-free mice in each treatment group from in vivo tumors

As shown in table 11, mice treated with the triple combination of mVV1 with anti-PD-1 and four doses of anti-CTLA 4 antibody were very effective at controlling and clearing large tumors during the course of the study. Mice treated with triple combination mVV1 with anti-PD-1 and one dose of anti-CTLA 4 antibody administered simultaneously with virus exhibited similar tumor volume reduction compared to four doses. In contrast, if one dose of anti-CTLA 4 antibody is administered three days after the virus and anti-PD-1, the efficacy of the triple combination is lost. This data shows that simultaneous administration of one dose of anti-CTLA 4 with continuous administration of anti-PD-1 with mVV1 can be used to achieve strong anti-tumor efficacy.

Example 7: is carried by 150mm ³ In mice with mean MC38 tumors, the drug was dissolved with intravenous deliveryAnti-tumor efficacy of combination treatment of anti-PD-1, one dose of anti-CTLA 4 administered concurrently with oncological VSV-mIFNb-NIS

This example describes the anti-tumor efficacy of using the oncolytic virus Vesicular Stomatitis Virus (VSV) encoding IFNb and NIS in combination with a triple combination of anti-PD-1 and a dose of anti-CTLA 4 in wild-type mice implanted with MC38 tumors. The VSV used in this study was a genetically attenuated virus designated VSV-mIFNb-NIS (or mVV 1) because it encodes mouse interferon beta (IFNb) inserted between M and G viral genes and encodes sodium/iodine symporter (NIS) inserted between G and L viral genes. The anti-PD-1 antibody used in this study was clone 29f1.a12 rat IgG2a from Bioxcell, and the anti-CTLA 4 antibody used was clone 9D9 in the form of mIgG2a from invitrogen.

Subcutaneous implantation of MC38 cells (3×10) on day 0 in background mice of the C57BL/6 strain from Jackson laboratories ⁵ Individual cells/mice). Tumors were measured using calipers and combined with the formula (L ² Tumor volume was calculated by x W)/2, where L is the smallest dimension. When the average tumor size reached 150mm on day 12 ³ At this time, mice were randomly aliquoted into four treatment groups. On days 12, 15, 19 and 22, mice received 200 μl of intravenous injection of 1×10 resuspended in PBS ⁹ The TCID50 dose of mVV1 or PBS served as control, and/or 250 μg isotype control antibody (mIgG 2a and/or rat IgG2 a) and/or anti-mouse PD-1 rat IgG2a antibody (29 f 1.a12), and/or 250 μg anti-mouse CTLA4-mIgG2a antibody (clone 9D 9) served as a single dose on day 12 or 15. Tumor volumes were monitored twice weekly by caliper measurements until the end of the study on day 60. The tumor volume average over time for each group indicated that one dose of anti-CTLA 4 antibody required concomitant administration with the virus, as the group receiving a-CTLA4 on day 15 had reduced anti-tumor control compared to the group receiving a-CTLA4 on day 12 (fig. 17).

In summary, the combined treatment of intravenously delivered mVV1 with anti-PD-1 and one dose of anti-mcctla 4 antibody with viral administration resulted in strong anti-tumor efficacy.

Example 8: is carried by 100mm ³ Average B16F10 tumorAntitumor efficacy in mice treated with anti-PD-1, one dose of anti-CTLA 4 in combination with intravenous delivery of oncolytic virus VSV-mIFNb-NIS

This example describes the anti-tumor efficacy of using the oncolytic virus Vesicular Stomatitis Virus (VSV) encoding IFNb and NIS in combination with a triple combination of anti-PD-1 and a dose of anti-CTLA 4 in wild-type mice implanted with B16F10 tumors. The VSV used in this study was a genetically attenuated virus designated VSV-mIFNb-NIS (or mVV 1) because it encodes mouse interferon beta (IFNb) inserted between M and G viral genes and encodes sodium/iodine symporter (NIS) inserted between G and L viral genes. The anti-PD-1 antibody used in this study was clone 29f1.a12 rat IgG2a from Bioxcell, and the anti-CTLA 4 antibody used was clone 9D9 in the form of mIgG2a from invitrogen.

B16F10 cells were subcutaneously implanted on day 0 in background mice of the C57BL/6 strain from Jackson laboratories (5X 10) ⁵ Individual cells/mice). Tumors were measured using calipers and combined with the formula (L ² Tumor volume was calculated by x W)/2, where L is the smallest dimension. When the average tumor size reached 100mm on day 10 ³ At this time, mice were randomly aliquoted into seven treatment groups. On days 12, 15, 19 and 22, mice received 200 μl of 1×10 resuspended in PBS intravenously ⁹ Or 5X 10 ⁷ Or 1X 10 ⁷ Or 1X 10 ⁶ The TCID50 dose of mVV1 or PBS served as control, and/or 250 μg isotype control antibody (mIgG 2a and/or rat IgG2 a) and/or anti-mouse PD-1 rat IgG2a antibody (29 f 1.a12), and/or 250 μg anti-mouse CTLA4-mIg a antibody (clone 9D 9) served as a single dose on day 10. Tumor volumes were monitored twice weekly by caliper measurements until the end of the study on day 60. The tumor volume average over time for each group indicated that one dose of anti-CTLA 4 antibody showed strong tumor growth inhibition in the B16F10 model compared to PBS or single agent (fig. 18). Notably, when the viral dose is from 1X 10 ⁹ Reduced to 5X 10 ⁷ Or 1X 10 ⁷ Or 1X 10 ⁶ The triple combination of intravenously delivered mVV1 with one dose of anti-CTLA 4 and anti-PD-1 antibody treatment also resulted in an improved survival class at TCID50Similar efficacy (fig. 18).

In summary, treatment with intravenously delivered mVV1 in combination with anti-PD-1 and one dose of anti-mCTLA 4 antibody resulted in strong anti-tumor efficacy in B16F10 anti-PD-1 resistant tumor models, and a broad range of viral titers could still achieve strong combined efficacy.

Table 12: experimental dosing and treatment regimen for groups of mice

Table 13: average tumor volume, percent survival, and number of tumor-free mice in each treatment group from in vivo tumors using the B16F10 melanoma tumor model.

As shown in table 13, mice treated with mVV1 or with anti-PD-1 in combination with one dose of anti-CTLA 4 antibody had a very modest effect on tumor growth in this high standard (high bar) immune checkpoint resistant tumor model using the B16F10 subcutaneous tumor model. However, the triple combination of mVV1 with anti-PD-1 in combination with one dose of anti-CTLA 4 antibody significantly increased anti-tumor efficacy compared to the other groups. This data suggests that VSV can sensitize checkpoint resistant tumors to immunotherapy.

Example 9: is carried by 150mm ³ Antitumor efficacy in mice with average CMT64 lung tumors treated with anti-PD-1, one dose of anti-CTLA 4 in combination with intravenous delivery of oncolytic virus VSV-mIFNb-NIS

This example describes the anti-tumor efficacy of using the oncolytic virus encoding IFNb and NIS Vesicular Stomatitis Virus (VSV) in combination with a triple combination of anti-PD-1 and a dose of anti-CTLA 4 in wild-type mice implanted with CMT64 tumors resistant to treatment with anti-PD-1. The VSV used in this study was a genetically attenuated virus designated VSV-mIFNb-NIS (or mVV 1) because it encodes mouse interferon beta (IFNb) inserted between M and G viral genes and encodes sodium/iodine symporter (NIS) inserted between G and L viral genes. The anti-PD-1 antibody used in this study was clone 29f1.a12 rat IgG2a from Bioxcell, and the anti-CTLA 4 antibody used was clone 9D9 in the form of mIgG2a from invitrogen.

B16F10 cells were subcutaneously implanted on day 0 in background mice of the C57BL/6 strain from Jackson laboratories (5X 10) ⁵ Individual cells/mice). Tumors were measured using calipers and combined with the formula (L ² Tumor volume was calculated by x W)/2, where L is the smallest dimension. When the average tumor size reached 100mm on day 10 ³ At this time, mice were randomly aliquoted into four treatment groups. On days 12, 15, 19 and 22, mice received 200 μl of intravenous injection of 1×10 resuspended in PBS ⁹ The TCID50 dose of mVV1 or PBS served as control, and/or 250 μg isotype control antibody (mIgG 2a and/or rat IgG2 a) and/or anti-mouse PD-1 rat IgG2a antibody (29 f 1.a12), and/or 50 μg anti-mouse CTLA4-mIg a antibody (clone 9D 9) as a single dose on day 10. Tumor volumes were monitored twice weekly by caliper measurements until the end of the study on day 60. The tumor volume average over time for each group indicated that one dose of anti-CTLA 4 antibody showed strong tumor growth inhibition in CMT64 tumor model compared to PBS or anti-PD-1 and anti-CTLA 4 (fig. 19). Figure 20 shows the average Spot Forming Units (SFU) of IFNg released from CD8 TIL harvested from tumors and again overnight exposed to the indicated tumor antigen or VSV-NP in each treatment group on day 17 after receiving VSV on day 12 and two doses of anti-PD-1 and a-CTLA4 on days 12 and 14. DMSO and PMA/ionomycin were used as negative and positive controls, respectively, at time points after implantation of multiple tumors, with treatment days indicated by arrows.

In summary, treatment with intravenously delivered mVV1 in combination with anti-PD-1 and one dose of anti-mcta 4 antibody resulted in unexpectedly strong anti-tumor efficacy in CMT64 anti-PD-1 resistant tumor models, indicating that this triple combined efficacy is applicable to a variety of tumor environments.

Table 14: experimental dosing and treatment regimen for groups of mice

Table 15: triple combinations of intravenous mVV1 with anti-ctla4+ anti-PD-1 antibodies were tested in CMT64 lung adenocarcinoma model, average tumor volume, percent survival, and number of tumor-free mice in each treatment group from in vivo tumors.

As shown in table 15, mice treated with a triple combination of intravenous mVV1 with anti-PD-1 and one dose of anti-CTLA 4 antibody were very effective in controlling CMT64 tumor growth.

Example 10: is carried by 150mm ³ In mice with average CMT64 lung tumors, combined treatment with anti-PD-1, anti-CTLA 4 and intravenous delivery of oncolytic virus VSV-mIFNb-NIS elicited a broad polyclonal anti-tumor T cell response

This example describes the mechanism of action of using the oncolytic virus Vesicular Stomatitis Virus (VSV) encoding IFNb and NIS in combination with a triple combination of anti-PD-1 and a dose of anti-CTLA 4 in wild-type mice implanted with CMT64 tumors resistant to treatment with anti-PD-1. The VSV used in this study was a genetically attenuated virus designated VSV-mIFNb-NIS (or mVV 1) because it encodes mouse interferon beta (IFNb) inserted between M and G viral genes and encodes sodium/iodine symporter (NIS) inserted between G and L viral genes. The anti-PD-1 antibody used in this study was clone 29f1.a12 rat IgG2a from Bioxcell, and the anti-CTLA 4 antibody used was clone 9D9 in the form of mIgG2a from invitrogen.

CMT64 cells were subcutaneously implanted on day 0 in background mice of the C57BL/6 strain from Jackson laboratories (5×10 ⁶ Individual cells/mice). Tumors were measured using calipers and combined with the formula (L ² Tumor volume was calculated by x W)/2, where L is the smallest dimension. When the average tumor size reached 100mm on day 10 ³ At this time, the mice were randomly aliquotedSeven treatment groups. On days 10 and 14, mice received 200 μl of 1×10 resuspended in PBS intravenously ⁹ The TCID50 dose of mVV1 or PBS served as control and/or 250 μg isotype control antibody (mIgG 2a and/or rat IgG2 a) and/or anti-mouse PD-1 rat IgG2a antibody (29 f 1.a12) and/or 10 μg anti-mouse CTLA4-mIgG2a antibody (clone 9D 9) was intraperitoneally injected. Tumors were harvested on day 17. Purified CD8 TIL and primary spleen cells were co-incubated at a 1:1 ratio by plating 10,000 cells/well and incubated overnight with the corresponding peptide antigen for IFNg ELISPOT assay. In the group receiving VSV, the large reactivity of CD8 TIL was detected specific for the VSV-NP antigen. Notably, in the re-exposed CD8 TIL collected from the group receiving VSV, many tumor neoantigens induced signals, and very limited responses were detected for the groups treated with anti-PD-1 and anti-CTLA 4 alone. Triple combination of VSV with a-PD-1 and a-CTLA4 induced a large polyclonal anti-tumor T cell response compared to the other groups, with only some neoantigen reactivity (e.g., NAIP2 and hx 2) detected in the triple combination.

This data indicates that triple combined efficacy is driven by the generation of polyclonal anti-tumor T cells that function within the tumor and induce an anti-tumor T cell response.

Table 16: experimental dosing and treatment regimen for groups of mice

Table 17: IFNg Elispot data generated from TIL isolated from CMT64 tumors harvested from seven day post-treatment mice (10,000 TIL:10,000 spleen cells)

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The scope of the present disclosure is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A method of treating a tumor or inhibiting tumor growth comprising:

(a) Selecting a patient with cancer; and

(b) Administering to the patient in need thereof: a combination of (i) a therapeutically effective amount of an oncolytic virus with (ii) a therapeutically effective amount of an inhibitor of the programmed death 1 (PD-1) pathway and (iii) a therapeutically effective amount of an inhibitor of the cytotoxic T lymphocyte antigen-4 (CTLA 4).

2. The method of claim 1, wherein the oncolytic virus comprises an oncolytic vesicular virus.

3. The method of claim 1 or 2, wherein the oncolytic vesicular virus comprises an oncolytic Vesicular Stomatitis Virus (VSV).

4. The method of claim 3, wherein the VSV comprises a recombinant VSV.

5. The method of claim 4, wherein the recombinant VSV comprises an M51R substitution.

6. The method of any one of claims 3 to 5, wherein the recombinant VSV expresses a cytokine.

7. The method of claim 6, wherein the cytokine comprises interferon-beta (IFNb).

8. The method of claim 7, wherein the nucleic acid sequence encoding the IFNb is located between M and G viral genes.

9. The method of any one of claims 4 to 8, wherein the recombinant VSV further expresses sodium/iodine symporter (NIS).

10. The method of claim 9, wherein the nucleic acid sequence encoding the NIS is located between G and L viral genes.

11. The method of any one of claims 1 to 10, wherein the oncolytic virus is Voyager V1.

12. The method of any one of claims 1 to 11, wherein the oncolytic virus, the PD-1 pathway inhibitor, and the CTLA4 inhibitor are administered to the patient simultaneously.

13. The method of any one of claims 1 to 11, wherein one or more doses of the oncolytic virus are administered in sequence in combination with one or more doses of the PD-1 pathway inhibitor and one or more doses of the CTLA4 inhibitor.

14. The method of claim 13, wherein one or more doses of the CTLA4 inhibitor comprise a single dose of the CTLA4 inhibitor, and wherein administration of a single dose of the CTLA4 inhibitor results in an anti-tumor efficacy comparable to that of a combination therapy comprising two or more doses of the CTLA4 inhibitor.

15. The method of claim 14, wherein the anti-tumor efficacy is characterized by the following decreases: tumor volume mean or average tumor volume, percent survival, number of tumor-free patients in each treatment group, or a combination thereof.

16. The method of any one of claims 1 to 15, wherein the oncolytic virus is administered to the patient in one or more of the following doses: 10 ⁴ To 10 ¹⁴ TCID ₅₀ 、10 ⁴ To 10 ¹² TCID ₅₀ 、10 ⁶ To 10 ¹² TCID ₅₀ 、10 ⁸ To 10 ¹⁴ TCID ₅₀ 、10 ⁸ To 10 ¹² TCID ₅₀ Or 10 ¹⁰ To 10 ¹² TCID ₅₀ 。

17. The method of any one of claims 1 to 16, wherein the PD-1 pathway inhibitor is administered to the patient at one or more doses of about 0.1mg/kg to about 20mg/kg of patient body weight.

18. The method of any one of claims 1 to 17, wherein the PD-1 pathway inhibitor is administered to the patient in one or more doses of about 1mg to about 1000 mg.

19. The method of any one of claims 1 to 18, wherein the CTLA4 inhibitor is administered to the patient at one or more doses of about 0.1mg/kg to about 15mg/kg of patient body weight.

20. The method of any one of claims 1 to 19, wherein the CTLA4 inhibitor is administered to the patient in a single dose of about 0.1mg/kg to about 15mg/kg of patient body weight.

21. The method of any one of claims 1 to 20, wherein the CTLA4 inhibitor is administered to the patient in one or more doses of about 1mg to about 600 mg.

22. The method of any one of claims 1 to 21, wherein the oncolytic virus is administered intratumorally or intravenously to the patient.

23. The method of any one of claims 1 to 22, wherein the PD-1 pathway inhibitor and the CTLA4 inhibitor are administered to the patient intravenously, subcutaneously, or intraperitoneally.

24. The method of any one of claims 1 to 23, wherein the cancer is selected from adrenal tumor, cholangiocarcinoma, bladder cancer, brain cancer, breast cancer, epithelial cancer, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer or hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, li-fomein tumor, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple neuroendocrine type I and type II tumors, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma tumor, ovarian cancer, pancreatic cancer, islet cell cancer, parathyroid cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, renal cancer, respiratory cancer, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, tracheal cancer, genitourinary cancer, and uterine cancer.

25. The method of any one of claims 1 to 24, wherein the cancer is resistant to treatment with at least one anti-PD-1 agent or treatment.

26. The method of any one of claims 1 to 25, wherein the PD-1 pathway inhibitor comprises an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, or an anti-PD-L2 antibody or antigen-binding fragment thereof.

27. The method of claim 26, wherein the anti-PD-1 antibody is selected from the group consisting of cimetidine Li Shan antibody, nivolumab, pembrolizumab, MEDI0608, BI 754091, PF-06801591, swadamascent, carlizumab, JNJ-63723283, and MCLA-134.

28. The method of any one of claims 26 to 27, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (HCDR) comprising a Heavy Chain Variable Region (HCVR) of the amino acid sequence of SEQ ID No. 1 and a light chain complementarity determining region (LCDR) comprising a Light Chain Variable Region (LCVR) of the amino acid sequence of SEQ ID No. 2.

29. The method of any one of claims 26 to 28, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR) (HCDR 1, HCDR2 and HCDR 3) comprising the corresponding amino acid sequences of SEQ ID NOs 3, 4 and 5; and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) comprising the corresponding amino acid sequences of SEQ ID NOS: 6, 7 and 8.

30. The method of any one of claims 26 to 29, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) comprising the amino acid sequence of SEQ ID No. 1; and a Light Chain Variable Region (LCVR) comprising the amino acid sequence of SEQ ID NO. 2.

31. The method of any one of claims 26 to 30, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy and light chain sequence pair of SEQ ID NOs 9 and 10.

32. The method of claim 26, wherein the anti-PD-L1 antibody is selected from REGN3504, avilamab, atuzumab, divaliab, MDX-1105, LY3300054, FAZ053, STI-1014, CX-072, KN035, and CK-301.

33. The method of claim 26, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) comprising the amino acid sequence of SEQ ID No. 11; and a Light Chain Variable Region (LCVR) comprising the amino acid sequence of SEQ ID NO. 12.

34. The method of any one of claims 26 to 33, wherein the anti-PD-L1 antibody comprises REGN3504.

35. The method of any one of claims 1 to 34, wherein the CTLA4 inhibitor comprises an anti-CTLA 4 antibody or antigen-binding fragment thereof.

36. The method of claim 35, wherein the anti-CTLA 4 antibody is selected from ipilimumab, tremelimumab, and REGN4659.

37. The method of claim 35 or 36, wherein the anti-CTLA 4 antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region (HCDR) comprising a Heavy Chain Variable Region (HCVR) of the amino acid sequence of SEQ ID No. 13 and a light chain complementarity determining region (LCDR) comprising a Light Chain Variable Region (LCVR) of the amino acid sequence of SEQ ID No. 14.

38. The method of any one of claims 35 to 37, wherein the anti-CTLA 4 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR) (HCDR 1, HCDR2 and HCDR 3) comprising the corresponding amino acid sequences of SEQ ID NOs 15, 16 and 17; and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) comprising the corresponding amino acid sequences of SEQ ID NOS: 18, 19 and 20.

39. The method of any one of claims 35 to 38, wherein the anti-CTLA 4 antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) comprising the amino acid sequence of SEQ ID No. 13; and a Light Chain Variable Region (LCVR) comprising the amino acid sequence of SEQ ID NO. 14.

40. The method of any one of claims 35 to 39, wherein the anti-CTLA 4 antibody or antigen-binding fragment thereof comprises the heavy and light chain sequence pairs of SEQ ID NOs 21 and 22.

41. The method of any one of claims 1 to 40, wherein the treatment produces a therapeutic effect selected from one or more of the following: delay in tumor growth, reduced tumor cell number, tumor regression, increased survival, partial response, and complete response.

42. The method of any one of claims 1 to 41, wherein the tumor growth is inhibited by at least 50% compared to an untreated patient.

43. The method of any one of claims 1 to 42, wherein the tumor growth is inhibited by at least 50% compared to a patient administered the oncolytic virus, the PD-1 pathway inhibitor, or the CTLA4 inhibitor as monotherapy.

44. The method of any one of claims 1 to 43, wherein the tumor growth is inhibited by at least 50% as compared to a patient administered any two of the oncolytic virus, the PD-1 pathway inhibitor, and the CTLA4 inhibitor.

45. The method of any one of claims 1 to 44, further comprising administering an additional therapeutic agent or treatment to the patient.

46. The method of claim 45, wherein the additional therapeutic agent or treatment is selected from the group consisting of: radiation, surgery, chemotherapeutics, cancer vaccines, B7-H3 inhibitors, B7-H4 inhibitors, lymphocyte activation gene 3 (LAG 3) inhibitors, inhibitors containing T cell immunoglobulin and mucin domain-3 (TIM 3), galectin 9 (GAL 9) inhibitors, inhibitors of T cell activated V domain-containing immunoglobulin (Ig) inhibitors (VISTA), killer cell immunoglobulin-like receptor (KIR) inhibitors, B and T Lymphocyte Attenuators (BTLA) inhibitors, T cell immune receptor (TIGIT) inhibitors having Ig and ITIM domains, CD47 inhibitors indoleamine-2, 3-dioxygenase (IDO) inhibitors, vascular Endothelial Growth Factor (VEGF) antagonists, angiopoietin-2 (Ang 2) inhibitors, transforming growth factor beta (tgfβ) inhibitors, epidermal Growth Factor Receptor (EGFR) inhibitors, antibodies to tumor-specific antigens, bcg, granulocyte-macrophage colony-stimulating factor (GM-CSF), cytotoxins, interleukin 6 receptor (IL-6R) inhibitors, interleukin 4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-12, IL-21, IL-15, antibody-drug conjugates, anti-inflammatory drugs, and combinations thereof.

47. A combination of an oncolytic virus, a PD-1 pathway inhibitor and a CTLA4 inhibitor for use in a method of treating a tumor or inhibiting tumor growth, the method comprising:

(a) Selecting a patient with cancer; and

(b) Administering to the patient in need thereof: a combination of (i) a therapeutically effective amount of the oncolytic virus with (ii) a therapeutically effective amount of the PD-1 pathway inhibitor and (iii) a therapeutically effective amount of the CTLA inhibitor.

48. A kit comprising an oncolytic virus, a PD-1 pathway inhibitor, and a CTLA4 inhibitor in combination with: written instructions for using a therapeutically effective amount of a combination of the oncolytic virus, the PD-1 pathway inhibitor, and the CTLA4 inhibitor to treat a tumor or inhibit tumor growth in a patient.