Patent Application
20220143116
This document relates to methods and materials for treating cancer. For example, this document provides engineered viruses (e.g., oncolytic viruses) containing nucleic acid encoding one or more inhibitors of apolipoprotein B editing complex 3B (APOBEC3B) polypeptide activity or expression and methods for using such viruses as an oncolytic agent (e.g., to treat cancer). For example, one or more engineered oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having cancer to treat that mammal.
Oncolytic virotherapy has been developed for the treatment of cancer as it combines tumor-tropic cytotoxicity with a highly inflammatory anti-viral response that can activate cellular anti-tumor responses. Strategies such as tropism targeting and arming of the virus with immune stimulatory cytokines to promote the recruitment of immune cells to the tumor have significantly improved the inherent anti-cancer properties of viral therapy (Jhawar et al. Front Oncol 7:202 (2017); and Lichty et al., Nat Rev Cancer 14:559-567 (2014)). The FDA approval of the Herpes simplex virus (HSV)-based viral therapy Talimogene laherparepvec (Tvec), Imlygic, in 2015 demonstrates the clinical significance of these viral-based platforms (Lawler et al., JAMA Oncol 3:841-849 (2017); and Corrigan et al., Ann Pharmacother 51:675-681 (2017)). Despite the ability to enhance viral-mediated tumor cell killing and immune activation, clinical responses are observed in only a subset of patients (Pol et al. Oncoimmunology 3:e28694 (2014); and Pol et al. Oncoimmunology 5:e1117740 (2016)).
Suboptimal (e.g., incomplete) T cell activation and limited effector function induces APOBEC3B upregulation in targeted tumor cells (e.g., tumor cells directly in contact with T cell activity) and in bystander tumor cells (e.g., tumor cells which are physically separated from the T cells themselves yet are exposed to T cell-derived factors). Upregulation of APOBEC3B in a cell can induce mutations that can provide that cell with a selective advantage to develop immunotherapy (e.g., oncolytic immunotherapy) escape and/or resistance.
This document provides methods and materials for treating cancer. For example, this document provides viral nucleic acid (e.g., viral vectors) as well as viruses (e.g., oncolytic viruses) that encode (e.g., that are engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression. In some cases, viral nucleic acid provided herein and oncolytic viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be used as an oncolytic agent (e.g., to treat cancer). For example, one or more oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having cancer to treat that mammal.
APOBEC3B is a factor that can restrict the potency of oncolytic viruses (e.g., oncolytic VSVs). For example, VSV infection of cancer cells (e.g., B16 murine melanoma cells) can upregulate APOBEC3B expression in an IFNβ-dependent manner, which is responsible for the evolution of virus-resistant cancer cell populations. As demonstrated herein, in vivo administration of oncolytic virus particles having nucleic acid encoding an inhibitor of APOBEC3B expression or activity (e.g., short hairpin RNA (shRNA) designed to inhibit APOBEC3B expression) to a mammal having cancer can result in an increased level of in vivo oncolytic activity against the cancer cells and/or a reduced level of resistance to the oncolytic therapy by cancer cells within the mammal (as compared to the levels observed using comparable oncolytic virus particles lacking the nucleic acid encoding an inhibitor of APOBEC3B expression or activity).
In some cases, an inhibitor of APOBEC3B polypeptide activity or expression (or nucleic acid constructs or viruses encoding an inhibitor of APOBEC3B polypeptide activity or expression) can be used in combination with immunotherapy (e.g., therapy involving the use of engineered T cells such as chimeric antigen receptor (CAR) T cells) to increase the level of anti-cancer activity against the cancer cells and/or to reduce the level of resistance to the immunotherapy by cancer cells within the mammal (as compared to the levels observed using the immunotherapy without inhibiting APOBEC3B expression or activity). For example, a virus (e.g., an oncolytic virus) encoding an inhibitor of APOBEC3B polypeptide activity or expression can be used in combination with CART cell therapy to treat cancer within a mammal (e.g., a human) in a manner that results in a reduced level of cancer cell resistance to the CAR T cell therapy within the mammal (as compared to the level observed using a comparable virus lacking nucleic acid encoding an inhibitor of APOBEC3B polypeptide activity or expression in combination with the CAR T cell therapy in a comparable mammal).
In general, one aspect of this document features methods for treating a mammal having cancer where the methods can include, or consist essentially of, administering a composition including an oncolytic virus to the mammal, thereby reducing the number of cancer cells within the mammal, where the oncolytic virus can include nucleic acid encoding an inhibitor of APOBEC3B polypeptide activity or expression, and where the level of cancer cell resistance development to the oncolytic virus within the mammal is reduced as compared to the level that develops in a comparable mammal administered a comparable oncolytic virus lacking the nucleic acid encoding the inhibitor. The mammal can be a human. The cancer can be breast cancer, brain cancer, prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, bladder cancer, colorectal cancer, osteosarcoma, myeloma, leukemia, or lymphoma. The oncolytic virus can be a vesicular stomatitis virus (VSV), a Maraba virus (MARAV), a herpes simplex virus (HSV), a vaccinia virus (VV), a measles virus (MV), or a poliovirus (PV). For example, the oncolytic virus can be a VSV. The inhibitor of APOBEC3B polypeptide activity or expression can be a short hairpin RNA (shRNA) that can target nucleic acid encoding the APOBEC3B polypeptide. The nucleic acid encoding the shRNA can include a nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The composition can include from about 103 plaque-forming units (PFUs) to about 1013 PFUs of oncolytic viruses. The composition can include oncolytic viruses at a multiplicity of infection (MOI) of from about 0.0000001 to about 10000.
In another aspect, this document features methods for treating a mammal having cancer where the methods can include, or consist essentially of, administering an oncolytic virus to the mammal, thereby reducing the number of cancer cells within the mammal, and administering nucleic acid or a virus to the mammal, where the nucleic acid or a virus includes nucleic acid encoding an inhibitor of APOBEC3B polypeptide activity or expression, where a reduced level of cancer cell resistance to the oncolytic virus develops within the mammal as compared to the level that develops in a comparable mammal administered the oncolytic virus in the absence of the nucleic acid encoding the inhibitor and in the absence of the virus containing the nucleic acid. The mammal can be a human. The cancer can be breast cancer, brain cancer, prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, bladder cancer, colorectal cancer, osteosarcoma, myeloma, leukemia, or lymphoma. The virus can be a retrovirus, a lentivirus, an adenoviruses, an adeno-associated virus, a VSV, a MARAV, a HSV, a VV, a MV, or a PV. For example, the virus can be a VSV. The oncolytic virus can be a VSV, a HSV, a VV, an AV, a MV, or a PV. For example, the oncolytic virus can be a VSV. The inhibitor of APOBEC3B polypeptide activity or expression can be a shRNA that can target nucleic acid encoding the APOBEC3B polypeptide. The nucleic acid encoding the shRNA can include a nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The composition can include from about 103 PFUs to about 1013 PFUs of oncolytic viruses. The composition can include oncolytic viruses at a MOI of from about 0.0000001 to about 10000.
In another aspect, this document features methods for treating a mammal having cancer where the methods can include, or consist essentially of, administering a composition including a virus to the mammal, thereby reducing the number of cancer cells within the mammal, where the virus can include nucleic acid encoding an inhibitor of APOBEC3B polypeptide activity or expression, and where the level of cancer cell resistance development to an oncolytic virus within the mammal is reduced as compared to the level that develops in a comparable mammal administered a comparable oncolytic virus lacking the nucleic acid encoding the inhibitor. The mammal can be a human. The cancer can be breast cancer, brain cancer, prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, bladder cancer, colorectal cancer, osteosarcoma, myeloma, leukemia, or lymphoma. The virus can be a VSV, a MARAV, a HSV, a VV, a MV, or a PV. The virus can be an oncolytic virus. The virus can be a non-oncolytic virus. The inhibitor of APOBEC3B polypeptide activity or expression can be a shRNA that can target nucleic acid encoding the APOBEC3B polypeptide. The nucleic acid encoding the shRNA can include a nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The composition can include from about 103 PFUs to about 1013 PFUs of oncolytic viruses. The composition can include oncolytic viruses at a MOI of from about 0.0000001 to about 10000.
In another aspect, this document features methods for treating a mammal having cancer where the methods can include, or consist essentially of, administering T cells to the mammal, thereby reducing the number of cancer cells within the mammal, and administering nucleic acid or a virus to the mammal, where the nucleic acid or a virus includes nucleic acid encoding an inhibitor of APOBEC3B polypeptide activity or expression, where a reduced level of cancer cell resistance to the T cells develops within the mammal as compared to the level that develops in a comparable mammal administered the T cells in the absence of the nucleic acid encoding the inhibitor and in the absence of the virus containing said nucleic acid. The mammal can be a human. The cancer can be breast cancer, brain cancer, prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, bladder cancer, colorectal cancer, osteosarcoma, myeloma, leukemia, or lymphoma. The T cells can be CAR T cells. The inhibitor of APOBEC3B polypeptide activity or expression can be a shRNA that can target nucleic acid encoding the APOBEC3B polypeptide. The nucleic acid encoding the shRNA can include a nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The composition comprises from about 103 PFUs to about 1013 PFUs of said oncolytic viruses. The composition can include oncolytic viruses at a MOI of from about 0.0000001 to about 10000.
Unless otherwise defined, 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials for treating cancer. For example, this document provides methods and materials for treating cancer using viral nucleic acid (e.g., viral vectors) and/or viruses (e.g., oncolytic viruses) that encode (e.g., that are engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression. In some cases, this document provides engineered oncolytic viruses that contain nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression. For example, an oncolytic virus encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can include nucleic acid encoding shRNA that can target APOBEC3B and inhibit its expression.
In some cases, this document provides methods for using viral nucleic acid (e.g., viral vectors) and/or viruses (e.g., oncolytic viruses) that encode (e.g., that are engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression to treat a mammal having, or at risk of developing, cancer. For example, engineered viral nucleic acid provided herein and/or oncolytic viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk of developing, cancer to reduce the number of cancer cells (e.g., by infecting cancer cells in the mammal and stimulating anti-cancer immune responses in the mammal) in the mammal (e.g., a human). In some cases, one or more viral vectors encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses can be administered to a mammal having, or at risk of developing, cancer to reduce the number of cancer cells in the mammal. In some cases, one or more oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk of developing, cancer to reduce the number of cancer cells in the mammal. In some cases, one or more viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses can be administered to a mammal having, or at risk of developing, cancer to reduce the number of cancer cells in the mammal.
Viral nucleic acid (e.g., a viral vector) encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be any appropriate nucleic acid (e.g., DNA, RNA, or a combination thereof). In some cases, viral nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be a nucleic acid construct.
A virus (e.g., an oncolytic virus particle) containing nucleic acid encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be any appropriate virus. For example, a virus having nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression as described herein can be an oncolytic virus or a non-oncolytic virus. Examples of types of viruses that can be designed to have nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression as described herein include, without limitation, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, rhabdoviruses (e.g., vesicular stomatitis virus (VSV) and Maraba virus (MARAV)), a herpes simplex virus (HSV), a vaccinia virus (VV), a measles virus (MV), and a poliovirus (PV), and hybrids thereof. Examples of oncolytic viruses that can be designed to encode one or more inhibitors of APOBEC3B polypeptide activity or expression as described herein include, without limitation, VSV, HSV, VV, MV, and PV.
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression described herein can be (or can contain in the case of viruses) double stranded nucleic acid.
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression described herein can be (or can contain in the case of viruses) single stranded nucleic acid.
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression described herein can be replication competent.
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression described herein can be replication defective.
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression described herein can be non-pathogenic (e.g., to a mammal being treated as described herein). For example, a virus particle provided herein containing nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be genetically modified to render it non-pathogenic to a mammal to be treated.
In some cases, viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression described herein can infect dividing cells (e.g., can infect only dividing cells).
In some cases, viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression described herein can infect non-dividing cells (e.g., can infect only non-dividing cells).
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression described herein are not destroyed a mammal's immune system. For example, viral nucleic acid provided herein and viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression described herein are not destroyed by antigen presenting cells (APCs), macrophages, and/or other immune cells in a mammal that the viral nucleic acid provided herein and/or the viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression are administered to.
Viral nucleic acid (e.g., viral vectors) provided herein and viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can encode any appropriate inhibitor(s) of APOBEC3B polypeptide activity or expression. An inhibitor of an APOBEC3B polypeptide activity or expression can be any molecule that inhibits (e.g., reduces or eliminates) APOBEC3B polypeptide activity or expression. An inhibitor of an APOBEC3B polypeptide activity or expression can be any appropriate type of molecule (e.g., nucleic acids such as siRNA molecules, shRNA molecules, antisense molecules, targeting guide RNA molecules of a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, miRNAs (e.g., natural miRNA and artificial miRNA); and polypeptides such as antibodies and transcription activator-like effector nucleases (TALENs)).
In some cases, RNA interference can be used reduce or eliminate APOBEC3B polypeptide activity or expression. For example, nucleic acid molecules designed to induce RNA interference of APOBEC3B (e.g., a siRNA molecule or a shRNA molecule) can be used as described herein to reduce or eliminate APOBEC3B polypeptide expression. Examples of nucleic acid molecules that can be used as described herein to reduce or eliminate APOBEC3B polypeptide expression include, without limitation, nucleic acid sequences encoding shRNA molecules that can target nucleic acid encoding an APOBEC3B polypeptide. Exemplary shRNA molecules that can target nucleic acid encoding an APOBEC3B polypeptide include, without limitation, those sequences set forth Table 1.
In some cases, a CRISPR/Cas9 system can be used as described herein to reduce or eliminate APOBEC3B polypeptide activity or expression. For example, guide RNA molecules of a CRISPR-Cas9 system can be designed to target nucleic acid encoding an APOBEC3B polypeptide such that the Cas9 of the CRISPR/Cas9 system can cleave the nucleic acid that encodes an APOBEC3B polypeptide to reduce or eliminate APOBEC3B polypeptide activity or expression. The CRISPR/Cas9 system can be as described elsewhere (Adli, Nat Commun. 9(1):1911 (2018)).
In some cases, one or more TALENs can be used as described herein to reduce or eliminate APOBEC3B polypeptide activity or expression. For example, TALENs can be designed to target nucleic acid encoding an APOBEC3B polypeptide such that the TALENs cleave the nucleic acid encoding an APOBEC3B polypeptide to reduce or eliminate APOBEC3B polypeptide activity or expression. The nucleic acid or polypeptide sequence of such genome editing molecules can be as described elsewhere (Campbell et al., Circulation Research, 113:571-587 (2013)).
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be as described in Example 1 or Example 2. In some cases, viral nucleic acid provided herein and viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be as described elsewhere (see, e.g., Huff et al., Mol. Ther. Oncolytics, 11:1-13 (2018)).
This document also provides methods for using viral nucleic acid (e.g., viral vectors) provided herein and viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression provided herein. In some cases, viral nucleic acid provided herein and/or viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be used to treat a mammal (e.g., a human) having, or at risk of developing, cancer. For example, viral nucleic acid provided herein and/or viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk of developing, cancer to reduce the number of cancer cells (e.g., by infecting and killing cancer cells) in the mammal. For example, viral nucleic acid provided herein and/or viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk of developing, cancer to reduce the size (e.g., the volume) of one or more tumors in the mammal. In some cases, viral nucleic acid provided herein and/or viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk of developing, cancer such that cancer cells within the mammal can develop resistance to an oncolytic virus at a reduced level (e.g., as compared to a level that develops in a comparable mammal administered a comparable oncolytic virus in the absence of any inhibitor of APOBEC3B polypeptide activity or expression). For example, when oncolytic viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression are administered to a mammal having, or at risk of developing, cancer, the level of cancer cell resistance development to the oncolytic virus within the mammal can be reduced (e.g., as compared to a level that develops in a comparable mammal administered a comparable oncolytic virus lacking nucleic acid encoding an inhibitor of APOBEC3B polypeptide activity or expression).
In some cases, methods described herein also can include identifying a mammal as having, or at risk of developing, cancer. Examples of methods for identifying a mammal as having, or at risk of developing, cancer include, without limitation, physical examination, laboratory tests (e.g., blood and/or urine), biopsy, imaging tests (e.g., X-ray, PET/CT, MRI, and/or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy, and/or genetic tests.
Once identified as having, or at risk of developing, cancer, a mammal can be administered or instructed to self-administer viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) encoding one or more inhibitors of APOBEC3B polypeptide activity or expression described herein (e.g., one or more oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression). In some cases, one or more viral vectors encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses can be administered to a mammal having, or at risk of developing, cancer to treat the mammal. In some cases, one or more oncolytic viruses and one or more viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk of developing, cancer to treat the mammal. In some cases, one or more oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk of developing, cancer to treat the mammal.
In cases where one or more viral vectors and/or one or more non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses are administered to a mammal having, or at risk of developing, cancer to treat the mammal, the one or more viral vectors and/or one or more non-oncolytic viruses and the one or more oncolytic viruses can be administered at the same time or independently. For example, one or more viral vectors and/or one or more non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses can be administered to a mammal at the same time (e.g., can be formulated together to form a single composition to be administered to a mammal). In some cases, one or more viral vectors and/or one or more non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered first, and the one or more oncolytic viruses administered second, or vice versa.
In cases where one or more viral vectors and/or one or more non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses are administered to a mammal having, or at risk of developing, cancer to treat the mammal, the one or more viral vectors and/or one or more non-oncolytic viruses and the one or more oncolytic viruses can be administered in any appropriate ratio (e.g., a ratio of one or more viral vectors and/or one or more non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression to one or more oncolytic viruses). For example, one or more viral vectors and/or one or more non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses can be administered to a mammal having, or at risk of developing, cancer at a ratio of from about 0.001:1 to about 100:1.
Any appropriate mammal having, or at risk of developing, cancer can be treated as described herein (e.g., by administering viral nucleic acid such as viral vectors provided herein and/or viruses such as oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression). For example, humans, non-human primates (e.g., monkeys), horses, bovine species, porcine species, dogs, cats, mice, and rats having, or at risk of developing, cancer can be treated for cancer as described herein. In some cases, a human having cancer can be treated as described herein.
A mammal having any type of cancer can be treated as described herein. In some cases, a cancer can include one or more solid tumors. In some cases, a cancer can be a blood cancer. Examples of cancers that can be treated as described herein include, without limitation, breast cancers (e.g., estrogen receptor positive breast cancer or estrogen receptor negative breast cancer), brain cancers (e.g., glioma), prostate cancers, ovarian cancers, lung cancers, hepatocellular carcinomas, pancreatic cancers, kidney cancers, melanomas, bladder cancers, colorectal cancers, sarcomas (e.g., osteosarcomas), and blood cancers (e.g., myelomas, leukemias, lymphomas).
In some cases, when administering viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression provided herein to a mammal (e.g., a human) as described herein, the viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can stimulate anti-cancer immune responses in the mammal.
In some cases, when administering viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression provided herein to a mammal (e.g., a human) as described herein, the viral nucleic acid and/or viruses encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can stimulate an optimal T cell activation. For example, administering viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression provided herein to a mammal as described herein can be effective to stimulate T cell responses in the mammal that are cytotoxic. In some cases, T cell responses in a mammal that are stimulated by administering viral nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression to the mammal can be effective to kill greater than about 75% (e.g., 75%, 80%, 85%, 90%, 93%, 95%, 98%, 99%, or 100%) of cancer cells within the mammal. In some cases, less than about 1% (e.g., about 1%, about 0.7%, about 0.5%, about 0.25%, or 0%) of cancer cells within a mammal can escape T cell responses in the mammal (e.g., can escape T cell responses and form one or more recurrent tumors in the mammal) that are stimulated by administering viral nucleic acid provided herein and/or viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression to the mammal.
In some cases, when administering viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression provided herein to a mammal (e.g., a human) as described herein, the viral nucleic acid and/or viruses encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be effective to reduce or eliminate APOBEC3B polypeptide activity or expression in infected cells and/or uninfected nearby cells. For example, administering viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression provided herein to a mammal as described herein can be effective to reduce or eliminate APOBEC3B polypeptide activity or expression in infected cancer cells and to kill those infected cancer cells within the mammal. In some cases, administering viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression provided herein to a mammal as described herein can be effective to reduce or eliminate APOBEC3B polypeptide activity or expression in infected cancer cells and to kill those infected cancer cells within the mammal, while being effective to reduce or eliminate APOBEC3B polypeptide activity or expression in nearby uninfected cancer cells influenced by one or more of the inhibitor(s) generated within the infected cells.
In some cases, when administering viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression provided herein to a mammal (e.g., a human) as described herein, the viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be effective to reduce or eliminate APOBEC3B polypeptide activity or expression in non-infected cells (e.g., non-infected bystander cells). In some cases, a non-infected bystander cell can be in direct contact (e.g., cell-cell contact) with an infected cell. In some cases, a non-infected bystander cell can be not in direct contact with an infected cell. When a non-infected bystander cell is not in direct contact with an infected cell, the non-infected bystander cell can in any appropriate location within a mammal (e.g., one or more inhibitors of APOBEC3B polypeptide activity or expression can be released from an infected cell and can travel anywhere in a mammals' body via, for example, the bloodstream to reduce or eliminate APOBEC3B polypeptide activity or expression in a bystander cell). For example, administering viral nucleic acid provided herein and/or viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression to a mammal as described herein can be effective to reduce or eliminate APOBEC3B polypeptide activity or expression in non-infected cancer cells that can be killed via subsequent oncolytic infection or subsequent anti-cancer treatment.
In some cases, oncolytic viruses lacking nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and viral nucleic acid (e.g., viral vectors) provided herein and/or non-oncolytic viruses provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression are administered to a mammal having, or at risk of developing, cancer to treat the mammal, the oncolytic viruses and the viral nucleic acid and/or non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to the mammal at any appropriate time. In some cases, oncolytic viruses containing nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be used. In some cases, one or more viral vectors encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and the one or more oncolytic viruses can be administered to the mammal at the same time. For example, one or more viral vectors encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses can be administered to the mammal at the same time. For example, one or more viral vectors encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and including one or more oncolytic viruses can be administered (e.g., as a single composition) to the mammal. In some cases, one or more viral vectors encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and the one or more oncolytic viruses can be administered to the mammal at the different times. For example, one or more viral vectors encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to the mammal before or after the administration of one or more oncolytic viruses.
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e.g., a human) having, or at risk of developing, cancer. For example, viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. In some cases where viral nucleic acid and/or non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses are administered to a mammal, the viral nucleic acid and/or non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and the one or more oncolytic viruses can be independently formulated with one or more pharmaceutically acceptable carriers (additives) and/or diluents in separate compositions (e.g., a first composition including viral nucleic acid and/or non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and a second composition including one or more oncolytic viruses). In some cases where viral nucleic acid and/or non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression is a viral vector encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and where one or more viral vectors encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses are administered to a mammal, both the viral nucleic acid and/or non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and the one or more oncolytic viruses can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents in a single composition (e.g., a composition including viral nucleic acid and/or non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and including one or more oncolytic viruses). Pharmaceutically acceptable carriers, fillers, and vehicles that may be used in a pharmaceutical composition described herein include, without limitation, dimethyl sulfoxide (DMSO), ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and wool fat.
A composition including viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be designed for administered by any appropriate route. In some cases, administration can be local administration. In some cases, administration can be systemic administration. Examples of routes of administration include, without limitation, intravenous, intramuscular, subcutaneous, oral, intranasal, inhalation, transdermal, parenteral, and intratumoral administration. For example, a composition including viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered locally to a mammal having, or at risk of developing, cancer by injection into or near a tumor within the mammal. For example, a composition including viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered systemically by oral administration to a mammal having, or at risk of developing, cancer. In cases where multiple compositions are administered, a first composition and a second composition can be administered by the same route or can be administered by different routes. For example, a first composition including viral nucleic acid and/or non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and a second composition including one or more oncolytic viruses described herein can be administered to the mammal by a same route (e.g., intratumorally). For example, a first composition including viral nucleic acid and/or non-oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression described herein can be administered to a mammal (e.g., a human) by a first route (e.g., intratumorally), and a second composition including one or more oncolytic viruses described herein can be administered to the mammal by a second route (e.g., intramuscularly).
A composition including viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be any appropriate route type of composition. Examples of compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. Examples of compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
A composition including viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal (e.g., a human) having, or at risk of developing, cancer in any appropriate amount (e.g., any appropriate dose). For example, any appropriate amount of oncolytic virus can be administered to a mammal as described herein. Effective amounts can vary depending on the severity of the cancer, the risk of developing cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician. For example, in cases where a composition includes one or more oncolytic viruses, the composition can include from about 103 plaque-forming units (PFUs) to about 1013 PFUs (e.g., from about 103 to about 1013, from about 105 to about 1013, from about 107 to about 1013, from about 108 to about 1013, from about 1010 to about 1013, from about 103 to about 1010, from about 103 to about 108, from about 103 to about 105, from about 105 to about 1010, from about 104 to about 108, from about 108 to about 1010, or from about 1010 to about 1012 PFUs) of oncolytic viruses can be administered to a mammal as described herein. For example, in cases where a composition includes one or more oncolytic viruses, the composition can include oncolytic viruses at a multiplicity of infection (MOI) of from about 0.0000001 to about 10000 (e.g., from about 0.0000001 to about 1000, from about 0.0000001 to about 100, from about 0.0000001 to about 10, from about 0.0000001 to about 1, from about 0.000001 to about 10000, from about 0.00001 to about 10000, from about 0.0001 to about 10000, from about 0.001 to about 10000, from about 0.01 to about 10000, from about 0.1 to about 10000, from about 1 to about 10000, from about 10 to about 10000, from about 100 to about 10000, from about 1000 to about 10000, from about 0.001 to about 1000, or from about 0.1 to about 100) can be administered to a mammal as described herein. An effective amount of a composition including one or more oncolytic viruses can be any amount that reduces the severity of the cancer without producing significant toxicity to the mammal. The effective amount can remain constant or can be adjusted as a sliding scale or variable amount depending on the mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, severity of the cancer, and risk of developing cancer may require an increase or decrease in the actual effective amount administered.
A composition including viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal (e.g., a human) having, or at risk of developing, cancer in any appropriate frequency. The frequency of administration can be any frequency that reduces the severity of the cancer without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a day to about once a week, from about once a week to about once every two weeks, or from about once every two weeks to about once a month. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, severity of the cancer, and risk of developing cancer may require an increase or decrease in administration frequency.
A composition including viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal (e.g., a human) having, or at risk of developing, cancer for any appropriate duration. An effective duration for administering a composition including viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be any duration that reduces the severity of the cancer without producing significant toxicity to the mammal. For example, the effective duration can vary from several days to several months or years to a lifetime. In some cases, the effective duration for the treatment of a cancer can range in duration from about a month to about a year. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, severity of the cancer, and risk of developing cancer.
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal (e.g., a human) having, or at risk for developing, cancer as the sole active ingredient(s). For example, one or more viral vectors encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more oncolytic viruses can be administered to a mammal having, or at risk for developing, cancer as the sole active ingredients used to treat the mammal. For example, one or more oncolytic viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk of developing, cancer as the sole active ingredient used to treat the mammal.
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal (e.g., a human) having, or at risk for developing, cancer with one or more inhibitors of TNFα polypeptide activity or expression. For example, viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered with one or more inhibitors of TNFα polypeptide activity or expression to a mammal in need thereof. For example, viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk for developing, cancer with one or more inhibitors of TNFα polypeptide activity or expression used to treat the mammal. The one or more inhibitors of TNFα polypeptide activity or expression used to treat a mammal having, or at risk of developing, cancer can include any appropriate inhibitor of TNFα polypeptide activity or expression. An inhibitor of TNFα polypeptide activity or expression can be any molecule that inhibits (e.g., reduces or eliminates) TNFα polypeptide activity or expression. An inhibitor of TNFα polypeptide activity or expression can be any appropriate type of molecule (e.g., nucleic acids such as siRNA molecules, shRNA molecules, antisense molecules, and targeting guide RNA molecules of a CRISPR/Cas9 system; and polypeptides such as antibodies and TALENs). For example, a mammal having cancer can be administered viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and can be administered one or more additional cancer treatments. In cases where a mammal having cancer is treated with viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and is treated with one or more inhibitors of TNFα polypeptide activity or expression, the one or more inhibitors of TNFα polypeptide activity or expression can be administered to a mammal at the same time or independently. For example, viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more inhibitors of TNFα polypeptide activity or expression can be administered to a mammal together (e.g., can be formulated together to form a single composition to be administered to a mammal). In some cases, viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered first, and the one or more inhibitors of TNFα polypeptide activity or expression administered second, or vice versa.
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal (e.g., a human) having, or at risk for developing, cancer together with one or more T cells and/or one or more agents that can stimulate one or more T cells. For example, viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered together with one or more T cells and/or one or more agents that can stimulate one or more T cells to a mammal in need thereof. For example, viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk for developing, cancer together with one or more T cells and/or one or more agents that can stimulate one or more T cells to treat the mammal. The one or more T cells used to treat a mammal having, or at risk of developing, cancer can be any appropriate T cell. In some cases, a T cell can be a tumor reactive T cell. In some cases, a T cell can be a CD8+ T cell or a CD4+ T cell. In some cases, a T cell can be obtained from a mammal to be treated. In some cases, a T cell can be an engineered T cell. For example, a T cell can include (e.g., can be engineered to include) an engineered T cell receptor (TCR) such as a chimeric antigen receptor (CAR). A T cell including a CAR can also be referred to as a CAR T cell. When a T cell is a CAR T cell, the CAR T cell can be any appropriate CART cell (e.g., a CART cell targeting CD19, CD20, and/or CD22). An agent that can stimulate one or more T cells can include any appropriate agent. T cells can be stimulated (e.g., can be contacted with one more agents that can stimulate the T cells) in vivo or in vitro. For example, a mammal having cancer can be administered viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and can be administered one or more T cells. In cases where a mammal having cancer is treated with viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and is treated with one or more T cells, the one or more T cells can be administered to a mammal at the same time or independently. For example, viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more T cells can be administered to a mammal at the same time (e.g., can be formulated together to form a single composition to be administered to a mammal). For example, oncolytic viruses containing nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more CAR T cells can be administered to a mammal at the same time (e.g., can be formulated together to form a single composition to be administered to the mammal). In some cases, viral nucleic acid and/or viruses encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered first, and the one or more T cells administered second, or vice versa. For example, oncolytic viruses containing nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered first, and then the one or more CAR T cells administered second, or vice versa.
In some cases, when viral nucleic acid provided herein and/or viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression are administered to a mammal having, or at risk of developing, cancer together with one or more T cells, cancer cells within the mammal can develop resistance to a T cell therapy at a reduced level (e.g., as compared to a level that develops in a comparable mammal administered a comparable T cell therapy in the absence of any inhibitor of APOBEC3B polypeptide activity or expression).
In some cases, when viral nucleic acid provided herein and/or viruses provided herein encoding one or more inhibitors of APOBEC3B polypeptide activity or expression are administered to a mammal having, or at risk of developing, cancer, cancer cells within the mammal can develop resistance to any other cancer treatment (e.g., radiation therapies, chemotherapies, hormone therapies, targeted therapies, and/or cytotoxic therapies) at a reduced level (e.g., as compared to a level that develops in a comparable mammal administered a comparable chemotherapy and/or a radiation therapy in the absence of any inhibitor of APOBEC3B polypeptide activity or expression).
In some cases, viral nucleic acid (e.g., viral vectors) provided herein and/or viruses (e.g., oncolytic viruses) provided herein encoding (e.g., engineered to encode) one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal (e.g., a human) having, or at risk for developing, cancer with one or more additional cancer treatments. For example, virus particles containing nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered with one or more additional cancer treatments to a mammal in need thereof. For example, virus particles containing nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered to a mammal having, or at risk for developing, cancer together with one or more additional cancer treatments used to treat the mammal. The one or more additional cancer treatments used to treat a mammal having, or at risk of developing, cancer can include any appropriate cancer treatment. In some cases, a cancer treatment can include surgery. In some cases, a cancer treatment can include radiation therapy. In some cases, a cancer treatment can include administration of one or more anti-cancer agents such as a chemotherapies, hormone therapies, targeted therapies, and/or cytotoxic therapies. For example, a mammal having cancer can be administered virus particles containing nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and can be administered one or more additional cancer treatments. In cases where a mammal having cancer is treated with virus particles containing nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and is treated with one or more additional cancer treatments, the one or more additional cancer treatments can be administered to a mammal at the same time or independently. For example, virus particles containing nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression and one or more anti-cancer agents can be administered to a mammal together (e.g., can be formulated together to form a single composition to be administered to a mammal). In some cases, virus particles containing nucleic acid encoding one or more inhibitors of APOBEC3B polypeptide activity or expression can be administered first, and the one or more additional cancer treatments administered second, or vice versa.
In certain instances, a course of treatment and the severity of a cancer can be monitored. Any appropriate method can be used to determine whether or not the severity of a cancer is reduced. For example, the severity of a cancer can be assessed using any appropriate methods and/or techniques, and can be assessed at different time points. For example, physical examination, laboratory tests (e.g., blood and/or urine), biopsy, imaging tests (e.g., X-ray, PET/CT, MRI, and/or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy, and/or genetic tests can be used to determine the severity of a cancer.
In some cases, the level of resistance development (if any) to a particular treatment (e.g., to oncolytic virus treatment or T cell therapy such as CAR T cell therapy) can be monitored following the treatment methods described herein.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
This Example identifies APOBEC3 as an important factor which restricts the potency of oncolytic Vesicular Stomatitis Virus (VSV) in oncolytic virus therapy.
Resistant Tumor Cell Populations Emerge after Oncolytic Virus Treatment
To investigate how tumor cells acquire resistance to viral therapy, murine B16 melanoma cells were infected with repetitive cycles of VSV-GFP at a low MOI (0.01) in vitro (
Oncolytic VSV Resistance is Associated with an IFN-Dependent Upregulation of APOBEC3
VSV-GFP infection of B16 cells induced moderate levels of type I IFN expression 48 hours post infection (
Taken together, these data led us to hypothesize that VSV-mediated induction of both type I interferon signaling and APOBEC3 expression is associated with enhanced tumor cell escape from viral oncolysis.
Consistent with a role for type I interferon induced genes being a key component in the de novo generation of oncolysis-resistant tumor cells, low MOI infection of B16 cells for 7 days with VSV-IFN-β generated more VSV-resistant colonies than did infection with VSV-GFP (
To demonstrate that APOBEC3 contributed to the acquisition of resistance to VSV infection, B16 cells in which expression of APOBEC3 had been knocked down with shRNA, as shown by ELISA and validated by western blot and qrtPCR (
APOBEC3 Inhibition Reduces Escape from VSV Therapy
To investigate whether APOBEC3 played an important role in the in vivo generation of resistance to viral oncolytic therapy, mice bearing 3 day established B16 parental, B16 (scrambled RNA), or B16(shAPOBEC3) tumors were injected with nine doses of VSV-GFP at the site of tumor cell injection. Over two separate experiments, fewer than 10% of mice injected with B16(shAPOBEC3) tumors escaped VSV therapy to form recurrent tumors (
Whether human APOBEC3B may recapitulate the function of murine APOBEC3 to drive the development of VSV-resistant populations in a human Mel888 xenograft tumor model was examined. VSV vectors expressing four shRNAs against hAPOBEC3B individually, or in combination (shRNA 1-4), were generated and knock down of human APOBEC3B in Mel888 cells infected with each vector was confirmed by western blot (
Tumors Overexpressing hAPOBEC3B Readily Escape VSV Therapy
A mutated, enzymatically non-functional (
Consistent with the in vitro data (
hAPOBEC3B Overexpression Reduces VSV Fitness
The data has shown that APOBEC3 contributed to generation of a virus-resistant phenotype of infected tumor cells (
Murine melanoma B16 cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS). B16TK cells were derived from a B16.F1 clone transfected with a plasmid expressing the Herpes Simplex Virus thymidine kinase (HSV-1 TK) gene. Following stable selection in 1.25 μg/mL puromycin, these cells were shown to be sensitive to Ganciclovir (Cymevene) at 5 μg/ml. B16-APOBEC3B and B16-APOBEC3B MUT cells were maintained in DMEM 10% FBS at 37° C. 10% CO2, and selected in hygromycin (200 μg/mL). Baby hamster kidney (BHK) cells were cultures in DMEM supplemented with 10% Fetal Bovine Serum (FBS). All cell lines were maintained at 37° C. 10% CO2 and regularly shown to be free of Mycoplasma infection.
6-8 week old female C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Me.).
VSV was generated from pXN2 cDNA plasmid using the established reverse genetics system in BHK cells as described elsewhere (see, e.g., Whelan et al., Proc Natl Acad Sci USA 92:8388-8392 (1995)). All transgenes were inserted between the viral G and L protein using the XhoI and NheI restriction sites. Virus titers were determined by plaque assay on BHK cells.
B16 cells were infected at an MOI of 0.01 (VSV) for 1 hour, washed with phosphate buffer (PBS), and then incubated for 7 days. Dead cells were removed every 2 days by washing with PBS. After 7 days, the cells were collected by detachment with trypsin, and replated. These cells were subjected to two repeated rounds of infection as previously described. After 21 days, or three total rounds of infection, the remaining virus-escape cells were collected. This protocol was performed in the presence or absence of anti-IFNβ antibody (Rabbit polyclonal anti-mouse interferon beta, (01 interferon source, Piscataway, N.J.), PKC signaling inhibitor (AEB071; 10 μM) (MedChemExpress, Monmouth Junction, NJ), or control IgG (Chrome Pure anti-rabbit IgG; catalog no. 011-000-003; The Jackson Laboratory).
Murine APOBEC3 was measured by Western Blot using a rabbit monoclonal anti-APOBEC3 (184990, Abcam, San Francisco, Calif.); human APOBEC3B was measured by Western Blot using a rabbit polyclonal anti APOBEC3B PA5-11430 (Thermo Fisher). Murine APOBEC3 was measured by rabbit monoclonal anti-human APOBEC3B ELISA (Abcam, San Francisco, Calif.) which reacts with both human APOBEC3B and murine APOBEC3 according to the manufacturer's instructions. Murine IFN-β was measured by direct ELISA (R&D systems) according to the manufacturer's instructions.
RNA was prepared with the QIAGEN-RNeasy-MiniKit (Qiagen, Valencia, Calif.). 1 μg total RNA was reverse-transcribed in a 20 μl volume using oligo-(dT) primers using the First Strand cDNA Synthesis Kit (Roche, Indianapolis, Ind.). A cDNA equivalent of 1 ng RNA was amplified by PCR with gene-specific primers using GAPDH as loading control (mgapdh sense: TCATGACCACAGTCCATGCC (SEQ ID NO:5); mgapdh antisense: TCAGCTCTGGGATGACCTTG (SEQ ID NO:6); APOBEC3 sense: ATGGGACCATTCTGTCTGGGA (SEQ ID NO:7); APOBEC3 antisense: TCAAGACACGGGGGTCCAAG (SEQ ID NO:8)). qRT-PCR was carried out using a LightCycler480 SYBRGreenI Master kit and a LightCycler480 instrument (Roche) according to the manufacturer's instructions. The ΔΔCT method was used to calculate the fold change in expression level of APOBEC3 relative to GAPDH and normalized to an untreated calibrator sample.
Four separate mouse unique 29mer shRNA retroviral constructs, or a single scrambled shRNA encoding retroviral construct (Origene Technologies, Rockville, Md.) were transfected into the GP+E86 ecotropic packaging cell line and supernatant was used to infect B16TK cells to generate the B16TK (shRNA APOBEC3) and B16TK scrambled shRNA populations, respectively. In addition, a single scrambled negative control non-effective shRNA cassette was similarly packaged and used to infect B16TK cells to generate B16TK (scrambled shRNA) cells.
B16TK tumor cell lines were engineered to over express human APOBEC3B, or a catalytically inactive form of the protein APOBEC3B MUT, following infection with either pBABE-Hygro APOBEC3B or pBABE-Hygro APOBEC3B MUT (see, e.g., Pak et al., J Virol 85:8538-8547 (2011)) (See
B16-APOBEC3B MUT or B16-APOBEC3B overexpressing cells were infected with VSV-GFP at an MOI of 0.01 and incubated for 24 hours. Supernatant was collected and 1 mL of supernatant was used to infect a fresh monolayer of cells. This was repeated out to five passages. The DIP assay was done by serially diluting passage 1 and passage 5 of VSV-GFP from B16-APOBEC3B MUT or B16-APOBEC3B cells (1:10 to 1:100,000). Fresh BHK cells were seeded the day before in triplicate wells, and diluted viral supernatants were allowed to adsorb for 1 hour. Stock VSV-GFP virus was then added at an MOI of 20 (8×105 pfu/well) and incubated for 1 hour. Cells were then washed 3× in PBS and fresh supernatant was added. Supernatant was collected 24 hours post infection and tittered by plaque assay by limiting dilution on BHK cells.
All in vivo studies were approved by the Institutional Animal Care and Use Committee at Mayo Clinic. Mice were challenged subcutaneously with 2×105 B16TK melanoma cells, in 100 μL PBS (HyClone, Logan, Utah). All virus injections were delivered intratumorally in 50 μl volume. Tumors were measured 3 times per week, and mice were euthanized when tumors reached 1.0 cm in diameter. Mice were sacrificed upon emergence of neurological symptoms or weight loss.
Survival curves were analyzed by the Log-Rank test. Student's T tests, one way ANOVA and two way ANOVA were applied for in vitro assays as appropriate. Statistical significance was set at p≤0.05 for all experiments.
These results show that oncolytic viral infection with VSV induces a type I IFN-dependent upregulation of APOBEC3 that promotes the generation of virus-resistant tumor cell populations and reduces the oncolytic fitness of the virus itself. For example, these results identify APOBEC3 as a target to improve the efficacy of oncolytic platforms and other viral based therapies. APOBEC3 overexpression generates significant levels of cellular genomic mutations and at least some of these can act to decrease viral replication. Accordingly, APOBEC3 can be targeted during virus infection to improve the therapeutic outcome of oncolytic viral therapy.
This Example shows that weak affinity/low frequency T cell responses against tumor antigens may actively contribute to the ability of tumor cells to evolve away from frontline therapies. Therefore, immunotherapies need to be optimized as early as possible so that, if they do not kill the tumor completely, they do not promote treatment resistance.
Tumor Cell Escape from Therapy is Enhanced by the Presence of Tumor Reactive CD8 T Cells.
B16 cells expressing the HSV-1 thymidine kinase (B16TK) were sensitive to treatment with ganciclovir (GCV) at 5 μg/mL (
A CD8 T Cell Mutator Phenotype is Associated with C-T Mutation of Target Antigen
To investigate this phenomenon in a model with a defined antigenic target, the potential of B16OVA cells to escape therapy when co-cultured with in vitro activated CD8 OT-I T cells was evaluated. At high E:T ratios (50:1 and 10:1), no discrete surviving escape colonies of B16OVA were observed (Table 2). At lower ratios (5:1 and 1:1) individual colonies of B16OVA cells could be isolated as escape variants, and were subsequently resistant to further OT-I killing, even at high E:T ratios (
The first hotspot C to T transition mutation in the ova gene was consistent with the previously reported murine APOBEC3 motif TXC, and both hotspots were consistent with that of the APOBEC3B cytosine deaminase with an A in the +1 position (TCA) (Roberts et al., Nat Genet. 45(9):970-6 (2013); MacMillan et al., J Virol. 87(9):4808-17 (2013); Shi et al., Nat Struct Mol Biol. 24(2):131-139 (2017); Nair et al., J Virol. 88(7):3850-60 (2014); and Chen et al., PLoS Comput Biol. 13(3):e1005471 (2017)). It was therefore hypothesized that T cell interaction may induce an equivalent murine APOBEC3B-like activity in tumor cells, which plays a role in generating cellular mutations that allow for escape from therapy. The expression of mAPOBEC3 was evaluated by qRT-PCR in tumor cells following co-culture with tumor reactive T cells at effector to target ratios at which escape variants were observed.
mAPOBEC3 mRNA expression rose sharply after 12 hours of co-culture with OT-I or T.E. CD8 T cells, as well as following treatment with the PKC activator PMA (
Upregulation of APOBEC3 in tumor cells at suboptimal effector to target ratios inversely correlated with the secretion of the effector cytokine IFNγ (
To show the relationship between the E:T ratio of T cell killing, TNF-α levels, and mAPOBEC3 induction, a transwell co-culture system was used in which B16OVA target tumor cells were co-cultured with effector OT-I T cells in the upper chambers, and (bystander) B16OVA cells were plated in the lower chambers (
Those bystander B16OVA cells which survived in the lower chambers following exposure to TNFα, and in which mAPOBEC3 had been induced (
It was also confirmed that a low E:T ratio of tumor antigen specific T cells to tumor cells induced mAPOBEC3 not only in the B16 cell line but also in both the GL261 glioma line (
mAPOBEC3 induction and tumor cell outgrowth from GCV and T cell therapy was dependent on MHC class I recognition of tumor cells, TNFα secretion, and activation of PKC signaling, as antibody blockade of H-2 Kb, TNFα, or pharmacologic inhibition of PKC by AEB071 ablated the effect (
To confirm that mAPOBEC3 was required for the outgrowth of escape variants in this model, a stable B16TK cell line was generated expressing 4 unique 29mer shRNA constructs targeting mAPOBEC3 as well as a stable B16TK cell line with a single scrambled shRNA construct. mAPOBEC3 expression was significantly reduced in B16TK shRNA mAPOBEC3 cells, both at basal levels, and upon induction with PMA (
These results were validated in vivo where B16TK cells transduced with the scrambled shRNA or the mAPOBEC3-targeting shRNA were implanted subcutaneously and treated with a suboptimal course of GCV. While B16TK (scrambled shRNA) tumors all eventually escaped therapy (0/7 long term survivors) (
As the knockdown of mAPOBEC3 significantly reduced the ability of B16TK cells to escape GCV treatment (
The mechanism of GCV escape was investigated by sequencing the bulk populations from each condition in
hAPOBEC3B Overexpression Drives Tumor Escape
The role of APOBEC3B in the acquisition of therapeutic resistance was evaluated using the retroviral overexpression system of human APOBEC3B used in
Finally, when implanted intracranially in a model of metastatic melanoma, B16TK-hAPOBEC3B tumors grew significantly more quickly following GCV treatment compared to B16TK-hAPOBEC3B MUT tumors (
B16 murine melanoma cells were obtained from the ATCC prior to being modified with the relevant transgenes. Cell lines were authenticated by morphology, growth characteristics, PCR for melanoma specific gene expression (gp100, TYRP-1 and TYRP-2) and biologic behavior, tested mycoplasma-free and frozen. Cells were cultured less than 3 months after thawing. The B16OVA cell line was derived from a B16.F1 clone transfected with a pcDNA3.1ova plasmid described elsewhere (see, e.g., Kaluza et al., Hum Gene Ther. 131:844-854 (2012); and Kaluza et al., Int J Cancer. 131(4):844-54 (2012)). B16OVA cells were grown in DMEM (HyClone, Logan, Utah, USA)+10% FBS (Life Technologies)+5 mg/mL G418 (Mediatech, Manassas, Va., USA) until challenge. GL2610VA was obtained by transfection of parental GL261 cells with pcDNA3.1 OVA. LLCOVA was obtained by transfection of parental LLC cells with pcDNA3.1 OVA. B16TK cells were derived from a B16.F1 clone transfected with a plasmid expressing the Herpes Simplex Virus thymidine kinase (HSV-1 TK) as described elsewhere (see, e.g., Boichard et al., Oncoimmunology. 6(3):e1284719 (2017)). Following stable selection in 1.25 μg/mL puromycin, these cells were shown to be sensitive to Ganciclovir (Cymevene) at 5 μg/ml. Cells were tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza Rockland, Inc. ME, USA).
6-8 week old female C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Me.). The OT-I mouse strain is on a C57Bl/6 background and expresses a transgenic T cell receptor Vα2/Vβ5 specific for the SIINFEKL (SEQ ID NO:9) peptide of ovalbumin in the context of MHC class I, H-2 Kb. The Pmel mouse strain is on a C57Bl/6 background and express a transgenic T cell receptor Vα1/Vβ13 that recognizes amino acids 25-33 of gp100 presented by H2-db.
Wild-type Reovirus type 3 (Dearing strain) was obtained from Oncolytics Biotech (Calgary, AB, Canada) and stock titers were measured by plaque assay on L929 cells.
B16TK cells were seeded in 96 well plates in triplicate and treated with reovirus (MOI 0.1) or with GCV (Cymevene) at 5 μg/ml. Cell titer blue (Promega, Madison, Wis.) was added to wells at 10% v/v and fluorescence was measured after approximately 4 hours incubation (560Ex/590Em). Relative viability of experimental conditions was normalized to untreated cells.
Spleens were immediately excised from euthanized C57Bl/6, OT-I mice and dissociated in vitro to achieve single-cell suspensions. Red blood cells were lysed with ACK lysis buffer. CD8 T cells were prepared using the CD8a T Cell Isolation kit (Miltenyi, Auburn, Calif.) and co-cultured with target tumor cells at various effector to target ratios as described in the text. Supernatants were assayed for IFNγ by ELISA as directed in the manufacturer's instructions (Mouse IFN-γ ELISA Kit, OptEIA, BD Biosciences, San Diego, Calif.).
OT-I or Pmel T cells were activated in IMDM (Gibco, Grand Island, N.Y., USA)+5% FBS+1% Pen/Strep+40 μM 2-ME. Media was supplemented with the SIINFEKL (SEQ ID NO:9) or KVPRNQDWL (SEQ ID NO:10) peptides respectively at 1 μg/mL and human IL2 at 50 U/mL. Cells were used for in vitro assays following 4 days of activation.
CD8 T cells were prepared as described above from C57BL/6 mice that had been cured of subcutaneous B16TK tumors following three weekly courses of GCV (50 mg/kg on days 5-9, 12-16, and 19-23). Cells were harvested between 60 and 80 days post tumor implantation.
B16TK or B16OVA cells were plated in triplicate wells in the presence of GCV (Cymevene) at 5 μg/ml, reovirus (MOI 0.1) or 4-day in vitro activated OT-I CD8T cells or T.E. CD8 T cells (E:T ratio of 5:1) for 7 days in Iscove's Modified Dulbecco's Medium (IMDM; Gibco, Grand Island, N.Y.)+5% FBS+1% Pen-Strep+40 μL β-mercaptoethanol. Wells were washed 3 times with PBS and cultured in normal medium for a further 7 days. Surviving cells were then cultured again in the presence of PBS, GCV, reovirus (MOI 0.1) or 4-day in vitro activated OT-I CD8 T cells or T.E. CD8 T cells (various effector to target ratios) for 7 days.
These co-culture systems were also performed with the anti-H-2 Kb antibody (AF6-88.5; 0.5 μg/mL) (Biolegend, San Diego, Calif.), the inhibitor of PKC signaling (AEB071; 10 μM) (MedChemExpress, Monmouth Junction, NJ) or the anti-TNFα antibody (AF-410-NA; 0.5 μg/ml) (R&D Systems; Minneapolis, Minn.) or the anti-IFNγ antibody (MAB485; 0.5 μg/ml) (R&D Systems; Minneapolis, Minn.).
RNA was prepared with the QIAGEN-RNeasy-MiniKit (Qiagen, Valencia, Calif.). 1 μg total RNA was reverse-transcribed in a 20 μl volume using oligo-(dT) primers using the First Strand cDNA Synthesis Kit (Roche, Indianapolis, Ind.). A cDNA equivalent of 1 ng RNA was amplified by PCR with gene-specific primers using GAPDH as loading control (mgapdh sense: TCATGACCACAGTCCATGCC (SEQ ID NO:5); mgapdh antisense: TCAGCTCTGGGATGACCTTG (SEQ ID NO:6); APOBEC3 sense: ATGGGACCATTCTGTCTGGGA (SEQ ID NO:7); APOBEC3 antisense: TCAAGACACGGGGGTCCAAG (SEQ ID NO:8)). qRT-PCR was carried out using a LightCycler480 SYBRGreenI Master kit and a LightCycler480 instrument (Roche) according to the manufacturer's instructions. The ΔΔCT method was used to calculate the fold change in expression level of APOBEC3 and GAPDH as an endogenous control for all treated samples relative to an untreated calibrator sample.
The OVA transgene was sequenced using the following primers:
Sense: ATGGGCTCCATCGGCGCAGC (SEQ ID NO:11) and antisense: CCGTCTACACAAAGGGGAATT (SEQ ID NO:12) and aligned to the reference sequence CAA23682.1. The HSV TK transgene was sequenced using the following primers: CACGCAGATGCAGTCGGGGCGGCG (SEQ ID NO:13) (Downstream of the EcoR1 site in the 5′UTR), CTGGTGGCCCTGGGTTCGCGCGA (SEQ ID NO:14), GCGTTCGTGGCCCTCATCCC (SEQ ID NO:15), GCCTGGGCCTTGGACGTCTTGG (SEQ ID NO:16), and AGGGCGCAACGCCGTACGTCG (SEQ ID NO:17) and aligned to the reference sequence AB009254.2.
Murine APOBEC3 was measured by western blotting with a rabbit polyclonal (PA511430, Thermo Fisher) or a rabbit monoclonal anti-human APOBEC3B (184990, Abcam, San Francisco, Calif.) which react with both human APOBEC3B and murine APOBEC3 (Thermo Fisher) or by ELISA according to the manufacturer's instructions (Antibody Research Corporation, St Charles, Mo.). B16TK cells were treated with recombinant murine TNFα (R&D Systems, Minneapolis, Minn.). HSV TK protein was detected by western blotting tumor cell lysates with a goat polyclonal antibody (28038; Santa Cruz, Dallas, Tex.). β-actin was detected using an HRP conjugated mouse monoclonal antibody (clone AC-15; Sigma, St. Louis, Mo.).
Mouse unique 29mer shRNA retroviral constructs (Origene Technologies, Rockville, Md.) were tested individually, or as a combination, for their ability to reduce expression of murine APOBEC3 in B16 cells compared to a single scrambled shRNA encoding retroviral construct. Optimal knockdown for periods of more than two weeks in culture was achieved using all four constructs pre-packaged as retroviral particles in the GP+E86 ecotropic packaging cell line and used to infect B16 cells at an MOI of ˜10 per retroviral construct. In addition, a single scrambled negative control non-effective shRNA cassette was similarly packaged and used to infect B16TK cells to generate B16TK (scrambled shRNA) cells.
B16TK cells were infected with a retroviral vector encoding either full length functional APOBEC3B or a mutated, non-functional form of APOBEC3B as a negative control. Infected populations were selected for 7 days in hygromycin to generate B16TK (APOBEC3B) or B16TK (APOBEC3B MUT) cell lines and used for experiments as described. In populations of B16TK (APOBEC3B) cells selected for more than 7-10 days in hygromycin expression of APOBEC3B returned to basal levels associated with the toxicity of prolonged APOBEC3B expression. Murine APOBEC3 (Accession: BC003314) was expressed from the pCMV-SPORT6 plasmid obtained from Dharmacon, Lafayette, Colo.)
All in vivo studies were approved by the Institutional Animal Care and Use Committee at Mayo Clinic. Mice were challenged subcutaneously with 2×105 B16TK melanoma cells, in 100 μL PBS (HyClone, Logan, Utah, USA) or with 1×104 cells in 2 uL intracranially into the frontal lobe as described elsewhere (see, e.g., Carlson et al., Curr Protoc Pharmacol. Chapter 14:Unit 14-16 (2011)). Subcutaneous tumors were treated with a two or three-week course of GCV (50 mg/kg) administered IP daily. Tumors were measured 3 times per week, and mice were euthanized when tumors reached 1.0 cm in diameter. Intracranial tumors were treated with a three-week courses of GCV (50 mg/kg) administered IP daily. Mice were sacrificed upon emergence of neurological symptoms or weight loss.
Survival curves were analyzed by the Log-Rank test. Student's T tests, one-way ANOVA and two-way ANOVA were applied for in vitro assays as appropriate. Statistical significance was set at p≤0.05 for all experiments.
These results suggest that the generation of weak affinity and/or low frequency, sub-optimal T cell responses against TAAs may actively drive a mutator phenotype in tumors through APOBEC3 activity and promote the emergence of treatment resistant tumor populations. Hence, whenever potentially immunogenic frontline therapies are being administered to patients, it will be important to try to optimize the generation of potent CD8 T cell responses, with high frequencies of high affinity circulating anti-tumor T cells in order to reduce any T cell mediated induction of tumor escape and recurrence.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/819,333, filed Mar. 15, 2019. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/022252 | 3/12/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62819333 | Mar 2019 | US |
